WO2022006278A1 - Reducing fibrosis and treating related diseases, disorders, and conditions - Google Patents

Reducing fibrosis and treating related diseases, disorders, and conditions Download PDF

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Publication number
WO2022006278A1
WO2022006278A1 PCT/US2021/039899 US2021039899W WO2022006278A1 WO 2022006278 A1 WO2022006278 A1 WO 2022006278A1 US 2021039899 W US2021039899 W US 2021039899W WO 2022006278 A1 WO2022006278 A1 WO 2022006278A1
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compound
patient
mean
population
disease
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PCT/US2021/039899
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French (fr)
Inventor
Shakil ASLAM
III John Francis NEYLAN
Teresa M. ALVAREZ-DIEZ
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Angion Biomedica Corp.
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Publication of WO2022006278A1 publication Critical patent/WO2022006278A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/4965—Non-condensed pyrazines
    • A61K31/497—Non-condensed pyrazines containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys

Definitions

  • Fibrotic diseases contribute to as much as 45% of deaths in the industrialized world (Wynn, T. A. J. Pathol. 2008;214:199-210). Fibrosis, i.e., excessive tissue scarring, is a common feature of many chronic diseases, as well as acute injuries, where accumulation of scar tissue can lead to loss of organ function and, ultimately, organ failure (Friedman, S. U, et al. Sci. Transl. Med. 2019 Jan 9;5(167):167srl).
  • the present disclosure provides certain technologies for reducing fibrosis.
  • the present disclosure provides certain technologies for reducing fibrosis of, e.g., gastrointestinal tract, heart, kidney, lung, liver, muscle, pancreas, and/or skin.
  • the present disclosure provides methods for treating (e.g., lessening the severity of, such as by delaying onset and/or reducing degree and/or frequency of one or more features of) a disease, disorder, or condition associated with fibrosis.
  • such methods comprise, for example, administering an antifibrotic agent (e.g., a small molecule, such as Compound 1 described herein).
  • an antifibrotic agent e.g., a small molecule, such as Compound 1 described herein.
  • the present disclosure also provides methods of administering an antifibrotic agent (e.g., Compound 1 described herein) to a subject or a population of subjects in need thereof.
  • an antifibrotic agent e.g., Compound 1 described herein
  • the present disclosure encompasses the recognition that particular modes of administering an antifibrotic agent (e.g., Compound 1 described herein) achieve one or more particular effects, e.g., in a population of healthy subjects or in a population of subjects suffering from primary glomerular disease and/or persistent proteinuria.
  • the present disclosure demonstrates that administration of an antifibrotic agent (e.g., Compound 1 described herein) achieves different pharmacokinetic properties (e.g., higher mean Cmax and/or mean AUCo-iast) than what was expected based on standard animal models.
  • the present disclosure demonstrates that an antifibrotic agent (e.g., Compound 1 described herein) is suitable for administration once daily and/or twice daily. In some embodiments, the present disclosure demonstrates that an antifibrotic agent (e.g., Compound 1 described herein) is well-tolerated, e.g., in healthy subjects, e.g., at doses suitable to achieve one or more particular effects (e.g., certain PK parameters).
  • an antifibrotic agent e.g., Compound 1 described herein
  • the present disclosure demonstrates that administration of an antifibrotic agent (e.g., Compound 1 described herein) achieves one or more particular effects (e.g., higher mean Cmax and/or comparable mean AUCo-iast) when administered to subjects in one fed/fasted state, compared to subjects in another fed/fasted state.
  • administration of an antifibrotic agent e.g., Compound 1 described herein
  • achieves different effects e.g., outcomes indicative of reduced fibrosis
  • the present disclosure encompasses the recognition that one or more properties of an antifibrotic agent (e.g., Compound 1 described herein) make it particularly suitable for treating subjects with certain fibrotic diseases or disorders.
  • an antifibrotic agent e.g., Compound 1 described herein
  • the present disclosure demonstrates that Compound 1 may be suitable to treat such subjects, e.g., as indicated by one or more properties described herein.
  • FIG. 1 is a graph of mean Compound 1 blood concentration over time from SAD cohorts in a fasted state.
  • FIG. 2 is a graph of mean Compound 1 plasma concentration over time in SAD food effect cohorts receiving 200 mg Compound 1.
  • FIG. 3 depicts a Western analysis demonstrating inhibition of phosphorylation of PDGFRp at tyrosine 751 by Compound 1 in serum-starved hepatic stellate cells.
  • FIG. 4 depicts a Western analysis demonstrating inhibition of phosphorylation of VEGFR2 at tyrosine 1175 by Compound 1 in serum-starved HUVEC cells.
  • FIG. 5 depicts results of a KINOMEscan profile of Compound 1 in small airway epithelial cells and lung fibroblasts (SAEMyF); lung fibroblasts only (MyoF); and renal proximal tubular epithelial cells and lung fibroblasts (REMyoF).
  • SAEMyF small airway epithelial cells and lung fibroblasts
  • MyoF lung fibroblasts only
  • REMyoF renal proximal tubular epithelial cells and lung fibroblasts
  • FIG. 6 is a graph showing soluble collagen concentrations in normal human lung fibroblasts (NHLF) treated with TGFpi and Compound 1.
  • FIG. 7A is a graph showing lung-to-body weight ratio of mice treated with Compound 1 in a bleomycin-induced injury model of idiopathic pulmonary fibrosis (IPF).
  • FIG. 7B is a graph showing hydroxyproline levels in mice treated with Compound 1 in a bleomycin- induced injury model of IPF.
  • FIG. 7C is a graph showing histopathological damage in lung tissue sections using the Ashcroft scale from mice treated with Compound 1 in a bleomycin- induced injury model of IPF.
  • FIG. 7D is a graph showing extent of picrosirius red staining in lung tissue sections from mice treated with Compound 1 in a bleomycin-induced injury model of IPF.
  • FIG. 7E is a graph showing extent of immunohistological staining for TGFpi in lung tissue sections from mice treated with Compound 1 in a bleomycin-induced injury model of IPF.
  • FIG. 8A is a graph showing lung fibrosis score using Ashcroft scale based on H&E staining in lung tissue sections from mice treated with Compound 1 in an inducible TGFpi mouse model of lung fibrosis.
  • FIG. 8B is a graph hydroxyproline levels in mice treated with Compound 1 in an inducible TGFpi mouse model of lung fibrosis.
  • FIG. 8C is a graph showing extent of picrosirius red staining in lung tissue sections from mice treated with Compound 1 in an inducible TGFpi mouse model of lung fibrosis.
  • FIG. 8A is a graph showing lung fibrosis score using Ashcroft scale based on H&E staining in lung tissue sections from mice treated with Compound 1 in an inducible TGFpi mouse model of lung fibrosis.
  • FIG. 8B is a graph hydroxyproline levels in mice treated with Compound 1 in an inducible TGFpi mouse model of lung fibrosis.
  • FIG. 8C is
  • FIG. 8D is a graph showing extent of aSMA staining in lung tissue sections from mice treated with Compound 1 in an inducible TGFpi mouse model of lung fibrosis.
  • FIG. 9A is a graph showing dermal thickness, measured as the distance between epidermal-dermal junction and dermal-subcutaneous fat junction in H&E-stained slides prepared from a skin biopsy from mice treated with Compound 1 in a bleomycin systemic sclerosis mouse model.
  • FIG. 9B is a graph showing skin fibrotic score, judged based on H&E-stained slides prepared from a skin biopsy from mice treated with Compound 1 in a bleomycin systemic sclerosis mouse model.
  • FIG. 9A is a graph showing dermal thickness, measured as the distance between epidermal-dermal junction and dermal-subcutaneous fat junction in H&E-stained slides prepared from a skin biopsy from mice treated with Compound 1 in a bleomycin systemic sclerosis mouse model.
  • FIG. 9B is
  • FIG. 9C is a graph showing lung hydroxyproline levels from mice treated with Compound 1 in a bleomycin systemic sclerosis mouse model.
  • FIG. 9D is a graph showing lung fibrosis score using Ashcroft scale based on H&E staining in lung tissue sections from mice treated with Compound 1 in a bleomycin systemic sclerosis mouse model.
  • FIG. 9E is a graph showing kidney hydroxyproline levels from mice treated with Compound 1 in a bleomycin systemic sclerosis mouse model.
  • FIG. 9F is a graph showing extent of picrosirius red staining in kidney tissue sections from mice treated with Compound 1 in a bleomycin systemic sclerosis mouse model.
  • FIG. 10A is a graph showing urine protein levels in rats treated with Compound 1 in a FSGS-relevant model of PAN-induced proteinuria.
  • FIG. 10B is a graph showing intraperitoneal fluid volume in rats treated with Compound 1 in a FSGS-relevant model of PAN- induced proteinuria.
  • FIG. IOC is a graph showing GFR, measured using FITC-sinistrin decay kinetics, in rats treated with Compound 1 in a FSGS-relevant model of PAN-induced proteinuria.
  • FIG. 10D is a graph showing glomerular diameter, measured using histopathological analysis of P AS-stained renal coronal sections from rats treated with Compound 1 in a FSGS-relevant model of PAN-induced proteinuria.
  • FIG. 10E is a graph showing collagen-3 expression, determined in glomeruli using quantitative immunohistochemistry, in rats treated with Compound 1 in a FSGS- relevant model of PAN-induced proteinuria.
  • FIG. 11A is a graph showing urine protein levels in rats treated with Compound 1 in a DOCA/salt model of renal fibrosis and injury.
  • FIG. 1 IB is a graph showing urine albumin levels in rats treated with Compound 1 in a DOCA/salt model of renal fibrosis and injury.
  • FIG. llC is a graph showing urine albumin to creatinine ratio in rats treated with Compound 1 in a DOCA/salt model of renal fibrosis and injury.
  • FIG. 1 ID is a graph showing urine kidney injury molecule-1 (KIM1) levels in rats treated with Compound 1 in a DOCA/salt model of renal fibrosis and injury.
  • KIM1 urine kidney injury molecule-1
  • FIG. 11E is a graph showing renal hydroxyproline levels in rats treated with Compound 1 in a DOCA/salt model of renal fibrosis and injury.
  • FIG. 1 IF is a graph showing renal damage scores, based on H&E stained slides of kidney tissue sections from rats treated with Compound 1 in a DOCA/salt model of renal fibrosis and injury.
  • FIG. 11G is a graph showing extent of picrosirius red staining from slides of kidney tissue sections from rats treated with Compound 1 in a DOCA/salt model of renal fibrosis and injury.
  • FIG. 11H is a graph showing extent of aSMA staining in kidney tissue sections from rats treated with Compound 1 in a DOCA/salt model of renal fibrosis and injury.
  • EIUO unilateral ureteral obstruction
  • FIG. 13A is a graph showing serum creatinine (SCr) levels in rats treated with Compound 1 in a PCK rat model of polycystic kidney disease.
  • FIG. 13B is a graph showing BUN levels in rats treated with Compound 1 in a PCK rat model of polycystic kidney disease.
  • FIG. 13C is a graph showing kidney weight at sacrifice in rats treated with Compound 1 in a PCK rat model of polycystic kidney disease.
  • FIG. 13D is a graph showing kidney weight as a percentage of body weight at sacrifice rats treated with Compound 1 in a PCK rat model of polycystic kidney disease.
  • FIG. 13E is a graph showing kidney hydroxyproline levels in rats treated with Compound 1 in a PCK rat model of polycystic kidney disease.
  • FIG. 13F is a graph showing cystic index, quantified in H&E stained kidney tissue sections using digital planimetry, in rats treated with Compound 1 in a PCK rat model of polycystic kidney disease.
  • FIG. 13G is a graph showing 24-hour urine volume in rats treated with Compound 1 in a PCK rat model of polycystic kidney disease.
  • FIG. 13H is a graph showing urine protein levels in rats treated with Compound 1 in a PCK rat model of polycystic kidney disease.
  • FIG. 131 is a graph showing urine albumin levels in rats treated with Compound 1 in a PCK rat model of polycystic kidney disease.
  • FIG. 13J is a graph showing urine neutrophil gelatinase-associated lipocalin (NGAL) levels in rats treated with Compound 1 in a PCK rat model of polycystic kidney disease.
  • FIG. 13K is a graph showing urine KIM1 levels in rats treated with Compound 1 in a PCK rat model of polycystic kidney disease.
  • FIG. 13L is a graph showing urine interleukin 18 (IL18) levels in rats treated with Compound 1 in a PCK rat model of polycystic kidney disease.
  • FIG. 13M is a graph showing urine cystatin C levels in in rats treated with Compound 1 in a PCK rat model of polycystic kidney disease.
  • FIG. 14A is a graph showing body weight after sacrifice in mice treated with Compound 1 in a TNBS model of inflammatory bowel disease/acute colitis.
  • FIG. 14B is a graph showing colon weight after sacrifice in mice treated with Compound 1 in a TNBS model of inflammatory bowel disease/acute colitis.
  • FIG. 14C is a graph showing colon length after sacrifice in mice treated with Compound 1 in a TNBS model of inflammatory bowel disease/acute colitis.
  • FIG. 14D is a graph showing colon damage score in mice treated with Compound 1 in a TNBS model of inflammatory bowel disease/acute colitis.
  • FIG. 14A is a graph showing body weight after sacrifice in mice treated with Compound 1 in a TNBS model of inflammatory bowel disease/acute colitis.
  • FIG. 14B is a graph showing colon weight after sacrifice in mice treated with Compound 1 in a TNBS model of inflammatory bowel disease/acute colitis.
  • FIG. 14C is a graph showing
  • FIG. 14E is a graph showing colon histopathological scores of H&E-stained colon tissue sections from mice treated with Compound 1 in a TNBS model of inflammatory bowel disease/acute colitis. Each of (a) colon architecture, (b) degree of inflammatory cell infiltration, (c) muscle thickening, and (d) crypt damage and goblet cells loss was assessed.
  • FIG. 14F is a graph showing composite colon histopathological scores of H&E-stained colon tissue sections from mice treated with Compound 1 in a TNBS model of inflammatory bowel disease/acute colitis.
  • FIG. 14E is a graph showing colon histopathological scores of H&E-stained colon tissue sections from mice treated with Compound 1 in a TNBS model of inflammatory bowel disease/acute colitis.
  • FIG. 14G is a graph showing extent of Alcian blue staining in slides of colon tissue sections from mice treated with Compound 1 in a TNBS model of inflammatory bowel disease/acute colitis.
  • FIG. 14H is a graph showing extent of myeloperoxidase staining in slides of colon tissue sections from mice treated with Compound 1 in a TNBS model of inflammatory bowel disease/acute colitis.
  • FIG. 141 is a graph showing extent of F4/80 staining in slides of colon tissue sections from mice treated with Compound 1 in a TNBS model of inflammatory bowel disease/acute colitis.
  • FIG. 15A is a graph showing body weight in mice prior to treatment with Compound 1 in an acetic acid induced colitis model.
  • FIG. 15B is a graph showing colon weight in mice prior to treatment with Compound 1 in an acetic acid induced colitis model.
  • FIG. 15C is a graph showing colon length in mice prior to treatment with Compound 1 in an acetic acid induced colitis model.
  • FIG. 15D is a graph showing gross morphological colon damage score in mice prior to treatment with Compound 1 in an acetic acid induced colitis model.
  • FIG. 15E is a graph showing colon length in mice treated with Compound 1 in an acetic acid induced colitis model.
  • FIG. 15F is a graph showing gross morphological colon damage score in mice treated with Compound 1 in an acetic acid induced colitis model.
  • FIG. 15G is a graph showing histopathological colon damage score in mice treated with Compound 1 in an acetic acid induced colitis model.
  • FIG. 15H is a graph showing extent of Alcian blue staining in slides of colon tissue sections from mice treated with Compound 1 in an acetic acid induced colitis model.
  • FIG. 16A is a graph showing colon length in mice treated with Compound 1 in a dextran sulfate sodium model of chronic colitis in inflammatory bowel disease.
  • FIG. 16B is a graph showing macroscopic colon damage score in mice treated with Compound 1 in a dextran sulfate sodium model of chronic colitis in inflammatory bowel disease.
  • FIG. 16C is a graph showing colon hydroxyproline levels in mice treated with Compound 1 in a dextran sulfate sodium model of chronic colitis in inflammatory bowel disease.
  • FIG. 16D is a graph showing histopathological injury score from H&E-stained slides of colon tissue section from mice treated with Compound 1 in a dextran sulfate sodium model of chronic colitis in inflammatory bowel disease.
  • FIG. 17A is a graph showing lung hydroxyproline levels in mice treated with Compound 1 or pirfenidone (PFD) in a bleomycin-induce lung fibrosis model.
  • FIG. 17B is a graph showing Ashcroft lung histopathological score in lung tissue sections from mice treated with Compound 1 or pirfenidone (PFD) in a bleomycin-induce lung fibrosis model.
  • FIG. 17C is a graph showing extent of picrosirius red staining in lung tissue sections from mice treated with Compound 1 or pirfenidone (PFD) in a bleomycin-induce lung fibrosis model.
  • FIG. 17D is a graph showing extent of TGFpi staining in lung tissue sections from mice treated with Compound 1 or pirfenidone (PFD) in a bleomycin-induce lung fibrosis model.
  • FIG. 18 provides a cartoon schematic of varying relationships between genomic responses and kidney disease outcomes.
  • FIG. 19 provides proteinuria of PAN treated model rats treated with Compound 1.
  • FIG. 20 provides fold change in expression for COL1 A1 mRNA rat PAN model with treatment of Compound 1.
  • FIG. 21 provides fold change in expression for COL3 A1 mRNA rat PAN model with treatment of Compound 1.
  • FIG. 22 depicts a Compound 1 interactome built using in vitro activity data.
  • FIG. 23 provides urine protein levels in PAN treated model rats.
  • FIG. 24A provides COL3 A1 mRNA levels in PAN-treated model rats.
  • FIG. 24B provides an association of COL3 A1 mRNA levels and urine protein values.
  • FIG. 25A depicts a glomerular COL3 A1 interactome.
  • FIG. 25B depicts a tubular COL3A1 interactome.
  • FIG. 26A is a graph showing that, prior to randomization, proteinuria of Alport mice is elevated compared to wild-type mice.
  • FIG. 26B is a Kaplan Myer survival graph of Alport mice treated with Compound 1 vs. Alport mice treated with vehicle.
  • FIG. 26C and FIG. 26D are graphs of body weight and kidney weight, respectively, for mice in an Alport model study.
  • FIG. 26E is a graph of proteinuria in Alport mice treated with Compound 1 vs. Alport mice treated with vehicle.
  • FIG. 26F is a graph of protein-to-creatinine ratio in Alport mice treated with Compound 1 vs. Alport mice treated with vehicle.
  • 26G is a graph of serum BUN levels in Alport mice treated with Compound 1 vs. Alport mice treated with vehicle.
  • FIG. 26H is a graph of serum creatinine levels in Alport mice treated with Compound 1 vs. Alport mice treated with vehicle.
  • FIG. 261 is a graph of kidney hydroxyproline levels in Alport mice treated with Compound 1 vs. Alport mice treated with vehicle.
  • FIG. 26 J is a graph of kidney injury scores based on H&E stained kidney samples in Alport mice treated with Compound 1 vs. Alport mice treated with vehicle.
  • FIG. 26K is a graph of renal fibrosis scores based on Masson’s Trichome- stained kidney samples in Alport mice treated with Compound 1 vs. Alport mice treated with vehicle.
  • 26L is a graph of IHC staining for TGFpi in Alport mice treated with Compound 1 vs. Alport mice treated with vehicle.
  • FIG. 26M is a graph of IHC staining for aSMA in Alport mice treated with Compound 1 vs. Alport mice treated with vehicle.
  • FIG. 26N is a graph of IHC staining for Collagen- 1 in Alport mice treated with Compound 1 vs. Alport mice treated with vehicle.
  • FIG. 260 is a graph of picrosirius red staining for collagen in Alport mice treated with Compound 1 vs. Alport mice treated with vehicle.
  • FIG. 26P depicts a Western blot analysis for collagen 1, aSMA, TGFpi, and PDGFR in kidney tissue samples from sacrificed Alport mice.
  • FIG. 26Q is a graph of densitometric analysis of a Western blot for collagen 1, aSMA, TGFpi, and PDGFR in Alport mice treated with Compound 1 vs. Alport mice treated with vehicle.
  • FIG. 27A is a Kaplan Meier curve showing survival analysis of rats in a Passive Heymann nephritis model.
  • FIG. 27B is a graph of body mass of rats in a Passive Heymann nephritis model.
  • FIG. 27C is a graph of kidney mass of rats in a Passive Heymann nephritis model.
  • FIG. 27D is a graph of kidney mass to body mass ratio of rats in a Passive Heymann nephritis model.
  • FIG. 27E is a graph of protein-to-creatinine ratio (PCR) of rats in a Passive Heymann nephritis model.
  • FIG. 27F is a graph plotting PCR of rats in a Passive Heymann nephritis model from Day 7 until end of study.
  • FIG. 27G is a graph of serum cholesterol of rats in a Passive Heymann nephritis model.
  • FIG. 27H is a graph of serum triglycerides of rats in a Passive Heymann nephritis model.
  • FIG. 271 is a graph of serum creatinine of rats in a Passive Heymann nephritis model.
  • FIG. 27J is a graph of BUN of rats in a Passive Heymann nephritis model.
  • FIG. 27K is a graph of hydroxyproline content per kidney in rats in a Passive Heymann nephritis model.
  • FIG. 27L depicts representative Trichrome-stained kidney tissue samples from rats in a Passive Heymann nephritis model.
  • FIG. 27M depicts representative PAS-stained kidney tissue samples from rats in a Passive Heymann nephritis model.
  • FIG. 27N is a graph of glomerular damage score in rats in a Passive Heymann nephritis model.
  • FIG. 270 is a Western blot analysis on total kidney lysates from rats in a Passive Heymann nephritis model.
  • FIG. 27P is a graph of PDGFRP levels, normalized to GAPDH, from rats in a Passive Heymann nephritis model.
  • FIG. 28 summarizes pre-screening and screening processes for a Phase 2
  • FIG. 29 is a graph of mean oral blood PK profile of Compound 1 over time from SAD cohorts in a fasted state.
  • FIG. 30A is a graph of mean oral blood PK profile of Compound 1 over time from BID MAD cohorts in a fasted state at Day 1.
  • FIG. 30B is a graph of mean oral blood PK profile of Compound 1 over time from BID MAD cohorts in a fasted state at Day 14.
  • FIG. 31 is a graph of mean Compound 1 blood concentration vs. time profiles at steady state for QD MAD cohorts in the fed state at Day 14.
  • FIG. 32 is a graph of Compound 1 blood concentration vs. time in a food effect cross-over study with a 600 mg single dose.
  • administering typically refers to administration of a composition to a subject to achieve delivery of an active agent to a site of interest (e.g., a target site which may, in some embodiments, be a site of disease or damage, and/or a site of responsive processes, cells, tissues, etc.)
  • a site of interest e.g., a target site which may, in some embodiments, be a site of disease or damage, and/or a site of responsive processes, cells, tissues, etc.
  • a site of interest e.g., a target site which may, in some embodiments, be a site of disease or damage, and/or a site of responsive processes, cells, tissues, etc.
  • a site of interest e.g., a target site which may, in some embodiments, be a site of disease or damage, and/or a site of responsive processes, cells, tissues, etc.
  • one or more particular routes of administration may be feasible and/or useful in the practice of the present disclosure.
  • administration may be parenteral
  • the term “comparable” refers to two or more agents, entities, situations, sets of conditions, circumstances, individuals, or populations, etc. that may not be identical to one another but that are sufficiently similar to permit comparison there between so that one skilled in the art will appreciate that conclusions may reasonably be drawn based on differences or similarities observed.
  • comparable agents, entities, situations, sets of conditions, circumstances, individuals, or populations are characterized by a plurality of substantially identical features and one or a small number of varied features.
  • dosage form may be used to refer to a physically discrete unit of an active agent (e.g., a therapeutic agent) for administration to a subject.
  • an active agent e.g., a therapeutic agent
  • each such unit contains a predetermined quantity of active agent.
  • such quantity is a unit dosage amount (or a whole fraction thereof) appropriate for administration in accordance with a dosing regimen that has been determined to correlate with a desired or beneficial outcome when administered to a relevant population (i.e., with a therapeutic dosing regimen).
  • the total amount of a therapeutic composition or agent administered to a particular subject is determined by one or more attending physicians and may involve administration of multiple dosage forms.
  • salt form refers to a form of a relevant compound as a salt appropriate for use in pharmaceutical contexts, i.e., salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and/or lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio.
  • Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge, et al. describes pharmaceutically acceptable salts in detail in ./. Pharmaceutical Sciences , 66: 1-19 (1977).
  • the term “reference” describes a standard or control relative to which a comparison is performed. For example, in some embodiments, an agent, individual, population, sample, sequence or value of interest is compared with a reference or control agent, individual, population, sample, sequence or value. In some embodiments, a reference or control is tested and/or determined substantially simultaneously with the testing or determination of interest. In some embodiments, a reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, as would be understood by those skilled in the art, a reference or control is determined or characterized under comparable conditions or circumstances to those under assessment. Those skilled in the art will appreciate when sufficient similarities are present to justify reliance on and/or comparison to a particular possible reference or control.
  • risk of a disease, disorder, and/or condition refers to a likelihood that a particular individual will develop the disease, disorder, and/or condition. In some embodiments, risk is expressed as a percentage. In some embodiments, risk is from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 up to 100%. In some embodiments risk is expressed as a risk relative to a risk associated with a reference sample or group of reference samples. In some embodiments, a reference sample or group of reference samples have a known risk of a disease, disorder, condition and/or event. In some embodiments a reference sample or group of reference samples are from individuals comparable to a particular individual. In some embodiments, relative risk is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.
  • the term “subject” refers an organism, typically a mammal (e.g., a human). In some embodiments, a subject is suffering from a relevant disease, disorder or condition. In some embodiments, a human subject is an adult, adolescent, or pediatric subject.
  • a subject is at risk of (e.g., susceptible to), e.g., at elevated risk of relative to an appropriate control individual or population thereof, a disease, disorder, or condition.
  • a subject displays one or more symptoms or characteristics of a disease, disorder or condition.
  • a subject does not display any symptom or characteristic of a disease, disorder, or condition.
  • a subject is someone with one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition.
  • a subject is an individual to whom diagnosis and/or therapy and/or prophylaxis is and/or has been administered.
  • the terms “subject” and “patient” are used interchangeably herein.
  • the term “treat” refers to any administration of a therapy that partially or completely alleviates, ameliorates, relieves, inhibits, delays onset of, reduces severity of, and/or reduces incidence of one or more symptoms, features, and/or causes of a particular disease, disorder, and/or condition.
  • such treatment may be of a subject who does not exhibit signs of the relevant disease, disorder and/or condition and/or of a subject who exhibits only early signs of the disease, disorder, and/or condition.
  • such treatment may be of a subject who exhibits one or more established signs of the relevant disease, disorder and/or condition.
  • treatment may be of a subject who has been diagnosed as suffering from the relevant disease, disorder, and/or condition.
  • Compound 1 is in a pharmacological class of tyrosine kinase inhibitors (TKI).
  • TKI tyrosine kinase inhibitors
  • Compound 1 is an orally bioavailable small molecule dual kinase inhibitor of platelet-derived growth factor receptors (PDGFR) and vascular endothelial growth factor receptors (VEGFR2).
  • PDGFR platelet-derived growth factor receptors
  • VEGFR2 vascular endothelial growth factor receptors
  • Compound 1 is provided and/or utilized (e.g., for inclusion in, e.g., during one or more steps of manufacturing of, a composition and/or for delivery to a subject) in accordance with the present disclosure in a form such as a salt form (e.g., a pharmaceutically acceptable salt form).
  • a salt form e.g., a pharmaceutically acceptable salt form
  • pharmaceutically acceptable salt forms are well known in the art.
  • Compound 1 is provided and/or utilized in a hydrochloride salt form, a maleate salt form, a mesylate salt form, or a tosylate salt form.
  • Compound 1 is provided and/or utilized (e.g., for inclusion in, e.g., during one or more steps of manufacturing of, a composition and/or for delivery to a subject) in accordance with the present disclosure in a form such as a solid form.
  • Compound 1 is provided and/or utilized in accordance with the present disclosure in an amorphous solid form, in a crystalline solid form, or in a mixture thereof.
  • a crystalline solid form may be or comprise a solvate, hydrate, or an unsolvated form. The use of any and all such forms are contemplated by the present disclosure.
  • Compound 1 is provided and/or utilized (e.g., for inclusion in, e.g., during one or more steps of manufacturing of, a composition and/or for delivery to a subject) as a hydrochloride salt form (e.g., a channel hydrate comprising up to about 4 equivalents of water, up to about 3 equivalents of water, up to about 2 equivalents of water, or up to about 1 equivalent of water).
  • a hydrochloride salt form of Compound 1 is characterized by one or more peaks in its XRPD selected from those at about one or more peaks in its XRPD selected from those at about 5.28, 10.63, 11.54, 17.05, and 20.98 degrees 2-theta.
  • such a hydrochloride salt form of Compound 1 is characterized by two or more peaks in its XRPD selected from those at about one or more peaks in its XRPD selected from those at about 5.28, 10.63, 11.54, 17.05, and 20.98 degrees 2-theta. In some embodiments, such a hydrochloride salt form of Compound 1 is characterized by one or more peaks in its XRPD selected from those at about three or more peaks in its XRPD selected from those at about 5.28, 10.63, 11.54, 17.05, and 20.98 degrees 2-theta.
  • such a hydrochloride salt form of Compound 1 is characterized by the following peaks in its XRPD pattern at about 5.28, 10.63, 11.54, 17.05, and 20.98 degrees 2-theta. In some embodiments, such a hydrochloride salt form of Compound 1 is characterized by peaks in its XRPD pattern at about 5.28, 10.63, 11.54, 17.05, and 20.98 degrees 2-theta, corresponding to d- spacing of about 16.74, 8.33, 7.67, 5.20, and 4.23 angstroms, respectively.
  • such a hydrochloride salt form of Compound 1 is characterized by substantially all of the peaks in its XRPD pattern selected from 5.28, 5.76, 10.63, 11.54, 12.73, 13.13, 14.08, 15.34, 15.64, 16.00, 16.55, 17.05, 17.78, 18.86, 19.08, 20.16, 20.68, 20.98, 21.62, 22.05, 22.82, 23.97, 24.94, 25.23, 25.61, 26.58, 27.01, 27.78, 29.89, 30.51, 30.91, 31.72, and 33.39 degrees 2- theta.
  • Compound 1 refers to Compound 1 (i.e., methyl (Z)-3-(((4-(N-methyl-2-(4-methylpiperazin-l- yl)acetamido)phenyl)amino)(phenyl)methylene)-2-oxo-2,3-dihydro-lH-pyrrolo[2,3-b]pyridine- 6-carboxylate) in any available form, such as, e.g., a salt form and/or solid form. It will be understood, therefore, that reference to an amount (e.g., in mg) of Compound 1 means the amount of Compound 1 in free base form.
  • Compound 1 may be provided and/or utilized as, e.g., a salt form of Compound 1 such that the amount of the salt (or other form) is an amount that corresponds to the “free base equivalent” of Compound 1.
  • “50 mg Compound 1” means, e.g., approx. 53.4 mg of Compound 1 Hydrochloride anhydrate, approx. 58.4 mg of Compound 1 Hydrochloride trihydrate, and approx. 58.9 mg of Compound 1 Mesylate anhydrate, etc.
  • compositions that delivers Compound 1 e.g., in some embodiments, a composition that is or comprises Compound 1, or a composition that otherwise delivers Compound 1 - e.g., that is or comprises a prodrug of Compound 1, a complex or other entity that releases Compound 1 upon administration, etc.
  • a composition that delivers Compound 1 e.g., in some embodiments, a composition that is or comprises Compound 1, or a composition that otherwise delivers Compound 1 - e.g., that is or comprises a prodrug of Compound 1, a complex or other entity that releases Compound 1 upon administration, etc.
  • the present disclosure provides methods of administering Compound 1 to a subject or population of subjects, according to a regimen established to achieve one or more particular effects.
  • a regimen has been established to achieve one or more particular effects, relative to that observed for a comparable reference population that has not received Compound 1 (e.g., that has received a reference composition which does not deliver Compound 1).
  • a regimen has been established to achieve one or more particular effects in a population of healthy subjects.
  • a regimen has been established to achieve one or more particular effects in a population of healthy subjects, relative to that observed for a comparable reference population that has not received Compound 1 (e.g., that has received a reference composition which does not deliver Compound 1).
  • certain parameters may be evaluated to determine if a particular effect is achieved. For example, in some embodiments, certain measures of, e.g., TEAEs, Cmax, Tmax, AUCo-iast, AUCo-inf, Vz/F, CL/F, and/or ti/2, can be obtained. Additionally, in some embodiments, certain measure of, e.g., Tiast, AUCo-12, AUCo-24, C12, C24, Ctrough, RaccCmax, RaccAUC, CLss/F, and/or Vz ss /F, etc. can be obtained.
  • certain measures of e.g., TEAEs, Cmax, Tmax, AUCo-iast, AUCo-inf, Vz/F, CL/F, and/or ti/2, can be obtained.
  • a reference population has not received a composition providing Compound 1.
  • a reference population has received an otherwise comparable reference composition that does not provide Compound 1 (e.g., a placebo and/or another therapy).
  • a placebo as used herein is a dosage form that matches that of an active study compound, but does not deliver the active study compound (e.g., Compound 1).
  • a placebo can be a capsule that visually matches an active study drug and is composed of the same capsule shell but is filled with a pharmaceutical excipient (and lacking the active study drug), e.g., silicified microcrystalline cellulose.
  • a reference composition may be or may have been administered at the same intervals and/or in the same amounts as a composition providing Compound 1.
  • composition providing Compound 1 is administered according to a regimen established to achieve a particular effect, e.g., at a particular time point.
  • a composition providing Compound 1 is administered according to a regimen established to achieve a particular effect, e.g., at a particular time point, relative to an appropriate reference as described herein (e.g., as is observed in a comparable population who has not received a composition providing Compound 1).
  • the present disclosure provides methods of administering to a subject or population of subjects in need thereof an amount of Compound 1, wherein said amount, when administered as a single oral dose to a population of healthy subjects (e.g., healthy subjects in a fed or fasted state), was established to achieve one or more particular effects.
  • a particular effect is or comprises a particular mean maximum concentration (Cmax).
  • Cmax mean maximum concentration
  • a mean Cmax is measured in a population of healthy subjects (e.g., healthy subjects in a fed or fasted state) who received a single oral dose of Compound 1.
  • a mean Cmax is measured in a population of healthy subjects (e.g., healthy subjects in a fed or fasted state) who received multiple oral doses of Compound 1 (e.g., once daily (QD) or twice daily (BID) for, e.g., 14 days).
  • QD once daily
  • BID twice daily
  • a particular effect is or comprises a mean Cmax of from about 30 ng/mL to about 800 ng/mL, from about 85 ng/mL to about 800 ng/mL, from about 230 ng/mL to about 800 ng/mL, from about 400 ng/mL to about 800 ng/mL, from about 30 ng/mL to about 400 ng/mL, or from about 85 ng/mL to about 400 ng/mL.
  • a particular effect is or comprises a mean Cmax of less than about 800 ng/mL, less than about 400 ng/mL, less than about 235 ng/mL, or less than about 90 ng/mL.
  • a particular effect is or comprises a mean Cmax of greater than about 30 ng/mL, greater than about 85 ng/mL, greater than about 230 ng/mL, or greater than about 400 ng/mL.
  • a particular effect is or comprises a particular median time to maximum concentration (Tmax).
  • Tmax is measured in a population of healthy subjects (e.g., healthy subjects in a fed or fasted state) who received a single oral dose of Compound 1.
  • a median Tmax is measured in a population of healthy subjects (e.g., healthy subjects in a fed or fasted state) who received multiple oral doses of Compound 1 (e.g., once daily (QD) or twice daily (BID) for, e.g., 14 days).
  • QD once daily
  • BID twice daily
  • a particular effect is or comprises a median Tmax of from about 1 hour to about 4 hours, from about 1 hour to about 2 hours, or from about 1 hour to about 1.5 hours. In some embodiments, a particular effect is or comprises a median Tmax of from about 0.5 hours to about 4 hours, from about 1 hour to about 4 hours, from about 1 hour to about 2 hours, or from about 1 hour to about 1.5 hours. In some embodiments, a particular effect is or comprises a median Tmax of less than about 4 hours, less than about 2 hours, or less than about 1.5 hours. In some embodiments, a particular effect is or comprises a median Tmax of greater than about 1 hour, greater than about 1.5 hours, or greater than about 2 hours.
  • a particular effect is or comprises a particular mean area under drug concentration-time curve (AUCo-iast).
  • AUCo-iast mean area under drug concentration-time curve
  • a mean AUCo-iast is measured in a population of healthy subjects (e.g., healthy subjects in a fed or fasted state) who received a single oral dose of Compound 1.
  • a particular effect is or comprises a mean AUCo-iast of from about 60 ng*h/mL to about 4000 ng*h/mL, from about 60 ng*h/mL to about 2200 ng*h/mL, from about 170 ng*h/mL to about 2200 ng*h/mL, from about 400 ng*h/mL to about 2200 ng*h/mL, from about 1260 ng*h/mL to about 2200 ng*h/mL, from about 60 ng*h/mL to about 1260 ng*h/mL, or from about 170 ng*h/mL to about 1260 ng*h/mL.
  • a particular effect is or comprises a mean AUCo-iast of from about 100 ng*h/mL to about 4000 ng*h/mL, from about 1000 ng*h/mL to about 2400 ng*h/mL, from about 180 ng*h/mL to about 2400 ng*h/mL, from about 430 ng*h/mL to about 2400 ng*h/mL, from about 1260 ng*h/mL to about 2400 ng*h/mL, from about 100 ng*h/mL to about 1260 ng*h/mL, or from about 180 ng*h/mL to about 1260 ng*h/mL.
  • a particular effect is or comprises a mean AUCo-iast of less than about 4000 ng*h/mL, less than about 2200 ng*h/mL, less than about 1270 ng*h/mL, less than about 420 ng*h/mL, or less than about 180 ng*h/mL. In some embodiments, a particular effect is or comprises a mean AUCo-iast of less than about 4000 ng*h/mL, less than about 2400 ng*h/mL, less than about 1270 ng*h/mL, less than about 440 ng*h/mL, or less than about 190 ng*h/mL.
  • a particular effect is or comprises a mean AUCo-iast of greater than about 60 ng*h/mL, greater than about 170 ng*h/mL, greater than about 400 ng*h/mL, or greater than about 1260 ng*h/mL.
  • a particular effect is or comprises a particular mean area under drug concentration-time curve (AUCo-inf).
  • AUCo-inf mean area under drug concentration-time curve
  • a mean AUCo-inf is measured in a population of healthy subjects (e.g., healthy subjects in a fed or fasted state) who received a single oral dose of Compound 1.
  • a particular effect is or comprises a mean AUCo-inf of from about 60 ng*h/mL to about 4000 ng*h/mL, from about 60 ng*h/mL to about 2410 ng*h/mL, from about 190 ng*h/mL to about 2410 ng*h/mL, from about 490 ng*h/mL to about 2410 ng*h/mL, from about 1300 ng*h/mL to about 2410 ng*h/mL, or from about 190 ng*h/mL to about 1300 ng*h/mL.
  • a particular effect is or comprises a mean AUCo-inf of from about 60 ng*h/mL to about 4000 ng*h/mL, from about 60 ng*h/mL to about 2580 ng*h/mL, from about 180 ng*h/mL to about 2580 ng*h/mL, from about 550 ng*h/mL to about 2580 ng*h/mL, from about 1300 ng*h/mL to about 2580 ng*h/mL, or from about 180 ng*h/mL to about 1300 ng*h/mL.
  • a particular effect is or comprises a mean AUCo-inf of less than about 4000 ng*h/mL, less than about 2410 ng*h/mL, less than about 1300 ng*h/mL, less than about 500 ng*h/mL, or less than about 200 ng*h/mL. In some embodiments, a particular effect is or comprises a mean AUCo-inf of less than about 4000 ng*h/mL, less than about 2580 ng*h/mL, less than about 1310 ng*h/mL, less than about 560 ng*h/mL, or less than about 190 ng*h/mL.
  • a particular effect is or comprises a mean AUCo-inf of greater than about 60 ng*h/mL, greater than about 190 ng*h/mL, greater than about 490 ng*h/mL, or greater than about 1300 ng*h/mL.
  • a particular effect is or comprises a particular mean area under drug concentration-time curve (AUCo-12).
  • AUCo-12 is measured in a population of healthy subjects (e.g., healthy subjects in a fed or fasted state) who received multiple oral doses of Compound 1 (e.g., once daily (QD) or twice daily (BID) for, e.g., 14 days).
  • QD once daily
  • BID twice daily
  • a particular effect is or comprises a mean AUCo-12 of from about 100 ng*h/mL to about 1500 ng*h/mL, from about 120 ng*h/mL to about 1340 ng*h/mL, from about 250 ng*h/mL to about 1340 ng*h/mL, from about 920 ng*h/mL to about 1340 ng*h/mL, from about 120 ng*h/mL to about 930 ng*h/mL, or from about 250 ng*h/mL to about 930 ng*h/mL.
  • a particular effect is or comprises a mean AUCo-12 of less than about 1500 ng*h/mL, less than about 1300 ng*h/mL, less than about 930 ng*h/mL, or less than about 260 ng*h/mL. In some embodiments, a particular effect is or comprises a mean AUCo-12 of greater than about 115 ng*h/mL, greater than about 250 ng*h/mL, or greater than about 920 ng*h/mL.
  • a particular effect is or comprises a particular mean apparent volume of distribution (Vz/F).
  • a mean Vz/F is measured in a population of healthy subjects (e.g., healthy subjects in a fed or fasted state) who received a single oral dose of Compound 1.
  • a particular effect is or comprises a mean Vz/F of from about 2000 L to about 5500 L, from about 2200 L to about 5500 L, or from about 2100 L to about 2300 L.
  • a particular effect is or comprises a mean Vz/F of from about 2000 L to about 7800 L, from about 2200 L to about 5500 L, or from about 5400 L to about 7800 L.
  • a particular effect is or comprises a mean Vz/F of less than about 5500 L, less than about 2300 L, or less than about 2200 L. In some embodiments, a particular effect is or comprises a mean Vz/F of less than about 7800L, less than about 5500 L, less than about 2300 L, or less than about 2200 L. In some embodiments, a particular effect is or comprises a mean Vz/F of greater than about 2100 L, greater than about 2200 L, or greater than about 5100 L.
  • a particular effect is or comprises a particular mean apparent total clearance (CL/F).
  • a mean CL/F is measured in a population of healthy subjects (e.g., healthy subjects in a fed or fasted state) who received a single oral dose of Compound 1.
  • a particular effect is or comprises a mean CL/F of from about 300 L/h to about 600 L/h, from about 300 L/h to about 450 L/h, or from about 400 L/h to about 600 L/h.
  • a particular effect is or comprises a mean CL/F of from about 270 L/h to about 590 L/h, from about 270 L/h to about 370 L/h, or from about 340 L/h to about 590 L/h. In some embodiments, a particular effect is or comprises a mean CL/F of less than about 600 L/h, less than about 450 L/h, or less than about 350 L/h. In some embodiments, a particular effect is or comprises a mean CL/F of greater than about 300 L/h, greater than about 400 L/h, or greater than about 575 L/h. In some embodiments, a particular effect is or comprises a mean CL/F of greater than about 250 L/h, greater than about 340 L/h, or greater than about 370 L/h.
  • a particular effect is or comprises a particular mean half-life (ti/2).
  • a mean ti/2 is measured in a population of healthy subjects (e.g., healthy subjects in a fed or fasted state) who received a single oral dose of Compound 1.
  • a mean ti/2 is measured in a population of healthy subjects (e.g., healthy subjects in a fed or fasted state) who received multiple oral doses of Compound 1 (e.g., once daily (QD) or twice daily (BID) for, e.g., 14 days).
  • QD once daily
  • BID twice daily
  • a particular effect is or comprises a mean ti/2 of from about 2 hours to about 30 hours, from about 3 hours to about 30 hours, from about 10 hours to about 30 hours, from about 2 hours to about 11 hours, or from about 3 hours to about 11 hours.
  • a particular effect is or comprises a mean ti/2 of from about 12 hours to about 21 hours.
  • a particular effect is or comprises a mean ti/2 of less than about 30 hours, less than about 11 hours, less than about 4 hours, or less than about 3 hours.
  • a particular effect is or comprises a mean ti/2 of less than about 22 hours or less than about 15 hours.
  • a particular effect is or comprises a mean ti/2 of greater than about 2 hours, greater than about 3 hours, or greater than about 10 hours.
  • a particular effect is or comprises a particular mean accumulation ratio of Cmax (RaccCmax).
  • a mean RaccCmax is measured in a population of healthy subjects (e.g., healthy subjects in a fed or fasted state) who received multiple oral doses of Compound 1 (e.g., once daily (QD) or twice daily (BID) for, e.g., 14 days).
  • a particular effect is or comprises a mean RaccCmax of from about 0.6 to about 2.7, from about 0.6 to about 1.6, or from about 1.0 to about 1.6.
  • a particular effect is or comprises a mean RaccCmax of less than about 2.0, less than about 1.6, or less than about 1.4. In some embodiments, a particular effect is or comprises a mean RaccCmax of greater than about 0.5, greater than about 1.0, or greater than about 1.2.
  • a particular effect is or comprises a particular mean accumulation ratio of AUC (RaccAUC).
  • a mean RaccAUC is measured in a population of healthy subjects (e.g., healthy subjects in a fed or fasted state) who received multiple oral doses of Compound 1 (e.g., once daily (QD) or twice daily (BID) for, e.g., 14 days).
  • a particular effect is or comprises a mean RaccAUC of from about 0.8 to about 1.6 or from about 0.8 to about 1.2.
  • a particular effect is or comprises a mean RaccAUC of less than about 2.0, less than about 1.2, or less than about 1.0.
  • a particular effect is or comprises a mean RaccAUC of greater than about 0.5, greater than about 0.8, or greater than about 1.0.
  • the present disclosure provides methods of administering Compound 1 to a subject or population of subjects, according to a regimen established to achieve one or more particular effects in a population of subjects in one fed/fasted state, relative to a comparable population of subjects in another fed/fasted state.
  • a regimen is established to achieve one or more particular effects in a population of healthy subjects in one fed/fasted state, relative to a comparable population of healthy subjects in another fed/fasted state.
  • a regimen is established to achieve one or more particular effects in a population of healthy subjects in a fasted state, relative to a comparable population of healthy subjects in a fed state.
  • a regimen is established to achieve one or more particular effects in a population of healthy subjects in a fed state, relative to a comparable population of healthy subjects in a fasted state.
  • a reference population e.g., in a fasted or fed state
  • a composition providing Compound 1 e.g., the same composition providing Compound 1).
  • a population in a fasted state has received a composition providing Compound 1 after a period of fasting (e.g., an overnight fast of at least 10 hours).
  • a population in a fed state has received a composition providing Compound 1 after consumption of a meal (e.g., a high fat meal) within a period of time (e.g., within 30 minutes of receiving Compound 1).
  • the present disclosure provides methods of administering to a subject or population of subjects in need thereof an amount of Compound 1, wherein said amount, when administered as a single oral dose to a population of healthy subjects in one fed/fasted state, was established to achieve one or more particular effects relative to a comparable population of healthy subjects in another fed/fasted state.
  • the present disclosure provides methods of administering to a subject or population of subjects in need thereof an amount of Compound 1, wherein said amount, when administered as a single oral dose to a population of healthy subjects in a fasted state, was established to achieve one or more particular effects relative to a comparable population of healthy subjects in a fed state.
  • a particular effect is or comprises a difference in mean Cmax in a population of subjects (e.g., healthy subjects) in one fed/fasted state relative to a comparable population of subjects (e.g., healthy subjects) in another fed/fasted state.
  • a particular effect is or comprises an increased mean Cmax in a population of subjects in a fasted state relative to a comparable population of subjects in a fed state.
  • a particular effect is or comprises a mean Cmax in a population of subjects in a fasted state that is about 3 fold, about 3.5 fold, or about 4 fold greater than a mean Cmax in a population of subjects in a fed state.
  • a particular effect is or comprises a comparable mean Cmax in a population of subjects in a fasted state relative to a comparable population of subjects in a fed state. In some embodiments, a particular effect is or comprises a mean Cmax in a population of subjects in a fasted state that is within about 50%, about 40%, or about 25% of a mean Cmax in a population of subjects in a fed state.
  • a particular effect is or comprises a difference in median Tmax in a population of subjects (e.g., healthy subjects) in one fed/fasted state relative to a comparable population of subjects (e.g., healthy subjects) in another fed/fasted state.
  • a particular effect is or comprises a decreased median Tmax in a population of subjects in a fasted state relative to a comparable population of subjects in a fed state.
  • a particular effect is or comprises a median Tmax in a population of subjects in a fasted state that is about 2 fold, about 2.5 fold, or about 3 fold less than a median Tmax in a population of subjects in a fed state.
  • a particular effect is or comprises a lack of meaningful difference in mean AUCo-iast in a population of subjects (e.g., healthy subjects) in one fed/fasted state relative to a comparable population of subjects (e.g., healthy subjects) in another fed/fasted state.
  • a particular effect is or comprises a comparable mean AUCo-iast in a population of subjects in a fasted state relative to a comparable population of subjects in a fed state.
  • a particular effect is or comprises a mean AUCo-iast in a population of subjects in a fasted state that is within about 10%, about 20%, or about 30% of a mean AUCo- iast in a population of subjects in a fed state.
  • a particular effect is or comprises a lack of meaningful difference in mean AUCo-inf in a population of subjects (e.g., healthy subjects) in one fed/fasted state relative to a comparable population of subjects (e.g., healthy subjects) in another fed/fasted state.
  • a particular effect is or comprises a comparable mean AUCo-inf in a population of subjects in a fasted state relative to a comparable population of subjects in a fed state. In some embodiments, a particular effect is or comprises a mean AUCo-inf in a population of subjects in a fasted state that is within about 10%, about 20%, or about 30% of a mean AUCo- inf in a population of subjects in a fed state.
  • a particular effect is or comprises a difference in mean Vz/F in a population of subjects (e.g., healthy subjects) in one fed/fasted state relative to a comparable population of subjects (e.g., healthy subjects) in another fed/fasted state.
  • a particular effect is or comprises a decreased mean Vz/F in a population of subjects in a fasted state relative to a comparable population of subjects in a fed state.
  • a particular effect is or comprises a mean Vz/F in a population of subjects in a fasted state that is about 3 fold, about 3.5 fold, or about 4 fold less than a mean Vz/F in a population of subjects in a fed state.
  • a particular effect is or comprises a lack of meaningful difference in mean CL/F in a population of subjects (e.g., healthy subjects) in one fed/fasted state relative to a comparable population of subjects (e.g., healthy subjects) in another fed/fasted state.
  • a particular effect is or comprises a comparable mean CL/F in a population of subjects in a fasted state relative to a comparable population of subjects in a fed state.
  • a particular effect is or comprises a mean CL/F in a population of subjects in a fasted state that is within about 10%, about 20%, or about 30% of a mean CL/F in a population of subjects in a fed state.
  • a particular effect is or comprises a difference in mean tm in a population of subjects (e.g., healthy subjects) in one fed/fasted state relative to a comparable population of subjects (e.g., healthy subjects) in another fed/fasted state.
  • a particular effect is or comprises a decreased mean tm in a population of subjects in a fasted state relative to a comparable population of subjects in a fed state.
  • a particular effect is or comprises a mean tm in a population of subjects in a fasted state that is about 3 fold, about 3.5 fold, or about 4 fold less than a mean tm in a population of subjects in a fed state.
  • a particular effect is or comprises a comparable mean tm in a population of subjects in a fasted state relative to a comparable population of subjects in a fed state. In some embodiments, a particular effect is or comprises a mean tm in a population of subjects in a fasted state that is within about 10%, about 20%, or about 30% of a mean tm in a population of subjects in a fed state.
  • the present disclosure provides methods of administering Compound 1 to a subject or population of subjects, according to a regimen has been established to achieve one or more particular effects in a population of subjects suffering from a disease or disorder described herein (e.g., suffering from primary proteinuric kidney disease and/or primary glomerular disease and/or persistent proteinuria).
  • a regimen has been established to achieve one or more particular effects in a population of subjects suffering from a disease or disorder described herein (e.g., suffering from primary proteinuric kidney disease and/or primary glomerular disease and/or persistent proteinuria), relative to that observed for a comparable reference population that has not received Compound 1 (e.g., that has received a reference composition which does not deliver Compound 1).
  • a reference population has received an otherwise comparable reference composition that does not provide Compound 1 (e.g., a placebo as described herein).
  • composition providing Compound 1 is administered according to a regimen established to achieve a particular effect, e.g., at a particular time point.
  • a composition providing Compound 1 is administered according to a regimen established to achieve a particular effect, e.g., at a particular time point, relative to an appropriate reference as described herein (e.g., as is observed in a comparable population who has not received a composition providing Compound 1).
  • the present disclosure provides methods of administering to a subject or population of subjects in need thereof an amount of Compound 1, wherein said amount, when administered to a population of subjects suffering from a disease or disorder described herein (e.g., suffering from primary proteinuric kidney disease and/or primary glomerular disease and/or persistent proteinuria), has been established to achieve one or more particular effects.
  • a disease or disorder described herein e.g., suffering from primary proteinuric kidney disease and/or primary glomerular disease and/or persistent proteinuria
  • a particular effect is or comprises a particular mean or median percentage change in 24-hour urinary protein excretion (e.g., at Week 12). In some embodiments, a particular effect is or comprises a particular mean or median percent reduction in 24-hour urinary protein excretion (e.g., at Week 12). In some embodiments, a particular effect is or comprises a greater mean or median percent reduction in 24-hour urinary protein excretion (e.g., at Week 12) in a population of subjects suffering from primary glomerular disease and/or persistent proteinuria administered a composition providing Compound 1 relative to a comparable population who has not received a composition providing Compound 1. In some embodiments, a particular effect is or comprises a mean or median percent reduction in 24-hour urinary protein excretion (e.g., at Week 12) of about 20% to about 40%.
  • a particular effect is or comprises a particular mean or median percentage change in 24-hour urinary albumin (e.g., at Week 12). In some embodiments, a particular effect is or comprises a particular mean or median percent reduction in 24-hour urinary albumin (e.g., at Week 12). In some embodiments, a particular effect is or comprises a greater mean or median percent reduction in 24-hour urinary albumin (e.g., at Week 12) in a population of subjects suffering from primary glomerular disease and/or persistent proteinuria administered a composition providing Compound 1 relative to a comparable population who has not received a composition providing Compound 1.
  • a particular effect is or comprises a particular proportion of subjects with complete remission in proteinuria.
  • a complete remission in proteinuria is defined as a 24-hour urinary protein excretion of less than 300 mg (e.g., at Week 12).
  • a particular effect is or comprises a greater proportion of subjects with complete remission in proteinuria in a population of subjects suffering from primary glomerular disease and/or persistent proteinuria administered a composition providing Compound 1 relative to a comparable population who has not received a composition providing Compound 1.
  • a particular effect is or comprises a particular proportion of subjects with partial remission in proteinuria.
  • a partial remission in proteinuria is defined as (e.g., at Week 12) a 24-hour urinary protein excretion reduction of less than or equal to 50% from baseline and/or a 24-hour urinary protein excretion of less than 3.5 g/day if baseline 24-hour urinary protein excretion was greater than 3.5 g.
  • a particular effect is or comprises a greater proportion of subjects with partial remission in proteinuria in a population of subjects suffering from primary glomerular disease and/or persistent proteinuria administered a composition providing Compound 1 relative to a comparable population who has not received a composition providing Compound 1.
  • a particular effect is or comprises a greater proportion of subjects with complete or partial remission in proteinuria in a population of subjects suffering from primary glomerular disease and/or persistent proteinuria administered a composition providing Compound 1 relative to a comparable population who has not received a composition providing Compound 1.
  • a particular effect is or comprises a particular proportion of subjects with greater than or equal to 50% reduction in 24-hour urinary protein excretion from baseline (e.g., at Week 12). In some embodiments, a particular effect is or comprises a greater proportion of subjects with greater than or equal to 50% reduction in 24-hour urinary protein excretion from baseline (e.g., at Week 12) in a population of subjects suffering from primary glomerular disease and/or persistent proteinuria administered a composition providing Compound 1 relative to a comparable population who has not received a composition providing Compound 1.
  • a particular effect is or comprises a particular proportion of subjects with greater than or equal to 50% reduction in 24-hour urinary albumin excretion from baseline (e.g., at Week 12). In some embodiments, a particular effect is or comprises a greater proportion of subjects with greater than or equal to 50% reduction in 24-hour urinary albumin excretion from baseline (e.g., at Week 12) in a population of subjects suffering from primary glomerular disease and/or persistent proteinuria administered a composition providing Compound 1 relative to a comparable population who has not received a composition providing Compound 1.
  • a particular effect is or comprises a particular mean or median percentage change from baseline in creatinine clearance (e.g., at Week 12). In some embodiments, a particular effect is or comprises a particular mean or median percent increase from baseline in creatinine clearance (e.g., at Week 12). In some embodiments, a particular effect is or comprises a greater mean or median percent increase from baseline in creatinine clearance (e.g., at Week 12) in a population of subjects suffering from primary glomerular disease and/or persistent proteinuria administered a composition providing Compound 1 relative to a comparable population who has not received a composition providing Compound 1.
  • a particular effect is or comprises a particular mean or median change from baseline in serum albumin (e.g., at Week 12). In some embodiments, a particular effect is or comprises a particular mean or median increase from baseline in serum albumin (e.g., at Week 12). In some embodiments, a particular effect is or comprises a greater mean or median increase from baseline in serum albumin (e.g., at Week 12) in a population of subjects suffering from primary glomerular disease and/or persistent proteinuria administered a composition providing Compound 1 relative to a comparable population who has not received a composition providing Compound 1.
  • a particular effect is or comprises a particular mean or median change from baseline in fasting triglyceride levels (e.g., at Week 12). In some embodiments, a particular effect is or comprises a particular mean or median decrease from baseline in fasting triglyceride levels (e.g., at Week 12). In some embodiments, a particular effect is or comprises a greater mean or median decrease from baseline in fasting triglyceride levels (e.g., at Week 12) in a population of subjects suffering from primary glomerular disease and/or persistent proteinuria administered a composition providing Compound 1 relative to a comparable population who has not received a composition providing Compound 1.
  • a particular effect is or comprises a particular mean or median change from baseline in cholesterol levels (e.g., at Week 12). In some embodiments, a particular effect is or comprises a particular mean or median decrease from baseline in cholesterol levels (e.g., at Week 12). In some embodiments, a particular effect is or comprises a greater mean or median decrease from baseline in cholesterol levels (e.g., at Week 12) in a population of subjects suffering from primary glomerular disease and/or persistent proteinuria administered a composition providing Compound 1 relative to a comparable population who has not received a composition providing Compound 1.
  • a particular effect is or comprises a particular mean or median level of one or more plasma or urine biomarkers selected from plasma soluble TNF receptor 2 (sTNFR2), kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), urinary fibronectin, high molecular weight type IV collagen (HMW collagen IV), monocyte chemotactic protein-1 (MCP-1), and matrix metalloproteinase-7 (MMP-7).
  • plasma or urine biomarkers selected from plasma soluble TNF receptor 2 (sTNFR2), kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), urinary fibronectin, high molecular weight type IV collagen (HMW collagen IV), monocyte chemotactic protein-1 (MCP-1), and matrix metalloproteinase-7 (MMP-7).
  • a particular effect is or comprises a particular mean or median change in the level(s) of one or more plasma or urine biomarkers selected from plasma soluble TNF receptor 2 (sTNFR2), kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), urinary fibronectin, high molecular weight type IV collagen (HMW collagen IV), monocyte chemotactic protein- 1 (MCP-1), and matrix metalloproteinase-7 (MMP-7) in a population of subjects suffering from primary glomerular disease and/or persistent proteinuria administered a composition providing Compound 1 relative to a comparable population who has not received a composition providing Compound 1.
  • plasma or urine biomarkers selected from plasma soluble TNF receptor 2 (sTNFR2), kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), urinary fibronectin, high molecular weight type IV collagen (HMW collagen IV), monocyte chemotactic protein- 1 (MCP-1),
  • a particular effect is or comprises a particular mean maximum concentration (Cmax).
  • Cmax mean maximum concentration
  • a mean or median Cmax is measured in a population of subjects suffering from primary glomerular disease and/or persistent proteinuria administered a composition providing Compound 1.
  • a particular effect is or comprises a particular median time to maximum concentration (Tmax).
  • Tmax median time to maximum concentration
  • a median Tmax is measured in a population of subjects suffering from primary glomerular disease and/or persistent proteinuria administered a composition providing Compound 1.
  • a particular effect is or comprises a particular mean area under drug concentration-time curve from time zero to last measurable concentration (AUCo-iast).
  • AUCo-iast mean area under drug concentration-time curve from time zero to last measurable concentration
  • a mean AUCo-iast is measured in a population of subjects suffering from primary glomerular disease and/or persistent proteinuria administered a composition providing Compound 1.
  • a particular effect is or comprises a particular mean area under drug concentration-time curve from time zero to infinity (AUCo-inf).
  • AUCo-inf mean area under drug concentration-time curve from time zero to infinity
  • a mean AUCo-inf is measured in a population of subjects suffering from primary glomerular disease and/or persistent proteinuria administered a composition providing Compound 1.
  • a particular effect is or comprises a particular mean area under the drug concentration-time curve over the dosing interval (AUCtau).
  • AUCtau is measured in a population of subjects suffering from primary glomerular disease and/or persistent proteinuria administered a composition providing Compound 1.
  • a particular effect is or comprises a particular mean half-life (ti/2). In some embodiments, a mean ti/2 is measured in a population of subjects suffering from primary glomerular disease and/or persistent proteinuria administered a composition providing Compound 1. [0103] In some embodiments, a particular effect is or comprises a particular mean or median terminal elimination rate constant (K ei ). In some embodiments, a mean or median K ei is measured in a population of subjects suffering from primary glomerular disease and/or persistent proteinuria administered a composition providing Compound 1.
  • a particular effect is or comprises a particular mean or median trough plasma concentration at steady-state measured at the end of a dosing interval before next administration (Ctrough).
  • a mean or median Ctrough is measured in a population of subjects suffering from primary glomerular disease and/or persistent proteinuria administered a composition providing Compound 1.
  • a particular effect is or comprises a particular mean apparent total clearance (CL/F) on Day 1.
  • a mean CL/F is measured in a population of subjects suffering from primary glomerular disease and/or persistent proteinuria administered a composition providing Compound 1.
  • a particular effect is or comprises a particular mean apparent total clearance at steady state (CL/Fss).
  • CL/Fss is measured in a population of subjects suffering from primary glomerular disease and/or persistent proteinuria administered a composition providing Compound 1.
  • a particular effect is or comprises a particular mean apparent volume of distribution (Vz/F) on Day 1.
  • a mean Vz/F is measured in a population of subjects suffering from primary glomerular disease and/or persistent proteinuria administered a composition providing Compound 1.
  • a particular effect is or comprises a particular mean apparent volume of distribution at steady state (Vz/Fss).
  • a mean Vz/Fss is measured in a population of subjects suffering from primary glomerular disease and/or persistent proteinuria administered a composition providing Compound 1.
  • a particular effect is or comprises a particular mean or median accumulation ratio.
  • a mean or median accumulation ratio is measured in a population of subjects suffering from primary glomerular disease and/or persistent proteinuria administered a composition providing Compound 1.
  • the present disclosure provides amounts of Compound 1 suitable to achieve one or more particular effects.
  • Compound 1 is administered in a daily dose of from about 50 mg to about 600 mg, from about 100 mg to about 600 mg, from about 200 mg to about 600 mg, from about 400 mg to about 600 mg, from about 50 mg to about 500 mg, from about 100 mg to about 500 mg, from about 250 mg to about 500 mg, from about 50 mg to about 250 mg, from about 100 mg to about 1000 mg, from about 200 mg to about 1000 mg, from about 500 mg to about 1000 mg, or from about 200 mg to about 500 mg.
  • Compound 1 is administered in a dose of about 50 mg, about 100 mg, about 200 mg, about 250 mg, about 400 mg, about 500 mg, or about 600 mg. In some embodiments, Compound 1 is administered in a dose of about 50 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg, or about 600 mg. In some embodiments, Compound 1 is administered in a daily dose of about 50 mg, about 100 mg, about 200 mg, about 400 mg, about 500 mg, or about 600 mg. In some embodiments, Compound 1 is administered in a twice daily dose of about 50 mg, about 100 mg, about 250 mg, or about 500 mg.
  • Compound 1 is administered in a dose of about 100 mg once daily. In some embodiments, Compound 1 is administered in a dose of about 200 mg once daily. In some embodiments, Compound 1 is administered in a dose of about 300 mg once daily. In some embodiments, Compound 1 is administered in a dose of about 400 mg once daily. In some embodiments, Compound 1 is administered in a dose of about 500 mg once daily. In some embodiments, Compound 1 is administered in a dose of about 600 mg once daily.
  • Compound 1 is administered in a dose of about 50 mg twice daily. In some embodiments, Compound 1 is administered in a dose of about 100 mg twice daily. In some embodiments, Compound 1 is administered in a dose of about 150 mg twice daily. In some embodiments, Compound 1 is administered in a dose of about 200 mg twice daily. In some embodiments, Compound 1 is administered in a dose of about 250 mg twice daily. In some embodiments, Compound 1 is administered in a dose of about 300 mg twice daily. In some embodiments, Compound 1 is administered in a dose of about 350 mg twice daily. In some embodiments, Compound 1 is administered in a dose of about 400 mg twice daily.
  • provided methods further comprise reducing a dose of Compound 1 if a subject experiences a side effect (e.g., a gastrointestinal or liver side effect) and/or if a subject’s side effect persists.
  • one or more doses of Compound 1 are reduced by 100 mg if a subject experiences a side effect (e.g., a gastrointestinal or liver side effect).
  • a Compound 1 dosing regimen of 200 mg QD is reduced to 100 mg QD.
  • a Compound 1 dosing regimen of 400 mg QD is reduced to 300 mg QD.
  • a Compound 1 dosing regimen of 300 mg BID is reduced to 200 mg BID.
  • a Compound 1 dosing regimen of 300 mg BID is reduced to a once daily dose of 200 mg and a once daily dose of 300 mg (e.g., said doses separated by approximately 12 hours).
  • provided methods further comprise discontinuing administration of Compound 1 if a subject experiences a side effect and/or if a subject’s side effect persists after a dose reduction.
  • gastrointestinal side effects include diarrhea, nausea, vomiting, and/or abdominal cramps.
  • provided methods further comprise reducing the dose of Compound 1 if a subject experiences mild diarrhea (e.g., 4-6 extra stools per day) for 8 days or longer.
  • mild diarrhea e.g., 4-6 extra stools per day
  • anti motility therapy e.g., loperamide
  • provided methods further comprise discontinuing administration of Compound 1 if a subject experiences mild diarrhea (e.g., 4-6 extra stools per day) for an additional 7 days or longer after dose reduction.
  • provided methods further comprise reducing the dose of Compound 1 if a subject experiences severe diarrhea (e.g., more than 6 extra stools per day with clinical evidence of volume depletion or impact on activities of daily living). In some embodiments, such a subject is also treated with anti-motility therapy (e.g., loperamide). In some embodiments, provided methods further comprise discontinuing administration of Compound 1 if a subject experiences severe diarrhea (e.g., more than 6 extra stools per day with clinical evidence of volume depletion or impact on activities of daily living) for 14 days or longer.
  • severe diarrhea e.g., more than 6 extra stools per day with clinical evidence of volume depletion or impact on activities of daily living
  • provided methods further comprise reducing the dose of Compound 1 is a subject experiences nausea and/or vomiting for 8 days or longer. In some embodiments, such subjects are also treated with ondansetron. In some embodiments, provided methods further comprise discontinuing administration of Compound 1 if a subject experiences nausea and/or vomiting for 14 days or longer.
  • liver side effects include elevated liver function tests (e.g., AST or ALT increase to > 3x upper limit of normal or AST or ALT increase to > 2x baseline value if elevated at baseline).
  • provided methods comprise discontinuing administration of Compound 1 if a subject has elevated liver function tests for 2 or more days.
  • the present disclosure provides methods of treating diseases, disorders, and conditions (e.g., according to methods provided herein).
  • provided methods are useful for reducing fibrosis in a subject in need thereof.
  • provided methods are useful for treating a disease, disorder, or condition characterized by or otherwise associated with fibrosis.
  • the present disclosure encompasses the recognition that treating fibrosis (e.g., using provided methods) instead of the underlying etiology may allow for broadly applicable antifibrotic therapies. It will be appreciated that provided methods may be suitable for reducing fibrosis in a variety of tissues and/or organs; the present disclosure contemplates use of Compound 1 for treating diseases, disorders, and conditions characterized by or otherwise associated with fibrosis in any suitable tissue and/or organ. For example, in some embodiments, provided methods are suitable for treating diseases, disorders and conditions that are or comprise fibrosis of gastrointestinal tract, heart, kidney, lung, liver, muscle, pancreas, and/or skin.
  • provided methods are suitable for treating diseases, disorders, and conditions characterized by or otherwise associated with cysts (e.g., in the kidney, liver, pancreas, ovaries, spermatic duct, etc.). It will be appreciated that provided methods may be suitable for treating diseases, disorders, and conditions in which fibrosis is the sole or a predominant component, as well as those in which fibrosis is a secondary component (e.g., a symptom and/or result of an underlying disease, disorder, or condition). It will also be appreciated that there are a variety of sources or causes of fibrosis.
  • certain injuries can progress to development of fibrosis.
  • provided methods are useful for treating acute injuries (e.g., acute organ injuries, such as acute lung injury, acute liver injury, or acute kidney injury), as well as for treating chronic injuries (e.g., chronic organ injuries, such as chronic lung injury, chronic liver injury, or chronic kidney injury).
  • provided methods are useful for treating fibrosis associated with an acute injury, such as that incurred from trauma and/or surgery and/or infection (e.g., a viral infection).
  • provided methods are useful for acceleration of wound healing, reduction of post-surgical scarring, and/or reduction of adhesion formation.
  • provided methods are useful for treating damaged and/or ischemic organs, transplants, or grafts, as well as ischemia/reperfusion injury or post- surgical scarring.
  • provided methods are useful for promoting vascularization of a damaged and/or ischemic organ, transplant, or graft, ameliorating ischemia/reperfusion injury (e.g., in brain, heart, liver, or kidney), normalizing myocardial perfusion resulting from chronic cardiac ischemia or myocardial infarction, and/or developing or augmenting collateral vessel development after vascular occlusion or to ischemic tissues or organs.
  • provided methods are useful for treating pulmonary diseases, disorders, or conditions. In some embodiments, provided methods are useful for treating pulmonary fibrosis. In some embodiments, provided methods are useful for treating pulmonary fibrosis secondary to, or otherwise associated with, an underlying indication. In some embodiments, provided methods are useful for treating interstitial lung diseases (e.g., fibrosing interstitial lung diseases). In some embodiments, provided methods are useful for treating pneumonias (e.g., idiopathic interstitial pneumonias). In some embodiments, provided methods are useful for treating idiopathic pulmonary fibrosis (IPF).
  • IPF idiopathic pulmonary fibrosis
  • provided methods are useful for treating pulmonary fibrosis associated with an infection (e.g., a bacterial, viral, or fungal infection).
  • an infection e.g., a bacterial, viral, or fungal infection.
  • provided methods are useful for treating pulmonary fibrosis associated with a viral infection (e.g., an influenza or coronavirus infection, such as COVID-19).
  • a fibrotic disease to be treated by methods of the present disclosure is pulmonary fibrosis.
  • Pulmonary fibrosis is a chronic, progressive, and ultimately fatal interstitial lung disease resulting from epithelial cell injury due to many factors.
  • activation of inflammatory cells and fibroblasts/myofibroblasts involves a cascade of cytokines/chemokines, growth factor network and deposit extracellular matrix, including collagen), which leads to pulmonary fibrosis and respiratory failure.
  • Pulmonary fibrosis causes high morbidity and mortality. At least five million people worldwide and -200,000 people in the United States suffer from pulmonary fibrosis. There is an unmet critical need for effective and affordable treatments for acute and chronic lung injuries.
  • pulmonary fibrosis Numerous endogenous and exogenous factors can provide primary stimuli for pulmonary fibrosis. Dust, silica, smoke, aerosolized toxins, infections and certain medicines have the potential to injure the lung and set the stage for the development of chronic pulmonary fibrosis. For example, viral infections may cause lung damage and/or otherwise develop into pulmonary fibrosis.
  • ARDS acute respiratory distress syndrome
  • Pulmonary fibrosis is associated with pronounced morbidity with high impact on economic burden.
  • the prevalence of pneumoconiosis (a disease caused by inhalation of dust and silica that causes inflammation and lung fibrosis) caused direct and indirect economic losses of around 28 billion yuan in China (4.3 billion US dollars) for 1 year.
  • a fibrotic disease to be treated by methods of the present disclosure is idiopathic pulmonary fibrosis.
  • Idiopathic pulmonary fibrosis is a chronic, irreversible, and progressive fibrotic disorder of the lower respiratory tract that typically affects adults over the age of 40.
  • Idiopathic interstitial pneumonias are diffuse parenchymal lung diseases, of which IPF is the most common and severe type of fibrotic lung disease. It is anatomically characterized by scarring of the lungs with a pattern of usual interstitial pneumonia (UIP) on high resolution computed tomography or histologic appearance on lung biopsy.
  • IPF Intra-pulmonary disease characterized by exertional dyspnea and cough.
  • Median survival following diagnosis of IPF ranges between 2 and 5 years, lower than that for many common cancers (Ley, B., et al. Am. J. Respir. Crit. Care Med. 2011;183:431-440; Seigel, R. L., et al. CA. Cancer J. Clin. 2016;66:7- 30).
  • Fibrotic process in IPF is progressive and, regardless of the nature of the initial injury, may follow a common pathway characterized by alveolar epithelial cell (AEC) dysfunction.
  • AEC alveolar epithelial cell
  • AECsl type I epithelial cells
  • AECs2 type II epithelial cells
  • TGF-bI transforming growth factor beta 1
  • PDGF platelet-derived growth factor
  • VEGF vascular endothelial growth factor
  • FGF fibroblast growth factor
  • the progression of fibrosis in IPF follows a common and complex path in which the AECs, fibroblasts, and endothelial cells produce an array of cytokines and growth factors that stimulate fibroblast proliferation and matrix synthesis.
  • provided methods are useful for treating hepatic diseases, disorders, or conditions. In some embodiments, provided methods are useful for treating hepatic fibrosis (e.g., fibrotic liver disease). In some embodiments, provided methods are useful for treating cirrhosis. In some embodiments, provided methods are useful for treating hepatic fibrosis and/or cirrhosis secondary to, or otherwise associated with, an underlying indication.
  • hepatic fibrosis e.g., fibrotic liver disease
  • provided methods are useful for treating cirrhosis.
  • provided methods are useful for treating hepatic fibrosis and/or cirrhosis secondary to, or otherwise associated with, an underlying indication.
  • provided methods are useful for treating hepatic fibrosis associated with hepatitis C, hepatitis B, delta hepatitis, chronic alcoholism, nonalcoholic steatohepatitis (NASH), extrahepatic obstructions (e.g., stones in bile duct), cholangiopathies (e.g., primary biliary cirrhosis or sclerosing cholangitis), autoimmune liver disease, or inherited metabolic disorders (e.g., Wilson’s disease, hemochromatosis, or alpha-1 antitrypsin deficiency).
  • NASH nonalcoholic steatohepatitis
  • a fibrotic disease to be treated by methods of the present disclosure is liver fibrosis.
  • Liver fibrosis is a scarring response of the liver to chronic liver injury; when fibrosis progresses to cirrhosis, morbid complications can develop.
  • end- stage liver fibrosis or cirrhosis is the seventh leading cause of death in the United States, and afflicts hundreds of millions of people worldwide; deaths from end-stage liver disease in the United States are expected to increase, mainly due to the hepatitis C epidemic.
  • liver disease In addition to the hepatitis C virus, many other forms of chronic liver injury also lead to end-stage liver disease and cirrhosis, including other viruses such as hepatitis B and delta hepatitis, chronic alcoholism, non-alcoholic steatohepatitis, extrahepatic obstructions (e.g., stones in the bile duct), cholangiopathies (e.g., primary biliary cirrhosis and sclerosing cholangitis), autoimmune liver disease, and inherited metabolic disorders (e.g., Wilson's disease, hemochromatosis, and alpha- 1 anti trypsin deficiency).
  • viruses such as hepatitis B and delta hepatitis, chronic alcoholism, non-alcoholic steatohepatitis, extrahepatic obstructions (e.g., stones in the bile duct), cholangiopathies (e.g., primary biliary cirrhosis and sclerosing
  • liver fibrosis has traditionally focused on eliminating a primary injury. For extrahepatic obstructions, biliary decompression is the recommended mode of treatment whereas patients with Wilson's disease are treated with zinc acetate. Treatments for other chronic liver diseases such as hepatitis B, autoimmune hepatitis and Wilson's disease are also associated with many side effects, while primary biliary cirrhosis, primary sclerosing cholangitis and non alcoholic fatty liver disease have no effective treatment other than liver transplantation.
  • provided methods are useful for treating renal diseases, disorders, or conditions. In some embodiments, provided methods are useful for treating renal fibrosis. In some embodiments, provided methods are useful for treating renal fibrosis secondary to, or otherwise associated with, an underlying indication.
  • provided methods are useful for treating renal fibrosis associated with renal failure, renal obstruction, renal trauma, renal transplantation, chronic kidney disease, diabetes, hypertension, radiocontrast nephropathy, immune-mediated glomerulonephri tides (e.g., lupus nephritis, ANCA-associated glomerulonephritides (e.g., Wegener’s granulomatosis, microscopic polyangiitis, or renal limited vasculitis), anti-GBM nephropathy, IgA nephropathy, membranous glomerulonephritis, or focal and segmental glomerulosclerosis), non-immune-mediated glomerulonephritides (e.g., polycystic kidney disease, collagen type III glomerulopathy, nail-patella syndrome, or Alport syndrome), minimal change disease, or nephrotic syndrome (e.g., steroid-resistant nephrotic syndrome).
  • provided methods are useful for treating nephrotic syndrome and/or diseases, disorders, or conditions associated with nephrotic syndrome (e.g., focal and segmental glomerulosclerosis, minimal change disease, and membranous nephropathy).
  • nephrotic syndrome e.g., focal and segmental glomerulosclerosis, minimal change disease, and membranous nephropathy.
  • provided methods are useful for treating a fibrotic disease of the kidney that is or comprises: focal segmental glomerulosclerosis (FSGS), steroid resistant nephrotic syndrome (SRNS), proteinuria, lupus nephritis, minimal change disease, an anti-neutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis, Alport syndrome, anti-globular basement membrane (anti-GBM) nephropathy, IgA nephropathy, membranous glomerulonephritis (MG), autosomal dominant polycystic kidney disease (ADPKD), collagen type III glomerulopathy, nail- patella syndrome, or chronic kidney disease.
  • FSGS focal segmental glomerulosclerosis
  • SRNS steroid resistant nephrotic syndrome
  • proteinuria lupus nephritis
  • minimal change disease an anti-neutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis
  • ANCA
  • provided methods are useful for treating a fibrotic disease of the kidney that is or comprises an anti-neutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis.
  • ANCA-associated glomerulonephritis is selected from Wegener’s granulomatosis, microscopic polyangiitis (MPA), or renal limited vasculitis.
  • provided methods are useful for treating focal and segmental glomerulosclerosis (FSGS).
  • FSGS focal and segmental glomerulosclerosis
  • provided methods are useful for treating Alport syndrome.
  • provided methods are useful for treating polycystic kidney disease (e.g., autosomal dominant polycystic kidney disease or autosomal recessive polycystic kidney disease).
  • provided methods are useful for treating primary proteinuric kidney disease (PPKD).
  • PPKD primary proteinuric kidney disease
  • PGDs primary glomerular diseases
  • PGDs are among the leading causes of chronic kidney disease and end-stage kidney disease in the world. PGDs predominantly affect younger patients, significantly reducing their quality of life, productivity, and longevity.
  • FSGS, membranous nephropathy (MN), and IgA nephropathy are among the three most common primary glomerular diseases in adults. Accordingly, in some embodiments, provided methods are useful for treating FSGS. In some embodiments, provided methods are useful for treating MN. In some embodiments, provided methods are useful for treating IgA nephropathy.
  • CNIs calcineurin inhibitors
  • Some of the drugs have a narrow therapeutic index necessitating close monitoring of the drug levels.
  • Long-term use of CNIs is associated with hypertension, nephrotoxicity, and metabolic abnormalities such as diabetes and dyslipidemia.
  • cessation of calcineurin inhibitors results in the relapse of proteinuria (Meyrier, A. et al., Kidney International. 1994;45(5): 1446-56).
  • a significant number of patients eventually become either resistant to or dependent on these toxic agents.
  • Some of these glomerular diseases also recur after renal transplantation posing unique management problems (Choy, B.Y., et al., Am. J. Transplant. 2006;6(11):2535-42).
  • provided methods are useful for treating patients with proteinuria (e.g., persistent proteinuria). It is well established that higher rates of urinary protein excretion are associated with worse prognosis, and therapies that reduce proteinuria are desirable for improving renal outcomes. Patients with persistent proteinuria (e.g., who continue to have >
  • ESKD end-stage kidney disease
  • eGFR estimated glomerular filtration rate
  • Patients with persistent proteinuria also develop further complications of chronic kidney disease (CKD) such as dyslipidemia, cardiovascular disease, abnormalities in mineral-bone metabolism, and hypertension, resulting in significant increases in morbidity and mortality and utilization of health care resources.
  • CKD chronic kidney disease
  • RAAS renin-angiotensin-aldosterone system
  • ARB angiotensin-receptor blockers
  • RAAS blockers reduce proteinuria and improve clinical outcomes in proteinuric renal diseases regardless of the etiology.
  • Other standard of care recommendations include aggressive blood pressure control ( ⁇ 130/80), and HMG-CoA reductase inhibitors (e.g., statins) in patients with hyperlipidemia.
  • statins HMG-CoA reductase inhibitors
  • the inhibitors of the mineralocorticoid receptor and sodium glucose co-transporter-2 (SGLT-2) are increasingly being used in these patients as well.
  • provided methods are useful for treating primary glomerular diseases (e.g., FSGS, membranous nephropathy, or IgA nephropathy) and persistent proteinuria.
  • primary glomerular diseases e.g., FSGS, membranous nephropathy, or IgA nephropathy
  • persistent proteinuria e.g., FSGS, membranous nephropathy, or IgA nephropathy
  • PDGFRP Platelet- derived growth factor receptor beta
  • a kidney disease to be treated by methods of the present disclosure is nephrotic syndrome (NS).
  • NS is a group of rare renal diseases, including focal and segmental glomerulosclerosis (FSGS), minimal change disease (MCD), and membranous nephropathy.
  • FSGS focal and segmental glomerulosclerosis
  • MCD minimal change disease
  • membranous nephropathy is a rare disease that attacks the kidney’s filtering units (glomeruli) causing serious scarring which leads to permanent kidney damage and even failure (Fogo, A.B. Nat. Rev. Nephrol. 2015 Feb;l l(2):76-87, PMCID:PMC4772430). It will be appreciated that there are at least three types of FSGS.
  • Primary FSGS is FSGS that has no known cause (also referred to as idiopathic FSGS). Secondary FSGS is caused by one or more factors such as infection, drug toxicity, diseases such as diabetes or sickle cell disease, obesity, or other kidney diseases. Genetic FSGS (also called familial FSGS) is caused by one or more genetic mutations. Primary FSGS is idiopathic in nature. Manifestations of this disease include hypoalbuminemia and edema, lipid abnormalities and nephrotic range proteinuria. More than 5400 patients are diagnosed with FSGS every year (O’Shaughnessy, M.M., et al. Nephrol. Dial. Transplant 2018 Apr l;33(4):661-9).
  • a kidney disease to be treated by methods of the present disclosure is minimal change disease (MCD).
  • MCD is a kidney disease in which large amounts of protein are lost in the urine. It is one of the most common causes of the nephrotic syndrome worldwide. In children, MCD is usually primary (or idiopathic), but in adults, the disease is usually secondary. Secondary causes for MCD include allergic reactions, use of certain painkillers such as non-steroidal anti-inflammatory drugs (NSAIDs), tumors, or viral infections.
  • a kidney disease to be treated by methods of the present disclosure is membranous glomerulonephritis (MG or MGN), also known as membranous nephropathy (MN).
  • MG or MGN is a slowly progressive renal disease caused by immune complex formation in the glomerulus.
  • Immune complexes are formed by binding of antibodies to antigens in the glomerular basement membrane.
  • the antigens may be part of the basement membrane, or deposited from elsewhere by the systemic circulation.
  • a kidney disease to be treated by methods of the present disclosure is anti -neutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis.
  • Anti neutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis is a rapidly progressive renal disease and includes, e.g., Wegener's granulomatosis, microscopic polyangiitis, and renal limited vasculitis.
  • Wegener's granulomatosis is an organ- and/or life-threatening autoimmune disease of unknown etiology.
  • the classical clinical triad consists of necrotizing granulomatous inflammation of the upper and/or lower respiratory tract, necrotizing glomerulonephritis, and an autoimmune necrotizing systemic vasculitis affecting predominantly small vessels.
  • the detection of anti-neutrophil cytoplasmic antibodies directed against proteinase 3 (PR3-ANCA) is a highly specific indicator for Wegener's granulomatosis.
  • Microscopic polyangiitis is a disorder that causes blood vessel inflammation (vasculitis), which can lead to organ damage.
  • the kidneys, lungs, nerves, skin, and joints are the most commonly affected areas of the body.
  • MPA is diagnosed in people of all ages, all ethnicities, and both genders. The cause of this disorder is unknown.
  • Renal limited vasculitis is a type of anti-neutrophil cytoplasmic antibody (ANCA)- associated vasculitis that presents with only a renal manifestation; no other organs, including lungs, are involved.
  • ANCA anti-neutrophil cytoplasmic antibody
  • a kidney disease to be treated by methods of the present disclosure is lupus nephritis.
  • Lupus nephritis is inflammation of the kidney that is caused by the autoimmune disease systemic lupus erythematous (SLE). With lupus, the body's immune system targets its own body tissues; lupus nephritis occurs when lupus involves the kidneys.
  • a kidney disease to be treated by methods of the present disclosure is anti-globular basement membrane (anti-GBM) nephropathy.
  • Anti-globular basement membrane (anti-GBM) nephropathy is a disease that occurs as a result of injury to small blood vessels (capillaries) in the kidneys and/or lungs.
  • anti-GBM disease autoantibodies are targeted to the basement membrane in capillary blood vessels of the kidneys and lung, where they target and damage GBM.
  • a kidney disease to be treated by methods of the present disclosure is IgA nephropathy, also known as Berger’s disease.
  • IgA nephropathy is a kidney disease that occurs when IgA deposits build up in the kidneys, causing inflammation that damages kidney tissues.
  • IgA nephropathy affects the kidneys by attacking the glomeruli. The buildup of IgA deposits inflames and damages the glomeruli, causing the kidneys to leak blood and protein into the urine. The damage may lead to scarring of the nephrons that progresses slowly over many years. Eventually, IgA nephropathy can lead to end-stage kidney disease.
  • a kidney disease to be treated by methods of the present disclosure is collagen type III glomerulopathy.
  • Collagen type III glomerulopathy also known as collagenic or collagenofibrotic glomerulopathy, is characterized by pathological accumulation of collagen type III in glomeruli.
  • Collagen type III glomerulopathy presents either in childhood, often with a family history suggesting autosomal recessive inheritance, or in adults as a sporadic occurrence.
  • Proteinuria is a typical manifestation, with progression to end stage renal disease (ESRD) in approximately 10 years.
  • ESRD end stage renal disease
  • a kidney disease to be treated by methods of the present disclosure is nail-patella syndrome.
  • Nail-patella syndrome is a multi-organ disorder caused by mutations in the LMX1B gene. Nail-patella syndrome manifests with orthopedic and cutaneous deformities, as well as kidney complications due to development of structural lesions of collagen type III within glomerular basement membranes. Although the structural lesions may be asymptomatic, they are usually accompanied by proteinuria.
  • a kidney disease to be treated by methods of the present disclosure is Alport syndrome (AS).
  • AS is a genetic condition characterized by kidney disease, hearing loss, and eye abnormalities. Most affected individuals experience progressive loss of kidney function, usually resulting in end-stage kidney disease.
  • Alport syndrome is inherited in an X-linked manner and is caused by mutation(s) in the COL4A5 gene. In other cases, it can be inherited in either an autosomal recessive, or rarely in an autosomal dominant manner, and is caused by mutation(s) in the COL4A3 and/or COL4A4 genes.
  • Current therapies include hearing aid, hemodialysis, peritoneal dialysis and kidney transplantation.
  • a kidney disease to be treated by methods of the present disclosure is polycystic kidney disease (e.g., autosomal recessive polycystic kidney disease (ARPKD) - congenital hepatic fibrosis (CHF)).
  • ARPKD-CHF is a highly aggressive fibropolycystic disease that is characterized by the formation and expansion of fluid-filled cysts in the kidneys, enlargement of the kidneys and progressive fibrosis of both the kidney and the liver (Hartung, E.A., and Guay -Woodford, L.M. Pediatrics 2014 Sep;134(3):e833-e845; Gunay- Aygun, M., et al. J.
  • ARPKD-CHF congenital hepatic fibrosis
  • a kidney disease to be treated by methods of the present disclosure is or includes renal cysts.
  • Aberrant signaling by tyrosine kinases including platelet- derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) and their receptors (R), PDGFR and VEGFR/KDR, respectively, has been implicated in the formation and expansion of renal cysts.
  • PDGF platelet- derived growth factor
  • VEGF vascular endothelial growth factor
  • R vascular endothelial growth factor
  • a PDGF-driven ciliopathy and/or overexpression of PDGF in the cyst lining and adjacent tubules are thought to, in part, drive renal cystic disease (Torres, V.E., et al. Lancet 2007 Apr 14;369(9569): 1287-301; Park. J.H. et al.
  • VEGF-driven angiogenesis is also thought to contribute to the growth of renal cysts, and inhibition of VEGFR/KDR signaling is associated with decreased tubule cell proliferation, decreased cystogenesis, and blunted renal enlargement (Bello-Ruess, E., et al. Kidney Int. 2001 Jul;60(l):37-45; Schrijvers, B.F., et al. Kidney Int. 2004 Jun;65(6):2003-17). Nevertheless, the role of VEGF in fibropoly cystic disease is more controversial, with at least some reports suggesting that this growth factor might be associated with disease mitigation (Spirli, C., et al. Gastroenterology 2010 Jan;138(l):360-71).
  • provided methods are useful for treating dermal diseases, disorders, or conditions. In some embodiments, provided methods are useful for treating dermal fibrosis. In some embodiments, provided methods are useful for treating dermal fibrosis secondary to, or otherwise associated with, an underlying indication. In some embodiments, provided methods are useful for treating scleroderma and/or systemic sclerosis (e.g., diffuse systemic sclerosis or limited systemic sclerosis).
  • systemic sclerosis e.g., diffuse systemic sclerosis or limited systemic sclerosis.
  • a fibrotic disease to be treated by methods of the present disclosure is scleroderma and/or systemic sclerosis (SSc).
  • Scleroderma which literally means hard skin, is a chronic fibrotic disorder of unknown etiology that affects the skin and other internal organs (SSc) (www.scleroderma.org).
  • SSc systemic sclerosis
  • Many patients who suffer from scleroderma/SSc also have loss of lung function.
  • Scleroderma/SSc and related diseases afflict approximately 400,000 to 990,000 people in the USA every year. Mortality and morbidity in scleroderma/SSc are very high and directly related to the extent of fibrosis of the involved organs (Hinchcliff, M.
  • Scleroderma/SSc can be classified in terms of the degree and location of the skin involvement and has been categorized into two major groups - diffuse and limited.
  • the diffuse form of scleroderma/SSc involves symmetric thickening of skin of the extremities, face and trunk. Organs affected include the esophagus, intestines, lungs, heart, and kidneys (Mayes, M. D. Semin. Cutan. Med. Surg. 1998 Mar;17(l):22-6; Jacobsen, L. et al. J. Am. Acad. Dermatol. 2003 Aug;49(2):323-5).
  • the limited form of scleroderma/SSc tends to be confined to the skin of fingers and face.
  • scleroderma/SSc The limited form of scleroderma/SSc is the CREST variant of scleroderma/SSc based on the clinical pattern of calcinosis with tiny deposits of calcium in the skin, Raynaud's phenomenon in the fingers, toes, nose, tongue, or ears, poor functioning of muscle of esophagus, sclerodactyly of the skin of the fingers or toes, and telangiectasias on the face, hands and mouth (Winterbauer, R.H. Bull. Johns Hopkins Hospital 1964;114:361-83; Wollheim, F.A. Classification of systemic sclerosis. Visions and reality. Rheumatology (Oxford) 2005).
  • fibrotic pathways are activated in scleroderma/SSc for reasons that are not completely understood.
  • the pathogenesis of fibrosis in scleroderma/SSc involves a complex set of interactions involving immune activation, microvascular damage and the activation of fibroblasts.
  • Scleroderma/SSc is characterized by excessive deposition of collagen in the skin and other involved organs and abnormalities of blood vessels (Jimenez, S. A., et al. Rheum. Dis.
  • TGFpi a multifunctional cytokine
  • PDGF downregulating growth factor
  • TGFp and PDGF are the most potent proteins involved in fibroblast proliferation, collagen gene expression and connective tissue (collagen) accumulation (Antoniades, H.N. Baillieres Clin. Endocrinol. Metab. 1991 Dec;5(4):595-613). Numerous other cytokines including VEGF, as well as cell-matrix interactions, also modify collagen expression and can influence the effects of TGFpi and PDGF (Trojanowska, M. Rheumatology (Oxford) 2008 Oct;47 Suppl 5:v2-4).
  • Persistent overproduction of collagen and other connective tissue results in excessive accumulation of ECM components leading to the formation of scar tissue (fibrosis) in the skin and other organs and is responsible for the progressive nature of scleroderma/SSc (Mauch, C. Rheum. Dis. Clin. North Am. 1990 Feb;16(l):93-107). This leads to thickness and firmness of involved areas.
  • the pathogenic cascade at different stages of scleroderma/SSc may have autoimmune, inflammatory, fibrotic and vascular components with systemic fibrosis and vasculopathy.
  • provided methods are useful for treating gastrointestinal diseases, disorders, or conditions.
  • provided methods are useful for treating gastrointestinal fibrosis (e.g., fibrosis of esophagus, stomach, intestines, and/or colon).
  • provided methods are useful for treating gastrointestinal fibrosis secondary to, or otherwise associated with, an underlying indication.
  • provided methods are useful for treating inflammatory bowel disease (e.g., ulcerative colitis or Crohn’s disease), e.g., treating gastrointestinal fibrosis associated with inflammatory bowel disease.
  • IBD is primarily an inflammatory autoimmune disease
  • anti-fibrotic agents e.g., Compound 1 could be useful in treating IBD and, further could provide benefits beyond mere anti-fibrotic effects.
  • a disease to be treated by methods of the present disclosure is inflammatory bowel disease (IBD).
  • IBD is an inflammatory condition that comprises both ulcerative colitis (UC) and Crohn's disease (CD). While UC affects the entire colon, CD typically affects the ileum but can occur in any part of the GI tract.
  • IBD can manifest as acute or chronic colitis, characterized by recurrent intestinal inflammation accompanied by diarrhea and abdominal pain (Arivarasu, N., et al. Tissue Barriers 2018;6(2):el463897; Ponder, A. and Long, M.D. Clin. Epidemiol. 2013;5:237-47).
  • IBD Incidence of IBD is increasing worldwide and is an expanding global health problem (Amosy, E., et al. Clin. Med. Insights Gastroenterol. 2013;6:33-47). An estimated 2.5-3 million people in Europe are affected by IBD (Burisch, J., et al. J. Crohns Colitis 2013 May;7(4):322- 37). According to the Centers for Disease Control and Prevention (CDC), 3.1 million adults in this country were diagnosed with IBD in 2015, a substantial increase from the - 1.4 million adults diagnosed per 2008 reports (www.cdc.gov/IBD; www.cdc.gov/ibd/pdf/inflammatory- bowel-disease-an-expensive-disease.pdf).
  • IBD accounts for -1,300,000 physician visits and -92,000 hospitalizations each year in the United States. Of these, 75% patients diagnosed with CD and 25% patients diagnosed with UC and require surgery. Risk factors associated with IBD include environmental, genetic and immunologic factors (Abegunde, A.T., et al. World J. Gastroenterol. 2016 Jul 21;22(27):6296-6317; Frolkis, A., et al. Can. J. Gastroenterol. 2013 Mar; 27(3 ) : e28 -24) .
  • IBD ulcerative colitis
  • IBD is an autoimmune disease with excessive activation of the adaptive immune response.
  • Various factors including genetic factors alter the intestinal flora and trigger an inflammatory reaction, activate T cells, B cells, mast cells, macrophages and microglia, smooth muscle cells and fibroblasts in the colon, inducing mucosal disruption (Hildner, K., et al. Dig. Dis. 2016;34Suppl 1:40-7; Curciarello, R., et al. Front Med. (Lausanne) 2017 Aug 7;4:126).
  • Epithelial and endothelial damage release chemotactic factors promoting recruitment and activation of inflammatory cells, and release various cytokines including TNFa, and activate fibroblasts via TGFpi.
  • Activated fibroblasts i.e. myofibroblasts, secrete growth factors including platelet derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) (Scaldaferri, et al. Gastroenterology 2009 Feb;136(2):585-95.e5).
  • PDGF platelet derived growth factor
  • VEGF vascular endothelial growth factor
  • angiogenesis is also an important part of IBD pathogenesis in the colon of IBD patients.
  • Alkim, et al. demonstrated enhanced microvessel density in the intestinal tissue of both UC and CD patients, which correlated both the level of local VEGF expression and disease activity (Int. J. Inflam. 2015;2015:970890).
  • Anti-inflammatory drugs including 5-aminosalicylic acid (5-ASA)-based preparations, are often the first line of therapy in IBD (Segars, L.W., et al. Clin. Pharm. 1992 Jun;ll(6):514-28).
  • Anti-TNFa antibodies such as infliximab and adalimumab are also being used. Nevertheless, patients treated with adalimumab are at increased risk for serious infections and lymphoma (Dulai, P.S., et al. Clin. Gastroenterol. Hepatol. 2014 Sep; 12(9): 1443-51).
  • PDGF activates fibroblasts and IBD-fibroblasts proliferate more rapidly than normal fibroblasts; collagen secretion from IBD patients’ fibroblasts was increased compared to collagen secretion by normal fibroblasts.
  • IBD is also associated with increased circulating PDGF and the level of this growth factor has been reported to correspond with disease severity (Andrae, J., et al. Genes Dev. 2008 May 15;22(10): 1276-1312).
  • angiogenesis is a novel component of IBD pathogenesis and angiogenic activity is increased in IBD patients.
  • Serum VEGF levels were significantly higher in IBD patients compared to controls in several studies.
  • Griga et al. demonstrated that sources of increased serum VEGF were from inflamed intestinal tissue of IBD patients (Scand. J. Gastroenterol. 1998 May;33(5):504-8; Hepatogastroenterology 2002 Jan-Feb;49(43): 116-23; Hepatogastroenterology 1999 Mar-Apr;46(26):920-3; Eur. J. Gastroenterol. Hepatol. 1999 Feb; 11(2): 175-9).
  • VEGF expression was markedly increased in the inflamed mucosa of both CD and UC patients, when compared with normal mucosa of the same patient. Studies also showed that VEGF expression was increased in colon and was higher across all IBD groups (both CD and UC) when compared with healthy controls. Scaldaferri, et al. (2009) reported that VEGF receptor (VEGFR/KDR) levels were increased in intestinal samples of IBD patients, and in mice with experimental colitis.
  • VEGFR/KDR VEGF receptor
  • provided methods are useful for treating certain other diseases, disorders, or conditions.
  • provided methods are useful for treating cardiac fibrosis and/or fibrosis associated with cardiovascular system.
  • provided methods are useful for treating cardiac fibrosis secondary to, or otherwise associated with, an underlying indication.
  • provide methods are useful for treating cardiac and/or cardiovascular fibrosis associated with ischemic heart disease, myocardial ischemia, athereosclerosis, myocardial perfusion (e.g., as a consequence of chronic cardiac ischemia or myocardial infarction), vascular occlusion, or restenosis.
  • a disease to be treated by methods of the present disclosure is ischemic heart disease.
  • Ischemic heart disease is a leading cause of morbidity and mortality in the US, afflicting millions of Americans each year at a cost expected to exceed $300 billion/year.
  • Numerous pharmacological and interventional approaches are being developed to improve treatment of ischemic heart disease including reduction of modifiable risk factors, improved revascularization procedures, and therapies to halt progression and/or induce regression of atherosclerosis.
  • atherosclerosis comprises a fibrotic component.
  • provided methods are useful for treating fibrosis associated with central nervous system (CNS) and/or one or more CNS-related diseases, disorders, or conditions.
  • CNS central nervous system
  • provided methods are useful for treating CNS-associated fibrosis secondary to, or otherwise associated with, an underlying indication.
  • provided methods are useful for treating fibrosis associated with cerebral infarction, stroke, or amyotrophic lateral sclerosis.
  • provided methods are useful for treating fibrosis associated with musculoskeletal system and/or one or more musculoskeletal diseases, disorders, or conditions. In some embodiments, provided methods are useful for treating musculoskeletal- associated fibrosis secondary to, or otherwise associated with, an underlying indication. In some embodiments, provided methods are useful for treating fibrosis associated with muscular dystrophy.
  • provided methods are useful for treating pancreatic fibrosis.
  • provided methods are useful for treating pancreatic fibrosis secondary to, or otherwise associated with, an underlying indication. In some embodiments, provided methods are useful for treating fibrosis associated with pancreatitis.
  • one or more subjects or populations are selected to receive Compound 1 as described herein based on one or more markers and/or characteristics such as, for example, one or more risk factors of fibrosis or an associated disease, disorder or condition and/or one or more biomarkers, etc.
  • a subject or population thereof is selected to receive Compound 1 using technologies provided herein (e.g., based on assessment of one or more markers and/or characteristics, such as one or more biomarkers).
  • technologies are used to inform or determine one or more features of a therapeutic regimen (e.g., selection of subject(s) to receive a particular therapy (e.g., Compound 1 therapy) and/or dose thereof and/or timing of administration of such therapy).
  • assessment of one or more markers and/or characteristics is performed with respect to the same subject at a plurality of different time points. In some embodiments, assessment of one or more markers and/or characteristics is performed with respect to a particular patient prior to initiation of a particular therapeutic regimen (e.g., a Compound 1 therapeutic regimen) and/or prior to administration of a particular dose of therapy (e.g., Compound 1 therapy) in accordance with such therapeutic regimen.
  • a particular therapeutic regimen e.g., a Compound 1 therapeutic regimen
  • a particular dose of therapy e.g., Compound 1 therapy
  • a subject or population thereof is suffering from or susceptible to a disease, disorder, or condition described herein. In some embodiments, a subject or population thereof is suffering from or is susceptible to fibrosis. In some embodiments, a subject or population thereof is suffering from or is susceptible to a disease, disorder, or condition characterized by or otherwise associated with fibrosis. In some embodiments, a subject or population thereof is suffering from or is susceptible to fibrosis of gastrointestinal tract, heart, kidney, lung, liver, muscle, pancreas, and/or skin.
  • a subject or population thereof is suffering from or is susceptible to an acute injury (e.g., an acute organ injury, such as acute lung injury, acute liver injury, or acute kidney injury).
  • a subject or population thereof is suffering from or is susceptible to a chronic injury (e.g., a chronic organ injury, such as chronic lung injury, chronic liver injury, or chronic kidney injury).
  • a subject or population thereof is suffering from a traumatic injury.
  • a subject or population thereof has undergone, is undergoing, or will undergo an organ transplantation.
  • a subject or population thereof is suffering from or susceptible to a damaged and/or ischemic organ, transplant, or graft.
  • a subject or population thereof is suffering from or susceptible to ischemia/reperfusion injury. In some embodiments, a subject or population thereof is suffering from or susceptible to post-surgical scarring. In some embodiments, a subject or population thereof is suffering from a wound.
  • a subject or population thereof is suffering from or is susceptible to a pulmonary disease, disorder, or condition. In some embodiments, a subject or population thereof is suffering from or is susceptible to pulmonary fibrosis. In some embodiments, a subject or population thereof is suffering from or is susceptible to pulmonary fibrosis secondary to, or otherwise associated with, an underlying indication. In some embodiments, a subject or population thereof is suffering from or is susceptible to interstitial lung disease (e.g., fibrosing interstitial lung disease). In some embodiments, a subject or population thereof is suffering from or is susceptible to idiopathic interstitial pneumonia. In some embodiments, a subject or population thereof is suffering from or is susceptible to idiopathic pulmonary fibrosis.
  • interstitial lung disease e.g., fibrosing interstitial lung disease
  • a subject or population thereof is suffering from or is susceptible to idiopathic interstitial pneumonia. In some embodiments, a subject or population thereof is suffering from or is susceptible to idiopathic
  • a subject or population thereof is suffering from or is susceptible to a hepatic disease, disorder, or condition.
  • a subject or population thereof is suffering from or is susceptible to hepatic fibrosis (e.g., fibrotic liver disease).
  • a subject or population thereof is suffering from or is susceptible to cirrhosis.
  • a subject or population thereof is suffering from or is susceptible to hepatic fibrosis secondary to, or otherwise associated with, an underlying indication.
  • a subject or population thereof is suffering from or is susceptible to hepatitis C, hepatitis B, delta hepatitis, chronic alcoholism, nonalcoholic steatohepatitis (NASH), extrahepatic obstructions (e.g., stones in bile duct), cholangiopathies (e.g., primary biliary cirrhosis or sclerosing cholangitis), autoimmune liver disease, or inherited metabolic disorders (e.g., Wilson’s disease, hemochromatosis, or alpha-1 antitrypsin deficiency).
  • a subject or population thereof is suffering from or is susceptible to a renal disease, disorder, or condition.
  • a subject or population thereof is suffering from or is susceptible to renal fibrosis. In some embodiments, a subject or population thereof is suffering from or is susceptible to renal fibrosis secondary to, or otherwise associated with, an underlying indication. In some embodiments, a subject or population thereof is suffering from or is susceptible to renal failure, renal obstruction, renal trauma, renal transplantation, chronic kidney disease, diabetes, hypertension, radiocontrast nephropathy, immune-mediated glomerulonephri tides (e.g., lupus nephritis, ANCA-associated glomerulonephritides (e.g., Wegener’s granulomatosis, microscopic polyangiitis, or renal limited vasculitis), anti-GBM nephropathy, IgA nephropathy, membranous glomerulonephritis, or focal and segmental glomerulosclerosis), non-immune-mediated glomerulonephritides (e.g., polyc
  • a subject or population thereof is suffering from or is susceptible to a fibrotic disease of the kidney that is or comprises: focal segmental glomerulosclerosis (FSGS), steroid resistant nephrotic syndrome (SRNS), proteinuria, lupus nephritis, minimal change disease, an anti-neutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis, Alport syndrome, anti-globular basement membrane (anti-GBM) nephropathy, IgA nephropathy, membranous glomerulonephritis (MG), autosomal dominant polycystic kidney disease (ADPKD), collagen type III glomerulopathy, nail-patella syndrome, or chronic kidney disease.
  • FSGS focal segmental glomerulosclerosis
  • SRNS steroid resistant nephrotic syndrome
  • proteinuria lupus nephritis
  • minimal change disease an anti-neutrophil cytoplasmic antibody (ANCA)-associated glomeruloneph
  • a subject or population thereof is suffering from or is susceptible to a fibrotic disease of the kidney that is or comprises an anti-neutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis.
  • ANCA-associated glomerulonephritis is selected from Wegener’s granulomatosis, microscopic polyangiitis (MPA), or renal limited vasculitis.
  • a subject or population thereof is suffering from or is susceptible to collagen type III glomerulopathy or nail-patella syndrome.
  • a subject or population thereof is suffering from or is susceptible to nephrotic syndrome and/or diseases, disorders, or conditions associated with nephrotic syndrome (e.g., focal and segmental glomerulosclerosis, minimal change disease, and membranous nephropathy).
  • a subject or population thereof is suffering from or is susceptible to focal and segmental glomerulosclerosis.
  • a subject or population thereof is suffering from or is susceptible to Alport syndrome.
  • a subject or population thereof is suffering from or is susceptible to polycystic kidney disease (e.g., autosomal dominant polycystic kidney disease or autosomal recessive polycystic kidney disease).
  • a subject or population thereof is suffering from or is susceptible to primary proteinuric kidney disease (e.g., as confirmed from a renal biopsy).
  • a subject or population thereof is suffering from or is susceptible to primary glomerular diseases (e.g., as confirmed from a renal biopsy).
  • a subject or population thereof is suffering from or susceptible to persistent proteinuria.
  • a subject or population thereof is suffering from or susceptible to primary glomerular disease (e.g., as confirmed from a renal biopsy) and persistent proteinuria.
  • a subject or population thereof is suffering from or susceptible to proteinuric chronic kidney disease.
  • a subject or population thereof is suffering from or is susceptible to focal and segmental glomerulosclerosis. In some embodiments, a subject or population thereof is suffering from or is susceptible to membranous nephropathy. In some embodiments, a subject or population thereof is suffering from or susceptible to IgA nephropathy.
  • a subject or population thereof has one or more risk factors for primary glomerular disease and/or primary proteinuric kidney disease.
  • a subject or population thereof has one or more risk factors for primary glomerular disease and/or primary proteinuric kidney disease selected from proteinuria, renal dysfunction, hypertension, interstitial fibrosis on renal biopsy, and lack of response to therapy or relapse in proteinuria.
  • a subject or population thereof has one or more risk factors for primary glomerular disease (e.g., FSGS) selected from serum creatinine > 1.3 mg/dL, proteinuria > 3.5 g/day, lack of response to therapy, and/or a collapsing variant.
  • a subject or population thereof has one or more risk factors for primary glomerular disease (e.g., IgA nephropathy) selected from serum creatinine > 1.26 mg/dL, hypertension (e.g., >140/90 mmHg), and/or persistent (e.g., > 6 months) protein excretion > 1000 mg/day.
  • IgA nephropathy selected from serum creatinine > 1.26 mg/dL, hypertension (e.g., >140/90 mmHg), and/or persistent (e.g., > 6 months) protein excretion > 1000 mg/day.
  • a subject or population thereof has one or more risk factors for primary glomerular disease (e.g., membranous nephropathy) selected from serum creatinine > 1.5 mg/dL, progressive decline in eGFR > 25% over prior 2 years, and/or severe disabling or life-threatening nephrotic syndrome (with e.g., serum albumin ⁇ 2.5 g/dL, refractory edema, and/or thromboembolic event).
  • primary glomerular disease e.g., membranous nephropathy
  • serum creatinine > 1.5 mg/dL e.g., progressive decline in eGFR > 25% over prior 2 years
  • severe disabling or life-threatening nephrotic syndrome with e.g., serum albumin ⁇ 2.5 g/dL, refractory edema, and/or thromboembolic event.
  • a subject or population thereof has a risk factor for primary glomerular disease (e.g., FSGS, IgA nephropathy, and/or membranous nephropathy) of eGFR ⁇ 70 mL/min/1.73m 2 .
  • FSGS primary glomerular disease
  • IgA nephropathy IgA nephropathy
  • membranous nephropathy membranous nephropathy
  • a subject or population thereof has an estimated glomerular filtration rate (eGFR) of greater than or equal to 40 mL/min/1.73m 2 , e.g., calculated using Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI). In some embodiments, a subject or population thereof has an estimated glomerular filtration rate (eGFR) of greater than or equal to 30 mL/min/1.73m 2 , e.g., calculated using Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI).
  • CKD-EPI Chronic Kidney Disease Epidemiology Collaboration
  • a subject or population thereof has a urinary protein excretion of greater than or equal to 1 g/day on a 24-hour urine collection.
  • a subject or population thereof has stable blood pressure (e.g., less than or equal to 140/90 mmHg) for at least 12 weeks. In some embodiments, a subject or population thereof is on a stable antihypertensive regimen and has stable blood pressure (e.g., less than or equal to 140/90 mmHg) for at least 12 weeks.
  • a subject or population thereof is receiving or has received standard of care therapy. In some embodiments, a subject or population thereof is receiving or has received a maximally tolerated or recommended dose of standard of care therapy. In some embodiments, a subject or population is receiving or has received standard of care therapy and has been stable on said therapy for at least 12 weeks. In some embodiments, a subject or population thereof is resistant to standard of care therapy. In some embodiments, a subject or population thereof has relapsed after receiving standard of care therapy. In some embodiments, standard of care therapy comprises immunosuppressive or immunomodulatory agents.
  • standard of care therapy comprises renin-angiotensin-aldosterone system (RAAS) blockers, such as, e.g., ACE inhibitors or angiotensin-receptor blockers (ARBs).
  • RAAS renin-angiotensin-aldosterone system
  • standard of care therapy comprises immunosuppressive agents and RAAS blockers (e.g., ACE inhibitors or ARBs).
  • standard of care comprises blood pressure control (e.g., to ⁇ 130/80 mmHg) and/or HMG-CoA reductase inhibitors (i.e., statins), particularly in patients with hyperlipidemia.
  • a subject or population thereof is receiving or has received an immunosuppressive or immunomodulatory agent. In some embodiments, a subject or population thereof is receiving or has received therapy comprising an immunosuppressive or immunomodulatory agent and has been stable on said therapy for at least 12 weeks.
  • a subject or population thereof is receiving or has received a RAAS blocker. In some embodiments, a subject or population thereof is receiving or has received an ACE inhibitor. In some embodiments, a subject or population thereof is receiving or has received an ARB. In some embodiments, a subject or population thereof is receiving or has received a maximally tolerated or recommended dose of a RAAS blocker (e.g., an ACE inhibitor or ARB). In some embodiments, a subject or population thereof is receiving or has received an inhibitor of mineralocorticoid receptor. In some embodiments, a subject or population thereof is receiving or has received an inhibitor of sodium glucose co-transporter-2 (SGLT-2). In some embodiments, a subject or population thereof is receiving or has received therapy comprising a RAAS blocker (e.g., an ACE inhibitor or ARB) or SGLT-2 inhibitor and has been stable on said therapy for at least 12 weeks.
  • a RAAS blocker e.g., an ACE inhibitor or ARB
  • SGLT-2 inhibitor
  • a subject or population thereof is receiving or has received therapy comprising an immunosuppressive or immunomodulatory agent and one or more of a RAAS blocker (e.g., ACE inhibitor or ARB), mineralocorticoid receptor inhibitor or SGLT-2 inhibitor.
  • a RAAS blocker e.g., ACE inhibitor or ARB
  • mineralocorticoid receptor inhibitor e.g., mineralocorticoid receptor inhibitor or SGLT-2 inhibitor.
  • the subject or population thereof has been stable on said therapy for at least 12 weeks.
  • a subject or population thereof is not receiving non-steroidal anti-inflammatory agents (NSAIDS). In some embodiments, a subject or population thereof is not receiving non-steroidal anti-inflammatory agents (NSAIDS) chronically.
  • NSAIDS non-steroidal anti-inflammatory agents
  • a subject or population thereof is negative for hepatitis B virus, hepatitis C virus, and/or human immunodeficiency virus. In some embodiments, a subject or population thereof does not have a history of treated hepatitis C virus.
  • a subject or population thereof does not have any one or more hematologic abnormalities selected from hemoglobin ⁇ 8 g/dL, platelets ⁇ 50,000, and absolute neutrophil count (ANC) ⁇ 1000 cells/pL. In some embodiments, a subject or population thereof does not have hemoglobin AIC > 8.5%.
  • a subject or population thereof does not have any one or more liver function results selected from aspartate aminotransferase (AST), alanine aminotransferase (ALT), or total bilirubin > 2x upper limit of normal (ULN).
  • AST aspartate aminotransferase
  • ALT alanine aminotransferase
  • UPN upper limit of normal
  • a subject or population thereof has no known predisposition to bleeding.
  • a subject or population thereof does not require fibrinolysis, full-dose therapeutic anti coagulation (e.g., vitamin K antagonists, dabigatran, heparin, hirudin, etc.), or high dose antiplatelet therapy.
  • a subject or population thereof has no history of hemorrhagic central nervous system events, e.g., within 12 months. In some embodiments, a subject or population thereof has no history of active gastrointestinal bleeding, e.g., within 6 months. In some embodiments, a subject or population thereof has no history of thrombotic events (including, e.g., stroke or transient ischemic attack), e.g., within 12 months.
  • a subject or population thereof is not diagnosed with Type I diabetes mellitus. In some embodiments, a subject or population thereof has never had (e.g., in the last 5 years) a renal biopsy showing histopathological evidence of diabetic kidney disease. In some embodiments, a subject or population thereof is not diagnosed with anti-PLA2R antibody- associated membranous nephropathy.
  • a subject or population thereof has not had a myocardial infarction or unstable angina, e.g., within 6 months.
  • a subject or population thereof has no history of solid organ or hematopoietic cell transplantation. In some embodiments, a subject or population thereof is not on an organ transplant waiting list.
  • a subject or population thereof has no history or presence of any form of cancer except excised basal or squamous cell carcinoma of the skin, e.g., within 2 years.
  • a subject or population thereof is not suffering from renal disease secondary to systemic disease, including but not limited to systemic lupus erythematosus, anti-neutrophil cytoplasmic antibodies-associated diseases, anti-glomerular basement disease, secondary forms of focal segmental glomerulosclerosis, renal diseases associated with para- proteinemias, C3 glomerulopathy, and diabetic kidney disease.
  • a subject or population thereof is not suffering from a known systemic disorder that requires or is expected to require systemic glucocorticoids or immune modulators.
  • a subject or population thereof has not received treatment with anti-CD20 monoclonal antibodies, e.g., within 6 months.
  • a subject or population thereof has a BMI of greater than or equal to 40 kg/m 2 .
  • a subject or population thereof is suffering from or is susceptible to a dermal disease, disorder, or condition. In some embodiments, a subject or population thereof is suffering from or is susceptible to dermal fibrosis. In some embodiments, a subject or population thereof is suffering from or is susceptible to dermal fibrosis secondary to, or otherwise associated with, an underlying indication. In some embodiments, a subject or population thereof is suffering from or is susceptible to scleroderma and/or systemic sclerosis (e.g., diffuse systemic sclerosis or limited systemic sclerosis).
  • systemic sclerosis e.g., diffuse systemic sclerosis or limited systemic sclerosis
  • a subject or population thereof is suffering from or is susceptible to a gastrointestinal disease, disorder, or condition.
  • a subject or population thereof is suffering from or is susceptible to gastrointestinal fibrosis (e.g., fibrosis of esophagus, stomach, intestines, and/or colon).
  • a subject or population thereof is suffering from or is susceptible to gastrointestinal fibrosis secondary to, or otherwise associated with, an underlying indication.
  • a subject or population thereof is suffering from or is susceptible to inflammatory bowel disease (e.g., ulcerative colitis or Crohn’s disease).
  • a subject or population thereof is suffering from or is susceptible to cardiac fibrosis and/or fibrosis associated with cardiovascular system. In some embodiments, a subject or population thereof is suffering from or is susceptible to cardiac fibrosis secondary to, or otherwise associated with, an underlying indication. In some embodiments, a subject or population thereof is suffering from or is susceptible to ischemic heart disease, myocardial ischemia, atherosclerosis, myocardial perfusion (e.g., as a consequence of chronic cardiac ischemia or myocardial infarction), vascular occlusion, or restenosis.
  • a subject or population thereof is suffering from or is susceptible to fibrosis associated with central nervous system (CNS) and/or one or more CNS- related diseases, disorders, or conditions.
  • CNS central nervous system
  • a subject or population thereof is suffering from or is susceptible to CNS-associated fibrosis secondary to, or otherwise associated with, an underlying indication.
  • a subject or population thereof is suffering from or is susceptible to cerebral infarction, stroke, or amyotrophic lateral sclerosis.
  • a subject or population thereof is suffering from or is susceptible to fibrosis associated with musculoskeletal system and/or one or more musculoskeletal diseases, disorders, or conditions.
  • a subject or population thereof is suffering from or is susceptible to musculoskeletal-associated fibrosis secondary to, or otherwise associated with, an underlying indication. In some embodiments, a subject or population thereof is suffering from or is susceptible to muscular dystrophy.
  • a subject or population thereof is suffering from or is susceptible to pancreatic fibrosis. In some embodiments, a subject or population thereof is suffering from or is susceptible to pancreatic fibrosis secondary to, or otherwise associated with, an underlying indication. In some embodiments, a subject or population thereof is suffering from or is susceptible to pancreatitis.
  • the present disclosure also provides methods related to treatment of fibrotic and related disease(s) (e.g., as described herein, such as kidney fibrotic disease(s) or lung fibrotic disease(s)) and selecting, identifying, and/or characterizing patients likely to benefit from a treatment with Compound 1 as described herein.
  • fibrotic and related disease(s) e.g., as described herein, such as kidney fibrotic disease(s) or lung fibrotic disease(s)
  • the present disclosure is based in part on the discovery that certain biomarkers can distinguish patients who are likely to respond to therapy, for example because the drivers of their disease (e.g., as described herein, such as kidney disease or lung disease) correspond with a mechanism of action of Compound 1.
  • a patient to be treated with a method of the present disclosure has an altered level of one or more gene products or one or more proteins (or fragments thereof) that are part of the mechanism of action of Compound 1 (e.g., down regulated by Compound 1).
  • a biomarker is a component of a biological sample that may be detected and/or quantified when present in the biological sample.
  • a patient to be treated with a method of the present disclosure has an elevated level of gene expression (e.g., COL3A1 expression and/or COL1A1 expression).
  • a patient to be treated with a method of the present disclosure has an elevated level of protein expression (e.g., COL3A1 protein and/or COL1 A1 protein) or elevated level of a fragment thereof.
  • an elevated level of expression e.g., of COL3A1 and/or COL1 Al
  • a gene product e.g., mRNA expressed from a COL3A1 gene and/or COL1A1 gene
  • protein e.g., COL3A1 protein and/or COL1 Al protein
  • a non-italicized name will be used to refer to both the gene and protein product.
  • a level of COL3A1 and/or COL1A1 corresponds to a level of gene expression (e.g., RNA expression, e.g., mRNA expression).
  • a level of COL3A1 and/or COL1 Al corresponds to a level of collagen protein expression.
  • collagen protein expression includes expression of a protein in any form, including, a procollagen polypeptide (e.g., type III procollagen or type I procollagen), a collagen protein (e.g., type III collagen or type I collagen), a preprocollagen polypeptide, an amino-terminal procollagen polypeptide (e.g., pN-type III collagen (“PIIINP”) or pN-type I collagen (“PINP”)), collagen fibril, collagen fiber, and/or any fragment or degradation product thereof.
  • a procollagen polypeptide e.g., type III procollagen or type I procollagen
  • collagen protein e.g., type III collagen or type I collagen
  • preprocollagen polypeptide e.g., an amino-terminal procollagen polypeptide (e.g., pN-type III collagen (“PIIINP”) or pN-type I collagen (“PINP”)), collagen fibril, collagen fiber, and/or any fragment or degradation product thereof.
  • PIIINP pN
  • a level of COL3 Al refers to a gene product (e.g., RNA, e.g., mRNA) or a protein product (e.g., a prepropolypeptide, propolypeptide, amino- terminal propolypeptide, protein, fibril, fiber, or any fragment or degradation product thereof).
  • a gene product e.g., RNA, e.g., mRNA
  • a protein product e.g., a prepropolypeptide, propolypeptide, amino- terminal propolypeptide, protein, fibril, fiber, or any fragment or degradation product thereof.
  • a level of COL1A1 refers to a gene product (e.g., RNA, e.g., mRNA) or a protein product (e.g., a prepropolypeptide, propolypeptide, amino-terminal propolypeptide, protein, fibril, fiber, or any fragment or degradation product thereof).
  • a gene product e.g., RNA, e.g., mRNA
  • a protein product e.g., a prepropolypeptide, propolypeptide, amino-terminal propolypeptide, protein, fibril, fiber, or any fragment or degradation product thereof.
  • a method for treating a patient diagnosed with, suspected of having, or at risk for a fibrotic disease comprising: (i) obtaining or determining a level of expression of a biomarker (e.g., COL3 Al and/or COL1 Al) in a biological sample from the patient; (ii) comparing the level of biomarker expression with a threshold level (e.g., a predetermined mean or median level of a population of healthy subjects), wherein if the expression level of the biomarker is different from the threshold level (e.g., above the threshold level or below the threshold level), administering to the patient an effective amount of Compound 1, or a pharmaceutical composition thereof.
  • obtaining a level of expression of a biomarker may comprise obtaining knowledge of a level that has been determined previously (e.g., obtaining said level from a report
  • a method for treating a patient diagnosed with, suspected of having, or at risk for a fibrotic disease comprising: (i) obtaining or determining a level of expression of a biomarker (e.g., COL3A1 and/or COL1 Al) in a biological sample from the patient; (ii) comparing the level of biomarker expression with a threshold level (e.g., a predetermined mean or median level of a population of healthy subjects), wherein if the expression level of the biomarker is above the threshold level, administering to the patient an effective amount of Compound 1, or a pharmaceutical composition thereof.
  • a biomarker e.g., COL3A1 and/or COL1 Al
  • a method for treating a patient diagnosed with, suspected of having, or at risk for a fibrotic disease comprising administering an effective amount of Compound 1, or a pharmaceutical composition thereof, to a patient that has been determined to have an altered (e.g., elevated or reduced) level of a biomarker (e.g., COL3 Al and/or COL1 Al).
  • a biomarker e.g., COL3 Al and/or COL1 Al
  • a method for treating a patient diagnosed with, suspected of having, or at risk for a fibrotic disease comprising administering an effective amount of Compound 1, or a pharmaceutical composition thereof, to a patient that has been determined to have an elevated level of a biomarker (e.g., COL3 Al and/or COL1 Al).
  • a biomarker e.g., COL3 Al and/or COL1 Al.
  • an elevated level of a biomarker comprises a level that is above that of a corresponding threshold level.
  • a reduced level of COL3A1 and/or COL1 Al comprises a level that is below a corresponding threshold level.
  • the level of biomarker (e.g., COL3A1 and/or COL1 Al) was obtained from or previously determined from a biological sample from the patient.
  • the method further comprises obtaining or determining a level of a biomarker (e.g., COL3A1 and/or COL1 Al) in a biological sample from the patient.
  • a method for treating a patient diagnosed with, suspected of having, or at risk for a fibrotic disease e.g., a fibrotic disease described herein, such as a fibrotic disease of the kidney or the lung
  • a fibrotic disease e.g., a fibrotic disease described herein, such as a fibrotic disease of the kidney or the lung
  • a biomarker e.g., COL3A1 and/or COL1A1
  • an elevated level of a biomarker comprises a level that is above a corresponding threshold level.
  • the level of biomarker (e.g., COL3A1 and/or COL1A1) was obtained from or previously determined from a biological sample from the patient.
  • the method further comprises obtaining or determining a level of a biomarker (e.g., COL3A1 and/or COL1 Al) in a biological sample from the patient.
  • a threshold expression level corresponds to a predetermined mean or median level of COL3A1 and/or COL1A1 of a population of healthy subjects (e.g., healthy human subjects). In some embodiments, a threshold expression level corresponds to a predetermined normal range of COL3A1 and/or COL1A1 of a population of healthy subjects (e.g., healthy human subjects).
  • the expression level of COL3 Al is at least 20% higher than the corresponding threshold expression level. In some embodiments, the expression level of COL1 Al is at least 20% higher than the corresponding threshold expression level. In some embodiments, the expression levels of both COL3A1 and COL1 Al are at least 20% higher than the respective corresponding threshold expression levels.
  • the expression level of COL3 Al is at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% higher than the corresponding threshold expression level.
  • the expression level of COL1 Al is at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% higher than the corresponding threshold expression level.
  • the expression levels of both COL3A1 and COL1 Al are at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% higher than the respective corresponding threshold expression levels.
  • a biological fluid may be or comprise an intracellular fluid, an extracellular fluid, an intravascular fluid (blood plasma), an interstitial fluid, a lymphatic fluid, and/or a transcellular fluid.
  • a biological tissue or sample may be obtained, for example, by aspirate, biopsy (e.g., fine needle or tissue biopsy), swab (e.g., oral, nasal, skin, or vaginal swab), scraping, surgery, washing or lavage (e.g., bronchoalveolar, ductal, nasal, ocular, oral, uterine, vaginal, or other washing or lavage).
  • the biological sample (e.g., from which an expression level of a biomarker is obtained or has been determined) is a renal biopsy sample, a urine sample or a blood sample.
  • an expression level of a protein biomarker e.g., a prepropeptide, propeptide, amino-terminal propeptide, protein, fibril or fiber, e.g., of COL3A1 and/or of COL1 Al
  • a protein biomarker e.g., a prepropeptide, propeptide, amino-terminal propeptide, protein, fibril or fiber, e.g., of COL3A1 and/or of COL1 Al
  • an expression level of a genetic biomarker e.g., a gene product, e.g., RNA, e.g., mRNA, e.g., mRNA encoding COL3A1 and/or COL1A1
  • a genetic biomarker e.g., a gene product, e.g., RNA, e.g., mRNA, e.g., mRNA encoding COL3A1 and/or COL1A1
  • RNA e.g., mRNA, e.g., mRNA encoding COL3A1 and/or COL1A1
  • the biological sample (e.g., from which an expression level of a biomarker is or has been determined) is a lung biopsy sample, a bronchoalveolar lavage fluid (BALF) sample or a blood sample.
  • a protein biomarker e.g., a prepropeptide, propeptide, amino-terminal propeptide, protein, fibril or fiber, e.g., of COL3A1 and/or of COL1 Al
  • a protein biomarker e.g., a prepropeptide, propeptide, amino-terminal propeptide, protein, fibril or fiber, e.g., of COL3A1 and/or of COL1 Al
  • an expression level of a genetic biomarker e.g., a gene product, e.g., RNA, e.g., mRNA, e.g., mRNA encoding COL3A1 and/or COL1A1
  • a genetic biomarker e.g., a gene product, e.g., RNA, e.g., mRNA, e.g., mRNA encoding COL3A1 and/or COL1A1
  • a genetic biomarker e.g., a gene product, e.g., RNA, e.g., mRNA, e.g., mRNA encoding COL3A1 and/or COL1A1
  • an expression level of a biomarker is obtained from or determined in a renal biopsy sample from a patient.
  • an expression level of a biomarker e.g., COL3A1 and/or COL1 Al
  • a gene biomarker e.g., RNA, e.g., mRNA
  • the biological sample is a renal biopsy sample and the expression level of COL3 Al is an mRNA level and/or the expression level of COL1A1 is an mRNA level.
  • the biological sample is a renal biopsy sample and the expression level of COL3 Al is an mRNA level and/or the expression level of COL1 A1 is an mRNA level.
  • a biomarker e.g., mRNA level
  • Methods for measuring and/or determining levels of a biomarker (e.g., mRNA level) in renal biopsy samples are known in the art, for example, Genovese et ah, Biomark Insights . 2016 May 22;ll:77-84.
  • an expression level of a biomarker is obtained from or determined in a urine sample from a patient.
  • the biological sample is a urine sample and the expression level of COL3 A1 is a level of COL3 A1 protein or fragments thereof and/or the expression level of COL1 A1 is a level of COL1 A1 protein or fragments thereof.
  • Methods for measuring and/or determining levels of a biomarker (e.g., a level of collagen protein and/or fragment thereof) in urine samples are known in the art, for example, Soylemezoglu et ah, Nephrol Dial Transplant. 1997 Sep;12(9):1883-9.
  • an expression level of a biomarker is obtained from or determined in a blood sample from a patient.
  • the biological sample is a blood sample and the expression level of COL3 A1 is a level of COL3 A1 protein or fragments thereof and/or the expression level of COL1 A1 is a level of COL1 A1 protein or fragments thereof.
  • Methods for measuring and/or determining levels of a biomarker (e.g., a level of collagen protein and/or fragment thereof) in blood samples are known in the art.
  • an expression level of a biomarker is obtained from or determined in a lung biopsy sample from a patient.
  • an expression level of a biomarker e.g., COL3A1 and/or COL1 Al
  • a gene biomarker e.g., RNA, e.g., mRNA
  • the biological sample is a lung biopsy sample and the expression level of COL3 Al is an mRNA level and/or the expression level of COL1A1 is an mRNA level.
  • the biological sample is a lung biopsy sample and the expression level of COL3 Al is an mRNA level and/or the expression level of COL1 Al is an mRNA level.
  • Methods for measuring and/or determining levels of a biomarker (e.g., mRNA level) in lung biopsy samples are known in the art.
  • an expression level of a biomarker is obtained from or determined in a BALF sample from a patient.
  • the biological sample is a BALF sample and the expression level of COL3 Al is a level of COL3 Al protein or fragments thereof and/or the expression level of COL1 Al is a level of COL1 Al protein or fragments thereof.
  • Methods for measuring and/or determining levels of a biomarker (e.g., a level of collagen protein and/or fragment thereof) in BALF samples are known in the art, for example, Wattiez, R. and Falmagne, P., J Chromatogr B Analyt Technol Biomed Life Sci. 2005 Feb 5;57(9):2005-l 1.
  • the present disclosure provides methods of administering Compound 1 to a subject or population of subjects described herein, according to a regimen established to achieve one or more desirable outcomes.
  • the fibrotic disease e.g., a fibrotic disease described herein, such as a fibrotic disease of the kidney or the lung
  • the fibrotic disease is stabilized (i.e., does not worsen) and/or is ameliorated (i.e., one or more symptoms improve) in a patient treated with Compound 1.
  • treatment of a patient with Compound 1 reduces a level of one or more biomarkers (e.g., COL1 A1 and/or COL3A1).
  • treatment of a patient with Compound 1 reduces proteinuria.
  • a regimen has been established to achieve one or more desirable outcomes, relative to that observed for a comparable reference population that has not received Compound 1 (e.g., that has received a placebo), such as those described herein.
  • a reference composition may be or may have been administered at the same intervals and/or in the same amounts as a composition providing Compound 1.
  • a regimen has been established to achieve one or more desirable outcomes, relative to that observed for a comparable reference population that does not have an altered level of a biomarker (e.g., an elevated level of COL3A1 and/or COL1 Al).
  • a patient that expresses a biomarker treated with Compound 1 has an improved outcome (e.g., improved stability and/or amelioration of the fibrotic disease) relative to a patient with the same disease that does not express an elevated level of the biomarker.
  • treatment of a patient with Compound 1 reduces a level of one or more biomarkers (e.g., COL1 Al and/or COL3A1).
  • treatment of a patient with Compound 1 reduces proteinuria.
  • a composition providing Compound 1 is administered according to a regimen established to achieve a particular effect, e.g., at a particular time point, such as regimens described herein. In some embodiments, a composition providing Compound 1 is administered according to a regimen established to achieve a particular effect, such as regimens described herein. [0230] In some embodiments, a patient with a fibrotic disease of the kidney exhibits increased renal COL3 A1 expression that is correlated with urine protein to creatinine ratio. In some embodiments, a patient with a fibrotic disease of the kidney exhibits COL3 A1 expression that is inversely correlated with eGFR.
  • a kidney disease that is or comprises focal and segmental glomerulosclerosis (FSGS).
  • a patient has FSGS and an elevated level of COL3A1 and/or COL1 A1 (e.g., mRNA and/or protein), for example, as determined in or obtained from a biological sample from the patient (e.g., renal biopsy sample, urine sample, blood sample, etc.).
  • a patient has FSGS and also exhibits (i) increased renal COL3 A1 expression that is correlated with urine protein to creatinine ratio and/or (ii) COL3 A1 expression that is inversely correlated with eGFR.
  • provided methods are useful in treating a kidney disease that is or comprises primary proteinuric kidney disease (PPKD).
  • PPKD primary proteinuric kidney disease
  • a patient has PPKD and an elevated level of COL3A1 and/or COL1 A1 (e.g., mRNA and/or protein), for example, as determined in or obtained from a biological sample from the patient (e.g., renal biopsy sample, urine sample, blood sample, etc.).
  • provided methods are useful in treating a kidney disease that is or comprises primary glomerular disease (PGD).
  • PGD primary glomerular disease
  • a patient has PGD and an elevated level of COL3A1 and/or COL1 A1 (e.g., mRNA and/or protein), for example, as determined in or obtained from a biological sample from the patient (e.g., renal biopsy sample, urine sample, blood sample, etc.).
  • a biological sample from the patient e.g., renal biopsy sample, urine sample, blood sample, etc.
  • provided methods are useful in treating a kidney disease that is or comprises steroid resistant nephrotic syndrome (SRNS).
  • SRNS steroid resistant nephrotic syndrome
  • a patient has SRNS and an elevated level of COL3A1 and/or COL1 A1 (e.g., mRNA and/or protein), for example, as determined in or obtained from a biological sample from the patient (e.g., renal biopsy sample, urine sample, blood sample, etc.).
  • provided methods are useful in treating a kidney disease that is or comprises proteinuria.
  • a patient has proteinuria and an elevated level of COL3A1 and/or COL1 A1 (e.g., mRNA and/or protein), for example, as determined in or obtained from a biological sample from the patient (e.g., renal biopsy sample, urine sample, blood sample, etc.).
  • a biological sample e.g., renal biopsy sample, urine sample, blood sample, etc.
  • provided methods are useful in treating a kidney disease that is or comprises lupus nephritis.
  • a patient has lupus nephritis and an elevated level of COL3A1 and/or COL1A1 (e.g., mRNA and/or protein), for example, as determined in or obtained from a biological sample from the patient (e.g., renal biopsy sample, urine sample, blood sample, etc.).
  • a biological sample from the patient e.g., renal biopsy sample, urine sample, blood sample, etc.
  • provided methods are useful in treating a kidney disease that is or comprises minimal change disease.
  • a patient has minimal change disease and an elevated level of COL3A1 and/or COL1 A1 (e.g., mRNA and/or protein), for example, as determined in or obtained from a biological sample from the patient (e.g., renal biopsy sample, urine sample, blood sample, etc.).
  • a biological sample from the patient e.g., renal biopsy sample, urine sample, blood sample, etc.
  • provided methods are useful in treating a kidney disease that is or comprises ANCA-associated glomerulonephritis.
  • a patient has ANCA-associated glomerulonephritis and an elevated level of COL3A1 and/or COL1A1 (e.g., mRNA and/or protein), for example, as determined in or obtained from a biological sample from the patient (e.g., renal biopsy sample, urine sample, blood sample, etc.).
  • a biological sample from the patient e.g., renal biopsy sample, urine sample, blood sample, etc.
  • provided methods are useful in treating a kidney disease that is or comprises anti-GBM nephropathy.
  • a patient has anti-GBM nephropathy and an elevated level of COL3A1 and/or COL1 A1 (e.g., mRNA and/or protein), for example, as determined in or obtained from a biological sample from the patient (e.g., renal biopsy sample, urine sample, blood sample, etc.).
  • a biological sample from the patient e.g., renal biopsy sample, urine sample, blood sample, etc.
  • provided methods are useful in treating a kidney disease that is or comprises IgA nephropathy.
  • a patient has IgA nephropathy and an elevated level of COL3A1 and/or COL1A1 (e.g., mRNA and/or protein), for example, as determined in or obtained from a biological sample from the patient (e.g., renal biopsy sample, urine sample, blood sample, etc.).
  • a biological sample from the patient e.g., renal biopsy sample, urine sample, blood sample, etc.
  • provided methods are useful in treating a kidney disease that is or comprises membranous glomerulonephritis (MG).
  • MG membranous glomerulonephritis
  • a patient has MG and an elevated level of COL3A1 and/or COL1 A1 (e.g., mRNA and/or protein), for example, as determined in or obtained from a biological sample from the patient (e.g., renal biopsy sample, urine sample, blood sample, etc.).
  • a biological sample from the patient e.g., renal biopsy sample, urine sample, blood sample, etc.
  • provided methods are useful in treating a kidney disease that is or comprises polycystic kidney disease (e.g., autosomal dominant polycystic kidney disease (ADPKD)).
  • ADPKD autosomal dominant polycystic kidney disease
  • a patient has polycystic kidney disease (e.g., ADPKD) and an elevated level of COL3A1 and/or COL1 A1 (e.g., mRNA and/or protein), for example, as determined in or obtained from a biological sample from the patient (e.g., renal biopsy sample, urine sample, blood sample, etc.).
  • ADPKD polycystic kidney disease
  • COL3A1 and/or COL1 A1 e.g., mRNA and/or protein
  • provided methods are useful in treating a kidney disease that is or comprises chronic kidney disease.
  • a patient has chronic kidney disease and an elevated level of COL3A1 and/or COL1 A1 (e.g., mRNA and/or protein), for example, as determined in or obtained from a biological sample from the patient (e.g., renal biopsy sample, urine sample, blood sample, etc.).
  • COL3A1 and/or COL1 A1 e.g., mRNA and/or protein
  • provided methods are useful in treating a kidney disease that is or comprises collagen type III glomerulopathy.
  • a patient has collagen type III glomerulopathy and an elevated level of COL3A1 and/or COL1 A1 (e.g., mRNA and/or protein), for example, as determined in or obtained from a biological sample from the patient (e.g., renal biopsy sample, urine sample, blood sample, etc.).
  • a biological sample from the patient e.g., renal biopsy sample, urine sample, blood sample, etc.
  • provided methods are useful in treating a kidney disease that is or comprises nail-patella syndrome.
  • a patient has nail-patella syndrome and an elevated level of COL3A1 and/or COL1 A1 (e.g., mRNA and/or protein), for example, as determined in or obtained from a biological sample from the patient (e.g., renal biopsy sample, urine sample, blood sample, etc.).
  • COL3A1 and/or COL1 A1 e.g., mRNA and/or protein
  • provided methods are useful in treating a lung disease that is or comprises idiopathic pulmonary fibrosis.
  • a patient has idiopathic pulmonary fibrosis and an elevated level of one or more biomarkers, e.g., identified as described in Example 22 (optionally, e.g., COL3A1 and/or COL1 A1 (e.g., mRNA and/or protein)), for example, as determined in or obtained from a biological sample from the patient (e.g., lung biopsy sample, BALF sample, blood sample, etc.).
  • biomarkers e.g., identified as described in Example 22
  • COL3A1 and/or COL1 A1 e.g., mRNA and/or protein
  • a method for treating a patient diagnosed with, suspected of having, or at risk for a fibrotic disease comprising: (i) obtaining or determining a level of expression of one or more gene products or proteins in a biological sample from the patient, wherein the one or more gene products or proteins are selected from: COL3A1 and COL1A1; (ii) comparing the expression level of the one or more gene products or proteins with that of a corresponding threshold level, and (iii) if the expression level of COL3 A1 and/or the expression level of COL1 A1 is above the threshold expression level, then administering to the patient an effective amount of Compound 1 or a pharmaceutical composition thereof, and if the expression level of COL3A1
  • a fibrotic disease e.g., a fibrotic disease as described herein, e.g., a fibrotic disease of the kidney or the lung
  • a fibrotic disease e.g., a fibrotic disease as described herein, e.g., a fibrotic disease of the kidney or the lung
  • a biomarker e.g., COL3A1 and/or COL1 Al
  • the method comprising: (i) administering an effective amount of Compound 1 or a pharmaceutical composition thereof, and (ii) monitoring a level of biomarker (e.g., COL3A1 and/or COL1A1) subsequent to administration (e.g., after a duration of a day, week, two weeks, month, two months, 3 months, etc.).
  • the level of biomarker e.g., COL3A1 and/or COL1 Al
  • further treatment with Compound 1 is discontinued.
  • the level of biomarker e.g., COL3A1 and/or COL1 Al
  • the dose of Compound 1 administered to the patient is increased.
  • the level of biomarker e.g., COL3A1 and/or COL1 Al
  • treatment with Compound 1 is continued (e.g., subsequent doses are administered).
  • a fibrotic disease e.g., a fibrotic disease as described herein, e.g., a fibrotic disease of the kidney or the lung
  • a fibrotic disease is stabilized (i.e., does not worsen) and/or is ameliorated (i.e., one or more symptoms improve) in a patient treated with Compound 1.
  • treatment of a patient with Compound 1 reduces a level of one or more biomarkers (e.g., COL1 Al and/or COL3A1).
  • administration of Compound 1 to the patient (i) reduces the expression of renal COL3A1 , (ii) reduces the expression of renal COL1A1 , (iii) reduces renal COL3A1 accumulation, (iv) reduces renal COL1A1 accumulation, or (v) any combination thereof.
  • administration of Compound 1 to the patient (i) reduces the expression of pulmonary COL3A1 , (ii) reduces the expression of pulmonary COL1A1 , (iii) reduces pulmonary COL3A1 accumulation, (iv) reduces pulmonary COL1 A1 accumulation, or (v) any combination thereof.
  • a patient to be treated with a method of the present disclosure exhibits proteinuria.
  • a method for treatment with Compound 1 that includes: (i) receiving a report listing the expression level of one or more biomarkers (e.g., COL3A1 and/or COL1 Al) for a patient with a fibrotic disease (e.g., a fibrotic disease described herein, such as a fibrotic disease of the kidney or the lung), (ii) receiving a request for reimbursement of the screening and/or of a particular therapeutic regimen; and (iii) approving payment and/or reimbursement for Compound 1 therapy if the report indicates the level of biomarker is above a threshold level.
  • a fibrotic disease e.g., a fibrotic disease described herein, such as a fibrotic disease of the kidney or the lung
  • the present disclosure provides certain biomarkers that can distinguish subjects (e.g., subjects suffering from or at risk of fibrosis or associated diseases, disorders, and conditions) who are more likely than others to respond to therapy with Compound 1.
  • the present disclosure provides insight that certain biomarkers can distinguish patients who are likely to respond to therapy, for example because the drivers of their fibrotic disease correspond with the mechanism of action of Compound 1.
  • an altered level of one or more gene products or proteins that are part of the mechanism of action of Compound 1 e.g., down- or up-regulated by Compound 1).
  • one or more biomarkers comprise an elevated level of COL3A1 expression and/or COL1 Al expression.
  • a patient with an altered level of one or more biomarkers may have an improved response to treatment with Compound 1 relative to a patient that does not have a level of the biomarker that meets the threshold criteria.
  • one biomarker is used to characterize subjects; in some embodiments, more than one biomarker (e.g., two, three, etc.) is used to characterize subjects.
  • a biomarker is differentially present in a sample taken from a subject of one status as compared with another status (e.g., more responsive to Compound 1 therapy vs. less responsive to Compound 1 therapy).
  • a biomarker is differentially present in a sample taken from the same subject at two or more different time points, i.e., when the status of the subject has changed from one time point to another.
  • a biomarker is detected and/or quantified in a tissue sample (e.g., from a biopsy, such as a kidney or lung biopsy) and/or in a biological fluid (e.g., blood, urine, BALF, etc.).
  • a biomarker is a level of mRNA that is detected and/or quantified in a kidney tissue sample, e.g., obtained from a kidney biopsy.
  • a biomarker is detected and/or quantified in a urine sample (e.g., a level of a protein or protein fragment).
  • a biomarker is detected and/or quantified in a blood sample (e.g., a level of a protein or protein fragment). In some embodiments, a biomarker is detected and/or quantified in a BALF sample (e.g., a level of a protein or protein fragment). In some embodiments, a biomarker is a level of mRNA that is detected and/or quantified in a lung tissue sample, e.g., obtained from a lung biopsy.
  • a biomarker may include one or more of a peptide, protein, nucleic acid (e.g., polynucleotide, DNA, RNA, etc.), polysaccharide (e.g., lectins or sugars), lipid, enzyme, small molecule, ligand, receptor, antigen, or antibody.
  • a biomarker comprises a protein.
  • a biomarker comprises a nucleic acid (e.g., mRNA).
  • detection of a threshold level of one or more biomarkers are used to select and/or characterize patients who may be responsive to Compound 1 therapy.
  • levels of one or more biomarkers in a sample from a subject are compared to a threshold level.
  • a biomarker is considered increased if the level is increased relative to a threshold level (e.g., increased by at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%, or more).
  • a threshold level is determined from a population of healthy volunteers (e.g., a mean or median level from a population of healthy volunteers).
  • a method includes an in vitro method for determining a level of a biomarker.
  • in vitro methods for determining a level of a biomarker include, but are not limited to, a chemiluminescence assay, enzymatic assay, enzyme immunoassay, multiplex immunoassay, ELISA, chromatographic immunoassay, electrophoresis assay, radioimmunoassay, colorimetric assay, chromatography/mass spectrometry (e.g., GC/MS, LC/MS, LC/MS/MS, etc.), High Performance Liquid Chromatography (“HPLC”), and/or PCR (e.g., real-time PCR).
  • HPLC High Performance Liquid Chromatography
  • a method for detecting a level of a biomarker includes chromatographic and/or MS methods.
  • Exemplary methods include, but are not limited to, gas chromatography (GC), liquid chromatography/mass spectroscopy (LC-MS), gas chromatography/mass spectroscopy (GC-MS), nuclear magnetic resonance (NMR), magnetic resonance imaging (MRI), Fourier Transform InfraRed (FT-IR), and inductively coupled plasma mass spectrometry (ICP-MS).
  • the present disclosure encompasses the recognition that COL1 A1 and/or COL3A1 are useful biomarkers in the methods provided herein. In some embodiments, the present disclosure provides insight that increased levels of COL1 A1 and/or COL3 A1 expression may be useful in selecting and/or characterizing patients for Compound 1 therapy.
  • COL1 A1 is a biomarker useful in the methods provided herein.
  • COL3 A1 is a biomarker useful in the methods provided herein.
  • an elevated level of COL1A1 and/or COL3A1 corresponds to a level of gene expression (e.g., RNA, e.g., mRNA).
  • an elevated level of COL1A1 and/or COL3A1 corresponds to a level of collagen protein expression.
  • an elevated level of COL1A1 and/or COL3A1 protein is or includes, e.g., a corresponding procollagen polypeptide, collagen protein, preprocollagen polypeptide, amino- terminal procollagen polypeptide, collagen fibril, collagen fiber, and/or any fragment or degradation product thereof.
  • an increased level of COL1 A1 and/or COL3A1 is above a threshold level (e.g., a predetermined median or mean level). In some embodiments, an increased level of COL1 A1 and/or COL3A1 is more than about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%, or more of a threshold level. In some embodiments, an increased level of COL1 A1 and/or COL3A1 is more than about 0.5, about 1.0, about 1.5, or about 2.0, or more standard deviations above a threshold level.
  • a threshold level e.g., a predetermined median or mean level. In some embodiments, an increased level of COL1 A1 and/or COL3A1 is more than about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%, or more of a threshold level. In some embodiments, an
  • subjects who are selected for Compound 1 therapy based on increased levels of COL1 A1 and/or COL3A1 achieve improved outcomes (e.g., compared to subjects who do not meet one or more selection criteria for Compound 1 therapy).
  • the present disclosure provides methods of treating a disease, disorder, or condition characterized by increased expression of COL1 A1 and/or COL3A1, comprising administering Compound 1 to a subject or population of subjects in need thereof as described herein.
  • such a disease, disorder, or condition is a renal disease, disorder, or condition (e.g., a renal disease, disorder, or condition associated with fibrosis) as described herein.
  • a renal disease, disorder, or condition is characterized by increased expression of COL1A1 and/or COL3A1.
  • such a disease, disorder, or condition is a pulmonary disease, disorder, or condition (e.g., a pulmonary disease, disorder, or condition associated with fibrosis) as described herein.
  • a pulmonary disease, disorder, or condition is characterized by increased expression of COL1A1 and/or COL3A1.
  • the present disclosure provides methods comprising administering Compound 1 to a subject in need thereof, wherein the subject has been determined to have increased level(s) of COL1A1 and/or COL3A1.
  • a subject has been determined to have increased level(s) of COL1 A1 and/or COL3A1 in a renal biopsy sample and/or a urine sample.
  • a subject has been determined to have increased level(s) of COL1 A1 and/or COL3 A1 in a biological sample other than a renal biopsy sample and/or a urine sample.
  • a subject has been determined to have increased level(s) of COL1A1 and/or COL3A1 in aBALF sample.
  • the present disclosure provides methods comprising determining level(s) of COL1A1 and/or COL3A1 by: obtaining a biological sample from a subject; performing an assay on the sample to determine level(s) of COL1 A1 and/or COL3A1 in the sample; and comparing the determined level(s) to a threshold level.
  • a sample has increased level(s) of COL1 A1 and/or COL3A1 compared to a threshold level
  • Compound 1 is administered to the subject.
  • if a sample does not have increased level(s) of COL1 A1 and/or COL3A1 compared to a threshold level then Compound 1 is not administered to the subject.
  • the present disclosure provides methods comprising determining level(s) of COL1 A1 and/or COL3A1 in a biological sample from a subject; comparing the determined level(s) to a threshold level; and identifying the subject as in need of therapeutic intervention when the sample is determined to have increased level(s) of COL1 A1 and/or COL3A1 compared to the threshold level.
  • a subject is identified as in need of therapeutic intervention with Compound 1 therapy as described herein.
  • a provided method further comprises administering Compound 1 to a subject (e.g., a subject identified as in need of therapeutic intervention) as described herein.
  • provided technologies are useful for monitoring subjects (e.g., monitoring status of subjects over time and/or monitoring therapy).
  • the present disclosure provides methods comprising determining level(s) of COL1A1 and/or COL3A1 in each of a plurality of biological samples obtained at different time points from a single patient; and comparing the determined level(s) from a first time point with that from at least one later time point.
  • the present disclosure provides methods comprising determining level(s) of COL1 A1 and/or COL3A1 from a biological sample obtained from a subject for whom level(s) of COL1 A1 and/or COL3A1 have previously been obtained at least once; and comparing the determined level(s) with the previously obtained level(s).
  • a first time point and one or more later time points are separated from one another by a reasonably consistent interval.
  • a significant change in the determined level over time indicates a change in the subject’s status.
  • a significant change in a determined level over time is a change (e.g., an increase or a decrease) of at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%, or more compared to a threshold level.
  • a significant change in a determined level over time is a change of more than about 0.5, about 1.0, about 1.5, or about 2.0, or more standard deviations away from a threshold level.
  • such methods further comprise administering Compound 1 therapy to a subject (e.g., a subject who has been determined to have moved from a non- responsive to a responsive state) as described herein.
  • a subject e.g., a subject who has been determined to have moved from a non- responsive to a responsive state
  • provided methods are useful for monitoring therapy (e.g., efficacy and/or other indicators of response).
  • a sample from a first time point was obtained from a subject prior to administration of Compound 1
  • a sample from a second time point was obtained from a subject after administration of Compound 1.
  • Compound 1 therapy is continued.
  • Compound 1 therapy is discontinued, or dosage amount and/or frequency of Compound 1 therapy is increased.
  • a method includes an in vitro method for determining a level of a biomarker.
  • a method for determining a level of a biomarker can be or include a chemiluminescence assay, enzymatic assay, enzyme immunoassay, multiplex immunoassay, ELISA, chromatographic immunoassay, electrophoresis assay, radioimmunoassay, colorimetric assay, UV spectroscopy, chromatography/mass spectrometry (e.g., GC/MS, LC/MS, LC/MS/MS, etc.), High Performance Liquid Chromatography (“HPLC”), and/or PCR (e.g., quantitative PCR and/or real-time PCR).
  • a chemiluminescence assay e.g., enzymatic assay, enzyme immunoassay, multiplex immunoassay, ELISA, chromatographic immunoassay, electrophoresis assay, radioimmunoassay,
  • a level of a biomarker corresponds to a level of gene expression (e.g., RNA, e.g., mRNA) and is quantified using methods known in the art.
  • a method of determining a level of expression of a biomarker gene can be or include a chemiluminescence assay, UV spectroscopy, hybridization assay (e.g., Fluorescent in Situ Hybridization (FISH), e.g., RNA- FISH), enzymatic assay, enzyme immunoassay (e.g., ELISA), multiplex assay, electrophoresis assay, radioassay, colorimetric assay, chromatography/mass spectrometry (e.g., GC/MS, LC/MS, LC/MS/MS, etc.), High Performance Liquid Chromatography (“HPLC”), and/or PCR (e.g., quantitative PCR and/or real-time PCR).
  • FISH Fluorescent in Situ Hybridization
  • RNA- FISH e.g., RNA- FISH
  • enzymatic assay e.g., enzyme immunoassay (e.g., ELISA), multiplex
  • a level of a biomarker corresponds to a level of protein (e.g., procollagen polypeptide, collagen protein, preprocollagen polypeptide, amino-terminal procollagen polypeptide, collagen fibril, collagen fiber, and/or any fragment or degradation product thereof) and is quantified using methods known in the art.
  • protein e.g., procollagen polypeptide, collagen protein, preprocollagen polypeptide, amino-terminal procollagen polypeptide, collagen fibril, collagen fiber, and/or any fragment or degradation product thereof
  • a method of determining a level of expression of a biomarker protein can be or include a chemiluminescence assay, enzymatic assay, enzyme immunoassay, multiplex immunoassay, ELISA, chromatographic immunoassay, electrophoresis assay, radioimmunoassay, colorimetric assay, UV spectroscopy, chromatography/mass spectrometry (e.g., GC/MS, LC/MS, LC/MS/MS, etc.), or High Performance Liquid Chromatography (“HPLC”).
  • HPLC High Performance Liquid Chromatography
  • a method for treating a fibrotic disease of the kidney comprising: administering an effective amount of a Compound 1 or a pharmaceutical composition thereof, to a patient that has been determined to have an elevated level of COL3 A1 and/or COL1A1.
  • a method for treating a patient diagnosed with or at risk for a fibrotic disease of the kidney comprising:
  • the expression level of COL3A1 is at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% higher than the corresponding threshold expression level and/or the expression level of COL1 A1 is at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% higher than the corresponding threshold expression level.
  • fibrotic disease of the kidney is or comprises: focal segmental glomerulosclerosis (FSGS), steroid resistant nephrotic syndrome (SRNS), proteinuria, lupus nephritis, minimal change disease, an anti-neutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis, anti-globular basement membrane (anti-GBM) nephropathy, IgA nephropathy, membranous glomerulonephritis (MG), autosomal dominant polycystic kidney disease (ADPKD), collagen type III glomerulopathy, nail-patella syndrome, or chronic kidney disease.
  • FSGS focal segmental glomerulosclerosis
  • SRNS steroid resistant nephrotic syndrome
  • proteinuria lupus nephritis
  • minimal change disease an anti-neutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis
  • anti-GBM anti-globular basement membrane
  • ANCA anti-neutrophil cytoplasmic antibody
  • MPA microscopic polyangiitis
  • fibrotic disease of the kidney is or comprises focal and segmental glomerulosclerosis (FSGS).
  • FSGS focal and segmental glomerulosclerosis
  • a method for treating a patient diagnosed with, suspected of having, or at risk for a fibrotic disease of the kidney comprising:
  • fibrotic disease of the kidney is or comprises: focal segmental glomerulosclerosis (FSGS), steroid resistant nephrotic syndrome (SRNS), proteinuria, lupus nephritis, minimal change disease, an anti-neutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis, anti-globular basement membrane (anti-GBM) nephropathy, IgA nephropathy, membranous glomerulonephritis (MG), autosomal dominant polycystic kidney disease (ADPKD), collagen type III glomerulopathy, nail-patella syndrome, or chronic kidney disease.
  • FSGS focal segmental glomerulosclerosis
  • SRNS steroid resistant nephrotic syndrome
  • proteinuria lupus nephritis
  • minimal change disease an anti-neutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis
  • anti-GBM anti-globular basement membrane
  • composition providing Compound 1, as described herein, can be administered in accordance with methods provided herein.
  • a composition providing Compound 1 is a composition comprising Compound 1 (in a pharmaceutically acceptable form as described herein), formulated together with one or more pharmaceutically acceptable carriers.
  • a composition providing Compound 1 is or comprises Compound 1 present in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population.
  • compositions providing Compound 1 may be specially formulated for administration in solid or liquid form, including those adapted for the following: oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), capsules, tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream, or foam; sublingually; ocularly; transdermally; or nasally, pulmonary, and to other mucosal surfaces.
  • oral administration for example, drenches (aqueous or non
  • composition providing Compound 1 is formulated for oral administration (e.g., in a capsule form). In some embodiments, a composition providing Compound 1 is administered orally.
  • a composition providing Compound 1 is administered as one or more unit dosage forms.
  • a composition providing Compound 1 is administered as one or more solid unit dosage forms (e.g., one or more capsules or tablets).
  • a composition providing Compound 1 is administered as one or more immediate release solid unit dosage forms.
  • Compound 1 is administered as one or more oral unit dosage forms.
  • a composition providing Compound 1 is a capsule. In some embodiments, a composition providing Compound l is a tablet.
  • Compound 1 is administered as a capsule comprising 10 mg, 50 mg, or 250 mg of Compound 1 In some embodiments, Compound 1 is administered as a capsule comprising 10 mg, 50 mg, or 250 mg of Compound 1 with no excipients. In some embodiments, Compound 1 is administered as a capsule comprising 50 mg of Compound 1 (e.g., with no excipients). In some embodiments, Compound 1 is administered as a capsule comprising 250 mg of Compound 1 (e.g., with no excipients). [0287] In some embodiments, Compound 1 is administered as a capsule comprising 100 mg of Compound 1 (e.g., a Form A Compound 1 Hydrochloride Trihydrate). In some embodiments, Compound 1 is administered as a capsule comprising 200 mg of Compound 1 (e.g., a Form A Compound 1 Hydrochloride Trihydrate). In some such embodiments, a capsule comprising Compound 1 has no excipients.
  • Compound 1 is administered as a capsule comprising 10 mg
  • unit dosage forms e.g., tablets or capsules
  • one unit dosage form e.g., tablet or capsule
  • a suitable dose as described herein e.g., a dose of about 50 mg, about 100 mg, about 200 mg, about 250 mg, about 400 mg, about 500 mg, or about 600 mg
  • more than one (e.g., 2, 3, 4, 5, etc.) unit dosage forms e.g., tablets or capsules
  • a suitable dose as described herein e.g., a dose of about 50 mg, about 100 mg, about 200 mg, about 250 mg, about 400 mg, about 500 mg, or about 600 mg.
  • each unit dosage form when multiple unit dosage forms are administered, each unit dosage form contains the same amount of Compound 1, in order to provide a suitable dose as described herein; in some embodiments, when multiple unit dosage forms are administered, each unit dosage form contains different amounts of Compound 1, in order to provide a suitable dose as described herein.
  • a suitable number of unit dosage forms are administered in order to provide a dose of about 50 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg, or about 600 mg.
  • one unit dosage form is administered to provide a dose of about 50 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg, or about 600 mg.
  • more than one (e.g., 2, 3, or 4) unit dosage forms are administered to provide a dose of about 50 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg, or about 600 mg.
  • a composition providing Compound 1 is administered as a single dose. In some embodiments, a composition providing Compound 1 is administered at regular intervals. Administration at an “interval,” as used herein, indicates that the therapeutically effective amount is administered periodically (as distinguished from a one-time dose). In some embodiments, a composition providing Compound 1 is administered bimonthly (Q2M), monthly (QM), twice monthly (BIM), triweekly (Q3W), biweekly (Q2W), weekly (QW), twice weekly (BIW), thrice weekly (TIW), daily (QD), twice daily (BID), thrice daily (TID), or four times a day (QID) in accordance with methods provided herein. In some embodiments, a composition providing Compound 1 is administered twice daily (BID). In some embodiments, a composition providing Compound 1 is administered once daily (QD).
  • the present disclosure encompasses the recognition that, in some embodiments, administration of Compound 1 once daily is desirable for, e.g., patient convenience and/or compliance. Alternatively and/or additionally, the present disclosure encompasses the recognition that, in some embodiments, administration of Compound 1 twice daily may be desirable to achieve certain outcomes (e.g., to meet a particular threshold blood concentration, etc.).
  • a composition providing Compound 1 is administered in a suitable number of unit dosage forms (e.g., tablets or capsules) in order to provide a dose of about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, or about 600 mg once daily.
  • a composition providing Compound 1 is administered in a suitable number of unit dosage forms (e.g., tablets or capsules) in order to provide a dose of about 50 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, or about 400 mg twice daily.
  • a composition providing Compound 1 is administered at regular intervals indefinitely. In some embodiments, a composition providing Compound 1 is administered at regular intervals for a defined period of time. In some embodiments, a composition providing Compound 1 is administered at regular intervals for at least 12 weeks. [0294] In some embodiments, a composition providing Compound 1 is administered to a subject in a fed state (e.g., after a meal, such as within 1 hour, 45 minutes, 30 minutes, or 15 minutes of a meal). In some embodiments, Compound 1 is administered to a subject in a fasted state (e.g., after a fast of at least 4 hours, at least 6 hours, at least 8 hours, at least 10 hours, at least 12 hours, or at least 16 hours).
  • composition providing Compound 1 is administered to a subject at least 30 minutes apart from any other medications.
  • Nintedanib is approved by the FDA for treatment of idiopathic pulmonary fibrosis, for treatment chronic fibrosing interstitial lung diseases with a progressive phenotype and for slowing the rate of decline in pulmonary function in patients with systemic sclerosis-associated interstitial lung disease (OFEV ® [package insert] Ridgefield, CT: Boehringer Ingelheim Pharmaceuticals, Inc.; March 2020).
  • Nintedanib is an oral tyrosine kinase inhibitor and has a recommended dosage of 150 mg twice daily approximately 12 hours apart taken with food.
  • Nintedanib is also referred to as liT-indole-6-carboxylic acid, 2,3-dihydro-3-[[[4-[methyl[(4- methyl-l-piperazinyl)acetyl]amino]phenyl]amino]phenylmethylene]-2-oxo-, methyl ester, (3Z)-, ethanesulfonate.
  • Nintedanib is reported to reach maximum plasma concentrations approximately 2 to 4 hours after oral administration as a soft gelatin capsule under fed conditions.
  • the absolute bioavailability of a 100 mg dose was 4.7% (90% CL 3.62 to 6.08) in healthy volunteers.
  • nintedanib exposure increased by approximately 20% compared to administration under fasted conditions (90% CL 95.3% to 152.5%) and absorption was delayed (median tmax fasted: 2.00 hours; fed: 3.98 hours), irrespective of food type.
  • the effective half-life of nintedanib in patients with IPF was 9.5 hours (gCV 31.9%). Total plasma clearance after intravenous infusion was high (CL: 1390 mL/min; gCV 28.8%).
  • Urinary excretion of unchanged drug within 48 hours was about 0.05% of the dose after oral and about 1.4% of the dose after intravenous administration; the renal clearance was 20 mL/min.
  • Fig. 2 of Wind, S., et al. Clin. Pharmacokinet. 2019;58:1131-47 A trough concentration for producing 80% of Umax (ECxo) on FVC decline was estimated to be > 10 ng/mL.
  • Fig. 6 of Wind, S., et al. Clin. Pharmacokinet. 2019;58:1131-47 was estimated to be > 10 ng/mL.
  • Nintedanib is not recommended for use in patients with moderate or severe hepatic impairment. Elevated liver enzymes (e.g., ALT, AST, and bilirubin) and drug-induced liver injury have occurred with nintedanib. Diarrhea, nausea, and vomiting have occurred with nintedanib. Nintedanib may cause fetal harm. Arterial thromboembolic events have occurred with nintedanib. Bleeding events have occurred with nintedanib. Gastrointestinal perforation has been reported with nintedanib. See OFEV ® [package insert] Ridgefield, CT: Boehringer Ingelheim Pharmaceuticals, Inc.; March 2020.
  • the present disclosure also encompasses the recognition that provided technologies may provide different (e.g., improved) properties and/or outcomes, compared to one or more other known therapies (e.g., nintedanib).
  • Compound 1 may display different (e.g., improved) pharmacokinetic, pharmacodynamic, safety and/or efficacy characteristics, compared to one or more other known therapies (e.g., nintedanib).
  • a representative procedure for obtaining Compound 1 is as follows.
  • the quantities of materials used are approximate and may be increased or decreased in unison to obtain a larger or smaller lot size.
  • Conditions such as time or temperature are approximate and may be used as targets.
  • Step 1 Acetic anhydride (5.40 kg) and methyl 2-oxo-2,3-dihydro-lH-pyrrolo[2, 3- b]pyridine-6-carboxylate (1.0 kg) were added to a reactor at room temperature and stirred to combine. Trimethylorthobenzoate (1.90 kg) was added to the reaction mixture. The mixture was then heated to 105 °C and stirred for 1 hr. The reaction was cooled to 40 °C and isopropyl alcohol (3.14 kg) was added. The reaction was cooled further to 5 °C and stirred for 4 hr. The mixture was then filtered, and the product washed with isopropyl alcohol twice.
  • Step 2 Methanol (7.12 kg), methyl (E)-l -acetyl-3 -(methoxy(phenyl)methylene)-2- oxo-2, 3-dihydro-lH-pyrrolo[2,3-b]pyridine-6-carboxylate (1.0 kg), and A f -(4-aminophenyl)-N- methyl-2-(4-methylpiperazin-l-yl)acetamide (0.78 kg) in methanol (0.79 kg) were added to a reactor at room temperature and stirred to combine. The reaction mixture was then heated to 63 °C and stirred for 4 hrs. The reaction mixture was cooled to 5 °C and stirred for 4 hrs.
  • Step 3 Purified water (2.50 kg) and methyl (Z)-3-(((4-(N-methyl-2-(4- methylpiperazin-l-yl)acetamido)phenyl)amino)(phenyl)methylene)-2-oxo-2,3-dihydro-lH- pyrrolo[2,3-b]pyridine-6-carboxylate (1.0 kg) were added to a reactor at room temperature and stirred to combine. Acetone (1.975 kg) was then added, followed by iron-free HC1 (1.20 mol eq.). The mixture was stirred for 1 hr at 30 °C, and then filtered through a micron filter and washed with purified water (0.2 kg).
  • Compound 1 was formulated in a capsule for oral administration.
  • the capsule formulation included a Size 00 Swedish orange capsule containing Compound 1 (10 mg, 50 mg, or 250 mg) with no excipients.
  • Ingredients of the capsule shell were hypromellose (hydroxypropylmethyl cellulose), iron oxide as a coloring agent, and titanium dioxide as an opacifier.
  • Capsule formulations were prepared as follows. First, an optional sieving step was performed to deagglomerate the active agent if needed. Then, Compound 1 was filled into HPMC capsules, using either an automated Xelodose machine (e.g., for 10 mg and 50 mg capsules) or a semiautomated process (e.g., for 250 mg capsules). All capsules were polished or dedusted, either by an inline deduster (e.g., for 10 mg and 50 mg capsules) or a separate capsule polisher (e.g., for 250 mg capsules).
  • an automated Xelodose machine e.g., for 10 mg and 50 mg capsules
  • a semiautomated process e.g., for 250 mg capsules. All capsules were polished or dedusted, either by an inline deduster (e.g., for 10 mg and 50 mg capsules) or a separate capsule polisher (e.g., for 250 mg capsules).
  • Compound 1 Hydrochloride Form A was formulated in a capsule for oral administration.
  • the capsule formulation included a Swedish Orange capsule containing Compound 1 Hydrochloride Form A (100 mg or 200 mg dose) with no excipients.
  • Ingredients of the capsule shell were hypromellose (hydroxypropylmethyl cellulose), iron oxide as a coloring agent, and titanium dioxide as an opacifier.
  • Capsule formulations were prepared as described above.
  • Example 2 A Phase 1, Randomized, Double-Blind, Placebo-controlled, Single and Multiple Ascending Dose Study to Determine the Safety, Tolerability, Pharmacokinetics, and Food Effect of Compound 1 in Healthy Adult Participants Objectives
  • Primary Endpoint The frequency and severity of treatment-emergent adverse events (TEAEs), including clinically significant abnormal vital signs, electrocardiograms (ECGs), laboratory test results, and physical examination findings.
  • TEAEs treatment-emergent adverse events
  • ECGs electrocardiograms
  • Plasma PK endpoints include:
  • Urine PK endpoints include:
  • Cohort A3 was a food effect cohort, and participants in Cohort A3 only returned to the CRU on Day 14 and, following a 14-day washout, received a second single dose of their assigned treatment on Day 15 following consumption of a high fat meal. For Cohort A3 only, a second period of confinement applied from Day 14 until completion of the 72-hour post-dose assessments on Day 18.
  • Part A participants returned to the CRU for a follow-up visit 7 days ( ⁇ 1 day) after their final dose of study drug.
  • SAD the decision to escalate a dose or modify a dose was determined by the SMC following review of the 7-day blinded safety and available PK data from the preceding cohort.
  • Compound 1 dose will not exceed 1200 mg or a dose level that leads to mean C max >800 ng/mL or mean AUCo-iast > 4000 ng*h mL.
  • Compound 1 dose will not exceed 1000 mg per day or a dose level that leads to mean C max >800 ng/mL or mean AUCo-iast > 4000 ng*h mL.
  • Part A up to 48 healthy volunteers; Part B: up to 32 healthy volunteers).
  • Participants must have a minimum body weight of 50 kg and a body mass index (BMI) between >18.0 and ⁇ 32.0 kg/m 2 at screening.
  • BMI body mass index
  • Participants must have clinical laboratory values within normal range as specified by the testing laboratory, unless deemed not clinically significant by the investigator or delegate.
  • Double contraception is defined as a condom and one other form of the following:
  • FSH follicle-stimulating hormone
  • Periodic abstinence e.g., calendar, ovulation, symptothermal, post-ovulation- methods
  • withdrawal are not considered highly effective methods of birth control. Participant complete abstinence for the duration of the study and for 1 months after last study treatment is acceptable.
  • WOCBP must have negative pregnancy test at screening and Day 1 and be willing to have additional pregnancy test as required.
  • GI gastrointestinal
  • atrophic gastritis atrophic gastritis
  • lactose intolerance atrophic gastritis
  • H. Helicobacter
  • Fever body temperature > 38 °C
  • symptomatic viral or bacterial infection within 2 weeks prior to screening.
  • HCV hepatitis C antibody
  • HBV hepatitis B surface antigen
  • HCV human immunodeficiency virus
  • Participants with a positive toxicology screening panel urine test including qualitative identification of barbiturates, tetrahydrocannabinol (THC), amphetamines, benzodiazepines, opiates, and ***e
  • Alcohol breath test Participants with a history of substance abuse or dependency or history of recreational intravenous (IV) drug use over the last 5 years (by self-declaration).
  • Participant is unwilling to abstain from alcohol beginning 48 hours prior to admission to the CRU and during confinement period.
  • Use of any prescription drugs other than hormonal contraception: OCPs, long-acting implantable hormones, injectable hormones, a vaginal ring or an HID), over-the- counter (OTC) medication, herbal remedies, supplements or vitamins within 1 week prior to dosing and during course of study without prior approval of the investigator and medical monitor.
  • Simple analgesia nonsteroidal anti-inflammatory drug (NSAID)
  • paracetamol may be permitted at discretion of investigator.
  • Compound 1 was provided as a powder in capsule formulation for oral administration.
  • the formulation was a Size 00 Swedish orange capsule containing drug substance (10 mg, 50 mg, or 250 mg) with no excipients.
  • Ingredients of the capsule shell were hypromellose (hydroxypropylmethyl cellulose (HPMC)), iron oxide as coloring agent, and titanium dioxide as an opacifier.
  • HPMC hydroxypropylmethyl cellulose
  • the drug product was stored at room temperature (15 °C - 25 °C).
  • Compound 1 is in a pharmacological class of tyrosine kinase inhibitors (TKI).
  • TKI tyrosine kinase inhibitors
  • Compound 1 is an orally bioavailable small molecule dual kinase inhibitor of platelet-derived growth factor receptors (PDGFR) and vascular endothelial growth factor receptors (VEGFR2). Duration of Treatment
  • Part A In Part A (SAD), participants randomized to active treatment in cohorts Al, A2, A4, A5, and A6 received a single oral dose of Compound 1 administered once on Day 1 only. Participants randomized to active treatment in Cohort A3 (Food Effect) received a single oral dose of Compound 1 on Days 1 and 15.
  • PartB In Part B (MAD), participants randomized to active treatment received oral Compound 1 administered twice daily for 7 consecutive days (Day 1 to Day 7) or once daily for 14 consecutive days (Day 1 to Day 14), to be determined on completion of Part A (SAD) of the study.
  • the placebo capsules were visually matching to active study drug and were composed of the same capsule shell but were filled with silicified microcrystalline cellulose.
  • Silicified microcrystalline cellulose is a pharmaceutical excipient composed of co-process microcrystalline cellulose and colloidal silicon dioxide.
  • Safety The safety and tolerability of single and repeat-doses of Compound 1 was investigated according to the following specific assessments: vital signs (systolic and diastolic blood pressure, pulse rate, body temperature, and respiratory rate), 12-lead ECG, clinical laboratory tests (hematology, biochemistry, coagulation, and urinalysis), physical examination, and assessment of TEAEs.
  • ITT Intent-to-Treat
  • Safety Population All participants who received any amount of study drug (Compound 1 or placebo) were included in the Safety population. The Safety population was used for the summaries of all safety assessments. Participants were analyzed according to treatment received.
  • Pharmacokinetic Population All participants who received any amount of active study drug (Compound 1) and have sufficiently evaluable concentration-time profile to allow determination of at least one PK parameter were included in the PK population. An evaluable PK profile was determined at the discretion of the pharmacokineticist following examination of subjects with dosing or protocol deviations that could potentially affect the PK profile. The PK population was used for the summaries of all PK data.
  • Concomitant medications were coded using the World Health Organization (WHO) drug dictionary Version B3 September 2018 Drug Global. Concomitant medications were listed by participant and summarized by anatomical therapeutic class and preferred name.
  • WHO World Health Organization
  • Pharmacokinetics Plasma Compound 1 concentrations, actual blood sampling times, and PK parameters were listed by treatment and protocol specified time point and summarized using descriptive statistics for PK data as outlined above for each scheduled time point by treatment arm. Individual and mean Compound 1 concentration-time profiles were also presented graphically for each treatment. Pharmacokinetic parameters were computed from the individual plasma Compound 1 concentrations using a non-compartmental approach.
  • the following plasma Compound 1 non-compartmental PK parameters were estimated, as appropriate: Cmax, Tmax, AUCo-iast, AUCo-inf, AUCtau (calculated for MAD cohorts only), Kei, ti/2, CL/F (SAD cohorts), CL/Fss (MAD cohorts), Vz/F (SAD cohorts, Vz/Fss (MAD cohorts), RA (calculated for MAD cohorts only).
  • Urine collection time, volume collected, and Compound 1 concentration (Aeu-a) duration of each sampling interval was listed for each participant and summarized by nominal sampling time point and treatment using descriptive statistics (Number of subjects [N], arithmetic mean, SD, CV%, geometric mean, median, minimum and maximum). Individual and mean Compound 1 cumulative urinary excretion-time profiles for each treatment were also presented graphically. Where urine was collected for PK analysis, the following parameters are calculated, as appropriate: Aeu-t2, CLR, CL/F, and Feu-t2.
  • Standard extrapolation for administration to humans would predict that a mean Cmax > 800 ng/mL or mean AUCo-iast > 4000 ng*h/mL would be observed at a human dose of approximately 24 mg/kg/day (i.e., approximately 1440 mg/day for a 60 kg human). Yet, during the clinical study described above, Cmax and AUCo-iast approached their limits at doses approx. 2.4-fold lower than predicted using standard techniques, and Compound 1 exposure in humans was approximately 6-8 times greater than in monkeys. For example, as described below, subjects given a 600 mg dose (i.e., approx.
  • Example 24 describes a protocol for a Phase I study with different cohorts based on certain preliminary PK findings.
  • FIG. 1 summarizes mean oral blood PK profile over time from SAD cohorts in a fasted state.
  • Table 4 summarizes mean oral blood PK parameters from SAD cohorts in a fasted state as of a first data cutoff.
  • Compound 1 was rapidly absorbed with a median Tmax of 1-2 h post-dose. With 400 mg and 600 mg doses, a decline in plasma concentration was observed with mean ti/2 of approx. 10-30 h. Moderate inter-subject variability was observed in 400 mg and 600 mg doses, with approx. 28-44% variability in Cmax and AUC. Cmax increased dose-proportionally up to 400 mg and slightly over-proportionally from 400 mg to 600 mg.
  • Plasma AUC increased 1.6-fold with a 1.5-fold increase in dose (i.e., from 400 mg to 600 mg).
  • Blood Cmax in human healthy volunteers given a 600 mg dose was greater than Cmax of maximum tolerated dose in a monkey 7-day QD study, where blood Cmax in monkey was 605 ng/mL (male) and 780 ng/mL (female) and blood AUC in monkey was 5720 ng*h/mL (male) and 6450 ng*h/mL (female).
  • FIG. 2 and Table 5 summarize results of food effect cohorts (3 A and 3B) as of a first data cutoff.
  • FIG. 2 shows mean oral plasma PK profiles over time in SAD food effect cohorts receiving 200 mg Compound 1. A negative food effect was observed, with 3.6-fold lower Cmax when Compound 1 was administered with food, yet little change in AUCo-iast was observed.
  • Compound 1 pharmacokinetics were characterized following single and multiple oral doses to male Sprague Dawley rats and to male and female cynomolgus monkeys in pharmacokinetic and toxicology studies. Single intravenous doses were administered to rats and monkeys for the purpose of estimating bioavailability of oral doses of Compound 1. Table 6 summarizes pharmacokinetics of Compound 1 observed in rats and monkeys following single intravenous or oral doses.
  • Exposure as assessed by AUCo-iast increased with increasing dose; however, the increase was greater than dose proportional in rats and nearly dose proportional in the monkey.
  • the oral bioavailability of Compound 1 across a dose range of 10 to 100 mg/kg ranged from 4 to 10% in both species.
  • Compound 1 When administered intravenously, Compound 1 was highly distributed outside of the systemic circulation and its volume of distribution values of 2.3 to 18.2 L/kg exceeded total body water in rats and monkeys.
  • Example 4 Compound 1 Inhibits PDGFRjl Phosphorylation in Hepatic Stellate Cells
  • Human Hepatic Stellate Cells (ScienCell Research Labs, Carlsbad, CA, Cat# 5300) were grown in Stellate Cell Medium (ScienCell Research Labs, Carlsbad, CA) in a 37 °C incubator with an atmosphere of 5% C02/95% air. Cells were maintained in tissue culture flasks, plated in 6-well tissue culture plates (at 1 million cells per well), allowed to adhere and grown to 80% confluence. Cells were starved overnight in Stellate Cell Medium without serum. From a 10 mM stock solution of Compound 1 in DMSO, 10-fold serial compound dilutions were prepared. Diluted Compound 1 solutions in DMSO were added at final concentrations of 10 mM, 1 pM,
  • PDGF-BB R&D Systems Catalog # 220-BB
  • lysis buffer RIPA lysis buffer, Sigma R0278
  • the lysis buffer also contained Phosphatase Inhibitor Cocktail 1 (Sigma P2850) and Phosphatase Inhibitor Cocktail 2 Sigma P5726).
  • Example 5 Compound 1 Inhibits KDR Phosphorylation in HUVEC Cells
  • HUVEC Human Umbilical Vein Endothelial Cells (HUVEC) (ATCC Catalog # CRL1730) cells were grown in EBM-2 (Endothelial Cell Growth Basal Medium-2, Lonza Catalog # 00190860) supplemented with EGM-2 Endothelial SingleQuots Kit (Lonza Catalog # CC-4176) in a 37°C incubator with an atmosphere of 5% C02/95% air. Cells were maintained in tissue culture flasks, plated in 6-well tissue culture plates (at 1 million cells per well), allowed to adhere and grown to 80% confluence. Cells were starved overnight in EBM-2 without serum. From a 10 mM stock solution of Compound 1 in DMSO, 10-fold serial compound dilutions were prepared. Diluted Compound 1 solutions in DMSO were added at final concentrations of 10 mM, 1 pM,
  • VEGF165 R&D Systems Catalog # 293-VE
  • lysis buffer RIPA lysis buffer, Sigma R0278.
  • the lysis buffer also contained Phosphatase Inhibitor Cocktail 1 (Sigma P2850) and Phosphatase Inhibitor Cocktail 2 (Sigma P5726).
  • Protein concentrations in the lysates were determined using the DC protein determination kit (Biorad 500-0113 and 500-0114) and 25 pg protein was loaded for Western analysis on 10 lane gels (Invitrogen Catalog # NP0321). In a control lane, MagicMarkTM XP Western Protein Standard was loaded (Invitrogen Catalog # LC5602). Proteins were transferred to Nitrocellulose membranes (Invitrogen Catalog # 77010).
  • Blots were blocked with non-fat dry-milk (Biorad Catalog # 70-6404) and incubated overnight at 2-8 °C with primary antibodies against phospho-VEGF Receptor 2 (Tyrl 175) (Cell Signaling Catalog # 2478) or GAPDH (Cell Signaling # 2118) at a 1 : 1000 dilution. Blots were washed several times and then incubated with an anti-rabbit IgG, HRP-linked secondary antibody at a 1:1000 fold dilution (Cell Signaling Catalog # 7074).
  • Example 6 Compound 1 Decreases Expression of Certain Fibrosis and Inflammation Related Markers
  • Compound 1 is in a pharmacological class of tyrosine kinase inhibitors (TKI) and has a formula of: (Z)-methyl 3-(((4-(N-methyl-2-(4-methylpiperazin-l-yl)acetamido)phenyl)amino) (phenyl)methylene)-2-oxo-2,3-dihydro-lH-pyrrolo[2,3-b]pyridine-6-carboxylate as yellow solid.
  • TKI tyrosine kinase inhibitors
  • the present example describes Bio-Map studies of Compound 1 in cell culture systems using DiscoverX technology platform and that were profiled in a KINOMEscan (Ambit/Di scoveRx/Eurofms, CA) biochemical assay.
  • Exemplary culture systems analyzed were: (1) Small airway epithelial cells + lung fibroblasts (SAEMyoF); (2) Lung fibroblasts only (MyoF); and (3) Renal proximal tubular epithelial cells + lung fibroblasts (REMyoF). When activated, these cell systems mimic pathological conditions such as chronic inflammation, fibrosis, and matrix remodeling.
  • Co-cultures of epithelial cells and fibroblasts or fibroblasts alone were activated with TGFpi (10 ng/mL) + TNF alpha (lOng/mL) and treated with Compound 1 at 100 nM, 500 nM, 2.5mM and 13mM concentrations and incubated for 48 hours. Expression of numerous critical anti-inflammatory and anti-fibrotic marker proteins were evaluated.
  • Compound 1 treatment decreased levels of several inflammation-related markers including monocyte chemotactic protein (MCP-1), macrophage colony stimulating factor (M-CSF), soluble interleukin 8 (sIL-8), Interferon induced T cell alpha chemoattractant (I-TAC), IL-8, Interferon gamma induced Protein- 10 (IP- 10).
  • MCP-1 monocyte chemotactic protein
  • M-CSF macrophage colony stimulating factor
  • sIL-8 soluble interleukin 8
  • I-TAC Interferon induced T cell alpha chemoattractant
  • IL-8 Interferon gamma induced Protein- 10
  • Compound 1 treatment also decreased fibrosis-related markers including N-cadherin, a-SMA, Collagen I and III, TIMP-1, plasminogen activator inhibitor (PAI-1), matrix metalloprotease, MMP-1, MMP-9, tissue and urokinase plasminogen activator (tPA and uPA), soluble VEGF and epidermal growth factor (EGFR) as shown in FIG.
  • fibrosis-related markers including N-cadherin, a-SMA, Collagen I and III, TIMP-1, plasminogen activator inhibitor (PAI-1), matrix metalloprotease, MMP-1, MMP-9, tissue and urokinase plasminogen activator (tPA and uPA), soluble VEGF and epidermal growth factor (EGFR) as shown in FIG.
  • the present example demonstrates that Compound 1 induced dose-dependent decrease in expression of a number of different inflammation and fibrosis-related markers.
  • the present example demonstrates that a mixed cell culture comprising renal epithelial cells and activated myofibroblasts treated with Compound 1 (13 mM) reduced expression of COL1 and COL3.
  • Example 7 TGFpi-Induced Collagen Production in Normal Human Lung Fibroblasts
  • NHLF Normal Human Lung Fibroblasts
  • FGBM Fibroblast Growth Basal Medium
  • FGM2 Fibroblast Growth Medium-2 SingleQuots Supplements and Growth Factors
  • FGM2 Fibroblast Growth Medium-2 SingleQuots Supplements and Growth Factors
  • TGFpi R&D Systems; Catalog 240-B
  • Treatments were performed in duplicate wells and cells that were not treated with compound and/or TGFpi were used as controls.
  • the percentage inhibition was calculated using cells treated with TGFpi as 0% inhibition and cells not treated with compound and TGFpi as 100% inhibition.
  • collagen production was inhibited by 104% and 86%, respectively.
  • the inhibition of collagen production at both Compound 1 concentrations was statistically significant (p ⁇ 0.01).
  • Example 8 Compound 1 Ameliorates Fibrosis in Bleomycin-induced Injury Model of Idiopathic Pulmonary Fibrosis
  • mice Male C57BL/6 mice ( ⁇ 20 to 25 g) were anaesthetized for a short period of time using ketamine (100 mg/kg) and xylazine (10 mg/kg).
  • mice Two weeks after bleomycin instillation, a subgroup of animals was sacrificed and the establishment of lung fibrosis was confirmed (initially as determined by increased lung weight).
  • FIG. 7A is a graph showing lung to body weight ratio (mg/g).
  • Compound 1 significantly reduced hydroxyproline, a major component and marker of collagen deposition and fibrosis.
  • Two weeks after bleomycin exposure animals treated with bleomycin had increased lung hydroxyproline levels compared to sham-treated animals, which indicates development of pulmonary fibrosis. Animals then treated with Compound 1 for three weeks had reduced hydroxyproline levels compared to vehicle-treated animals (FIG. 7B).
  • Compound 1 also significantly reduced fibrosis score (Ashcroft) on histopathology. Lung tissue sections were stained with H&E, and slides were scored for histopathological damage using the Ashcroft scale. Two weeks after bleomycin exposure, animals treated with bleomycin had increased histopathological damage compared to sham-treated animals, indicative of development of pulmonary fibrosis. Animals treated with Compound 1 for three weeks had reduced histopathological damage compared to vehicle-treated animals (FIG. 7C).
  • TGFpi transforming growth factor beta
  • Example 9 Compound 1 Reduces Fibrosis in Inducible TGFpi Mouse Model of Lung Fibrosis
  • mice which expresses TGFpi in the lung via an externally regulatable, triple transgenic system using a doxycycline-inducible promoter.
  • a doxycycline-inducible promoter See Lee, C. G., et al. Proc. Am. Thorac. Soc. 2006 Jul;3(5):418-23; Lee C. G., et al. J. Exp. Med. 2004 Aug 2;200(3)377-89.
  • Eight-to-ten week old TGFpi positive female mice (approximately 25 g of body weight) were induced (fed) with doxycycline (dox) (0.5 mg/ml dox and 20 g/L sucrose in drinking water) for 4 weeks.
  • dox doxycycline
  • the TGFpi transgene was under the control of a dox-inducible promoter.
  • age and gender matched TGFpi negative mice from the transgenic mouse breeding colony not fed with dox were included as control mice (sham).
  • Marked loss of normal bronchial tree architecture due to TGFpi overexpression was evidenced after 4 weeks of dox compared to sham mice using micro-CT images.
  • lung hydroxyproline (HYP) for collagen content, picrosirius red (PSR) staining for collagen signal, and the histopathological observations from (H&E) slides by Ashcroft scoring and IHC staining for alpha SMA were performed. All histological quantitations were performed using a Bioquant image analysis program.
  • Compound 1 reduced lung fibrosis score (Ashcroft) in dox-fed TGFpi transgenic mice.
  • Dox feeding of TGFpi transgenic mice for 4 weeks resulted in a significant increase in lung fibrotic score (Ashcroft score based on H&E staining), compared to normal mice (FIG. 8A).
  • Further dox feeding for an additional 4 weeks resulted in more increases in lung fibrotic score, as shown by the vehicle group.
  • a reduction in hydroxyproline, a component of collagen is indicative of a reduction in fibrosis.
  • Compound 1 significantly decreases aSMA, an early lung fibrosis marker.
  • aSMA is a marker of myofibroblast activation, an early step in fibrosis development.
  • Example 10 Compound 1 Has Anti-Fibrotic Effects Across Multiple Organ Systems in Bleomycin Systemic Sclerosis Mouse Model
  • SC subcutaneous
  • the right lung and right kidney were also fixed in formalin for histopathological evaluation.
  • Dermal thickness measurements were made from H&E-stained tissue sections by measuring the distance from the epidermis to the dermal junction using Bioquant planimetric software.
  • Lung and kidney hydroxyproline (HYP) assays were performed to determine tissue collagen content. Skin, lung and kidney histopathological fibrotic scores were determined by two independent observers, and the scores were averaged.
  • Picrosirius red (PSR) staining of kidney sections was also performed to determine renal collagen deposition. A subset of animals were randomly chosen from each group for histological staining. All histological quantitations were performed using Bioquant image analysis software.
  • Scleroderma is characterized by thickening of the skin caused by accumulation of collagen.
  • dermal thickness was determined by measuring the distance between the epidermal-dermal junction and the dermal-subcutaneous fat junction. This was done in H&E-stained slides prepared from a skin biopsy. After 4 weeks of subcutaneous bleomycin injections (Bleo Pre-Rx Group), dermal thickness was significantly increased compared to sham-injected animals (Sham Group). This indicates significant skin thickening and scleroderma at the time of initiation of compound treatment.
  • Therapeutic treatment of Compound 1 for 5 weeks 25 mg/kg, PO, BID) (Bleo Compound 1 Group) significantly decreased dermal thickness compared to the bleomycin- exposed vehicle cohort (Bleo Vehicle Group).
  • FIG. 9A summarizes these results.
  • Hydroxyproline is a major component of collagen, and it is used to indicate tissue collagen deposition and the development of fibrosis.
  • pulmonary hydroxyproline levels were determined. After 4 weeks of subcutaneous bleomycin injections, lung hydroxyproline levels were significantly increased compared to sham treated animal (Sham vs Bleo PreRx Groups), indicating the development of pulmonary fibrosis.
  • Treatment of bleomycin exposed animals with Compound 1 for 5 weeks (25 mg/kg, PO, BID) (Bleo Compound 1 Group) significantly decreased lung hydroxyproline levels compared to bleomycin exposed vehicle treated animals (Bleo Vehicle), indicating anti -fibrotic activity of Compound 1.
  • FIG. 9C summarizes these results.
  • Urine was again collected (for 24 hours) from day 10-11 after first PAN injection (referred to as Day 11 urines).
  • Day 11 urines On day 11 after first PAN administration GFR was measured by computing FITC-sinistrin decay (Medibeacon) and animals were sacrificed. The left kidney was cut coronally, immersed in formalin (4%) and submitted for sectioning and mounting on slides for subsequent analysis (PAS stain for determining glomerular diameter) and anti-COL 3 antibody IHC. All microscopic analyses (40X) were conducted by an observer blinded to the treatment groups. Glomerular diameter was determined from digital microscopic pictures using ImageJ image analysis software to perform planimetry. Proteinuria was determined from urine samples.
  • every endpoint was not measurable in each animal within a group due to logistical limitations. In such cases, randomly selected animals were queried for data. For instance, no urine was collected for certain animals due to the limited number of metabolic cages available, and only a few animals in each cohort were randomly selected for GFR measurement due to the limited number of probes available for this measurement. The missing animals are marked as NA (not available) in the relevant data tables.
  • the highly sensitive FITC-sinistrin clearance method was employed in conscious animals. This method is capable of measuring the elimination kinetics of FITC-sinistrin using an optical device (Medibeacon, Germany) that can monitor fluorescence trans-cutaneously.
  • FITC-sinistrin clearance is a readout of glomerular filtration rate.
  • the optical device measures fluorescence emitted by FITC-sinistrin through the skin and was temporarily secured on the back of the animal.
  • a single dose of FITC-sinistrin (5 mg/100 g body weight FITC-sinistrin) was injected into the tail vein and the measurement with the optical device was performed for 1.5 hr. The data were downloaded and analyzed using the MPD lab software (Medibeacom, Germany).
  • Glomerular Filtration Rate determination by the FITC- sinistrin method was determined for three animals per experimental group.
  • Glomerular filtration rate was determined immediately prior to terminal sacrifice using FITC-sinistrin decay kinetics as a measurement of renal clearance.
  • GFR Glomerular filtration rate
  • FIG. IOC in Sham (Control) animals, administration of FITC-sinistrin results in gradual decline in fluorescence, indicative of normal renal clearance.
  • animals treated with PAN and Vehicle a significant decrease in the clearance of FITC-sinistrin was observed, showing marked renal dysfunction in the PAN Vehicle animals. Animals treated with PAN and Compound 1 had markedly enhanced clearance of FITC-sinistrin compared to PAN Vehicle animals.
  • Collagen 3 (COL-3) expression a measure of scar, was determined in glomeruli by quantitative immuno-histochemistry.
  • the COL-3 expression level was markedly elevated in glomeruli in the kidneys from PAN Vehicle animals compared to Sham treated animals (FIG. 10E).
  • the COL-3 level was significantly reduced with Compound 1 treatment (FIG. 10E) compared to PAN Vehicle treatment.
  • Example 12 Compound 1 Reduces Proteinuria and Fibrosis in DOCA/Salt Model of Renal Injury and Fibrosis
  • Induction of renal dysfunction was started on day 1, at which point experimental animals received a first subcutaneous dose of deoxycorticosterone acetate (DOCA) at 30 mg/kg and animals were switched from normal drinking water (tap water) to drinking water with 1% NaCl. Animals were kept on the 1% NaCl drinking water for the remainder of the experiment. Animals received weekly subcutaneous doses of DOCA; 30 mg/kg in weeks 1, 2, 3 and 4 and 15 mg/kg in weeks 5 and 6. Control groups were subcutaneously injected with PEG400 and received regular tap water ad libitum. After two weeks, overnight urine was collected in metabolic cages and a subset of animals was sacrificed to establish baseline values.
  • DOCA deoxycorticosterone acetate
  • Animals were monitored several times daily. Three animals in the DOCA + Salt + Vehicle group (Group 3) had unscheduled sacrifice (one each on days 29, 34, and 35). Body weight, serum and kidneys were collected for those animals and samples were processed along with the samples from other animals in the group.
  • kidneys were collected and their weights recorded. At terminal sacrifice, the kidney weights from animals of DOCA + Vehicle group (Group 4) were significantly more than the kidney weights in Sham animals (Group 3). The kidney weight in DOCA + Compound 1 group (Group 5) was not significantly different from the DOCA + Vehicle group (Group 4).
  • the weight of the kidney was also expressed as a percentage of the total body weight of each individual animal.
  • the % kidney weight from animals of DOCA + Vehicle group (Group 4) was significantly higher than the % kidney weights in Sham animals (Group 3).
  • Compound 1 increased body weight and decreased kidney weight compared to vehicle, albeit not statistically significant.
  • the % kidney weight (taken both body weight and kidney weight into consideration) in DOCA + Compound 1 group (Group 5) was significantly reduced compared to the DOCA + Vehicle group (Group 4).
  • Urines were analyzed for protein content and the total amount of protein produced over 24 hours was calculated from the protein concentration and the urine volume produced in 24 hours (FIG. 11 A).
  • a statistically significant elevation in proteinuria was observed in DOCA + Salt animals (Group 2) compared to Sham animals (Group 1), indicating renal dysfunction in the DOCA + Salt animals at the start of compound or vehicle treatment.
  • a statistically significant elevation in proteinuria was again observed in DOCA + Vehicle animals (Group 4) compared to Sham animals (Group 3), indicative of continued renal dysfunction.
  • DOCA-exposed animals treated with Compound 1 (Group 5) were found to have statistically significant reduction in proteinuria compared to DOCA + Vehicle treated animals (Group 4). This is consistent with an improvement in renal function by Compound 1 treatment.
  • Urines were analyzed for albumin concentration and the total amount of albumin produced over 24 hours was calculated from the albumin concentration and the urine volume produced in 24 hours (FIG. 1 IB).
  • elevated albuminuria was observed in DOCA + Salt animals (Group 2) compared to Sham animals (Group 1), albeit that this effect was not statistically significant.
  • a statistically significant elevation in albuminuria was observed in DOCA + Vehicle animals (Group 4) compared to Sham animals (Group 3), indicative of renal dysfunction.
  • DOCA-exposed animals treated with Compound 1 (Group 5) were found to have statistically significant reduction in albuminuria compared to DOCA + Vehicle treated animals (Group 4). This is consistent with the proteinuria data and with an improvement in renal function by Compound 1 treatment.
  • Urines were also analyzed for creatinine levels and the albumin-to-creatinine-ratio (ACR) was calculated (FIG. 11C). Data for ACR were similar to the overall albumin production data. As before, DOCA-exposed animals treated with Compound 1 (Group 5), were found to have statistically significant reduction in ACR compared to DOCA + Vehicle treated animals (Group 4). This is consistent with the proteinuria data and the albuminuria data, and with an improvement in renal function by Compound 1 treatment.
  • Kidney Injury Molecule- 1 (KIM1) (also known as Hepatitis A virus cellular receptor
  • HAVcr-1 T-cell immunoglobulin and mucin domain 1
  • TIM1 T-cell immunoglobulin and mucin domain 1
  • Hydroxyproline is a major component of collagen, and tissue hydroxyproline levels are considered a good indicator of tissue collagen content and therefore of tissue fibrosis.
  • the hydroxyproline content of renal samples was determined and expressed as the total hydroxyproline content per kidney (FIG. 1 IE).
  • a statistically significant elevation in renal hydroxyproline content was seen in DOCA + Vehicle animals (Group 4) compared to Sham animals (Group 3), consistent with marked renal fibrosis at study end.
  • DOCA-exposed animals treated with Compound 1 were found to have statistically significant reduction in renal hydroxyproline content compared to DOCA + Vehicle treated animals (Group 4). This indicates that Compound 1 treatment prevents the development of renal fibrosis in DOCA + Salt treated animals.
  • Picrosirius red-staining in the slides was quantified using Bioquant Image Analysis software. The data was expressed as arbitrary units of staining intensity and also expressed as a % of control in relation to the average staining intensity of the control group (Group 1). As shown in FIG. 11G, in pre-randomization animals, no increase in picrosirius red staining intensity was observed in DOCA + Salt animals (Group 2) compared to Sham control animals (Group 1). This indicates lack of renal fibrosis at the time of compound of vehicle treatment initiation. This lack of histologically observable fibrosis in the renal samples from Group 2 is consistent with the hydroxyproline data in this group.
  • DOCA + Vehicle treated animals (Group 4) had markedly increased insterstitial picrosirius red staining compared to Sham control animals (Group 3). This indicates marked collagen deposition and renal fibrosis (tubulointerstitial fibrosis) as a result of DOCA + Salt treatment.
  • animals that received DOCA + Salt that were treated with Compound 1 (Group 5) showed statistically significantly reduced picrosirius red staining intensity compared to DOCA + Vehicle animals (Group 4). This is consistent with the observed differences in renal hydroxyproline in the various experimental groups and shows anti-fibrotic effects of Compound 1 in this model.
  • the weight of the obstructed kidney was also expressed as a percentage of the body weight of each individual animal.
  • the percentage kidney weight for animals from the UUO + Vehicle group (Group 2) was elevated compared to Control animals (Group 1). There was no statistically significant difference between the UUO + Compound 1 group (Group 3) and the other experimental groups.
  • kidneys were fixed in 10% neutral buffered formalin for at least 48 hours before preparation for histology. Kidneys tissues were grossed, processed, dehydrated, embedded in paraffin and cut into 5 pm sections.
  • Compound 1 was evaluated in the Unilateral Ureteral Obstruction (UUO) model of renal fibrosis in the mouse.
  • Primary endpoints for evaluating compound effects were renal damage score, collagen deposition (as determined by picrosirius red staining) and formation of myofibroblasts (as determined by a-smooth muscle actin staining).
  • Compound 1 had statistically significant beneficial effects on renal damage, picrosirius red staining and alpha-SMA staining.
  • the PCK rat which has a mutation in PKHD1 (ARPKD gene/ phenotypically resembles both human ARPKD-CHF and human PKD in that it exhibits large renal cysts, and renal and hepatic scarring.
  • the PCK rat model is a well-established and well-characterized model that resembles human polycystic kidney and liver disease (Lager, D. L, et al. Kidney Int. 2001 Jan;59(l): 126-36). Results described in this Example are also described in Paka, P., et al. World J. Nephrol. 2018 Sept 8;7(5):96-107).
  • PCK rats that were treated with either vehicle or with Compound 1 did not differ in their body weights from age and sex matched wild-type (WT) Sprague Dawley rats.
  • SCr Serum creatinine
  • BUN Serum creatinine
  • WT wild-type rats
  • Kidneys were weighed at sacrifice and kidney weights are given in absolute weight (g) and as a percentage of body weight (FIG. 13C and FIG. 13D, respectively). Kidneys from PCK Vehicle rats were markedly larger than kidneys from wild-type (WT) rats. Compound 1 treatment in PCK rats reduced kidney size and the % kidney weight compared to PCK Vehicle animals.
  • Hydroxyproline is a major component of collagen, and it is used to indicate tissue collagen deposition and fibrosis. Hydroxyproline levels were determined in kidney samples of 14-week old animals (FIG. 13E). Kidney hydroxyproline levels were markedly increased in PCK Vehicle rats compared to wild-type (WT) rats. Compound 1 treatment significantly reduced renal hydroxyproline levels compared to PCK Vehicle animals, indicating reduced renal collagen deposition as a result of Compound 1 treatment.
  • Increased urine protein excretion can be due to diseases of the glomerulus, an increased quantity of proteins in serum (overflow proteinuria) or low reabsorption at proximal tubule.
  • Urine protein excretion was determined in urines collected over 24 hours immediately prior to sacrifice (FIG. 13H). Protein excretion was found to be elevated in PCK Vehicle rats compared to wild-type (WT) rats, and Compound 1 treatment markedly reduced proteinuria in PCK rats compared to PCK Vehicle animals.
  • Albumin is a major plasma protein. Usually, only trace amounts of it are present in urine, whereas larger amounts occur in the urine of patients with kidney disease. Urine albumin concentrations were determined in urines collected over 24 hours immediately prior to sacrifice (FIG. 131). Urine albumin production was found to be strongly elevated in PCK Vehicle rats compared to wild-type (WT) rats, and Compound 1 treatment markedly reduced urine albumin production in PCK rats compared to Vehicle treated PCK animals.
  • Lipocalin-2 (LCN2) is also known as neutrophil gelatinase-associated lipocalin (NGAL) and is a protein that is expressed in neutrophils and at low levels in normal kidneys (Cowland, J. B., et al. Genomics 1997 Oct 1;45(1): 17-23).
  • NGAL neutrophil gelatinase-associated lipocalin
  • Urine NGAL excretion was determined for 14-week old animals (FIG. 13J).
  • NGAL excretion was found to be markedly elevated in PCK Vehicle rats compared to wild-type (WT) rats, albeit that the increase was not statistically significant by one way ANOVA followed by Tukey’s test. This is probably the result of high variability in NGAL excretion amongst the PCK Vehicle animals.
  • Compound 1 treatment reduced NGAL excretion in PCK rats compared to Vehicle treated animals, albeit that this decrease was also not statistically significant when tested by ANOVA followed by Tukey’s test.
  • HAVcr-1 also known as KIM-1 (KIM1, kidney injury molecule 1) is highly upregulated in injured kidneys in response to various types of renal insults (Bonventre, J. V. Trans. Am. Clin.
  • KIM-1 excretion was found to be strongly elevated in PCK Vehicle rats compared to wild-type (WT) rats, indicating severe renal injury, in particular tubular injury (Bonventre 2014). The elevated KIM-1 excretion is consistent with tubular cyst formation and tubular damage known to exist in the PCK rat (Lager 2001).
  • Compound 1 treatment markedly reduced KIM-1 excretion in PCK rats compared to Vehicle treated animals (FIG. 13K), which indicates mitigation of renal damage, and in particular tubular damage.
  • Interleukin 18 (IL-18 or IL18) is also known as interferon-gamma inducing factor and is a pro-inflammatory cytokine.
  • Urine levels of IL-18 are associated with urine protein excretion and have been used as a biomarker for the progression of diabetic nephropathy (Zhang, D., et ak, PeerJ. 2019;7:e7079; Nakamura, A., et al. Diabetes Care 2005 Dec;28(12):2890-5). Urine IL-18 excretion was determined for 14-week old WT and PCK animals (FIG. 13L).
  • IL-18 excretion was found to be elevated in PCK Vehicle rats compared to wild-type (WT) rats, and Compound 1 treatment markedly reduced IL-18 excretion in PCK rats compared to Vehicle treated animals.
  • Cystatin C or cystatin 3 is used as a biomarker of kidney function (Dharnidharka, V. R., et al. Am. J. Kidney Dis. 2002 Aug;40(2):221-6; Roos, J. F., et al. Clin. Biochem. 2007 Mar;40(5-6):383-91).
  • Urine Cystatin C excretion was determined for 14-week old animals (FIG. 13M).
  • Cystatin C excretion was found to be elevated in PCK Vehicle rats compared to wild-type (WT) rats, and Compound 1 treatment markedly reduced Cystatin C excretion in PCK rats compared to Vehicle treated animals. These results are indicative of an improvement of renal function, consistent with the observation that Compound 1 reduced serum BUN and SCr levels in PCK rats compared to PCK Vehicle treated animals.
  • TNBS (2,4,6-trinitrobenzenesulfonic acid)/ethanol induces a severe illness characterized by bloody diarrhea and a dramatic loss of body weight during the first week with increased colon damage score (Mateus, V., et al. Clin. Exp. Gastroenterol. 2018;11:325-334).
  • TNBS-induced colitis characteristic of acute colitis and the early phase of IBD, is a commonly utilized animal model in rodents that shares significant properties with human Crohn's disease. Advantages of this model include reproducibility and technical simplicity (Filipescu, I. E., et al. PLoS One 2018 Aug 23;13(8):e0202929).
  • TNBS-induced colitis is a well-validated animal model to assess compounds with potential therapeutic effects such as anti-TNFa, corticosteroids, natural compounds and traditional medicine (Mateus 2018; Filipescu 2018; Wirtz, S. et al. Nat. Protoc. 2017 Jul; 12(7): 1295-1309). Compound 1 was therefore tested in the well-established model of TNBS-induced IBD/ Acute Colitis.
  • 5 TNBS-treated mice, as well as 2 sham mice were sacrificed to demonstrate significant TNBS-induced colonic inflammation and pathology, including decreased body weight, reduced colon length and colon gross morphological macroscopic damage.
  • mice were randomized to vehicle and Compound 1 at 5, 15 and 45 mg/kg Compound 1 Hydrochloride trihydrate, PO, BID for 4 days. After 4 days of Compound 1 or vehicle treatment, mice were sacrificed. Body weight, colon length and colon weight were recorded. The middle and distal part of colon were fixed in 10% neutral buffered formalin.
  • Colon histopathology was determined from H&E-stained tissue sections by two independent observers using a previously published scoring system.
  • the score for each individual component (0-3) of the Histopathological Score is shown in FIG. 14E and the composite (total) Colon Histopathological Score (the sum of individual component scores; 0-12) is shown in FIG. 14F. Histopathological Scores showed that Compound 1 treatment at 15 and 45 mg/kg markedly improved colon histology compared to pre-treatment and vehicle treated animals.
  • Colon tissue sections were stained with Alcian blue to evaluate crypt architecture/goblet cells. The degree of Alcian blue staining was quantified to determine crypt damage and goblet cell loss (FIG. 14G, group averages shown). Compound 1 treatment at 15 and 45 mg/kg significantly reduced TNBS-induced goblet cell loss compared to pre-treatment and vehicle treated animals.
  • MPO Myeloperoxidase
  • Example 16 Compound 1 Decreases Acetic Acid (AA) Induced Colitis in Mice
  • Male CD-I mice were infused with 4% AA in saline in 150 m ⁇ volume through rectal enema under isoflurane anesthesia.
  • AA or sham
  • Compound 1 treatment significantly mitigated the decrease in colon length (FIG. 15E), gross morphological colon damage (FIG. 15F) and histopathological colon damage (FIG. 15G) by decreasing mucosal wall thickness, and preserving and crypt/villi architecture compared to vehicle cohort (FIG. 15H).
  • Example 17 Compound 1 Decreases Chronic Colitis in IBD Mouse Model [0468] Efficacy of Compound 1 in a dextran sulfate sodium (DSS) induced chronic colitis/IBD model was evaluated.
  • DSS dextran sulfate sodium
  • Ratio Efficacy of Compound 1 in a dextran sulfate sodium (DSS) induced chronic colitis/IBD model was evaluated.
  • Adult male CD-I mice were fed with 3% DSS in drinking water for 5 days with alternate cycles of normal drinking water for 5 days and continued a total of 3 DSS cycles in 4 weeks. This model has been established to induce chronic colitis/IBD.
  • a small group (n 6) with normal drinking water was included as a sham control. Gross morphological and histopath
  • DSS-Vehicle cohort had the shortest colon length; DSS-Compound 1 cohort had significantly increased colon length compared to the DSS-vehicle cohort (FIG. 16A).
  • Compound 1 treatment decreased macroscopic colon damage score in terms of appearance, inflammation and rectal bleeding (FIG. 16B), decreased colonic hydroxyproline (FIG. 16C) and histopathological injury score based on crypt damage, epithelial erosion and inflammatory cell infiltration as seen in H&E stained colon sections (FIG. 16D). These results indicate that Compound 1 could prevent progression of inflammation-induced fibrosis.
  • PCK rat which has a mutation in PKHD1 (ARPKD gene) phenotypically resembles both human ARPKD-CHF and human PKD in that it exhibits large renal cysts, and renal and hepatic scarring, and is recognized model for polycystic kidney disease (e.g., as described in Example 14).
  • Male PCK rats (PCK/CrljCrl-pkhdlpck/Crl; Charles River Laboratories were randomized to vehicle or test compounds (25 mg/kg Compound 1 Hydrochloride trihydrate, BID, PO) at 6.5 weeks of age following confirmation of frank disease and then sacrificed at 13.5 weeks of age.
  • Age-matched male Sprague-Dawley rats served as wild-type controls. Proteinuria was recorded daily and the results are summarized in Table 13.
  • Compound 1 demonstrated a 2.9 fold greater reduction in proteinuria than nintedanib.
  • mice Streptozotocin (STZ)-induced diabetic mice were randomized to vehicle or test compounds 12 weeks after STZ induction. Results of kidney hydroxyproline analysis are summarized in Table 15. Compound 1 demonstrated 8.3 fold greater reduction in kidney hydroxyproline than nintedanib.
  • FIG. 17C Sirius red staining (FIG. 17C) and TGFpi staining (FIG. 17D) were reduced significantly compared to the vehicle cohort and showed better efficacy even when compared to the pirfenidone (PFD) cohort (an advanced clinical compound).
  • the present disclosure encompasses a recognition that in different patients, disease driving pathways may have divergent outcomes or converge on a common disease related outcome. For example, as depicted in FIG. 18, in some instances, different individuals may have distinct genomic responses that all result in a common kidney disease endpoint, while in other instances, different individuals may have a shared genomic response that leads to different kidney disease endpoints. For example, distinct disease-driving pathways in different individuals may lead to a common outcome of a fibrotic kidney disease, such as, e.g., FSGS. As such, different patients may respond to different therapies and interventions for the same disease.
  • the present disclosure provides the insight that identifying and neutralizing the particular disease drivers of a given patient may provide an early and effective way to manage kidney disease endpoints. Specifically, the present example determined the signalosome in various models of Focal and Segmental Glomerulosclerosis (FSGS) to identify biomarkers for treatment with Compound 1.
  • FSGS Focal and Segmental Glomerulosclerosis
  • Exemplary and etiologically distinct murine models of FSGS were used to characterize the kidney transcriptome and proteome that are associated with therapeutic activity of Compound 1.
  • kidneys were harvested.
  • kidney tissue was preserved in RNAlater (Sigma Aldrich) for glomerular isolation by manual microdissection.
  • RNAlater Sigma Aldrich
  • glomeruli were isolated by magnetic bead extraction.
  • RNA was extracted from glomerular preparations.
  • RNA extraction, RNA sequencing, data normalization and filtering were then performed. Differential expression analysis was performed using DESeq2 and EdgeR (M. F Love, et ak, (December 5, 2014) Genome Biol. 15: 550; M. D.
  • Hierarchical cluster dendrograms were generated to determine the ability of overall transcriptional profiles to recapitulate treatment groups. Rat and murine genes were converted to the corresponding human orthologs using the NCBI homolog (Build 64) and Genomatix annotated ortholog databases. Signalosome of rodent models treated with Compound 1 was analyzed and compared with transcriptome of renal dysfunction in human patients to thereby identify biomarkers of patients to be treated with Compound 1.
  • PAN puromycin aminonucleoside
  • rat puromycin aminonucleoside nephropathy (PAN) model of proteinuric kidney disease exhibit proteinuria that is mitigated by treatment with Compound 1, but not with sham.
  • Animals were sacrificed on Day 21 after PAN administration.
  • Mean arterial pressure (MAP) was evaluated prior to sacrifice and was not substantially different between vehicle treated and Compound 1 treated animals (data not shown).
  • MAP Mean arterial pressure
  • Kidneys were harvested and one of the two kidneys was used for analysis of the glomerular transcriptome. The other kidneys were homogenized for determination of COL1 A1 and COL3 A1 expression.
  • Characteristics of this model include the development of severe proteinuria and glomerulosclerosis, which reflects several aspects of the pathogenesis of focal and segmental glomerulosclerosis (FSGS).
  • FSGS focal and segmental glomerulosclerosis
  • the present disclosure provides the insight that disease-driving networks in animal (e.g., rodent) models of kidney disease may prove beneficial in those human patients that share the same transcriptional elements.
  • animal e.g., rodent
  • lupus nephritis lupus nephritis
  • COL1A1 and COL3A1 have been identified as disease-associated nodes in human FSGS. See, e.g., Canadas-Garre, M., et al. J. Transl. Med. (2016) 16:292; Grgic, T, et al. Kidney Int. 2014 Dec; 86(6): 1116-1129; Schwab, K., et al. Am. J. Nephrol. 2004;24:438-447. Furthermore, increased renal COL3 Al expression is associated with increased renal dysfunction in human FSGS.
  • Compound 1 interactome network include MAP2K5, MAP3K3, MAPK7, PRKAR1A, PIK3R1, SMPD1, AXL, FYN, PLXND1, CDC42PB, VAT1, RPS6KA2, RBPMS, CAV1, BGN, NPR1, FGFR1, EFEMP2, COL6A1, HEG1, PCGF2, COL6A2, MYH11, PDGFRA, PDGFRB, and KIT.
  • the present disclosure identified biomarkers in rodent models of kidney disease and in human patients that correspond with the Compound 1 mechanism of action.
  • the present disclosure encompasses a recognition that patients with fibrotic kidney diseases who have an altered level of one or more of these biomarkers may benefit from treatment with Compound 1.
  • the present example describes treatment of fibrotic disease of the kidney in a patient.
  • a nephropathy patient with suspected glomerular disease i.e., suspected of having FSGS or MCD
  • a renal biopsy For example, mRNA is extracted from single glomeruli (see Menon et ah, JCI Insight. 2020, 5(6):el33267), and sequenced to quantify levels of certain biomarkers associated with Compound l’s mechanism of action.
  • mRNA is extracted from single glomeruli (see Menon et ah, JCI Insight. 2020, 5(6):el33267), and sequenced to quantify levels of certain biomarkers associated with Compound l’s mechanism of action.
  • COL1 A1 and COL3A1 expression are quantified. If the subject has a level of COL3A1 and/or COL1A1 that is above a threshold level, then the subject is to be treated with Compound 1.
  • An exemplary threshold level is a level that is about 20% or more above a predetermined normal range or mean or median level in a healthy patient. If a subject does not have a level of biomarker above the threshold level, then alternate treatment is determined.
  • a patient to be treated with Compound 1 the kidney disease is stabilized and/or ameliorated.
  • Example 21 Puromycin Aminonucleoside Administration in Rat is Associated with Collagen Type III Glomerulopathy
  • Urine protein was determine using the Bradford assay.
  • Renal homogenates were subjected to analysis for COL3A1 and peptidylprolyl isomerase A (housekeeping gene) mRNA.
  • qPCR was performed on a Thermofisher Quant-Studio 3 Real-Time PCR system, each sample was diluted three-fold, and qPCR reaction was performed in triplicate for all tissue samples. Renal slices were stained with periodic acid Schiff for morphometric analysis or with Collagen Type III (Col3al) Antibody (BioCompare) for immunohistochemical analysis.
  • COL3 A1 mRNA level was elevated ⁇ 4-fold in the PAN cohort (FIG. 24A). There was as significant and direct association between the fold-increase in renal COL3 A1 mRNA level and the corresponding urine protein value (FIG. 24B). Immunohistochemical analysis indicated deposition of collagen type III restricted to the glomerular mesangium.
  • the present example demonstrates that an increase in COL3A1 mRNA is directly associated with an increase in urine protein in a rat model of kidney disease.
  • collagen type III deposition is restricted to the glomerulus, which may be indicative of a robust COL3A1 transcriptomic network in that compartment, compared to, e.g., the tubulointerstitium.
  • the present disclosure encompasses a recognition that in different patients, disease driving pathways may have divergent outcomes or converge on a common disease related outcome. For example, in some instances, different individuals may have distinct genomic responses that all result in a common lung disease endpoint, while in other instances, different individuals may have a shared genomic response that leads to different lung disease endpoints. For example, distinct disease-driving pathways in different individuals may lead to a common outcome of a fibrotic lung disease, such as, e.g., IPF. As such, different patients may respond to different therapies and interventions for the same disease.
  • the present disclosure provides the insight that identifying and neutralizing the particular disease drivers of a given patient may provide an early and effective way to manage lung disease endpoints. Specifically, the present example determined the signalosome in various models of idiopathic pulmonary fibrosis (IPF) to identify biomarkers for treatment with Compound 1
  • IPPF idiopathic pulmonary fibrosis
  • Two exemplary and etiologically distinct models of pulmonary fibrosis are used to characterize the lung transcriptome and proteome that are associated with therapeutic activity of Compound 1. Dosing of Compound 1 starts 2 weeks after disease induction with bleomycin or S1O2 instillation, and dosing is continued via the oral route for 4 weeks. Lung hydroxyproline (HYP) and histopathology are determined. BAL inflammatory cell infiltrates, BAL protein, MPO, BAL cytokines, tissue cytokines, and lung fibrotic markers are evaluated by IHC stainings and transcriptomic analysis by performing real-time PCR. A cytokine/chemokine/growth factor array on minimally invasive BALF samples from the animal models.
  • HEP Lung hydroxyproline
  • BAL inflammatory cell infiltrates, BAL protein, MPO, BAL cytokines, tissue cytokines, and lung fibrotic markers are evaluated by IHC stainings and transcriptomic analysis by performing real-time
  • the present example describes treatment of fibrotic disease of the lung in a patient.
  • a sample of BALF is obtained from a patient with suspected or confirmed pulmonary disease (i.e., suspected of having idiopathic pulmonary fibrosis).
  • mRNA is extracted from the sample, and sequenced to quantify levels of certain biomarkers associated with Compound l’s mechanism of action.
  • expression of biomarkers identified using the method of Example 22 are quantified. If the subject has a level of a biomarker that is above a threshold level, then the subject is to be treated with Compound 1.
  • An exemplary threshold level is a level that is about 20% or more above a predetermined normal range or median level in a healthy patient. If a subject does not have a level of biomarker above the threshold level, then alternate treatment is determined.
  • the lung disease is stabilized and/or ameliorated.
  • Example 24 A Phase 1, Randomized, Double-Blind, Placebo-controlled, Single and Multiple Ascending Dose Study to Determine the Safety, Tolerability, Pharmacokinetics, and Food Effect of Compound 1 in Healthy Adult Participants
  • Primary Endpoint The frequency and severity of treatment-emergent adverse events (TEAEs), including clinically significant abnormal vital signs, electrocardiograms (ECGs), laboratory test results, and physical examination findings.
  • TEAEs treatment-emergent adverse events
  • ECGs electrocardiograms
  • Plasma PK endpoints include:
  • Urine PK endpoints include:
  • Cohort A3 was a food effect cohort, and participants in Cohort A3 only returned to the CRU on Day 14 and, following a 14-day washout, received a second single dose of their assigned treatment on Day 15 following consumption of a high fat meal. For Cohort A3 only, a second period of confinement applied from Day 14 until completion of the 72-hour post-dose assessments on Day 18. [0510] All Part A participants returned to the CRU for a follow-up visit 7 days ( ⁇ 1 day) after their final dose of study drug. For all cohorts in Part A (SAD), the decision to escalate a dose or modify a dose was determined by the SMC following review of the 7-day blinded safety and available PK data from the preceding cohort.
  • SAD Part A
  • Compound 1 dose will not exceed 1200 mg or a dose level that leads to mean whole blood C max >600 ng/mL or mean whole blood AUCo-i ast > 2000 ng*h/mL.
  • C max and AUCi ast values were extrapolated from the mean plasma C max of 800 ng/mL and the mean plasma AUCi ast of 4000 ng*hr/mL observed in non-human primates at the NOAEL of 75 mg/kg/day.
  • Part B Multiple Ascending Dose (MAD)
  • the Compound 1 dose level tested in Part B did not exceed 1000 mg per day or a dose level that led to a mean whole blood Cmax > 600 ng/mL or mean whole blood AUCo-iast > 2000 ng*h/mL.
  • Cmax and AUCo-iast values were extrapolated from the mean plasma Cmax of 800 ng/mL and the mean plasma AUCiast of 4000 ng*h/mL observed in non-human primates at the no observed adverse effect level (NOAEL) of 75 mg/kg/day.
  • Compound 1 dose will not exceed 1000 mg per day or a dose level that leads to mean whole blood C max >600 ng/mL or mean whole blood AUCo-ias t > 2000 ng*h/mL.
  • C max and AUCi ast values were extrapolated from the mean plasma C max of 800 ng/mL and the mean plasma AUCias t of 4000 ng*hr/mL observed in non-human primates at the NOAEL of 75 mg/kg/day.
  • Part D Single-Dose Food-Effect Cohort
  • Part A up to 48 healthy volunteers; Part B: up to 32 healthy volunteers; Part C: up to 16 healthy volunteers; Part D: up to 8 healthy volunteers).
  • Participants must have a minimum body weight of 50 kg and a body mass index (BMI) between >18.0 and ⁇ 32.0 kg/m 2 at screening.
  • BMI body mass index
  • Participants must have clinical laboratory values within normal range as specified by the testing laboratory, unless deemed not clinically significant by the investigator or delegate.
  • Double contraception is defined as a condom and one other form of the following:
  • FSH follicle-stimulating hormone
  • Periodic abstinence e.g., calendar, ovulation, symptothermal, post-ovulation- methods
  • withdrawal are not considered highly effective methods of birth control. Participant complete abstinence for the duration of the study and for 1 months after last study treatment is acceptable.
  • WOCBP must have negative pregnancy test at screening and Day 1 and be willing to have additional pregnancy test as required.
  • Acceptable methods of contraception include the use of condoms and the use of an effective contraceptive for the female partner that includes: OCPs, long-acting implantable hormones, injectable hormones, a vaginal ring or an IUD. Participants with same sex partners (abstinence from penile-vaginal intercourse) are eligible when this is their preferred and usual lifestyle.
  • GI gastrointestinal
  • atrophic gastritis atrophic gastritis
  • lactose intolerance atrophic gastritis
  • H. Helicobacter
  • Fever body temperature > 38 °C
  • symptomatic viral or bacterial infection within 2 weeks prior to screening.
  • HCV hepatitis C antibody
  • HBVAg hepatitis B surface antigen
  • HAV human immunodeficiency virus
  • Participants with a positive toxicology screening panel urine test including qualitative identification of barbiturates, tetrahydrocannabinol (THC), amphetamines, benzodiazepines, opiates, and ***e), or alcohol breath test.
  • any prescription drugs other than hormonal contraception: OCPs, long-acting implantable hormones, injectable hormones, a vaginal ring or an HID
  • over-the- counter (OTC) medication herbal remedies, supplements or vitamins within 1 week prior to dosing and during course of study without prior approval of the investigator and medical monitor.
  • Simple analgesia nonsteroidal anti-inflammatory drug (NSAID)
  • NSAID nonsteroidal anti-inflammatory drug
  • paracetamol may be permitted at discretion of investigator.
  • CYP3 A4 cytochrome P450 3 A4
  • any strong inhibitors or inducers of CYP3 A4 starting from 2 weeks prior to first dose of study drug and until end of study assessments.
  • Compound 1 was provided as a powder in capsule formulation for oral administration.
  • the formulation was a Size 00 Swedish orange capsule containing drug substance (50 mg or 250 mg) with no excipients.
  • Ingredients of the capsule shell were hypromellose (hydroxypropylmethyl cellulose (HPMC)), iron oxide as coloring agent, and titanium dioxide as an opacifier.
  • HPMC hydroxypropylmethyl cellulose
  • the drug product was stored at room temperature (15 °C - 25 °C).
  • Compound 1 is in a pharmacological class of tyrosine kinase inhibitors (TKI).
  • TKI tyrosine kinase inhibitors
  • Compound 1 is an orally bioavailable small molecule dual kinase inhibitor of platelet-derived growth factor receptors (PDGFR) and vascular endothelial growth factor receptors (VEGFR2). Duration of Treatment
  • Part A In Part A (SAD), participants randomized to active treatment in cohorts Al, A2, A4, A5, and A6 received a single oral dose of Compound 1 administered once on Day 1 only. Participants randomized to active treatment in Cohort A3 (Food Effect) received a single oral dose of Compound 1 on Days 1 and 15.
  • PartB In Part B (MAD), participants randomized to active treatment received oral Compound 1 administered twice daily for 7 consecutive days (Day 1 to Day 7) or twice daily for 14 consecutive days (Day 1 to Day 14), to be determined on completion of Part A (SAD) of the study.
  • Part C In Part C (MAD QD), participants randomized to active treatment received oral Compound 1 administered once daily for 14 consecutive days (Day 1 to Day 14).
  • the placebo capsules visually matched to active study drug and were composed of the same capsule shell but were filled with silicified microcrystalline cellulose.
  • Silicified microcrystalline cellulose is a pharmaceutical excipient composed of co-process microcrystalline cellulose and colloidal silicon dioxide.
  • Safety The safety and tolerability of single and repeat-doses of Compound 1 was investigated according to the following specific assessments: vital signs (systolic and diastolic blood pressure, pulse rate, body temperature, and respiratory rate), 12-lead ECG, clinical laboratory tests (hematology, biochemistry, coagulation, and urinalysis), physical examination, and assessment of TEAEs.
  • descriptive statistics e.g. arithmetic mean, standard deviation [SD], median, minimum and maximum
  • SD standard deviation
  • Frequency summaries e.g. number of observed and percentage of each categories
  • ITT Intent-to-Treat
  • Safety Population All participants who received any amount of study drug (Compound 1 or placebo) were included in the Safety population. The Safety population was used for the summaries of all safety assessments. Participants were analyzed according to treatment received.
  • Pharmacokinetic Population All participants who received any amount of active study drug (Compound 1) and have sufficiently evaluable concentration-time profile to allow determination of at least one PK parameter were included in the PK population. An evaluable PK profile was determined at the discretion of the pharmacokineticist following examination of subjects with dosing or protocol deviations that could potentially affect the PK profile. The PK population was used for the summaries of all PK data.
  • Concomitant medications were coded using the World Health Organization (WHO) drug dictionary Version B3 September 2018 Drug Global. Concomitant medications were listed by participant and summarized by anatomical therapeutic class and preferred name. [0550] Physical examinations at each visit were listed for each participant and summarized using descriptive statistics at each visit by treatment arm.
  • WHO World Health Organization
  • Pharmacokinetics Plasma Compound 1 concentrations, actual blood sampling times, and PK parameters were listed by treatment and protocol specified time point and summarized using descriptive statistics for PK data as outlined above for each scheduled time point by treatment arm. Individual and mean Compound 1 concentration-time profiles were also presented graphically for each treatment. Pharmacokinetic parameters were computed from the individual plasma Compound 1 concentrations using a non-compartmental approach.
  • the following plasma Compound 1 non-compartmental PK parameters were estimated, as appropriate: Cmax, Tmax, AUCo-iast, AUCo-inf, AUCtau (calculated for MAD cohorts only), Kei, ti/2, CL/F (SAD cohorts), CL/Fss (MAD cohorts), Vz/F (SAD cohorts, Vz/Fss (MAD cohorts), RA (calculated for MAD cohorts only).
  • Urine collection time, volume collected, and Compound 1 concentration (Aeu-t2) duration of each sampling interval was listed for each participant and summarized by nominal sampling time point and treatment using descriptive statistics (Number of subjects [N], arithmetic mean, SD, CV%, geometric mean, median, minimum and maximum). Individual and mean Compound 1 cumulative urinary excretion-time profiles for each treatment were also presented graphically. Where urine was collected for PK analysis, the following parameters are calculated, as appropriate: Aeu-t2, CLR, CL/F, and Feu-t2.
  • Example 25 Compound 1 Ameliorates Fibrosis in Alport Syndrome Mouse Model
  • Protein-to-creatinine ratio (PCR). Creatinine standard was prepared at 20, 10, 5, 2.5, 1.25, 0.625 and 0.3125 mg/dL. Urine samples were diluted 20-fold with water. Then 25 pL of samples were added to the 96 well plate and pipet 50 pL of samples, water as the blank, or standards into wells in the clear plate. 50 pL of the DetectX® Creatinine Reagent was added according to manufacturer instructions, (Arbor Assays). Urine protein was calculated for mg/mL. Then, urine protein of the sample was divided by the urine creatinine to determine the protein to creatinine ratio (PCR) and expressed as mg/mg.
  • PCR Protein-to-creatinine ratio
  • Serum BUN Serum samples were diluted 5-fold with distilled water. BUN standard was prepared at 10, 5, 2.5, 1.25, 0.625, 0.3125, and 0.156 mg/dL. 50 pL of samples or appropriate standards into 96 wells in the plate. 50 pL of water was used as the zero standard. Then, 75 pL of Color Reagent A to each well and then added 75 pL of Color Reagent B to each well using a multichannel pipet. The plates were incubated at room temperature for 30 minutes and then the optical density was read at 450 nm according to manufacturer's instructions (Arbor Assays).
  • Serum Creatinine 20 pL of the serum samples were analyzed for serum creatinine levels using LC-MS/MS. Briefly, after spiking in creatinine-d3 as internal standard, mouse serum samples were purified by protein precipitation with acetonitrile. Creatinine was analyzed by LC- MS/MS. Chromatographic separation was performed with a Shimadzu Prominence system on a Primesep 200 (SIELC Technologies) column with a gradient elution (0.1% formic acid in water as mobile phase A and 0.1% formic acid in methanol as mobile phase B). MRM detection (114.05 to 44.2 for creatinine and 117.05 to 47.2 for creatinine-d3) was carried out with AB Sciex API-3200 triple quadrupole mass spectrometer. Creatinine was expressed as mg/dL.
  • hydroxyproline Assay The hydroxyproline (HYP) content from kidney tissues was determined as described by Woessner et al (Archives of Biochemistry and Biophysics, Vol. 93, Issue , May 1961, 440-447).
  • Kidney sections were fixed in 10% formalin for at least 48 hr before preparation for histology. Kidney tissues were processed, dehydrated, embedded in paraffin, and cut into sequential 5 pm sections. Kidney sections were subjected to haematoxylin- eosin (H&E) and Masson’s Trichrome staining using a Trichrome Stain Vector Kit (Sigma). IHC staining using mouse antibodies for Collagen- 1 (Sigma), TGFpi (Cell Signaling), and aSMA (Sigma) were performed by following standard laboratory procedures. Photomicrographs were taken using a microscope (Leica) equipped with a computerized digital camera.
  • Trichrome Staining Working Weigeret’s Iron Hematoxylin Solution was prepared according to label instructions. The slides were deparaffmized and hydrated with deionized water. Then slides were placed in pre-Bouin’s Solution, at room temperature overnight. Slides in a Coplin Jar were washed in running tap water to remove yellow color from sections. The slides were then stained in Working Weigert’s Iron Hematoxylin solution for 5 minutes. The slides were washed in running tap water for 5 minutes. The slides were then stained in Trichrome Stain AB Solution (Sigma) for 5 minutes and placed in 0.5% Acetic acid for 1 minute. The slides were rinsed, dehydrated through alcohol, cleared in xylenes and then mounted.
  • PSR Picrosirius Red Staining. Sections of paraffin-embedded tissue were deparaffmized by sequential washes in xylene (2 times for 5 minutes), 100% ethanol (2 times for 5 minutes), 90% ethanol (once for 5 minutes), 80% ethanol (once for 5 minutes) and 70% ethanol (once for 5 minutes). Samples were then washed in water for 5 minutes. Samples were then incubated in a solution of saturated picric acid containing 0.2% Sirius Red dye at room temperature for 35 min with shaking. Samples were washed with tap water until the wash was colorless. Samples were then dehydrated with serial 5 minute washes in water, 70% ethanol,
  • IHC Stainings for Fibrotic Markers Formalin fixed kidney tissue sections were used for IHC staining. Endogenous peroxidases were quenched with 3.0% hydrogen peroxide in methanol for 30 min. Sections were further blocked with 3.0% bovine serum albumin (BSA) in PBS, exposed to 0.5 % Triton X-100 for 1 h to reduce non-specific antibody binding and incubated with primary antibodies of anti -mouse mouse aSMA antibody (Sigma), Collagen- 1 antibody (Sigma) and TGFB1 (Santa Cruz) at 4 °C overnight.
  • BSA bovine serum albumin
  • the sections were washed with PBS three times, incubated with HRP-conjugated secondary antibodies (Cell Signaling) for 1 hour followed by washing 3 times with lx PBS. The sections were then incubated with a HRP substrate (Invitrogen) for 2-3-minutes to obtain clean staining. After dehydration with a series of increasingly concentrated ethanol solutions, sections were mounted with neutral gum. Images were captured using a Nikon microscope equipped with a computer.
  • Renal injury was evaluated in H&E stained kidney sections, and a renal damage score was assigned on a numerical scale from 0 (normal/no injury) to 4 (severe injury) by two independent observers.
  • analysis consisted of estimating the total amount of tubular and glomerular damages in the cortical regions of the entire kidney section. Injuries included the disruption of normal tissue micro architecture, hemorrhage and monocyte infiltration, inflammation, casts formation, and necrosis and sclerosis.
  • Compound 1 did not decrease serum BUN or serum creatinine (SCr) in Alport mice (FIG. 26G and FIG. 26H, respectively). Treatment with Compound 1 for five weeks significantly decreased kidney fibrosis, as judged by hydroxyproline (HYP) levels, in Alport mice (FIG. 261). Compound 1 also significantly reduced renal histopathological injury score in Alport mice (FIG. 26J).
  • Compound 1 treatment decreased kidney Trichrome staining, an indication of decreased collagen and fibrosis, significantly compared to the vehicle cohort (FIG. 26K).
  • IHC stainings for kidney fibrotic markers demonstrated that Compound 1 decreased TGFpi, aSMA, and Collagen- 1 in Alport mice compared to vehicle (FIG. 26L, FIG. 26M, and FIG. 26N, respectively).
  • Picrosirius red (PSR) staining for collagen showed that Alport mice treated with Compound 1 had significantly reduced collagen as compared to Alport mice treated with vehicle (FIG. 260).
  • Example 26 Effect of Compound 1 in a Passive Heymann Nephritis Model
  • Compound 1 was tested in the Passive Heymann Nephritis model of renal dysfunction in rats.
  • renal dysfunction was mediated by intravenous administration of Anti-FXl A serum to rats, resulting in podocyte-localized deposition of antibodies, whereby immune deposits accumulate and obscure the slit diaphragm (Spicer et ah, J. Immunol. 2007 Jul 1; 179(1): 172-8). This results in podocyte foot process effacement and proteinuria.
  • the model has nearly identical pathology to human membranous nephropathy and gl omerul onephriti s .
  • Urine Analysis Overnight urines were collected using Tecniplast Metabolic Cage Systems for rodents (Tecniplast, Catalogue #3700M071). Animals were kept in metabolic cages for 24 hours. Urine samples were analyzed for protein content using the Bradford Assay (Sigma Cat #B6916-500ML) according to the manufacturer’s protocols. Urine samples were undiluted or diluted to 10-fold or 20-fold with water to be within the assay’s acceptable range. Creatinine was measured using the Quantichrom Creatinine Assay Kit (Bioassay Systems). A standard curve was generated to determine urine sample concentrations. The protein to creatinine ratio was determined and expressed as mg/mg.
  • Serum Analysis Sera were collected at sacrifice and processed in BD Microtainer serum collection tubes according to the manufacturer’s instructions. Sera were stored at -20 °C until analysis. Serum creatinine, cholesterol, and triglycerides were analyzed using validated analytical procedures for human samples. Serum BUN was measured according to the manufacturer’s protocols (Arbor Assays, Catalogue # K024-H).
  • Kidney tissue was collected, and mass determined at the time of tissue collection. Samples were stored at -80 °C until analysis. The hydroxyproline (HYP) content from kidney tissues were determined. Kidney tissue was subjected to hydrolysis in 10 N HC1 for 18 hr at 110 °C, and aliquots were incubated with chloramine T solution, perchloric acid and Ehrlich's reagent at 60 °C for 20 minutes. Absorbance was measured at 550 nm, and the amount of HYP was determined using a hydroxyproline standard curve. Total hydroxyproline is expressed as micrograms per kidney.
  • Body mass was measured at time of sacrifice, day 93.
  • the nintedanib-treated animals had significantly reduced body mass, reduced kidney mass and increased kidney mass to body mass ratio compared to the vehicle cohort (***p ⁇ 0.0001, One-way ANOVA, Tukey).
  • the Compound 1 treatment groups did not have a significant change in body mass (FIG. 27B), kidney mass (FIG. 27C), or the kidney to body mass ratio (FIG. 27D) compared to vehicle or sham.
  • Serum cholesterol was significantly increased (p ⁇ 0.05) in the nintedanib group compared to Sham, 50 mg/kg Compound 1, and 15 mg/kg Compound 1 groups (p ⁇ 0.05, One Way ANOVA) (FIG. 27G). No statistically significant changes in serum cholesterol were noted with Compound 1 treatment. Serum triglycerides were significantly reduced in the 50 mg/kg group compared to vehicle (*p ⁇ 0.05, One way ANOVA, Tukey), whereas the 100 mg/kg and 15 mg/kg groups did not have a statistically significant change in serum triglycerides (FIG. 27H). Serum creatinine (FIG. 271) and BUN (FIG. 27J) did not change significantly in the vehicle or Compound 1 treatment groups compared to sham. However, nintedanib treatment caused a significant increase in serum creatinine and BUN.
  • PAS staining was performed to semi-quantitatively determine the level of glomerulosclerosis of glomeruli. Representative images are shown in FIG. 27M, and glomerular damage scores are shown in FIG. 27N. There was a significant increase in glomerular injury in the vehicle group compared to sham. There was a significant reduction in glomerular damage with Compound 1 treatment at 50 mg/kg and 15 mg/kg. The Nintedanib group had global glomerulosclerosis.
  • Example 27 A Phase 2, Multicenter, Double-Blind, Randomized, Placebo-controlled Study of Safety and Efficacy of Compound 1 in Patients with Primary Glomerular Disease and Persistent Proteinuria
  • the primary objective of this study is to evaluate efficacy of Compound 1 in patients with primary glomerular disease and persistent proteinuria while on standard of care (SOC), as measured by a reduction in the 24-hour urinary protein excretion.
  • SOC standard of care
  • the secondary objective of this study is to evaluate the safety and tolerability of Compound 1 in patients with primary glomerular disease and persistent proteinuria.
  • the eligible patients are randomized in a 1 : 1 : 1 ratio to three dose levels of Compound 1 (200 mg once daily (QD), 400 mg QD, 300 mg twice daily (BID)) or placebo administered daily for 12 weeks.
  • Compound 1 200 mg once daily (QD), 400 mg QD, 300 mg twice daily (BID)
  • BID 300 mg twice daily
  • the study drugs are administered QD or BID orally to patients according to their assignment to QD or BID dosing schedule. All patients take the study drug either once-daily in the morning or twice-daily within 30 minutes of meals and at least 30 minutes apart from any other drugs. The study ends when the last patients have completed 12 weeks of study drug and the follow-up period of 4 weeks.
  • the study period is from the time of signing of the Informed Consent until their last visit.
  • Adverse events are collected during all the study periods and are considered under treatment until 4 weeks after the drug discontinuation.
  • Inclusion Criteria Approximately 100 patients (25 patients per study arm) are planned for this study. Diagnosis and Main Criteria for Inclusion: Inclusion Criteria:
  • ACEi Angiotensin-converting enzyme inhibitor
  • ARB Angiotensin II receptor blocker
  • Renin-angiotensin-aldosterone system (RAAS) blockers and Sodium glucose co transporter 2 (SGLT-2) inhibitors must be stable for at least 12 weeks prior to screening and projected to remain stable through week 16.
  • RAAS Renin-angiotensin-aldosterone system
  • SGLT-2 Sodium glucose co transporter 2
  • Immunosuppressive or immunomodulatory therapy must be stable for at least 12 weeks prior to screening and projected to remain stable through study week 16.
  • HBV Hepatitis B
  • HBV Hepatitis C
  • HBV human immunodeficiency virus
  • ANC absolute neutrophil count
  • NSAIDS non-steroidal anti-inflammatory agents
  • antiplatelet therapy e.g. acetyl salicylic acid up to 325 mg/d, or clopidogrel at 75 mg/d, or equivalent doses of other antiplatelet therapy.
  • Renal disease secondary to systemic disease including but not limited to: systemic lupus erythematosus, anti -neutrophil cytoplasmic antibodies-associated diseases, anti- glomerular basement disease, secondary forms of focal segmental glomerulosclerosis, renal diseases associated with para-proteinemias, C3 glomerulopathy, and diabetic kidney disease
  • Compound 1 is an orally active tyrosine kinase inhibitor. Three dose levels (200 and 400 mg QD and 300 mg BID) are tested in this study. The study drug and the placebo are administered once or twice daily orally after breakfast and dinner and at least 30 minutes apart from any concomitant medications.
  • Compound 1 is administered as a Form A hydrochloride trihydrate salt in a Hypromellose Swedish Orange-colored capsule containing no excipients.
  • Ingredients of the capsule shell are hypromellose (hydroxypropylmethyl cellulose also known as HPMC), iron oxide as a coloring agent, and titanium dioxide as an opacifier.
  • hypromellose hydroxypropylmethyl cellulose also known as HPMC
  • iron oxide as a coloring agent
  • titanium dioxide as an opacifier
  • Reference Therapy Patients receive the standard of care including maximally tolerated/recommended dose of ACEi or ARB but not both. Where applicable, the doses of mineralocorticoid receptor blockers, direct renin inhibitors, and SGLT-2 inhibitor doses must be stable for at least 12 weeks and anticipated to remain stable during the study period.
  • Placebo capsules are composed of the same capsule shell used for the active but are filled with silicified microcrystalline cellulose.
  • Silicified microcrystalline cellulose is a pharmaceutical excipient composed of co-processed microcrystalline cellulose and colloidal silicon dioxide.

Abstract

Provided herein are methods of reducing fibrosis by administering methyl (Z)-3-(((4- (N-methyl-2-(4-methylpiperazin-1-yl)acetamido)phenyl)amino)(phenyl)methylene)-2-oxo-2,3- dihydro-1H-pyrrolo[2,3-b]pyridine-6-carboxylate.

Description

REDUCING FIBROSIS AND TREATING RELATED DISEASES, DISORDERS, AND
CONDITIONS
RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Patent Application No. 63/046,925, filed July 1, 2020, U.S. Provisional Patent Application No. 63/089,917, filed October 9, 2020, and U.S. Provisional Patent Application No. 63/194,539, filed May 28, 2021, the entire contents of each of which are hereby incorporated by reference.
BACKGROUND
[0002] Fibrotic diseases contribute to as much as 45% of deaths in the industrialized world (Wynn, T. A. J. Pathol. 2008;214:199-210). Fibrosis, i.e., excessive tissue scarring, is a common feature of many chronic diseases, as well as acute injuries, where accumulation of scar tissue can lead to loss of organ function and, ultimately, organ failure (Friedman, S. U, et al. Sci. Transl. Med. 2019 Jan 9;5(167):167srl).
SUMMARY
[0003] The present disclosure provides certain technologies for reducing fibrosis. In some embodiments, the present disclosure provides certain technologies for reducing fibrosis of, e.g., gastrointestinal tract, heart, kidney, lung, liver, muscle, pancreas, and/or skin.
[0004] In some embodiments, the present disclosure provides methods for treating (e.g., lessening the severity of, such as by delaying onset and/or reducing degree and/or frequency of one or more features of) a disease, disorder, or condition associated with fibrosis. In some embodiments, such methods comprise, for example, administering an antifibrotic agent (e.g., a small molecule, such as Compound 1 described herein).
[0005] The present disclosure also provides methods of administering an antifibrotic agent (e.g., Compound 1 described herein) to a subject or a population of subjects in need thereof. The present disclosure encompasses the recognition that particular modes of administering an antifibrotic agent (e.g., Compound 1 described herein) achieve one or more particular effects, e.g., in a population of healthy subjects or in a population of subjects suffering from primary glomerular disease and/or persistent proteinuria. For example, the present disclosure demonstrates that administration of an antifibrotic agent (e.g., Compound 1 described herein) achieves different pharmacokinetic properties (e.g., higher mean Cmax and/or mean AUCo-iast) than what was expected based on standard animal models. In some embodiments, the present disclosure demonstrates that an antifibrotic agent (e.g., Compound 1 described herein) is suitable for administration once daily and/or twice daily. In some embodiments, the present disclosure demonstrates that an antifibrotic agent (e.g., Compound 1 described herein) is well-tolerated, e.g., in healthy subjects, e.g., at doses suitable to achieve one or more particular effects (e.g., certain PK parameters). In some embodiments, the present disclosure demonstrates that administration of an antifibrotic agent (e.g., Compound 1 described herein) achieves one or more particular effects (e.g., higher mean Cmax and/or comparable mean AUCo-iast) when administered to subjects in one fed/fasted state, compared to subjects in another fed/fasted state. In addition, for example, the present disclosure demonstrates that administration of an antifibrotic agent (e.g., Compound 1 described herein) achieves different effects (e.g., outcomes indicative of reduced fibrosis) in animal models, compared to relevant references, such as compared to controls and/or other therapies.
[0006] In some embodiments, the present disclosure encompasses the recognition that one or more properties of an antifibrotic agent (e.g., Compound 1 described herein) make it particularly suitable for treating subjects with certain fibrotic diseases or disorders. For example, in some embodiments, when treating a subject suffering from a disease with minimal symptoms and/or with minimal impact on daily life, a therapeutic agent with high tolerability is desirable. In some embodiments, the present disclosure demonstrates that Compound 1 may be suitable to treat such subjects, e.g., as indicated by one or more properties described herein.
[0007] The present disclosure also provides insight that certain subjects or populations of subjects can be selected for therapy with an antifibrotic agent (e.g., Compound 1 as described herein) based on certain markers and/or characteristics. In some embodiments, a subject or population of subjects is selected based on risk factors of (e.g., susceptibility to) fibrosis and/or diseases, disorders, or conditions associated with fibrosis. In some embodiments, a subject or population of subjects is selected based on one or more biomarkers as described herein. BRIEF DESCRIPTION OF THE DRAWINGS [0008] FIG. 1 is a graph of mean Compound 1 blood concentration over time from SAD cohorts in a fasted state.
[0009] FIG. 2 is a graph of mean Compound 1 plasma concentration over time in SAD food effect cohorts receiving 200 mg Compound 1.
[0010] FIG. 3 depicts a Western analysis demonstrating inhibition of phosphorylation of PDGFRp at tyrosine 751 by Compound 1 in serum-starved hepatic stellate cells.
[0011] FIG. 4 depicts a Western analysis demonstrating inhibition of phosphorylation of VEGFR2 at tyrosine 1175 by Compound 1 in serum-starved HUVEC cells.
[0012] FIG. 5 depicts results of a KINOMEscan profile of Compound 1 in small airway epithelial cells and lung fibroblasts (SAEMyF); lung fibroblasts only (MyoF); and renal proximal tubular epithelial cells and lung fibroblasts (REMyoF).
[0013] FIG. 6 is a graph showing soluble collagen concentrations in normal human lung fibroblasts (NHLF) treated with TGFpi and Compound 1.
[0014] FIG. 7A is a graph showing lung-to-body weight ratio of mice treated with Compound 1 in a bleomycin-induced injury model of idiopathic pulmonary fibrosis (IPF). FIG. 7B is a graph showing hydroxyproline levels in mice treated with Compound 1 in a bleomycin- induced injury model of IPF. FIG. 7C is a graph showing histopathological damage in lung tissue sections using the Ashcroft scale from mice treated with Compound 1 in a bleomycin- induced injury model of IPF. FIG. 7D is a graph showing extent of picrosirius red staining in lung tissue sections from mice treated with Compound 1 in a bleomycin-induced injury model of IPF. FIG. 7E is a graph showing extent of immunohistological staining for TGFpi in lung tissue sections from mice treated with Compound 1 in a bleomycin-induced injury model of IPF.
[0015] FIG. 8A is a graph showing lung fibrosis score using Ashcroft scale based on H&E staining in lung tissue sections from mice treated with Compound 1 in an inducible TGFpi mouse model of lung fibrosis. FIG. 8B is a graph hydroxyproline levels in mice treated with Compound 1 in an inducible TGFpi mouse model of lung fibrosis. FIG. 8C is a graph showing extent of picrosirius red staining in lung tissue sections from mice treated with Compound 1 in an inducible TGFpi mouse model of lung fibrosis. FIG. 8D is a graph showing extent of aSMA staining in lung tissue sections from mice treated with Compound 1 in an inducible TGFpi mouse model of lung fibrosis. [0016] FIG. 9A is a graph showing dermal thickness, measured as the distance between epidermal-dermal junction and dermal-subcutaneous fat junction in H&E-stained slides prepared from a skin biopsy from mice treated with Compound 1 in a bleomycin systemic sclerosis mouse model. FIG. 9B is a graph showing skin fibrotic score, judged based on H&E-stained slides prepared from a skin biopsy from mice treated with Compound 1 in a bleomycin systemic sclerosis mouse model. FIG. 9C is a graph showing lung hydroxyproline levels from mice treated with Compound 1 in a bleomycin systemic sclerosis mouse model. FIG. 9D is a graph showing lung fibrosis score using Ashcroft scale based on H&E staining in lung tissue sections from mice treated with Compound 1 in a bleomycin systemic sclerosis mouse model. FIG. 9E is a graph showing kidney hydroxyproline levels from mice treated with Compound 1 in a bleomycin systemic sclerosis mouse model. FIG. 9F is a graph showing extent of picrosirius red staining in kidney tissue sections from mice treated with Compound 1 in a bleomycin systemic sclerosis mouse model.
[0017] FIG. 10A is a graph showing urine protein levels in rats treated with Compound 1 in a FSGS-relevant model of PAN-induced proteinuria. FIG. 10B is a graph showing intraperitoneal fluid volume in rats treated with Compound 1 in a FSGS-relevant model of PAN- induced proteinuria. FIG. IOC is a graph showing GFR, measured using FITC-sinistrin decay kinetics, in rats treated with Compound 1 in a FSGS-relevant model of PAN-induced proteinuria. FIG. 10D is a graph showing glomerular diameter, measured using histopathological analysis of P AS-stained renal coronal sections from rats treated with Compound 1 in a FSGS-relevant model of PAN-induced proteinuria. FIG. 10E is a graph showing collagen-3 expression, determined in glomeruli using quantitative immunohistochemistry, in rats treated with Compound 1 in a FSGS- relevant model of PAN-induced proteinuria.
[0018] FIG. 11A is a graph showing urine protein levels in rats treated with Compound 1 in a DOCA/salt model of renal fibrosis and injury. FIG. 1 IB is a graph showing urine albumin levels in rats treated with Compound 1 in a DOCA/salt model of renal fibrosis and injury. FIG. llC is a graph showing urine albumin to creatinine ratio in rats treated with Compound 1 in a DOCA/salt model of renal fibrosis and injury. FIG. 1 ID is a graph showing urine kidney injury molecule-1 (KIM1) levels in rats treated with Compound 1 in a DOCA/salt model of renal fibrosis and injury. FIG. 11E is a graph showing renal hydroxyproline levels in rats treated with Compound 1 in a DOCA/salt model of renal fibrosis and injury. FIG. 1 IF is a graph showing renal damage scores, based on H&E stained slides of kidney tissue sections from rats treated with Compound 1 in a DOCA/salt model of renal fibrosis and injury. FIG. 11G is a graph showing extent of picrosirius red staining from slides of kidney tissue sections from rats treated with Compound 1 in a DOCA/salt model of renal fibrosis and injury. FIG. 11H is a graph showing extent of aSMA staining in kidney tissue sections from rats treated with Compound 1 in a DOCA/salt model of renal fibrosis and injury.
[0019] FIG. 12A is a graph showing renal damage scores, based on H&E stained slides of kidney tissue sections from mice treated with Compound 1 in a unilateral ureteral obstruction (UUO) model of renal fibrosis (*** = p <0.001). FIG. 12B is a graph showing extent of picrosirius red staining from slides of kidney tissue sections from mice treated with Compound 1 in a unilateral ureteral obstruction (EIUO) model of renal fibrosis (*** = p <0.001; ns = not significant). FIG. 12C is a graph showing extent of aSMA staining in kidney tissue sections from mice treated with Compound 1 in a unilateral ureteral obstruction (EIUO) model of renal fibrosis (*** = p <0.001; ** = p <0.01; ns = not significant).
[0020] FIG. 13A is a graph showing serum creatinine (SCr) levels in rats treated with Compound 1 in a PCK rat model of polycystic kidney disease. FIG. 13B is a graph showing BUN levels in rats treated with Compound 1 in a PCK rat model of polycystic kidney disease. FIG. 13C is a graph showing kidney weight at sacrifice in rats treated with Compound 1 in a PCK rat model of polycystic kidney disease. FIG. 13D is a graph showing kidney weight as a percentage of body weight at sacrifice rats treated with Compound 1 in a PCK rat model of polycystic kidney disease. FIG. 13E is a graph showing kidney hydroxyproline levels in rats treated with Compound 1 in a PCK rat model of polycystic kidney disease. FIG. 13F is a graph showing cystic index, quantified in H&E stained kidney tissue sections using digital planimetry, in rats treated with Compound 1 in a PCK rat model of polycystic kidney disease. FIG. 13G is a graph showing 24-hour urine volume in rats treated with Compound 1 in a PCK rat model of polycystic kidney disease. FIG. 13H is a graph showing urine protein levels in rats treated with Compound 1 in a PCK rat model of polycystic kidney disease. FIG. 131 is a graph showing urine albumin levels in rats treated with Compound 1 in a PCK rat model of polycystic kidney disease. FIG. 13J is a graph showing urine neutrophil gelatinase-associated lipocalin (NGAL) levels in rats treated with Compound 1 in a PCK rat model of polycystic kidney disease. FIG. 13K is a graph showing urine KIM1 levels in rats treated with Compound 1 in a PCK rat model of polycystic kidney disease. FIG. 13L is a graph showing urine interleukin 18 (IL18) levels in rats treated with Compound 1 in a PCK rat model of polycystic kidney disease. FIG. 13M is a graph showing urine cystatin C levels in in rats treated with Compound 1 in a PCK rat model of polycystic kidney disease.
[0021] FIG. 14A is a graph showing body weight after sacrifice in mice treated with Compound 1 in a TNBS model of inflammatory bowel disease/acute colitis. FIG. 14B is a graph showing colon weight after sacrifice in mice treated with Compound 1 in a TNBS model of inflammatory bowel disease/acute colitis. FIG. 14C is a graph showing colon length after sacrifice in mice treated with Compound 1 in a TNBS model of inflammatory bowel disease/acute colitis. FIG. 14D is a graph showing colon damage score in mice treated with Compound 1 in a TNBS model of inflammatory bowel disease/acute colitis. FIG. 14E is a graph showing colon histopathological scores of H&E-stained colon tissue sections from mice treated with Compound 1 in a TNBS model of inflammatory bowel disease/acute colitis. Each of (a) colon architecture, (b) degree of inflammatory cell infiltration, (c) muscle thickening, and (d) crypt damage and goblet cells loss was assessed. FIG. 14F is a graph showing composite colon histopathological scores of H&E-stained colon tissue sections from mice treated with Compound 1 in a TNBS model of inflammatory bowel disease/acute colitis. FIG. 14G is a graph showing extent of Alcian blue staining in slides of colon tissue sections from mice treated with Compound 1 in a TNBS model of inflammatory bowel disease/acute colitis. FIG. 14H is a graph showing extent of myeloperoxidase staining in slides of colon tissue sections from mice treated with Compound 1 in a TNBS model of inflammatory bowel disease/acute colitis. FIG. 141 is a graph showing extent of F4/80 staining in slides of colon tissue sections from mice treated with Compound 1 in a TNBS model of inflammatory bowel disease/acute colitis.
[0022] FIG. 15A is a graph showing body weight in mice prior to treatment with Compound 1 in an acetic acid induced colitis model. FIG. 15B is a graph showing colon weight in mice prior to treatment with Compound 1 in an acetic acid induced colitis model. FIG. 15C is a graph showing colon length in mice prior to treatment with Compound 1 in an acetic acid induced colitis model. FIG. 15D is a graph showing gross morphological colon damage score in mice prior to treatment with Compound 1 in an acetic acid induced colitis model. FIG. 15E is a graph showing colon length in mice treated with Compound 1 in an acetic acid induced colitis model. FIG. 15F is a graph showing gross morphological colon damage score in mice treated with Compound 1 in an acetic acid induced colitis model. FIG. 15G is a graph showing histopathological colon damage score in mice treated with Compound 1 in an acetic acid induced colitis model. FIG. 15H is a graph showing extent of Alcian blue staining in slides of colon tissue sections from mice treated with Compound 1 in an acetic acid induced colitis model.
[0023] FIG. 16A is a graph showing colon length in mice treated with Compound 1 in a dextran sulfate sodium model of chronic colitis in inflammatory bowel disease. FIG. 16B is a graph showing macroscopic colon damage score in mice treated with Compound 1 in a dextran sulfate sodium model of chronic colitis in inflammatory bowel disease. FIG. 16C is a graph showing colon hydroxyproline levels in mice treated with Compound 1 in a dextran sulfate sodium model of chronic colitis in inflammatory bowel disease. FIG. 16D is a graph showing histopathological injury score from H&E-stained slides of colon tissue section from mice treated with Compound 1 in a dextran sulfate sodium model of chronic colitis in inflammatory bowel disease.
[0024] FIG. 17A is a graph showing lung hydroxyproline levels in mice treated with Compound 1 or pirfenidone (PFD) in a bleomycin-induce lung fibrosis model. FIG. 17B is a graph showing Ashcroft lung histopathological score in lung tissue sections from mice treated with Compound 1 or pirfenidone (PFD) in a bleomycin-induce lung fibrosis model. FIG. 17C is a graph showing extent of picrosirius red staining in lung tissue sections from mice treated with Compound 1 or pirfenidone (PFD) in a bleomycin-induce lung fibrosis model. FIG. 17D is a graph showing extent of TGFpi staining in lung tissue sections from mice treated with Compound 1 or pirfenidone (PFD) in a bleomycin-induce lung fibrosis model.
[0025] FIG. 18 provides a cartoon schematic of varying relationships between genomic responses and kidney disease outcomes.
[0026] FIG. 19 provides proteinuria of PAN treated model rats treated with Compound 1.
[0027] FIG. 20 provides fold change in expression for COL1 A1 mRNA rat PAN model with treatment of Compound 1.
[0028] FIG. 21 provides fold change in expression for COL3 A1 mRNA rat PAN model with treatment of Compound 1.
[0029] FIG. 22 depicts a Compound 1 interactome built using in vitro activity data.
[0030] FIG. 23 provides urine protein levels in PAN treated model rats. [0031] FIG. 24A provides COL3 A1 mRNA levels in PAN-treated model rats. FIG. 24B provides an association of COL3 A1 mRNA levels and urine protein values.
[0032] FIG. 25A depicts a glomerular COL3 A1 interactome. FIG. 25B depicts a tubular COL3A1 interactome.
[0033] FIG. 26A is a graph showing that, prior to randomization, proteinuria of Alport mice is elevated compared to wild-type mice. FIG. 26B is a Kaplan Myer survival graph of Alport mice treated with Compound 1 vs. Alport mice treated with vehicle. FIG. 26C and FIG. 26D are graphs of body weight and kidney weight, respectively, for mice in an Alport model study. FIG. 26E is a graph of proteinuria in Alport mice treated with Compound 1 vs. Alport mice treated with vehicle. FIG. 26F is a graph of protein-to-creatinine ratio in Alport mice treated with Compound 1 vs. Alport mice treated with vehicle. FIG. 26G is a graph of serum BUN levels in Alport mice treated with Compound 1 vs. Alport mice treated with vehicle. FIG. 26H is a graph of serum creatinine levels in Alport mice treated with Compound 1 vs. Alport mice treated with vehicle. FIG. 261 is a graph of kidney hydroxyproline levels in Alport mice treated with Compound 1 vs. Alport mice treated with vehicle. FIG. 26 J is a graph of kidney injury scores based on H&E stained kidney samples in Alport mice treated with Compound 1 vs. Alport mice treated with vehicle. FIG. 26K is a graph of renal fibrosis scores based on Masson’s Trichome- stained kidney samples in Alport mice treated with Compound 1 vs. Alport mice treated with vehicle. FIG. 26L is a graph of IHC staining for TGFpi in Alport mice treated with Compound 1 vs. Alport mice treated with vehicle. FIG. 26M is a graph of IHC staining for aSMA in Alport mice treated with Compound 1 vs. Alport mice treated with vehicle. FIG. 26N is a graph of IHC staining for Collagen- 1 in Alport mice treated with Compound 1 vs. Alport mice treated with vehicle. FIG. 260 is a graph of picrosirius red staining for collagen in Alport mice treated with Compound 1 vs. Alport mice treated with vehicle. FIG. 26P depicts a Western blot analysis for collagen 1, aSMA, TGFpi, and PDGFR in kidney tissue samples from sacrificed Alport mice. FIG. 26Q is a graph of densitometric analysis of a Western blot for collagen 1, aSMA, TGFpi, and PDGFR in Alport mice treated with Compound 1 vs. Alport mice treated with vehicle.
[0034] FIG. 27A is a Kaplan Meier curve showing survival analysis of rats in a Passive Heymann nephritis model. FIG. 27B is a graph of body mass of rats in a Passive Heymann nephritis model. FIG. 27C is a graph of kidney mass of rats in a Passive Heymann nephritis model. FIG. 27D is a graph of kidney mass to body mass ratio of rats in a Passive Heymann nephritis model. FIG. 27E is a graph of protein-to-creatinine ratio (PCR) of rats in a Passive Heymann nephritis model. FIG. 27F is a graph plotting PCR of rats in a Passive Heymann nephritis model from Day 7 until end of study. FIG. 27G is a graph of serum cholesterol of rats in a Passive Heymann nephritis model. FIG. 27H is a graph of serum triglycerides of rats in a Passive Heymann nephritis model. FIG. 271 is a graph of serum creatinine of rats in a Passive Heymann nephritis model. FIG. 27J is a graph of BUN of rats in a Passive Heymann nephritis model. FIG. 27K is a graph of hydroxyproline content per kidney in rats in a Passive Heymann nephritis model. FIG. 27L depicts representative Trichrome-stained kidney tissue samples from rats in a Passive Heymann nephritis model. FIG. 27M depicts representative PAS-stained kidney tissue samples from rats in a Passive Heymann nephritis model. FIG. 27N is a graph of glomerular damage score in rats in a Passive Heymann nephritis model. FIG. 270 is a Western blot analysis on total kidney lysates from rats in a Passive Heymann nephritis model. FIG. 27P is a graph of PDGFRP levels, normalized to GAPDH, from rats in a Passive Heymann nephritis model.
[0035] FIG. 28 summarizes pre-screening and screening processes for a Phase 2,
Multicenter, Double-Blind, Randomized, Placebo-controlled Study of Safety and Efficacy of Compound 1 in Patients with Primary Glomerular Disease and Persistent Proteinuria.
[0036] FIG. 29 is a graph of mean oral blood PK profile of Compound 1 over time from SAD cohorts in a fasted state.
[0037] FIG. 30A is a graph of mean oral blood PK profile of Compound 1 over time from BID MAD cohorts in a fasted state at Day 1. FIG. 30B is a graph of mean oral blood PK profile of Compound 1 over time from BID MAD cohorts in a fasted state at Day 14.
[0038] FIG. 31 is a graph of mean Compound 1 blood concentration vs. time profiles at steady state for QD MAD cohorts in the fed state at Day 14.
[0039] FIG. 32 is a graph of Compound 1 blood concentration vs. time in a food effect cross-over study with a 600 mg single dose.
DETAILED DESCRIPTION
Definitions
[0040] The term “about”, when used herein in reference to a value, refers to a value that is similar, in context to the referenced value. In general, those skilled in the art, familiar with the context, will appreciate the relevant degree of variance encompassed by “about” in that context. For example, in some embodiments, the term “about” may encompass a range of values that is within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referred value.
[0041] As used herein, the term “administering” or “administration” typically refers to administration of a composition to a subject to achieve delivery of an active agent to a site of interest (e.g., a target site which may, in some embodiments, be a site of disease or damage, and/or a site of responsive processes, cells, tissues, etc.) As will be understood by those skilled in the art, reading the present disclosure, in some embodiments, one or more particular routes of administration may be feasible and/or useful in the practice of the present disclosure. For example, in some embodiments, administration may be parenteral. In some embodiments, administration may be oral. In some embodiments, administration may involve only a single dose. In some embodiments, administration may involve application of a fixed number of doses. In some embodiments, administration may involve dosing that is intermittent (e.g., a plurality of doses separated in time) and/or periodic (e.g., individual doses separated by a common period of time) dosing.
[0042] As used herein, the term “comparable” refers to two or more agents, entities, situations, sets of conditions, circumstances, individuals, or populations, etc. that may not be identical to one another but that are sufficiently similar to permit comparison there between so that one skilled in the art will appreciate that conclusions may reasonably be drawn based on differences or similarities observed. In some embodiments, comparable agents, entities, situations, sets of conditions, circumstances, individuals, or populations are characterized by a plurality of substantially identical features and one or a small number of varied features. Those of ordinary skill in the art will understand, in context, what degree of identity is required in any given circumstance for two or more such agents, entities, situations, sets of conditions, circumstances, individuals, or populations, etc. to be considered comparable. For example, those of ordinary skill in the art will appreciate that sets of circumstances, agents, entities, situations, individuals, or populations are comparable to one another when characterized by a sufficient number and type of substantially identical features to warrant a reasonable conclusion that differences in results obtained or phenomena observed under or with different agents, entities, situations, sets of circumstances, individuals, or populations are caused by or indicative of the variation in those features that are varied.
[0043] Those skilled in the art will appreciate that the term “dosage form” may be used to refer to a physically discrete unit of an active agent (e.g., a therapeutic agent) for administration to a subject. Typically, each such unit contains a predetermined quantity of active agent. In some embodiments, such quantity is a unit dosage amount (or a whole fraction thereof) appropriate for administration in accordance with a dosing regimen that has been determined to correlate with a desired or beneficial outcome when administered to a relevant population (i.e., with a therapeutic dosing regimen). Those of ordinary skill in the art appreciate that the total amount of a therapeutic composition or agent administered to a particular subject is determined by one or more attending physicians and may involve administration of multiple dosage forms. [0044] The term “pharmaceutically acceptable salt form,” as used herein, refers to a form of a relevant compound as a salt appropriate for use in pharmaceutical contexts, i.e., salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and/or lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge, et al. describes pharmaceutically acceptable salts in detail in ./. Pharmaceutical Sciences , 66: 1-19 (1977).
[0045] As used herein, the term “reference” describes a standard or control relative to which a comparison is performed. For example, in some embodiments, an agent, individual, population, sample, sequence or value of interest is compared with a reference or control agent, individual, population, sample, sequence or value. In some embodiments, a reference or control is tested and/or determined substantially simultaneously with the testing or determination of interest. In some embodiments, a reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, as would be understood by those skilled in the art, a reference or control is determined or characterized under comparable conditions or circumstances to those under assessment. Those skilled in the art will appreciate when sufficient similarities are present to justify reliance on and/or comparison to a particular possible reference or control.
[0046] As will be understood from context, “risk” of a disease, disorder, and/or condition refers to a likelihood that a particular individual will develop the disease, disorder, and/or condition. In some embodiments, risk is expressed as a percentage. In some embodiments, risk is from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 up to 100%. In some embodiments risk is expressed as a risk relative to a risk associated with a reference sample or group of reference samples. In some embodiments, a reference sample or group of reference samples have a known risk of a disease, disorder, condition and/or event. In some embodiments a reference sample or group of reference samples are from individuals comparable to a particular individual. In some embodiments, relative risk is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.
[0047] As used herein, the term “subject” refers an organism, typically a mammal (e.g., a human). In some embodiments, a subject is suffering from a relevant disease, disorder or condition. In some embodiments, a human subject is an adult, adolescent, or pediatric subject.
In some embodiments, a subject is at risk of (e.g., susceptible to), e.g., at elevated risk of relative to an appropriate control individual or population thereof, a disease, disorder, or condition. In some embodiments, a subject displays one or more symptoms or characteristics of a disease, disorder or condition. In some embodiments, a subject does not display any symptom or characteristic of a disease, disorder, or condition. In some embodiments, a subject is someone with one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition. In some embodiments, a subject is an individual to whom diagnosis and/or therapy and/or prophylaxis is and/or has been administered. The terms “subject” and “patient” are used interchangeably herein.
[0048] As used herein, the term “treat” (also “treatment” or “treating”) refers to any administration of a therapy that partially or completely alleviates, ameliorates, relieves, inhibits, delays onset of, reduces severity of, and/or reduces incidence of one or more symptoms, features, and/or causes of a particular disease, disorder, and/or condition. In some embodiments, such treatment may be of a subject who does not exhibit signs of the relevant disease, disorder and/or condition and/or of a subject who exhibits only early signs of the disease, disorder, and/or condition. Alternatively or additionally, such treatment may be of a subject who exhibits one or more established signs of the relevant disease, disorder and/or condition. In some embodiments, treatment may be of a subject who has been diagnosed as suffering from the relevant disease, disorder, and/or condition.
[0049] References cited herein are hereby incorporated by reference in their entirety. Compound 1
[0050] PCT Application No. PCT/US2013/023324, filed January 26, 2013 and published as WO 2013/112959 on August 1, 2013, the entirety of which is hereby incorporated by reference, describes certain antifibrotic compounds. Such compounds include Compound 1:
Figure imgf000014_0001
1
Compound 1 is in a pharmacological class of tyrosine kinase inhibitors (TKI). Compound 1 is an orally bioavailable small molecule dual kinase inhibitor of platelet-derived growth factor receptors (PDGFR) and vascular endothelial growth factor receptors (VEGFR2). Compound 1 is useful in methods provided herein.
[0051] Synthesis of Compound 1, i.e., methyl (Z)-3-(((4-(N-methyl-2-(4-methylpiperazin-l- yl)acetamido)phenyl)amino)(phenyl)methylene)-2-oxo-2,3-dihydro-lH-pyrrolo[2,3-b]pyridine- 6-carboxylate, is described in detail in Example 1 of WO 2013/112959, as well as in Example 1 herein.
[0052] In some embodiments, Compound 1 is provided and/or utilized (e.g., for inclusion in, e.g., during one or more steps of manufacturing of, a composition and/or for delivery to a subject) in accordance with the present disclosure in a form such as a salt form (e.g., a pharmaceutically acceptable salt form). As already noted herein, pharmaceutically acceptable salt forms are well known in the art. In some embodiments, Compound 1 is provided and/or utilized in a hydrochloride salt form, a maleate salt form, a mesylate salt form, or a tosylate salt form.
[0053] In some embodiments, Compound 1 is provided and/or utilized (e.g., for inclusion in, e.g., during one or more steps of manufacturing of, a composition and/or for delivery to a subject) in accordance with the present disclosure in a form such as a solid form. In some embodiments, Compound 1 is provided and/or utilized in accordance with the present disclosure in an amorphous solid form, in a crystalline solid form, or in a mixture thereof. In some embodiments, a crystalline solid form may be or comprise a solvate, hydrate, or an unsolvated form. The use of any and all such forms are contemplated by the present disclosure.
[0054] In some embodiments, Compound 1 is provided and/or utilized (e.g., for inclusion in, e.g., during one or more steps of manufacturing of, a composition and/or for delivery to a subject) as a hydrochloride salt form (e.g., a channel hydrate comprising up to about 4 equivalents of water, up to about 3 equivalents of water, up to about 2 equivalents of water, or up to about 1 equivalent of water). In some embodiments, such a hydrochloride salt form of Compound 1 is characterized by one or more peaks in its XRPD selected from those at about one or more peaks in its XRPD selected from those at about 5.28, 10.63, 11.54, 17.05, and 20.98 degrees 2-theta. In some embodiments, such a hydrochloride salt form of Compound 1 is characterized by two or more peaks in its XRPD selected from those at about one or more peaks in its XRPD selected from those at about 5.28, 10.63, 11.54, 17.05, and 20.98 degrees 2-theta. In some embodiments, such a hydrochloride salt form of Compound 1 is characterized by one or more peaks in its XRPD selected from those at about three or more peaks in its XRPD selected from those at about 5.28, 10.63, 11.54, 17.05, and 20.98 degrees 2-theta. In some embodiments, such a hydrochloride salt form of Compound 1 is characterized by the following peaks in its XRPD pattern at about 5.28, 10.63, 11.54, 17.05, and 20.98 degrees 2-theta. In some embodiments, such a hydrochloride salt form of Compound 1 is characterized by peaks in its XRPD pattern at about 5.28, 10.63, 11.54, 17.05, and 20.98 degrees 2-theta, corresponding to d- spacing of about 16.74, 8.33, 7.67, 5.20, and 4.23 angstroms, respectively. In some embodiments, such a hydrochloride salt form of Compound 1 is characterized by substantially all of the peaks in its XRPD pattern selected from 5.28, 5.76, 10.63, 11.54, 12.73, 13.13, 14.08, 15.34, 15.64, 16.00, 16.55, 17.05, 17.78, 18.86, 19.08, 20.16, 20.68, 20.98, 21.62, 22.05, 22.82, 23.97, 24.94, 25.23, 25.61, 26.58, 27.01, 27.78, 29.89, 30.51, 30.91, 31.72, and 33.39 degrees 2- theta.
[0055] Unless otherwise indicated, as used herein “Compound 1” refers to Compound 1 (i.e., methyl (Z)-3-(((4-(N-methyl-2-(4-methylpiperazin-l- yl)acetamido)phenyl)amino)(phenyl)methylene)-2-oxo-2,3-dihydro-lH-pyrrolo[2,3-b]pyridine- 6-carboxylate) in any available form, such as, e.g., a salt form and/or solid form. It will be understood, therefore, that reference to an amount (e.g., in mg) of Compound 1 means the amount of Compound 1 in free base form. Accordingly, Compound 1 may be provided and/or utilized as, e.g., a salt form of Compound 1 such that the amount of the salt (or other form) is an amount that corresponds to the “free base equivalent” of Compound 1. For example, “50 mg Compound 1” means, e.g., approx. 53.4 mg of Compound 1 Hydrochloride anhydrate, approx. 58.4 mg of Compound 1 Hydrochloride trihydrate, and approx. 58.9 mg of Compound 1 Mesylate anhydrate, etc.
Provided Methods
[0056] Provided herein are methods of treating a subject or a population of subjects comprising administering Compound 1 (e.g., by administering a composition that comprises and/or delivers Compound 1 as described herein) to the subject(s) in need thereof. In some embodiments, such administering is achieved by administering a composition that delivers Compound 1 (e.g., in some embodiments, a composition that is or comprises Compound 1, or a composition that otherwise delivers Compound 1 - e.g., that is or comprises a prodrug of Compound 1, a complex or other entity that releases Compound 1 upon administration, etc.). [0057] In some embodiments, the present disclosure provides methods of administering Compound 1 to a subject or population of subjects, according to a regimen established to achieve one or more particular effects. In some embodiments, a regimen has been established to achieve one or more particular effects, relative to that observed for a comparable reference population that has not received Compound 1 (e.g., that has received a reference composition which does not deliver Compound 1). In some embodiments, a regimen has been established to achieve one or more particular effects in a population of healthy subjects. In some embodiments, a regimen has been established to achieve one or more particular effects in a population of healthy subjects, relative to that observed for a comparable reference population that has not received Compound 1 (e.g., that has received a reference composition which does not deliver Compound 1).
[0058] In some embodiments, certain parameters may be evaluated to determine if a particular effect is achieved. For example, in some embodiments, certain measures of, e.g., TEAEs, Cmax, Tmax, AUCo-iast, AUCo-inf, Vz/F, CL/F, and/or ti/2, can be obtained. Additionally, in some embodiments, certain measure of, e.g., Tiast, AUCo-12, AUCo-24, C12, C24, Ctrough, RaccCmax, RaccAUC, CLss/F, and/or Vzss/F, etc. can be obtained. Any one or more of parameters such as these may, in some embodiments, be useful for determining, e.g., efficacy and/or safety of Compound 1. [0059] In some embodiments, in methods provided herein, a reference population has not received a composition providing Compound 1. In some embodiments, in methods provided herein, a reference population has received an otherwise comparable reference composition that does not provide Compound 1 (e.g., a placebo and/or another therapy). A placebo as used herein is a dosage form that matches that of an active study compound, but does not deliver the active study compound (e.g., Compound 1). For example, a placebo can be a capsule that visually matches an active study drug and is composed of the same capsule shell but is filled with a pharmaceutical excipient (and lacking the active study drug), e.g., silicified microcrystalline cellulose. In some embodiments, in methods provided herein, a reference composition may be or may have been administered at the same intervals and/or in the same amounts as a composition providing Compound 1.
[0060] In some embodiments, a composition providing Compound 1 is administered according to a regimen established to achieve a particular effect, e.g., at a particular time point.
In some embodiments, a composition providing Compound 1 is administered according to a regimen established to achieve a particular effect, e.g., at a particular time point, relative to an appropriate reference as described herein (e.g., as is observed in a comparable population who has not received a composition providing Compound 1).
[0061] In some embodiments, the present disclosure provides methods of administering to a subject or population of subjects in need thereof an amount of Compound 1, wherein said amount, when administered as a single oral dose to a population of healthy subjects (e.g., healthy subjects in a fed or fasted state), was established to achieve one or more particular effects.
[0062] In some embodiments, a particular effect is or comprises a particular mean maximum concentration (Cmax). In some embodiments, a mean Cmax is measured in a population of healthy subjects (e.g., healthy subjects in a fed or fasted state) who received a single oral dose of Compound 1. In some embodiments, a mean Cmax is measured in a population of healthy subjects (e.g., healthy subjects in a fed or fasted state) who received multiple oral doses of Compound 1 (e.g., once daily (QD) or twice daily (BID) for, e.g., 14 days). In some embodiments, a particular effect is or comprises a mean Cmax of from about 30 ng/mL to about 800 ng/mL, from about 85 ng/mL to about 800 ng/mL, from about 230 ng/mL to about 800 ng/mL, from about 400 ng/mL to about 800 ng/mL, from about 30 ng/mL to about 400 ng/mL, or from about 85 ng/mL to about 400 ng/mL. In some embodiments, a particular effect is or comprises a mean Cmax of less than about 800 ng/mL, less than about 400 ng/mL, less than about 235 ng/mL, or less than about 90 ng/mL. In some embodiments, a particular effect is or comprises a mean Cmax of greater than about 30 ng/mL, greater than about 85 ng/mL, greater than about 230 ng/mL, or greater than about 400 ng/mL.
[0063] In some embodiments, a particular effect is or comprises a particular median time to maximum concentration (Tmax). In some embodiments, a median Tmax is measured in a population of healthy subjects (e.g., healthy subjects in a fed or fasted state) who received a single oral dose of Compound 1. In some embodiments, a median Tmax is measured in a population of healthy subjects (e.g., healthy subjects in a fed or fasted state) who received multiple oral doses of Compound 1 (e.g., once daily (QD) or twice daily (BID) for, e.g., 14 days). In some embodiments, a particular effect is or comprises a median Tmax of from about 1 hour to about 4 hours, from about 1 hour to about 2 hours, or from about 1 hour to about 1.5 hours. In some embodiments, a particular effect is or comprises a median Tmax of from about 0.5 hours to about 4 hours, from about 1 hour to about 4 hours, from about 1 hour to about 2 hours, or from about 1 hour to about 1.5 hours. In some embodiments, a particular effect is or comprises a median Tmax of less than about 4 hours, less than about 2 hours, or less than about 1.5 hours. In some embodiments, a particular effect is or comprises a median Tmax of greater than about 1 hour, greater than about 1.5 hours, or greater than about 2 hours.
[0064] In some embodiments, a particular effect is or comprises a particular mean area under drug concentration-time curve (AUCo-iast). In some embodiments, a mean AUCo-iast is measured in a population of healthy subjects (e.g., healthy subjects in a fed or fasted state) who received a single oral dose of Compound 1. In some embodiments, a particular effect is or comprises a mean AUCo-iast of from about 60 ng*h/mL to about 4000 ng*h/mL, from about 60 ng*h/mL to about 2200 ng*h/mL, from about 170 ng*h/mL to about 2200 ng*h/mL, from about 400 ng*h/mL to about 2200 ng*h/mL, from about 1260 ng*h/mL to about 2200 ng*h/mL, from about 60 ng*h/mL to about 1260 ng*h/mL, or from about 170 ng*h/mL to about 1260 ng*h/mL. In some embodiments, a particular effect is or comprises a mean AUCo-iast of from about 100 ng*h/mL to about 4000 ng*h/mL, from about 1000 ng*h/mL to about 2400 ng*h/mL, from about 180 ng*h/mL to about 2400 ng*h/mL, from about 430 ng*h/mL to about 2400 ng*h/mL, from about 1260 ng*h/mL to about 2400 ng*h/mL, from about 100 ng*h/mL to about 1260 ng*h/mL, or from about 180 ng*h/mL to about 1260 ng*h/mL. In some embodiments, a particular effect is or comprises a mean AUCo-iast of less than about 4000 ng*h/mL, less than about 2200 ng*h/mL, less than about 1270 ng*h/mL, less than about 420 ng*h/mL, or less than about 180 ng*h/mL. In some embodiments, a particular effect is or comprises a mean AUCo-iast of less than about 4000 ng*h/mL, less than about 2400 ng*h/mL, less than about 1270 ng*h/mL, less than about 440 ng*h/mL, or less than about 190 ng*h/mL. In some embodiments, a particular effect is or comprises a mean AUCo-iast of greater than about 60 ng*h/mL, greater than about 170 ng*h/mL, greater than about 400 ng*h/mL, or greater than about 1260 ng*h/mL.
[0065] In some embodiments, a particular effect is or comprises a particular mean area under drug concentration-time curve (AUCo-inf). In some embodiments, a mean AUCo-inf is measured in a population of healthy subjects (e.g., healthy subjects in a fed or fasted state) who received a single oral dose of Compound 1. In some embodiments, a particular effect is or comprises a mean AUCo-inf of from about 60 ng*h/mL to about 4000 ng*h/mL, from about 60 ng*h/mL to about 2410 ng*h/mL, from about 190 ng*h/mL to about 2410 ng*h/mL, from about 490 ng*h/mL to about 2410 ng*h/mL, from about 1300 ng*h/mL to about 2410 ng*h/mL, or from about 190 ng*h/mL to about 1300 ng*h/mL. In some embodiments, a particular effect is or comprises a mean AUCo-inf of from about 60 ng*h/mL to about 4000 ng*h/mL, from about 60 ng*h/mL to about 2580 ng*h/mL, from about 180 ng*h/mL to about 2580 ng*h/mL, from about 550 ng*h/mL to about 2580 ng*h/mL, from about 1300 ng*h/mL to about 2580 ng*h/mL, or from about 180 ng*h/mL to about 1300 ng*h/mL. In some embodiments, a particular effect is or comprises a mean AUCo-inf of less than about 4000 ng*h/mL, less than about 2410 ng*h/mL, less than about 1300 ng*h/mL, less than about 500 ng*h/mL, or less than about 200 ng*h/mL. In some embodiments, a particular effect is or comprises a mean AUCo-inf of less than about 4000 ng*h/mL, less than about 2580 ng*h/mL, less than about 1310 ng*h/mL, less than about 560 ng*h/mL, or less than about 190 ng*h/mL. In some embodiments, a particular effect is or comprises a mean AUCo-inf of greater than about 60 ng*h/mL, greater than about 190 ng*h/mL, greater than about 490 ng*h/mL, or greater than about 1300 ng*h/mL.
[0066] In some embodiments, a particular effect is or comprises a particular mean area under drug concentration-time curve (AUCo-12). In some embodiments, a mean AUCo-12 is measured in a population of healthy subjects (e.g., healthy subjects in a fed or fasted state) who received multiple oral doses of Compound 1 (e.g., once daily (QD) or twice daily (BID) for, e.g., 14 days). In some embodiments, a particular effect is or comprises a mean AUCo-12 of from about 100 ng*h/mL to about 1500 ng*h/mL, from about 120 ng*h/mL to about 1340 ng*h/mL, from about 250 ng*h/mL to about 1340 ng*h/mL, from about 920 ng*h/mL to about 1340 ng*h/mL, from about 120 ng*h/mL to about 930 ng*h/mL, or from about 250 ng*h/mL to about 930 ng*h/mL. In some embodiments, a particular effect is or comprises a mean AUCo-12 of less than about 1500 ng*h/mL, less than about 1300 ng*h/mL, less than about 930 ng*h/mL, or less than about 260 ng*h/mL. In some embodiments, a particular effect is or comprises a mean AUCo-12 of greater than about 115 ng*h/mL, greater than about 250 ng*h/mL, or greater than about 920 ng*h/mL.
[0067] In some embodiments, a particular effect is or comprises a particular mean apparent volume of distribution (Vz/F). In some embodiments, a mean Vz/F is measured in a population of healthy subjects (e.g., healthy subjects in a fed or fasted state) who received a single oral dose of Compound 1. In some embodiments, a particular effect is or comprises a mean Vz/F of from about 2000 L to about 5500 L, from about 2200 L to about 5500 L, or from about 2100 L to about 2300 L. In some embodiments, a particular effect is or comprises a mean Vz/F of from about 2000 L to about 7800 L, from about 2200 L to about 5500 L, or from about 5400 L to about 7800 L. In some embodiments, a particular effect is or comprises a mean Vz/F of less than about 5500 L, less than about 2300 L, or less than about 2200 L. In some embodiments, a particular effect is or comprises a mean Vz/F of less than about 7800L, less than about 5500 L, less than about 2300 L, or less than about 2200 L. In some embodiments, a particular effect is or comprises a mean Vz/F of greater than about 2100 L, greater than about 2200 L, or greater than about 5100 L.
[0068] In some embodiments, a particular effect is or comprises a particular mean apparent total clearance (CL/F). In some embodiments, a mean CL/F is measured in a population of healthy subjects (e.g., healthy subjects in a fed or fasted state) who received a single oral dose of Compound 1. In some embodiments, a particular effect is or comprises a mean CL/F of from about 300 L/h to about 600 L/h, from about 300 L/h to about 450 L/h, or from about 400 L/h to about 600 L/h. In some embodiments, a particular effect is or comprises a mean CL/F of from about 270 L/h to about 590 L/h, from about 270 L/h to about 370 L/h, or from about 340 L/h to about 590 L/h. In some embodiments, a particular effect is or comprises a mean CL/F of less than about 600 L/h, less than about 450 L/h, or less than about 350 L/h. In some embodiments, a particular effect is or comprises a mean CL/F of greater than about 300 L/h, greater than about 400 L/h, or greater than about 575 L/h. In some embodiments, a particular effect is or comprises a mean CL/F of greater than about 250 L/h, greater than about 340 L/h, or greater than about 370 L/h.
[0069] In some embodiments, a particular effect is or comprises a particular mean half-life (ti/2). In some embodiments, a mean ti/2 is measured in a population of healthy subjects (e.g., healthy subjects in a fed or fasted state) who received a single oral dose of Compound 1. In some embodiments, a mean ti/2 is measured in a population of healthy subjects (e.g., healthy subjects in a fed or fasted state) who received multiple oral doses of Compound 1 (e.g., once daily (QD) or twice daily (BID) for, e.g., 14 days). In some embodiments, a particular effect is or comprises a mean ti/2 of from about 2 hours to about 30 hours, from about 3 hours to about 30 hours, from about 10 hours to about 30 hours, from about 2 hours to about 11 hours, or from about 3 hours to about 11 hours. In some embodiments, a particular effect is or comprises a mean ti/2 of from about 12 hours to about 21 hours. In some embodiments, a particular effect is or comprises a mean ti/2 of less than about 30 hours, less than about 11 hours, less than about 4 hours, or less than about 3 hours. In some embodiments, a particular effect is or comprises a mean ti/2 of less than about 22 hours or less than about 15 hours. In some embodiments, a particular effect is or comprises a mean ti/2 of greater than about 2 hours, greater than about 3 hours, or greater than about 10 hours.
[0070] In some embodiments, a particular effect is or comprises a particular mean accumulation ratio of Cmax (RaccCmax). In some embodiments, a mean RaccCmax is measured in a population of healthy subjects (e.g., healthy subjects in a fed or fasted state) who received multiple oral doses of Compound 1 (e.g., once daily (QD) or twice daily (BID) for, e.g., 14 days). In some embodiments, a particular effect is or comprises a mean RaccCmax of from about 0.6 to about 2.7, from about 0.6 to about 1.6, or from about 1.0 to about 1.6. In some embodiments, a particular effect is or comprises a mean RaccCmax of less than about 2.0, less than about 1.6, or less than about 1.4. In some embodiments, a particular effect is or comprises a mean RaccCmax of greater than about 0.5, greater than about 1.0, or greater than about 1.2.
[0071] In some embodiments, a particular effect is or comprises a particular mean accumulation ratio of AUC (RaccAUC). In some embodiments, a mean RaccAUC is measured in a population of healthy subjects (e.g., healthy subjects in a fed or fasted state) who received multiple oral doses of Compound 1 (e.g., once daily (QD) or twice daily (BID) for, e.g., 14 days). In some embodiments, a particular effect is or comprises a mean RaccAUC of from about 0.8 to about 1.6 or from about 0.8 to about 1.2. In some embodiments, a particular effect is or comprises a mean RaccAUC of less than about 2.0, less than about 1.2, or less than about 1.0. In some embodiments, a particular effect is or comprises a mean RaccAUC of greater than about 0.5, greater than about 0.8, or greater than about 1.0.
[0072] In some embodiments, the present disclosure provides methods of administering Compound 1 to a subject or population of subjects, according to a regimen established to achieve one or more particular effects in a population of subjects in one fed/fasted state, relative to a comparable population of subjects in another fed/fasted state. In some embodiments, a regimen is established to achieve one or more particular effects in a population of healthy subjects in one fed/fasted state, relative to a comparable population of healthy subjects in another fed/fasted state. In some embodiments, a regimen is established to achieve one or more particular effects in a population of healthy subjects in a fasted state, relative to a comparable population of healthy subjects in a fed state. In some embodiments, a regimen is established to achieve one or more particular effects in a population of healthy subjects in a fed state, relative to a comparable population of healthy subjects in a fasted state. In some such embodiments, a reference population (e.g., in a fasted or fed state) has received a composition providing Compound 1 (e.g., the same composition providing Compound 1).
[0073] In some embodiments, a population in a fasted state has received a composition providing Compound 1 after a period of fasting (e.g., an overnight fast of at least 10 hours). In some embodiments, a population in a fed state has received a composition providing Compound 1 after consumption of a meal (e.g., a high fat meal) within a period of time (e.g., within 30 minutes of receiving Compound 1).
[0074] In some embodiments, the present disclosure provides methods of administering to a subject or population of subjects in need thereof an amount of Compound 1, wherein said amount, when administered as a single oral dose to a population of healthy subjects in one fed/fasted state, was established to achieve one or more particular effects relative to a comparable population of healthy subjects in another fed/fasted state. In some embodiments, the present disclosure provides methods of administering to a subject or population of subjects in need thereof an amount of Compound 1, wherein said amount, when administered as a single oral dose to a population of healthy subjects in a fasted state, was established to achieve one or more particular effects relative to a comparable population of healthy subjects in a fed state. [0075] In some embodiments, a particular effect is or comprises a difference in mean Cmax in a population of subjects (e.g., healthy subjects) in one fed/fasted state relative to a comparable population of subjects (e.g., healthy subjects) in another fed/fasted state. In some embodiments, a particular effect is or comprises an increased mean Cmax in a population of subjects in a fasted state relative to a comparable population of subjects in a fed state. In some embodiments, a particular effect is or comprises a mean Cmax in a population of subjects in a fasted state that is about 3 fold, about 3.5 fold, or about 4 fold greater than a mean Cmax in a population of subjects in a fed state. In some embodiments, a particular effect is or comprises a comparable mean Cmax in a population of subjects in a fasted state relative to a comparable population of subjects in a fed state. In some embodiments, a particular effect is or comprises a mean Cmax in a population of subjects in a fasted state that is within about 50%, about 40%, or about 25% of a mean Cmax in a population of subjects in a fed state.
[0076] In some embodiments, a particular effect is or comprises a difference in median Tmax in a population of subjects (e.g., healthy subjects) in one fed/fasted state relative to a comparable population of subjects (e.g., healthy subjects) in another fed/fasted state. In some embodiments, a particular effect is or comprises a decreased median Tmax in a population of subjects in a fasted state relative to a comparable population of subjects in a fed state. In some embodiments, a particular effect is or comprises a median Tmax in a population of subjects in a fasted state that is about 2 fold, about 2.5 fold, or about 3 fold less than a median Tmax in a population of subjects in a fed state.
[0077] In some embodiments, a particular effect is or comprises a lack of meaningful difference in mean AUCo-iast in a population of subjects (e.g., healthy subjects) in one fed/fasted state relative to a comparable population of subjects (e.g., healthy subjects) in another fed/fasted state. In some embodiments, a particular effect is or comprises a comparable mean AUCo-iast in a population of subjects in a fasted state relative to a comparable population of subjects in a fed state. In some embodiments, a particular effect is or comprises a mean AUCo-iast in a population of subjects in a fasted state that is within about 10%, about 20%, or about 30% of a mean AUCo- iast in a population of subjects in a fed state. [0078] In some embodiments, a particular effect is or comprises a lack of meaningful difference in mean AUCo-inf in a population of subjects (e.g., healthy subjects) in one fed/fasted state relative to a comparable population of subjects (e.g., healthy subjects) in another fed/fasted state. In some embodiments, a particular effect is or comprises a comparable mean AUCo-inf in a population of subjects in a fasted state relative to a comparable population of subjects in a fed state. In some embodiments, a particular effect is or comprises a mean AUCo-inf in a population of subjects in a fasted state that is within about 10%, about 20%, or about 30% of a mean AUCo- inf in a population of subjects in a fed state.
[0079] In some embodiments, a particular effect is or comprises a difference in mean Vz/F in a population of subjects (e.g., healthy subjects) in one fed/fasted state relative to a comparable population of subjects (e.g., healthy subjects) in another fed/fasted state. In some embodiments, a particular effect is or comprises a decreased mean Vz/F in a population of subjects in a fasted state relative to a comparable population of subjects in a fed state. In some embodiments, a particular effect is or comprises a mean Vz/F in a population of subjects in a fasted state that is about 3 fold, about 3.5 fold, or about 4 fold less than a mean Vz/F in a population of subjects in a fed state.
[0080] In some embodiments, a particular effect is or comprises a lack of meaningful difference in mean CL/F in a population of subjects (e.g., healthy subjects) in one fed/fasted state relative to a comparable population of subjects (e.g., healthy subjects) in another fed/fasted state. In some embodiments, a particular effect is or comprises a comparable mean CL/F in a population of subjects in a fasted state relative to a comparable population of subjects in a fed state. In some embodiments, a particular effect is or comprises a mean CL/F in a population of subjects in a fasted state that is within about 10%, about 20%, or about 30% of a mean CL/F in a population of subjects in a fed state.
[0081] In some embodiments, a particular effect is or comprises a difference in mean tm in a population of subjects (e.g., healthy subjects) in one fed/fasted state relative to a comparable population of subjects (e.g., healthy subjects) in another fed/fasted state. In some embodiments, a particular effect is or comprises a decreased mean tm in a population of subjects in a fasted state relative to a comparable population of subjects in a fed state. In some embodiments, a particular effect is or comprises a mean tm in a population of subjects in a fasted state that is about 3 fold, about 3.5 fold, or about 4 fold less than a mean tm in a population of subjects in a fed state. In some embodiments, a particular effect is or comprises a comparable mean tm in a population of subjects in a fasted state relative to a comparable population of subjects in a fed state. In some embodiments, a particular effect is or comprises a mean tm in a population of subjects in a fasted state that is within about 10%, about 20%, or about 30% of a mean tm in a population of subjects in a fed state.
[0082] In some embodiments, the present disclosure provides methods of administering Compound 1 to a subject or population of subjects, according to a regimen has been established to achieve one or more particular effects in a population of subjects suffering from a disease or disorder described herein (e.g., suffering from primary proteinuric kidney disease and/or primary glomerular disease and/or persistent proteinuria). In some embodiments, a regimen has been established to achieve one or more particular effects in a population of subjects suffering from a disease or disorder described herein (e.g., suffering from primary proteinuric kidney disease and/or primary glomerular disease and/or persistent proteinuria), relative to that observed for a comparable reference population that has not received Compound 1 (e.g., that has received a reference composition which does not deliver Compound 1). In some such embodiments, a reference population has received an otherwise comparable reference composition that does not provide Compound 1 (e.g., a placebo as described herein).
[0083] In some embodiments, a composition providing Compound 1 is administered according to a regimen established to achieve a particular effect, e.g., at a particular time point.
In some embodiments, a composition providing Compound 1 is administered according to a regimen established to achieve a particular effect, e.g., at a particular time point, relative to an appropriate reference as described herein (e.g., as is observed in a comparable population who has not received a composition providing Compound 1).
[0084] In some embodiments, the present disclosure provides methods of administering to a subject or population of subjects in need thereof an amount of Compound 1, wherein said amount, when administered to a population of subjects suffering from a disease or disorder described herein (e.g., suffering from primary proteinuric kidney disease and/or primary glomerular disease and/or persistent proteinuria), has been established to achieve one or more particular effects.
[0085] In some embodiments, a particular effect is or comprises a particular mean or median percentage change in 24-hour urinary protein excretion (e.g., at Week 12). In some embodiments, a particular effect is or comprises a particular mean or median percent reduction in 24-hour urinary protein excretion (e.g., at Week 12). In some embodiments, a particular effect is or comprises a greater mean or median percent reduction in 24-hour urinary protein excretion (e.g., at Week 12) in a population of subjects suffering from primary glomerular disease and/or persistent proteinuria administered a composition providing Compound 1 relative to a comparable population who has not received a composition providing Compound 1. In some embodiments, a particular effect is or comprises a mean or median percent reduction in 24-hour urinary protein excretion (e.g., at Week 12) of about 20% to about 40%.
[0086] In some embodiments, a particular effect is or comprises a particular mean or median percentage change in 24-hour urinary albumin (e.g., at Week 12). In some embodiments, a particular effect is or comprises a particular mean or median percent reduction in 24-hour urinary albumin (e.g., at Week 12). In some embodiments, a particular effect is or comprises a greater mean or median percent reduction in 24-hour urinary albumin (e.g., at Week 12) in a population of subjects suffering from primary glomerular disease and/or persistent proteinuria administered a composition providing Compound 1 relative to a comparable population who has not received a composition providing Compound 1.
[0087] In some embodiments, a particular effect is or comprises a particular proportion of subjects with complete remission in proteinuria. In some embodiments, a complete remission in proteinuria is defined as a 24-hour urinary protein excretion of less than 300 mg (e.g., at Week 12). In some embodiments, a particular effect is or comprises a greater proportion of subjects with complete remission in proteinuria in a population of subjects suffering from primary glomerular disease and/or persistent proteinuria administered a composition providing Compound 1 relative to a comparable population who has not received a composition providing Compound 1.
[0088] In some embodiments, a particular effect is or comprises a particular proportion of subjects with partial remission in proteinuria. In some embodiments, a partial remission in proteinuria is defined as (e.g., at Week 12) a 24-hour urinary protein excretion reduction of less than or equal to 50% from baseline and/or a 24-hour urinary protein excretion of less than 3.5 g/day if baseline 24-hour urinary protein excretion was greater than 3.5 g. In some embodiments, a particular effect is or comprises a greater proportion of subjects with partial remission in proteinuria in a population of subjects suffering from primary glomerular disease and/or persistent proteinuria administered a composition providing Compound 1 relative to a comparable population who has not received a composition providing Compound 1.
[0089] In some embodiments, a particular effect is or comprises a greater proportion of subjects with complete or partial remission in proteinuria in a population of subjects suffering from primary glomerular disease and/or persistent proteinuria administered a composition providing Compound 1 relative to a comparable population who has not received a composition providing Compound 1.
[0090] In some embodiments, a particular effect is or comprises a particular proportion of subjects with greater than or equal to 50% reduction in 24-hour urinary protein excretion from baseline (e.g., at Week 12). In some embodiments, a particular effect is or comprises a greater proportion of subjects with greater than or equal to 50% reduction in 24-hour urinary protein excretion from baseline (e.g., at Week 12) in a population of subjects suffering from primary glomerular disease and/or persistent proteinuria administered a composition providing Compound 1 relative to a comparable population who has not received a composition providing Compound 1.
[0091] In some embodiments, a particular effect is or comprises a particular proportion of subjects with greater than or equal to 50% reduction in 24-hour urinary albumin excretion from baseline (e.g., at Week 12). In some embodiments, a particular effect is or comprises a greater proportion of subjects with greater than or equal to 50% reduction in 24-hour urinary albumin excretion from baseline (e.g., at Week 12) in a population of subjects suffering from primary glomerular disease and/or persistent proteinuria administered a composition providing Compound 1 relative to a comparable population who has not received a composition providing Compound 1.
[0092] In some embodiments, a particular effect is or comprises a particular mean or median percentage change from baseline in creatinine clearance (e.g., at Week 12). In some embodiments, a particular effect is or comprises a particular mean or median percent increase from baseline in creatinine clearance (e.g., at Week 12). In some embodiments, a particular effect is or comprises a greater mean or median percent increase from baseline in creatinine clearance (e.g., at Week 12) in a population of subjects suffering from primary glomerular disease and/or persistent proteinuria administered a composition providing Compound 1 relative to a comparable population who has not received a composition providing Compound 1. [0093] In some embodiments, a particular effect is or comprises a particular mean or median change from baseline in serum albumin (e.g., at Week 12). In some embodiments, a particular effect is or comprises a particular mean or median increase from baseline in serum albumin (e.g., at Week 12). In some embodiments, a particular effect is or comprises a greater mean or median increase from baseline in serum albumin (e.g., at Week 12) in a population of subjects suffering from primary glomerular disease and/or persistent proteinuria administered a composition providing Compound 1 relative to a comparable population who has not received a composition providing Compound 1.
[0094] In some embodiments, a particular effect is or comprises a particular mean or median change from baseline in fasting triglyceride levels (e.g., at Week 12). In some embodiments, a particular effect is or comprises a particular mean or median decrease from baseline in fasting triglyceride levels (e.g., at Week 12). In some embodiments, a particular effect is or comprises a greater mean or median decrease from baseline in fasting triglyceride levels (e.g., at Week 12) in a population of subjects suffering from primary glomerular disease and/or persistent proteinuria administered a composition providing Compound 1 relative to a comparable population who has not received a composition providing Compound 1.
[0095] In some embodiments, a particular effect is or comprises a particular mean or median change from baseline in cholesterol levels (e.g., at Week 12). In some embodiments, a particular effect is or comprises a particular mean or median decrease from baseline in cholesterol levels (e.g., at Week 12). In some embodiments, a particular effect is or comprises a greater mean or median decrease from baseline in cholesterol levels (e.g., at Week 12) in a population of subjects suffering from primary glomerular disease and/or persistent proteinuria administered a composition providing Compound 1 relative to a comparable population who has not received a composition providing Compound 1.
[0096] In some embodiments, a particular effect is or comprises a particular mean or median level of one or more plasma or urine biomarkers selected from plasma soluble TNF receptor 2 (sTNFR2), kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), urinary fibronectin, high molecular weight type IV collagen (HMW collagen IV), monocyte chemotactic protein-1 (MCP-1), and matrix metalloproteinase-7 (MMP-7). In some embodiments, a particular effect is or comprises a particular mean or median change in the level(s) of one or more plasma or urine biomarkers selected from plasma soluble TNF receptor 2 (sTNFR2), kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), urinary fibronectin, high molecular weight type IV collagen (HMW collagen IV), monocyte chemotactic protein- 1 (MCP-1), and matrix metalloproteinase-7 (MMP-7) in a population of subjects suffering from primary glomerular disease and/or persistent proteinuria administered a composition providing Compound 1 relative to a comparable population who has not received a composition providing Compound 1.
[0097] In some embodiments, a particular effect is or comprises a particular mean maximum concentration (Cmax). In some embodiments, a mean or median Cmax is measured in a population of subjects suffering from primary glomerular disease and/or persistent proteinuria administered a composition providing Compound 1.
[0098] In some embodiments, a particular effect is or comprises a particular median time to maximum concentration (Tmax). In some embodiments, a median Tmax is measured in a population of subjects suffering from primary glomerular disease and/or persistent proteinuria administered a composition providing Compound 1.
[0099] In some embodiments, a particular effect is or comprises a particular mean area under drug concentration-time curve from time zero to last measurable concentration (AUCo-iast). In some embodiments, a mean AUCo-iast is measured in a population of subjects suffering from primary glomerular disease and/or persistent proteinuria administered a composition providing Compound 1.
[0100] In some embodiments, a particular effect is or comprises a particular mean area under drug concentration-time curve from time zero to infinity (AUCo-inf). In some embodiments, a mean AUCo-inf is measured in a population of subjects suffering from primary glomerular disease and/or persistent proteinuria administered a composition providing Compound 1.
[0101] In some embodiments, a particular effect is or comprises a particular mean area under the drug concentration-time curve over the dosing interval (AUCtau). In some embodiments, a mean AUCtau is measured in a population of subjects suffering from primary glomerular disease and/or persistent proteinuria administered a composition providing Compound 1.
[0102] In some embodiments, a particular effect is or comprises a particular mean half-life (ti/2). In some embodiments, a mean ti/2 is measured in a population of subjects suffering from primary glomerular disease and/or persistent proteinuria administered a composition providing Compound 1. [0103] In some embodiments, a particular effect is or comprises a particular mean or median terminal elimination rate constant (Kei). In some embodiments, a mean or median Kei is measured in a population of subjects suffering from primary glomerular disease and/or persistent proteinuria administered a composition providing Compound 1.
[0104] In some embodiments, a particular effect is or comprises a particular mean or median trough plasma concentration at steady-state measured at the end of a dosing interval before next administration (Ctrough). In some embodiments, a mean or median Ctrough is measured in a population of subjects suffering from primary glomerular disease and/or persistent proteinuria administered a composition providing Compound 1.
[0105] In some embodiments, a particular effect is or comprises a particular mean apparent total clearance (CL/F) on Day 1. In some embodiments, a mean CL/F is measured in a population of subjects suffering from primary glomerular disease and/or persistent proteinuria administered a composition providing Compound 1.
[0106] In some embodiments, a particular effect is or comprises a particular mean apparent total clearance at steady state (CL/Fss). In some embodiments, a mean CL/Fss is measured in a population of subjects suffering from primary glomerular disease and/or persistent proteinuria administered a composition providing Compound 1.
[0107] In some embodiments, a particular effect is or comprises a particular mean apparent volume of distribution (Vz/F) on Day 1. In some embodiments, a mean Vz/F is measured in a population of subjects suffering from primary glomerular disease and/or persistent proteinuria administered a composition providing Compound 1.
[0108] In some embodiments, a particular effect is or comprises a particular mean apparent volume of distribution at steady state (Vz/Fss). In some embodiments, a mean Vz/Fss is measured in a population of subjects suffering from primary glomerular disease and/or persistent proteinuria administered a composition providing Compound 1.
[0109] In some embodiments, a particular effect is or comprises a particular mean or median accumulation ratio. In some embodiments, a mean or median accumulation ratio is measured in a population of subjects suffering from primary glomerular disease and/or persistent proteinuria administered a composition providing Compound 1.
[0110] In some embodiments, the present disclosure provides amounts of Compound 1 suitable to achieve one or more particular effects. In some embodiments, Compound 1 is administered in a daily dose of from about 50 mg to about 600 mg, from about 100 mg to about 600 mg, from about 200 mg to about 600 mg, from about 400 mg to about 600 mg, from about 50 mg to about 500 mg, from about 100 mg to about 500 mg, from about 250 mg to about 500 mg, from about 50 mg to about 250 mg, from about 100 mg to about 1000 mg, from about 200 mg to about 1000 mg, from about 500 mg to about 1000 mg, or from about 200 mg to about 500 mg. In some embodiments, Compound 1 is administered in a dose of about 50 mg, about 100 mg, about 200 mg, about 250 mg, about 400 mg, about 500 mg, or about 600 mg. In some embodiments, Compound 1 is administered in a dose of about 50 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg, or about 600 mg. In some embodiments, Compound 1 is administered in a daily dose of about 50 mg, about 100 mg, about 200 mg, about 400 mg, about 500 mg, or about 600 mg. In some embodiments, Compound 1 is administered in a twice daily dose of about 50 mg, about 100 mg, about 250 mg, or about 500 mg.
[0111] In some embodiments, Compound 1 is administered in a dose of about 100 mg once daily. In some embodiments, Compound 1 is administered in a dose of about 200 mg once daily. In some embodiments, Compound 1 is administered in a dose of about 300 mg once daily. In some embodiments, Compound 1 is administered in a dose of about 400 mg once daily. In some embodiments, Compound 1 is administered in a dose of about 500 mg once daily. In some embodiments, Compound 1 is administered in a dose of about 600 mg once daily.
[0112] In some embodiments, Compound 1 is administered in a dose of about 50 mg twice daily. In some embodiments, Compound 1 is administered in a dose of about 100 mg twice daily. In some embodiments, Compound 1 is administered in a dose of about 150 mg twice daily. In some embodiments, Compound 1 is administered in a dose of about 200 mg twice daily. In some embodiments, Compound 1 is administered in a dose of about 250 mg twice daily. In some embodiments, Compound 1 is administered in a dose of about 300 mg twice daily. In some embodiments, Compound 1 is administered in a dose of about 350 mg twice daily. In some embodiments, Compound 1 is administered in a dose of about 400 mg twice daily.
[0113] In some embodiments, provided methods further comprise reducing a dose of Compound 1 if a subject experiences a side effect (e.g., a gastrointestinal or liver side effect) and/or if a subject’s side effect persists. In some embodiments, one or more doses of Compound 1 are reduced by 100 mg if a subject experiences a side effect (e.g., a gastrointestinal or liver side effect). For example, in some embodiments, a Compound 1 dosing regimen of 200 mg QD is reduced to 100 mg QD. In some embodiments, a Compound 1 dosing regimen of 400 mg QD is reduced to 300 mg QD. In some embodiments, a Compound 1 dosing regimen of 300 mg BID is reduced to 200 mg BID. In some embodiments, a Compound 1 dosing regimen of 300 mg BID is reduced to a once daily dose of 200 mg and a once daily dose of 300 mg (e.g., said doses separated by approximately 12 hours). In some embodiments, provided methods further comprise discontinuing administration of Compound 1 if a subject experiences a side effect and/or if a subject’s side effect persists after a dose reduction.
[0114] In some embodiments, gastrointestinal side effects include diarrhea, nausea, vomiting, and/or abdominal cramps. In some embodiments, provided methods further comprise reducing the dose of Compound 1 if a subject experiences mild diarrhea (e.g., 4-6 extra stools per day) for 8 days or longer. In some embodiments, such a subject is also treated with anti motility therapy (e.g., loperamide). In some embodiments, provided methods further comprise discontinuing administration of Compound 1 if a subject experiences mild diarrhea (e.g., 4-6 extra stools per day) for an additional 7 days or longer after dose reduction. In some embodiments, provided methods further comprise reducing the dose of Compound 1 if a subject experiences severe diarrhea (e.g., more than 6 extra stools per day with clinical evidence of volume depletion or impact on activities of daily living). In some embodiments, such a subject is also treated with anti-motility therapy (e.g., loperamide). In some embodiments, provided methods further comprise discontinuing administration of Compound 1 if a subject experiences severe diarrhea (e.g., more than 6 extra stools per day with clinical evidence of volume depletion or impact on activities of daily living) for 14 days or longer.
[0115] In some embodiments, provided methods further comprise reducing the dose of Compound 1 is a subject experiences nausea and/or vomiting for 8 days or longer. In some embodiments, such subjects are also treated with ondansetron. In some embodiments, provided methods further comprise discontinuing administration of Compound 1 if a subject experiences nausea and/or vomiting for 14 days or longer.
[0116] In some embodiments, liver side effects include elevated liver function tests (e.g., AST or ALT increase to > 3x upper limit of normal or AST or ALT increase to > 2x baseline value if elevated at baseline). In some embodiments, provided methods comprise discontinuing administration of Compound 1 if a subject has elevated liver function tests for 2 or more days. [0117] In some embodiments, the present disclosure provides methods of treating diseases, disorders, and conditions (e.g., according to methods provided herein). In some embodiments, provided methods are useful for reducing fibrosis in a subject in need thereof. In some embodiments, provided methods are useful for treating a disease, disorder, or condition characterized by or otherwise associated with fibrosis. The present disclosure encompasses the recognition that treating fibrosis (e.g., using provided methods) instead of the underlying etiology may allow for broadly applicable antifibrotic therapies. It will be appreciated that provided methods may be suitable for reducing fibrosis in a variety of tissues and/or organs; the present disclosure contemplates use of Compound 1 for treating diseases, disorders, and conditions characterized by or otherwise associated with fibrosis in any suitable tissue and/or organ. For example, in some embodiments, provided methods are suitable for treating diseases, disorders and conditions that are or comprise fibrosis of gastrointestinal tract, heart, kidney, lung, liver, muscle, pancreas, and/or skin. In some embodiments, provided methods are suitable for treating diseases, disorders, and conditions characterized by or otherwise associated with cysts (e.g., in the kidney, liver, pancreas, ovaries, spermatic duct, etc.). It will be appreciated that provided methods may be suitable for treating diseases, disorders, and conditions in which fibrosis is the sole or a predominant component, as well as those in which fibrosis is a secondary component (e.g., a symptom and/or result of an underlying disease, disorder, or condition). It will also be appreciated that there are a variety of sources or causes of fibrosis.
[0118] In some embodiments, certain injuries can progress to development of fibrosis. In some embodiments, provided methods are useful for treating acute injuries (e.g., acute organ injuries, such as acute lung injury, acute liver injury, or acute kidney injury), as well as for treating chronic injuries (e.g., chronic organ injuries, such as chronic lung injury, chronic liver injury, or chronic kidney injury). In some embodiments, provided methods are useful for treating fibrosis associated with an acute injury, such as that incurred from trauma and/or surgery and/or infection (e.g., a viral infection). For example, in some embodiments, provided methods are useful for acceleration of wound healing, reduction of post-surgical scarring, and/or reduction of adhesion formation. In some embodiments, provided methods are useful for treating damaged and/or ischemic organs, transplants, or grafts, as well as ischemia/reperfusion injury or post- surgical scarring. For example, in some embodiments, provided methods are useful for promoting vascularization of a damaged and/or ischemic organ, transplant, or graft, ameliorating ischemia/reperfusion injury (e.g., in brain, heart, liver, or kidney), normalizing myocardial perfusion resulting from chronic cardiac ischemia or myocardial infarction, and/or developing or augmenting collateral vessel development after vascular occlusion or to ischemic tissues or organs.
Pulmonary Diseases, Disorders, and Conditions
[0119] In some embodiments, provided methods are useful for treating pulmonary diseases, disorders, or conditions. In some embodiments, provided methods are useful for treating pulmonary fibrosis. In some embodiments, provided methods are useful for treating pulmonary fibrosis secondary to, or otherwise associated with, an underlying indication. In some embodiments, provided methods are useful for treating interstitial lung diseases (e.g., fibrosing interstitial lung diseases). In some embodiments, provided methods are useful for treating pneumonias (e.g., idiopathic interstitial pneumonias). In some embodiments, provided methods are useful for treating idiopathic pulmonary fibrosis (IPF). In some embodiments, provided methods are useful for treating pulmonary fibrosis associated with an infection (e.g., a bacterial, viral, or fungal infection). In some embodiments, provided methods are useful for treating pulmonary fibrosis associated with a viral infection (e.g., an influenza or coronavirus infection, such as COVID-19).
[0120] In some embodiments, a fibrotic disease to be treated by methods of the present disclosure is pulmonary fibrosis. Pulmonary fibrosis is a chronic, progressive, and ultimately fatal interstitial lung disease resulting from epithelial cell injury due to many factors. Upon epithelial injury, activation of inflammatory cells and fibroblasts/myofibroblasts involves a cascade of cytokines/chemokines, growth factor network and deposit extracellular matrix, including collagen), which leads to pulmonary fibrosis and respiratory failure. Pulmonary fibrosis causes high morbidity and mortality. At least five million people worldwide and -200,000 people in the United States suffer from pulmonary fibrosis. There is an unmet critical need for effective and affordable treatments for acute and chronic lung injuries.
[0121] Numerous endogenous and exogenous factors can provide primary stimuli for pulmonary fibrosis. Dust, silica, smoke, aerosolized toxins, infections and certain medicines have the potential to injure the lung and set the stage for the development of chronic pulmonary fibrosis. For example, viral infections may cause lung damage and/or otherwise develop into pulmonary fibrosis. In a study of patients diagnosed with H1N1 infection, it was found that 10% of patients developed post-ARDS fibrosis (Mineo, G., et al. Radiol. Med. 2012;117:185-200). In COVID-19 patients, acute respiratory distress syndrome (ARDS) developed in 17-29% of hospitalized patients (Huang C, e al. The Lancet. 2020 Jan 24).
[0122] Pulmonary fibrosis is associated with pronounced morbidity with high impact on economic burden. A clinical study indicated that the total direct costs for patients with pulmonary fibrosis were $26,378 per person and other study indicated that the mean Medicare costs for a lung transplant recipient was $131,352. The prevalence of pneumoconiosis (a disease caused by inhalation of dust and silica that causes inflammation and lung fibrosis) caused direct and indirect economic losses of around 28 billion yuan in China (4.3 billion US dollars) for 1 year.
[0123] In some embodiments, a fibrotic disease to be treated by methods of the present disclosure is idiopathic pulmonary fibrosis. Idiopathic pulmonary fibrosis (IPF) is a chronic, irreversible, and progressive fibrotic disorder of the lower respiratory tract that typically affects adults over the age of 40. Idiopathic interstitial pneumonias (IIP) are diffuse parenchymal lung diseases, of which IPF is the most common and severe type of fibrotic lung disease. It is anatomically characterized by scarring of the lungs with a pattern of usual interstitial pneumonia (UIP) on high resolution computed tomography or histologic appearance on lung biopsy. Clinically IPF follows a relentless course of progressive, irreversible, and debilitating disease characterized by exertional dyspnea and cough. Median survival following diagnosis of IPF ranges between 2 and 5 years, lower than that for many common cancers (Ley, B., et al. Am. J. Respir. Crit. Care Med. 2011;183:431-440; Seigel, R. L., et al. CA. Cancer J. Clin. 2016;66:7- 30). Fibrotic process in IPF is progressive and, regardless of the nature of the initial injury, may follow a common pathway characterized by alveolar epithelial cell (AEC) dysfunction. The injury to epithelial cells and basement membrane results in complex cell and cytokine interactions that extend the fibrotic process to the alveolar walls, alveolar lumen, and then adjacent areas of lung parenchyma. Both epithelial and basement membrane injury appear necessary for the development of intraluminal fibrosis (Crapo, J. D., et al. Am. Rev. Respir. Dis. 1982;126:332-7). [0124] Normal alveolar epithelium is comprised predominantly of type I epithelial cells (AECsl), with a relatively small number of type II epithelial cells (AECs2). After injury, AECs2 proliferate and differentiate into AECsl and are normally responsible for re-epithelialization of injured alveoli. This is accomplished by several processes involving coagulation cascade, angiogenesis, fibroblast activation and migration, and collagen synthesis and proper alignment (Betensley, A., et al. J. Clin. Med. 2016;6:2). Many chemokines such as transforming growth factor beta 1 (TGF-bI), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and fibroblast growth factor (FGF) play a key role in these processes.
Regardless of the initial injury, the progression of fibrosis in IPF follows a common and complex path in which the AECs, fibroblasts, and endothelial cells produce an array of cytokines and growth factors that stimulate fibroblast proliferation and matrix synthesis.
Hepatic Diseases, Disorders, and Conditions
[0125] In some embodiments, provided methods are useful for treating hepatic diseases, disorders, or conditions. In some embodiments, provided methods are useful for treating hepatic fibrosis (e.g., fibrotic liver disease). In some embodiments, provided methods are useful for treating cirrhosis. In some embodiments, provided methods are useful for treating hepatic fibrosis and/or cirrhosis secondary to, or otherwise associated with, an underlying indication. In some embodiments provided methods are useful for treating hepatic fibrosis associated with hepatitis C, hepatitis B, delta hepatitis, chronic alcoholism, nonalcoholic steatohepatitis (NASH), extrahepatic obstructions (e.g., stones in bile duct), cholangiopathies (e.g., primary biliary cirrhosis or sclerosing cholangitis), autoimmune liver disease, or inherited metabolic disorders (e.g., Wilson’s disease, hemochromatosis, or alpha-1 antitrypsin deficiency).
[0126] In some embodiments, a fibrotic disease to be treated by methods of the present disclosure is liver fibrosis. Liver fibrosis is a scarring response of the liver to chronic liver injury; when fibrosis progresses to cirrhosis, morbid complications can develop. In fact, end- stage liver fibrosis or cirrhosis is the seventh leading cause of death in the United States, and afflicts hundreds of millions of people worldwide; deaths from end-stage liver disease in the United States are expected to increase, mainly due to the hepatitis C epidemic. In addition to the hepatitis C virus, many other forms of chronic liver injury also lead to end-stage liver disease and cirrhosis, including other viruses such as hepatitis B and delta hepatitis, chronic alcoholism, non-alcoholic steatohepatitis, extrahepatic obstructions (e.g., stones in the bile duct), cholangiopathies (e.g., primary biliary cirrhosis and sclerosing cholangitis), autoimmune liver disease, and inherited metabolic disorders (e.g., Wilson's disease, hemochromatosis, and alpha- 1 anti trypsin deficiency).
[0127] Treatment of liver fibrosis has traditionally focused on eliminating a primary injury. For extrahepatic obstructions, biliary decompression is the recommended mode of treatment whereas patients with Wilson's disease are treated with zinc acetate. Treatments for other chronic liver diseases such as hepatitis B, autoimmune hepatitis and Wilson's disease are also associated with many side effects, while primary biliary cirrhosis, primary sclerosing cholangitis and non alcoholic fatty liver disease have no effective treatment other than liver transplantation.
[0128] While transplantation may currently be the most effective cure for liver fibrosis, mounting evidence indicates that not only fibrosis, but even cirrhosis is reversible.
Unfortunately, patients often present with advanced stages of fibrosis and cirrhosis, when many therapies such as antivirals can no longer be safely used due to their side effect profile. Such patients would benefit enormously from effective antifibrotic therapy, because attenuating or reversing fibrosis may prevent many late stage complications such as infection, ascites, and loss of liver function and preclude the need for liver transplantation.
Renal Diseases, Disorders, and Conditions
[0129] In some embodiments, provided methods are useful for treating renal diseases, disorders, or conditions. In some embodiments, provided methods are useful for treating renal fibrosis. In some embodiments, provided methods are useful for treating renal fibrosis secondary to, or otherwise associated with, an underlying indication. In some embodiments, provided methods are useful for treating renal fibrosis associated with renal failure, renal obstruction, renal trauma, renal transplantation, chronic kidney disease, diabetes, hypertension, radiocontrast nephropathy, immune-mediated glomerulonephri tides (e.g., lupus nephritis, ANCA-associated glomerulonephritides (e.g., Wegener’s granulomatosis, microscopic polyangiitis, or renal limited vasculitis), anti-GBM nephropathy, IgA nephropathy, membranous glomerulonephritis, or focal and segmental glomerulosclerosis), non-immune-mediated glomerulonephritides (e.g., polycystic kidney disease, collagen type III glomerulopathy, nail-patella syndrome, or Alport syndrome), minimal change disease, or nephrotic syndrome (e.g., steroid-resistant nephrotic syndrome). In some embodiments, provided methods are useful for treating nephrotic syndrome and/or diseases, disorders, or conditions associated with nephrotic syndrome (e.g., focal and segmental glomerulosclerosis, minimal change disease, and membranous nephropathy). In some embodiments, provided methods are useful for treating a fibrotic disease of the kidney that is or comprises: focal segmental glomerulosclerosis (FSGS), steroid resistant nephrotic syndrome (SRNS), proteinuria, lupus nephritis, minimal change disease, an anti-neutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis, Alport syndrome, anti-globular basement membrane (anti-GBM) nephropathy, IgA nephropathy, membranous glomerulonephritis (MG), autosomal dominant polycystic kidney disease (ADPKD), collagen type III glomerulopathy, nail- patella syndrome, or chronic kidney disease. In some embodiments, provided methods are useful for treating a fibrotic disease of the kidney that is or comprises an anti-neutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis. In some embodiments, ANCA-associated glomerulonephritis is selected from Wegener’s granulomatosis, microscopic polyangiitis (MPA), or renal limited vasculitis. In some embodiments, provided methods are useful for treating focal and segmental glomerulosclerosis (FSGS). In some embodiments, provided methods are useful for treating Alport syndrome. In some embodiments, provided methods are useful for treating polycystic kidney disease (e.g., autosomal dominant polycystic kidney disease or autosomal recessive polycystic kidney disease).
[0130] In some embodiments, provided methods are useful for treating primary proteinuric kidney disease (PPKD). In some embodiments, provided methods are useful for treating primary glomerular diseases (PGDs). PGDs are among the leading causes of chronic kidney disease and end-stage kidney disease in the world. PGDs predominantly affect younger patients, significantly reducing their quality of life, productivity, and longevity. FSGS, membranous nephropathy (MN), and IgA nephropathy are among the three most common primary glomerular diseases in adults. Accordingly, in some embodiments, provided methods are useful for treating FSGS. In some embodiments, provided methods are useful for treating MN. In some embodiments, provided methods are useful for treating IgA nephropathy.
[0131] Currently, there are no therapies approved specifically for PPKDs and/or PGDs. Various immunosuppressive and/or cytotoxic agents primarily approved for other indications are first-line therapy for patients considered to be at a high risk of progression. Such therapies aim to induce remission, defined as a normalization of the urinary protein excretion. The patients who undergo a complete or a partial remission have significantly improved renal prognosis. Unfortunately, remission rates with immunosuppressive agents and cytotoxic therapy range only between 30-60% depending on the underlying disease, and up to 50% of these patients subsequently experience a relapse. Relapses in proteinuria are frequently treated with repeat administration of immunosuppressive and/or cytotoxic therapy; however, long-term treatment with these agents is limited by their significant dose-limiting toxicities. Some of the drugs, such as calcineurin inhibitors (CNIs), have a narrow therapeutic index necessitating close monitoring of the drug levels. Long-term use of CNIs is associated with hypertension, nephrotoxicity, and metabolic abnormalities such as diabetes and dyslipidemia. In most patients, cessation of calcineurin inhibitors results in the relapse of proteinuria (Meyrier, A. et al., Kidney International. 1994;45(5): 1446-56). A significant number of patients eventually become either resistant to or dependent on these toxic agents. Some of these glomerular diseases also recur after renal transplantation posing unique management problems (Choy, B.Y., et al., Am. J. Transplant. 2006;6(11):2535-42).
[0132] In some embodiments, provided methods are useful for treating patients with proteinuria (e.g., persistent proteinuria). It is well established that higher rates of urinary protein excretion are associated with worse prognosis, and therapies that reduce proteinuria are desirable for improving renal outcomes. Patients with persistent proteinuria (e.g., who continue to have >
1 gram of proteinuria per day) are at high risk of progressing to end-stage kidney disease (ESKD). Risk of progression is significantly increased in the presence of a reduced estimated glomerular filtration rate (eGFR) at the time of diagnosis or during the course of the disease. Patients with persistent proteinuria also develop further complications of chronic kidney disease (CKD) such as dyslipidemia, cardiovascular disease, abnormalities in mineral-bone metabolism, and hypertension, resulting in significant increases in morbidity and mortality and utilization of health care resources.
[0133] In addition to immunosuppressive agents, standard of care for patients with persistent proteinuria includes treatment with the renin-angiotensin-aldosterone system (RAAS) blockers, most commonly ACE inhibitors or angiotensin-receptor blockers (ARBs). The RAAS blockers reduce proteinuria and improve clinical outcomes in proteinuric renal diseases regardless of the etiology. Other standard of care recommendations include aggressive blood pressure control (< 130/80), and HMG-CoA reductase inhibitors (e.g., statins) in patients with hyperlipidemia. The inhibitors of the mineralocorticoid receptor and sodium glucose co-transporter-2 (SGLT-2) are increasingly being used in these patients as well.
[0134] In some embodiments, provided methods are useful for treating primary glomerular diseases (e.g., FSGS, membranous nephropathy, or IgA nephropathy) and persistent proteinuria. [0135] Several growth factor receptors have been implicated in the development of fibrosis of the kidney (Liu, F., et al. Int. J. Mol. Sci. 2016 Jun 20; 17(5), PMCID:PMC4926504). Platelet- derived growth factor receptor beta (PDGFRP) is postulated to play a particularly important role in the development of renal fibrosis (Floege, J., et al. J. Am. Soc. Nephrol. 2008 Jan; 19(1): 12-23; Ostendorf, T., et al. Pediartr. Nephrol. 2012 Jul;27(7): 1041-50; Ostendorf. T., et al. Kidney Int. Suppl. (2011) 2014 Nov;4(l):65-9, PMCID:PMC4536969; Abbound, H E. Annu. Rev. Physiol. 1995;57:297-309).
[0136] In some embodiments, a kidney disease to be treated by methods of the present disclosure is nephrotic syndrome (NS). NS is a group of rare renal diseases, including focal and segmental glomerulosclerosis (FSGS), minimal change disease (MCD), and membranous nephropathy. FSGS is a rare disease that attacks the kidney’s filtering units (glomeruli) causing serious scarring which leads to permanent kidney damage and even failure (Fogo, A.B. Nat. Rev. Nephrol. 2015 Feb;l l(2):76-87, PMCID:PMC4772430). It will be appreciated that there are at least three types of FSGS. Primary FSGS is FSGS that has no known cause (also referred to as idiopathic FSGS). Secondary FSGS is caused by one or more factors such as infection, drug toxicity, diseases such as diabetes or sickle cell disease, obesity, or other kidney diseases. Genetic FSGS (also called familial FSGS) is caused by one or more genetic mutations. Primary FSGS is idiopathic in nature. Manifestations of this disease include hypoalbuminemia and edema, lipid abnormalities and nephrotic range proteinuria. More than 5400 patients are diagnosed with FSGS every year (O’Shaughnessy, M.M., et al. Nephrol. Dial. Transplant 2018 Apr l;33(4):661-9). However, this is considered an underestimate because a limited number of biopsies are performed, and the number of FSGS cases is rising more than any other cause of NS. Standard of care for this patient population is steroid therapy. Current treatments for FSGS include corticosteroids, calcineurin inhibitors, mycophenolate mofetil, adrenocorticotropic hormone (ATCH), and rituximab; these are effective in at most 25-40% of patients. A subset of this population is resistant to steroids (steroid-resistant, or SR), and proteinuria, which is toxic to renal tubules, remains uncorrected. Consequently, this subset proceeds relatively rapidly to end-stage renal disease (ESRD). There is therefore an urgent need to develop therapies that reduce proteinuria in primary SR-FSGS (Nourbakhsh, N. and Mak,
R.H. Pediatric Health Med. Ther. 2017;8:29-37, PMCID:PMC5774596).
[0137] In some embodiments, a kidney disease to be treated by methods of the present disclosure is minimal change disease (MCD). MCD is a kidney disease in which large amounts of protein are lost in the urine. It is one of the most common causes of the nephrotic syndrome worldwide. In children, MCD is usually primary (or idiopathic), but in adults, the disease is usually secondary. Secondary causes for MCD include allergic reactions, use of certain painkillers such as non-steroidal anti-inflammatory drugs (NSAIDs), tumors, or viral infections. [0138] In some embodiments, a kidney disease to be treated by methods of the present disclosure is membranous glomerulonephritis (MG or MGN), also known as membranous nephropathy (MN). MG or MGN is a slowly progressive renal disease caused by immune complex formation in the glomerulus. Immune complexes are formed by binding of antibodies to antigens in the glomerular basement membrane. The antigens may be part of the basement membrane, or deposited from elsewhere by the systemic circulation.
[0139] In some embodiments, a kidney disease to be treated by methods of the present disclosure is anti -neutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis. Anti neutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis is a rapidly progressive renal disease and includes, e.g., Wegener's granulomatosis, microscopic polyangiitis, and renal limited vasculitis. Wegener's granulomatosis is an organ- and/or life-threatening autoimmune disease of unknown etiology. The classical clinical triad consists of necrotizing granulomatous inflammation of the upper and/or lower respiratory tract, necrotizing glomerulonephritis, and an autoimmune necrotizing systemic vasculitis affecting predominantly small vessels. The detection of anti-neutrophil cytoplasmic antibodies directed against proteinase 3 (PR3-ANCA) is a highly specific indicator for Wegener's granulomatosis. Microscopic polyangiitis is a disorder that causes blood vessel inflammation (vasculitis), which can lead to organ damage. The kidneys, lungs, nerves, skin, and joints are the most commonly affected areas of the body. MPA is diagnosed in people of all ages, all ethnicities, and both genders. The cause of this disorder is unknown. Renal limited vasculitis is a type of anti-neutrophil cytoplasmic antibody (ANCA)- associated vasculitis that presents with only a renal manifestation; no other organs, including lungs, are involved. [0140] In some embodiments, a kidney disease to be treated by methods of the present disclosure is lupus nephritis. Lupus nephritis is inflammation of the kidney that is caused by the autoimmune disease systemic lupus erythematous (SLE). With lupus, the body's immune system targets its own body tissues; lupus nephritis occurs when lupus involves the kidneys.
[0141] In some embodiments, a kidney disease to be treated by methods of the present disclosure is anti-globular basement membrane (anti-GBM) nephropathy. Anti-globular basement membrane (anti-GBM) nephropathy is a disease that occurs as a result of injury to small blood vessels (capillaries) in the kidneys and/or lungs. In anti-GBM disease, autoantibodies are targeted to the basement membrane in capillary blood vessels of the kidneys and lung, where they target and damage GBM.
[0142] In some embodiments, a kidney disease to be treated by methods of the present disclosure is IgA nephropathy, also known as Berger’s disease. IgA nephropathy is a kidney disease that occurs when IgA deposits build up in the kidneys, causing inflammation that damages kidney tissues. IgA nephropathy affects the kidneys by attacking the glomeruli. The buildup of IgA deposits inflames and damages the glomeruli, causing the kidneys to leak blood and protein into the urine. The damage may lead to scarring of the nephrons that progresses slowly over many years. Eventually, IgA nephropathy can lead to end-stage kidney disease. [0143] In some embodiments, a kidney disease to be treated by methods of the present disclosure is collagen type III glomerulopathy. Collagen type III glomerulopathy, also known as collagenic or collagenofibrotic glomerulopathy, is characterized by pathological accumulation of collagen type III in glomeruli. Collagen type III glomerulopathy presents either in childhood, often with a family history suggesting autosomal recessive inheritance, or in adults as a sporadic occurrence. Proteinuria is a typical manifestation, with progression to end stage renal disease (ESRD) in approximately 10 years. Although there is markedly elevated serum precursor collagen type III protein in the circulation, the usual manner of diagnosis is with kidney biopsy, which discloses type III collagen in subendothelial aspects of capillary walls and often in the mesangial matrix.
[0144] In some embodiments, a kidney disease to be treated by methods of the present disclosure is nail-patella syndrome. Nail-patella syndrome is a multi-organ disorder caused by mutations in the LMX1B gene. Nail-patella syndrome manifests with orthopedic and cutaneous deformities, as well as kidney complications due to development of structural lesions of collagen type III within glomerular basement membranes. Although the structural lesions may be asymptomatic, they are usually accompanied by proteinuria.
[0145] In some embodiments, a kidney disease to be treated by methods of the present disclosure is Alport syndrome (AS). AS is a genetic condition characterized by kidney disease, hearing loss, and eye abnormalities. Most affected individuals experience progressive loss of kidney function, usually resulting in end-stage kidney disease. In 80% of cases, Alport syndrome is inherited in an X-linked manner and is caused by mutation(s) in the COL4A5 gene. In other cases, it can be inherited in either an autosomal recessive, or rarely in an autosomal dominant manner, and is caused by mutation(s) in the COL4A3 and/or COL4A4 genes. Current therapies include hearing aid, hemodialysis, peritoneal dialysis and kidney transplantation.
[0146] In some embodiments, a kidney disease to be treated by methods of the present disclosure is polycystic kidney disease (e.g., autosomal recessive polycystic kidney disease (ARPKD) - congenital hepatic fibrosis (CHF)). ARPKD-CHF is a highly aggressive fibropolycystic disease that is characterized by the formation and expansion of fluid-filled cysts in the kidneys, enlargement of the kidneys and progressive fibrosis of both the kidney and the liver (Hartung, E.A., and Guay -Woodford, L.M. Pediatrics 2014 Sep;134(3):e833-e845; Gunay- Aygun, M., et al. J. Pediatr. 2006 Aug; 149(2): 159-64). Adults with polycystic kidney disease often also develop asymptomatic cysts in the liver, pancreas, ovaries and spermatic duct, in addition to cysts in the kidney. Caroli’s disease, which manifests as cystic dilatation of the intrahepatic ducts, often accompanies ARPKD-CHF (Sung, J.M., et al. Clin. Nephrol. 1992 Dec;38(6):324-8). In some embodiments, a subject is suffering from, susceptible to, or at risk of Caroli’s disease. Afflicted children that survive past two years of age more often than not require renal and/or hepatic transplantation by age ten. The need for transplantation is driven as much by progressive organ dysfunction as by significant enlargement of the diseased organ(s) accompanied by severe pain (www.arpkdchf.org).
[0147] In some embodiments, a kidney disease to be treated by methods of the present disclosure is or includes renal cysts. Aberrant signaling by tyrosine kinases, including platelet- derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) and their receptors (R), PDGFR and VEGFR/KDR, respectively, has been implicated in the formation and expansion of renal cysts. A PDGF-driven ciliopathy and/or overexpression of PDGF in the cyst lining and adjacent tubules are thought to, in part, drive renal cystic disease (Torres, V.E., et al. Lancet 2007 Apr 14;369(9569): 1287-301; Park. J.H. et al. Polycystic Kidney Disease Brisbane; 2015:375-96; Nakamura, T., et al. J. Am. Soc. Nephrol. 1993 Jan;3(7): 1378-86). Cowley et al. posited that elevated and abnormal c-myc proto-oncogene expression drives ARPKD (Proc. Natl. Acad. Sci. U.S.A. 1987 Dec:84(23):8394-8); c-myc expression is controlled by PDGF (Frick, K.K., et al. C. J. Biol. Chem. 1988 Feb 25;263(6):2948-52).
[0148] VEGF-driven angiogenesis is also thought to contribute to the growth of renal cysts, and inhibition of VEGFR/KDR signaling is associated with decreased tubule cell proliferation, decreased cystogenesis, and blunted renal enlargement (Bello-Ruess, E., et al. Kidney Int. 2001 Jul;60(l):37-45; Schrijvers, B.F., et al. Kidney Int. 2004 Jun;65(6):2003-17). Nevertheless, the role of VEGF in fibropoly cystic disease is more controversial, with at least some reports suggesting that this growth factor might be associated with disease mitigation (Spirli, C., et al. Gastroenterology 2010 Jan;138(l):360-71). Aside from their roles in renal cyst formation and expansion, it is being recognized in ARPKD-CHF that aberrant PDGF and VEGF signaling are also associated with extracellular matrix deposition in the liver and kidney (Rajekar, FL, et al. J. Clin. Exp. Hepatol. 2011 Sep;l(2):94-108; Jiang, L., et al. Biomed. Res. Int.
2016;2016:4918798; Tao, Y., et al. Kidney Int. 2007 Dec;72(l l);1358-66).
Dermal Diseases, Disorders, and Conditions
[0149] In some embodiments, provided methods are useful for treating dermal diseases, disorders, or conditions. In some embodiments, provided methods are useful for treating dermal fibrosis. In some embodiments, provided methods are useful for treating dermal fibrosis secondary to, or otherwise associated with, an underlying indication. In some embodiments, provided methods are useful for treating scleroderma and/or systemic sclerosis (e.g., diffuse systemic sclerosis or limited systemic sclerosis).
[0150] In some embodiments, a fibrotic disease to be treated by methods of the present disclosure is scleroderma and/or systemic sclerosis (SSc). Scleroderma, which literally means hard skin, is a chronic fibrotic disorder of unknown etiology that affects the skin and other internal organs (SSc) (www.scleroderma.org). Many patients who suffer from scleroderma/SSc also have loss of lung function. Scleroderma/SSc and related diseases afflict approximately 400,000 to 990,000 people in the USA every year. Mortality and morbidity in scleroderma/SSc are very high and directly related to the extent of fibrosis of the involved organs (Hinchcliff, M. and Varga, J. Am. Fam. Physician 2008 Oct;78(8):961-8). A number of international studies suggest that scleroderma/SSc occurs much more frequently in the USA than elsewhere and that it occurs three to four times more frequently among women (Mayes, M.D., et al. Arthritis Rheum. 2003 Aug;48(8):2246-55).
[0151] According to several studies, the total economic cost of scleroderma/SSc in the USA reaches $1.5 billion annually. Morbidity represents the major cost burden, associated with $820 million (55%) of total cost. The high cost of scleroderma/SSc reflects the burden of chronic disease affecting an early age of disease onset and its high morbidity (Wilson, L. Semin. Arthritis Rheum. 1997 Oct;27(2):73-84). Hence, there is a critical need for effective and affordable therapies.
[0152] Scleroderma/SSc can be classified in terms of the degree and location of the skin involvement and has been categorized into two major groups - diffuse and limited. The diffuse form of scleroderma/SSc involves symmetric thickening of skin of the extremities, face and trunk. Organs affected include the esophagus, intestines, lungs, heart, and kidneys (Mayes, M. D. Semin. Cutan. Med. Surg. 1998 Mar;17(l):22-6; Jacobsen, L. et al. J. Am. Acad. Dermatol. 2003 Aug;49(2):323-5). The limited form of scleroderma/SSc tends to be confined to the skin of fingers and face. The limited form of scleroderma/SSc is the CREST variant of scleroderma/SSc based on the clinical pattern of calcinosis with tiny deposits of calcium in the skin, Raynaud's phenomenon in the fingers, toes, nose, tongue, or ears, poor functioning of muscle of esophagus, sclerodactyly of the skin of the fingers or toes, and telangiectasias on the face, hands and mouth (Winterbauer, R.H. Bull. Johns Hopkins Hospital 1964;114:361-83; Wollheim, F.A. Classification of systemic sclerosis. Visions and reality. Rheumatology (Oxford) 2005).
[0153] Multiple fibrotic pathways are activated in scleroderma/SSc for reasons that are not completely understood. The pathogenesis of fibrosis in scleroderma/SSc involves a complex set of interactions involving immune activation, microvascular damage and the activation of fibroblasts. Scleroderma/SSc is characterized by excessive deposition of collagen in the skin and other involved organs and abnormalities of blood vessels (Jimenez, S. A., et al. Rheum. Dis.
Clin. North Am. 1996 Nov;22(4):647-74; Sakkas, L. I. Autoimmunity 2005 Mar;38(2): 113-6). TGFpi, a multifunctional cytokine, is an indirect mitogen for human fibroblasts, which through upregulating PDGF, is capable of inducing normal fibroblasts into a pathogenic myofibroblast phenotype that mediates ECM (collagen) accumulation (Mauch, C., et al. J. Invest. Dermatol. 1993 Jan;100(l):92S-96S; Hummers, L. K., et al. J. Rheumatol. 2009 Mar;36(3):576-82). The ubiquitous growth factors TGFp and PDGF are the most potent proteins involved in fibroblast proliferation, collagen gene expression and connective tissue (collagen) accumulation (Antoniades, H.N. Baillieres Clin. Endocrinol. Metab. 1991 Dec;5(4):595-613). Numerous other cytokines including VEGF, as well as cell-matrix interactions, also modify collagen expression and can influence the effects of TGFpi and PDGF (Trojanowska, M. Rheumatology (Oxford) 2008 Oct;47 Suppl 5:v2-4). Persistent overproduction of collagen and other connective tissue results in excessive accumulation of ECM components leading to the formation of scar tissue (fibrosis) in the skin and other organs and is responsible for the progressive nature of scleroderma/SSc (Mauch, C. Rheum. Dis. Clin. North Am. 1990 Feb;16(l):93-107). This leads to thickness and firmness of involved areas. Overall, the pathogenic cascade at different stages of scleroderma/SSc may have autoimmune, inflammatory, fibrotic and vascular components with systemic fibrosis and vasculopathy. Studies indicate that severe fibrosis and abnormal vascular remodeling were detected and the systemic vasculopathy is a hallmark in the pathogenesis of scleroderma/SSc (Yamamoto, T. Autoimmune mechanisms of scleroderma and a role of oxidative stress. 2011 Jan;2(l):4-10).
[0154] Other findings suggest that the pathology of scleroderma/SSc is driven by PDGF, and elevated expression of PDGF and its receptors have been found in scleroderma skin and lung tissues (Mauch 1993). Studies indicate that abnormal vascular remodeling with significant elevations of VEGF and PDGF in SSc patients and systemic vasculopathy is the most striking feature of SSc (Ou, X.M., et al. Int. Immunopharmacol. 2009 Jan;9(l):70-9; Pytel, D., et al. Anticancer Agents Med. Chem. 2009 Jan;9(l):66-76). PDGF and VEGF, together with their cognate receptors, have been shown to be upregulated in the skin of SSc patients.
[0155] The clinical management of patients with scleroderma/SSc remains a challenge and involves several therapeutic approaches. Methotrexate, cyclophosphamide, calcium channel blockers, ACE inhibitors, prostacyclin analogues and D-penicillamine are the most widely studied treatments for SSc. IV gamma globulins, mycophenolate mophetil, rituximab, fluoxetine, pirfenidone, relaxin, halofuginone, and anti-TGF-beta antibodies await more solid data, and side effects are common (Sapadin, A.N., et al. Arch. Dermatol. 2002 Jan;138(l):99-105; Stummvoll G.H. Acta Med. Austriaca 2002;29(l):14-9; Zandman-Goddard, G., et al. Clin. Dev. Immunol. 2005; 12(3): 165-73; Grassegger, A., et al. Clin. Exp. Dermatol. 2004 Nov;29(6):584-8; Nash, R.A., et al. Blood 2007; 110(4): 1388-96; Gavino, E.S. and FurstD.E. BioDrugs 2001;15(9):609- 14; Au, K., et al. Curr. Rhemuatol. Rep. 2009 Apr;l 1(2): 111). A combination of immunosuppressive agents and imatinib was tested in SSc patients for treating SSc-related lung disease (Kay, J. Arthritis Rheum. 2008 Aug;58(8):2543-8; Sabnani, I. Rheumatology (Oxford) 2009 Jan;58(l):49-52). Overall, the result of current research is mixed, with limited positive reports.
Gastrointestinal Diseases, Disorders, and Conditions
[0156] In some embodiments, provided methods are useful for treating gastrointestinal diseases, disorders, or conditions. In some embodiments, provided methods are useful for treating gastrointestinal fibrosis (e.g., fibrosis of esophagus, stomach, intestines, and/or colon). In some embodiments, provided methods are useful for treating gastrointestinal fibrosis secondary to, or otherwise associated with, an underlying indication. In some embodiments, provided methods are useful for treating inflammatory bowel disease (e.g., ulcerative colitis or Crohn’s disease), e.g., treating gastrointestinal fibrosis associated with inflammatory bowel disease. As described in Examples herein, while IBD is primarily an inflammatory autoimmune disease, it was surprisingly found that anti-fibrotic agents (e.g., Compound 1) could be useful in treating IBD and, further could provide benefits beyond mere anti-fibrotic effects.
[0157] In some embodiments, a disease to be treated by methods of the present disclosure is inflammatory bowel disease (IBD). IBD is an inflammatory condition that comprises both ulcerative colitis (UC) and Crohn's disease (CD). While UC affects the entire colon, CD typically affects the ileum but can occur in any part of the GI tract. IBD can manifest as acute or chronic colitis, characterized by recurrent intestinal inflammation accompanied by diarrhea and abdominal pain (Arivarasu, N., et al. Tissue Barriers 2018;6(2):el463897; Ponder, A. and Long, M.D. Clin. Epidemiol. 2013;5:237-47). Recurring bouts of inflammation can lead to tissue remodeling and is a serious presentation in IBD and a major cause of morbidity, often requiring hospitalization and surgical intervention (Wendelsdorf, K., et al. J. Theor. Biol. 2010 Jun 21;264(4): 1225-39; Fornaro, R., et al. J. Dig. Dis. 2015 Oct;16(10):558-67).
[0158] Incidence of IBD is increasing worldwide and is an expanding global health problem (Amosy, E., et al. Clin. Med. Insights Gastroenterol. 2013;6:33-47). An estimated 2.5-3 million people in Europe are affected by IBD (Burisch, J., et al. J. Crohns Colitis 2013 May;7(4):322- 37). According to the Centers for Disease Control and Prevention (CDC), 3.1 million adults in this country were diagnosed with IBD in 2015, a substantial increase from the - 1.4 million adults diagnosed per 2008 reports (www.cdc.gov/IBD; www.cdc.gov/ibd/pdf/inflammatory- bowel-disease-an-expensive-disease.pdf). IBD accounts for -1,300,000 physician visits and -92,000 hospitalizations each year in the United States. Of these, 75% patients diagnosed with CD and 25% patients diagnosed with UC and require surgery. Risk factors associated with IBD include environmental, genetic and immunologic factors (Abegunde, A.T., et al. World J. Gastroenterol. 2016 Jul 21;22(27):6296-6317; Frolkis, A., et al. Can. J. Gastroenterol. 2013 Mar; 27(3 ) : e28 -24) .
[0159] IBD is a major cause of morbidity in patients and is a major consumer of the health care budget. A European study estimated that direct healthcare costs for IBD in Europe are -5 billion Euros/year (Bursich 2013). In 2008, CDC reports indicate that direct treatment costs with IBD were estimated ~$6.3 billion and indirect costs were estimated to cost an additional $5.5 billion (www.cdc.gov/IBD). A study in 2017 indicated that the annual direct and indirect costs related to ulcerative colitis (UC) are estimated to be as high as €12.5-29.1 billion in Europe and US$8.1-14.9 billion in the USA (Ungaro, R., et al. Lancet 2017 Apr 29;389(10080): 1756-1770). Thus, IBD is an expensive disease without a cure.
[0160] IBD is an autoimmune disease with excessive activation of the adaptive immune response. Various factors including genetic factors alter the intestinal flora and trigger an inflammatory reaction, activate T cells, B cells, mast cells, macrophages and microglia, smooth muscle cells and fibroblasts in the colon, inducing mucosal disruption (Hildner, K., et al. Dig. Dis. 2016;34Suppl 1:40-7; Curciarello, R., et al. Front Med. (Lausanne) 2017 Aug 7;4:126). Epithelial and endothelial damage release chemotactic factors promoting recruitment and activation of inflammatory cells, and release various cytokines including TNFa, and activate fibroblasts via TGFpi. Activated fibroblasts, i.e. myofibroblasts, secrete growth factors including platelet derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) (Scaldaferri, et al. Gastroenterology 2009 Feb;136(2):585-95.e5). Studies indicate that angiogenesis is also an important part of IBD pathogenesis in the colon of IBD patients. In fact, Alkim, et al. demonstrated enhanced microvessel density in the intestinal tissue of both UC and CD patients, which correlated both the level of local VEGF expression and disease activity (Int. J. Inflam. 2015;2015:970890). [0161] Anti-inflammatory drugs, including 5-aminosalicylic acid (5-ASA)-based preparations, are often the first line of therapy in IBD (Segars, L.W., et al. Clin. Pharm. 1992 Jun;ll(6):514-28). Anti-TNFa antibodies such as infliximab and adalimumab are also being used. Nevertheless, patients treated with adalimumab are at increased risk for serious infections and lymphoma (Dulai, P.S., et al. Clin. Gastroenterol. Hepatol. 2014 Sep; 12(9): 1443-51). Corticosteroids, other immuno-suppressants, and antibiotics exhibit multiple side effects with relatively poor treatment responses (Kopylov, U., et al. Adv. Gastroenterol. 2016 Jul:9(4):513- 26; Waljee, A.K., et al. PLoS One 2016 Jun 23;ll(6):e0158017; Cosnes, J., et al. Gut 2005;54:237-241).
[0162] Studies indicate that PDGF and its receptors are highly expressed in areas of ongoing inflammation and fibrosis in IBD (Zeisberg, M. and Kalluri, R. Am. J. Physiol. Cell Physiol.
2013 Feb 1;304(3):C216-C225). PDGF activates fibroblasts and IBD-fibroblasts proliferate more rapidly than normal fibroblasts; collagen secretion from IBD patients’ fibroblasts was increased compared to collagen secretion by normal fibroblasts. IBD is also associated with increased circulating PDGF and the level of this growth factor has been reported to correspond with disease severity (Andrae, J., et al. Genes Dev. 2008 May 15;22(10): 1276-1312).
[0163] Studies indicate that angiogenesis is a novel component of IBD pathogenesis and angiogenic activity is increased in IBD patients. Serum VEGF levels were significantly higher in IBD patients compared to controls in several studies. Griga et al. demonstrated that sources of increased serum VEGF were from inflamed intestinal tissue of IBD patients (Scand. J. Gastroenterol. 1998 May;33(5):504-8; Hepatogastroenterology 2002 Jan-Feb;49(43): 116-23; Hepatogastroenterology 1999 Mar-Apr;46(26):920-3; Eur. J. Gastroenterol. Hepatol. 1999 Feb; 11(2): 175-9). Furthermore, they found VEGF expression was markedly increased in the inflamed mucosa of both CD and UC patients, when compared with normal mucosa of the same patient. Studies also showed that VEGF expression was increased in colon and was higher across all IBD groups (both CD and UC) when compared with healthy controls. Scaldaferri, et al. (2009) reported that VEGF receptor (VEGFR/KDR) levels were increased in intestinal samples of IBD patients, and in mice with experimental colitis. Other Diseases, Disorders, and Conditions
[0164] In some embodiments, provided methods are useful for treating certain other diseases, disorders, or conditions. In some embodiments, provided methods are useful for treating cardiac fibrosis and/or fibrosis associated with cardiovascular system. In some embodiments, provided methods are useful for treating cardiac fibrosis secondary to, or otherwise associated with, an underlying indication. In some embodiments, provide methods are useful for treating cardiac and/or cardiovascular fibrosis associated with ischemic heart disease, myocardial ischemia, athereosclerosis, myocardial perfusion (e.g., as a consequence of chronic cardiac ischemia or myocardial infarction), vascular occlusion, or restenosis.
[0165] In some embodiments, a disease to be treated by methods of the present disclosure is ischemic heart disease. Ischemic heart disease is a leading cause of morbidity and mortality in the US, afflicting millions of Americans each year at a cost expected to exceed $300 billion/year. Numerous pharmacological and interventional approaches are being developed to improve treatment of ischemic heart disease including reduction of modifiable risk factors, improved revascularization procedures, and therapies to halt progression and/or induce regression of atherosclerosis. Furthermore, atherosclerosis comprises a fibrotic component.
[0166] In some embodiments, provided methods are useful for treating fibrosis associated with central nervous system (CNS) and/or one or more CNS-related diseases, disorders, or conditions. In some embodiments, provided methods are useful for treating CNS-associated fibrosis secondary to, or otherwise associated with, an underlying indication. In some embodiments, provided methods are useful for treating fibrosis associated with cerebral infarction, stroke, or amyotrophic lateral sclerosis.
[0167] In some embodiments, provided methods are useful for treating fibrosis associated with musculoskeletal system and/or one or more musculoskeletal diseases, disorders, or conditions. In some embodiments, provided methods are useful for treating musculoskeletal- associated fibrosis secondary to, or otherwise associated with, an underlying indication. In some embodiments, provided methods are useful for treating fibrosis associated with muscular dystrophy.
[0168] In some embodiments, provided methods are useful for treating pancreatic fibrosis.
In some embodiments, provided methods are useful for treating pancreatic fibrosis secondary to, or otherwise associated with, an underlying indication. In some embodiments, provided methods are useful for treating fibrosis associated with pancreatitis.
Subjects to Be Treated
[0169] In some embodiments, one or more subjects or populations are selected to receive Compound 1 as described herein based on one or more markers and/or characteristics such as, for example, one or more risk factors of fibrosis or an associated disease, disorder or condition and/or one or more biomarkers, etc.
[0170] In some embodiments, a subject or population thereof is selected to receive Compound 1 using technologies provided herein (e.g., based on assessment of one or more markers and/or characteristics, such as one or more biomarkers). In some embodiments, such technologies are used to inform or determine one or more features of a therapeutic regimen (e.g., selection of subject(s) to receive a particular therapy (e.g., Compound 1 therapy) and/or dose thereof and/or timing of administration of such therapy).
[0171] In some embodiments, assessment of one or more markers and/or characteristics is performed with respect to the same subject at a plurality of different time points. In some embodiments, assessment of one or more markers and/or characteristics is performed with respect to a particular patient prior to initiation of a particular therapeutic regimen (e.g., a Compound 1 therapeutic regimen) and/or prior to administration of a particular dose of therapy (e.g., Compound 1 therapy) in accordance with such therapeutic regimen.
[0172] In some embodiments, a subject or population thereof is suffering from or susceptible to a disease, disorder, or condition described herein. In some embodiments, a subject or population thereof is suffering from or is susceptible to fibrosis. In some embodiments, a subject or population thereof is suffering from or is susceptible to a disease, disorder, or condition characterized by or otherwise associated with fibrosis. In some embodiments, a subject or population thereof is suffering from or is susceptible to fibrosis of gastrointestinal tract, heart, kidney, lung, liver, muscle, pancreas, and/or skin.
[0173] In some embodiments, a subject or population thereof is suffering from or is susceptible to an acute injury (e.g., an acute organ injury, such as acute lung injury, acute liver injury, or acute kidney injury). In some embodiments, a subject or population thereof is suffering from or is susceptible to a chronic injury (e.g., a chronic organ injury, such as chronic lung injury, chronic liver injury, or chronic kidney injury). In some embodiments, a subject or population thereof is suffering from a traumatic injury. In some embodiments, a subject or population thereof has undergone, is undergoing, or will undergo an organ transplantation. In some embodiments, a subject or population thereof is suffering from or susceptible to a damaged and/or ischemic organ, transplant, or graft. In some embodiments, a subject or population thereof is suffering from or susceptible to ischemia/reperfusion injury. In some embodiments, a subject or population thereof is suffering from or susceptible to post-surgical scarring. In some embodiments, a subject or population thereof is suffering from a wound.
[0174] In some embodiments, a subject or population thereof is suffering from or is susceptible to a pulmonary disease, disorder, or condition. In some embodiments, a subject or population thereof is suffering from or is susceptible to pulmonary fibrosis. In some embodiments, a subject or population thereof is suffering from or is susceptible to pulmonary fibrosis secondary to, or otherwise associated with, an underlying indication. In some embodiments, a subject or population thereof is suffering from or is susceptible to interstitial lung disease (e.g., fibrosing interstitial lung disease). In some embodiments, a subject or population thereof is suffering from or is susceptible to idiopathic interstitial pneumonia. In some embodiments, a subject or population thereof is suffering from or is susceptible to idiopathic pulmonary fibrosis.
[0175] In some embodiments, a subject or population thereof is suffering from or is susceptible to a hepatic disease, disorder, or condition. In some embodiments, a subject or population thereof is suffering from or is susceptible to hepatic fibrosis (e.g., fibrotic liver disease). In some embodiments, a subject or population thereof is suffering from or is susceptible to cirrhosis. In some embodiments, a subject or population thereof is suffering from or is susceptible to hepatic fibrosis secondary to, or otherwise associated with, an underlying indication. In some embodiments, a subject or population thereof is suffering from or is susceptible to hepatitis C, hepatitis B, delta hepatitis, chronic alcoholism, nonalcoholic steatohepatitis (NASH), extrahepatic obstructions (e.g., stones in bile duct), cholangiopathies (e.g., primary biliary cirrhosis or sclerosing cholangitis), autoimmune liver disease, or inherited metabolic disorders (e.g., Wilson’s disease, hemochromatosis, or alpha-1 antitrypsin deficiency). [0176] In some embodiments, a subject or population thereof is suffering from or is susceptible to a renal disease, disorder, or condition. In some embodiments, a subject or population thereof is suffering from or is susceptible to renal fibrosis. In some embodiments, a subject or population thereof is suffering from or is susceptible to renal fibrosis secondary to, or otherwise associated with, an underlying indication. In some embodiments, a subject or population thereof is suffering from or is susceptible to renal failure, renal obstruction, renal trauma, renal transplantation, chronic kidney disease, diabetes, hypertension, radiocontrast nephropathy, immune-mediated glomerulonephri tides (e.g., lupus nephritis, ANCA-associated glomerulonephritides (e.g., Wegener’s granulomatosis, microscopic polyangiitis, or renal limited vasculitis), anti-GBM nephropathy, IgA nephropathy, membranous glomerulonephritis, or focal and segmental glomerulosclerosis), non-immune-mediated glomerulonephritides (e.g., polycystic kidney disease, collagen type III glomerulopathy, nail-patella syndrome, or Alport syndrome), minimal change disease, or nephrotic syndrome (e.g., steroid-resistant nephrotic syndrome). In some embodiments, a subject or population thereof is suffering from or is susceptible to a fibrotic disease of the kidney that is or comprises: focal segmental glomerulosclerosis (FSGS), steroid resistant nephrotic syndrome (SRNS), proteinuria, lupus nephritis, minimal change disease, an anti-neutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis, Alport syndrome, anti-globular basement membrane (anti-GBM) nephropathy, IgA nephropathy, membranous glomerulonephritis (MG), autosomal dominant polycystic kidney disease (ADPKD), collagen type III glomerulopathy, nail-patella syndrome, or chronic kidney disease.
In some embodiments, a subject or population thereof is suffering from or is susceptible to a fibrotic disease of the kidney that is or comprises an anti-neutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis. In some embodiments, ANCA-associated glomerulonephritis is selected from Wegener’s granulomatosis, microscopic polyangiitis (MPA), or renal limited vasculitis. In some embodiments, a subject or population thereof is suffering from or is susceptible to collagen type III glomerulopathy or nail-patella syndrome. In some embodiments, a subject or population thereof is suffering from or is susceptible to nephrotic syndrome and/or diseases, disorders, or conditions associated with nephrotic syndrome (e.g., focal and segmental glomerulosclerosis, minimal change disease, and membranous nephropathy). In some embodiments, a subject or population thereof is suffering from or is susceptible to focal and segmental glomerulosclerosis. In some embodiments, a subject or population thereof is suffering from or is susceptible to Alport syndrome. In some embodiments, a subject or population thereof is suffering from or is susceptible to polycystic kidney disease (e.g., autosomal dominant polycystic kidney disease or autosomal recessive polycystic kidney disease).
[0177] In some embodiments, a subject or population thereof is suffering from or is susceptible to primary proteinuric kidney disease (e.g., as confirmed from a renal biopsy). In some embodiments, a subject or population thereof is suffering from or is susceptible to primary glomerular diseases (e.g., as confirmed from a renal biopsy). In some embodiments, a subject or population thereof is suffering from or susceptible to persistent proteinuria. In some embodiments, a subject or population thereof is suffering from or susceptible to primary glomerular disease (e.g., as confirmed from a renal biopsy) and persistent proteinuria. In some embodiments, a subject or population thereof is suffering from or susceptible to proteinuric chronic kidney disease. In some embodiments, a subject or population thereof is suffering from or is susceptible to focal and segmental glomerulosclerosis. In some embodiments, a subject or population thereof is suffering from or is susceptible to membranous nephropathy. In some embodiments, a subject or population thereof is suffering from or susceptible to IgA nephropathy.
[0178] In some embodiments, a subject or population thereof has one or more risk factors for primary glomerular disease and/or primary proteinuric kidney disease. In some embodiments, a subject or population thereof has one or more risk factors for primary glomerular disease and/or primary proteinuric kidney disease selected from proteinuria, renal dysfunction, hypertension, interstitial fibrosis on renal biopsy, and lack of response to therapy or relapse in proteinuria. For example, in some embodiments, a subject or population thereof has one or more risk factors for primary glomerular disease (e.g., FSGS) selected from serum creatinine > 1.3 mg/dL, proteinuria > 3.5 g/day, lack of response to therapy, and/or a collapsing variant. In some embodiments, a subject or population thereof has one or more risk factors for primary glomerular disease (e.g., IgA nephropathy) selected from serum creatinine > 1.26 mg/dL, hypertension (e.g., >140/90 mmHg), and/or persistent (e.g., > 6 months) protein excretion > 1000 mg/day. In some embodiments, a subject or population thereof has one or more risk factors for primary glomerular disease (e.g., membranous nephropathy) selected from serum creatinine > 1.5 mg/dL, progressive decline in eGFR > 25% over prior 2 years, and/or severe disabling or life-threatening nephrotic syndrome (with e.g., serum albumin < 2.5 g/dL, refractory edema, and/or thromboembolic event). In some embodiments, a subject or population thereof has a risk factor for primary glomerular disease (e.g., FSGS, IgA nephropathy, and/or membranous nephropathy) of eGFR < 70 mL/min/1.73m2.
[0179] In some embodiments, a subject or population thereof has an estimated glomerular filtration rate (eGFR) of greater than or equal to 40 mL/min/1.73m2, e.g., calculated using Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI). In some embodiments, a subject or population thereof has an estimated glomerular filtration rate (eGFR) of greater than or equal to 30 mL/min/1.73m2, e.g., calculated using Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI).
[0180] In some embodiments, a subject or population thereof has a urinary protein excretion of greater than or equal to 1 g/day on a 24-hour urine collection.
[0181] In some embodiments, a subject or population thereof has stable blood pressure (e.g., less than or equal to 140/90 mmHg) for at least 12 weeks. In some embodiments, a subject or population thereof is on a stable antihypertensive regimen and has stable blood pressure (e.g., less than or equal to 140/90 mmHg) for at least 12 weeks.
[0182] In some embodiments, a subject or population thereof is receiving or has received standard of care therapy. In some embodiments, a subject or population thereof is receiving or has received a maximally tolerated or recommended dose of standard of care therapy. In some embodiments, a subject or population is receiving or has received standard of care therapy and has been stable on said therapy for at least 12 weeks. In some embodiments, a subject or population thereof is resistant to standard of care therapy. In some embodiments, a subject or population thereof has relapsed after receiving standard of care therapy. In some embodiments, standard of care therapy comprises immunosuppressive or immunomodulatory agents. In some embodiments, standard of care therapy comprises renin-angiotensin-aldosterone system (RAAS) blockers, such as, e.g., ACE inhibitors or angiotensin-receptor blockers (ARBs). In some embodiments, standard of care therapy comprises immunosuppressive agents and RAAS blockers (e.g., ACE inhibitors or ARBs). In some embodiments, standard of care comprises blood pressure control (e.g., to < 130/80 mmHg) and/or HMG-CoA reductase inhibitors (i.e., statins), particularly in patients with hyperlipidemia.
[0183] In some embodiments, a subject or population thereof is receiving or has received an immunosuppressive or immunomodulatory agent. In some embodiments, a subject or population thereof is receiving or has received therapy comprising an immunosuppressive or immunomodulatory agent and has been stable on said therapy for at least 12 weeks.
[0184] In some embodiments, a subject or population thereof is receiving or has received a RAAS blocker. In some embodiments, a subject or population thereof is receiving or has received an ACE inhibitor. In some embodiments, a subject or population thereof is receiving or has received an ARB. In some embodiments, a subject or population thereof is receiving or has received a maximally tolerated or recommended dose of a RAAS blocker (e.g., an ACE inhibitor or ARB). In some embodiments, a subject or population thereof is receiving or has received an inhibitor of mineralocorticoid receptor. In some embodiments, a subject or population thereof is receiving or has received an inhibitor of sodium glucose co-transporter-2 (SGLT-2). In some embodiments, a subject or population thereof is receiving or has received therapy comprising a RAAS blocker (e.g., an ACE inhibitor or ARB) or SGLT-2 inhibitor and has been stable on said therapy for at least 12 weeks.
[0185] In some embodiments, a subject or population thereof is receiving or has received therapy comprising an immunosuppressive or immunomodulatory agent and one or more of a RAAS blocker (e.g., ACE inhibitor or ARB), mineralocorticoid receptor inhibitor or SGLT-2 inhibitor. In some such embodiments, the subject or population thereof has been stable on said therapy for at least 12 weeks.
[0186] In some embodiments, a subject or population thereof is not receiving non-steroidal anti-inflammatory agents (NSAIDS). In some embodiments, a subject or population thereof is not receiving non-steroidal anti-inflammatory agents (NSAIDS) chronically.
[0187] In some embodiments, a subject or population thereof is negative for hepatitis B virus, hepatitis C virus, and/or human immunodeficiency virus. In some embodiments, a subject or population thereof does not have a history of treated hepatitis C virus.
[0188] In some embodiments, a subject or population thereof does not have any one or more hematologic abnormalities selected from hemoglobin < 8 g/dL, platelets < 50,000, and absolute neutrophil count (ANC) < 1000 cells/pL. In some embodiments, a subject or population thereof does not have hemoglobin AIC > 8.5%.
[0189] In some embodiments, a subject or population thereof does not have any one or more liver function results selected from aspartate aminotransferase (AST), alanine aminotransferase (ALT), or total bilirubin > 2x upper limit of normal (ULN). [0190] In some embodiments, a subject or population thereof has no known predisposition to bleeding. In some embodiments, a subject or population thereof does not require fibrinolysis, full-dose therapeutic anti coagulation (e.g., vitamin K antagonists, dabigatran, heparin, hirudin, etc.), or high dose antiplatelet therapy.
[0191] In some embodiments, a subject or population thereof has no history of hemorrhagic central nervous system events, e.g., within 12 months. In some embodiments, a subject or population thereof has no history of active gastrointestinal bleeding, e.g., within 6 months. In some embodiments, a subject or population thereof has no history of thrombotic events (including, e.g., stroke or transient ischemic attack), e.g., within 12 months.
[0192] In some embodiments, a subject or population thereof is not diagnosed with Type I diabetes mellitus. In some embodiments, a subject or population thereof has never had (e.g., in the last 5 years) a renal biopsy showing histopathological evidence of diabetic kidney disease. In some embodiments, a subject or population thereof is not diagnosed with anti-PLA2R antibody- associated membranous nephropathy.
[0193] In some embodiments, a subject or population thereof has not had a myocardial infarction or unstable angina, e.g., within 6 months.
[0194] In some embodiments, a subject or population thereof has no history of solid organ or hematopoietic cell transplantation. In some embodiments, a subject or population thereof is not on an organ transplant waiting list.
[0195] In some embodiments, a subject or population thereof has no history or presence of any form of cancer except excised basal or squamous cell carcinoma of the skin, e.g., within 2 years.
[0196] In some embodiments, a subject or population thereof is not suffering from renal disease secondary to systemic disease, including but not limited to systemic lupus erythematosus, anti-neutrophil cytoplasmic antibodies-associated diseases, anti-glomerular basement disease, secondary forms of focal segmental glomerulosclerosis, renal diseases associated with para- proteinemias, C3 glomerulopathy, and diabetic kidney disease. In some embodiments, a subject or population thereof is not suffering from a known systemic disorder that requires or is expected to require systemic glucocorticoids or immune modulators.
[0197] In some embodiments, a subject or population thereof has not received treatment with anti-CD20 monoclonal antibodies, e.g., within 6 months. [0198] In some embodiments, a subject or population thereof has a BMI of greater than or equal to 40 kg/m2.
[0199] In some embodiments, a subject or population thereof is suffering from or is susceptible to a dermal disease, disorder, or condition. In some embodiments, a subject or population thereof is suffering from or is susceptible to dermal fibrosis. In some embodiments, a subject or population thereof is suffering from or is susceptible to dermal fibrosis secondary to, or otherwise associated with, an underlying indication. In some embodiments, a subject or population thereof is suffering from or is susceptible to scleroderma and/or systemic sclerosis (e.g., diffuse systemic sclerosis or limited systemic sclerosis).
[0200] In some embodiments, a subject or population thereof is suffering from or is susceptible to a gastrointestinal disease, disorder, or condition. In some embodiments, a subject or population thereof is suffering from or is susceptible to gastrointestinal fibrosis (e.g., fibrosis of esophagus, stomach, intestines, and/or colon). In some embodiments, a subject or population thereof is suffering from or is susceptible to gastrointestinal fibrosis secondary to, or otherwise associated with, an underlying indication. In some embodiments, a subject or population thereof is suffering from or is susceptible to inflammatory bowel disease (e.g., ulcerative colitis or Crohn’s disease).
[0201] In some embodiments, a subject or population thereof is suffering from or is susceptible to cardiac fibrosis and/or fibrosis associated with cardiovascular system. In some embodiments, a subject or population thereof is suffering from or is susceptible to cardiac fibrosis secondary to, or otherwise associated with, an underlying indication. In some embodiments, a subject or population thereof is suffering from or is susceptible to ischemic heart disease, myocardial ischemia, atherosclerosis, myocardial perfusion (e.g., as a consequence of chronic cardiac ischemia or myocardial infarction), vascular occlusion, or restenosis.
[0202] In some embodiments, a subject or population thereof is suffering from or is susceptible to fibrosis associated with central nervous system (CNS) and/or one or more CNS- related diseases, disorders, or conditions. In some embodiments, a subject or population thereof is suffering from or is susceptible to CNS-associated fibrosis secondary to, or otherwise associated with, an underlying indication. In some embodiments, a subject or population thereof is suffering from or is susceptible to cerebral infarction, stroke, or amyotrophic lateral sclerosis. [0203] In some embodiments, a subject or population thereof is suffering from or is susceptible to fibrosis associated with musculoskeletal system and/or one or more musculoskeletal diseases, disorders, or conditions. In some embodiments, a subject or population thereof is suffering from or is susceptible to musculoskeletal-associated fibrosis secondary to, or otherwise associated with, an underlying indication. In some embodiments, a subject or population thereof is suffering from or is susceptible to muscular dystrophy.
[0204] In some embodiments, a subject or population thereof is suffering from or is susceptible to pancreatic fibrosis. In some embodiments, a subject or population thereof is suffering from or is susceptible to pancreatic fibrosis secondary to, or otherwise associated with, an underlying indication. In some embodiments, a subject or population thereof is suffering from or is susceptible to pancreatitis.
Biomarkers
[0205] In some embodiments, the present disclosure also provides methods related to treatment of fibrotic and related disease(s) (e.g., as described herein, such as kidney fibrotic disease(s) or lung fibrotic disease(s)) and selecting, identifying, and/or characterizing patients likely to benefit from a treatment with Compound 1 as described herein.
[0206] In some embodiments, the present disclosure is based in part on the discovery that certain biomarkers can distinguish patients who are likely to respond to therapy, for example because the drivers of their disease (e.g., as described herein, such as kidney disease or lung disease) correspond with a mechanism of action of Compound 1. For example, in some embodiments, a patient to be treated with a method of the present disclosure has an altered level of one or more gene products or one or more proteins (or fragments thereof) that are part of the mechanism of action of Compound 1 (e.g., down regulated by Compound 1).
[0207] Generally, a biomarker is a component of a biological sample that may be detected and/or quantified when present in the biological sample.
[0208] In some embodiments, a patient to be treated with a method of the present disclosure has an elevated level of gene expression (e.g., COL3A1 expression and/or COL1A1 expression). In some embodiments, a patient to be treated with a method of the present disclosure has an elevated level of protein expression (e.g., COL3A1 protein and/or COL1 A1 protein) or elevated level of a fragment thereof. As used herein, an elevated level of expression, e.g., of COL3A1 and/or COL1 Al, may refer to either to an elevated level of a gene product (e.g., mRNA expressed from a COL3A1 gene and/or COL1A1 gene) and/or protein (e.g., COL3A1 protein and/or COL1 Al protein), or a fragment thereof. For simplicity, a non-italicized name will be used to refer to both the gene and protein product.
[0209] In some embodiments, a level of COL3A1 and/or COL1A1 corresponds to a level of gene expression (e.g., RNA expression, e.g., mRNA expression). In some embodiments, a level of COL3A1 and/or COL1 Al corresponds to a level of collagen protein expression. As used herein, collagen protein expression includes expression of a protein in any form, including, a procollagen polypeptide (e.g., type III procollagen or type I procollagen), a collagen protein (e.g., type III collagen or type I collagen), a preprocollagen polypeptide, an amino-terminal procollagen polypeptide (e.g., pN-type III collagen (“PIIINP”) or pN-type I collagen (“PINP”)), collagen fibril, collagen fiber, and/or any fragment or degradation product thereof. It will be appreciated that “type III collagen” may also be referred to as “collagen type III” or “collagen III”, and that “type I collagen” may also be referred to as “collagen type I” or “collagen I”. [0210] In some embodiments provided herein, a level of COL3 Al refers to a gene product (e.g., RNA, e.g., mRNA) or a protein product (e.g., a prepropolypeptide, propolypeptide, amino- terminal propolypeptide, protein, fibril, fiber, or any fragment or degradation product thereof).
In some embodiments provided herein, a level of COL1A1 refers to a gene product (e.g., RNA, e.g., mRNA) or a protein product (e.g., a prepropolypeptide, propolypeptide, amino-terminal propolypeptide, protein, fibril, fiber, or any fragment or degradation product thereof).
[0211] In some embodiments, a method for treating a patient diagnosed with, suspected of having, or at risk for a fibrotic disease (e.g., a disease described herein, such as a fibrotic disease of the kidney or the lung), is provided, the method comprising: (i) obtaining or determining a level of expression of a biomarker (e.g., COL3 Al and/or COL1 Al) in a biological sample from the patient; (ii) comparing the level of biomarker expression with a threshold level (e.g., a predetermined mean or median level of a population of healthy subjects), wherein if the expression level of the biomarker is different from the threshold level (e.g., above the threshold level or below the threshold level), administering to the patient an effective amount of Compound 1, or a pharmaceutical composition thereof. It will be appreciated that obtaining a level of expression of a biomarker may comprise obtaining knowledge of a level that has been determined previously (e.g., obtaining said level from a report, database, etc. containing the value of a previously determined level).
[0212] In some embodiments, a method for treating a patient diagnosed with, suspected of having, or at risk for a fibrotic disease (e.g., a fibrotic disease described herein, such as a fibrotic disease of the kidney or the lung), is provided, the method comprising: (i) obtaining or determining a level of expression of a biomarker (e.g., COL3A1 and/or COL1 Al) in a biological sample from the patient; (ii) comparing the level of biomarker expression with a threshold level (e.g., a predetermined mean or median level of a population of healthy subjects), wherein if the expression level of the biomarker is above the threshold level, administering to the patient an effective amount of Compound 1, or a pharmaceutical composition thereof.
[0213] In some embodiments, a method for treating a patient diagnosed with, suspected of having, or at risk for a fibrotic disease (e.g., a disease described herein, such as a fibrotic disease of the kidney or the lung), is provided, the method comprising administering an effective amount of Compound 1, or a pharmaceutical composition thereof, to a patient that has been determined to have an altered (e.g., elevated or reduced) level of a biomarker (e.g., COL3 Al and/or COL1 Al). In some embodiments, a method for treating a patient diagnosed with, suspected of having, or at risk for a fibrotic disease (e.g., a fibrotic disease described herein, such as a fibrotic disease of the kidney or the lung), is provided, the method comprising administering an effective amount of Compound 1, or a pharmaceutical composition thereof, to a patient that has been determined to have an elevated level of a biomarker (e.g., COL3 Al and/or COL1 Al). In some embodiments, an elevated level of a biomarker comprises a level that is above that of a corresponding threshold level. In some embodiments, a reduced level of COL3A1 and/or COL1 Al comprises a level that is below a corresponding threshold level. In some embodiments, the level of biomarker (e.g., COL3A1 and/or COL1 Al) was obtained from or previously determined from a biological sample from the patient. In some embodiments, the method further comprises obtaining or determining a level of a biomarker (e.g., COL3A1 and/or COL1 Al) in a biological sample from the patient.
[0214] In some embodiments, a method for treating a patient diagnosed with, suspected of having, or at risk for a fibrotic disease (e.g., a fibrotic disease described herein, such as a fibrotic disease of the kidney or the lung) in a patient characterized by an elevated level of a biomarker (e.g., COL3A1 and/or COL1A1) is provided, the method comprising administering an effective amount of Compound 1 or a pharmaceutical composition thereof, to the patient. In some embodiments, an elevated level of a biomarker comprises a level that is above a corresponding threshold level. In some embodiments, the level of biomarker (e.g., COL3A1 and/or COL1A1) was obtained from or previously determined from a biological sample from the patient. In some embodiments, the method further comprises obtaining or determining a level of a biomarker (e.g., COL3A1 and/or COL1 Al) in a biological sample from the patient.
[0215] In some embodiments, a threshold expression level corresponds to a predetermined mean or median level of COL3A1 and/or COL1A1 of a population of healthy subjects (e.g., healthy human subjects). In some embodiments, a threshold expression level corresponds to a predetermined normal range of COL3A1 and/or COL1A1 of a population of healthy subjects (e.g., healthy human subjects).
[0216] In some embodiments, the expression level of COL3 Al is at least 20% higher than the corresponding threshold expression level. In some embodiments, the expression level of COL1 Al is at least 20% higher than the corresponding threshold expression level. In some embodiments, the expression levels of both COL3A1 and COL1 Al are at least 20% higher than the respective corresponding threshold expression levels.
[0217] In some embodiments, the expression level of COL3 Al is at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% higher than the corresponding threshold expression level. In some embodiments, the expression level of COL1 Al is at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% higher than the corresponding threshold expression level. In some embodiments, the expression levels of both COL3A1 and COL1 Al are at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% higher than the respective corresponding threshold expression levels.
[0218] It will be appreciated that a variety of biological samples are suitable for the methods provided herein, including amniotic fluid, aqueous humor, ascites, bile, bone marrow, blood, breast milk, bronchoalveolar lavage fluid, cerebrospinal fluid, cerumen, chyle, chime, ejaculate, endolymph, exudate, feces, gastric acid, gastric juice, lymph, mucus, pericardial fluid, perilymph, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum, semen, serum, smegma, sputum, synovial fluid, sweat, tears, urine, vaginal secretions, vitreous humour, vomit, and/or combinations or component(s) thereof. In some embodiments, a biological fluid may be or comprise an intracellular fluid, an extracellular fluid, an intravascular fluid (blood plasma), an interstitial fluid, a lymphatic fluid, and/or a transcellular fluid. In some embodiments, a biological tissue or sample may be obtained, for example, by aspirate, biopsy (e.g., fine needle or tissue biopsy), swab (e.g., oral, nasal, skin, or vaginal swab), scraping, surgery, washing or lavage (e.g., bronchoalveolar, ductal, nasal, ocular, oral, uterine, vaginal, or other washing or lavage).
[0219] In some embodiments, the biological sample (e.g., from which an expression level of a biomarker is obtained or has been determined) is a renal biopsy sample, a urine sample or a blood sample. In some embodiments, an expression level of a protein biomarker (e.g., a prepropeptide, propeptide, amino-terminal propeptide, protein, fibril or fiber, e.g., of COL3A1 and/or of COL1 Al) is obtained from or has been determined in a renal biopsy sample, a urine sample and/or a blood sample. In some embodiments, an expression level of a genetic biomarker (e.g., a gene product, e.g., RNA, e.g., mRNA, e.g., mRNA encoding COL3A1 and/or COL1A1) is obtained from or has been determined in a renal biopsy sample, a urine sample, and/or a blood sample.
[0220] In some embodiments, the biological sample (e.g., from which an expression level of a biomarker is or has been determined) is a lung biopsy sample, a bronchoalveolar lavage fluid (BALF) sample or a blood sample. In some embodiments, an expression level of a protein biomarker (e.g., a prepropeptide, propeptide, amino-terminal propeptide, protein, fibril or fiber, e.g., of COL3A1 and/or of COL1 Al) is obtained from or has been determined in a lung biopsy sample, a BALF sample or a blood sample. In some embodiments, an expression level of a genetic biomarker (e.g., a gene product, e.g., RNA, e.g., mRNA, e.g., mRNA encoding COL3A1 and/or COL1A1 ) is obtained from or has been determined in a lung biopsy sample, a BALF sample or a blood sample.
[0221] In some embodiments, an expression level of a biomarker is obtained from or determined in a renal biopsy sample from a patient. In some embodiments, an expression level of a biomarker (e.g., COL3A1 and/or COL1 Al) in a renal biopsy sample is a gene biomarker (e.g., RNA, e.g., mRNA) and/or a protein biomarker. In some embodiments, the biological sample is a renal biopsy sample and the expression level of COL3 Al is an mRNA level and/or the expression level of COL1A1 is an mRNA level. In some embodiments, the biological sample is a renal biopsy sample and the expression level of COL3 Al is an mRNA level and/or the expression level of COL1 A1 is an mRNA level. Methods for measuring and/or determining levels of a biomarker (e.g., mRNA level) in renal biopsy samples are known in the art, for example, Genovese et ah, Biomark Insights . 2016 May 22;ll:77-84.
[0222] In some embodiments, an expression level of a biomarker is obtained from or determined in a urine sample from a patient. In some embodiments, the biological sample is a urine sample and the expression level of COL3 A1 is a level of COL3 A1 protein or fragments thereof and/or the expression level of COL1 A1 is a level of COL1 A1 protein or fragments thereof. Methods for measuring and/or determining levels of a biomarker (e.g., a level of collagen protein and/or fragment thereof) in urine samples are known in the art, for example, Soylemezoglu et ah, Nephrol Dial Transplant. 1997 Sep;12(9):1883-9.
[0223] In some embodiments, an expression level of a biomarker is obtained from or determined in a blood sample from a patient. In some embodiments, the biological sample is a blood sample and the expression level of COL3 A1 is a level of COL3 A1 protein or fragments thereof and/or the expression level of COL1 A1 is a level of COL1 A1 protein or fragments thereof. Methods for measuring and/or determining levels of a biomarker (e.g., a level of collagen protein and/or fragment thereof) in blood samples are known in the art.
[0224] In some embodiments, an expression level of a biomarker is obtained from or determined in a lung biopsy sample from a patient. In some embodiments, an expression level of a biomarker (e.g., COL3A1 and/or COL1 Al) in a lung biopsy sample is a gene biomarker (e.g., RNA, e.g., mRNA) and/or a protein biomarker. In some embodiments, the biological sample is a lung biopsy sample and the expression level of COL3 Al is an mRNA level and/or the expression level of COL1A1 is an mRNA level. In some embodiments, the biological sample is a lung biopsy sample and the expression level of COL3 Al is an mRNA level and/or the expression level of COL1 Al is an mRNA level. Methods for measuring and/or determining levels of a biomarker (e.g., mRNA level) in lung biopsy samples are known in the art.
[0225] In some embodiments, an expression level of a biomarker is obtained from or determined in a BALF sample from a patient. In some embodiments, the biological sample is a BALF sample and the expression level of COL3 Al is a level of COL3 Al protein or fragments thereof and/or the expression level of COL1 Al is a level of COL1 Al protein or fragments thereof. Methods for measuring and/or determining levels of a biomarker (e.g., a level of collagen protein and/or fragment thereof) in BALF samples are known in the art, for example, Wattiez, R. and Falmagne, P., J Chromatogr B Analyt Technol Biomed Life Sci. 2005 Feb 5;57(9):2005-l 1.
[0226] In some embodiments, the present disclosure provides methods of administering Compound 1 to a subject or population of subjects described herein, according to a regimen established to achieve one or more desirable outcomes. In some embodiments, the fibrotic disease (e.g., a fibrotic disease described herein, such as a fibrotic disease of the kidney or the lung) is stabilized (i.e., does not worsen) and/or is ameliorated (i.e., one or more symptoms improve) in a patient treated with Compound 1. In some embodiments, treatment of a patient with Compound 1, reduces a level of one or more biomarkers (e.g., COL1 A1 and/or COL3A1).
In some embodiments, treatment of a patient with Compound 1 reduces proteinuria.
[0227] In some embodiments, a regimen has been established to achieve one or more desirable outcomes, relative to that observed for a comparable reference population that has not received Compound 1 (e.g., that has received a placebo), such as those described herein. In some embodiments, in methods provided herein, a reference composition may be or may have been administered at the same intervals and/or in the same amounts as a composition providing Compound 1.
[0228] In some embodiments, a regimen has been established to achieve one or more desirable outcomes, relative to that observed for a comparable reference population that does not have an altered level of a biomarker (e.g., an elevated level of COL3A1 and/or COL1 Al). In some embodiments, a patient that expresses a biomarker treated with Compound 1 has an improved outcome (e.g., improved stability and/or amelioration of the fibrotic disease) relative to a patient with the same disease that does not express an elevated level of the biomarker. In some embodiments, treatment of a patient with Compound 1, reduces a level of one or more biomarkers (e.g., COL1 Al and/or COL3A1). In some embodiments, treatment of a patient with Compound 1 reduces proteinuria.
[0229] In some embodiments, a composition providing Compound 1 is administered according to a regimen established to achieve a particular effect, e.g., at a particular time point, such as regimens described herein. In some embodiments, a composition providing Compound 1 is administered according to a regimen established to achieve a particular effect, such as regimens described herein. [0230] In some embodiments, a patient with a fibrotic disease of the kidney exhibits increased renal COL3 A1 expression that is correlated with urine protein to creatinine ratio. In some embodiments, a patient with a fibrotic disease of the kidney exhibits COL3 A1 expression that is inversely correlated with eGFR.
[0231] In some embodiments, provided methods are useful in treating a kidney disease that is or comprises focal and segmental glomerulosclerosis (FSGS). In some embodiments, a patient has FSGS and an elevated level of COL3A1 and/or COL1 A1 (e.g., mRNA and/or protein), for example, as determined in or obtained from a biological sample from the patient (e.g., renal biopsy sample, urine sample, blood sample, etc.). In some embodiments, a patient has FSGS and also exhibits (i) increased renal COL3 A1 expression that is correlated with urine protein to creatinine ratio and/or (ii) COL3 A1 expression that is inversely correlated with eGFR.
[0232] In some embodiments, provided methods are useful in treating a kidney disease that is or comprises primary proteinuric kidney disease (PPKD). In some embodiments, a patient has PPKD and an elevated level of COL3A1 and/or COL1 A1 (e.g., mRNA and/or protein), for example, as determined in or obtained from a biological sample from the patient (e.g., renal biopsy sample, urine sample, blood sample, etc.).
[0233] In some embodiments, provided methods are useful in treating a kidney disease that is or comprises primary glomerular disease (PGD). In some embodiments, a patient has PGD and an elevated level of COL3A1 and/or COL1 A1 (e.g., mRNA and/or protein), for example, as determined in or obtained from a biological sample from the patient (e.g., renal biopsy sample, urine sample, blood sample, etc.).
[0234] In some embodiments, provided methods are useful in treating a kidney disease that is or comprises steroid resistant nephrotic syndrome (SRNS). In some embodiments, a patient has SRNS and an elevated level of COL3A1 and/or COL1 A1 (e.g., mRNA and/or protein), for example, as determined in or obtained from a biological sample from the patient (e.g., renal biopsy sample, urine sample, blood sample, etc.).
[0235] In some embodiments, provided methods are useful in treating a kidney disease that is or comprises proteinuria. In some embodiments, a patient has proteinuria and an elevated level of COL3A1 and/or COL1 A1 (e.g., mRNA and/or protein), for example, as determined in or obtained from a biological sample from the patient (e.g., renal biopsy sample, urine sample, blood sample, etc.). [0236] In some embodiments, provided methods are useful in treating a kidney disease that is or comprises lupus nephritis. In some embodiments, a patient has lupus nephritis and an elevated level of COL3A1 and/or COL1A1 (e.g., mRNA and/or protein), for example, as determined in or obtained from a biological sample from the patient (e.g., renal biopsy sample, urine sample, blood sample, etc.).
[0237] In some embodiments, provided methods are useful in treating a kidney disease that is or comprises minimal change disease. In some embodiments, a patient has minimal change disease and an elevated level of COL3A1 and/or COL1 A1 (e.g., mRNA and/or protein), for example, as determined in or obtained from a biological sample from the patient (e.g., renal biopsy sample, urine sample, blood sample, etc.).
[0238] In some embodiments, provided methods are useful in treating a kidney disease that is or comprises ANCA-associated glomerulonephritis. In some embodiments, a patient has ANCA-associated glomerulonephritis and an elevated level of COL3A1 and/or COL1A1 (e.g., mRNA and/or protein), for example, as determined in or obtained from a biological sample from the patient (e.g., renal biopsy sample, urine sample, blood sample, etc.).
[0239] In some embodiments, provided methods are useful in treating a kidney disease that is or comprises anti-GBM nephropathy. In some embodiments, a patient has anti-GBM nephropathy and an elevated level of COL3A1 and/or COL1 A1 (e.g., mRNA and/or protein), for example, as determined in or obtained from a biological sample from the patient (e.g., renal biopsy sample, urine sample, blood sample, etc.).
[0240] In some embodiments, provided methods are useful in treating a kidney disease that is or comprises IgA nephropathy. In some embodiments, a patient has IgA nephropathy and an elevated level of COL3A1 and/or COL1A1 (e.g., mRNA and/or protein), for example, as determined in or obtained from a biological sample from the patient (e.g., renal biopsy sample, urine sample, blood sample, etc.).
[0241] In some embodiments, provided methods are useful in treating a kidney disease that is or comprises membranous glomerulonephritis (MG). In some embodiments, a patient has MG and an elevated level of COL3A1 and/or COL1 A1 (e.g., mRNA and/or protein), for example, as determined in or obtained from a biological sample from the patient (e.g., renal biopsy sample, urine sample, blood sample, etc.). [0242] In some embodiments, provided methods are useful in treating a kidney disease that is or comprises polycystic kidney disease (e.g., autosomal dominant polycystic kidney disease (ADPKD)). In some embodiments, a patient has polycystic kidney disease (e.g., ADPKD) and an elevated level of COL3A1 and/or COL1 A1 (e.g., mRNA and/or protein), for example, as determined in or obtained from a biological sample from the patient (e.g., renal biopsy sample, urine sample, blood sample, etc.).
[0243] In some embodiments, provided methods are useful in treating a kidney disease that is or comprises chronic kidney disease. In some embodiments, a patient has chronic kidney disease and an elevated level of COL3A1 and/or COL1 A1 (e.g., mRNA and/or protein), for example, as determined in or obtained from a biological sample from the patient (e.g., renal biopsy sample, urine sample, blood sample, etc.).
[0244] In some embodiments, provided methods are useful in treating a kidney disease that is or comprises collagen type III glomerulopathy. In some embodiments, a patient has collagen type III glomerulopathy and an elevated level of COL3A1 and/or COL1 A1 (e.g., mRNA and/or protein), for example, as determined in or obtained from a biological sample from the patient (e.g., renal biopsy sample, urine sample, blood sample, etc.).
[0245] In some embodiments, provided methods are useful in treating a kidney disease that is or comprises nail-patella syndrome. In some embodiments, a patient has nail-patella syndrome and an elevated level of COL3A1 and/or COL1 A1 (e.g., mRNA and/or protein), for example, as determined in or obtained from a biological sample from the patient (e.g., renal biopsy sample, urine sample, blood sample, etc.).
[0246] In some embodiments, provided methods are useful in treating a lung disease that is or comprises idiopathic pulmonary fibrosis. In some embodiments, a patient has idiopathic pulmonary fibrosis and an elevated level of one or more biomarkers, e.g., identified as described in Example 22 (optionally, e.g., COL3A1 and/or COL1 A1 (e.g., mRNA and/or protein)), for example, as determined in or obtained from a biological sample from the patient (e.g., lung biopsy sample, BALF sample, blood sample, etc.).
[0247] In some embodiments are provided methods that recognize when to adjust or discontinue treatment of a patient with Compound 1. For example, in some embodiments are provided a method for treating a patient diagnosed with, suspected of having, or at risk for a fibrotic disease (e.g., a fibrotic disease as described herein, e.g., a fibrotic disease of the kidney or the lung), the method comprising: (i) obtaining or determining a level of expression of one or more gene products or proteins in a biological sample from the patient, wherein the one or more gene products or proteins are selected from: COL3A1 and COL1A1; (ii) comparing the expression level of the one or more gene products or proteins with that of a corresponding threshold level, and (iii) if the expression level of COL3 A1 and/or the expression level of COL1 A1 is above the threshold expression level, then administering to the patient an effective amount of Compound 1 or a pharmaceutical composition thereof, and if the expression level of COL3A1 and/or the expression level of COL1 A1 is not above the threshold expression level, then the patient is not administered Compound 1.
[0248] In some embodiments, provided are methods for treating a patient diagnosed with, suspected of having, or at risk for a fibrotic disease (e.g., a fibrotic disease as described herein, e.g., a fibrotic disease of the kidney or the lung), which patient is characterized by an elevated level of a biomarker (e.g., COL3A1 and/or COL1 Al), the method comprising: (i) administering an effective amount of Compound 1 or a pharmaceutical composition thereof, and (ii) monitoring a level of biomarker (e.g., COL3A1 and/or COL1A1) subsequent to administration (e.g., after a duration of a day, week, two weeks, month, two months, 3 months, etc.). In some embodiments, if the level of biomarker (e.g., COL3A1 and/or COL1 Al) increases in a patient administered Compound 1, then further treatment with Compound 1 is discontinued. In some embodiments, if the level of biomarker (e.g., COL3A1 and/or COL1 Al) increases in a patient administered Compound 1, then the dose of Compound 1 administered to the patient is increased. In some embodiments, if the level of biomarker (e.g., COL3A1 and/or COL1 Al) decreases in a patient administered Compound 1, then treatment with Compound 1 is continued (e.g., subsequent doses are administered).
[0249] In some embodiments, a fibrotic disease (e.g., a fibrotic disease as described herein, e.g., a fibrotic disease of the kidney or the lung) is stabilized (i.e., does not worsen) and/or is ameliorated (i.e., one or more symptoms improve) in a patient treated with Compound 1. In some embodiments, treatment of a patient with Compound 1, reduces a level of one or more biomarkers (e.g., COL1 Al and/or COL3A1).
[0250] In some embodiments, administration of Compound 1 to the patient: (i) reduces the expression of renal COL3A1 , (ii) reduces the expression of renal COL1A1 , (iii) reduces renal COL3A1 accumulation, (iv) reduces renal COL1A1 accumulation, or (v) any combination thereof. In some embodiments, administration of Compound 1 to the patient: (i) reduces the expression of pulmonary COL3A1 , (ii) reduces the expression of pulmonary COL1A1 , (iii) reduces pulmonary COL3A1 accumulation, (iv) reduces pulmonary COL1 A1 accumulation, or (v) any combination thereof.
[0251] In some embodiments, a patient to be treated with a method of the present disclosure exhibits proteinuria.
[0252] In some embodiments are provided a method for treatment with Compound 1 that includes: (i) receiving a report listing the expression level of one or more biomarkers (e.g., COL3A1 and/or COL1 Al) for a patient with a fibrotic disease (e.g., a fibrotic disease described herein, such as a fibrotic disease of the kidney or the lung), (ii) receiving a request for reimbursement of the screening and/or of a particular therapeutic regimen; and (iii) approving payment and/or reimbursement for Compound 1 therapy if the report indicates the level of biomarker is above a threshold level.
[0253] In some embodiments, the present disclosure provides certain biomarkers that can distinguish subjects (e.g., subjects suffering from or at risk of fibrosis or associated diseases, disorders, and conditions) who are more likely than others to respond to therapy with Compound 1. The present disclosure provides insight that certain biomarkers can distinguish patients who are likely to respond to therapy, for example because the drivers of their fibrotic disease correspond with the mechanism of action of Compound 1. For example, in some embodiments, an altered level of one or more gene products or proteins that are part of the mechanism of action of Compound 1 (e.g., down- or up-regulated by Compound 1). In some embodiments, one or more biomarkers comprise an elevated level of COL3A1 expression and/or COL1 Al expression. [0254] In some embodiments, a patient with an altered level of one or more biomarkers may have an improved response to treatment with Compound 1 relative to a patient that does not have a level of the biomarker that meets the threshold criteria.
[0255] In some embodiments, one biomarker is used to characterize subjects; in some embodiments, more than one biomarker (e.g., two, three, etc.) is used to characterize subjects. [0256] In some embodiments, a biomarker is differentially present in a sample taken from a subject of one status as compared with another status (e.g., more responsive to Compound 1 therapy vs. less responsive to Compound 1 therapy). In some embodiments, a biomarker is differentially present in a sample taken from the same subject at two or more different time points, i.e., when the status of the subject has changed from one time point to another.
[0257] In some embodiments, a biomarker is detected and/or quantified in a tissue sample (e.g., from a biopsy, such as a kidney or lung biopsy) and/or in a biological fluid (e.g., blood, urine, BALF, etc.). In some embodiments, a biomarker is a level of mRNA that is detected and/or quantified in a kidney tissue sample, e.g., obtained from a kidney biopsy. In some embodiments, a biomarker is detected and/or quantified in a urine sample (e.g., a level of a protein or protein fragment). In some embodiments, a biomarker is detected and/or quantified in a blood sample (e.g., a level of a protein or protein fragment). In some embodiments, a biomarker is detected and/or quantified in a BALF sample (e.g., a level of a protein or protein fragment). In some embodiments, a biomarker is a level of mRNA that is detected and/or quantified in a lung tissue sample, e.g., obtained from a lung biopsy.
[0258] A biomarker may include one or more of a peptide, protein, nucleic acid (e.g., polynucleotide, DNA, RNA, etc.), polysaccharide (e.g., lectins or sugars), lipid, enzyme, small molecule, ligand, receptor, antigen, or antibody. In some embodiments, a biomarker comprises a protein. In some embodiments, a biomarker comprises a nucleic acid (e.g., mRNA).
[0259] In some embodiments, detection of a threshold level of one or more biomarkers are used to select and/or characterize patients who may be responsive to Compound 1 therapy. In some embodiments, levels of one or more biomarkers in a sample from a subject are compared to a threshold level. In some embodiments, a biomarker is considered increased if the level is increased relative to a threshold level (e.g., increased by at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%, or more). In some embodiments, a threshold level is determined from a population of healthy volunteers (e.g., a mean or median level from a population of healthy volunteers). [0260] Any suitable means can be used to determine levels of one or more biomarkers in accordance with the present disclosure. In some embodiments, a method includes an in vitro method for determining a level of a biomarker. For example, in vitro methods for determining a level of a biomarker include, but are not limited to, a chemiluminescence assay, enzymatic assay, enzyme immunoassay, multiplex immunoassay, ELISA, chromatographic immunoassay, electrophoresis assay, radioimmunoassay, colorimetric assay, chromatography/mass spectrometry (e.g., GC/MS, LC/MS, LC/MS/MS, etc.), High Performance Liquid Chromatography (“HPLC”), and/or PCR (e.g., real-time PCR).
[0261] In some embodiments, a method for detecting a level of a biomarker includes chromatographic and/or MS methods. Exemplary methods include, but are not limited to, gas chromatography (GC), liquid chromatography/mass spectroscopy (LC-MS), gas chromatography/mass spectroscopy (GC-MS), nuclear magnetic resonance (NMR), magnetic resonance imaging (MRI), Fourier Transform InfraRed (FT-IR), and inductively coupled plasma mass spectrometry (ICP-MS).
[0262] In some embodiments, the present disclosure encompasses the recognition that COL1 A1 and/or COL3A1 are useful biomarkers in the methods provided herein. In some embodiments, the present disclosure provides insight that increased levels of COL1 A1 and/or COL3 A1 expression may be useful in selecting and/or characterizing patients for Compound 1 therapy. In some embodiments, COL1 A1 is a biomarker useful in the methods provided herein. In some embodiments, COL3 A1 is a biomarker useful in the methods provided herein.
[0263] In some embodiments, an elevated level of COL1A1 and/or COL3A1 corresponds to a level of gene expression (e.g., RNA, e.g., mRNA). In some embodiments, an elevated level of COL1A1 and/or COL3A1 corresponds to a level of collagen protein expression. For example, in some embodiments, an elevated level of COL1A1 and/or COL3A1 protein is or includes, e.g., a corresponding procollagen polypeptide, collagen protein, preprocollagen polypeptide, amino- terminal procollagen polypeptide, collagen fibril, collagen fiber, and/or any fragment or degradation product thereof.
[0264] In some embodiments, an increased level of COL1 A1 and/or COL3A1 is above a threshold level (e.g., a predetermined median or mean level). In some embodiments, an increased level of COL1 A1 and/or COL3A1 is more than about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%, or more of a threshold level. In some embodiments, an increased level of COL1 A1 and/or COL3A1 is more than about 0.5, about 1.0, about 1.5, or about 2.0, or more standard deviations above a threshold level.
[0265] In some embodiments, subjects who are selected for Compound 1 therapy based on increased levels of COL1 A1 and/or COL3A1 achieve improved outcomes (e.g., compared to subjects who do not meet one or more selection criteria for Compound 1 therapy). [0266] In some embodiments, the present disclosure provides methods of treating a disease, disorder, or condition characterized by increased expression of COL1 A1 and/or COL3A1, comprising administering Compound 1 to a subject or population of subjects in need thereof as described herein.
[0267] In some embodiments, such a disease, disorder, or condition is a renal disease, disorder, or condition (e.g., a renal disease, disorder, or condition associated with fibrosis) as described herein. For example, in some embodiments, a renal disease, disorder, or condition is characterized by increased expression of COL1A1 and/or COL3A1.
[0268] In some embodiments, such a disease, disorder, or condition is a pulmonary disease, disorder, or condition (e.g., a pulmonary disease, disorder, or condition associated with fibrosis) as described herein. For example, in some embodiments, a pulmonary disease, disorder, or condition is characterized by increased expression of COL1A1 and/or COL3A1.
[0269] In some embodiments, the present disclosure provides methods comprising administering Compound 1 to a subject in need thereof, wherein the subject has been determined to have increased level(s) of COL1A1 and/or COL3A1. In some embodiments, a subject has been determined to have increased level(s) of COL1 A1 and/or COL3A1 in a renal biopsy sample and/or a urine sample. In some embodiments, a subject has been determined to have increased level(s) of COL1 A1 and/or COL3 A1 in a biological sample other than a renal biopsy sample and/or a urine sample. In some embodiments, a subject has been determined to have increased level(s) of COL1A1 and/or COL3A1 in aBALF sample.
[0270] In some embodiments, the present disclosure provides methods comprising determining level(s) of COL1A1 and/or COL3A1 by: obtaining a biological sample from a subject; performing an assay on the sample to determine level(s) of COL1 A1 and/or COL3A1 in the sample; and comparing the determined level(s) to a threshold level. In some embodiments, if a sample has increased level(s) of COL1 A1 and/or COL3A1 compared to a threshold level, then Compound 1 is administered to the subject. In some embodiments, if a sample does not have increased level(s) of COL1 A1 and/or COL3A1 compared to a threshold level, then Compound 1 is not administered to the subject.
[0271] In some embodiments, the present disclosure provides methods comprising determining level(s) of COL1 A1 and/or COL3A1 in a biological sample from a subject; comparing the determined level(s) to a threshold level; and identifying the subject as in need of therapeutic intervention when the sample is determined to have increased level(s) of COL1 A1 and/or COL3A1 compared to the threshold level. For example, in some embodiments, a subject is identified as in need of therapeutic intervention with Compound 1 therapy as described herein. [0272] In some embodiments, a provided method further comprises administering Compound 1 to a subject (e.g., a subject identified as in need of therapeutic intervention) as described herein.
[0273] In some embodiments, provided technologies are useful for monitoring subjects (e.g., monitoring status of subjects over time and/or monitoring therapy). In some embodiments, the present disclosure provides methods comprising determining level(s) of COL1A1 and/or COL3A1 in each of a plurality of biological samples obtained at different time points from a single patient; and comparing the determined level(s) from a first time point with that from at least one later time point. In some embodiments, the present disclosure provides methods comprising determining level(s) of COL1 A1 and/or COL3A1 from a biological sample obtained from a subject for whom level(s) of COL1 A1 and/or COL3A1 have previously been obtained at least once; and comparing the determined level(s) with the previously obtained level(s). In some embodiments, a first time point and one or more later time points are separated from one another by a reasonably consistent interval.
[0274] In some embodiments, a significant change in the determined level over time indicates a change in the subject’s status. In some embodiments, a significant change in a determined level over time is a change (e.g., an increase or a decrease) of at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%, or more compared to a threshold level. In some embodiments, a significant change in a determined level over time is a change of more than about 0.5, about 1.0, about 1.5, or about 2.0, or more standard deviations away from a threshold level.
[0275] In some embodiments, such methods further comprise administering Compound 1 therapy to a subject (e.g., a subject who has been determined to have moved from a non- responsive to a responsive state) as described herein.
[0276] In some embodiments, provided methods are useful for monitoring therapy (e.g., efficacy and/or other indicators of response). In some embodiments, a sample from a first time point was obtained from a subject prior to administration of Compound 1, and a sample from a second time point was obtained from a subject after administration of Compound 1. In some such embodiments, if a decrease in COL1 A1 and/or COL3A1 level(s) is observed in a later sample compared to a first sample, then Compound 1 therapy is continued. In some such embodiments, if no change or an increase in COL1A1 and/or COL3A1 level(s) is observed in a later sample compared to a first sample, then Compound 1 therapy is discontinued, or dosage amount and/or frequency of Compound 1 therapy is increased.
[0277] In some embodiments, a method includes an in vitro method for determining a level of a biomarker. In some embodiments, a method for determining a level of a biomarker can be or include a chemiluminescence assay, enzymatic assay, enzyme immunoassay, multiplex immunoassay, ELISA, chromatographic immunoassay, electrophoresis assay, radioimmunoassay, colorimetric assay, UV spectroscopy, chromatography/mass spectrometry (e.g., GC/MS, LC/MS, LC/MS/MS, etc.), High Performance Liquid Chromatography (“HPLC”), and/or PCR (e.g., quantitative PCR and/or real-time PCR).
[0278] In some embodiments, a level of a biomarker (e.g., COL1A1 and/or COL3A1 ) corresponds to a level of gene expression (e.g., RNA, e.g., mRNA) and is quantified using methods known in the art. In some embodiments, a method of determining a level of expression of a biomarker gene (e.g., RNA, e.g., mRNA) can be or include a chemiluminescence assay, UV spectroscopy, hybridization assay (e.g., Fluorescent in Situ Hybridization (FISH), e.g., RNA- FISH), enzymatic assay, enzyme immunoassay (e.g., ELISA), multiplex assay, electrophoresis assay, radioassay, colorimetric assay, chromatography/mass spectrometry (e.g., GC/MS, LC/MS, LC/MS/MS, etc.), High Performance Liquid Chromatography (“HPLC”), and/or PCR (e.g., quantitative PCR and/or real-time PCR).
[0279] In some embodiments, a level of a biomarker (e.g., COL1 A1 and/or COL3A1) corresponds to a level of protein (e.g., procollagen polypeptide, collagen protein, preprocollagen polypeptide, amino-terminal procollagen polypeptide, collagen fibril, collagen fiber, and/or any fragment or degradation product thereof) and is quantified using methods known in the art. In some embodiments, a method of determining a level of expression of a biomarker protein (e.g., COL1 A1 and/or COL3A1) can be or include a chemiluminescence assay, enzymatic assay, enzyme immunoassay, multiplex immunoassay, ELISA, chromatographic immunoassay, electrophoresis assay, radioimmunoassay, colorimetric assay, UV spectroscopy, chromatography/mass spectrometry (e.g., GC/MS, LC/MS, LC/MS/MS, etc.), or High Performance Liquid Chromatography (“HPLC”). [0280] The present disclosure also contemplates, among other things, the following numbered embodiments:
1. A method for treating a fibrotic disease of the kidney, the method comprising: administering an effective amount of a Compound 1 or a pharmaceutical composition thereof, to a patient that has been determined to have an elevated level of COL3 A1 and/or COL1A1.
2. A method for treating a fibrotic disease of the kidney in a patient characterized by an elevated level of COL3A1 and/or COL1A1, the method comprising: administering to the patient an effective amount of a Compound 1 or a pharmaceutical composition thereof.
3. The method of embodiment 1 or 2, wherein the elevated level of COL3A1 and/or COL1 A1 comprises a level that is above that of a corresponding threshold expression level.
4. The method of any one of embodiments 1 to 3, wherein the elevated level of COL3A1 and/or COL1 A1 was obtained from or determined in a biological sample from the patient.
5. The method of any one of embodiments 1 to 3, wherein the method further comprises obtaining or determining a level of COL3A1 and/or COL1 A1 in a biological sample from the patient.
6. A method for treating a patient diagnosed with or at risk for a fibrotic disease of the kidney, the method comprising:
(i) obtaining or determining a level of expression of one or more gene products or proteins in a biological sample from the patient, wherein the one or more gene products or proteins are selected from: COL3A1 and COL1A1;
(ii) comparing the expression level of the one or more gene products or proteins with that of a corresponding threshold level, wherein if the expression level of COL3A1 and/or the expression level of COL1 A1 is above the threshold expression level: administering to the patient an effective amount of Compound 1 or a pharmaceutical composition thereof. 7. The method of embodiment 3 or 6, wherein the threshold expression level corresponds to a predetermined mean or median level of COL3A1 and/or COL1 A1 of a population of healthy subjects.
8. The method of embodiment 7, wherein the expression level of COL3A1 is at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% higher than the corresponding threshold expression level and/or the expression level of COL1 A1 is at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% higher than the corresponding threshold expression level.
9. The method of any one of embodiments 1 to 8, wherein the biological sample is a renal biopsy sample, a urine sample or a blood sample.
10. The method of embodiment 9, wherein the biological sample is a renal biopsy sample and the expression level of COL3A1 is an mRNA level and/or the expression level of COL1 A1 is an mRNA level.
11. The method of embodiment 9, wherein the biological sample is a urine sample and the expression level of COL3 A1 is a level of COL3 A1 protein or fragments thereof and/or the expression level of COL1 A1 is a level of COL1 A1 protein or fragments thereof.
12. The method of any one of embodiments 1 to 11, wherein the fibrotic disease of the kidney is or comprises: focal segmental glomerulosclerosis (FSGS), steroid resistant nephrotic syndrome (SRNS), proteinuria, lupus nephritis, minimal change disease, an anti-neutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis, anti-globular basement membrane (anti-GBM) nephropathy, IgA nephropathy, membranous glomerulonephritis (MG), autosomal dominant polycystic kidney disease (ADPKD), collagen type III glomerulopathy, nail-patella syndrome, or chronic kidney disease.
13. The method of embodiment 12, wherein the anti-neutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis is Wegener’s granulomatosis, microscopic polyangiitis (MPA), or renal limited vasculitis.
14. The method of embodiment 12, wherein the fibrotic disease of the kidney is or comprises focal and segmental glomerulosclerosis (FSGS).
15. The method of embodiment 14, wherein:
(i) increased renal COL3 A1 expression is correlated with urine protein to creatinine ratio; and/or (ii) COL3 A1 expression is inversely correlated with eGFR.
16. The method of any one of embodiments 1 to 11, wherein the fibrotic disease of the kidney is or comprises primary proteinuric kidney disease.
17. The method of any one of embodiments 1 to 11, wherein the fibrotic disease of the kidney is or comprises primary glomerular disease.
18. The method of any one of embodiments 1 to 17, wherein administration of Compound 1 to the patient: (i) reduces the expression of renal COL3A1 , (ii) reduces the expression of renal COL1A1 , (iii) reduces renal COL3A1 accumulation, (iv) reduces renal COL1 A1 accumulation; or (v) any combination thereof.
19. The method of any one of embodiments 1 to 18, wherein the patient has proteinuria.
20. The method of any one of embodiments 1 to 19, wherein Compound 1 is administered in a pharmaceutically acceptable salt form.
21. A method compri sing :
(a) receiving a report listing the expression level of COL3A1 and COL1 A1 for a patient with a focal segmental glomerulosclerosis (FSGS);
(b) receiving a request for reimbursement of the screening and/or of a particular therapeutic regimen;
(c) approving payment and/or reimbursement for treatment with Compound 1 therapy if the report indicates the level of COL3A1 and/or COL1 A1 is above a threshold level.
22. A method for treating a patient diagnosed with, suspected of having, or at risk for a fibrotic disease of the kidney, the method comprising:
(i) obtaining or determining a level of expression of one or more gene products or proteins in a biological sample from the patient, wherein the one or more gene products or proteins are selected from: COL3A1 and COL1A1;
(ii) comparing the expression level of the one or more gene products or proteins with that of a corresponding threshold level, and
(iii) if the expression level of COL3A1 and/or the expression level of COL1 A1 is above the threshold expression level, then administering to the patient an effective amount of Compound 1 and if the expression level of COL3 A1 and/or the expression level of COL1 A1 is not above the threshold expression level, then the patient is not administered Compound 1. 23. A method for treating a patient diagnosed with, suspected of having, or at risk for a fibrotic disease of the kidney, which patient is characterized by an elevated level of COL3 A1 and/or COL1A1, the method comprising:
(i) administering an effective amount of Compound 1 or a pharmaceutical composition thereof, and
(ii) monitoring a level of COL3A1 and/or COL1A1.
24. The method of embodiment 23, wherein if the level of COL3A1 and/or COL1 A1 increases in a patient administered Compound 1, discontinuing further treatment with Compound 1
25. The method of embodiment 23, wherein if the level of COL3A1 and/or COL1 A1 increases in a patient administered Compound 1, increasing the dose of Compound 1 administered to the patient.
26. The method of any one of embodiments 22 to 25, wherein the fibrotic disease of the kidney is or comprises: focal segmental glomerulosclerosis (FSGS), steroid resistant nephrotic syndrome (SRNS), proteinuria, lupus nephritis, minimal change disease, an anti-neutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis, anti-globular basement membrane (anti-GBM) nephropathy, IgA nephropathy, membranous glomerulonephritis (MG), autosomal dominant polycystic kidney disease (ADPKD), collagen type III glomerulopathy, nail-patella syndrome, or chronic kidney disease.
27. The method of any one of claims 22 to 25, wherein the fibrotic disease of the kidney is or comprises primary proteinuric kidney disease.
28. The method of any one of claims 22 to 25, wherein the fibrotic disease of the kidney is or comprises primary glomerular disease.
29. The method of any one of claims 22 to 28, wherein Compound 1 is administered in a pharmaceutically acceptable salt form.
Administration
[0281] A composition providing Compound 1, as described herein, can be administered in accordance with methods provided herein.
[0282] In some embodiments, a composition providing Compound 1 is a composition comprising Compound 1 (in a pharmaceutically acceptable form as described herein), formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, a composition providing Compound 1 is or comprises Compound 1 present in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, compositions providing Compound 1 may be specially formulated for administration in solid or liquid form, including those adapted for the following: oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), capsules, tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream, or foam; sublingually; ocularly; transdermally; or nasally, pulmonary, and to other mucosal surfaces.
[0283] In some embodiments, as described herein, composition providing Compound 1 is formulated for oral administration (e.g., in a capsule form). In some embodiments, a composition providing Compound 1 is administered orally.
[0284] In some embodiments, a composition providing Compound 1 is administered as one or more unit dosage forms. In some embodiments, a composition providing Compound 1 is administered as one or more solid unit dosage forms (e.g., one or more capsules or tablets). In some embodiments, a composition providing Compound 1 is administered as one or more immediate release solid unit dosage forms. In some embodiments, Compound 1 is administered as one or more oral unit dosage forms.
[0285] In some embodiments, a composition providing Compound 1 is a capsule. In some embodiments, a composition providing Compound l is a tablet.
[0286] In some embodiments, Compound 1 is administered as a capsule comprising 10 mg, 50 mg, or 250 mg of Compound 1 In some embodiments, Compound 1 is administered as a capsule comprising 10 mg, 50 mg, or 250 mg of Compound 1 with no excipients. In some embodiments, Compound 1 is administered as a capsule comprising 50 mg of Compound 1 (e.g., with no excipients). In some embodiments, Compound 1 is administered as a capsule comprising 250 mg of Compound 1 (e.g., with no excipients). [0287] In some embodiments, Compound 1 is administered as a capsule comprising 100 mg of Compound 1 (e.g., a Form A Compound 1 Hydrochloride Trihydrate). In some embodiments, Compound 1 is administered as a capsule comprising 200 mg of Compound 1 (e.g., a Form A Compound 1 Hydrochloride Trihydrate). In some such embodiments, a capsule comprising Compound 1 has no excipients.
[0288] It will be appreciated that a suitable number of unit dosage forms (e.g., tablets or capsules) are administered in order to provide a suitable dose as described herein. For example, in some embodiments, one unit dosage form (e.g., tablet or capsule) is administered in order to provide a suitable dose as described herein (e.g., a dose of about 50 mg, about 100 mg, about 200 mg, about 250 mg, about 400 mg, about 500 mg, or about 600 mg); in some embodiments, more than one (e.g., 2, 3, 4, 5, etc.) unit dosage forms (e.g., tablets or capsules) are administered in order to provide a suitable dose as described herein (e.g., a dose of about 50 mg, about 100 mg, about 200 mg, about 250 mg, about 400 mg, about 500 mg, or about 600 mg). In some embodiments, when multiple unit dosage forms are administered, each unit dosage form contains the same amount of Compound 1, in order to provide a suitable dose as described herein; in some embodiments, when multiple unit dosage forms are administered, each unit dosage form contains different amounts of Compound 1, in order to provide a suitable dose as described herein.
[0289] In some embodiments, a suitable number of unit dosage forms (e.g., tablets or capsules) are administered in order to provide a dose of about 50 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg, or about 600 mg. For example, in some embodiments, one unit dosage form is administered to provide a dose of about 50 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg, or about 600 mg. In some embodiments, more than one (e.g., 2, 3, or 4) unit dosage forms are administered to provide a dose of about 50 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg, or about 600 mg.
[0290] In some embodiments, a composition providing Compound 1 is administered as a single dose. In some embodiments, a composition providing Compound 1 is administered at regular intervals. Administration at an “interval,” as used herein, indicates that the therapeutically effective amount is administered periodically (as distinguished from a one-time dose). In some embodiments, a composition providing Compound 1 is administered bimonthly (Q2M), monthly (QM), twice monthly (BIM), triweekly (Q3W), biweekly (Q2W), weekly (QW), twice weekly (BIW), thrice weekly (TIW), daily (QD), twice daily (BID), thrice daily (TID), or four times a day (QID) in accordance with methods provided herein. In some embodiments, a composition providing Compound 1 is administered twice daily (BID). In some embodiments, a composition providing Compound 1 is administered once daily (QD).
[0291] The present disclosure encompasses the recognition that, in some embodiments, administration of Compound 1 once daily is desirable for, e.g., patient convenience and/or compliance. Alternatively and/or additionally, the present disclosure encompasses the recognition that, in some embodiments, administration of Compound 1 twice daily may be desirable to achieve certain outcomes (e.g., to meet a particular threshold blood concentration, etc.).
[0292] In some embodiments, a composition providing Compound 1 is administered in a suitable number of unit dosage forms (e.g., tablets or capsules) in order to provide a dose of about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, or about 600 mg once daily. In some embodiments, a composition providing Compound 1 is administered in a suitable number of unit dosage forms (e.g., tablets or capsules) in order to provide a dose of about 50 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, or about 400 mg twice daily.
[0293] In some embodiments, a composition providing Compound 1 is administered at regular intervals indefinitely. In some embodiments, a composition providing Compound 1 is administered at regular intervals for a defined period of time. In some embodiments, a composition providing Compound 1 is administered at regular intervals for at least 12 weeks. [0294] In some embodiments, a composition providing Compound 1 is administered to a subject in a fed state (e.g., after a meal, such as within 1 hour, 45 minutes, 30 minutes, or 15 minutes of a meal). In some embodiments, Compound 1 is administered to a subject in a fasted state (e.g., after a fast of at least 4 hours, at least 6 hours, at least 8 hours, at least 10 hours, at least 12 hours, or at least 16 hours).
[0295] In some embodiments, a composition providing Compound 1 is administered to a subject at least 30 minutes apart from any other medications. Related Therapies
[0296] Nintedanib is approved by the FDA for treatment of idiopathic pulmonary fibrosis, for treatment chronic fibrosing interstitial lung diseases with a progressive phenotype and for slowing the rate of decline in pulmonary function in patients with systemic sclerosis-associated interstitial lung disease (OFEV® [package insert] Ridgefield, CT: Boehringer Ingelheim Pharmaceuticals, Inc.; March 2020). Nintedanib is an oral tyrosine kinase inhibitor and has a recommended dosage of 150 mg twice daily approximately 12 hours apart taken with food. Nintedanib is also referred to as liT-indole-6-carboxylic acid, 2,3-dihydro-3-[[[4-[methyl[(4- methyl-l-piperazinyl)acetyl]amino]phenyl]amino]phenylmethylene]-2-oxo-, methyl ester, (3Z)-, ethanesulfonate.
[0297] Nintedanib is reported to reach maximum plasma concentrations approximately 2 to 4 hours after oral administration as a soft gelatin capsule under fed conditions. The absolute bioavailability of a 100 mg dose was 4.7% (90% CL 3.62 to 6.08) in healthy volunteers. After food intake, nintedanib exposure increased by approximately 20% compared to administration under fasted conditions (90% CL 95.3% to 152.5%) and absorption was delayed (median tmax fasted: 2.00 hours; fed: 3.98 hours), irrespective of food type. The effective half-life of nintedanib in patients with IPF was 9.5 hours (gCV 31.9%). Total plasma clearance after intravenous infusion was high (CL: 1390 mL/min; gCV 28.8%). Urinary excretion of unchanged drug within 48 hours was about 0.05% of the dose after oral and about 1.4% of the dose after intravenous administration; the renal clearance was 20 mL/min. See OFEV® [package insert] Ridgefield, CT: Boehringer Ingelheim Pharmaceuticals, Inc.; March 2020. A 100 mg single dose of nintedanib given to healthy volunteers (n = 14) displayed a plasma Cmax of approx. 7 ng/mL. See , e.g., Fig. 2 of Wind, S., et al. Clin. Pharmacokinet. 2019;58:1131-47. A trough concentration for producing 80% of Umax (ECxo) on FVC decline was estimated to be > 10 ng/mL. See, e.g., Fig. 6 of Wind, S., et al. Clin. Pharmacokinet. 2019;58:1131-47.
[0298] Nintedanib is not recommended for use in patients with moderate or severe hepatic impairment. Elevated liver enzymes (e.g., ALT, AST, and bilirubin) and drug-induced liver injury have occurred with nintedanib. Diarrhea, nausea, and vomiting have occurred with nintedanib. Nintedanib may cause fetal harm. Arterial thromboembolic events have occurred with nintedanib. Bleeding events have occurred with nintedanib. Gastrointestinal perforation has been reported with nintedanib. See OFEV® [package insert] Ridgefield, CT: Boehringer Ingelheim Pharmaceuticals, Inc.; March 2020.
[0299] The present disclosure also encompasses the recognition that provided technologies may provide different (e.g., improved) properties and/or outcomes, compared to one or more other known therapies (e.g., nintedanib). For example, in some embodiments, Compound 1 may display different (e.g., improved) pharmacokinetic, pharmacodynamic, safety and/or efficacy characteristics, compared to one or more other known therapies (e.g., nintedanib).
EXAMPLES
Example 1. Preparation of a Composition that Provides Compound 1 Synthesis of Compound 1
[0300] A representative procedure for obtaining Compound 1 is as follows. The quantities of materials used are approximate and may be increased or decreased in unison to obtain a larger or smaller lot size. Conditions such as time or temperature are approximate and may be used as targets.
Figure imgf000084_0001
[0301] Step 1: Acetic anhydride (5.40 kg) and methyl 2-oxo-2,3-dihydro-lH-pyrrolo[2, 3- b]pyridine-6-carboxylate (1.0 kg) were added to a reactor at room temperature and stirred to combine. Trimethylorthobenzoate (1.90 kg) was added to the reaction mixture. The mixture was then heated to 105 °C and stirred for 1 hr. The reaction was cooled to 40 °C and isopropyl alcohol (3.14 kg) was added. The reaction was cooled further to 5 °C and stirred for 4 hr. The mixture was then filtered, and the product washed with isopropyl alcohol twice. The product was spin dried for 1 hr and for 4 hr at 50 °C under vacuum to give methyl (E)-l -acetyl-3 - (methoxy(phenyl)methylene)-2-oxo-2,3-dihydro-lH-pyrrolo[2,3-b]pyridine-6-carboxylate. [0302] Step 2: Methanol (7.12 kg), methyl (E)-l -acetyl-3 -(methoxy(phenyl)methylene)-2- oxo-2, 3-dihydro-lH-pyrrolo[2,3-b]pyridine-6-carboxylate (1.0 kg), and Af-(4-aminophenyl)-N- methyl-2-(4-methylpiperazin-l-yl)acetamide (0.78 kg) in methanol (0.79 kg) were added to a reactor at room temperature and stirred to combine. The reaction mixture was then heated to 63 °C and stirred for 4 hrs. The reaction mixture was cooled to 5 °C and stirred for 4 hrs. The reaction mixture was then filtered, and the product washed methanol (1.98 kg). The product was spin dried for 1 hr and for 4 hr at 50 °C under vacuum to give methyl (Z)-3-(((4-(N-methyl-2-(4- methylpiperazin-l-yl)acetamido)phenyl)amino)(phenyl)methylene)-2-oxo-2,3-dihydro-lH- pyrrolo[2,3-b]pyridine-6-carboxylate.
[0303] Step 3: Purified water (2.50 kg) and methyl (Z)-3-(((4-(N-methyl-2-(4- methylpiperazin-l-yl)acetamido)phenyl)amino)(phenyl)methylene)-2-oxo-2,3-dihydro-lH- pyrrolo[2,3-b]pyridine-6-carboxylate (1.0 kg) were added to a reactor at room temperature and stirred to combine. Acetone (1.975 kg) was then added, followed by iron-free HC1 (1.20 mol eq.). The mixture was stirred for 1 hr at 30 °C, and then filtered through a micron filter and washed with purified water (0.2 kg). The washings were transferred into a reactor and acetone was added (21 .72 kg). The mixture was stirred for 1 h at 30 °C, then cooled to 5 °C and stirred for 5 h. The product was isolated in centrifuge and washed with acetone (1.58 kg). The product was spin dried for 1 hr and for 8 hrs at 50 °C under vacuum to give Compound 1 as a Form A hydrochloride salt channel hydrate (e.g., with approx three equivalents of water).
[0304] The XRPD data of the material obtained from this Example is summarized below:
Figure imgf000085_0001
Figure imgf000086_0001
Capsule Formulation of Compound 1
[0305] Compound 1 was formulated in a capsule for oral administration. The capsule formulation included a Size 00 Swedish orange capsule containing Compound 1 (10 mg, 50 mg, or 250 mg) with no excipients. Ingredients of the capsule shell were hypromellose (hydroxypropylmethyl cellulose), iron oxide as a coloring agent, and titanium dioxide as an opacifier.
[0306] Capsule formulations were prepared as follows. First, an optional sieving step was performed to deagglomerate the active agent if needed. Then, Compound 1 was filled into HPMC capsules, using either an automated Xelodose machine (e.g., for 10 mg and 50 mg capsules) or a semiautomated process (e.g., for 250 mg capsules). All capsules were polished or dedusted, either by an inline deduster (e.g., for 10 mg and 50 mg capsules) or a separate capsule polisher (e.g., for 250 mg capsules).
Additional Capsule Formulations of Compound 1
[0307] Compound 1 Hydrochloride Form A was formulated in a capsule for oral administration. The capsule formulation included a Swedish Orange capsule containing Compound 1 Hydrochloride Form A (100 mg or 200 mg dose) with no excipients. Ingredients of the capsule shell were hypromellose (hydroxypropylmethyl cellulose), iron oxide as a coloring agent, and titanium dioxide as an opacifier. Capsule formulations were prepared as described above.
Example 2. A Phase 1, Randomized, Double-Blind, Placebo-controlled, Single and Multiple Ascending Dose Study to Determine the Safety, Tolerability, Pharmacokinetics, and Food Effect of Compound 1 in Healthy Adult Participants Objectives
[0308] Primary: To assess the safety and tolerability of single and multiple ascending doses of Compound 1 in healthy adult participants.
[0309] Secondary: To assess the pharmacokinetics (PK) of single and multiple ascending doses of Compound 1 and to evaluate the effect of a high fat meal on the PK of a single dose of Compound 1 administered to healthy adult participants.
Endpoints
[0310] Primary Endpoint: The frequency and severity of treatment-emergent adverse events (TEAEs), including clinically significant abnormal vital signs, electrocardiograms (ECGs), laboratory test results, and physical examination findings.
[0311] Secondary Endpoints:
[0312] Plasma PK endpoints include:
• Maximum concentration (Cmax)
• Time to maximum concentration (Tmax)
• Area under drug concentration-time curve, from time zero to the last measurable concentration (AUCo-iast)
• Area under drug concentration-time curve, from time zero to infinity (AUCo-inf)
• Area under drug concentration-time curve over inter-dosing interval (AUCtau); MAD cohorts only
• Apparent terminal half-life (ti/2)
• Apparent terminal elimination rate constant (Kei)
• Apparent clearance (SAD cohorts = CL/F; MAD cohorts = CL/Fss)
• Apparent terminal volume of distribution (SAD cohorts = Vz/F; MAD cohorts = Vz/Fss) • Accumulation ration (RA); MAD cohorts only [0313] Urine PK endpoints include:
• Amount of drug excreted in urine over time (Aeu-u), including cumulative Ae(o-72 h)
• Renal clearance (CLR)
• Fraction of systemic clearance (CL/F) represented by renal clearance (CLR/[CL/F])
• Fraction of administered dose excreted in urine over dosing intervals (Feu-u), including cumulative Fe(o-72 h)
Methodology
[0314] This was a first in human, single-center, double-blind, randomized, cross-over, SAD design followed by a MAD design study of Compound 1 conducted in healthy adult participants, designed to evaluate safety, tolerability, PK, and food effect of Compound 1 in healthy adults.
Up to 80 participants were enrolled into one of up to six SAD cohorts (n=8 per cohort) and four MAD cohorts (n=8 per cohort).
Part A: Single Ascending Dose (SAD)
[0315] Up to 48 participants were enrolled into one of up to six cohorts (Cohorts A1 to A6; n=8 per cohort). All Part A (SAD) participants were confined to a clinical research unit from Day -1 (pre-dose) until completion of the 72-hour post-dose assessments on Day 4.
[0316] Cohort A3 was a food effect cohort, and participants in Cohort A3 only returned to the CRU on Day 14 and, following a 14-day washout, received a second single dose of their assigned treatment on Day 15 following consumption of a high fat meal. For Cohort A3 only, a second period of confinement applied from Day 14 until completion of the 72-hour post-dose assessments on Day 18.
[0317] All Part A participants returned to the CRU for a follow-up visit 7 days (±1 day) after their final dose of study drug. For all cohorts in Part A (SAD), the decision to escalate a dose or modify a dose was determined by the SMC following review of the 7-day blinded safety and available PK data from the preceding cohort.
[0318] Cohorts AF A2. A4. A5. and A6: Participants (n=8) were randomized 3:1 to receive a single oral dose of either Compound 1 (n=6) or matching placebo (n=2) on Day 1 following an overnight fast of at least 10 hours and after all pre-dose assessments were performed. Two sentinel participants received a single oral dose of Compound 1 (n=l) or matching placebo (n=l) initially. If dosing of these sentinel participants proceeds without clinically significant safety signals in the first 48 hours post-dose (as adjudicated by the principal investigator or delegate), the remaining participants in each cohort received a single dose of Compound 1 (n=5) or matching placebo (n=l) according to the randomization schedule.
[0319] Participants were monitored for safety and blood and urine samples were collected for assessment of PK parameters at predefined time points pre and post-dose. Participants were discharged on Day 4 following completion of all specified study procedures. Participants returned to the CRU for an end of study Follow-up visit on Day 8 (±1 day).
[0320] Cohort A3 (food effect): Participants in the Food Effect Cohort (A3; n=8) received a single oral dose of either Compound 1 or matching placebo in the fasted state (Period 1) followed by the same assigned treatment in the fed state (Period 2) after a 14-day washout period as follows.
[0321] Cohort A3 (Period 1): Participants (n=8) were randomized to receive a single oral dose of either Compound 1 (n=6) or matching placebo (n=2) on Day 1 following an overnight fast of at least 10 hours and after all pre-dose assessments were performed. Two sentinel participants received a single oral dose of Compound 1 (n=l) or matching placebo (n=l) initially. If dosing of these sentinel participants proceeded without clinically significant safety signals in the first 48 hours post-dose (as adjudicated by the PI or delegate), the remaining participants received a single dose of Compound 1 (n=5) or placebo (n=l) according to the randomization schedule. Participants were monitored for safety and blood and urine samples were collected for assessment of PK parameters at predefined time points pre- and post-dose. Participants were discharged on Day 4 following completion of all specified study procedures. [0322] Cohort A3 (Period 2): Participants (n=8) completed a 14-day washout following dosing in Period 1 and returned and were admitted to the CRU on Day 14, after which they received a second single dose of their assigned treatment (oral dose of Compound 1 (n=6) or matching placebo (n=2)) on Day 15, following consumption of a high fat meal. The high fat meal was served 30 minutes prior to study drug administration and was consumed within 30 minutes. Participants were monitored for safety and blood and urine samples were collected for assessment of PK parameters at predefined time points pre-and post-dose. Participants were discharged on Day 18 following completion of all specified study procedures. Participants in Cohort A3 returned to the CRU for an end of study follow-up visit on Day 22 (±1 day). [0323] Compound 1 dose level tested in Part A (SAD) did not exceed 1200 mg or the dose level that led to a mean Cmax > 800 ng/mL or mean AUCo-iast > 4000 ng*h/mL. These Cmax and AUCo-iast values were observed in non-human primates at the no observed adverse effect level (NOAEL) of 75 mg/kg/day.
[0324] For Part A (SAD), single oral dosing was carried out according to Table 1.
Table 1.
Figure imgf000090_0001
* Subject to change; Compound 1 dose will not exceed 1200 mg or a dose level that leads to mean Cmax >800 ng/mL or mean AUCo-iast > 4000 ng*h mL.
** Day 15 doses for Cohort A3 (food effect) are administered following consumption of a high fat meal.
Part B: Multiple Ascending Dose (MAD j
[0325] Up to 32 participants were enrolled into up to four cohorts (Cohorts B 1 to B4; n=8 per cohort). Participants were administered Compound 1 (n=6 per cohort) or matching placebo (n=2 per cohort) twice daily for 7 consecutive days (Day 1 to Day 7) or once daily for 14 consecutive days (Day 1 to Day 14) determined based on the safety and tolerability data from the SAD portion of the study (Part A). All Part B (MAD) participants were confined to the CRU from Day -1 (pre-dose) to Day 10 or Day 17, depending on the dosing schedule following review of data from Part A (SAD). Participants were monitored for safety and blood and urine samples were collected for assessment of PK at predefined time points pre- and post-dose.
[0326] Participants were administered study drug in a standardized manner; i.e., either with or without food depending on preliminary bioavailability results from Part A (SAD). Participants were discharged from the CRU upon completion of final 72-hour post-dose assessments. Participants then returned to the CRU for an end of study follow-up visit 7 days after the final dose of study drug (±1 day). For all cohorts in Part B (MAD), the decision to escalate a dose or modify a dose was determined following review of the 10- or 17-day blinded safety and available PK data from the preceding cohort. [0327] The Compound 1 dose level tested in Part B (MAD) did not exceed 1000 mg per day or a dose level that led to a mean Cmax > 800 ng/mL or mean AUCo-iast > 4000 ng*h/mL. These Cmax and AUCo-iast values were observed in non-human primates at the no observed adverse effect level (NOAEL) of 75 mg/kg/day.
[0328] For Part B (MAD), once daily oral dosing was carried out according to Table 2.
Table 2.
Figure imgf000091_0001
* Subject to change; Compound 1 dose will not exceed 1000 mg per day or a dose level that leads to mean Cmax >800 ng/mL or mean AUCo-iast > 4000 ng*h mL.
** May be twice daily for 7 consecutive days (Day 1 to Day 7) or once daily for 14 consecutive days (Day 1 to Day 14).
[0329] Up to 80 participants were enrolled in the study (Part A: up to 48 healthy volunteers; Part B: up to 32 healthy volunteers).
Diagnosis and Main Criteria for Inclusion:
Inclusion Criteria:
[0330] To be eligible for the study, participants were required to meet all of the following inclusion criteria:
1. Healthy male or female volunteer, aged 18 to 65 years.
2. Participants must be in good health, with no significant medical history, have no clinically significant abnormalities on physical examination at screening and/or before administration of the initial dose of study drug.
3. Participants must have a minimum body weight of 50 kg and a body mass index (BMI) between >18.0 and <32.0 kg/m2 at screening.
4. Participants must have clinical laboratory values within normal range as specified by the testing laboratory, unless deemed not clinically significant by the investigator or delegate.
5. Participants who smoke no more than 2 cigarettes or equivalent per week can be included in study but must be willing to abstain from smoking during the confinement period. Participants must have no relevant dietary restrictions, and be willing to consume standard meals provided during the confinement period. Women of childbearing potential (WOCBP) must be non-pregnant and non-lactating, and must use an acceptable, highly effective double contraception from screening until study completion, including the follow-up period. Double contraception is defined as a condom and one other form of the following:
• Established hormonal contraception (oral contraceptive pills, long-acting implantable hormones, injectable hormones)
• A vaginal ring or an intrauterine device
• Documented evidence of surgical sterilization at least 6 months prior to screening (e.g., tubal occlusion, hysterectomy, bilateral salpingectomy, or bilateral oophorectomy for women or vasectomy for men (with appropriate post-vasectomy documentation of absence of sperm in semen) provided the male partner is a sole partner
Women not of childbearing potential must be postmenopausal for > 12 months. Postmenopausal status are confirmed through testing follicle-stimulating hormone (FSH) levels > 40 IU/mL at screening for amenorrhoeic female participants. Females who are abstinent from heterosexual intercourse are also eligible.
Periodic abstinence (e.g., calendar, ovulation, symptothermal, post-ovulation- methods) and withdrawal are not considered highly effective methods of birth control. Participant complete abstinence for the duration of the study and for 1 months after last study treatment is acceptable.
Female participants who are in same sex relationships are not required to use contraception.
WOCBP must have negative pregnancy test at screening and Day 1 and be willing to have additional pregnancy test as required.
Males must be surgically sterile (>30 days since vasectomy with no viable sperm), abstinent, or if engaged in sexual relations with a WOCBP, the participant and his partner must be surgically sterile (e.g., tubal occlusion, hysterectomy, bilateral salpingectomy, or bilateral oophorectomy) or using an acceptable, highly effective 8. Male participants must not donate sperm for at least 90 days after the last dose of study drug.
9. Participants must have the ability and willingness to attend necessary visits to the CRU.
10. Participants must be willing and able to provide written informed consent after the nature of the study has been explained and prior to the commencement of any study procedures.
[0331] A participant who met any of the following exclusion criteria were excluded from the study:
1. Pregnant or lactating at screening or planning to become pregnant (self or partner) at any time during the study, including the follow-up period.
2. Prior or ongoing medical conditions, medical history, physical findings, or laboratory abnormality that, in the investigator’s (or delegate’s) medical opinion, could adversely affect the safety of the participant.
3. History of gastrointestinal (GI) disorders such as celiac disease, atrophic gastritis, lactose intolerance, and Helicobacter (H.) pylori infection.
4. Presence of any underlying physical or psychological medical condition that, in the opinion of the investigator, would make it unlikely that the participant will comply with the protocol or complete the study per protocol.
5. Any surgical or medical condition that could interfere with the absorption, distribution, metabolism, or excretion of the study drug.
6. Blood donation or significant blood loss within 60 days prior to the first study drug administration.
7. Plasma donation within 7 days prior to the first study drug administration.
8. Fever (body temperature > 38 °C) or symptomatic viral or bacterial infection within 2 weeks prior to screening.
9. Any acute illness within 30 days prior to Day 1.
10. History of severe allergic or anaphylactic reaction.
11. History of malignancy except for non-melanoma skin cancer excised more than 2 years ago and cervical intraepithelial neoplasia (CIN) that has been successfully cured more than 5 years prior to screening. Abnormal ECG finding at screening that are considered by the investigator to be clinically significant. History or presence of a condition associated with significant immunosuppression. History of life-threatening infection (e.g., meningitis). Infections requiring parenteral antibiotics within 6 months prior to screening. Vaccination with a live vaccine within 4 weeks prior to screening or that is planned within 4 weeks of dosing. Exposure to any significantly immune suppressing drug (including experimental therapies as part of a clinical trial) within 4 months prior to screening or 5 half-lives, whichever is longer. Positive test for hepatitis C antibody (HCV), hepatitis B surface antigen (HBsAg), or human immunodeficiency virus (HIV) antibody at screening. Participants with a positive toxicology screening panel (urine test including qualitative identification of barbiturates, tetrahydrocannabinol (THC), amphetamines, benzodiazepines, opiates, and ***e), or alcohol breath test. Participants with a history of substance abuse or dependency or history of recreational intravenous (IV) drug use over the last 5 years (by self-declaration). Regular alcohol consumption defined as >21 alcohol units per week (where 1 unit = 284 mL of beer, 25 mL of 40% spirit or a 125 mL glass of wine). Participant is unwilling to abstain from alcohol beginning 48 hours prior to admission to the CRU and during confinement period. Use of any IP or investigational medical device within 30 days prior to screening, or 5 half-lives of the product (whichever is longest) or participation in more than four investigational drug studies within 1 year prior to screening. Use of any prescription drugs (other than hormonal contraception: OCPs, long-acting implantable hormones, injectable hormones, a vaginal ring or an HID), over-the- counter (OTC) medication, herbal remedies, supplements or vitamins within 1 week prior to dosing and during course of study without prior approval of the investigator and medical monitor. Simple analgesia (nonsteroidal anti-inflammatory drug (NSAID)) or paracetamol may be permitted at discretion of investigator. Use of fibrates for hyperlipidemia. 25. Consumption of any nutrients or concomitant medications known to modulate cytochrome P450 3 A4 (CYP3 A4) or any strong inhibitors or inducers of CYP3 A4, starting from 2 weeks prior to first dose of study drug and until end of study assessments.
26. Inability to refrain from consumption of grapefruit and Seville oranges or St. John’s Wort within 2 weeks prior to first dose of study drug and until final PK assessment.
27. Participant is unwilling to refrain from strenuous exercise from 72 hours prior to admission to CRU until completion of final follow-up visit.
Product and Mode of Administration
[0332] Compound 1 was provided as a powder in capsule formulation for oral administration. The formulation was a Size 00 Swedish orange capsule containing drug substance (10 mg, 50 mg, or 250 mg) with no excipients. Ingredients of the capsule shell were hypromellose (hydroxypropylmethyl cellulose (HPMC)), iron oxide as coloring agent, and titanium dioxide as an opacifier. The drug product was stored at room temperature (15 °C - 25 °C).
[0333] Compound 1 is in a pharmacological class of tyrosine kinase inhibitors (TKI). Compound 1 is an orally bioavailable small molecule dual kinase inhibitor of platelet-derived growth factor receptors (PDGFR) and vascular endothelial growth factor receptors (VEGFR2). Duration of Treatment
[0334] Depending on study part and cohort assignment, the duration of study participation for each participant ranged from approximately 36 days to 50 days, including up to 28 days of screening. Duration of treatment by cohort is summarized in Table 3.
Table 3.
Figure imgf000095_0001
[0335] Part A (SAD): In Part A (SAD), participants randomized to active treatment in cohorts Al, A2, A4, A5, and A6 received a single oral dose of Compound 1 administered once on Day 1 only. Participants randomized to active treatment in Cohort A3 (Food Effect) received a single oral dose of Compound 1 on Days 1 and 15.
[0336] PartB (MAD): In Part B (MAD), participants randomized to active treatment received oral Compound 1 administered twice daily for 7 consecutive days (Day 1 to Day 7) or once daily for 14 consecutive days (Day 1 to Day 14), to be determined on completion of Part A (SAD) of the study.
Reference Therapy and Mode of Administration
[0337] The placebo capsules were visually matching to active study drug and were composed of the same capsule shell but were filled with silicified microcrystalline cellulose. Silicified microcrystalline cellulose is a pharmaceutical excipient composed of co-process microcrystalline cellulose and colloidal silicon dioxide.
Criteria for Evaluation Part A (SAD) and Part B (MAD)
[0338] Safety: The safety and tolerability of single and repeat-doses of Compound 1 was investigated according to the following specific assessments: vital signs (systolic and diastolic blood pressure, pulse rate, body temperature, and respiratory rate), 12-lead ECG, clinical laboratory tests (hematology, biochemistry, coagulation, and urinalysis), physical examination, and assessment of TEAEs.
[0339] Pharmacokinetics: Blood and urine samples for PK analysis of Compound 1 was collected pre-dose and following oral administration of single and repeat-doses of Compound 1 in the fasted state. Blood and urine samples for PK analysis was collected pre-dose and following administration of single doses of Compound 1 immediately after ingestion of a high fat meal (Food Effect Cohort A3 only).
Statistical Methods
[0340] The number of participants was selected to allow for evaluation of safety/tolerability, PK and food effect of the single and multiple doses to be administered in this study and was consistent with standards of practice for Phase 1 studies.
[0341] In general, descriptive statistics (e.g. arithmetic mean, standard deviation [SD], median, minimum and maximum) were calculated for continuous data among treatment groups (or sequences), as well as difference from baseline by study part for each applicable scheduled time point, when appropriate. Frequency summaries (e.g. number of observed and percentage of each categories) were applied for categorical data among treatment groups (or sequences), by study part and for each scheduled time point.
[0342] For PK data the arithmetic mean, SD, median, minimum, maximum, coefficient of variation (CV%), geometric mean, geometric coefficient of variation (geo CV% or gCV) and the number of below the limit of quantification (BLQ) values was presented. No geometric statistics were computed for BLQ plasma concentrations.
[0343] No formal hypothesis testing was performed for this study.
[0344] Analysis Populations: Participant inclusion into each population was determined prior to the final analysis.
[0345] Intent-to-Treat (ITT) Population: All enrolled participants, regardless of whether they receive study drug or not, were included in the ITT population. Analysis is based on the treatment assigned to a participant, not what they actually received. The ITT population was used for all data listings and summaries involving disposition and enrollment.
[0346] Safety Population: All participants who received any amount of study drug (Compound 1 or placebo) were included in the Safety population. The Safety population was used for the summaries of all safety assessments. Participants were analyzed according to treatment received.
[0347] Pharmacokinetic Population: All participants who received any amount of active study drug (Compound 1) and have sufficiently evaluable concentration-time profile to allow determination of at least one PK parameter were included in the PK population. An evaluable PK profile was determined at the discretion of the pharmacokineticist following examination of subjects with dosing or protocol deviations that could potentially affect the PK profile. The PK population was used for the summaries of all PK data.
[0348] Safety and Tolerability: All adverse events (AEs) were coded using the Medical Dictionary for Regulatory Activities (MedDRA®) Version 22.0. A by participant AE data listing, including verbatim term, preferred term (PT), system organ class (SOC), treatment, severity, and relationship to study drug was provided. The number of participants experiencing TEAEs and number of individual TEAEs was summarized by SOC and PT. TEAEs were also summarized by severity and by relationship to study drug. [0349] Laboratory evaluations, vital signs assessments and ECG parameters were summarized for each scheduled visit by treatment arm. A summary of change from baseline at each protocol specified time point by treatment arm were also presented.
[0350] Concomitant medications were coded using the World Health Organization (WHO) drug dictionary Version B3 September 2018 Drug Global. Concomitant medications were listed by participant and summarized by anatomical therapeutic class and preferred name.
[0351] Physical examinations at each visit were listed for each participant and summarized using descriptive statistics at each visit by treatment arm.
[0352] Medical history, pregnancy test/FSH, urine drug screen/alcohol breath test, physical examinations and serology (HIV, Hepatitis B & C screen) were listed by participant.
[0353] Pharmacokinetics: Plasma Compound 1 concentrations, actual blood sampling times, and PK parameters were listed by treatment and protocol specified time point and summarized using descriptive statistics for PK data as outlined above for each scheduled time point by treatment arm. Individual and mean Compound 1 concentration-time profiles were also presented graphically for each treatment. Pharmacokinetic parameters were computed from the individual plasma Compound 1 concentrations using a non-compartmental approach.
[0354] The following plasma Compound 1 non-compartmental PK parameters were estimated, as appropriate: Cmax, Tmax, AUCo-iast, AUCo-inf, AUCtau (calculated for MAD cohorts only), Kei, ti/2, CL/F (SAD cohorts), CL/Fss (MAD cohorts), Vz/F (SAD cohorts, Vz/Fss (MAD cohorts), RA (calculated for MAD cohorts only).
[0355] Value for Kei, ti/2, AUCo-inf, CL/F (CL/Fss), or Vz/F (Vz/Fss) were not reported for cases that fail to exhibit a terminal log-linear phase in the concentration versus time profile. Additional analyses were performed as deemed necessary upon review of the data.
[0356] A food effect assessment was conducted on Compound 1 PK parameters in Cohort A3. Analyses of variance (ANOVA) were performed on the ln-transformed AUCo-iast, AUCo-inf and Cmax (fasting vs fed). Ratios of the geometric means were calculated using the exponentiation of the least squares mean (LSM) from the analyses on the ln-transformed AUCo- iast, AUCo-inf and Cmax. Ratios were expressed as a percentage relative to the fasting regimen. 90% Cl for the ratios were derived by exponentiation of the CIs obtained for the difference between regimen LSM resulting from the analyses on the ln-transformed AUCo-iast, AUCo-inf, and Cmax. [0357] The analysis of dose proportionality was conducted for AUC and Cmax of single agent Compound 1 using a power model on log-transformed scale. The log-transformed exposure parameters were each regressed onto a fixed factor for log (dose). The 90% Confidence Interval (Cl) of the slope for each exposure parameter was computed from the model and presented in a summary table.
[0358] Urine collection time, volume collected, and Compound 1 concentration (Aeu-a) duration of each sampling interval was listed for each participant and summarized by nominal sampling time point and treatment using descriptive statistics (Number of subjects [N], arithmetic mean, SD, CV%, geometric mean, median, minimum and maximum). Individual and mean Compound 1 cumulative urinary excretion-time profiles for each treatment were also presented graphically. Where urine was collected for PK analysis, the following parameters are calculated, as appropriate: Aeu-t2, CLR, CL/F, and Feu-t2.
Results
[0359] As described above, Compound 1 dose levels tested in Part A (SAD) were limited so as to not exceed 1200 mg or the dose level that leads to a mean Cmax > 800 ng/mL or mean AUCo-iast > 4000 ng*h/mL. These Cmax and AUCo-iast values were observed in non-human primates at the no observed adverse effect level (NOAEL) of 75 mg/kg/day (see, e.g., Example 3). Standard extrapolation for administration to humans would predict that a mean Cmax > 800 ng/mL or mean AUCo-iast > 4000 ng*h/mL would be observed at a human dose of approximately 24 mg/kg/day (i.e., approximately 1440 mg/day for a 60 kg human). Yet, during the clinical study described above, Cmax and AUCo-iast approached their limits at doses approx. 2.4-fold lower than predicted using standard techniques, and Compound 1 exposure in humans was approximately 6-8 times greater than in monkeys. For example, as described below, subjects given a 600 mg dose (i.e., approx. 10 mg/kg/day for a 60 kg human) of Compound 1 displayed a mean Cmax of about 800 ng/mL and a mean AUCo-iast of about 2130 ng*h/mL. The unexpectedly greater exposure achieved by Compound 1 in humans even prompted a change in dosing protocol, whereby the planned 800 mg and 1200 mg SAD cohorts were replaced with a 600 mg SAD cohort. For example, Example 24 below describes a protocol for a Phase I study with different cohorts based on certain preliminary PK findings.
[0360] FIG. 1 summarizes mean oral blood PK profile over time from SAD cohorts in a fasted state. Table 4 summarizes mean oral blood PK parameters from SAD cohorts in a fasted state as of a first data cutoff. As shown in Table 4, Compound 1 was rapidly absorbed with a median Tmax of 1-2 h post-dose. With 400 mg and 600 mg doses, a decline in plasma concentration was observed with mean ti/2 of approx. 10-30 h. Moderate inter-subject variability was observed in 400 mg and 600 mg doses, with approx. 28-44% variability in Cmax and AUC. Cmax increased dose-proportionally up to 400 mg and slightly over-proportionally from 400 mg to 600 mg. Plasma AUC increased 1.6-fold with a 1.5-fold increase in dose (i.e., from 400 mg to 600 mg). Blood Cmax in human healthy volunteers given a 600 mg dose was greater than Cmax of maximum tolerated dose in a monkey 7-day QD study, where blood Cmax in monkey was 605 ng/mL (male) and 780 ng/mL (female) and blood AUC in monkey was 5720 ng*h/mL (male) and 6450 ng*h/mL (female).
Table 4.†
Figure imgf000100_0001
†A11 data given as mean value ± SD (% C.V.), unless otherwise noted. **Tmax given as median (range). ***Tiast = 4 h (n = 4), 12 h (n = 1). a AUC0.24h since Tlast = 72 h (n = 4), 48 h (n = 2). b n = 3.
[0361] FIG. 2 and Table 5 summarize results of food effect cohorts (3 A and 3B) as of a first data cutoff. FIG. 2 shows mean oral plasma PK profiles over time in SAD food effect cohorts receiving 200 mg Compound 1. A negative food effect was observed, with 3.6-fold lower Cmax when Compound 1 was administered with food, yet little change in AUCo-iast was observed.
Table 5.†
Figure imgf000100_0002
Figure imgf000101_0001
†A11 data given as mean value ± SD (% C.V.), unless otherwise noted. *TmaX given as median (range).
[0362] As of a first data cutoff, no serious adverse events assessed to be drug-related were observed. AEs were mild and typical for the population (e.g., nausea, headache, and dizziness), and no dose dependent AEs were reported.
Example 3. Administration of Compound 1 to Rats and Monkeys
Pharmacokinetics of Compound 1 in Rats and Monkeys
[0363] Compound 1 pharmacokinetics were characterized following single and multiple oral doses to male Sprague Dawley rats and to male and female cynomolgus monkeys in pharmacokinetic and toxicology studies. Single intravenous doses were administered to rats and monkeys for the purpose of estimating bioavailability of oral doses of Compound 1. Table 6 summarizes pharmacokinetics of Compound 1 observed in rats and monkeys following single intravenous or oral doses.
Table 6.
Figure imgf000101_0002
a Dose expressed as free base. b AUCo-mf was not reported for all animals in some cases due to insufficient data in terminal phase. c Bioavailability determined using data from 30 mg/kg IV dose in rats and monkeys (Compound 1 pharmacokinetics assumed to be linear). [0364] Following a 5-minute intravenous infusion, Compound 1 whole blood concentrations generally declined in a bi-exponential manner with a terminal half-life of approximately 4 hours in rats and 4-5 hours in monkeys. The volume of distribution values exceeded total body water. [0365] After oral administration, Compound 1 was rapidly absorbed in rat and monkey as maximum whole blood concentrations were reached within 1 to 2 hours in rats and 1 to 3 hours in monkeys. Exposure as assessed by AUCo-iast increased with increasing dose; however, the increase was greater than dose proportional in rats and nearly dose proportional in the monkey. The oral bioavailability of Compound 1 across a dose range of 10 to 100 mg/kg ranged from 4 to 10% in both species.
[0366] Exposure to Compound 1, as assessed by mean Cmax and AUCo-iast, increased with increasing dose in Sprague Dawley rats and cynomolgus monkeys in the maximum tolerated (MTD) single dose, 7-day and 28-day oral toxicology studies. Exposure to Compound 1 on Day 7 or Day 28 after repeated dosing as assessed by AUCo-iast in rats and monkeys was similar to or greater by about 1.2- to 2.9-fold than AUCo-iast values on Day 1. No consistent gender-related differences in exposure were observed.
[0367] When administered intravenously, Compound 1 was highly distributed outside of the systemic circulation and its volume of distribution values of 2.3 to 18.2 L/kg exceeded total body water in rats and monkeys.
Twenty-Eight Day Oral GLP Toxicity Study of Compound 1 in Monkeys [0368] Five groups of cynomolgus monkeys (3/sex) were administered Compound 1 at 0, 3, 15, 75, and 250 mg/kg for at least 28 days. An additional 2 monkey s/sex/group were dosed at 0 and 250 mg/kg and allowed a two-week recovery following the final dose to determine reversibility. Monkeys were observed for clinical signs of toxicity, changes in body weight, food consumption, as well as hematology, coagulation, clinical chemistry, urinary parameters, electrocardiographic measurements, macroscopic pathologic findings, weight of selected organs, and microscopic examination of selected tissues.
[0369] In summary, animals tolerated oral dosing of Compound 1 for 28 days up to 250 mg/kg for males and 75 mg/kg for females. One female at 250 mg/kg had clinical signs of chronic watery diarrhea and dehydration and an increase in the incidence and severity of spontaneous gastrointestinal changes. Based on the results of this study, the no-observed- adverse-effect-level (NOAEL) was 250 mg/kg in males and 75 mg/kg in females. Plasma exposure on Day 28 at the NOAEL, as determined by Cmax and AUCo-iast, was 1240 and 796 ng/mL (Cmax), and 10600 and 4070 hr*ng/mL (AUCo-iast) for males and females respectively. Based on the results of this study, a human equivalent dose for the NOAEL in monkeys was determined to be 24 mg/kg/day. Employing a 28-fold safety factor, the clinical trial of Example 2 was designed to begin with a 50 mg dose of Compound 1.
Example 4. Compound 1 Inhibits PDGFRjl Phosphorylation in Hepatic Stellate Cells [0370] Human Hepatic Stellate Cells (ScienCell Research Labs, Carlsbad, CA, Cat# 5300) were grown in Stellate Cell Medium (ScienCell Research Labs, Carlsbad, CA) in a 37 °C incubator with an atmosphere of 5% C02/95% air. Cells were maintained in tissue culture flasks, plated in 6-well tissue culture plates (at 1 million cells per well), allowed to adhere and grown to 80% confluence. Cells were starved overnight in Stellate Cell Medium without serum. From a 10 mM stock solution of Compound 1 in DMSO, 10-fold serial compound dilutions were prepared. Diluted Compound 1 solutions in DMSO were added at final concentrations of 10 mM, 1 pM,
100 nM, 10 nM, 1 nM and 0.1 nM and at a final DMSO concentration in the assay of 0.1%. After 2 hours of incubation with compound or control, PDGF-BB (R&D Systems Catalog # 220-BB) was added to a final concentration of 25 ng/mL and after 15 minutes treatment with PDGF-BB, cells were lysed in lysis buffer (RIPA lysis buffer, Sigma R0278). The lysis buffer also contained Phosphatase Inhibitor Cocktail 1 (Sigma P2850) and Phosphatase Inhibitor Cocktail 2 Sigma P5726).
[0371] After addition of PDGF-BB, receptor activation was assessed by looking at receptor autophosphorylation at tyrosine residue 751. The autophosphorylation was determined by Western analysis, using GAPDH as a loading control. Protein concentrations in the lysates were determined using the DC protein determination kit (Biorad 500-0113 and 500-0114) and 25 pg protein was loaded for Western analysis on 10 lane gels (Invitrogen Catalog # NP0321). In a control lane, MagicMark™ XP Western Protein Standard was loaded (Invitrogen Catalog # LC5602). Proteins were transferred to Nitrocellulose membranes (Invitrogen Catalog # 77010). [0372] As shown in FIG. 3, treatment of serum-starved hepatic stellate cells with PDGFBB resulted in marked phosphorylation of PDGFRP at tyrosine 751. At 1 pM and higher concentrations of Compound 1, this PDGF-BB-stimulated Y751 phosphorylation was markedly inhibited.
Example 5. Compound 1 Inhibits KDR Phosphorylation in HUVEC Cells [0373] Human Umbilical Vein Endothelial Cells (HUVEC) (ATCC Catalog # CRL1730) cells were grown in EBM-2 (Endothelial Cell Growth Basal Medium-2, Lonza Catalog # 00190860) supplemented with EGM-2 Endothelial SingleQuots Kit (Lonza Catalog # CC-4176) in a 37°C incubator with an atmosphere of 5% C02/95% air. Cells were maintained in tissue culture flasks, plated in 6-well tissue culture plates (at 1 million cells per well), allowed to adhere and grown to 80% confluence. Cells were starved overnight in EBM-2 without serum. From a 10 mM stock solution of Compound 1 in DMSO, 10-fold serial compound dilutions were prepared. Diluted Compound 1 solutions in DMSO were added at final concentrations of 10 mM, 1 pM,
100 nM, 10 nM, 1 nM and 0.1 nM and at a final DMSO concentration in the assay of 0.1%. After 30 minutes incubation with compound or controls, VEGF165 (R&D Systems Catalog # 293-VE) was added to a final concentration of 25 ng/mL and after 15 minutes treatment, cells were lysed in lysis buffer (RIPA lysis buffer, Sigma R0278). The lysis buffer also contained Phosphatase Inhibitor Cocktail 1 (Sigma P2850) and Phosphatase Inhibitor Cocktail 2 (Sigma P5726).
[0374] Protein concentrations in the lysates were determined using the DC protein determination kit (Biorad 500-0113 and 500-0114) and 25 pg protein was loaded for Western analysis on 10 lane gels (Invitrogen Catalog # NP0321). In a control lane, MagicMark™ XP Western Protein Standard was loaded (Invitrogen Catalog # LC5602). Proteins were transferred to Nitrocellulose membranes (Invitrogen Catalog # 77010).
[0375] Blots were blocked with non-fat dry-milk (Biorad Catalog # 70-6404) and incubated overnight at 2-8 °C with primary antibodies against phospho-VEGF Receptor 2 (Tyrl 175) (Cell Signaling Catalog # 2478) or GAPDH (Cell Signaling # 2118) at a 1 : 1000 dilution. Blots were washed several times and then incubated with an anti-rabbit IgG, HRP-linked secondary antibody at a 1:1000 fold dilution (Cell Signaling Catalog # 7074). After several times washing, the blots were then incubated with Immobilon Western Chemiluminescent HRP Substrate (Millipore Catalog # WBKLSOIOO), exposed to X-ray film (Fujifilm Catalog # 47410-08399) and processed in a Kodak X-omat film processor (Eastman Kodak Company, Rochester, NY). Images were then scanned on a Digital Flatbed Scanner (Hewlett-Packard, Palo Alto, CA). [0376] As shown in FIG. 4, treatment of serum-starved HUVEC cells with VEGF resulted in marked phosphorylation of VEGFR2 at tyrosine 1175. At low concentrations of Compound 1, this phosphorylation was also observed, but at 1 mM and higher concentrations of Compound 1, this VEGF-stimulated autophosphorylation was completely inhibited.
Example 6. Compound 1 Decreases Expression of Certain Fibrosis and Inflammation Related Markers
[0377] The present example probed the effects of Compound 1 on expression of various anti inflammatory and anti-fibrotic marker proteins in a cell culture based model of fibrosis. Compound 1 is in a pharmacological class of tyrosine kinase inhibitors (TKI) and has a formula of: (Z)-methyl 3-(((4-(N-methyl-2-(4-methylpiperazin-l-yl)acetamido)phenyl)amino) (phenyl)methylene)-2-oxo-2,3-dihydro-lH-pyrrolo[2,3-b]pyridine-6-carboxylate as yellow solid. MS (ES+): m/z 541.1 (MH+). Compound 1 is an orally bioavailable small molecule dual kinase inhibitor of platelet-derived growth factor receptors (PDGFR) and vascular endothelial growth factor receptors (VEGFR2).
[0378] Specifically, the present example describes Bio-Map studies of Compound 1 in cell culture systems using DiscoverX technology platform and that were profiled in a KINOMEscan (Ambit/Di scoveRx/Eurofms, CA) biochemical assay. Exemplary culture systems analyzed were: (1) Small airway epithelial cells + lung fibroblasts (SAEMyoF); (2) Lung fibroblasts only (MyoF); and (3) Renal proximal tubular epithelial cells + lung fibroblasts (REMyoF). When activated, these cell systems mimic pathological conditions such as chronic inflammation, fibrosis, and matrix remodeling.
[0379] Co-cultures of epithelial cells and fibroblasts or fibroblasts alone were activated with TGFpi (10 ng/mL) + TNF alpha (lOng/mL) and treated with Compound 1 at 100 nM, 500 nM, 2.5mM and 13mM concentrations and incubated for 48 hours. Expression of numerous critical anti-inflammatory and anti-fibrotic marker proteins were evaluated. This revealed that Compound 1 treatment decreased levels of several inflammation-related markers including monocyte chemotactic protein (MCP-1), macrophage colony stimulating factor (M-CSF), soluble interleukin 8 (sIL-8), Interferon induced T cell alpha chemoattractant (I-TAC), IL-8, Interferon gamma induced Protein- 10 (IP- 10). Compound 1 treatment also decreased fibrosis-related markers including N-cadherin, a-SMA, Collagen I and III, TIMP-1, plasminogen activator inhibitor (PAI-1), matrix metalloprotease, MMP-1, MMP-9, tissue and urokinase plasminogen activator (tPA and uPA), soluble VEGF and epidermal growth factor (EGFR) as shown in FIG.
5.
[0380] Accordingly, the present example demonstrates that Compound 1 induced dose- dependent decrease in expression of a number of different inflammation and fibrosis-related markers. In particular, the present example demonstrates that a mixed cell culture comprising renal epithelial cells and activated myofibroblasts treated with Compound 1 (13 mM) reduced expression of COL1 and COL3.
Example 7. TGFpi-Induced Collagen Production in Normal Human Lung Fibroblasts [0381] Normal Human Lung Fibroblasts (NHLF) (Lonza; Catalog # CC-2512) were grown to confluence in Fibroblast Growth Basal Medium (FGBM; Lonza; Catalog # CC-3131) supplemented with Fibroblast Growth Medium-2 SingleQuots Supplements and Growth Factors (FGM2; Lonza; Catalog # CC-4126) in a 37 °C incubator with an atmosphere of 5% C02/95% air. Cells were maintained in tissue culture flasks, plated in a 24-well tissue culture plate (at 50 thousand cells per well), allowed to adhere and serum starved for 24 hours. From a 10 mM stock solution of Compound 1 in DMSO, diluted Compound 1 solutions were added at final concentrations of 1 mM and 100 nM of compound with a final DMSO concentration in the assay of 0.1%. TGFpi (R&D Systems; Catalog 240-B) was added at a final concentration of 10 ng/mL. Treatments were performed in duplicate wells and cells that were not treated with compound and/or TGFpi were used as controls. After 48 hours incubation, supernatants were collected and soluble collagen in the supernatant was determined using the Sircol Soluble Collagen Assay (Biocolor Life Science Assays; distributed by Accurate Chemical & Scientific Corporation, 300 Shames Drive, Westbury, NY, 11590, United States) following the manufacturer’s instructions. Collagen concentrations in the supernatant were expressed as pg/mL.
[0382] The concentrations of soluble collagen in the supernatants are shown in FIG. 6. TGFpi increased the collagen level by more than fivefold (5.93/1.16 = 5.1 fold). The percentage inhibition was calculated using cells treated with TGFpi as 0% inhibition and cells not treated with compound and TGFpi as 100% inhibition. At 1 pM and 100 nM Compound 1, collagen production was inhibited by 104% and 86%, respectively. The inhibition of collagen production at both Compound 1 concentrations was statistically significant (p < 0.01).
Example 8. Compound 1 Ameliorates Fibrosis in Bleomycin-induced Injury Model of Idiopathic Pulmonary Fibrosis
[0383] Male C57BL/6 mice (~20 to 25 g) were anaesthetized for a short period of time using ketamine (100 mg/kg) and xylazine (10 mg/kg). A single dose of bleomycin sulfate at 2.5 U/kg body weight in sterile isotonic saline (50 pL per animal) was intratracheally instilled by means of a 22 gauge plastic cannula coupled to a 1 mL syringe to each animal at the start of the study. The same volume of sterile saline was administered to control mice (sham, n=4). Two weeks after bleomycin instillation, a subgroup of animals was sacrificed and the establishment of lung fibrosis was confirmed (initially as determined by increased lung weight). The bleomycin mice (n=12/group) were then randomized to vehicle and Compound 1 (25 mg/kg Compound 1 Hydrochloride trihydrate, PO, BID) treatment groups and treated for 3 weeks. All mice were then sacrificed and lungs collected to determine lung hydroxyproline (HYP), picrosirius red (PSR) staining for collagen content, and the extent of lung fibrosis from haematoxylin-eosin (H&E) slides by histopathological scoring using the Ashcroft scale. IHC staining for TGFpi, a marker of fibrosis, was also performed. All histological quantitations were performed using a Bioquant image analysis program. Body weight and lung weight were also determined.
[0384] Animals treated with Compound 1 had a lower lung to body weight ratio than vehicle-treated animals. Lower lung weight likely reflects less pulmonary fibrosis and collagen deposition. FIG. 7A is a graph showing lung to body weight ratio (mg/g).
[0385] Compound 1 significantly reduced hydroxyproline, a major component and marker of collagen deposition and fibrosis. Two weeks after bleomycin exposure, animals treated with bleomycin had increased lung hydroxyproline levels compared to sham-treated animals, which indicates development of pulmonary fibrosis. Animals then treated with Compound 1 for three weeks had reduced hydroxyproline levels compared to vehicle-treated animals (FIG. 7B).
[0386] Compound 1 also significantly reduced fibrosis score (Ashcroft) on histopathology. Lung tissue sections were stained with H&E, and slides were scored for histopathological damage using the Ashcroft scale. Two weeks after bleomycin exposure, animals treated with bleomycin had increased histopathological damage compared to sham-treated animals, indicative of development of pulmonary fibrosis. Animals treated with Compound 1 for three weeks had reduced histopathological damage compared to vehicle-treated animals (FIG. 7C).
[0387] Lung sections were stained with picrosirius red (PSR), which stains for collagen.
Two weeks after bleomycin exposure, animals treated with bleomycin had increased PSR staining compared to sham-treated animals, indicating development of pulmonary fibrosis. Animals treated with Compound 1 for three weeks had reduced PSR staining compared to vehicle treated animals (FIG. 7D).
[0388] Compound 1 significantly reduced transforming growth factor beta (TGFpi) staining. Lung sections were immunohistologically stained for TGFpi. Two weeks after bleomycin exposure, animals treated with bleomycin had increased TGFpi staining (albeit not statistically significant) compared to sham-treated animals. Animals then treated with Compound 1 for three weeks had reduced TGFpi staining compared to vehicle-treated animals (FIG. 7E).
Example 9. Compound 1 Reduces Fibrosis in Inducible TGFpi Mouse Model of Lung Fibrosis
[0389] A line of mice were used which expresses TGFpi in the lung via an externally regulatable, triple transgenic system using a doxycycline-inducible promoter. (See Lee, C. G., et al. Proc. Am. Thorac. Soc. 2006 Jul;3(5):418-23; Lee C. G., et al. J. Exp. Med. 2004 Aug 2;200(3)377-89). Eight-to-ten week old TGFpi positive female mice (approximately 25 g of body weight) were induced (fed) with doxycycline (dox) (0.5 mg/ml dox and 20 g/L sucrose in drinking water) for 4 weeks. The TGFpi transgene was under the control of a dox-inducible promoter. In addition, age and gender matched TGFpi negative mice from the transgenic mouse breeding colony not fed with dox were included as control mice (sham). Marked loss of normal bronchial tree architecture due to TGFpi overexpression was evidenced after 4 weeks of dox compared to sham mice using micro-CT images.
[0390] Some mice not fed with dox (n=3, normal) and 4-week dox fed mice (n=4, Pre-Rx) were sacrificed to confirm that pathological changes had happened (increases in lung weight) in dox fed mice. The remaining dox fed mice were randomized to vehicle (n=l 1) and Compound 1 (n=l 1, at 25 mg/kg Compound 1 Hydrochloride trihydrate, PO, BID) for 4 weeks with continued dox feeding (0.25 mg/ml dox and 20 g/L sucrose in drinking water). All mice were then sacrificed and body weights and lung weights were taken. To determine lung fibrosis, lung hydroxyproline (HYP) for collagen content, picrosirius red (PSR) staining for collagen signal, and the histopathological observations from (H&E) slides by Ashcroft scoring and IHC staining for alpha SMA were performed. All histological quantitations were performed using a Bioquant image analysis program.
[0391] Compound 1 reduced lung fibrosis score (Ashcroft) in dox-fed TGFpi transgenic mice. Dox feeding of TGFpi transgenic mice for 4 weeks resulted in a significant increase in lung fibrotic score (Ashcroft score based on H&E staining), compared to normal mice (FIG. 8A). Further dox feeding for an additional 4 weeks resulted in more increases in lung fibrotic score, as shown by the vehicle group. Compound 1 treatment for 4 weeks following 4-week dox feeding of TGFpi transgenic mice significantly (p=0.005) decreased lung fibrotic score compared to vehicle treatment.
[0392] Similarly, Compound 1 treatment for 4 weeks following 4-week dox feeding significantly (p=0.005) decreased lung hydroxyproline levels compared to vehicle treatment (FIG. 8B). A reduction in hydroxyproline, a component of collagen is indicative of a reduction in fibrosis.
[0393] Compound 1 treatment for 4 weeks following 4-week dox feeding decreased picrosirius red staining signals in the lung (an indication of collagen levels) compared to vehicle treatment (FIG. 8C); the reduction was not statistically significant (p=0.053) although such a reduction is consistent with the reductions in lung fibrotic score and hydroxyproline levels.
[0394] Compound 1 significantly decreases aSMA, an early lung fibrosis marker.
Compound 1 treatment for 4 weeks following 4-week dox feeding of TGFpi transgenic mice significantly (p=0.026) decreased aSMA staining signals compared to vehicle treatment (FIG. 8D). aSMA is a marker of myofibroblast activation, an early step in fibrosis development.
Example 10. Compound 1 Has Anti-Fibrotic Effects Across Multiple Organ Systems in Bleomycin Systemic Sclerosis Mouse Model
[0395] Female C57BL/6 mice at 7 to 8 weeks old (~24 g) were used. An area of -1.5 cm diameter was shaved on the back. Bleomycin sulfate was dissolved in PBS at a concentration of 1 mg/mL. 100 pL of bleomycin solution was injected subcutaneously with a 27 gauge needle into the shaved back of the mice. Bleomycin administrations were done 5 times a week for 4 weeks. 100 pL of PBS was similarly given to sham-treated control animals (n=8). After 4 weeks of subcutaneous (SC) bleomycin injections, some mice were sacrificed (3 sham treated animals and 5 bleomycin treated animals). Body weight and lung weight (as a percentage of body weight) of bleomycin-treated mice were significantly increased compared to sham control animals. Then, bleomycin-treated mice were randomized to vehicle (n=20) and Compound 1 (n=20) treatment groups. Compound 1 was dosed orally twice a day at 25 mg/kg of Compound 1 Hydrochloride trihydrate. Subcutaneous bleomycin administrations were continued 3 days per week. After 5 weeks of vehicle or Compound 1 treatment, mice were sacrificed and body weight and lung weight were recorded. Tissue samples from the left lung and left kidney were snap frozen. A skin biopsy was taken from the bleomycin injected site (-1.5 cm diameter) and fixed in formalin. The right lung and right kidney were also fixed in formalin for histopathological evaluation. Dermal thickness measurements were made from H&E-stained tissue sections by measuring the distance from the epidermis to the dermal junction using Bioquant planimetric software. Lung and kidney hydroxyproline (HYP) assays were performed to determine tissue collagen content. Skin, lung and kidney histopathological fibrotic scores were determined by two independent observers, and the scores were averaged. Picrosirius red (PSR) staining of kidney sections was also performed to determine renal collagen deposition. A subset of animals were randomly chosen from each group for histological staining. All histological quantitations were performed using Bioquant image analysis software.
[0396] Scleroderma is characterized by thickening of the skin caused by accumulation of collagen. As a first indicator of the effect of compound treatment on scleroderma, dermal thickness was determined by measuring the distance between the epidermal-dermal junction and the dermal-subcutaneous fat junction. This was done in H&E-stained slides prepared from a skin biopsy. After 4 weeks of subcutaneous bleomycin injections (Bleo Pre-Rx Group), dermal thickness was significantly increased compared to sham-injected animals (Sham Group). This indicates significant skin thickening and scleroderma at the time of initiation of compound treatment. Therapeutic treatment of Compound 1 for 5 weeks (25 mg/kg, PO, BID) (Bleo Compound 1 Group) significantly decreased dermal thickness compared to the bleomycin- exposed vehicle cohort (Bleo Vehicle Group). FIG. 9A summarizes these results.
[0397] To determine the effect of compound treatment on the development of skin fibrosis, H&E stained slides were scored on a scale from 0-8, with 0 representing no fibrosis and 8 representing full fibrosis. After 4 weeks of subcutaneous bleomycin injections (Bleo Pre-Rx Group), the skin fibrosis score was significantly increased compared to sham-injected animals (Sham Group). This confirms marked collagen deposition underlying the skin thickening and scleroderma at the time of initiation of compound treatment. Therapeutic treatment of Compound 1 for 5 weeks (Bleo Compound 1 Group) significantly decreased the skin fibrotic score compared to the bleomycin-exposed vehicle cohort (Bleo Vehicle Group). FIG. 9B summarizes these results.
[0398] Hydroxyproline is a major component of collagen, and it is used to indicate tissue collagen deposition and the development of fibrosis. To directly evaluate the effect of compound treatment on fibrosis in the lung, pulmonary hydroxyproline levels were determined. After 4 weeks of subcutaneous bleomycin injections, lung hydroxyproline levels were significantly increased compared to sham treated animal (Sham vs Bleo PreRx Groups), indicating the development of pulmonary fibrosis. Treatment of bleomycin exposed animals with Compound 1 for 5 weeks (25 mg/kg, PO, BID) (Bleo Compound 1 Group) significantly decreased lung hydroxyproline levels compared to bleomycin exposed vehicle treated animals (Bleo Vehicle), indicating anti -fibrotic activity of Compound 1. FIG. 9C summarizes these results.
[0399] H&E-stained slides were scored on the Ashcroft scale (0-8). After 4 weeks of subcutaneous bleomycin injections, pulmonary fibrosis was manifest (Sham vs Bleo PreRx). Therapeutic treatment of bleomycin exposed animals with Compound 1 for 5 weeks (Bleo Compound 1) significantly decreased lung fibrotic score compared to bleomycin exposed vehicle treated animals (Bleo Vehicle), confirming anti-fibrotic activity of Compound 1. FIG. 9D summarizes these results.
[0400] Systemic sclerosis is often also manifested in the kidney. To evaluate the renal effects of Compound 1, kidneys were collected at terminal sacrifice and kidney collagen content was determined by hydroxyproline assay and histochemical staining with picrosirius red. Treatment of Compound 1 for 5 weeks (25 mg/kg, PO, BID) significantly decreased kidney hydroxyproline levels in bleomycin-exposed animals compared to the vehicle cohort (Bleo Vehicle vs Bleo A Compound 1 groups). FIG. 9E summarizes these results.
[0401] Treatment with Compound 1 for 5 weeks also significantly decreased kidney picrosirius red staining, confirming the systemic anti-fibrotic activity of Compound 1 (Bleo Compound 1 vs Bleo Vehicle). FIG. 9F summarizes these results. Example 11. Compound 1 Reduces Proteinuria and Sclerosis in FSGS-Relevant Rat Model of PAN-induced Proteinuria
[0402] Administration of puromycin aminonucleoside (PAN) to Wistar rats (a sensitive strain) produces overt proteinuria (Moreno-Manzano, V., et al. Br. J. Pharmacol. 2003 Jun;139(4):823-31). The study was designed to intervene with Compound 1 on day 4 after PAN administration. In addition to the effects of Compound 1 on proteinuria, effects of drug on clinically relevant endpoints including ascites and GFR were determined. Changes in glomerular microarchitecture are associated with proteinuria, as is accumulation of matrix in the glomerulus. Effects of drug on glomerular dimensions and glomerular collagen 3 (COL-3) accumulation were examined.
[0403] On Day 0, animals were injected with PAN (100 mg/kg, IP, dissolved in water, 1 mL administration volume) or water. Urine was collected for 24 hours between days 3-4 (referred to as Day 4 urines) by placing animals in metabolic cages. During urine collection, animals had access to water but not food. On Day 4, animals were re-injected with PAN (67 mg/kg, IP, 1 mL administration volume) and randomized to receive vehicle (water) or Compound 1 (50 mg/kg Compound 1 Hydrochloride trihydrate, PO, BID).
[0404] Urine was again collected (for 24 hours) from day 10-11 after first PAN injection (referred to as Day 11 urines). On day 11 after first PAN administration GFR was measured by computing FITC-sinistrin decay (Medibeacon) and animals were sacrificed. The left kidney was cut coronally, immersed in formalin (4%) and submitted for sectioning and mounting on slides for subsequent analysis (PAS stain for determining glomerular diameter) and anti-COL 3 antibody IHC. All microscopic analyses (40X) were conducted by an observer blinded to the treatment groups. Glomerular diameter was determined from digital microscopic pictures using ImageJ image analysis software to perform planimetry. Proteinuria was determined from urine samples.
[0405] In general, every endpoint was not measurable in each animal within a group due to logistical limitations. In such cases, randomly selected animals were queried for data. For instance, no urine was collected for certain animals due to the limited number of metabolic cages available, and only a few animals in each cohort were randomly selected for GFR measurement due to the limited number of probes available for this measurement. The missing animals are marked as NA (not available) in the relevant data tables. [0406] For determination of glomerular filtration rate, the highly sensitive FITC-sinistrin clearance method was employed in conscious animals. This method is capable of measuring the elimination kinetics of FITC-sinistrin using an optical device (Medibeacon, Germany) that can monitor fluorescence trans-cutaneously. FITC-sinistrin clearance is a readout of glomerular filtration rate. The optical device measures fluorescence emitted by FITC-sinistrin through the skin and was temporarily secured on the back of the animal. A single dose of FITC-sinistrin (5 mg/100 g body weight FITC-sinistrin) was injected into the tail vein and the measurement with the optical device was performed for 1.5 hr. The data were downloaded and analyzed using the MPD lab software (Medibeacom, Germany). The ti/2 obtained from this curve is used to calculate GFR using the formula: GFR (pL/min/100 g b wt) = 21.33 [pL/100 g b wt]/ti/2(FITc- sinistrin)[min]; b wt=body weight. Glomerular Filtration Rate determination by the FITC- sinistrin method was determined for three animals per experimental group.
[0407] Animals were randomized to PAN treatment or sham treatment on day 0 and the PAN cohort was subsequently randomized on day 4 to vehicle or Compound 1 treatment groups. Animals were weighed on day 0 (day of first PAN administration) and on day 4 (day of second PAN administration and randomization). While sham treated animals gained 4.9 grams of body weight on average from day 0 to day 4, animals that were treated with PAN lost 7.1 grams of body weight on average (p=0.002 for PAN vs Sham animals), indicating a deleterious effect of PAN administration on body weight gain.
[0408] Overnight (24-hour) urines were collected from day 3-4 (referred to as Day 4 urines) prior to initiation of compound treatment. Urine volumes for animals that were sham treated or animals that were PAN treated were not statistically different. The total amount of protein excreted in 24 hours was determined for these urine samples and was found to be only 15% higher in the PAN group compared to the sham group (p=0.127), indicating that animals were not significantly proteinuric at randomization and initiation of vehicle and Compound 1 treatment.
[0409] Animals received a second dose of PAN on Day 4, were then randomized to vehicle or Compound 1 treatment groups, and then treated for 7 days. Animals were weighed on the day of randomization and at sacrifice on Day 11 after the first PAN administration. Animals in the Sham group gained on average 70 grams from Day 4 to Day 11, while animals in the PAN Vehicle group gained 40 gram and animals PAN Compound 1 gained on average 48 grams of body weight during the same period. This indicates that on average, PAN administration continued to have a deleterious effect on body weight gain and that there was no statistically significant effect of Compound 1 treatment on body weight or body weight gain from day 4-11 relative to the vehicle treated group.
[0410] Overnight (24-hour) urines were collected on days 10-11, immediately prior to sacrifice. As shown in Table 7, Animals in the PAN Vehicle and PAN Compound 1 groups produced similar amounts of urine as those in the Sham exposed animals.
[0411] As a key efficacy endpoint for proteinuric kidney disease, protein excretion in the urine was measured in urines collected immediately prior to sacrifice. While urine protein was significantly increased by PAN Vehicle treatment group compared to the sham treatment group, it was markedly reduced in the PAN Compound 1 cohort compared to the PAN vehicle cohort (FIG. 10A). This shows a significant anti-proteinuric effect of Compound 1 treatment.
[0412] At sacrifice, ascites was evaluated in animals (n>4 per group) as a clinically relevant endpoint often associated with proteinuric kidney disease. Intraperitoneal fluid volume was determined as a quantitative measurement of ascites production. A marked increase in peritoneal ascites production was observed in PAN Vehicle animals, compared to Sham control animals. Treatment with Compound 1 was associated with a significant decrease to a near normal levels of peritoneal fluid / ascites accumulation (FIG. 10B).
[0413] Glomerular filtration rate (GFR) was determined immediately prior to terminal sacrifice using FITC-sinistrin decay kinetics as a measurement of renal clearance. As shown in FIG. IOC, in Sham (Control) animals, administration of FITC-sinistrin results in gradual decline in fluorescence, indicative of normal renal clearance. In animals treated with PAN and Vehicle, a significant decrease in the clearance of FITC-sinistrin was observed, showing marked renal dysfunction in the PAN Vehicle animals. Animals treated with PAN and Compound 1 had markedly enhanced clearance of FITC-sinistrin compared to PAN Vehicle animals. The difference in GFR between the Sham (Control) group and the PAN Vehicle group (p=0.0445) and between the PAN Vehicle and the PAN Compound 1 group (p=0.0495) were statistically significant. These data indicate that treatment with Compound 1 mitigates PAN-related decrease in renal function, restoring GFR to a normal level (FIG. IOC).
[0414] Histopathological analysis of PAS-stained renal coronal sections demonstrated that PAN treatment increased glomerular diameter, as determined by planimetry. This indicates glomerular hypertrophy, consistent with PAN-induced glomerular dysfunction. Compound 1 treatment reduced glomerular hypertrophy, as evidenced by a reduction in the PAN-induced increase in glomerular diameter (FIG. 10D).
[0415] Collagen 3 (COL-3) expression, a measure of scar, was determined in glomeruli by quantitative immuno-histochemistry. The COL-3 expression level was markedly elevated in glomeruli in the kidneys from PAN Vehicle animals compared to Sham treated animals (FIG. 10E). The COL-3 level was significantly reduced with Compound 1 treatment (FIG. 10E) compared to PAN Vehicle treatment.
Example 12. Compound 1 Reduces Proteinuria and Fibrosis in DOCA/Salt Model of Renal Injury and Fibrosis
[0416] Compound 1 was tested in the uninephrectomy (UNX) + DOC A + Salt model of renal dysfunction. The use of this model has been described extensively in the literature ( See Arai, K., et al. J. Pharm. Exp. Ther. 2016 Sep;358(3):548-57; Kretzler, M., et al. Virchows Arch. 1994;425(2): 181-93). Seven to eight week old male rats were uni-nephrectomized in the week prior to study start. Induction of renal dysfunction was started on day 1, at which point experimental animals received a first subcutaneous dose of deoxycorticosterone acetate (DOCA) at 30 mg/kg and animals were switched from normal drinking water (tap water) to drinking water with 1% NaCl. Animals were kept on the 1% NaCl drinking water for the remainder of the experiment. Animals received weekly subcutaneous doses of DOCA; 30 mg/kg in weeks 1, 2, 3 and 4 and 15 mg/kg in weeks 5 and 6. Control groups were subcutaneously injected with PEG400 and received regular tap water ad libitum. After two weeks, overnight urine was collected in metabolic cages and a subset of animals was sacrificed to establish baseline values. The remainder of the animals was randomized to compound treatment (Compound 1 in water, BID, PO, 50 mg/kg Compound 1 Hydrochloride trihydrate) or vehicle (water, BID, PO) treatment groups. Animals were treated for 4 weeks and immediately prior to terminal sacrifice, urines were again collected using metabolic cages. In total, there were 37 animals in the study, divided over five experimental groups (Group 1: UNX+sham PreTx); Group 2: UNX+DOCA+salt PreTx; Group 3: UNX+sham; Group 4: UNX+DOCA+salt+vehicle; Group 5: UNX+DOCA+salt+Compound 1). Groups 1 (n=3) and 2 (n=6) were sacrificed after two weeks and groups 3 (n=5), 4 (n=l 1) and 5 (n=12) were sacrificed at the end of the study period. [0417] Animals were monitored several times daily. Three animals in the DOCA + Salt + Vehicle group (Group 3) had unscheduled sacrifice (one each on days 29, 34, and 35). Body weight, serum and kidneys were collected for those animals and samples were processed along with the samples from other animals in the group. One animal in the DOCA + Salt + Compound 1 (Group 5) died on day 25 after initiation of DOCA + Salt. The death of this animal was not considered to be related to compound treatment. Observed mortality was not statistically significant compared to control groups (Kaplan-Meier Analysis using Graphpad Prism software). [0418] At pre-randomization and terminal sacrifice, body weights were recorded. The time of body weight measurement was at week 2 for Groups 1 and 2 and at week 6 for Groups 3, 4, and 5. All animals that were treated with DOCA + Salt had significantly reduced body weight compared to control animals that did not receive DOCA and were given regular tap water to drink. The body weight in the DOCA + Salt + Compound 1 treated group (Group 5) was not significantly different from animals in the DOCA + Salt + Vehicle (Group 4), indicating that there was no obvious deleterious effect of compound treatment on body weight.
[0419] At pre-randomization and terminal sacrifice kidneys were collected and their weights recorded. At terminal sacrifice, the kidney weights from animals of DOCA + Vehicle group (Group 4) were significantly more than the kidney weights in Sham animals (Group 3). The kidney weight in DOCA + Compound 1 group (Group 5) was not significantly different from the DOCA + Vehicle group (Group 4).
[0420] The weight of the kidney was also expressed as a percentage of the total body weight of each individual animal. At terminal sacrifice, the % kidney weight from animals of DOCA + Vehicle group (Group 4) was significantly higher than the % kidney weights in Sham animals (Group 3). Compound 1 increased body weight and decreased kidney weight compared to vehicle, albeit not statistically significant. The % kidney weight (taken both body weight and kidney weight into consideration) in DOCA + Compound 1 group (Group 5) was significantly reduced compared to the DOCA + Vehicle group (Group 4).
[0421] The sera were analyzed for serum creatinine (SCR) and blood urea nitrogen (BUN) levels. No statistically significant differences were found between any of the experimental groups for either SCR or BUN. It should be noted that some of the DOCA treated animals (either with Vehicle (Group 4) or with Compound 1 (Group 5)) appeared to have elevated BUN or SCR levels compared to Sham controls (Groups 1 and 3). [0422] Urines were collected from animals pre-randomization and immediately prior to study end. Animals from the DOCA + 1% NaCl in the drinking water groups (Groups 2, 4 and 5) showed markedly increased urine volumes compared to Sham control animals (Groups 1 and 3). Animals from the DOCA + Compound 1 group (Group 5) showed statistically reduced urine output compared to DOCA + Vehicle group (Group 4).
[0423] During urine collection, drink volumes were also recorded. Although none of the groups showed statistically significant differences in drink volume from each other, in general it appeared that animals from the DOCA + 1% NaCl in the drinking water groups (Groups 2, 4 and 5) showed increased drink volumes compared to Sham control animals (Groups 1 and 3). Drink volumes in the groups seem to roughly correspond to urine volumes in the same group.
[0424] Urines were analyzed for protein content and the total amount of protein produced over 24 hours was calculated from the protein concentration and the urine volume produced in 24 hours (FIG. 11 A). In urines collected from animals pre-randomization, a statistically significant elevation in proteinuria was observed in DOCA + Salt animals (Group 2) compared to Sham animals (Group 1), indicating renal dysfunction in the DOCA + Salt animals at the start of compound or vehicle treatment. In urines collected prior to study end, a statistically significant elevation in proteinuria was again observed in DOCA + Vehicle animals (Group 4) compared to Sham animals (Group 3), indicative of continued renal dysfunction. DOCA-exposed animals treated with Compound 1 (Group 5) were found to have statistically significant reduction in proteinuria compared to DOCA + Vehicle treated animals (Group 4). This is consistent with an improvement in renal function by Compound 1 treatment.
[0425] Urines were analyzed for albumin concentration and the total amount of albumin produced over 24 hours was calculated from the albumin concentration and the urine volume produced in 24 hours (FIG. 1 IB). In urines collected from animals pre-randomization, elevated albuminuria was observed in DOCA + Salt animals (Group 2) compared to Sham animals (Group 1), albeit that this effect was not statistically significant. In urines collected prior to study end, a statistically significant elevation in albuminuria was observed in DOCA + Vehicle animals (Group 4) compared to Sham animals (Group 3), indicative of renal dysfunction. DOCA-exposed animals treated with Compound 1 (Group 5) were found to have statistically significant reduction in albuminuria compared to DOCA + Vehicle treated animals (Group 4). This is consistent with the proteinuria data and with an improvement in renal function by Compound 1 treatment.
[0426] Urines were also analyzed for creatinine levels and the albumin-to-creatinine-ratio (ACR) was calculated (FIG. 11C). Data for ACR were similar to the overall albumin production data. As before, DOCA-exposed animals treated with Compound 1 (Group 5), were found to have statistically significant reduction in ACR compared to DOCA + Vehicle treated animals (Group 4). This is consistent with the proteinuria data and the albuminuria data, and with an improvement in renal function by Compound 1 treatment.
[0427] Kidney Injury Molecule- 1 (KIM1) (also known as Hepatitis A virus cellular receptor
1 (HAVcr-1) or T-cell immunoglobulin and mucin domain 1 (TIM1)) is a protein that is highly upregulated in injured kidneys by various types of insults (Bonventre, J. V. Trans. Am. Clin. Climatol. Assoc. 2014;125:293-9). In patients with Minimal Change Disease or Focal Segmental Glomerulosclerosis, elevated KIM1 levels are found in the urine (Zhang, Q., et al. Am. J. Med. Sci. 2018 Apr;355(4):314-21). Urines were analyzed for KIM1 and the total amount of KIM1 produced over 24 hours was calculated (FIG. 1 ID). In urines collected from animals pre randomization, elevated KIM1 production was observed in DOCA + Salt animals (Group 2) compared to Sham animals (Group 1), indicating renal injury at the start of compound or vehicle treatment. In urines collected prior to study end, a statistically significant elevation in KIM1 production was observed in DOCA + Vehicle animals (Group 4) compared to Sham animals (Group 3), indicative of continued renal injury. DOCA-exposed animals treated with Compound 1 (Group 5) were found to have statistically significant reduction in urinary KIM1 production compared to DOCA + Vehicle treated animals (Group 4). This is consistent with the notion that Compound 1 treatment reduces renal damage.
[0428] Hydroxyproline is a major component of collagen, and tissue hydroxyproline levels are considered a good indicator of tissue collagen content and therefore of tissue fibrosis. The hydroxyproline content of renal samples was determined and expressed as the total hydroxyproline content per kidney (FIG. 1 IE). In kidneys from pre-randomization DOCA + Salt treated animals (Group 2), no elevation in renal hydroxyproline content was observed, compared to pre-randomization Sham animals (Group 1). This indicates absence of renal fibrosis at the start of compound or vehicle treatment. In kidneys collected at study end, a statistically significant elevation in renal hydroxyproline content was seen in DOCA + Vehicle animals (Group 4) compared to Sham animals (Group 3), consistent with marked renal fibrosis at study end. DOCA-exposed animals treated with Compound 1 (Group 5), were found to have statistically significant reduction in renal hydroxyproline content compared to DOCA + Vehicle treated animals (Group 4). This indicates that Compound 1 treatment prevents the development of renal fibrosis in DOCA + Salt treated animals.
[0429] The H&E stained slides were scored for renal damage by two blinded observers and the scores were averaged. As shown in FIG. 1 IF, relatively mild (but statistically significant) renal damage was observed in pre-randomization DOCA + Salt animals (Group 2), compared to Sham control animals (Group 1). At study end, animals from the DOCA + Vehicle group (Group 4) had significant renal damage, compared to the Sham treatment group (Group 3). Animals from the DOCA + Compound 1 group (Group 5) showed significantly reduced renal damage compared to the DOCA + Vehicle group (Group 4).
[0430] Picrosirius red-staining in the slides was quantified using Bioquant Image Analysis software. The data was expressed as arbitrary units of staining intensity and also expressed as a % of control in relation to the average staining intensity of the control group (Group 1). As shown in FIG. 11G, in pre-randomization animals, no increase in picrosirius red staining intensity was observed in DOCA + Salt animals (Group 2) compared to Sham control animals (Group 1). This indicates lack of renal fibrosis at the time of compound of vehicle treatment initiation. This lack of histologically observable fibrosis in the renal samples from Group 2 is consistent with the hydroxyproline data in this group. At the end of the compound or vehicle treatment period, DOCA + Vehicle treated animals (Group 4) had markedly increased insterstitial picrosirius red staining compared to Sham control animals (Group 3). This indicates marked collagen deposition and renal fibrosis (tubulointerstitial fibrosis) as a result of DOCA + Salt treatment. By comparison, animals that received DOCA + Salt that were treated with Compound 1 (Group 5) showed statistically significantly reduced picrosirius red staining intensity compared to DOCA + Vehicle animals (Group 4). This is consistent with the observed differences in renal hydroxyproline in the various experimental groups and shows anti-fibrotic effects of Compound 1 in this model.
[0431] All a-Smooth Muscle Actin-stained slides were quantified using Bioquant Image Analysis software. The data was expressed as arbitrary units of staining intensity and also expressed as a % of control in relation to the average staining intensity of the control group (Group 1). As shown in FIG. 11H, in pre-randomization animals, an increase in a-Smooth Muscle Actin staining intensity was observed in DOCA + Salt animals (Group 2) compared to Sham control animals (Group 1), albeit that this increased staining intensity was not statistically significant. It should be noted that the increased a-Smooth Muscle Actin staining occurred while other fibrotic markers such as hydroxyproline and picrosirius red staining were not elevated. This is consistent with the notion that a-Smooth Muscle Actin staining as an indicator of myofibroblast activation, is an early marker of fibrosis. At the end of the compound or vehicle treatment period, DOCA + Vehicle treated animals (Group 4) had markedly increased a-Smooth Muscle Actin staining compared to Sham control animals (Group 3). By comparison, animals that received DOCA + Salt that were treated with Compound 1 (Group 5) showed statistically significantly reduced a-Smooth Muscle Actin staining intensity compared to DOCA + Vehicle animals (Group 4). This is consistent with the observed differences in renal hydroxyproline and picrosirius red staining in the various experimental groups and shows anti-fibrotic effects of Compound 1 in this model.
Example 13. Unilateral Ureteral Obstruction (UUO) Model of Renal Fibrosis in Mice [0432] Male C57BL/6 mice were anesthetized and placed on a heated surgical pad to maintain body temperature between 37 °C and 37.5 °C throughout the surgical procedure and recovery. The left ureter was visualized via a flank incision and ligated with a 6-0 suture.
Starting the day after UUO, animals were randomized to groups receiving vehicle or Compound 1
[0433] Animals with unilateral ureteral obstruction (UUO) were randomized into two experimental groups and animals that did not undergo surgery were used as a control group, as shown Table 7. In order to minimize the potential impact of variation in surgical procedure and execution, animals were randomized for the surgeon performing the UUO operation. Compound 1 or vehicle treatment was initiated the day after UUO surgery and maintained daily until terminal sacrifice on day 10 after UUO. Compound 1 was dosed orally, once a day, at 100 mg/kg. Table 7
Figure imgf000121_0001
a 100 mg/kg Compound 1 Hydrochloride trihydrate.
[0434] Animals were monitored several times daily. Two animals died in the UUO + Vehicle Group (Group 2) on days 2 and 4 after UUO. One animal died 5 days after UUO in the UUO + Compound 1 group (Group 3). Observed mortality was not statistically significant compared to Group 1 (analysis by the Log-rank (Mantel-Cox) test using Graphpad Prism 5.04 data analysis software). Deaths were not deemed to be related to compound treatment.
[0435] At terminal sacrifice, body weights were recorded. All animals that underwent UUO had significantly reduced body weight compared to control animals that did not undergo UUO (Control; Group 1). The body weight in each of the UUO + Compound 1 treated group (Group 3) was not significantly different from animals undergoing UUO that had been treated with Vehicle (Group 2), indicating that there was no obvious effect of compound treatment on body weight. [0436] At terminal sacrifice, obstructed kidneys were collected and their weights recorded. Kidney weights from animals of the experimental groups were not significantly different from each other.
[0437] The weight of the obstructed kidney was also expressed as a percentage of the body weight of each individual animal. The percentage kidney weight for animals from the UUO + Vehicle group (Group 2) was elevated compared to Control animals (Group 1). There was no statistically significant difference between the UUO + Compound 1 group (Group 3) and the other experimental groups.
[0438] The obstructed kidney was fixed in 10% neutral buffered formalin for at least 48 hours before preparation for histology. Kidneys tissues were grossed, processed, dehydrated, embedded in paraffin and cut into 5 pm sections.
[0439] All H&E stained slides were scored for renal damage by two blinded observers and the scores were averaged. As shown in FIG. 12A, severe renal damage was observed in UUO animals that were treated with Vehicle (Group 2). By comparison, animals that had undergone UUO and were treated with Compound 1 (Group 3) showed statistically significant improvements (reductions) in renal damage score.
[0440] All Picrosirius Red-stained slides were quantified using Bioquant Image Analysis software. The data was initially expressed as arbitrary units of staining intensity and then expressed as a % of control in relation to the average staining intensity of the control group (Group 1). As shown in FIG. 12B, a marked increase in picrosirius red staining intensity was observed in UUO animals that were treated with Vehicle (Group 2). This is consistent with marked collagen deposition and renal fibrosis (primarily tubulointerstitial fibrosis). By comparison, animals that had undergone UUO and were treated with Compound 1 (Group 3) showed statistically significantly reduced picrosirius red staining intensity compared to UUO + Vehicle animals (Group 2).
[0441] All a-Smooth Muscle Actin-stained slides were quantified using Bioquant Image Analysis software. The data was initially expressed as arbitrary units of staining intensity and then expressed as a % of control in relation to the average staining intensity of the control group (Group 1). As shown in FIG. 12C, a marked increase in a-Smooth Muscle Actin staining intensity was observed in UUO animals that were treated with Vehicle (Group 2). This is consistent with marked myofibroblast formation. By comparison, animals that had undergone UUO and were treated with Compound 1 (Group 3) showed statistically significantly reduced a- Smooth Muscle Actin staining intensity compared to UUO + Vehicle animals (Group 2).
[0442] The effect of Compound 1 was evaluated in the Unilateral Ureteral Obstruction (UUO) model of renal fibrosis in the mouse. Primary endpoints for evaluating compound effects were renal damage score, collagen deposition (as determined by picrosirius red staining) and formation of myofibroblasts (as determined by a-smooth muscle actin staining). As summarized in Table 8, Compound 1 had statistically significant beneficial effects on renal damage, picrosirius red staining and alpha-SMA staining.
Table 8
Figure imgf000122_0001
Figure imgf000123_0001
A = log-rank (Mantel-Cox) test; B = one-way ANOVA followed by Tukey’s test
Example 14. PCK Rat Model for Polycystic Kidney Disease
[0443] The PCK rat which has a mutation in PKHD1 (ARPKD gene/ phenotypically resembles both human ARPKD-CHF and human PKD in that it exhibits large renal cysts, and renal and hepatic scarring. The PCK rat model is a well-established and well-characterized model that resembles human polycystic kidney and liver disease (Lager, D. L, et al. Kidney Int. 2001 Jan;59(l): 126-36). Results described in this Example are also described in Paka, P., et al. World J. Nephrol. 2018 Sept 8;7(5):96-107).
[0444] One wild-type (WT) SD rat (Group 1) and one PCK rat (Group 2) were sacrificed at 6 weeks of age to confirm disease pathology. PCK rats were then randomized to vehicle (water,
PO, BID; n = 14, Group 4) or Compound 1 (25 mg/kg Compound 1 Hydrochloride trihydrate, PO, BID; n = 14, Group 5). A sham/wild-type SD cohort (n=3, Group 3) served as age-matched control. At 10 weeks of age (i.e., after 4 weeks of drug treatment) animals were anesthetized with isoflurane (2%), a midline incision was made and the left kidney was removed. The left kidney was removed to accelerate disease progression. Animals were then returned to their cages and allowed to recover. And treatment continued. At 14 weeks of age {i.e., after 8 weeks of drug dosing), 24-hour urine was collected, animals were anesthetized, and the right kidney was removed. One animal in Group 5 (PCK Compound 1) died during anesthesia. Experimental groups in this study are summarized in Table 9.
Table 9
Figure imgf000123_0002
[0445] At 6 weeks of age, the PCK rat kidney was enlarged and exhibited numerous cysts compared the kidney from the wild-type (WT) Sprague Dawley animal. At 6 weeks of age, PCK animals were randomized to Compound 1 and vehicle treatment groups and animals were treated for 8 weeks, before collection of 24-hour urine and sacrifice of animals at 14 weeks of age.
[0446] The 14-week old animals were weighed at sacrifice and body weights collected. PCK rats that were treated with either vehicle or with Compound 1 did not differ in their body weights from age and sex matched wild-type (WT) Sprague Dawley rats.
[0447] Serum creatinine (SCr) and BUN levels were determined in sera of 14-week old animals (FIG. 13 A and FIG. 13B, respectively). Both the SCr and BUN levels were elevated in PCK Vehicle rats compared to wild-type (WT) rats, indicating mild, but statistically significant level of renal dysfunction in the PCK animals. Compound 1 treatment for 8 weeks reduced both SCr and BUN levels compared to PCK Vehicle animals, indicating improvement in renal function as a result of Compound 1 treatment.
[0448] Kidneys were weighed at sacrifice and kidney weights are given in absolute weight (g) and as a percentage of body weight (FIG. 13C and FIG. 13D, respectively). Kidneys from PCK Vehicle rats were markedly larger than kidneys from wild-type (WT) rats. Compound 1 treatment in PCK rats reduced kidney size and the % kidney weight compared to PCK Vehicle animals.
[0449] Hydroxyproline is a major component of collagen, and it is used to indicate tissue collagen deposition and fibrosis. Hydroxyproline levels were determined in kidney samples of 14-week old animals (FIG. 13E). Kidney hydroxyproline levels were markedly increased in PCK Vehicle rats compared to wild-type (WT) rats. Compound 1 treatment significantly reduced renal hydroxyproline levels compared to PCK Vehicle animals, indicating reduced renal collagen deposition as a result of Compound 1 treatment.
[0450] The percentage of the renal parenchyma occupied by cysts (cystic index) was quantified in H&E-stained kidney sections using digital planimetry. In kidneys of 14-week old PCK Vehicle animals, there was a marked increase in the cystic index compared to wild-type (WT) rats (FIG. 13F). Compound 1 treatment markedly reduced cystic index compared to PCK Vehicle animals, indicating a significant improvement in renal pathology due to Compound 1 treatment. [0451] Prior to sacrifice, 24-hour urine was collected for individual animals using metabolic cages. In some instances, urine was contaminated by blood (toe bleed in metabolic cages) and the sample was discarded. PCK Vehicle animals produced more urine than age and sex matched Sprague Dawley WT animals (FIG. 13G). Compound 1 treatment in PCK rats reduced urine production to levels similar to the Sprague Dawley WT control animals.
[0452] Increased urine protein excretion can be due to diseases of the glomerulus, an increased quantity of proteins in serum (overflow proteinuria) or low reabsorption at proximal tubule. Urine protein excretion was determined in urines collected over 24 hours immediately prior to sacrifice (FIG. 13H). Protein excretion was found to be elevated in PCK Vehicle rats compared to wild-type (WT) rats, and Compound 1 treatment markedly reduced proteinuria in PCK rats compared to PCK Vehicle animals.
[0453] Albumin is a major plasma protein. Usually, only trace amounts of it are present in urine, whereas larger amounts occur in the urine of patients with kidney disease. Urine albumin concentrations were determined in urines collected over 24 hours immediately prior to sacrifice (FIG. 131). Urine albumin production was found to be strongly elevated in PCK Vehicle rats compared to wild-type (WT) rats, and Compound 1 treatment markedly reduced urine albumin production in PCK rats compared to Vehicle treated PCK animals.
[0454] Lipocalin-2 (LCN2) is also known as neutrophil gelatinase-associated lipocalin (NGAL) and is a protein that is expressed in neutrophils and at low levels in normal kidneys (Cowland, J. B., et al. Genomics 1997 Oct 1;45(1): 17-23). Upon injury of the kidneys, renal and urine NGAL are strongly upregulated, and NGAL is consequently used as a biomarker of kidney injury (Devarajan, P. Nephrology (Carlton) 2010 Jun;15(4):419-28). Urine NGAL excretion was determined for 14-week old animals (FIG. 13J). NGAL excretion was found to be markedly elevated in PCK Vehicle rats compared to wild-type (WT) rats, albeit that the increase was not statistically significant by one way ANOVA followed by Tukey’s test. This is probably the result of high variability in NGAL excretion amongst the PCK Vehicle animals. Compound 1 treatment reduced NGAL excretion in PCK rats compared to Vehicle treated animals, albeit that this decrease was also not statistically significant when tested by ANOVA followed by Tukey’s test. [0455] Hepatitis A virus cellular receptor 1 (HAVcr-1) also known as KIM-1 (KIM1, kidney injury molecule 1) is highly upregulated in injured kidneys in response to various types of renal insults (Bonventre, J. V. Trans. Am. Clin. Climatol. Assoc. 2014;125:293-9). Urine levels of KIM-1 were determined for 14-week old animals WT and PCK receiving vehicle or Compound 1 treatment (FIG. 13K). KIM-1 excretion was found to be strongly elevated in PCK Vehicle rats compared to wild-type (WT) rats, indicating severe renal injury, in particular tubular injury (Bonventre 2014). The elevated KIM-1 excretion is consistent with tubular cyst formation and tubular damage known to exist in the PCK rat (Lager 2001). Compound 1 treatment markedly reduced KIM-1 excretion in PCK rats compared to Vehicle treated animals (FIG. 13K), which indicates mitigation of renal damage, and in particular tubular damage.
[0456] Interleukin 18 (IL-18 or IL18) is also known as interferon-gamma inducing factor and is a pro-inflammatory cytokine. Urine levels of IL-18 are associated with urine protein excretion and have been used as a biomarker for the progression of diabetic nephropathy (Zhang, D., et ak, PeerJ. 2019;7:e7079; Nakamura, A., et al. Diabetes Care 2005 Dec;28(12):2890-5). Urine IL-18 excretion was determined for 14-week old WT and PCK animals (FIG. 13L). IL-18 excretion was found to be elevated in PCK Vehicle rats compared to wild-type (WT) rats, and Compound 1 treatment markedly reduced IL-18 excretion in PCK rats compared to Vehicle treated animals. [0457] Cystatin C or cystatin 3 is used as a biomarker of kidney function (Dharnidharka, V. R., et al. Am. J. Kidney Dis. 2002 Aug;40(2):221-6; Roos, J. F., et al. Clin. Biochem. 2007 Mar;40(5-6):383-91). Urine Cystatin C excretion was determined for 14-week old animals (FIG. 13M). Cystatin C excretion was found to be elevated in PCK Vehicle rats compared to wild-type (WT) rats, and Compound 1 treatment markedly reduced Cystatin C excretion in PCK rats compared to Vehicle treated animals. These results are indicative of an improvement of renal function, consistent with the observation that Compound 1 reduced serum BUN and SCr levels in PCK rats compared to PCK Vehicle treated animals.
Example 15. Model of Inflammatory Bowel Disease (IBD)/Acute Colitis Induced by TNBS in Mice
[0458] Intracolonic administration of TNBS (2,4,6-trinitrobenzenesulfonic acid)/ethanol induces a severe illness characterized by bloody diarrhea and a dramatic loss of body weight during the first week with increased colon damage score (Mateus, V., et al. Clin. Exp. Gastroenterol. 2018;11:325-334). TNBS-induced colitis, characteristic of acute colitis and the early phase of IBD, is a commonly utilized animal model in rodents that shares significant properties with human Crohn's disease. Advantages of this model include reproducibility and technical simplicity (Filipescu, I. E., et al. PLoS One 2018 Aug 23;13(8):e0202929). The use of TNBS-induced colitis has been valuable in elucidating the mechanisms that mediate disease immune-pathogenesis. TNBS-induced colitis is a well-validated animal model to assess compounds with potential therapeutic effects such as anti-TNFa, corticosteroids, natural compounds and traditional medicine (Mateus 2018; Filipescu 2018; Wirtz, S. et al. Nat. Protoc. 2017 Jul; 12(7): 1295-1309). Compound 1 was therefore tested in the well-established model of TNBS-induced IBD/ Acute Colitis.
[0459] Male CD-I mice (n=33) were starved overnight and then infused with 0.1 mL of 25 mg/mL TNBS (2.5 mg/mouse) in 50 % ethanol through rectal enema using a 4 cm long PE- catheter, under isoflurane anesthesia. A sham group (n=6) received an equal volume of 50% ethanol. At 72 hours (3 days) following TNBS (or sham) infusion, 5 TNBS-treated mice, as well as 2 sham mice, were sacrificed to demonstrate significant TNBS-induced colonic inflammation and pathology, including decreased body weight, reduced colon length and colon gross morphological macroscopic damage. The remaining Sham (n=4) and TNBS mice (n=7/group) were randomized to vehicle and Compound 1 at 5, 15 and 45 mg/kg Compound 1 Hydrochloride trihydrate, PO, BID for 4 days. After 4 days of Compound 1 or vehicle treatment, mice were sacrificed. Body weight, colon length and colon weight were recorded. The middle and distal part of colon were fixed in 10% neutral buffered formalin.
[0460] Three days after TNBS (or sham) infusion, one group of mice (n=5) was sacrificed to evaluate TNBS-induced effects on overall animal health and colon damage. Sham -injected animals (n=2) were used as controls. Briefly, a score (Colon Damage Score) of 0 (normal) to 4 (severe) was assigned based on gross morphological observations of colon appearance, colon length/shrinkage, stool consistency and rectal bleeding. TNBS infusion induced significant body weight loss, reduction in colon length and a marked increase in Colon Damage Score.
[0461] Following TNBS treatment, animals were treated for 4 days with vehicle or Compound 1 (5, 15 and 45 mg/kg, BID, PO), before being sacrificed. Animals were weighed (FIG. 14 A), colons were collected and colon weight (FIG. 14B) was recorded. Colon length was determined (FIG. 14C) and macroscopic colon damage was scored (FIG. 14D), using a Colon Damage Score. Compound 1 treatment at 15 and 45 mg/kg, BID, significantly restored body weight, colon weight, and colon length. At 15 and 45 mg/kg, there was also a marked reduction in colon damage, as indicated by significantly reduced Colon Damage Score. [0462] Colon histopathology was determined from H&E-stained tissue sections by two independent observers using a previously published scoring system. The colon Histopathological Score comprises the following four parameters: (a) colon architecture (normal = 0 to severe crypt distortion with loss of entire crypts = 3), (b) degree of inflammatory cell infiltration (normal = 0 to dense inflammatory infiltrate = 3), (c) muscle thickening (normal = 0 to marked muscle thickening present = 3), (d) crypt damage and goblet cells loss (0 to 3). The score for each individual component (0-3) of the Histopathological Score is shown in FIG. 14E and the composite (total) Colon Histopathological Score (the sum of individual component scores; 0-12) is shown in FIG. 14F. Histopathological Scores showed that Compound 1 treatment at 15 and 45 mg/kg markedly improved colon histology compared to pre-treatment and vehicle treated animals.
[0463] Colon tissue sections were stained with Alcian blue to evaluate crypt architecture/goblet cells. The degree of Alcian blue staining was quantified to determine crypt damage and goblet cell loss (FIG. 14G, group averages shown). Compound 1 treatment at 15 and 45 mg/kg significantly reduced TNBS-induced goblet cell loss compared to pre-treatment and vehicle treated animals.
[0464] Myeloperoxidase (MPO) is abundantly expressed in neutrophils and F4/80 expression is restricted to eosinophils. Staining for these markers is therefore indicative of tissue inflammation. Colon tissue was stained for MPO, with quantified data shown in FIG. 14H (group averages shown). Similarly, F4/80 was performed, with quantified staining intensity shown in FIG. 141 (group averages shown). Quantitative analysis of MPO-stained colon tissue showed that Compound 1 treatment at 5 mg/kg and 15 mg/kg significantly decreased MPO staining versus vehicle treated animals. Compound 1 treatment at 45 mg/kg also decreased MPO staining, but the reduction did not reach statistical significance. Quantitative analysis of F4/80-stained slides showed that Compound 1 treatment at 15 mg/kg and higher significantly decreased F4/80- staining versus TNBS plus vehicle treated animals.
[0465] Compound 1 treatment at doses of 15 mg/kg and higher, BID, significantly attenuates inflammatory bowel disease (IBD) in a mouse model. Example 16. Compound 1 Decreases Acetic Acid (AA) Induced Colitis in Mice [0466] Male CD-I mice were infused with 4% AA in saline in 150 mΐ volume through rectal enema under isoflurane anesthesia. A sham group (n=6) received an equivalent volume of saline. After 72 hours of AA (or sham) infusion, a few mice were sacrificed to demonstrate the significant AA-induced IBD pathology including decreased body weight (FIG. 15 A) and reduced colon length (FIG. 15B), increased colon weight (FIG. 15C) and increased gross morphological colon damage (FIG. 15D).
[0467] Remaining AA mice (n=9/ for each group) were then randomized to vehicle and/or Compound 1 at 50 mg/kg Compound 1 Hydrochloride trihydrate, PO, BID for 3 days.
Compound 1 treatment significantly mitigated the decrease in colon length (FIG. 15E), gross morphological colon damage (FIG. 15F) and histopathological colon damage (FIG. 15G) by decreasing mucosal wall thickness, and preserving and crypt/villi architecture compared to vehicle cohort (FIG. 15H).
Example 17. Compound 1 Decreases Chronic Colitis in IBD Mouse Model [0468] Efficacy of Compound 1 in a dextran sulfate sodium (DSS) induced chronic colitis/IBD model was evaluated. Adult male CD-I mice were fed with 3% DSS in drinking water for 5 days with alternate cycles of normal drinking water for 5 days and continued a total of 3 DSS cycles in 4 weeks. This model has been established to induce chronic colitis/IBD. Vehicle (n=10/group) and Compound 1 (50 mg/kg Compound 1 Hydrochloride trihydrate, PO, BID) were administered 72 hours after DSS and continued treatment for 4 weeks, then all mice were sacrificed. A small group (n=6) with normal drinking water was included as a sham control. Gross morphological and histopathological end points were evaluated. Additionally, tissue from the middle colon (lOOmg) from all groups was subjected to hydroxyproline assay for collagen estimation as a fibrotic end point.
[0469] DSS-Vehicle cohort had the shortest colon length; DSS-Compound 1 cohort had significantly increased colon length compared to the DSS-vehicle cohort (FIG. 16A). Compound 1 treatment decreased macroscopic colon damage score in terms of appearance, inflammation and rectal bleeding (FIG. 16B), decreased colonic hydroxyproline (FIG. 16C) and histopathological injury score based on crypt damage, epithelial erosion and inflammatory cell infiltration as seen in H&E stained colon sections (FIG. 16D). These results indicate that Compound 1 could prevent progression of inflammation-induced fibrosis.
Example 18. Compound 1 Reduces Fibrosis in Additional Animal Models
Unilateral Ureteral Obstruction (UUO) Mouse Model
[0470] Ligation of one ureter in a mouse caused renal dysfunction and fibrosis. As described in Example 13, extent of renal fibrosis was quantified by Sirius Red staining for collagen deposition of kidney sections, wherein less staining indicated less renal fibrosis. The non-ligated kidney was used as a control. Compounds were administered orally once a day in 100 mg/kg Compound 1 Hydrochloride trihydrate. Results are summarized in Table 10. Compound 1 demonstrated 1.9 fold greater inhibition of fibrosis compared to nintedanib.
Table 10
Figure imgf000130_0001
Uninephrectomized ( UNX ) Rat Model
[0471] One kidney of a rat was surgically removed. The remaining kidney developed pathology manifested by the appearance of protein in urine, increasing from about 225 mg/day in normal rats to about 450 mg/day in UNX rats. Reduced proteinuria indicated healthier kidney. Compounds were administered orally at 50 mg/kg Compound 1 Hydrochloride trihydrate twice a day. Results of proteinuria analysis are summarized in Table 11. Compound 1 demonstrated 2.3 fold better reduction in proteinuria than nintedanib.
Table 11
Figure imgf000130_0002
[0472] The extent of kidney injury was also evaluated via histopathological score (scored by a blinded individual). Results of histopathological analysis are summarized in Table 12. Compound 1 demonstrated 1.4 fold greater reduction in pathology compared to nintedanib.
Table 12
Figure imgf000131_0001
PCK Rat Model
[0473] The PCK rat which has a mutation in PKHD1 (ARPKD gene) phenotypically resembles both human ARPKD-CHF and human PKD in that it exhibits large renal cysts, and renal and hepatic scarring, and is recognized model for polycystic kidney disease (e.g., as described in Example 14). Male PCK rats (PCK/CrljCrl-pkhdlpck/Crl; Charles River Laboratories were randomized to vehicle or test compounds (25 mg/kg Compound 1 Hydrochloride trihydrate, BID, PO) at 6.5 weeks of age following confirmation of frank disease and then sacrificed at 13.5 weeks of age. Age-matched male Sprague-Dawley rats served as wild-type controls. Proteinuria was recorded daily and the results are summarized in Table 13. Compound 1 demonstrated a 2.9 fold greater reduction in proteinuria than nintedanib.
Table 13
Figure imgf000131_0002
[0474] Female PCK rats (PCK/CrljCrl-pkhdlpck/Crl) were randomized to vehicle or test compounds (25 mg/kg Compound 1 Hydrochloride trihydrate, BID, PO) at 6.5 weeks of age, following confirmation of frank disease and sacrificed at 13.5 weeks. Age-matched Sprague- Dawley rats served as wild-type controls. Proteinuria was recorded daily and the results are summarized in Table 14. Compound 1 demonstrated 3.8 fold greater reduction in proteinuria compared to nintedanib.
Table 14
Figure imgf000132_0001
Streytozotocin-Induced Diabetic Mouse Model
[0475] Streptozotocin (STZ)-induced diabetic mice were randomized to vehicle or test compounds 12 weeks after STZ induction. Results of kidney hydroxyproline analysis are summarized in Table 15. Compound 1 demonstrated 8.3 fold greater reduction in kidney hydroxyproline than nintedanib.
Table 15
Figure imgf000132_0002
[0476] Results of kidney weight analysis are summarized in Table 16. Compound 1 demonstrated 8.1 fold greater reduction in kidney weight than nintedanib.
Table 16
Figure imgf000132_0003
Bleomycin-Induced Pulmonary Fibrosis Mouse Model
[0477] Ten weeks old male C57BL/6 mice were instilled with bleomycin intratracheally (2.5 U/kg body weight in 50 pL volume). A sham group received equal volume of intratracheal saline. Three weeks after bleomycin instillation, a subgroup of animals was sacrificed and established lung fibrosis was confirmed as determined by lung hydroxyproline content and histopathology. Then the mice (n=12/group) were randomized to vehicle, Compound 1 and pirfenidone (PFD), each at 25 mg/kg (PO, BID) and treated for 3 weeks. (Compound 1 was administered as 25 mg/kg Compound 1 Hydrochloride trihydrate.) All mice were then sacrificed to determine the extent of lung fibrosis. Compound 1 treatment significantly decreased lung hydroxyproline (FIG. 17A), based on the Ashcroft lung histopathological score (FIG. 17B),
Sirius red staining (FIG. 17C) and TGFpi staining (FIG. 17D) were reduced significantly compared to the vehicle cohort and showed better efficacy even when compared to the pirfenidone (PFD) cohort (an advanced clinical compound).
Example 19. Identification of Biomarkers for Treatment of Kidney Fibrotic Diseases with Compound 1
[0478] The present disclosure encompasses a recognition that in different patients, disease driving pathways may have divergent outcomes or converge on a common disease related outcome. For example, as depicted in FIG. 18, in some instances, different individuals may have distinct genomic responses that all result in a common kidney disease endpoint, while in other instances, different individuals may have a shared genomic response that leads to different kidney disease endpoints. For example, distinct disease-driving pathways in different individuals may lead to a common outcome of a fibrotic kidney disease, such as, e.g., FSGS. As such, different patients may respond to different therapies and interventions for the same disease. The present disclosure provides the insight that identifying and neutralizing the particular disease drivers of a given patient may provide an early and effective way to manage kidney disease endpoints. Specifically, the present example determined the signalosome in various models of Focal and Segmental Glomerulosclerosis (FSGS) to identify biomarkers for treatment with Compound 1.
[0479] Exemplary and etiologically distinct murine models of FSGS were used to characterize the kidney transcriptome and proteome that are associated with therapeutic activity of Compound 1. At the end of the in-life component of each of the studies, kidneys were harvested. For the rat models, kidney tissue was preserved in RNAlater (Sigma Aldrich) for glomerular isolation by manual microdissection. For the mouse model, glomeruli were isolated by magnetic bead extraction. RNA was extracted from glomerular preparations. RNA extraction, RNA sequencing, data normalization and filtering were then performed. Differential expression analysis was performed using DESeq2 and EdgeR (M. F Love, et ak, (December 5, 2014) Genome Biol. 15: 550; M. D. Robinson, et ak, (January 1, 2010) Bioinformatics, 26(1): 139-140). Significantly regulated genes were analyzed by creating biological literature-based networks with Genomatix Pathway System software (GePS) (www.genomatix.de). Canonical pathways were analyzed using Ingenuity Pathway Analysis software (IP A)
(www.ingenuity.com). Hierarchical cluster dendrograms were generated to determine the ability of overall transcriptional profiles to recapitulate treatment groups. Rat and murine genes were converted to the corresponding human orthologs using the NCBI homolog (Build 64) and Genomatix annotated ortholog databases. Signalosome of rodent models treated with Compound 1 was analyzed and compared with transcriptome of renal dysfunction in human patients to thereby identify biomarkers of patients to be treated with Compound 1.
Exemplary Rodent Model of Kidney Disease
[0480] Administration of puromycin aminonucleoside (PAN) to Wistar rats (a sensitive strain) produces overt proteinuria (Moreno-Manzano, V., et al. Br. J. Pharmacol. 2003 Jun; 139(4): 823 -31). Adult Wistar rats were administered PAN (-100 mg/kg, IP). On Day 4 after PAN administration and after confirming increased urine protein (24 hr), animals were randomized to vehicle (n=14) or Compound 1 (45 mg/kg Compound 1 Hydrochloride trihydrate, PO, BID, n=14). Urine protein was determined again on Days 8, 12, and 21 (FIG. 19).
[0481] These data show that rat puromycin aminonucleoside nephropathy (PAN) model of proteinuric kidney disease exhibit proteinuria that is mitigated by treatment with Compound 1, but not with sham. Animals were sacrificed on Day 21 after PAN administration. Mean arterial pressure (MAP) was evaluated prior to sacrifice and was not substantially different between vehicle treated and Compound 1 treated animals (data not shown). Thus, therapeutic intervention with Compound 1 in this rodent model was associated with a decrease in urine protein with no change in MAP. [0482] Kidneys were harvested and one of the two kidneys was used for analysis of the glomerular transcriptome. The other kidneys were homogenized for determination of COL1 A1 and COL3 A1 expression. Therapeutic intervention with Compound 1 was associated with decreased renal COL1 A1 and COL3A1 expression, shown in FIG. 20 and FIG. 21, respectively. [0483] Similar results are observed using a uninephrectomy (UNX) + DOCA + Salt rat model of renal dysfunction. In this model, renal dysfunction is introduced by removing one kidney and administering subcutaneous injections of the aldosterone precursor deoxycorticosteone acetate (DOCA), while providing 1% NaCl in the drinking water. Characteristics of this model include the development of severe proteinuria and glomerulosclerosis, which reflects several aspects of the pathogenesis of focal and segmental glomerulosclerosis (FSGS).The use of this model has been described extensively in the literature (See Arai, K., et al. J. Pharm. Exp. Ther. 2016 Sep;358(3):548-57; Kretzler, M., et al. Virchows Arch. 1994;425(2): 181-93).
[0484] The present disclosure provides the insight that disease-driving networks in animal (e.g., rodent) models of kidney disease may prove beneficial in those human patients that share the same transcriptional elements. Using network level human-rodent transcriptome mapping strategies for diabetic kidney disease (Hodgin, J. B., et al. Diabetes 2013 Jan;62(l):299-308) and lupus nephritis (Berthier, C. C., et al. J. Immunol. 2012 Jul 15; 189(2):988-1001), human-model network intersects were generated using available transcriptional profiling in human subjects with minimal change disease (MCD) and/or FSGS. These network models were used to identify disease marker genes and signatures that respond to Compound 1 therapeutic intervention.
[0485] COL1A1 and COL3A1 have been identified as disease-associated nodes in human FSGS. See, e.g., Canadas-Garre, M., et al. J. Transl. Med. (2018) 16:292; Grgic, T, et al. Kidney Int. 2014 Dec; 86(6): 1116-1129; Schwab, K., et al. Am. J. Nephrol. 2004;24:438-447. Furthermore, increased renal COL3 Al expression is associated with increased renal dysfunction in human FSGS.
[0486] The therapeutic relevance of the Compound 1 interactome in clinical kidney disease has been established, with COL1 Al and COL3A1 identified as components of this network (FIG. 22).
[0487] Other components of the Compound 1 interactome network include MAP2K5, MAP3K3, MAPK7, PRKAR1A, PIK3R1, SMPD1, AXL, FYN, PLXND1, CDC42PB, VAT1, RPS6KA2, RBPMS, CAV1, BGN, NPR1, FGFR1, EFEMP2, COL6A1, HEG1, PCGF2, COL6A2, MYH11, PDGFRA, PDGFRB, and KIT.
[0488] Thus, the present disclosure identified biomarkers in rodent models of kidney disease and in human patients that correspond with the Compound 1 mechanism of action. The present disclosure encompasses a recognition that patients with fibrotic kidney diseases who have an altered level of one or more of these biomarkers may benefit from treatment with Compound 1.
Example 20. Therapeutic Treatment of Fibrotic Kidney Disease Patients Expressing Biomarkers for Compound 1
[0489] The present example describes treatment of fibrotic disease of the kidney in a patient. Specifically, a nephropathy patient with suspected glomerular disease (i.e., suspected of having FSGS or MCD) is subjected to a renal biopsy. For example, mRNA is extracted from single glomeruli (see Menon et ah, JCI Insight. 2020, 5(6):el33267), and sequenced to quantify levels of certain biomarkers associated with Compound l’s mechanism of action. In particular,
COL1 A1 and COL3A1 expression are quantified. If the subject has a level of COL3A1 and/or COL1A1 that is above a threshold level, then the subject is to be treated with Compound 1. An exemplary threshold level is a level that is about 20% or more above a predetermined normal range or mean or median level in a healthy patient. If a subject does not have a level of biomarker above the threshold level, then alternate treatment is determined. In some embodiments, a patient to be treated with Compound 1, the kidney disease is stabilized and/or ameliorated.
Example 21. Puromycin Aminonucleoside Administration in Rat is Associated with Collagen Type III Glomerulopathy
[0490] Adult male Wistar rats (~75 g) were administered PAN (100 mg/kg, intaperitoneally, n=8) and followed out to 21 days when they were sacrificed. A sham cohort (n=4) was injected with an equivalent volume of water. Twenty-four hour urine samples were obtained on Days 4,
8, 14 and 21 after PAN administration and from the sham cohort. Urine protein was determine using the Bradford assay.
[0491] Administration of PAN was associated with a steep increase in urine protein that peaked at ~ day 14 (FIG. 23). By Day 21 urine protein level had decreased although it was still elevated compared to the sham cohort. Histopathological analysis of the kidney showed an increased hypertrophy of the glomerulus in the PAN cohort accompanied by expansion of both the Bowman’s capsule and Bowman’s space.
[0492] At sacrifice the left kidney was retrieved. Renal homogenates were subjected to analysis for COL3A1 and peptidylprolyl isomerase A (housekeeping gene) mRNA. qPCR was performed on a Thermofisher Quant-Studio 3 Real-Time PCR system, each sample was diluted three-fold, and qPCR reaction was performed in triplicate for all tissue samples. Renal slices were stained with periodic acid Schiff for morphometric analysis or with Collagen Type III (Col3al) Antibody (BioCompare) for immunohistochemical analysis.
[0493] HumanBase was used to build glomerular (G) and tubular (T) COL3 A1 transcriptomic networks. Network analysis was restricted to 51 elements each, inclusive of COL3A1, with a minimum interaction confidence of 0.01. The Jaccard-Tanimoto similarity index was used to calculate common elements within the two compartments.
] (GX) = j n T ÷j u T
[0494] Data are expressed as average ± standard error of mean. Between group differences were calculated using Student’s T-test and a p value < 0.05 assumed significant.
[0495] COL3 A1 mRNA level was elevated ~4-fold in the PAN cohort (FIG. 24A). There was as significant and direct association between the fold-increase in renal COL3 A1 mRNA level and the corresponding urine protein value (FIG. 24B). Immunohistochemical analysis indicated deposition of collagen type III restricted to the glomerular mesangium.
[0496] Network analysis revealed a relative strong glomerular COL3 A1 interactome with an average strength of 0.8±0.08 (FIG. 25A) and a relatively weaker tubular COL3 A1 interactome with an average strength of 0.56±0.01 (FIG. 25B). The Jaccard-Tannimoto similarity index between the glomerular (G) and tubular (T) COL3A1 signaling elements was 5.1%.
[0497] The present example demonstrates that an increase in COL3A1 mRNA is directly associated with an increase in urine protein in a rat model of kidney disease. In particular, collagen type III deposition is restricted to the glomerulus, which may be indicative of a robust COL3A1 transcriptomic network in that compartment, compared to, e.g., the tubulointerstitium. Example 22. Identification of Biomarkers for Treatment of Pulmonary Fibrotic Diseases with Compound 1
[0498] The present disclosure encompasses a recognition that in different patients, disease driving pathways may have divergent outcomes or converge on a common disease related outcome. For example, in some instances, different individuals may have distinct genomic responses that all result in a common lung disease endpoint, while in other instances, different individuals may have a shared genomic response that leads to different lung disease endpoints. For example, distinct disease-driving pathways in different individuals may lead to a common outcome of a fibrotic lung disease, such as, e.g., IPF. As such, different patients may respond to different therapies and interventions for the same disease. The present disclosure provides the insight that identifying and neutralizing the particular disease drivers of a given patient may provide an early and effective way to manage lung disease endpoints. Specifically, the present example determined the signalosome in various models of idiopathic pulmonary fibrosis (IPF) to identify biomarkers for treatment with Compound 1
[0499] Two exemplary and etiologically distinct models of pulmonary fibrosis are used to characterize the lung transcriptome and proteome that are associated with therapeutic activity of Compound 1. Dosing of Compound 1 starts 2 weeks after disease induction with bleomycin or S1O2 instillation, and dosing is continued via the oral route for 4 weeks. Lung hydroxyproline (HYP) and histopathology are determined. BAL inflammatory cell infiltrates, BAL protein, MPO, BAL cytokines, tissue cytokines, and lung fibrotic markers are evaluated by IHC stainings and transcriptomic analysis by performing real-time PCR. A cytokine/chemokine/growth factor array on minimally invasive BALF samples from the animal models. It is expected that the results of such analysis will enable identification of biomarkers that correspond with Compound l’s mechanism of action. The present disclosure encompasses a recognition that patients with fibrotic lung disease who have an altered level of one or more of these biomarkers may benefit from treatment with Compound 1.
Example 23. Therapeutic Treatment of Fibrotic Lung Disease Patients Expressing Biomarkers for Compound 1
[0500] The present example describes treatment of fibrotic disease of the lung in a patient. Specifically, a sample of BALF is obtained from a patient with suspected or confirmed pulmonary disease (i.e., suspected of having idiopathic pulmonary fibrosis). For example, mRNA is extracted from the sample, and sequenced to quantify levels of certain biomarkers associated with Compound l’s mechanism of action. In particular, expression of biomarkers identified using the method of Example 22 are quantified. If the subject has a level of a biomarker that is above a threshold level, then the subject is to be treated with Compound 1. An exemplary threshold level is a level that is about 20% or more above a predetermined normal range or median level in a healthy patient. If a subject does not have a level of biomarker above the threshold level, then alternate treatment is determined. In some embodiments, in a patient to be treated with Compound 1, the lung disease is stabilized and/or ameliorated.
Example 24. A Phase 1, Randomized, Double-Blind, Placebo-controlled, Single and Multiple Ascending Dose Study to Determine the Safety, Tolerability, Pharmacokinetics, and Food Effect of Compound 1 in Healthy Adult Participants
Objectives
[0501] Primary: To assess the safety and tolerability of single and multiple ascending doses of Compound 1 in healthy adult participants.
[0502] Secondary: To assess the pharmacokinetics (PK) of single and multiple ascending doses of Compound 1 and to evaluate the effect of a high fat meal on the PK of a single dose of Compound 1 administered to healthy adult participants.
Endpoints
[0503] Primary Endpoint: The frequency and severity of treatment-emergent adverse events (TEAEs), including clinically significant abnormal vital signs, electrocardiograms (ECGs), laboratory test results, and physical examination findings.
[0504] Secondary Endpoints:
[0505] Plasma PK endpoints include:
• Maximum concentration (Cmax)
• Time to maximum concentration (Tmax)
• Area under drug concentration-time curve, from time zero to the last measurable concentration (AUCo-iast)
• Area under drug concentration-time curve, from time zero to infinity (AUCo-inf) • Area under drug concentration-time curve over inter-dosing interval (AUCtau); MAD cohorts only
• Apparent terminal half-life (ti/2)
• Apparent terminal elimination rate constant (Kei)
• Apparent clearance (SAD cohorts = CL/F; MAD cohorts = CL/Fss)
• Apparent terminal volume of distribution (SAD cohorts = Vz/F; MAD cohorts = Vz/Fss)
• Accumulation ration (RA); MAD cohorts only [0506] Urine PK endpoints include:
• Amount of drug excreted in urine over time (Aeu-u), including cumulative Ae(o-72 h)
• Renal clearance (CLR)
• Fraction of systemic clearance (CL/F) represented by renal clearance (CLR/[CL/F])
• Fraction of administered dose excreted in urine over dosing intervals (Feu-u), including cumulative Fe(o-72 h)
Methodology
[0507] This was a first in human, single-center, double-blind, randomized, cross-over, SAD design followed by a MAD design study of Compound 1 conducted in healthy adult participants, designed to evaluate safety, tolerability, PK, and food effect of Compound 1 in healthy adults.
Up to 104 participants were enrolled into one of up to six SAD cohorts (n=8 per cohort), four MAD cohorts (n=8 per cohort) for twice-daily dosing for two weeks, two MAD cohorts (n=8 per cohort) for once-daily dosing for two weeks, and one 500 - 600 mg single dose food-effect cross-over cohort (n=8 per cohort).
Part A: Single Ascending Dose (SAD)
[0508] Up to 48 participants were enrolled into one of up to six cohorts (Cohorts A1 to A6; n=8 per cohort). All Part A (SAD) participants were confined to a clinical research unit (CRU) from Day -1 (pre-dose) until completion of the 72-hour post-dose assessments on Day 4.
[0509] Cohort A3 was a food effect cohort, and participants in Cohort A3 only returned to the CRU on Day 14 and, following a 14-day washout, received a second single dose of their assigned treatment on Day 15 following consumption of a high fat meal. For Cohort A3 only, a second period of confinement applied from Day 14 until completion of the 72-hour post-dose assessments on Day 18. [0510] All Part A participants returned to the CRU for a follow-up visit 7 days (±1 day) after their final dose of study drug. For all cohorts in Part A (SAD), the decision to escalate a dose or modify a dose was determined by the SMC following review of the 7-day blinded safety and available PK data from the preceding cohort.
[0511] Cohorts AE A2. A4. A5. and A6: Participants (n=8) were randomized 3 : 1 to receive a single oral dose of either Compound 1 (n=6) or matching placebo (n=2) on Day 1 following an overnight fast of at least 10 hours and after all pre-dose assessments were performed. Two sentinel participants received a single oral dose of Compound 1 (n=l) or matching placebo (n=l) initially. If dosing of these sentinel participants proceeds without clinically significant safety signals in the first 48 hours post-dose (as adjudicated by the principal investigator or delegate), the remaining participants in each cohort received a single dose of Compound 1 (n=5) or matching placebo (n=l) according to the randomization schedule.
[0512] Participants were monitored for safety and blood and urine samples were collected for assessment of PK parameters at predefined time points pre and post-dose. Participants were discharged on Day 4 following completion of all specified study procedures. Participants returned to the CRU for an end of study Follow-up visit on Day 8 (±1 day).
[0513] Cohort A3 (food effect): Participants in the Food Effect Cohort (A3; n=8) received a single oral dose of either Compound 1 or matching placebo in the fasted state (Period 1) followed by the same assigned treatment in the fed state (Period 2) after a 14-day washout period as follows.
[0514] Cohort A3 (Period 1): Participants (n=8) were randomized to receive a single oral dose of either Compound 1 (n=6) or matching placebo (n=2) on Day 1 following an overnight fast of at least 10 hours and after all pre-dose assessments were performed. Two sentinel participants received a single oral dose of Compound 1 (n=l) or matching placebo (n=l) initially. If dosing of these sentinel participants proceeded without clinically significant safety signals in the first 48 hours post-dose (as adjudicated by the PI or delegate), the remaining participants received a single dose of Compound 1 (n=5) or placebo (n=l) according to the randomization schedule. Participants were monitored for safety and blood and urine samples were collected for assessment of PK parameters at predefined time points pre- and post-dose. Participants were discharged on Day 4 following completion of all specified study procedures. [0515] Cohort A3 (Period 2): Participants (n=8) completed a 14-day washout following dosing in Period 1 and returned and were admitted to the CRU on Day 14, after which they received a second single dose of their assigned treatment (oral dose of Compound 1 (n=6) or matching placebo (n=2)) on Day 15, following consumption of a high fat meal. The high fat meal was served 30 minutes prior to study drug administration and was consumed within 30 minutes. Participants were monitored for safety and blood and urine samples were collected for assessment of PK parameters at predefined time points pre-and post-dose. Participants were discharged on Day 18 following completion of all specified study procedures. Participants in Cohort A3 returned to the CRU for an end of study follow-up visit on Day 22 (±1 day).
[0516] Compound 1 dose level tested in Part A (SAD) did not exceed 1200 mg or the dose level that led to a mean whole blood Cmax > 600 ng/mL or mean whole blood AUCo-iast > 2000 ng*h/mL. These Cmax and AUCo-iast values were extrapolated from the mean plasma Cmax of 800 ng/mL and the mean plasma AUCiast of 4000 ng*h/mL observed in non-human primates at the no observed adverse effect level (NOAEL) of 75 mg/kg/day.
[0517] For Part A (SAD), single oral dosing was carried out according to Table 1.
Table 17.
Figure imgf000142_0001
* Compound 1 dose will not exceed 1200 mg or a dose level that leads to mean whole blood Cmax >600 ng/mL or mean whole blood AUCo-iast > 2000 ng*h/mL. These Cmax and AUCiast values were extrapolated from the mean plasma Cmax of 800 ng/mL and the mean plasma AUCiast of 4000 ng*hr/mL observed in non-human primates at the NOAEL of 75 mg/kg/day.
** Day 15 doses for Cohort A3 (food effect) were administered following consumption of a high fat meal.
Part B: Multiple Ascending Dose (MAD)
[0518] Up to 32 participants were enrolled into up to four cohorts (Cohorts B 1 to B4; n=8 per cohort). Participants were administered Compound 1 (n=6 per cohort) or matching placebo (n=2 per cohort) twice daily for 7 consecutive days (Day 1 to Day 7) or twice daily for 14 consecutive days (Day 1 to Day 14) determined based on the safety and tolerability data from the SAD portion of the study (Part A). All Part B (MAD) participants were confined to the CRU from Day -1 (pre-dose) to Day 10 or Day 17, depending on the dosing schedule determined following review of data from Part A (SAD). Participants were monitored for safety and blood and urine samples were collected for assessment of PK at predefined time points pre- and post dose.
[0519] Participants were administered study drug in a standardized manner; i.e., either with or without food depending on preliminary bioavailability results from Part A (SAD). Participants were discharged from the CRU upon completion of final 72-hour post-dose assessments. Participants then returned to the CRU for an end of study follow-up visit 7 days after the final dose of study drug (±1 day). For all cohorts in Part B (MAD), the decision to escalate a dose or modify a dose was determined following review of the 10- or 17-day blinded safety and available PK data from the preceding cohort.
[0520] The Compound 1 dose level tested in Part B (MAD) did not exceed 1000 mg per day or a dose level that led to a mean whole blood Cmax > 600 ng/mL or mean whole blood AUCo-iast > 2000 ng*h/mL. These Cmax and AUCo-iast values were extrapolated from the mean plasma Cmax of 800 ng/mL and the mean plasma AUCiast of 4000 ng*h/mL observed in non-human primates at the no observed adverse effect level (NOAEL) of 75 mg/kg/day.
[0521] For Part B (MAD), once daily oral dosing was carried out according to Table 18.
Table 18.
Figure imgf000143_0001
* Compound 1 dose will not exceed 1000 mg per day or a dose level that leads to mean whole blood Cmax >600 ng/mL or mean whole blood AUCo-iast > 2000 ng*h/mL. These Cmax and AUCiast values were extrapolated from the mean plasma Cmax of 800 ng/mL and the mean plasma AUCiast of 4000 ng*hr/mL observed in non-human primates at the NOAEL of 75 mg/kg/day.
** May be twice daily for 7 consecutive days (Day 1 to Day 7) or twice daily for 14 consecutive days (Day 1 to Day 14).
Part C: Multiple Ascending Dose Once Daily (MAD QD)
[0522] Up to 16 participants were enrolled into up to 2 cohorts (Cohorts Cl to C2; n=8 per cohort). Participants were administered Compound 1 (n=6 per cohort) or matching placebo (n=2 per cohort) once daily for 14 consecutive days (Day 1 to Day 14). All Part C participants were confined to the CRU from Day -1 (pre-dose) to Day 17. Participants were monitored for safety and blood and urine samples were collected for assessment of PK at predefined time points pre- and post-dose.
[0523] For Part C (MAD QD), once daily oral dosing was carried out according to Table 19.
Table 19.
Figure imgf000144_0001
* Compound 1 dose will not exceed dose level that leads to mean whole blood Cmax >600 ng/mL or mean whole blood AUCo-iast > 2000 ng*h/mL. These Cmax and AUCiast values were extrapolated from the mean plasma Cmax of 800 ng/mL and the mean plasma AUCiast of 4000 ng*hr/mL observed in non-human primates at the NOAEL of 75 mg/kg/day.
Part D: Single-Dose Food-Effect Cohort
[0524] Eight participants were enrolled into 1 cohort (Cohort Dl). Four participants were randomly administered a single dose of Compound 1 (n=3) or matching placebo (n=l) in fasting condition first, and four participants were administered a single dose of Compound 1 (n=3) or matching placebo (n=l) after a standard FDA high-fat meal in the Period 1. Participants completed the 72-hour post first dose assessments and began the Period 2 of Part D on Day 5, thus allowing a 4-day washout period from the first administered dose. In Period 2, in a cross over design, the group administered Compound 1 and placebo in the fasted condition during Period 1 received Compound 1 (n=3) and placebo (n=l) in a fed state. The group assigned to the fed state dosing during Period 1 crossed over to receive Compound 1 (n=3) and placebo (n=l) after fasting. All Part D participants were confined to the CRU from Day -1 (pre-dose) until the completion of the 72-hour post second single-dose assessments on Day 8. Participants were monitored for safety and blood and urine samples were collected for assessment of PK at predefined time points pre- and post-dose.
[0525] For Part D (Single Dose Food Effect), once daily oral dosing was carried out according to Table 20. Table 20.
Figure imgf000145_0001
* Compound 1 dose will not exceed dose level that leads to mean whole blood Cmax >600 ng/mL or mean whole blood AUCo-iast > 2000 ng*h/mL. These Cmax and AUCiast values were extrapolated from the mean plasma Cmax of 800 ng/mL and the mean plasma AUCiast of 4000 ng*hr/mL observed in non-human primates at the NOAEL of 75 mg/kg/day.
[0526] Up to 104 participants were enrolled in the study (Part A: up to 48 healthy volunteers; Part B: up to 32 healthy volunteers; Part C: up to 16 healthy volunteers; Part D: up to 8 healthy volunteers).
Diagnosis and Main Criteria for Inclusion:
Inclusion Criteria:
[0527] To be eligible for the study, participants were required to meet all of the following inclusion criteria:
1. Healthy male or female volunteer, aged 18 to 65 years.
2. Participants must be in good health, with no significant medical history, have no clinically significant abnormalities on physical examination at screening and/or before administration of the initial dose of study drug.
3. Participants must have a minimum body weight of 50 kg and a body mass index (BMI) between >18.0 and <32.0 kg/m2 at screening.
4. Participants must have clinical laboratory values within normal range as specified by the testing laboratory, unless deemed not clinically significant by the investigator or delegate.
5. Participants who smoke no more than 2 cigarettes or equivalent per week can be included in study but must be willing to abstain from smoking during the confinement period.
6. Participants must have no relevant dietary restrictions, and be willing to consume standard meals provided during the confinement period. Women of childbearing potential (WOCBP) must be non-pregnant and non-lactating, and must use an acceptable, highly effective double contraception from screening until study completion, including the follow-up period. Double contraception is defined as a condom and one other form of the following:
• Established hormonal contraception (oral contraceptive pills, long-acting implantable hormones, injectable hormones)
• A vaginal ring or an intrauterine device
• Documented evidence of surgical sterilization at least 6 months prior to screening (e.g., tubal occlusion, hysterectomy, bilateral salpingectomy, or bilateral oophorectomy for women or vasectomy for men (with appropriate post-vasectomy documentation of absence of sperm in semen) provided the male partner is a sole partner
Women not of childbearing potential must be postmenopausal for > 12 months. Postmenopausal status are confirmed through testing follicle-stimulating hormone (FSH) levels > 40 IU/mL at screening for amenorrhoeic female participants. Females who are abstinent from heterosexual intercourse are also eligible.
Periodic abstinence (e.g., calendar, ovulation, symptothermal, post-ovulation- methods) and withdrawal are not considered highly effective methods of birth control. Participant complete abstinence for the duration of the study and for 1 months after last study treatment is acceptable.
Female participants who are in same sex relationships are not required to use contraception.
WOCBP must have negative pregnancy test at screening and Day 1 and be willing to have additional pregnancy test as required.
Males must be surgically sterile (>30 days since vasectomy with no viable sperm), abstinent, or if engaged in sexual relations with a WOCBP, the participant and his partner must be surgically sterile (e.g., tubal occlusion, hysterectomy, bilateral salpingectomy, or bilateral oophorectomy) or using an acceptable, highly effective contraceptive method from Screening until study completion, including the follow-up period. Acceptable methods of contraception include the use of condoms and the use of an effective contraceptive for the female partner that includes: OCPs, long-acting implantable hormones, injectable hormones, a vaginal ring or an IUD. Participants with same sex partners (abstinence from penile-vaginal intercourse) are eligible when this is their preferred and usual lifestyle.
8. Male participants must not donate sperm for at least 90 days after the last dose of study drug.
9. Participants must have the ability and willingness to attend necessary visits to the CRU.
10. Participants must be willing and able to provide written informed consent after the nature of the study has been explained and prior to the commencement of any study procedures.
[0528] A participant who met any of the following exclusion criteria were excluded from the study:
1. Pregnant or lactating at screening or planning to become pregnant (self or partner) at any time during the study, including the follow-up period.
2. Prior or ongoing medical conditions, medical history, physical findings, or laboratory abnormality that, in the investigator’s (or delegate’s) medical opinion, could adversely affect the safety of the participant.
3. History of gastrointestinal (GI) disorders such as celiac disease, atrophic gastritis, lactose intolerance, and Helicobacter (H.) pylori infection.
4. Presence of any underlying physical or psychological medical condition that, in the opinion of the investigator, would make it unlikely that the participant will comply with the protocol or complete the study per protocol.
5. Any surgical or medical condition that could interfere with the absorption, distribution, metabolism, or excretion of the study drug.
6. Blood donation or significant blood loss within 60 days prior to the first study drug administration.
7. Plasma donation within 7 days prior to the first study drug administration.
8. Fever (body temperature > 38 °C) or symptomatic viral or bacterial infection within 2 weeks prior to screening.
9. Any acute illness within 30 days prior to Day 1.
10. History of severe allergic or anaphylactic reaction. History of malignancy except for non-melanoma skin cancer excised more than 2 years ago and cervical intraepithelial neoplasia (CIN) that has been successfully cured more than 5 years prior to screening. Abnormal ECG finding at screening that are considered by the investigator to be clinically significant. History or presence of a condition associated with significant immunosuppression. History of life-threatening infection (e.g., meningitis). Infections requiring parenteral antibiotics within 6 months prior to screening. Vaccination with a live vaccine within 4 weeks prior to screening or that is planned within 4 weeks of dosing. Exposure to any significantly immune suppressing drug (including experimental therapies as part of a clinical trial) within 4 months prior to screening or 5 half-lives, whichever is longer. Positive test for hepatitis C antibody (HCV), hepatitis B surface antigen (HBsAg), or human immunodeficiency virus (HIV) antibody at screening. Participants with a positive toxicology screening panel (urine test including qualitative identification of barbiturates, tetrahydrocannabinol (THC), amphetamines, benzodiazepines, opiates, and ***e), or alcohol breath test. Participants with a history of substance abuse or dependency or history of recreational intravenous (IV) drug use over the last 5 years (by self-declaration). Regular alcohol consumption defined as >21 alcohol units per week (where 1 unit = 284 mL of beer, 25 mL of 40% spirit or a 125 mL glass of wine). Participant is unwilling to abstain from alcohol beginning 48 hours prior to admission to the CRU and during confinement period. Use of any IP or investigational medical device within 30 days prior to screening, or 5 half-lives of the product (whichever is longest) or participation in more than four investigational drug studies within 1 year prior to screening. Use of any prescription drugs (other than hormonal contraception: OCPs, long-acting implantable hormones, injectable hormones, a vaginal ring or an HID), over-the- counter (OTC) medication, herbal remedies, supplements or vitamins within 1 week prior to dosing and during course of study without prior approval of the investigator and medical monitor. Simple analgesia (nonsteroidal anti-inflammatory drug (NSAID)) or paracetamol may be permitted at discretion of investigator.
24. Use of fibrates for hyperlipidemia.
25. Consumption of any nutrients or concomitant medications known to modulate cytochrome P450 3 A4 (CYP3 A4) or any strong inhibitors or inducers of CYP3 A4, starting from 2 weeks prior to first dose of study drug and until end of study assessments.
26. Inability to refrain from consumption of grapefruit and Seville oranges or St. John’s Wort within 2 weeks prior to first dose of study drug and until final PK assessment.
27. Participant is unwilling to refrain from strenuous exercise from 72 hours prior to admission to CRU until completion of final follow-up visit.
Product and Mode of Administration
[0529] Compound 1 was provided as a powder in capsule formulation for oral administration. The formulation was a Size 00 Swedish orange capsule containing drug substance (50 mg or 250 mg) with no excipients. Ingredients of the capsule shell were hypromellose (hydroxypropylmethyl cellulose (HPMC)), iron oxide as coloring agent, and titanium dioxide as an opacifier. The drug product was stored at room temperature (15 °C - 25 °C).
[0530] Compound 1 is in a pharmacological class of tyrosine kinase inhibitors (TKI). Compound 1 is an orally bioavailable small molecule dual kinase inhibitor of platelet-derived growth factor receptors (PDGFR) and vascular endothelial growth factor receptors (VEGFR2). Duration of Treatment
[0531] Depending on study part and cohort assignment, the duration of study participation for each participant ranged from approximately 36 days to 50 days, including up to 28 days of screening. Duration of treatment by cohort is summarized in Table 21. Table 21.
Figure imgf000150_0001
[0532] Part A (SAD): In Part A (SAD), participants randomized to active treatment in cohorts Al, A2, A4, A5, and A6 received a single oral dose of Compound 1 administered once on Day 1 only. Participants randomized to active treatment in Cohort A3 (Food Effect) received a single oral dose of Compound 1 on Days 1 and 15.
[0533] PartB (MAD): In Part B (MAD), participants randomized to active treatment received oral Compound 1 administered twice daily for 7 consecutive days (Day 1 to Day 7) or twice daily for 14 consecutive days (Day 1 to Day 14), to be determined on completion of Part A (SAD) of the study.
[0534] Part C (MAD QD): In Part C (MAD QD), participants randomized to active treatment received oral Compound 1 administered once daily for 14 consecutive days (Day 1 to Day 14). [0535] Part D (Single Dose Food Effect): In Part D (Single Dose Food Effect), participants randomized to active treatment received a single oral dose of Compound 1 under fasting conditions (n=3) or under fed conditions (n=3) in Study Period 1. On Day 5, after a total of 4 days of wash-out from the first dose, participants crossed-over to Period 2 where the participants received a single oral dose of Compound 1 under the feeding condition opposite to the meal condition in Period 1.
Reference Therapy and Mode of Administration
[0536] The placebo capsules visually matched to active study drug and were composed of the same capsule shell but were filled with silicified microcrystalline cellulose. Silicified microcrystalline cellulose is a pharmaceutical excipient composed of co-process microcrystalline cellulose and colloidal silicon dioxide.
Criteria for Evaluation Part A (SAD), Part B (MAD), Part C (MA I) QD) and Part D (Single Dose Food Effect
[0537] Safety: The safety and tolerability of single and repeat-doses of Compound 1 was investigated according to the following specific assessments: vital signs (systolic and diastolic blood pressure, pulse rate, body temperature, and respiratory rate), 12-lead ECG, clinical laboratory tests (hematology, biochemistry, coagulation, and urinalysis), physical examination, and assessment of TEAEs.
[0538] Pharmacokinetics: Blood and urine samples for PK analysis of Compound 1 was collected pre-dose and following oral administration of single and repeat-doses of Compound 1 in the fasted state. Blood and urine samples for PK analysis were collected pre-dose and following administration of single doses of Compound 1 immediately after ingestion of a high fat meal (Food Effect Cohort A3 and Cohort Dl).
Statistical Methods
[0539] The number of participants was selected to allow for evaluation of safety/tolerability, PK and food effect of the single and multiple doses administered in this study and was consistent with standards of practice for Phase 1 studies.
[0540] In general, descriptive statistics (e.g. arithmetic mean, standard deviation [SD], median, minimum and maximum) were calculated for continuous data among treatment groups (or sequences), as well as difference from baseline by study part for each applicable scheduled time point, when appropriate. Frequency summaries (e.g. number of observed and percentage of each categories) were applied for categorical data among treatment groups (or sequences), by study part and for each scheduled time point.
[0541] For PK data the arithmetic mean, SD, median, minimum, maximum, coefficient of variation (CV%), geometric mean, geometric coefficient of variation (geo CV% or gCV) and the number of below the limit of quantification (BLQ) values were presented. No geometric statistics were computed for BLQ plasma concentrations.
[0542] No formal hypothesis testing was performed for this study.
[0543] Analysis Populations: Participant inclusion into each population was determined prior to the final analysis.
[0544] Intent-to-Treat (ITT) Population: All enrolled participants, regardless of whether they receive study drug or not, were included in the ITT population. Analysis is based on the treatment assigned to a participant, not what they actually received. The ITT population was used for all data listings and summaries involving disposition and enrollment.
[0545] Safety Population: All participants who received any amount of study drug (Compound 1 or placebo) were included in the Safety population. The Safety population was used for the summaries of all safety assessments. Participants were analyzed according to treatment received.
[0546] Pharmacokinetic Population: All participants who received any amount of active study drug (Compound 1) and have sufficiently evaluable concentration-time profile to allow determination of at least one PK parameter were included in the PK population. An evaluable PK profile was determined at the discretion of the pharmacokineticist following examination of subjects with dosing or protocol deviations that could potentially affect the PK profile. The PK population was used for the summaries of all PK data.
[0547] Safety and Tolerability: All adverse events (AEs) were coded using the Medical Dictionary for Regulatory Activities (MedDRA®) Version 22.0. A by participant AE data listing, including verbatim term, preferred term (PT), system organ class (SOC), treatment, severity, and relationship to study drug was provided. The number of participants experiencing TEAEs and number of individual TEAEs was summarized by SOC and PT. TEAEs were also summarized by severity and by relationship to study drug.
[0548] Laboratory evaluations, vital signs assessments and ECG parameters were summarized for each scheduled visit by treatment arm. A summary of change from baseline at each protocol specified time point by treatment arm were also presented.
[0549] Concomitant medications were coded using the World Health Organization (WHO) drug dictionary Version B3 September 2018 Drug Global. Concomitant medications were listed by participant and summarized by anatomical therapeutic class and preferred name. [0550] Physical examinations at each visit were listed for each participant and summarized using descriptive statistics at each visit by treatment arm.
[0551] Medical history, pregnancy test/FSH, urine drug screen/alcohol breath test, physical examinations and serology (HIV, Hepatitis B & C screen) were listed by participant.
[0552] Pharmacokinetics: Plasma Compound 1 concentrations, actual blood sampling times, and PK parameters were listed by treatment and protocol specified time point and summarized using descriptive statistics for PK data as outlined above for each scheduled time point by treatment arm. Individual and mean Compound 1 concentration-time profiles were also presented graphically for each treatment. Pharmacokinetic parameters were computed from the individual plasma Compound 1 concentrations using a non-compartmental approach.
[0553] The following plasma Compound 1 non-compartmental PK parameters were estimated, as appropriate: Cmax, Tmax, AUCo-iast, AUCo-inf, AUCtau (calculated for MAD cohorts only), Kei, ti/2, CL/F (SAD cohorts), CL/Fss (MAD cohorts), Vz/F (SAD cohorts, Vz/Fss (MAD cohorts), RA (calculated for MAD cohorts only).
[0554] Value for Kei, ti/2, AUCo-inf, CL/F (CL/Fss), or Vz/F (Vz/Fss) were not reported for cases that fail to exhibit a terminal log-linear phase in the concentration versus time profile. Additional analyses were performed as deemed necessary upon review of the data.
[0555] A food effect assessment was conducted on Compound 1 PK parameters in Cohort A3. Analyses of variance (ANOVA) were performed on the ln-transformed AUCo-iast, AUCo-inf and Cmax (fasting vs fed). Ratios of the geometric means were calculated using the exponentiation of the least squares mean (LSM) from the analyses on the ln-transformed AUCo- iast, AUCo-inf and Cmax. Ratios were expressed as a percentage relative to the fasting regimen. 90% Cl for the ratios were derived by exponentiation of the CIs obtained for the difference between regimen LSM resulting from the analyses on the ln-transformed AUCo-iast, AUCo-inf, and Cmax. [0556] The analysis of dose proportionality was conducted for AUC and Cmax of single agent Compound 1 using a power model on log-transformed scale. The log-transformed exposure parameters were each regressed onto a fixed factor for log (dose). The 90% Confidence Interval (Cl) of the slope for each exposure parameter was computed from the model and presented in a summary table.
[0557] Urine collection time, volume collected, and Compound 1 concentration (Aeu-t2) duration of each sampling interval was listed for each participant and summarized by nominal sampling time point and treatment using descriptive statistics (Number of subjects [N], arithmetic mean, SD, CV%, geometric mean, median, minimum and maximum). Individual and mean Compound 1 cumulative urinary excretion-time profiles for each treatment were also presented graphically. Where urine was collected for PK analysis, the following parameters are calculated, as appropriate: Aeu-t2, CLR, CL/F, and Feu-t2.
Example 25. Compound 1 Ameliorates Fibrosis in Alport Syndrome Mouse Model [0558] A line of Col4a3-/~ mice on 129Xl/SvJ background (“Alport mice”), a known model for Alport syndrome ( see Andrews, et ak, Am. J. Pathol., 2002, 160(2), 721-30), was used to evaluate Compound 1. After genotyping analysis, four-week-old male and female Alport mice were randomized to vehicle (water, PO, BID; n = 12) or Compound 1 (25 mg/kg Compound 1 Hydrochloride trihydrate, PO, BID; n = 12). A sham/wild-type cohort (n = 9) served as age- matched control.
[0559] Mortality was monitored through 5 weeks of treatment. Spot urines were collected at 4 weeks and 5 weeks during treatment and prior to sacrifice to determine proteinuria and protein- to-creatinine ratio (PCR). All mice were then sacrificed and body weights and kidney weights were taken. Blood was drawn from all the mice and serum was collected to determine BUN and creatinine levels. One half kidney was snap frozen to determine hydroxyproline (HYP) and Western blot analysis for fibrotic markers. One kidney was fixed in 10% neutral buffered formalin to perform histopathological observations and IHC stainings for fibrotic markers.
[0560] Proteinuria. Urine samples were undiluted, or diluted to 10-fold or 20-fold with water. Then, 5 pL of samples were added to the 96 well plate and 200 pL of Bradford Reagent (Sigma) was added to the sample wells. Bovine serum albumin (BSA) standard samples 0 to 2 mg/mL were prepared and 5 pL of each standard added to the microwell plates. Both standard and samples were incubated with the Bradford reagent for 30 min, and the absorbance was read at 650 nM with a Tekan machine.
[0561] Protein-to-creatinine ratio (PCR). Creatinine standard was prepared at 20, 10, 5, 2.5, 1.25, 0.625 and 0.3125 mg/dL. Urine samples were diluted 20-fold with water. Then 25 pL of samples were added to the 96 well plate and pipet 50 pL of samples, water as the blank, or standards into wells in the clear plate. 50 pL of the DetectX® Creatinine Reagent was added according to manufacturer instructions, (Arbor Assays). Urine protein was calculated for mg/mL. Then, urine protein of the sample was divided by the urine creatinine to determine the protein to creatinine ratio (PCR) and expressed as mg/mg.
[0562] Serum BUN. Serum samples were diluted 5-fold with distilled water. BUN standard was prepared at 10, 5, 2.5, 1.25, 0.625, 0.3125, and 0.156 mg/dL. 50 pL of samples or appropriate standards into 96 wells in the plate. 50 pL of water was used as the zero standard. Then, 75 pL of Color Reagent A to each well and then added 75 pL of Color Reagent B to each well using a multichannel pipet. The plates were incubated at room temperature for 30 minutes and then the optical density was read at 450 nm according to manufacturer's instructions (Arbor Assays).
[0563] Serum Creatinine. 20 pL of the serum samples were analyzed for serum creatinine levels using LC-MS/MS. Briefly, after spiking in creatinine-d3 as internal standard, mouse serum samples were purified by protein precipitation with acetonitrile. Creatinine was analyzed by LC- MS/MS. Chromatographic separation was performed with a Shimadzu Prominence system on a Primesep 200 (SIELC Technologies) column with a gradient elution (0.1% formic acid in water as mobile phase A and 0.1% formic acid in methanol as mobile phase B). MRM detection (114.05 to 44.2 for creatinine and 117.05 to 47.2 for creatinine-d3) was carried out with AB Sciex API-3200 triple quadrupole mass spectrometer. Creatinine was expressed as mg/dL.
[0564] Western Blots and Densitometry Aliquots of tissue extracts from 4 or 5 kidneys of each group of animals (30 pg protein) were dissolved in SDS-sample buffer, separated by electrophoresis in a SDS-polyacrylamide gel (4 to 20%) under reducing conditions, transferred to a nitrocellulose membrane, and blocked for 60 minutes at room temperature with 5% milk- powder in a 0.2 mol/L Tris-HCl buffer, pH 7.6, containing 0.1% Tween 20 solution (TBST buffer). Mouse antibodies for Collagen-1 (EMD Millipore), TGFpi (Santa Cruz), PDGFR (Cell Signaling) and aSMA (Sigma) antibodies as the primary antibody was diluted in TBST and then added to the membrane and allowed to incubate for 2 hrs at room temperature or overnight at 40 °C. The membranes were washed three times with TBST and then incubated for 60 minutes with secondary antibodies conjugated with HRP antibodies (Sigma). The membranes were washed as above, and the blots were developed using chemiluminescence substrate (Thermo Fisher). Collagen-1, TGFpi, PDGFR and aSMA expression was analyzed through Image Analyzer (Cambridge Scientific Products) and densitometrically normalized to the GAPDH (Cell Signaling) loading control using an Image J Software. [0565] Hydroxyproline Assay. The hydroxyproline (HYP) content from kidney tissues was determined as described by Woessner et al (Archives of Biochemistry and Biophysics, Vol. 93, Issue , May 1961, 440-447). After weighing (~40 mg) and hydrolysis of the kidney tissue for 18 hr at 110 °C in 10 N HC1, samples were neutralized to pH 7 with 10 M NaOH, and aliquots were assayed by incubating with chloramine T solution, perchloric acid and Ehrlich's reagent at 60 °C for 20 minutes. Absorbance was measured at 550 nm, and the amount of HYP was determined using a standard hydroxyproline curve. Total HYP was expressed as micrograms per kidney tissue.
[0566] Histological Procedures. The kidneys were fixed in 10% formalin for at least 48 hr before preparation for histology. Kidney tissues were processed, dehydrated, embedded in paraffin, and cut into sequential 5 pm sections. Kidney sections were subjected to haematoxylin- eosin (H&E) and Masson’s Trichrome staining using a Trichrome Stain Vector Kit (Sigma). IHC staining using mouse antibodies for Collagen- 1 (Sigma), TGFpi (Cell Signaling), and aSMA (Sigma) were performed by following standard laboratory procedures. Photomicrographs were taken using a microscope (Leica) equipped with a computerized digital camera.
[0567] Trichrome Staining. Working Weigeret’s Iron Hematoxylin Solution was prepared according to label instructions. The slides were deparaffmized and hydrated with deionized water. Then slides were placed in pre-Bouin’s Solution, at room temperature overnight. Slides in a Coplin Jar were washed in running tap water to remove yellow color from sections. The slides were then stained in Working Weigert’s Iron Hematoxylin solution for 5 minutes. The slides were washed in running tap water for 5 minutes. The slides were then stained in Trichrome Stain AB Solution (Sigma) for 5 minutes and placed in 0.5% Acetic acid for 1 minute. The slides were rinsed, dehydrated through alcohol, cleared in xylenes and then mounted.
[0568] Picrosirius Red (PSR) Staining. Sections of paraffin-embedded tissue were deparaffmized by sequential washes in xylene (2 times for 5 minutes), 100% ethanol (2 times for 5 minutes), 90% ethanol (once for 5 minutes), 80% ethanol (once for 5 minutes) and 70% ethanol (once for 5 minutes). Samples were then washed in water for 5 minutes. Samples were then incubated in a solution of saturated picric acid containing 0.2% Sirius Red dye at room temperature for 35 min with shaking. Samples were washed with tap water until the wash was colorless. Samples were then dehydrated with serial 5 minute washes in water, 70% ethanol,
80% ethanol, 90% ethanol, 100% ethanol (twice) and 100% xylene (twice). Slides were mounted using Permount mounting medium and cover-slipped. Photomicrographs were taken using a bright-field microscope equipped with a computerized digital camera. Staining intensity was quantified using a Bioquant image analysis program. The Bioquant image analysis program (Nashville, TN) was used to quantify the degree of stained versus non-stained regions.
[0569] IHC Stainings for Fibrotic Markers. Formalin fixed kidney tissue sections were used for IHC staining. Endogenous peroxidases were quenched with 3.0% hydrogen peroxide in methanol for 30 min. Sections were further blocked with 3.0% bovine serum albumin (BSA) in PBS, exposed to 0.5 % Triton X-100 for 1 h to reduce non-specific antibody binding and incubated with primary antibodies of anti -mouse mouse aSMA antibody (Sigma), Collagen- 1 antibody (Sigma) and TGFB1 (Santa Cruz) at 4 °C overnight. The sections were washed with PBS three times, incubated with HRP-conjugated secondary antibodies (Cell Signaling) for 1 hour followed by washing 3 times with lx PBS. The sections were then incubated with a HRP substrate (Invitrogen) for 2-3-minutes to obtain clean staining. After dehydration with a series of increasingly concentrated ethanol solutions, sections were mounted with neutral gum. Images were captured using a Nikon microscope equipped with a computer.
[0570] Histological Examination. Renal injury was evaluated in H&E stained kidney sections, and a renal damage score was assigned on a numerical scale from 0 (normal/no injury) to 4 (severe injury) by two independent observers. For each animal, analysis consisted of estimating the total amount of tubular and glomerular damages in the cortical regions of the entire kidney section. Injuries included the disruption of normal tissue micro architecture, hemorrhage and monocyte infiltration, inflammation, casts formation, and necrosis and sclerosis. The scoring scale was defined as follows: 0 = normal architecture or <5% injury, 1 = 5-<25% of tissue is injured, 2 = 25-<50% of tissue is injured, 3 = 50-<75% of tissue is injured, and 4 = >75% of kidney is injured. Similarly, trichrome staining and IHC staining were scored on a scale of 0-4 (0 = normal or <5% staining; 1 = 5 to <25% area; 2 = 25 to <50% area, 3= 50 to <75% area; and 4 being > 75% area).
Results
[0571] Spot urines were collected from 4 week old WT (N=8) and Alport mice (n=10), and analyzed for proteinuria prior to randomization to vehicle and Compound 1 groups. Proteinuria was significantly increased in Alport mice compared to wild-type (FIG. 26A). [0572] Overall, Compound 1 significantly decreased mortality in Alport mice and improved survival benefit significantly compared to vehicle cohort, per Kaplan Myer’s survival analysis (FIG. 26B). Compound 1 had no significant effect on body weight or kidney weight of Alport mice (FIG. 26C and FIG. 26D). Compound 1 significantly decreased proteinuria (FIG. 26E) and PCR (FIG. 26F) in Alport mice at 5 weeks of treatment/9 weeks of age. Compound 1 did not decrease serum BUN or serum creatinine (SCr) in Alport mice (FIG. 26G and FIG. 26H, respectively). Treatment with Compound 1 for five weeks significantly decreased kidney fibrosis, as judged by hydroxyproline (HYP) levels, in Alport mice (FIG. 261). Compound 1 also significantly reduced renal histopathological injury score in Alport mice (FIG. 26J).
Additionally, Compound 1 treatment decreased kidney Trichrome staining, an indication of decreased collagen and fibrosis, significantly compared to the vehicle cohort (FIG. 26K). IHC stainings for kidney fibrotic markers demonstrated that Compound 1 decreased TGFpi, aSMA, and Collagen- 1 in Alport mice compared to vehicle (FIG. 26L, FIG. 26M, and FIG. 26N, respectively). Picrosirius red (PSR) staining for collagen showed that Alport mice treated with Compound 1 had significantly reduced collagen as compared to Alport mice treated with vehicle (FIG. 260). Lastly, densitometric analysis of Western blots for collagen 1, aSMA, TGFpi, PDGFR, and aSMA showed decreased expression of all four markers in Alport mice treated with Compound 1 (FIG. 26P and FIG. 26Q). GAPDH loading levels were normalized to WT groups to evaluate relative expression of fibrotic markers.
Example 26. Effect of Compound 1 in a Passive Heymann Nephritis Model [0573] In this study, Compound 1 was tested in the Passive Heymann Nephritis model of renal dysfunction in rats. In this model, renal dysfunction was mediated by intravenous administration of Anti-FXl A serum to rats, resulting in podocyte-localized deposition of antibodies, whereby immune deposits accumulate and obscure the slit diaphragm (Spicer et ah, J. Immunol. 2007 Jul 1; 179(1): 172-8). This results in podocyte foot process effacement and proteinuria. The model has nearly identical pathology to human membranous nephropathy and gl omerul onephriti s .
[0574] Sixty-five CD® rats (Sprague Dawley Rat) with an average mass of 300 g were acclimatized and administered anti-FXl A serum (tail vein, IV, 600 pL). Spot urines were collected 7 days later and proteinuria was measured. Animals were randomized based on proteinuria levels, ensuring equivalent average protein to creatinine ratio (PCR) and standard deviation in each group. Groups included: (1) 100 mg/kg Compound 1 (n=9); (2) 50 mg/kg Compound 1 (n=10); (3) 15 mg/kg Compound 1 (n=ll); (4) Vehicle (n=ll); (5) 50 mg/kg nintedanib; and (6) sham (n=5). Dosing started on day 10 and continued for 12 weeks (PO, BID). 24-hour urine collections and protein to creatinine ratio were determined approximately bi weekly. At the end of the study, animals were sacrificed, and body weights and kidney weights were measured. Blood was drawn from all rats and serum was collected. Half of the kidney was snap frozen to determine hydroxyproline (HYP) and Western blot analysis. One kidney was fixed in 10% neutral buffered formalin.
[0575] Urine Analysis. Overnight urines were collected using Tecniplast Metabolic Cage Systems for rodents (Tecniplast, Catalogue #3700M071). Animals were kept in metabolic cages for 24 hours. Urine samples were analyzed for protein content using the Bradford Assay (Sigma Cat #B6916-500ML) according to the manufacturer’s protocols. Urine samples were undiluted or diluted to 10-fold or 20-fold with water to be within the assay’s acceptable range. Creatinine was measured using the Quantichrom Creatinine Assay Kit (Bioassay Systems). A standard curve was generated to determine urine sample concentrations. The protein to creatinine ratio was determined and expressed as mg/mg.
[0576] Serum Analysis. Sera were collected at sacrifice and processed in BD Microtainer serum collection tubes according to the manufacturer’s instructions. Sera were stored at -20 °C until analysis. Serum creatinine, cholesterol, and triglycerides were analyzed using validated analytical procedures for human samples. Serum BUN was measured according to the manufacturer’s protocols (Arbor Assays, Catalogue # K024-H).
[0577] Hydroxyproline Assay. Kidney tissue was collected, and mass determined at the time of tissue collection. Samples were stored at -80 °C until analysis. The hydroxyproline (HYP) content from kidney tissues were determined. Kidney tissue was subjected to hydrolysis in 10 N HC1 for 18 hr at 110 °C, and aliquots were incubated with chloramine T solution, perchloric acid and Ehrlich's reagent at 60 °C for 20 minutes. Absorbance was measured at 550 nm, and the amount of HYP was determined using a hydroxyproline standard curve. Total hydroxyproline is expressed as micrograms per kidney.
[0578] Histological Procedures. At time of sacrifice, half (coronal section) of the kidney was fixed in 10% formalin for at least 48 hr before preparation for histology. Kidney tissues were processed, dehydrated, embedded in paraffin, and cut into sequential 5 pm sections. One set of slides was stained with Periodic Acid Schiff (PAS) and one set with Masson’s Trichrome staining using standard protocols. Photomicrographs were taken using a bright-field microscope (Leica) equipped with a computerized digital camera.
[0579] Histological Examination. Glomerular injury was evaluated from PAS-stained kidney sections on a numerical scale from 0 (normal/no injury) to 4 (severe injury) by two independent observers. For each animal, analysis consisted of estimating the total amount of glomerular damage as measured by sclerosis, which is indicated by dark pink staining. The scoring scale was defined as follows: 0 = normal architecture, 1 = 0-10% glomerular damage, 2 = 10-<25% glomerular damage, 3 = 25-<50% glomerular damage, 4 = 50-100% glomerular damage.
[0580] Western Blots and Densitometry Kidneys were lysed by tissue shedder (Biospec Products) in lx lysis buffer (Cell Signaling Catalog # 9803). The lysis buffer also contained PMSF (1 mM) and HALT protease/phosphatase inhibitor (Pierce Catalog # 36978). Protein concentrations in the lysates were determined using the DC protein determination kit (Biorad Catalog # 500-0113 and 500-0114). After centrifugation at 4 °C, 2X Laemmli (4% SDS, 20% glycerol, 120 mM Tris-HCl pH 6.8) buffer was added to an equal volume of clarified lysate and heated to 100 °C for 10 minutes. 25 pg Protein was loaded for Western analysis on 15 lane 4- 20% Tris-glycine gels (Thermo Catalog # XPO4205). In a control lane, BioRad Precision Plus Protein Standard was loaded (BioRad Catalog # 1610374). Proteins were transferred to Nitrocellulose membranes using Invitrogen iBlot 7-minute transfer unit (iBlot transfer stacks Catalog # IB301001).
[0581] Blots were blocked with 5% Carnation non-fat powdered milk in TBS (VWR Catalog # 51-17-01) plus 0.1% Tween-20 (BioRad Catalog # 170-6531)(TBST) and incubated overnight at 2-8 °C with primary antibodies against PDGFR (Cell Signaling Technology Inc., Danvers, MA, Catalog # 3169) or GAPDH (Cell Signaling Technology Inc., Danvers, MA, Catalog #
2118) at a 1 : 1000 dilution in 5% BSA/TBST. Blots were washed several times with TBST and then incubated with an anti -rabbit IgG, HRP-linked secondary antibody at a 1:1000 fold dilution (Cell Signaling Technology Inc., Danvers, MA, Catalog # 7074) diluted in above block buffer. After several times washing, the blots were then incubated with Supersignal West Femto chemiluminescent substrate (Thermo Catalog # 34094), and luminescence was detected using an Alpha-Innotech Gel Imaging system (Alpha Innotech Corporation, San Leandro, CA). Densitometry was measured using ImageJ software (NIH) using standard protocols.
Results
[0582] Animals were monitored closely throughout the experiment, and the survival analysis was plotted in a Kaplan Meier curve (FIG. 27A). Only 38.5% of animals in the nintedanib group survived the study, which was statistically significant compared to the Sham and Vehicle cohorts. 88.9% and 80% of animals remained at the end of the study from the 100 mg/kg and 50 mg/kg groups, respectively; this was not statistically different from the Vehicle or Sham groups (Mantel-Haenszel Log Rank Test, Graphpad Prism Software). Of note, there were no animals lost in the 15 mg/kg Compound 1, Vehicle and Sham cohorts.
[0583] Body mass was measured at time of sacrifice, day 93. The nintedanib-treated animals had significantly reduced body mass, reduced kidney mass and increased kidney mass to body mass ratio compared to the vehicle cohort (***p<0.0001, One-way ANOVA, Tukey). The Compound 1 treatment groups did not have a significant change in body mass (FIG. 27B), kidney mass (FIG. 27C), or the kidney to body mass ratio (FIG. 27D) compared to vehicle or sham.
[0584] Seven days after administration of the antiFXl A serum, animals were placed into metabolic cages and 24 hour urines collected. PCR was plotted over time from day 7 to the end of the study (day 93) (FIG. 27F). As shown in FIG. 27E, at day 93, PCR was reduced in the 100 mg/kg Compound 1 (p<0.05) and 15 mg/kg Compound 1 (p=0.05) groups compared to vehicle (*p<0.05, T-test). Animals treated with Compound 1 had a 52% reduction in the protein to creatinine ratio (p<0.05). The 50 mg/kg Compound 1 and nintedanib group was not significantly different from the vehicle group at day 93.
[0585] Serum cholesterol was significantly increased (p<0.05) in the nintedanib group compared to Sham, 50 mg/kg Compound 1, and 15 mg/kg Compound 1 groups (p<0.05, One Way ANOVA) (FIG. 27G). No statistically significant changes in serum cholesterol were noted with Compound 1 treatment. Serum triglycerides were significantly reduced in the 50 mg/kg group compared to vehicle (*p<0.05, One way ANOVA, Tukey), whereas the 100 mg/kg and 15 mg/kg groups did not have a statistically significant change in serum triglycerides (FIG. 27H). Serum creatinine (FIG. 271) and BUN (FIG. 27J) did not change significantly in the vehicle or Compound 1 treatment groups compared to sham. However, nintedanib treatment caused a significant increase in serum creatinine and BUN.
[0586] Compound 1 treatment at 50 mg/kg and 15 mg/kg significantly reduced hydroxyproline compared to vehicle (*p<0.05, **p<0.01, T-test) (FIG. 27K). When analyzed with Trichrome staining, Vehicle tissue contained more proteinaceous casts (bright red staining) and collagen deposition (blue staining) compared to Sham, 100 mg/kg, 50 mg/kg and 15 mg/kg (FIG. 27L). The Nintedanib treated group had significantly increased proteinaceous casts and increased collagen deposition compared to all groups.
[0587] PAS staining was performed to semi-quantitatively determine the level of glomerulosclerosis of glomeruli. Representative images are shown in FIG. 27M, and glomerular damage scores are shown in FIG. 27N. There was a significant increase in glomerular injury in the vehicle group compared to sham. There was a significant reduction in glomerular damage with Compound 1 treatment at 50 mg/kg and 15 mg/kg. The Nintedanib group had global glomerulosclerosis.
[0588] Western blot analysis was performed on total kidney lysates to determine and quantify PDGFRP (FIG. 270). PDGFRP expression levels were increased 3.8 fold in Vehicle group compared to Sham group. Compound 1 (15 mg/kg) treatment brought PDGFRP down to Sham levels in total kidney lysates (p<0.05) (FIG. 27P).
Example 27. A Phase 2, Multicenter, Double-Blind, Randomized, Placebo-controlled Study of Safety and Efficacy of Compound 1 in Patients with Primary Glomerular Disease and Persistent Proteinuria
Objectives
[0589] The primary objective of this study is to evaluate efficacy of Compound 1 in patients with primary glomerular disease and persistent proteinuria while on standard of care (SOC), as measured by a reduction in the 24-hour urinary protein excretion.
[0590] The secondary objective of this study is to evaluate the safety and tolerability of Compound 1 in patients with primary glomerular disease and persistent proteinuria.
Methodology
[0591] This is a randomized, prospective, double-blind, multicenter, multinational, placebo- controlled, parallel-group, dose-ranging study that will assess the efficacy and safety of three dose levels of Compound 1 in patients with primary proteinuric renal disease who continue to have urinary protein excretion > 1 g/day while on SOC therapy. Patients with a biopsy-proven primary glomerular disease on stable SOC therapy for at least 12 weeks prior to randomization who meet the inclusion criteria but none of the exclusion criteria are eligible to participate in this study. Patients with genetic forms of focal segmental glomerulosclerosis (FSGS) may be enrolled without a renal biopsy if their clinical presentation is consistent with the genetic testing results and they, otherwise, meet all eligibility criteria.
[0592] The eligible patients are randomized in a 1 : 1 : 1 : 1 ratio to three dose levels of Compound 1 (200 mg once daily (QD), 400 mg QD, 300 mg twice daily (BID)) or placebo administered daily for 12 weeks.
[0593] The study drugs are administered QD or BID orally to patients according to their assignment to QD or BID dosing schedule. All patients take the study drug either once-daily in the morning or twice-daily within 30 minutes of meals and at least 30 minutes apart from any other drugs. The study ends when the last patients have completed 12 weeks of study drug and the follow-up period of 4 weeks.
[0594] For each patient, the study period is from the time of signing of the Informed Consent until their last visit. Adverse events are collected during all the study periods and are considered under treatment until 4 weeks after the drug discontinuation.
[0595] Patients who discontinue the drug early (before the Week 12 visit) are asked to come for an early study discontinuation visit. A repeat 24-hour urine collection 4 weeks after the last study dose is performed in all patients who have received at least 2 weeks of the study drug. [0596] Screening for study eligibility is performed in two clinic visits; Visit 0a (pre screening) and Visit 0b (full screening). Additional visits to complete screening are allowed. Patients who sign the Informed Consent and meet the medical history-based eligibility criteria undergo a urinalysis and spot urinary protein to creatinine ratio testing on the first AM urine sample. Pre-screening and screening processes are summarized in FIG. 28.
[0597] All patients have PK samples obtained at Day 1 (Visit 1) and Week 2 (Visit 2); a subset of patients (8 patients/dose cohort) will have additional PK sample time-points on Day 1 and on Week 2.
[0598] Approximately 100 patients (25 patients per study arm) are planned for this study. Diagnosis and Main Criteria for Inclusion: Inclusion Criteria:
[0599] To be eligible for the study, participants are required to meet all of the following inclusion criteria:
• Male or female participants aged 18 and older.
• Diagnosis of a primary glomerular disease confirmed from a past renal biopsy. Participants with genetic forms of FSGS may be enrolled without a renal biopsy if the clinical picture is consistent with the genetic testing results.
• Estimated glomerular filtration rate (eGFR) by Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) > 40 mL/min/1.73m2.
• Urinary protein excretion > 1 g/day on a 24-hour urine collection.
• Stable blood pressure < 140/90 mmHg with stable antihypertensive regimen for at least 12 weeks prior to screening.
• All participants must be on the SOC therapy including the maximally tolerated/recommended doses of an Angiotensin-converting enzyme inhibitor (ACEi) or Angiotensin II receptor blocker (ARB), but not both.
• Renin-angiotensin-aldosterone system (RAAS) blockers and Sodium glucose co transporter 2 (SGLT-2) inhibitors must be stable for at least 12 weeks prior to screening and projected to remain stable through week 16.
• Immunosuppressive or immunomodulatory therapy must be stable for at least 12 weeks prior to screening and projected to remain stable through study week 16.
• Both genders of childbearing potential must agree to use adequate contraception, during and for at least 3 months after the last dose of study drug.
• Participants must be willing and able to give written Informed Consent and to comply with protocol requirements.
• Participants must be judged to be otherwise fit for the study by the Investigator. Exclusion Criteria:
[0600] A participant who meets any of the following exclusion criteria is excluded from the study:
• Positive Hepatitis B (HBV), Hepatitis C (HCV), or human immunodeficiency virus (HIV) viral screening test; historical or during the screening • Hematologic abnormalities as follows: Hemoglobin (Hb) < 8 g/dL, platelets < 50,000, and/or absolute neutrophil count (ANC) <1000 cells/pL at baseline
• Aspartate Aminotransferase (AST) or alanine Aminotransferase (ALT) or total bilirubin > 2 ULN
• Hemoglobin AIC > 8.5%
• Participants taking non-steroidal anti-inflammatory agents (NSAIDS) chronically (intermittent, i.e., occasional NSAIDS for pain or fever is discouraged, but is not excluded). No NSAIDS allowed within 72 hours of the scheduled lab work to determine eGFR and/or urinary protein and creatinine measurements.
• Known predisposition to bleeding
• Participants who require fibrinolysis, full-dose therapeutic anti coagulation (e.g. vitamin K antagonists, dabigatran, heparin, hirudin, etc.), or high dose antiplatelet therapy. Prophylactic use of antiplatelet therapy (e.g. acetyl salicylic acid up to 325 mg/d, or clopidogrel at 75 mg/d, or equivalent doses of other antiplatelet therapy) is not excluded.
• History of hemorrhagic central nervous system (CNS) event within 12 months of the screening visit
• History of active gastrointestinal bleeding within 6 months of the screening visit
• History of thrombotic event (including stroke and transient ischemic attacks) within 12 months of the screening visit
• Type I diabetes mellitus
• Renal biopsy performed no more than 5 years prior to screening showing histopathological evidence of diabetic kidney disease
• Anti-PLA2R antibody-associated membranous nephropathy
• Myocardial infarction or unstable angina within 6 months of the screening visit
• History of solid organ or hematopoietic cell transplantation
• On an organ transplant waiting list or anticipated organ transplant within 6 months of screening
• History of treated Hepatitis C (HCV)
• Participation in any clinical study of an investigational product within 12 weeks from the date of screening • History or presence of any form of cancer within 2 years of screening except excised basal or squamous cell carcinoma of the skin
• Renal disease secondary to systemic disease including but not limited to: systemic lupus erythematosus, anti -neutrophil cytoplasmic antibodies-associated diseases, anti- glomerular basement disease, secondary forms of focal segmental glomerulosclerosis, renal diseases associated with para-proteinemias, C3 glomerulopathy, and diabetic kidney disease
• Treatment with Anti-CD20 monoclonal antibodies within 6 months prior to screening
• A known systemic disorder that requires, or is expected to require, systemic glucocorticoids or immune modulators during the study
• BMI > 40 kg/m2
• Pregnant women or women who are breast feeding or of child-bearing potential not using 2 effective methods of birth control (1 barrier and 1 highly effective non-barrier) for at least 1 month prior to enrollment (and until 3 months after treatment end)
• Sexually active males not committing to using condoms during the study (except if their partner is not of childbearing potential) and 3 months after end of treatment
Investigational Product, Dosage, and Mode of Administration
[0601] Compound 1 is an orally active tyrosine kinase inhibitor. Three dose levels (200 and 400 mg QD and 300 mg BID) are tested in this study. The study drug and the placebo are administered once or twice daily orally after breakfast and dinner and at least 30 minutes apart from any concomitant medications.
[0602] Compound 1 is administered as a Form A hydrochloride trihydrate salt in a Hypromellose Swedish Orange-colored capsule containing no excipients. Ingredients of the capsule shell are hypromellose (hydroxypropylmethyl cellulose also known as HPMC), iron oxide as a coloring agent, and titanium dioxide as an opacifier. Two strengths (100 mg and 200 mg) and a visually matching placebo are used in this study.
Duration of Treatment
[0603] Once or twice daily for 12 weeks. The end of the study is reached when the last patient has completed 12 weeks of study drug and the follow-up period of 4 weeks.
Reference Therapy [0604] Patients receive the standard of care including maximally tolerated/recommended dose of ACEi or ARB but not both. Where applicable, the doses of mineralocorticoid receptor blockers, direct renin inhibitors, and SGLT-2 inhibitor doses must be stable for at least 12 weeks and anticipated to remain stable during the study period.
[0605] Placebo capsules are composed of the same capsule shell used for the active but are filled with silicified microcrystalline cellulose. Silicified microcrystalline cellulose is a pharmaceutical excipient composed of co-processed microcrystalline cellulose and colloidal silicon dioxide.
Criteria for Evaluation
[0606] Primary Efficacy: Percentage change in 24-hour urinary protein excretion at Week 12
[0607] Secondary Efficacy:
• Percentage change in 24-hour urinary albumin excretion at Week 12
• Number of patients with complete remission in proteinuria, defined as a 24-hour urinary protein excretion < 300 mg at Week 12
• Number of patients with partial remissions in proteinuria, defined as a 24-hour urinary protein excretion reduction of > 50% from the baseline and a 24-hour urinary protein excretion < 3.5 g/day if the baseline 24-hour urinary protein excretion > 3.5 g at Week 12
• Number of patients with > 50% reduction in 24-hour urinary protein excretion from the baseline at Week 12
• Number of patients with > 50% reduction in 24-hour urinary albumin excretion the baseline at Week 12
• Percentage change from baseline in creatinine clearance (CrCl) at Week 12
• Changes in serum albumin, fasting triglycerides, and cholesterol at Week 12 [0608] Exploratory:
• Plasma/urine Biomarkers include the following: plasma Soluble TNF receptor 2 (sTNFR2), kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), Urinary fibronectin, high molecular weight type IV collagen (HMW collagen IV), monocyte chemotactic protein- 1 (MCP-1), and matrix metalloproteinase-7 (MMP-7)
• PK including the following end-points: maximum concentration (Cmax), time to maximum concentration (Tmax), area under the drug concentration-time curve from time zero to the last measurable concentration (AUCo-iast), area under the drug concentration time curve from time zero to infinity (AUCo-inf), area under the drug concentration-time curve over the dosing interval (AUCtau), terminal half-life of elimination from plasma (t½), terminal elimination rate constant (Kei), trough plasma concentration at steady-state measured at the end of a dosing interval before the next administration (Ctrough), oral clearance (CL/F) on Day 1 and at steady-state CLSS/F, apparent terminal volume of distribution on Day 1 (Vz/F) and at steady-state (Vzss/F), and accumulation ratios
• Analysis of Compound 1 metabolites in plasma and/or urine [0609] Safety:
• Adverse events (AEs), serious adverse events (SAEs), and AEs leading to discontinuation of study treatment, emerging during treatment and within 4 weeks post-treatment
• Laboratory parameters (hematology, chemistry, hepatic, coagulation, urinalysis)
• Vital signs
• Electrocardiogram
• Proportion of patients showing an increase in mean arterial pressure (MAP) of > 10 mmHg from the baseline on at least two visits or requiring addition of new hypertensive medication or increases in the existing antihypertensive medications
Statistical Methods
[0610] Sample Size: The study plan is to enroll appropriately 100 patients at a 1 : 1 : 1 : 1 ratio to Compound 1 200 mg QD, Compound 1 400 mg QD, Compound 1 300 mg BID, or placebo (QD or BID) administered daily for 12 weeks. Without wishing to be bound by theory, in some embodiments, a mean reduction in 24-hour urinary protein excretion at Week 12 is expected to be, e.g., 20% to 40% for Compound 1, with no reduction in the placebo group stable on the SOC therapy other than some minor variability in protein excretion (e.g., in the range of ± 10 - 20%). [0611] High variability at baseline may be expected due to enrollment of patients of various glomerular diseases. Under a range of conservative assumptions for 24-hour urinary protein excretion, the study is most likely under 80% power to demonstrate an expected difference of 20% between any given dose of Compound 1 and placebo. A 20% difference could be considered clinically important, though the total sample size needed at 80% power is estimated to be large (-100 patients per group at a two-sided alpha level of 0.05). For this reason, this proof-of-concept Phase II study is designed to provide an assessment of safety and estimated beneficial effects incorporating the totality of the data across all endpoints and all dose levels to guide further clinical development of Compound 1 in patients with primary glomerular diseases with persistent proteinuria while on the SOC therapy.
[0612] Methods: Continuous variables are summarized with descriptive statistics (the number of non-missing values [n], mean, median, standard deviation [SD], minimum, and maximum). All categorical variables are summarized with frequency counts and percentages, as applicable. A Mixed Model Repeated Measures (MMRM) analysis or Analysis of Covariance (ANCOVA) is carried out for continuous variables, depending on the number of assessments post-baseline. For binary endpoints, difference in proportions between treatment groups is analyzed using chi-squared test. Analyses are carried out on the Full Analysis Set.
[0613] For the primary efficacy endpoint, a Mixed Model Repeated Measures (MMRM) analysis is performed including treatment group, visit, treatment group by visit, and 24-hour urinary protein excretion value at baseline as a covariate. A similar model is used for the analysis of 24-hour urinary albumin excretion. Non-parametric method is used if the data is not normally distributed. PK concentration and parameters, biomarkers, and all other assessments are summarized descriptively.
[0614] Interim Analysis: Formal interim analysis is not planned, but the safety data are reviewed on an ongoing basis by an independent Safety Review Committee/Data Monitoring Committee.
[0615] Safety: All patients randomized and who received at least one dose of study drug are evaluated for safety. The safety analyses include evaluation of the incidence of treatment- emergent AEs, SAEs, and AEs leading to discontinuation of study treatment. Laboratory and vital signs assessments are evaluated over time on study using descriptive statistics. Shift analyses of relevant clinical laboratory parameters are produced, showing shifts across low, normal, and high categories.
Study Design Rationale
[0616] In the non-clinical models of kidney disease in rats, an efficacious dose of Compound 1 ranged from 50 mg/kg/day to 200 mg/kg/day administered twice daily. Based on the extrapolation from the toxicokinetic data from the 28-day toxicity study in rats, these doses correspond to a mean plasma AUClast range of approximately 640-2500 ng. hr/ml. [0617] The proposed dose levels and schedules are expected to provide the efficacious range of exposure based on the data from the Phase 1 study in healthy volunteers. The proposed dosing regimens were well tolerated in the Phase 1 study. A total of 97 subjects received the study drug; 72 subjects received Compound 1 and 25 subjects received placebo. No dose- limiting toxicity was observed in the single ascending doses up to 600 mg once daily and multiple ascending doses up to 600 mg once daily or 500 mg twice daily for two weeks. More subjects in the placebo cohorts (17 subjects [68%]) experienced at least one TEAE than in the Compound 1 treatment cohorts (41 subjects [42.4%]). Overall, the most common TEAE and treatment-related TEAE was in the gastrointestinal disorders system organ class: TEAEs by Preferred Term included abdominal discomfort, abdominal pain, diarrhea, and nausea. There were no serious or severe adverse events.
[0618] Diarrhea was the most commonly reported AE. The highest incidence of diarrhea was observed in 500 mg BID and 600 mg QD dosing cohorts in the MAD part of the study. One subject in the 400 mg QD cohort reported mild diarrhea, which did not require any intervention. In the 250 mg BID cohort, two subjects reported nausea, and one subject reported diarrhea, both mild in severity and neither required any intervention. The dose of 300 mg instead of 250 mg twice daily was selected to achieve the highest exposure without increasing the risk of GI intolerability. The dose level of 300 mg also helps minimize dosing errors and increases convenience when using only two capsule strengths of 100 and 200 mg for all dose levels and dose titration.
[0619] The 12-week duration of dosing is supported by the 90-day GLP toxicology studies in rats and cynomolgus monkeys. The toxicokinetic data from these studies shows that the dosing regimens proposed in this Phase 2 study provide a 5- to 6-fold margin of safety based on the AUC observed at the NOAEL. The duration of 12 weeks is well-established and widely accepted for the proof-of-concept interventional studies in proteinuric renal diseases. In non-clinical efficacy models, Compound 1 was demonstrated to induce a sustained reduction in proteinuria for 12-weeks. This duration is sufficiently long to demonstrate a clinically meaningful reduction in proteinuria and to rule out any transient hemodynamically mediated reduction in proteinuria while avoiding any unacceptable safety risks to the study population.
[0620] In summary, limiting a single dose of Compound 1 to less than 400 mg provides optimum exposure without the increased risk of diarrhea. Administration of 300 mg BID provides maximum exposure with minimum risk of diarrhea based on the results of the Phase 1 study. The highest doses being tested in this study (400 mg QD and 300 mg BID) were well tolerated in healthy volunteers. The 12-week duration of dosing is supported by 90-d GLP tox studies in rats and monkeys and provide sufficient margin of safety based on the AUC. The study protocol allows down-titration in case of any intolerability not amenable to conservative management.
Dose Adjustment
[0621] One dose reduction is permitted, according to the following Table:
Figure imgf000171_0001
[0622] Compound 1 is expected to be predominantly cleared via hepato-biliary elimination in humans based on minor urinary excretion of the parent compound observed in healthy volunteers. In healthy volunteers only < 3% of the total administered dose of Compound 1 was excreted in the urine (for doses up to 500 mg BID and 600 mg QD). After 14 days of daily dosing (600 mg) the percentage of Compound 1 excreted in urine was 1.6 ± 0.41% of the dose. Dose adjustment in patients with mild to moderate renal impairment is, therefore, not required. Compound 1 should not be used in patients with severe renal impairment (CrCl < 30 mL/min). [0623] Nausea, abdominal cramps, and diarrhea were the most frequently reported AEs in healthy volunteers in the Phase 1 study. These events were generally mild and occurred most frequently when the dose of Compound 1 exceeded 400 mg per dose. Nausea was less frequent when Compound 1 was administered after meals. Diarrhea resolved spontaneously in most participants. Occasionally, temporary withholding of Compound 1 or treatment with antidiarrheal agents was required. Gastrointestinal adverse events have also been reported with other TKIs with similar pharmacological profile. If a patient experiences nausea, vomiting, or diarrhea, the study drug dose is reduced per the table above and details below.
[0624] Diarrhea: 4-6 extra stools per day with no clinical evidence of volume depletion or impact on the activities of daily living is treated with anti-motility drugs such as loperamide and increased oral fluids as clinically indicated. If diarrhea persists for 8 or more days despite symptomatic treatment, the dose of the study drug is reduced as outlined in the table above. The patients are continued on anti-motility agents if clinically indicated during this time. If diarrhea does not resolve in another 7 days, the study drug is discontinued, and a follow-up scheduled. [0625] More than 6 extra stools per day with clinical evidence of volume depletion or impact on the activities of daily living is treated with anti-motility drugs such as loperamide, increased oral fluids, and a decrease in dose. If diarrhea persists despite these measures for 14 or more days, the study drug is discontinued, and a follow-up scheduled.
[0626] Nausea and/or vomiting: Nausea and/or vomiting is treated by ensuring the study drug is taken after meals and/or treated with ondansetron (4-8 mg orally every 8 to 12 hours as clinically indicated). If nausea and/or vomiting continues despite the above measures for 8 or more days, the study drug dose is reduced as outlined in the table above. If nausea and/or vomiting persists despite these measures for 14 or more days, the study drug is discontinued, and a follow-up scheduled.
[0627] Liver enzyme elevation: If a patient experiences elevated liver function tests of AST or ALT increased to > 3x ULN or AST or ALT increased to > 2x baseline value in patients with elevated values at baseline, then testing is repeated within 48-72 hours, including total bilirubin and alkaline phosphatase and the study drug is continued at the same dose. If repeat AST or ALT elevation persists at same levels or progresses, then study drug is discontinued, and the patient is assessed for alert indicators of drug-induced liver injury and is monitored for liver safety.
Drug-Drug Interactions
[0628] In vitro studies indicate that Compound l is a CYP3 A4 substrate. The contribution of CYP3 A4 to the overall metabolism of Compound 1 has not been yet determined. Strong inhibitors and inducers of CYP3A4 could alter the PK of Compound 1. Patients are monitored closely for tolerability, specifically gastrointestinal tolerability, of Compound 1 if co administered with strong inhibitor or inducers of this enzyme (e.g., indinavir/ritonavir, tipranavir/ritonavir, ritonavir, indinavir, ketoconazole, troleandomycin, telaprevir , itraconazole, voriconazole, mibefradil, clarithromycin, posaconazole, telithromycin, conivaptan, nefazodone, nelfmavir, saquinavir, idelalisib, boceprevir, avasimibe, carbamazepine, phenytoin, rifampin, rifabutin, phenobarbital, mitotane, or enzalutamide). [0629] Compound 1 is a strong inhibitor of UGT1A9 and could increase exposure to coadministered substrates of this enzymes (e.g., propofol, canagliflozin, sorafenib, fibrates, irinoteca, or acetaminophen).
Example 28. A Phase 1, Randomized, Double-Blind, Placebo-controlled, Single and Multiple Ascending Dose Study to Determine the Safety, Tolerability, Pharmacokinetics, and Food Effect of Compound 1 in Healthy Adult Participants
Objectives
[0630] Primary: To assess the safety and tolerability of single and multiple ascending doses of Compound 1 in healthy adult participants.
[0631] Secondary: To assess the pharmacokinetics (PK) of single and multiple ascending doses of Compound 1 and to evaluate the effect of a high fat meal on the PK of a single dose of Compound 1 administered to healthy adult participants.
Endpoints
[0632] Primary Endpoint: The frequency and severity of treatment-emergent adverse events (TEAEs), including clinically significant abnormal vital signs, electrocardiograms (ECGs), laboratory test results, and physical examination findings.
[0633] Secondary Endpoints:
[0634] Blood PK endpoints include:
• Maximum concentration (Cmax)
• Time to maximum concentration (Tmax)
• Time of last quantifiable concentration (Tiast)
• Area under drug concentration-time curve, from time zero to the last measurable concentration (AUCo-iast)
• Area under drug concentration-time curve, from time zero to infinity (AUCo-inf)
• Area under drug concentration-time curve over inter-dosing interval (AUCtau); MAD cohorts only
• Apparent terminal half-life (ti/2)
• Apparent terminal elimination rate constant (Kei)
• Apparent clearance (SAD cohorts = CL/F; MAD cohorts = CLSs/F)
• Apparent terminal volume of distribution (SAD cohorts = Vz/F; MAD cohorts = Vzss/F) • Accumulation ration (RA); MAD cohorts only [0635] Urine PK endpoints include:
• Amount of drug excreted in urine over time (Aeu-u), including cumulative Ae(o-72 h)
• Renal clearance (CLR)
• Fraction of systemic clearance (CL/F) represented by renal clearance (CLR/[CL/F])
• Fraction of administered dose excreted in urine over dosing intervals (Feu-u), including cumulative Fe(o-72 h)
Methodology
[0636] This was a first in human, single-center, double-blind, randomized, cross-over, SAD design followed by a MAD design study of Compound 1 conducted in healthy adult participants, designed to evaluate safety, tolerability, PK, and food effect of Compound 1 in healthy adults.
Up to 104 participants were to be enrolled into one of up to six SAD cohorts (n=8 per cohort), four MAD cohorts (n=8 per cohort) for twice-daily dosing for two weeks, two MAD cohorts (n=8 per cohort) for once-daily dosing for two weeks, and a single dose food-effect cross-over cohort (n=8 per cohort). A total of 97 participants were enrolled into 1 of 12 cohorts (n=8 per cohort + 1 replacement participant in Cohort A3). The final SAD cohort (Cohort A6) was not initiated, and no participant was enrolled in more than one cohort.
Part A: Single Ascending Dose (SAD)
[0637] Up to 48 participants were to be enrolled into one of up to six cohorts (Cohorts A1 to A6; n=8 per cohort). A total of 41 participants were enrolled into 1 of 5 cohorts (Cohorts A1 to A5; n=8 per cohort + 1 replacement participant in Cohort A3). Cohort A6 was not initiated. [0638] All Part A (SAD) participants were confined to a clinical research unit (CRU) from Day -1 (pre-dose) until completion of the 72-hour post-dose assessments on Day 4.
[0639] Cohort A3 was a food effect cohort, and participants in Cohort A3 only returned to the CRU on Day 14 and, following a 14-day washout, received a second single dose of their assigned treatment on Day 15 following consumption of a high fat meal. For Cohort A3 only, a second period of confinement applied from Day 14 until completion of the 72-hour post-dose assessments on Day 18.
[0640] All Part A participants returned to the CRU for a follow-up visit 7 days (±1 day) after their final dose of study drug. For all cohorts in Part A (SAD), the decision to escalate a dose or modify a dose was determined by the SMC following review of the 7-day blinded safety and available PK data from the preceding cohort.
[0641] Cohorts Al. A2. A4. and A5: Participants (n=8) were randomized 3:1 to receive a single oral dose of either Compound 1 (n=6) or matching placebo (n=2) on Day 1 following an overnight fast of at least 10 hours and after all pre-dose assessments were performed. Two sentinel participants received a single oral dose of Compound 1 (n=l) or matching placebo (n=l) initially. If dosing of these sentinel participants proceeds without clinically significant safety signals in the first 48 hours post-dose (as adjudicated by the principal investigator or delegate), the remaining participants in each cohort received a single dose of Compound 1 (n=5) or matching placebo (n=l) according to the randomization schedule.
[0642] Participants were monitored for safety and blood and urine samples were collected for assessment of PK parameters at predefined time points pre and post-dose. Participants were discharged on Day 4 following completion of all specified study procedures. Participants returned to the CRU for an end of study Follow-up visit on Day 8 (±1 day).
[0643] Cohort A3 (food effect): Participants in the Food Effect Cohort (A3; n=8) received a single oral dose of either Compound 1 or matching placebo in the fasted state (Period 1) followed by the same assigned treatment in the fed state (Period 2) after a 14-day washout period as follows.
[0644] Cohort A3 (Period 1): Participants (n=8) were randomized to receive a single oral dose of either Compound 1 (n=6) or matching placebo (n=2) on Day 1 following an overnight fast of at least 10 hours and after all pre-dose assessments were performed. Two sentinel participants received a single oral dose of Compound 1 (n=l) or matching placebo (n=l) initially. If dosing of these sentinel participants proceeded without clinically significant safety signals in the first 48 hours post-dose (as adjudicated by the PI or delegate), the remaining participants received a single dose of Compound 1 (n=5) or placebo (n=l) according to the randomization schedule. Participants were monitored for safety and blood and urine samples were collected for assessment of PK parameters at predefined time points pre- and post-dose. Participants were discharged on Day 4 following completion of all specified study procedures. [0645] Cohort A3 (Period 2): Participants (n=8) completed a 14-day washout following dosing in Period 1 and returned and were admitted to the CRU on Day 14, after which they received a second single dose of their assigned treatment (oral dose of Compound 1 (n=6) or matching placebo (n=2)) on Day 15, following consumption of a high fat meal. The high fat meal was served 30 minutes prior to study drug administration and was consumed within 30 minutes. Participants were monitored for safety and blood and urine samples were collected for assessment of PK parameters at predefined time points pre-and post-dose. Participants were discharged on Day 18 following completion of all specified study procedures. Participants in Cohort A3 returned to the CRU for an end of study follow-up visit on Day 22 (±1 day).
[0646] Compound 1 dose levels tested in Part A (SAD) did not exceed 1200 mg or the dose level that led to a mean whole blood Cmax > 600 ng/mL or mean whole blood AUCo-iast > 2000 ng*h/mL. These Cmax and AUCo-iast values were extrapolated from the mean plasma Cmax of 800 ng/mL and the mean plasma AUCiast of 4000 ng*h/mL observed in non-human primates at the no observed adverse effect level (NOAEL) of 75 mg/kg/day.
[0647] For Part A (SAD), single oral dosing was carried out according to Table 22.
Table 22.
Figure imgf000176_0001
* Compound 1 dose did not exceed 1200 mg or a dose level that leads to mean whole blood Cmax >600 ng/mL or mean whole blood AUCo-iast > 2000 ng*h/mL. These Cmax and AUCiast values were extrapolated from the mean plasma Cmax of 800 ng/mL and the mean plasma AUCiast of 4000 ng*hr/mL observed in non-human primates at the NOAEL of 75 mg/kg/day.
** Day 15 doses for Cohort A3 (food effect) were administered following consumption of a high fat meal.
Part B: Multiple Ascending Dose (MAD j
[0648] Up to 32 participants were to be enrolled into up to four cohorts (Cohorts B1 to B4; n=8 per cohort). All 32 participants were enrolled as planned (n=8 per cohort). Participants were administered Compound 1 (n=6 per cohort) or matching placebo (n=2 per cohort) twice daily for 14 consecutive days (Day 1 to Day 14). All Part B (MAD) participants were confined to the CRU from Day -1 (pre-dose) to Day 17. Participants were monitored for safety and blood and urine samples were collected for assessment of PK at predefined time points pre- and post-dose. [0649] Participants were administered study drug in a standardized manner; i.e., either with or without food depending on preliminary bioavailability results from Part A (SAD). Participants were discharged from the CRU upon completion of final 72-hour post-dose assessments. Participants then returned to the CRU for an end of study follow-up visit 7 days after the final dose of study drug (±1 day). For all cohorts in Part B (MAD), the decision to escalate a dose or modify a dose was determined following review of the 17-day blinded safety and available PK data from the preceding cohort.
[0650] The Compound 1 dose level tested in Part B (MAD) did not exceed 1000 mg per day or a dose level that led to a mean whole blood Cmax > 600 ng/mL or mean whole blood AUCo-iast > 2000 ng*h/mL. These Cmax and AUCo-iast values were extrapolated from the mean plasma Cmax of 800 ng/mL and the mean plasma AUCiast of 4000 ng*h/mL observed in non-human primates at the no observed adverse effect level (NOAEL) of 75 mg/kg/day.
[0651] For Part B (MAD), once daily oral dosing was carried out according to Table 23.
Table 23.
Figure imgf000177_0001
* Compound 1 dose did not exceed 1000 mg per day or a dose level that leads to mean whole blood Cmax >600 ng/mL or mean whole blood AUCo-iast > 2000 ng*h/mL. These Cmax and AUCiast values were extrapolated from the mean plasma Cmax of 800 ng/mL and the mean plasma AUCiast of 4000 ng*hr/mL observed in non-human primates at the NOAEL of 75 mg/kg/day.
Part C: Multiple Ascending Dose Once Daily (MAD QD)
[0652] Up to 16 participants were to be enrolled into up to 2 cohorts (Cohorts Cl to C2; n=8 per cohort). All 16 participants were enrolled as planned (n=8 per cohort). Participants were administered Compound 1 (n=6 per cohort) or matching placebo (n=2 per cohort) once daily for 14 consecutive days (Day 1 to Day 14). All Part C participants were confined to the CRU from Day -1 (pre-dose) to Day 17. Participants were monitored for safety and blood and urine samples were collected for assessment of PK at predefined time points pre- and post-dose.
[0653] For Part C (MAD QD), once daily oral dosing was carried out according to Table 24. Table 24.
Figure imgf000177_0002
* Compound 1 dose did not exceed dose level that leads to mean whole blood CmaX >600 ng/mL or mean whole blood AUCo-iast > 2000 ng*h/mL. These CmaX and AUCiast values were extrapolated from the mean plasma Cmax of 800 ng/mL and the mean plasma AUCiast of 4000 ng*hr/mL observed in non-human primates at the NOAEL of 75 mg/kg/day.
Part D: Single-Dose Food-Effect Cohort
[0654] Eight participants were enrolled into 1 cohort (Cohort Dl). Four participants were randomly administered a single dose of Compound 1 (n=3) or matching placebo (n=l) in fasting condition first, and four participants were administered a single dose of Compound 1 (n=3) or matching placebo (n=l) after a standard FDA high-fat meal in the Period 1. Participants completed the 72-hour post first dose assessments and began the Period 2 of Part D on Day 5, thus allowing a 4-day washout period from the first administered dose. In Period 2, in a cross over design, the group administered Compound 1 and placebo in the fasted condition during Period 1 received Compound 1 (n=3) and placebo (n=l) in a fed state. The group assigned to the fed state dosing during Period 1 crossed over to receive Compound 1 (n=3) and placebo (n=l) after fasting. All Part D participants were confined to the CRU from Day -1 (pre-dose) until the completion of the 72-hour post second single-dose assessments on Day 8. Participants were monitored for safety and blood and urine samples were collected for assessment of PK at predefined time points pre- and post-dose.
[0655] For Part D (Single Dose Food Effect), once daily oral dosing was carried out according to Table 25.
Table 25.
Figure imgf000178_0001
* Compound 1 dose did not exceed dose level that leads to mean whole blood Cmax >600 ng/mL or mean whole blood AUCo-iast > 2000 ng*h mL. These Cmax and AUCiast values were extrapolated from the mean plasma Cmax of 800 ng/mL and the mean plasma AUCiast of 4000 ng*hr/mL observed in non-human primates at the NOAEL of 75 mg/kg/day.
[0656] Up to 104 participants were enrolled in the study (Part A: up to 48 healthy volunteers; Part B: up to 32 healthy volunteers; Part C: up to 16 healthy volunteers; Part D: up to 8 healthy volunteers). 97 participants were enrolled in the study. Diagnosis and Main Criteria for Inclusion:
Inclusion Criteria:
[0657] To be eligible for the study, participants were required to meet all of the following inclusion criteria:
1. Healthy male or female volunteer, aged 18 to 65 years.
2. Participants must be in good health, with no significant medical history, have no clinically significant abnormalities on physical examination at screening and/or before administration of the initial dose of study drug.
3. Participants must have a minimum body weight of 50 kg and a body mass index (BMI) between >18.0 and <32.0 kg/m2 at screening.
4. Participants must have clinical laboratory values within normal range as specified by the testing laboratory, unless deemed not clinically significant by the investigator or delegate.
5. Participants who smoke no more than 2 cigarettes or equivalent per week can be included in study but must be willing to abstain from smoking during the confinement period.
6. Participants must have no relevant dietary restrictions, and be willing to consume standard meals provided during the confinement period.
7. Women of childbearing potential (WOCBP) must be non-pregnant and non-lactating, and must use an acceptable, highly effective double contraception from screening until study completion, including the follow-up period. Double contraception is defined as a condom and one other form of the following:
• Established hormonal contraception (oral contraceptive pills, long-acting implantable hormones, injectable hormones)
• A vaginal ring or an intrauterine device
• Documented evidence of surgical sterilization at least 6 months prior to screening (e.g., tubal occlusion, hysterectomy, bilateral salpingectomy, or bilateral oophorectomy for women or vasectomy for men (with appropriate post-vasectomy documentation of absence of sperm in semen) provided the male partner is a sole partner Women not of childbearing potential must be postmenopausal for > 12 months. Postmenopausal status are confirmed through testing follicle-stimulating hormone (FSH) levels > 40 IU/mL at screening for amenorrhoeic female participants. Females who are abstinent from heterosexual intercourse are also eligible.
Periodic abstinence (e.g., calendar, ovulation, symptothermal, post-ovulation- methods) and withdrawal are not considered highly effective methods of birth control. Participant complete abstinence for the duration of the study and for 1 months after last study treatment is acceptable.
Female participants who are in same sex relationships are not required to use contraception.
WOCBP must have negative pregnancy test at screening and Day 1 and be willing to have additional pregnancy test as required.
Males must be surgically sterile (>30 days since vasectomy with no viable sperm), abstinent, or if engaged in sexual relations with a WOCBP, the participant and his partner must be surgically sterile (e.g., tubal occlusion, hysterectomy, bilateral salpingectomy, or bilateral oophorectomy) or using an acceptable, highly effective contraceptive method from Screening until study completion, including the follow-up period. Acceptable methods of contraception include the use of condoms and the use of an effective contraceptive for the female partner that includes: OCPs, long-acting implantable hormones, injectable hormones, a vaginal ring or an IUD. Participants with same sex partners (abstinence from penile-vaginal intercourse) are eligible when this is their preferred and usual lifestyle.
8. Male participants must not donate sperm for at least 90 days after the last dose of study drug.
9. Participants must have the ability and willingness to attend necessary visits to the CRU.
10. Participants must be willing and able to provide written informed consent after the nature of the study has been explained and prior to the commencement of any study procedures.
[0658] A participant who met any of the following exclusion criteria were excluded from the study: Pregnant or lactating at screening or planning to become pregnant (self or partner) at any time during the study, including the follow-up period. Prior or ongoing medical conditions, medical history, physical findings, or laboratory abnormality that, in the investigator’s (or delegate’s) medical opinion, could adversely affect the safety of the participant. History of gastrointestinal (GI) disorders such as celiac disease, atrophic gastritis, lactose intolerance, and Helicobacter (H.) pylori infection. Presence of any underlying physical or psychological medical condition that, in the opinion of the investigator, would make it unlikely that the participant will comply with the protocol or complete the study per protocol. Any surgical or medical condition that could interfere with the absorption, distribution, metabolism, or excretion of the study drug. Blood donation or significant blood loss within 60 days prior to the first study drug administration. Plasma donation within 7 days prior to the first study drug administration. Fever (body temperature > 38 °C) or symptomatic viral or bacterial infection within 2 weeks prior to screening. Any acute illness within 30 days prior to Day 1. History of severe allergic or anaphylactic reaction. History of malignancy except for non-melanoma skin cancer excised more than 2 years ago and cervical intraepithelial neoplasia (CIN) that has been successfully cured more than 5 years prior to screening. Abnormal ECG finding at screening that are considered by the investigator to be clinically significant. History or presence of a condition associated with significant immunosuppression. History of life-threatening infection (e.g., meningitis). Infections requiring parenteral antibiotics within 6 months prior to screening. Vaccination with a live vaccine within 4 weeks prior to screening or that is planned within 4 weeks of dosing. Exposure to any significantly immune suppressing drug (including experimental therapies as part of a clinical trial) within 4 months prior to screening or 5 half-lives, whichever is longer. Positive test for hepatitis C antibody (HCV), hepatitis B surface antigen (HBsAg), or human immunodeficiency virus (HIV) antibody at screening. Participants with a positive toxicology screening panel (urine test including qualitative identification of barbiturates, tetrahydrocannabinol (THC), amphetamines, benzodiazepines, opiates, and ***e), or alcohol breath test. Participants with a history of substance abuse or dependency or history of recreational intravenous (IV) drug use over the last 5 years (by self-declaration). Regular alcohol consumption defined as >21 alcohol units per week (where 1 unit = 284 mL of beer, 25 mL of 40% spirit or a 125 mL glass of wine). Participant is unwilling to abstain from alcohol beginning 48 hours prior to admission to the CRU and during confinement period. Use of any IP or investigational medical device within 30 days prior to screening, or 5 half-lives of the product (whichever is longest) or participation in more than four investigational drug studies within 1 year prior to screening. Use of any prescription drugs (other than hormonal contraception: OCPs, long-acting implantable hormones, injectable hormones, a vaginal ring or an HID), over-the- counter (OTC) medication, herbal remedies, supplements or vitamins within 1 week prior to dosing and during course of study without prior approval of the investigator and medical monitor. Simple analgesia (nonsteroidal anti-inflammatory drug (NSAID)) or paracetamol may be permitted at discretion of investigator. Use of fibrates for hyperlipidemia. Consumption of any nutrients or concomitant medications known to modulate cytochrome P450 3 A4 (CYP3 A4) or any strong inhibitors or inducers of CYP3 A4, starting from 2 weeks prior to first dose of study drug and until end of study assessments. Inability to refrain from consumption of grapefruit and Seville oranges or St. John’s Wort within 2 weeks prior to first dose of study drug and until final PK assessment. 27. Participant is unwilling to refrain from strenuous exercise from 72 hours prior to admission to CRU until completion of final follow-up visit.
Product and Mode of Administration
[0659] Compound 1 was provided as a powder in capsule formulation for oral administration. The formulation was a Size 00 Swedish orange capsule containing drug substance (50 mg or 250 mg) with no excipients. Ingredients of the capsule shell were hypromellose (hydroxypropylmethyl cellulose (HPMC)), iron oxide as coloring agent, and titanium dioxide as an opacifier. The drug product was stored at room temperature (15 °C - 25 °C).
[0660] Compound 1 is in a pharmacological class of tyrosine kinase inhibitors (TKI). Compound 1 is an orally bioavailable small molecule dual kinase inhibitor of platelet-derived growth factor receptors (PDGFR) and vascular endothelial growth factor receptors (VEGFR2). Duration of Treatment
[0661] Depending on study part and cohort assignment, the duration of study participation for each participant ranged from approximately 36 days to 50 days, including up to 28 days of screening. Duration of treatment by cohort is summarized in Table 26.
Table 26.
Figure imgf000183_0001
[0662] Part A (SAD): In Part A (SAD), participants randomized to active treatment in cohorts Al, A2, A4, and A5 received a single oral dose of Compound 1 administered once on Day 1 only. Participants randomized to active treatment in Cohort A3 (Food Effect) received a single oral dose of Compound 1 on Days 1 and 15.
[0663] Part B (MAD BID): In Part B (MAD), participants randomized to active treatment received oral Compound 1 administered twice daily for 14 consecutive days (Day 1 to Day 14). [0664] Part C (MAD QD): In Part C (MAD QD), participants randomized to active treatment received oral Compound 1 administered once daily for 14 consecutive days (Day 1 to Day 14). [0665] Part D (Single Dose Food Effect): In Part D (Single Dose Food Effect), participants randomized to active treatment received a single oral dose of Compound 1 under fasting conditions (n=3) or under fed conditions (n=3) in Study Period 1. On Day 5, after a total of 4 days of wash-out from the first dose, participants crossed-over to Period 2 where the participants received a single oral dose of Compound 1 under the feeding condition opposite to the meal condition in Period 1.
Reference Therapy and Mode of Administration
[0666] The placebo capsules visually matched to active study drug and were composed of the same capsule shell but were filled with silicified microcrystalline cellulose. Silicified microcrystalline cellulose is a pharmaceutical excipient composed of co-process microcrystalline cellulose and colloidal silicon dioxide.
Criteria for Evaluation Part A (SAD), Part B (MAD), Part C (MA I) QD) and Part D (Single Dose Food Effect
[0667] Safety: The safety and tolerability of single and repeat-doses of Compound 1 were investigated according to the following specific assessments: vital signs (systolic and diastolic blood pressure, pulse rate, body temperature, and respiratory rate), 12-lead ECG, clinical laboratory tests (hematology, biochemistry, coagulation, and urinalysis), physical examination, and assessment of TEAEs.
[0668] Pharmacokinetics: Blood and urine samples for PK analysis of Compound 1 were collected pre-dose and following oral administration of single and repeat-doses of Compound 1 in the fasted state. Blood and urine samples for PK analysis were collected pre-dose and following administration of single doses of Compound 1 immediately after ingestion of a high fat meal (Food Effect Cohort A3 and Cohort Dl). Statistical Methods
[0669] The number of participants was selected to allow for evaluation of safety/tolerability, PK and food effect of the single and multiple doses administered in this study and was consistent with standards of practice for Phase 1 studies.
[0670] In general, descriptive statistics (e.g. arithmetic mean, standard deviation [SD], median, minimum and maximum) were calculated for continuous data among treatment groups (or sequences), as well as difference from baseline by study part for each applicable scheduled time point, when appropriate. Frequency summaries (e.g. number of observed and percentage of each categories) were applied for categorical data among treatment groups (or sequences), by study part and for each scheduled time point.
[0671] For PK data the arithmetic mean, SD, median, minimum, maximum, coefficient of variation (CV%), geometric mean, geometric coefficient of variation (geo CV% or gCV) and the number of below the limit of quantification (BLQ) values were presented. No geometric statistics were computed for BLQ plasma concentrations.
[0672] No formal hypothesis testing was performed for this study.
[0673] Analysis Populations: Participant inclusion into each population was determined prior to the final analysis.
[0674] Intent-to-Treat (ITT) Population: All enrolled participants, regardless of whether they received study drug or not, were included in the ITT population. Analysis was based on the treatment assigned to a participant, not what they actually received. The ITT population was used for all data listings and summaries involving disposition and enrollment.
[0675] Safety Population: All participants who received any amount of study drug (Compound 1 or placebo) were included in the Safety population. The Safety population was used for the summaries of all safety assessments. Participants were analyzed according to treatment received.
[0676] Pharmacokinetic Population: All participants who received any amount of active study drug (Compound 1) and had sufficiently evaluable concentration-time profile to allow determination of at least one PK parameter were included in the PK population. An evaluable PK profile was determined at the discretion of the pharmacokineticist following examination of subjects with dosing or protocol deviations that could potentially affect the PK profile. The PK population was used for the summaries of all PK data. [0677] Safety and Tolerability: All adverse events (AEs) were coded using the Medical Dictionary for Regulatory Activities (MedDRA®) Version 22.0. A by participant AE data listing, including verbatim term, preferred term (PT), system organ class (SOC), treatment, severity, and relationship to study drug was provided. The number of participants experiencing TEAEs and number of individual TEAEs was summarized by SOC and PT. TEAEs were also summarized by severity and by relationship to study drug.
[0678] Laboratory evaluations, vital signs assessments and ECG parameters were summarized for each scheduled visit by treatment arm. A summary of change from baseline at each protocol specified time point by treatment arm was also presented.
[0679] Concomitant medications were coded using the World Health Organization (WHO) drug dictionary Version B3 September 2018 Drug Global. Concomitant medications were listed by participant and summarized by anatomical therapeutic class and preferred name.
[0680] Physical examinations at each visit were listed for each participant and summarized using descriptive statistics at each visit by treatment arm.
[0681] Medical history, pregnancy test/FSH, urine drug screen/alcohol breath test, physical examinations and serology (HIV, Hepatitis B & C screen) were listed by participant.
[0682] Pharmacokinetics: Blood Compound 1 concentrations, actual blood sampling times, and PK parameters were listed by treatment and protocol specified time point and summarized using descriptive statistics for PK data as outlined above for each scheduled time point by treatment arm. Individual and mean Compound 1 concentration-time profiles were also presented graphically for each treatment. Pharmacokinetic parameters were computed from the individual plasma Compound 1 concentrations using a non-compartmental approach.
[0683] The following blood Compound 1 non-compartmental PK parameters were estimated, as appropriate: Cmax, Tmax, Tiast, AUCo-iast, AUCo-inf, AUCtau (calculated for MAD cohorts only), Kei, ti/2, CL/F (SAD cohorts), CL/Fss (MAD cohorts), Vz/F (SAD cohorts, Vz/Fss (MAD cohorts), and RA (calculated for MAD cohorts only), as well as various PK parameters derived therefrom (e.g., AUCoWDose).
[0684] Value for Kei, ti/2, AUCo-inf, CL/F (CL/Fss), or Vz/F (Vz/Fss) were not reported for cases that fail to exhibit a terminal log-linear phase in the concentration versus time profile. Additional analyses were performed as deemed necessary upon review of the data. [0685] A food effect assessment was conducted on Compound 1 PK parameters in Cohort A3. Analyses of variance (ANOVA) were performed on the log-transformed AUCo-iast, AUCo-inf and Cmax (fasting vs fed). Ratios of the geometric means were calculated using the exponentiation of the least squares mean (LSM) from the analyses on the ln-transformed AUCo- iast, AUCo-inf and Cmax. Ratios were expressed as a percentage relative to the fasting regimen. 90% Cl for the ratios were derived by exponentiation of the CIs obtained for the difference between regimen LSM resulting from the analyses on the ln-transformed AUCo-iast, AUCo-inf, and Cmax. [0686] The analysis of dose proportionality was conducted for AUC and Cmax of single agent Compound 1 using a power model on log-transformed scale. The log-transformed exposure parameters were each regressed onto a fixed factor for log (dose). The 90% Confidence Interval (Cl) of the slope for each exposure parameter was computed from the model and presented in a summary table.
[0687] Urine collection time, volume collected, and Compound 1 concentration (Aeu-a) duration of each sampling interval was listed for each participant and summarized by nominal sampling time point and treatment using descriptive statistics (Number of subjects [N], arithmetic mean, SD, CV%, geometric mean, median, minimum and maximum). Individual and mean Compound 1 cumulative urinary excretion-time profiles for each treatment were also presented graphically. Where urine was collected for PK analysis, the following parameters are calculated, as appropriate: Aeu-t2, CLR, CL/F, and Feu-t2.
Results
[0688] FIG. 1 and FIG. 29 summarize mean oral blood PK profile over time from SAD cohorts in a fasted state. Table 27 summarizes mean oral blood PK parameters from SAD cohorts in a fasted state.
Table 27.†
Figure imgf000187_0001
Figure imgf000188_0001
†A11 data given as mean value ± SD (% C. V.), unless otherwise noted. *tm only estimated when the terminal slope’s R2 > 0.8 (12-48 h post-hoc). **Tmax given as median (range). ***Tiast = 4 h (n = 4), 12 h (n = 1). a Tiast = 72 h (n = 4), 48 h (n = 2): AUC0-24h = 2189 ng*h mL (28% Cl). b n = 2. c n =4.
[0689] FIG. 30A and FIG. 30B summarize mean oral blood PK profiles over time from BID MAD cohorts in the fasted state at Day 1 and Day 14, respectively. Table 28 summarizes mean oral blood PK parameters from MAD cohorts in a fasted state.
Table 28.
Figure imgf000188_0002
*TPHC given as median (range).
[0690] As shown in Tables 27 and 28, Compound 1 was rapidly absorbed with a median Tmax of about 1-2 h post-dose. Mean half-lives (T1/2) of 12-21 h were observed in the SAD, MAD QD, and food effect cohorts. Little or no accumulation in blood after 14 days of dosing indicated that QD and/or BID dosing can be used in further clinical studies (e.g., in the Phase 2 study of Example 27) and/or as part of a therapeutic regimen. Cmax, AUCo-t, and AUCo-inf increased slightly over-proportionally in the SAD cohort based on a Power Model assessment. Mean C24 levels after 400 mg and 600 mg dose were ~5 and 8 ng/mL, respectively. PK data from MAD BID fasted cohorts were consistent with PK observed in SAD cohort. Saturation of absorption was observed at the 500 mg BID dose level in MAD cohorts (see Table 28).
[0691] FIG. 31 summarizes mean blood concentration time profiles at steady state for QD MAD cohorts in the fed state at Day 14. Table 29 summarizes mean PK parameters on Day 14 from a 400 mg QD MAD cohort. Table 29.
Figure imgf000189_0001
* Tmax given as median. ** n=2; CLSS/F = Dose/AUC(0-t). *** n=3.
[0692] Median Tmax for 400 mg and 600 mg QD doses in the fed state (4 and 3.5 h, respectively) was longer than in the corresponding fasted SAD and BID cohorts (~l-2 h), indicating a delay in absorption when Compound 1 was administered with food. Mean Day 14 Cmax.ss was 221_± 91 ng/mL (41% CV) and 307 ± 244 ng/mL (79.6% CV) for 400 mg and 600 mg QD, respectively. Mean Day 14 AUCo-24 was 946 ng*h/mL (52.1%) and 1649 ng*h/mL (51.6%) for 400 mg and 600 mg QD, respectively. The mean half-life of elimination (T1/2) was ~21 h at 400 mg QD (n=4) and 15 h at 600 mg QD (n=4).
[0693] FIG. 32 summarizes Compound 1 blood concentration in a food effect cross-over study with 600 mg single dose. AUCo-iast, AUCo-inf and Cmax in fed and fasting states were compared using an Analysis of Variance model. Statistical analysis of the effect of food (high fat) on the PK of 600 mg dose showed a food effect on AUCo-inf and Cmax (90% C.I. of the ratios of the geometric least square means were > 60-140%). The difference in AUCo-iast under fed and fasted conditions was less than 25%. T1/2 of elimination was similar between the fed and fasted conditions (12.0 and 12.7 h, respectively). Food effect data indicated that there was evidence of a lower rate of absorption of Compound 1 in the fed state, e.g., with an 18% lower Cmax reached more slowly than in the fasting state. It will be appreciated that the observed food effect data indicate that Compound 1 is likely suitable for administration in either the fed or fasted state, though certain GI side effects may be mitigated by fed state administration.
[0694] Compound 1 was generally well-tolerated at all dose levels tested during this study. No serious adverse events were reported. The majority of TEAEs reported were Grade 1 (mild) or Grade 2 (moderate). Mild to moderate diarrhea was the most common adverse event reported and was seen only at high doses (> 400 mg per dose).
[0695] Compound 1 was rapidly absorbed, and across the SAD dose range (50 mg to 600 mg), the increase in exposure of Compound 1 following oral administration was slightly greater than dose proportional for Cmax, AUCo-t, and AUCo-inf. based on a Power Model assessment without wishing to be bound by theory, the overall lack of accumulation in blood and mean overall t½ range of 12 to 21 hours (estimated from SAD, Day 14 MAD QD and the Food effect cohort) would permit dosing of Compound 1 either BID or QD. Systemic clearance after oral dosing was high overall, > 2-fold the hepatic blood flow, suggesting additional non-hepatic mechanisms of clearance contributing to its elimination. Volumes of distribution were large (> 2000 L) indicating extensive distribution of Compound 1 into the tissues. Urinary excretion is a minor pathway in Compound 1 elimination. The single dose cross-over food effect study data (600 mg) indicated a lower rate of absorption of Compound 1 in the fed state as characterized by an 11% lower geometric mean Cmax, which was reached more slowly (1 hour later) than in the fasting state.
[0696] The embodiments of the disclosure described above are intended to be merely exemplary, numerous variations and modifications will be apparent to those skilled in the art.
All such variations and modifications are intended to be within the scope of the present invention as defined in any appended claims.

Claims

1. A method comprising administering to a patient in need thereof about 50 mg to about 600 mg daily of Compound 1 :
Figure imgf000191_0001
2. A method comprising administering to a patient in need thereof an amount of Compound 1:
Figure imgf000191_0002
wherein said amount, when administered as a single oral dose to a population of healthy subjects, was established to achieve one or more of:
(i) a mean maximum concentration (Cmax) of from about 30 ng/mL to about 800 ng/mL;
(ii) a median time to maximum concentration (Tmax) of from about 1 hour to about 4 hours;
(iii) a mean area under drug concentration-time curve (AUCo-iast) of from about 60 ng*h/mL to about 4000 ng*h/mL;
(iv) a mean area under drug concentration-time curve (AUCo-inf) of from about 60 ng*h/mL to about 4000 ng*h/mL; (v) a mean apparent volume of distribution (Vz/F) of from about 2000 L to about 5500 L;
(vi) a mean apparent total clearance (CL/F) of from about 300 L/h to about 600 L/h; and
(vii) a mean half-life (ti/2) of from about 2 hours to about 30 hours.
3. The method of claim 2, wherein the amount was established to achieve a mean half-life (ti/2) of from about 12 hours to about 21 hours.
4. A method comprising administering to a patient in need thereof an amount of Compound
1:
Figure imgf000192_0001
1 wherein said amount, when administered as multiple oral doses to a population of healthy subjects, was established to achieve one or more of:
(i) a mean maximum concentration (Cmax) of from about 30 ng/mL to about 800 ng/mL;
(ii) a median time to maximum concentration (Tmax) of from about 1 hour to about 2 hours;
(iii) a mean area under drug concentration-time curve (AUCo-12) of from about 100 ng*h/mL to about 1500 ng*h/mL;
(iv) a mean accumulation ratio of Cmax (RaccCmax) of from about 0.6 to about 2.7; and
(vi) a mean accumulation ratio of AUC (RaccAUC) of from about 0.8 to about 2.6.
5. A method comprising administering to a patient in need thereof an amount of Compound
1:
1 wherein said amount, when administered as a single oral dose to a population of healthy subjects in a fasted state, was established to achieve one or more of:
(i) an increased mean maximum concentration (Cmax);
(ii) a decreased median time to maximum concentration (Tmax);
(iii) a comparable mean area under drug concentration-time curve (AUCo-iast);
(iv) a comparable mean area under drug concentration-time curve (AUCo-inf);
(v) a decreased mean apparent volume of distribution (Vz/F);
(vi) a comparable mean apparent total clearance (CL/F); and
(vii) a decreased mean half-life (ti/2), relative to a comparable population of healthy subjects in a fed state.
6 A method comprising administering to a patient in need thereof an amount of Compound
1:
Figure imgf000193_0001
1 wherein said amount, when administered as a single oral dose to a population of healthy subjects in a fasted state, was established to achieve one or more of:
(i) an increased mean maximum concentration (Cmax); (ii) a decreased median time to maximum concentration (Tmax);
(iii) a comparable mean area under drug concentration-time curve (AUCo-iast);
(iv) a comparable mean area under drug concentration-time curve (AUCo-inf);
(v) a comparable mean apparent volume of distribution (Vz/F);
(vi) a comparable mean apparent total clearance (CL/F); and
(vii) a comparable mean half-life (ti/2), relative to a comparable population of healthy subjects in a fed state.
7. A method comprising administering to a patient in need thereof an amount of Compound 1:
Figure imgf000194_0001
wherein said amount, when administered to a population of subject suffering from primary glomerular disease and/or persistent proteinuria, was established to achieve one or more of:
(i) a greater mean percent reduction in 24-hour urinary protein excretion;
(ii) a greater mean percent reduction in 24-hour urinary albumin;
(iii) a greater proportion of subject with complete remission in proteinuria;
(iv) a greater proportion of subjects with partial remission in proteinuria;
(v) a greater proportion of subject with complete or partial remission in proteinuria;
(vi) a greater proportion of subjects with at least 50% reduction in 24-hour urinary protein excretion from baseline;
(vii) a greater proportion of subjects with at least 50% reduction in 24-hour urinary albumin excretion from baseline;
(viii) a greater mean percent increase from baseline in creatinine clearance;
(ix) a greater mean increase from baseline in serum albumin;
(x) a greater mean decrease from baseline in fasting triglycerides levels; (xi) a greater men decrease from baseline in cholesterol levels; and
(xii) a greater mean change in level(s) of one or more plasma or urine biomarkers selected from plasma soluble TNF receptor 2 (sTNFR2), kidney injury molecule- 1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), urinary fibronectin, high molecular weight type IV collagen (HMW collagen IV), monocyte chemotactic protein- 1 (MCP-1), and matrix metalloproteinase-7 (MMP-7), relative to a comparable population of subjects that did not receive Compound 1.
8. The method of any one of the preceding claims, wherein the method comprises administering to a patient in need thereof Compound 1 in a daily dose of from about 200 mg to about 600 mg.
9. The method of any one of the preceding claims, wherein the method comprises administering to a patient in need thereof about 200 mg of Compound 1 once daily.
10. The method of any one of the preceding claims, wherein the method comprises administering to a patient in need thereof about 400 mg of Compound 1 once daily.
11. The method of any one of the preceding claims, wherein the method comprises administering to a patient in need thereof about 300 mg of Compound 1 twice daily.
12. The method of any one of the preceding claims, wherein the method further comprises reducing the amount of Compound 1 administered if the patient experiences a gastrointestinal side effect.
13. The method of claim 12, wherein the gastrointestinal side effect is selected from diarrhea, nausea, and vomiting.
14. The method of claim 12 or 13, wherein the amount of Compound 1 is reduced by about
100 mg.
15. The method of any one of claims 12-14, wherein the method further comprises discontinuing administration of Compound 1 if the gastrointestinal side effect persists.
16. The method of any one of the preceding claims, wherein Compound 1 is in a pharmaceutically acceptable salt form.
17. The method of claim 16, wherein the pharmaceutically acceptable salt form is a hydrochloride salt form.
18. The method of claim 17, wherein the pharmaceutically acceptable salt form is a Form A hydrochloride trihydrate.
19. The method of any one of the preceding claims, wherein Compound 1 is administered orally.
20. The method of any one of the preceding claims, wherein Compound 1 is administered in a solid dosage form.
21. The method of any one of the preceding claims, wherein Compound 1 is administered in a capsule dosage form.
22. The method of any one of the preceding claims, wherein Compound 1 is administered in a capsule comprising about 10 mg, about 50 mg, or about 250 mg of Compound 1.
23. The method of any one of the preceding claims, wherein Compound 1 is administered in a capsule comprising about 100 mg Compound 1.
24. The method of any one of the preceding claims, wherein Compound 1 is administered in a capsule comprising about 200 mg Compound 1.
25. The method of claim any one of claims 21-24, wherein the capsule comprises Compound 1 with no excipients.
26. The method of any one of the preceding claims, wherein the patient is suffering from a disease, disorder, or condition characterized by or otherwise associated with fibrosis.
27. The method of claim 26, wherein the disease, disorder, or condition is or comprises fibrosis of gastrointestinal tract, heart, kidney, lung, liver, muscle, pancreas, or skin.
28. The method of any one of the preceding claims, wherein the patient is suffering from pulmonary fibrosis.
29. The method of any one of the preceding claims, wherein the patient is suffering from idiopathic pulmonary fibrosis.
30. The method of any one of the preceding claims, wherein the patient is suffering from renal fibrosis.
31. The method of any one of the preceding claims, wherein the patient is suffering from primary glomerular disease.
32. The method of claim 31, wherein the primary glomerular disease has been confirmed by a renal biopsy.
33. The method of any one of the preceding claims, wherein the patient has proteinuria.
34. The method of any one of the preceding claims, wherein the patient is suffering from persistent proteinuria.
35. The method of any one of the preceding claims, wherein the patient is suffering from membranous nephropathy.
36. The method of any one of the preceding claims, wherein the patient is suffering from IgA nephropathy.
37. The method of any one of the preceding claims, wherein the patient is suffering from primary proteinuric kidney disease.
38. The method of any one of the preceding claims, wherein the patient has the one or more risk factors for primary glomerular disease selected from proteinuria, renal dysfunction, hypertension, interstitial fibrosis on renal biopsy, and lack of response to therapy or relapse in proteinuria.
39. The method of any one of the preceding claims, wherein the patient has an estimated glomerular filtration rate (eGFR) of greater than or equal to 40 mL/min/1.73m2.
40. The method of any one of the preceding claims, wherein the patient has a urinary protein excretion of greater than or equal to 1 g/day on a 24-hour urine collection.
41. The method of any one of the preceding claims, wherein the patient is receiving or has received standard of care therapy.
42. The method of claim 41, wherein the patient is resistant to standard of care therapy.
43. The method of claim 41 or 42, wherein the patient has relapsed after receiving standard of care therapy.
44. The method of any one of claims 41-43, wherein standard of care therapy comprises an immunosuppressive or immunomodulatory agent and/or one or more of a RAAS blocker, mineralocorticoid receptor inhibitor or SGLT-2 inhibitor.
45. The method of any one of the preceding claims, wherein the patient is receiving or has received an immunosuppressive or immunomodulatory agent.
46. The method of any one of the preceding claims, wherein the patient is receiving or has received a RAAS blocker.
47. The method of any one of the preceding claims, wherein the patient is receiving or has received an ACE inhibitor.
48. The method of any one of the preceding claims, wherein the patient is receiving or has received an angiotensin-receptor blocker.
49. The method of any one of the preceding claims, wherein the patient is receiving or has received a sodium glucose co-transporter-2 (SGLT-2) inhibitor.
50. The method of any one of the preceding claims, wherein the patient is suffering from renal failure, renal obstruction, renal trauma, renal transplantation, chronic kidney disease, diabetes, hypertension, radiocontrast nephropathy, an immune-mediated glomerulonephritis, a non-immune-mediated glomerulonephritis, minimal change disease, or nephrotic syndrome.
51. The method of claim 50, wherein the immune-mediated glomerulonephritis is selected from lupus nephritis, ANCA-associated glomerulonephritides, anti-GBM nephropathy, IgA nephropathy, membranous glomerulonephritis, or focal and segmental glomerulosclerosis.
52. The method of claim 50, wherein the non-immune-mediated glomerulonephritis is selected from polycystic kidney disease, collagen type III glomerulopathy, nail-patella syndrome, or Alport syndrome.
53. The method of any one of the preceding claims, wherein the patient is suffering from nephrotic syndrome.
54. The method of any one of the preceding claims, wherein the patient is suffering from steroid-resistant nephrotic syndrome.
55. The method of any one of the preceding claims, wherein the patient is suffering from focal and segmental glomerulosclerosis.
56. The method of any one of the preceding claims, wherein the patient is suffering from Alport syndrome.
57. The method of any one of the preceding claims, wherein the patient is suffering from dermal fibrosis.
58. The method of any one of the preceding claims, wherein the patient is suffering from scleroderma or systemic sclerosis.
59. The method of any one of the preceding claims, wherein the patient in need thereof is suffering from inflammatory bowel disease.
60. The method of any one of the preceding claims, wherein the patient is in a fed state.
61. The method of any one of the preceding claims, wherein the patient is in a fasted state.
62. The method of any one of the preceding claims, wherein the patient has not received any other medications within 30 minutes of administration of Compound 1.
63. A method of treating focal and segmental glomerulosclerosis in a patient in need thereof, comprising administering to the patient Compound 1 : 1
64. A method of treating Alport syndrome in a patient in need thereof, comprising administering to the patient Compound 1 :
Figure imgf000201_0001
1
65. The method of claim 63 or 64, wherein Compound 1 is in a pharmaceutically acceptable salt form.
66. The method of any one of claims 63-65, wherein Compound 1 is administered orally.
67. A method of treating primary proteinuric kidney disease in a patient in need thereof, comprising administering to the patient Compound 1 : 1
68. A method of treating primary glomerular disease in a patient in need thereof, comprising administering to the patient Compound 1 :
Figure imgf000202_0001
1
69. The method of claim 68, wherein the primary glomerular disease is selected from focal and segmental glomerulosclerosis, membranous nephropathy, and IgA nephropathy.
70. The method of any one of claims 67-69, wherein the patient has proteinuria.
71. The method of claim 70, wherein the patient is suffering from persistent proteinuria.
72. The method of any one of claims 67-71, wherein the method comprises administering to a patient in need thereof Compound 1 in a daily dose of from about 200 mg to about 600 mg.
73. The method of any one of claims 67-72, wherein the method comprises administering to a patient in need thereof about 200 mg of Compound 1 once daily.
74. The method of any one of claims 67-73, wherein the method comprises administering to a patient in need thereof about 400 mg of Compound 1 once daily.
75. The method of any one of claims 67-74, wherein the method comprises administering to a patient in need thereof about 300 mg of Compound 1 twice daily.
76. The method of any one of claims 67-75, wherein Compound 1 is administered in an amount, which, when administered as a single oral dose to a population of healthy subjects, was established to achieve one or more of:
(i) a mean maximum concentration (Cmax) of from about 30 ng/mL to about 800 ng/mL;
(ii) a median time to maximum concentration (Tmax) of from about 1 hour to about 4 hours;
(iii) a mean area under drug concentration-time curve (AUCo-iast) of from about 60 ng*h/mL to about 4000 ng*h/mL;
(iv) a mean area under drug concentration-time curve (AUCo-inf) of from about 60 ng*h/mL to about 4000 ng*h/mL;
(v) a mean apparent volume of distribution (Vz/F) of from about 2000 L to about 5500 L;
(vi) a mean apparent total clearance (CL/F) of from about 300 L/h to about 600 L/h; and
(vii) a mean half-life (ti/2) of from about 2 hours to about 30 hours.
77. The method of claim 76, wherein the amount was established to achieve a mean half-life (ti/2) of from about 12 hours to about 21 hours.
78. The method of any one of claims 67-77, wherein Compound 1 is administered in an amount, which, when administered as multiple oral doses to a population of healthy subjects, was established to achieve one or more of: (i) a mean maximum concentration (Cmax) of from about 30 ng/mL to about 800 ng/mL;
(ii) a median time to maximum concentration (Tmax) of from about 1 hour to about 2 hours;
(iii) a mean area under drug concentration-time curve (AUCo-12) of from about 100 ng*h/mL to about 1500 ng*h/mL;
(iv) a mean accumulation ratio of Cmax (RaccCmax) of from about 0.6 to about 2.7; and (vi) a mean accumulation ratio of AUC (RaccAUC) of from about 0.8 to about 2.6.
79. The method of any one of claims 67-78, wherein Compound 1 is administered in an amount, which, when administered as a single oral dose to a population of healthy subjects in a fasted state, was established to achieve one or more of:
(i) an increased mean maximum concentration (Cmax);
(ii) a decreased median time to maximum concentration (Tmax);
(iii) a comparable mean area under drug concentration-time curve (AUCo-iast);
(iv) a comparable mean area under drug concentration-time curve (AUCo-inf);
(v) a decreased mean apparent volume of distribution (Vz/F);
(vi) a comparable mean apparent total clearance (CL/F); and
(vii) a decreased mean half-life (ti/2), relative to a comparable population of healthy subjects in a fed state.
80. The method of any one of claims 67-79, wherein Compound 1 is administered in an amount, which, when administered as a single oral dose to a population of healthy subjects in a fasted state, was established to achieve one or more of:
(i) an increased mean maximum concentration (Cmax);
(ii) a decreased median time to maximum concentration (Tmax);
(iii) a comparable mean area under drug concentration-time curve (AUCo-iast);
(iv) a comparable mean area under drug concentration-time curve (AUCo-inf);
(v) a comparable mean apparent volume of distribution (Vz/F);
(vi) a comparable mean apparent total clearance (CL/F); and
(vii) a comparable mean half-life (ti/2), relative to a comparable population of healthy subjects in a fed state.
81. The method of any one of claims 67-80, wherein Compound 1 is administered in an amount, which, when administered to a population of subject suffering from primary glomerular disease and/or persistent proteinuria, was established to achieve one or more of:
(i) a greater mean percent reduction in 24-hour urinary protein excretion;
(ii) a greater mean percent reduction in 24-hour urinary albumin;
(iii) a greater proportion of subject with complete remission in proteinuria;
(iv) a greater proportion of subjects with partial remission in proteinuria;
(v) a greater proportion of subject with complete or partial remission in proteinuria;
(vi) a greater proportion of subjects with at least 50% reduction in 24-hour urinary protein excretion from baseline;
(vii) a greater proportion of subjects with at least 50% reduction in 24-hour urinary albumin excretion from baseline;
(viii) a greater mean percent increase from baseline in creatinine clearance;
(ix) a greater mean increase from baseline in serum albumin;
(x) a greater mean decrease from baseline in fasting triglycerides levels;
(xi) a greater men decrease from baseline in cholesterol levels; and
(xii) a greater mean change in level(s) of one or more plasma or urine biomarkers selected from plasma soluble TNF receptor 2 (sTNFR2), kidney injury molecule- 1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), urinary fibronectin, high molecular weight type IV collagen (HMW collagen IV), monocyte chemotactic protein- 1 (MCP-1), and matrix metalloproteinase-7 (MMP-7), relative to a comparable population of subjects that did not receive Compound 1.
82. The method of any one of claims 67-81, wherein the method further comprises reducing the amount of Compound 1 administered if the patient experiences a gastrointestinal side effect.
83. The method of claim 82, wherein the gastrointestinal side effect is selected from diarrhea, nausea, and vomiting.
84. The method of claim 82 or 83, wherein the amount of Compound 1 is reduced by about 100 mg.
85. The method of any one of claims 82-84, wherein the method further comprises discontinuing administration of Compound 1 if the gastrointestinal side effect persists.
86. The method of any one of claims 67-85, wherein Compound 1 is in a pharmaceutically acceptable salt form.
87. The method of claim 86, wherein the pharmaceutically acceptable salt form is a hydrochloride salt form.
88. The method of claim 87, wherein the pharmaceutically acceptable salt form is a Form A hydrochloride trihydrate.
89. The method of any one of claims 67-88, wherein Compound 1 is administered orally.
90. The method of any one of claims 67-89, wherein Compound 1 is administered in a solid dosage form.
91. The method of any one of claims 67-90, wherein Compound 1 is administered in a capsule dosage form.
92. The method of any one of claims 67-91, wherein Compound 1 is administered in a capsule comprising about 100 mg Compound 1.
93. The method of any one of claims 67-92, wherein Compound 1 is administered in a capsule comprising about 200 mg Compound 1.
94. The method of claim any one of claims 90-93, wherein the capsule comprises Compound 1 with no excipients.
95. The method of any one of claims 67-94, wherein the primary glomerular disease has been confirmed by a renal biopsy.
96. The method of any one of claims 67-95, wherein the patient has proteinuria.
97. The method of any one of claims 67-96, wherein the patient is suffering from persistent proteinuria.
98. The method of any one of claims 67-97, wherein the patient is suffering from membranous nephropathy.
99. The method of any one of claims 67-98, wherein the patient is suffering from IgA nephropathy.
100. The method of any one of claims 67-99, wherein the patient has one or more risk factors for primary glomerular disease.
101. The method of claim 100, wherein the one or more risk factors for primary glomerular disease are selected from proteinuria, renal dysfunction, hypertension, interstitial fibrosis on renal biopsy, and lack of response to therapy or relapse in proteinuria.
102. The method of any one of claims 67-101, wherein the patient has an estimated glomerular filtration rate (eGFR) of greater than or equal to 40 mL/min/1.73m2.
103. The method of any one of claims 67-102, wherein the patient has a urinary protein excretion of greater than or equal to 1 g/day on a 24-hour urine collection.
104. The method of any one of claims 67-103, wherein the patient is receiving or has received standard of care therapy.
105. The method of claim 104, wherein the patient is resistant to standard of care therapy.
106. The method of claim 104 or 105, wherein the patient has relapsed after receiving standard of care therapy.
107. The method of any one of claims 104-106, wherein standard of care therapy comprises an immunosuppressive or immunomodulatory agent and/or one or more of a RAAS blocker, mineralocorticoid receptor inhibitor or SGLT-2 inhibitor.
108. The method of any one of claims 67-107, wherein the patient is receiving or has received an immunosuppressive or immunomodulatory agent.
109. The method of any one of claims 67-108, wherein the patient is receiving or has received a RAAS blocker.
110. The method of any one of claims 67-109, wherein the patient is receiving or has received an ACE inhibitor.
111. The method of any one of claims 67-110, wherein the patient is receiving or has received an angiotensin-receptor blocker.
112. The method of any one of claims 67-111, wherein the patient is receiving or has received a sodium glucose co-transporter-2 (SGLT-2) inhibitor.
113. The method of any one of claims 67-112, wherein the patient is in a fed state.
114. The method of any one of claims 67-113, wherein the patient is in a fasted state.
115. The method of any one of claims 67-114, wherein the patient has not received any other medications within 30 minutes of administration of Compound 1.
PCT/US2021/039899 2020-07-01 2021-06-30 Reducing fibrosis and treating related diseases, disorders, and conditions WO2022006278A1 (en)

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