WO2022165092A1

WO2022165092A1 - Methods for treatment of fibrotic diseases

Info

Publication number: WO2022165092A1
Application number: PCT/US2022/014185
Authority: WO
Inventors: Prakash Narayan
Original assignee: Angion Biomedica Corp.
Priority date: 2021-01-29
Filing date: 2022-01-28
Publication date: 2022-08-04

Abstract

The present disclosure provides biomarkers and methods that are useful in treating, screening, and/or evaluating treatment of fibrotic diseases with 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.

Description

METHODS FOR TREATMENT OF FIBROTIC DISEASES

RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Application Nos. 63/143,630 filed January 29, 2021, 63/143,637 filed January 29, 2021, 63/296,690 filed January 5, 2022, and 63/296,692 filed January 5, 2022, the entire contents of each of which are hereby incorporated by reference.

GOVERNMENT SUPPORT

[0002] This invention was made with U.S. government support under Grant No. PR180780/W81X1H1910448 awarded by the United States Department of Defense. The U.S. government has certain rights in the invention.

BACKGROUND

[0003] 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. L., et al. Sci. Transl. Med. 2019 Jan 9;5(167): 167srl).

[0004] In particular, fibrosis of the kidney can result in progressive loss of renal function, which can lead to end stage renal failure. Renal failure is fatal without regular dialysis or a kidney transplant. There exists a continuing need for improved treatments for fibrotic diseases of the kidney.

SUMMARY

[0005] The present disclosure provides methods related to treatment of fibrotic 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 treatment with Compound 1 :

[0006] The present disclosure is based in part on the recognition 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. In some embodiments, the drivers of their kidney disease correspond with the 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 and/or one or more proteins (or fragments thereof) that are associated with the mechanism of action of Compound 1.

[0007] The present disclosure also encompasses the recognition that certain biomarkers can distinguish patients (e.g., patients with certain symptoms related to a fibrotic disease described herein, e.g., a kidney disease) who are likely to respond to therapy, regardless of disease pathology. For example, patients who present with nephrotic syndrome and/or proteinuria and/or hypoalbuminemia and/or hyperlipidemia and/or edema may be suffering from or susceptible to a variety of different kidney diseases or conditions. In some embodiments, the present disclosure provides methods of identifying and/or selecting among patients with such symptoms those patients who are likely to respond to Compound 1 therapy.

[0008] In another aspect, the present disclosure provides insights that a “signature” comprising levels of a plurality of biomarkers can be useful for methods provided herein (e.g., selecting, identifying and/or characterizing patients who are likely to respond to Compound 1 therapy). For example, in some embodiments, when a patient’s signature comprises levels of certain biomarkers that are different from corresponding threshold levels, that patient is likely to respond to Compound 1. In some embodiments, when a patient’s signature comprises levels of at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% of biomarkers that are altered relative to corresponding threshold levels, that patient is likely to respond to Compound 1. In some embodiments, the plurality of biomarkers that comprise a patient’s signature are selected from those described herein, including classes and subclasses described herein, both singly and in combination. In some embodiments, such methods further comprise administering an effective amount of Compound 1.

[0009] In some embodiments, the present disclosure encompasses the recognition that one or more collagen biomarkers may be useful for methods provided herein (e.g., selecting, identifying and/or characterizing patients who are likely to respond to Compound 1 therapy). For example, in some embodiments, when a patient has been determined to have an elevated level of one or more collagen biomarkers (including, e.g., a collagen 6 biomarker), that patient is likely to respond to Compound 1. In some embodiments, when a patient has been determined to have an elevated level of a collagen 6 biomarker and an altered level of one or more other biomarkers (e.g., a collagen 1 biomarker, a collagen 3 biomarker, and/or a biomarker selected from Table 1), that patient is likely to respond to Compound 1. In some embodiments, such methods further comprise administering an effective amount of Compound 1.

[0010] In some embodiments, the present disclosure provides methods of treating patients diagnosed with, suspected of having, or at risk of a fibrotic disease (e.g., of the kidney), comprising (i) obtaining or determining a level of one or more biomarkers (e.g., biomarkers described herein) in a biological sample obtained from the patients; and (ii) comparing the level with that of a corresponding threshold level. In some embodiments, methods further comprise administering Compound 1 therapy if the level of one or more biomarkers is different from the corresponding threshold level.

[0011] In some embodiments, the present disclosure provides methods of monitoring therapy with Compound 1. For example, in some embodiments, methods are provided comprising (i) administering an effective amount of Compound 1; and (ii) monitoring the levels of one or more biomarkers (e.g., biomarkers described herein). In some embodiments, such methods further comprise adjusting Compound 1 therapy based on the results (e.g., discontinuing Compound 1 therapy, changing dose and/or dosing frequency of Compound 1, etc.).

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] FIG. 1 is a graph showing results of Compound 1 treatment on urine protein in a PAN and uninephrectomy rat model of kidney disease. [0013] FIG. 2A and FIG. 2B are graphs showing change from baseline in urine protein per animal on Study Day 17 (Treatment Day 14).

[0014] FIG. 3A 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. 3B is a graph showing hydroxyproline levels in mice treated with Compound 1 in a bleomycin- induced injury model of IPF. FIG. 3C 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. 3D 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. 3E is a graph showing extent of immunohistological staining for TGFβ1 in lung tissue sections from mice treated with Compound 1 in a bleomycin-induced injury model of IPF.

[0015] FIG. 4A 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 TGFβ1 mouse model of lung fibrosis. FIG. 4B is a graph hydroxyproline levels in mice treated with Compound 1 in an inducible TGFβ1 mouse model of lung fibrosis. FIG. 4C is a graph showing extent of picrosirius red staining in lung tissue sections from mice treated with Compound 1 in an inducible TGFβ1 mouse model of lung fibrosis. FIG. 4D is a graph showing extent of aSMA staining in lung tissue sections from mice treated with Compound 1 in an inducible TGFβ1 mouse model of lung fibrosis.

[0016] FIG. 5A 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. 5B 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. 5C is a graph showing lung hydroxyproline levels from mice treated with Compound 1 in a bleomycin systemic sclerosis mouse model. FIG. 5D 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. 5E is a graph showing kidney hydroxyproline levels from mice treated with Compound 1 in a bleomycin systemic sclerosis mouse model. FIG. 5F 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. 6A is a graph showing urine protein levels in rats treated with Compound 1 in a FSGS-relevant model of PAN-induced proteinuria. FIG. 6B is a graph showing intraperitoneal fluid volume in rats treated with Compound 1 in a FSGS-relevant model of PAN-induced proteinuria. FIG. 6C 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. 6D is a graph showing glomerular diameter, measured using histopathological analysis of PAS- stained renal coronal sections from rats treated with Compound 1 in a FSGS-relevant model of PAN-induced proteinuria. FIG. 6E 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. 7A is a graph showing urine protein levels in rats treated with Compound 1 in a DOCA/salt model of renal fibrosis and injury. FIG. 7B is a graph showing urine albumin levels in rats treated with Compound 1 in a DOCA/salt model of renal fibrosis and injury. FIG. 7C 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. 7D is a graph showing urine kidney injury molecule- 1 (KIMI) levels in rats treated with Compound 1 in a DOCA/salt model of renal fibrosis and injury. FIG. 7E is a graph showing renal hydroxyproline levels in rats treated with Compound 1 in a DOCA/salt model of renal fibrosis and injury. FIG. 7F 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. 7G 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. 7H 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. 8A 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. 8B 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 (UUO) model of renal fibrosis (*** = p <0.001; ns = not significant). FIG. 8C is a graph showing extent of aSMA staining in kidney tissue sections from mice treated with Compound 1 in a unilateral ureteral obstruction (UUO) model of renal fibrosis (*** = p <0.001; ** = p <0.01; ns = not significant).

[0020] FIG. 9A is a graph showing serum creatinine (SCr) levels in rats treated with Compound 1 in a PCK rat model of polycystic kidney disease. FIG. 9B is a graph showing BUN levels in rats treated with Compound 1 in a PCK rat model of polycystic kidney disease. FIG. 9C is a graph showing kidney weight at sacrifice in rats treated with Compound 1 in a PCK rat model of polycystic kidney disease. FIG. 9D is a graph showing kidney weight as a percentage of body weight at sacrifice of rats treated with Compound 1 in a PCK rat model of polycystic kidney disease. FIG. 9E is a graph showing kidney hydroxyproline levels in rats treated with Compound 1 in a PCK rat model of polycystic kidney disease. FIG. 9F 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. 9G is a graph showing 24-hour urine volume in rats treated with Compound 1 in a PCK rat model of polycystic kidney disease. FIG. 9H is a graph showing urine protein levels in rats treated with Compound 1 in a PCK rat model of polycystic kidney disease. FIG. 91 is a graph showing urine albumin levels in rats treated with Compound 1 in a PCK rat model of polycystic kidney disease. FIG. 9J 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. 9K is a graph showing urine KIMI levels in rats treated with Compound 1 in a PCK rat model of polycystic kidney disease. FIG. 9L is a graph showing urine interleukin 18 (IL 18) levels in rats treated with Compound 1 in a PCK rat model of polycystic kidney disease. FIG. 9M 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. 10A 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. 10B 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. 10C 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. 10D is a graph showing colon damage score in mice treated with Compound 1 in a TNBS model of inflammatory bowel disease/acute colitis. FIG. 10E 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. 10F 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. 10G 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. 10H 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. 101 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. 11A is a graph showing body weight in mice prior to treatment with Compound 1 in an acetic acid induced colitis model. FIG. 11B is a graph showing colon weight in mice prior to treatment with Compound 1 in an acetic acid induced colitis model. FIG. 11C is a graph showing colon length in mice prior to treatment with Compound 1 in an acetic acid induced colitis model. FIG. 11D 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. HE is a graph showing colon length in mice treated with Compound 1 in an acetic acid induced colitis model. FIG. 11F is a graph showing gross morphological colon damage score in mice treated with Compound 1 in an acetic acid induced colitis model. FIG. 11G is a graph showing histopathological colon damage score in mice treated with Compound 1 in an acetic acid induced colitis model. FIG. 11H 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. 12A 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. 12B 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. 12C 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. 12D 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. 13A is a graph showing serum pro-collagen 6 levels in animals treated with Compound 1 compared to sham and control animals. FIG. 13B is a graph showing a correlation between serum pro-collagen 6 and TGF-β levels in animals evaluated in this Example 17. FIG. 13C is a graph showing serum pro-collagen 6 levels in animals treated with Compound 1 compared to sham and control animals in the PANX model. FIG. 13D is graph showing serum pro-collagen 6 levels in animals treated with Compound 1 compared to sham and control animals in the DOC A model.

DETAILED DESCRIPTION

Definitions

[0025] 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 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.

[0026] 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 invention. 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. [0027] 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.

[0028] 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. [0029] 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 J. Pharmaceutical Sciences, 66: 1-19 (1977).

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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.

Compound 1

[0034] 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 anti-fibrotic compounds. Such compounds include Compound 1 :

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 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. [0035] 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.

[0036] In some embodiments, Compound 1 is provided and/or utilized (e.g., for inclusion in a composition and/or for delivery to a subject) in accordance with the present disclosure in a form such as 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.

[0037] 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.

[0038] 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 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 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 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 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.

[0039] 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.

Biomarkers

[0040] In some embodiments, the present disclosure provides certain biomarkers that can distinguish subjects (e.g., subjects suffering from or at risk of fibrosis, such as renal fibrosis, or associated diseases, disorders, and conditions) who are more likely than others to respond to therapy with Compound 1. The present disclosure provides the 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. In some embodiments, the drivers of their kidney disease correspond with the mechanism of action of Compound 1. For example, in some embodiments, an altered level (e.g., an expression 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) is indicative of patients who are likely to respond to Compound 1 therapy. 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.

[0041] Generally, as used herein, a biomarker is a component of a biological sample that may be detected and/or quantified when present in the biological sample. 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). In some embodiments, a level of a biomarker corresponds to a level of gene expression (e.g., RNA expression, e.g., mRNA expression). In some certain embodiments, a level of a biomarker corresponds to a level of protein expression, including any fragment or degradation product thereof.

[0042] 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 (B ALF), 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).

[0043] In some embodiments, a biomarker is detected and/or quantified in a tissue sample (e.g., from a biopsy, such as a liver or kidney or lung biopsy) and/or in a biological fluid (e.g., blood, urine, BALF, etc.). In some embodiments, a biomarker (e.g., a level of mRNA) is detected and/or quantified in a tissue sample, e.g., obtained from a biopsy, e.g., a kidney biopsy. In some embodiments, a biomarker (e.g., a level of mRNA) is detected and/or quantified in a kidney tissue sample, e.g., obtained from a kidney biopsy. In some embodiments, a biomarker (e.g., a level of mRNA) is detected and/or quantified in a liver tissue sample, e.g., obtained from a liver biopsy. In some embodiments, a biomarker (e.g., a level of mRNA) is detected and/or quantified in a lung tissue sample, e.g., obtained from a lung biopsy. In some embodiments, a biomarker (e.g., a level of a protein or protein fragment) is detected and/or quantified in a urine sample. In some embodiments, a biomarker (e.g., a level of a protein or protein fragment) is detected and/or quantified in a blood sample. In some embodiments, a biomarker (e.g., a level of a protein or protein fragment) is detected and/or quantified in a BALF sample.

[0044] 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. In some embodiments, a “signature” comprising levels of a plurality of biomarkers is used to characterize subjects.

[0045] It will be appreciated that biomarkers (e.g., genes and/or proteins) identified using non-human animal models can be predictive of biomarkers relevant to treatment of human subjects (e.g., according to methods described herein). For example, a corresponding human analog of a biomarker (e.g., genes and/or proteins) identified using a non-human animal model can be determined; in some embodiments, such corresponding human analogs are useful in the treatment of human subjects as described herein. In some embodiments, a rodent (e.g., rat or mouse) model is used to identify biomarkers expected to be relevant to treatment of human subjects (e.g., according to methods described herein). Non-limiting examples of human analogs that correspond with certain rat biomarkers described herein (e.g., those in Table 1, Table 2, Table 3, and/or Table 5) are provided in Table 1 A. In some embodiments, a biomarker described herein is selected from the human analogs in Table 1 A. In some embodiments, a human analog described herein is the corresponding human analog listed in Table 1 A.

Table 1A.

[0046] It will also be appreciated that biomarkers (e.g., genes and/or proteins) identified using a disease model for fibrosis of one organ may be predictive of biomarkers relevant to treatment of fibrotic diseases of other organs. For example, biomarkers identified using a model of a kidney fibrotic disease may be predictive of biomarkers relevant to treatment of fibrotic diseases of, for example, the lung, liver, and/or skin.

[0047] In some embodiments, one or more biomarkers are differentially present in a sample taken from a subject of one status as compared with a subject of another status (e.g., more responsive to Compound 1 therapy vs less responsive to Compound 1 therapy). In some embodiments, one or more biomarkers are 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.

[0048] In some embodiments, detection of levels 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 obtained from a subject are compared to a threshold level. In some embodiments, a biomarker is considered altered if the level is altered relative to a threshold level (e.g., altered by at least about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, or about 20-fold, or more). In some embodiments, an altered biomarker is elevated relative to a threshold level (e.g., elevated by at least about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, or about 20-fold, or more). In some embodiments, an altered biomarker is reduced relative to a threshold level (e.g., reduced by at least about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, or about 20-fold, or more). In some embodiments, a biomarker is considered altered if the level is altered relative to a threshold level (e.g., altered by at least 0.5, about 1.0, about 1.5, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, or about 5.0, or more standard deviations). In some embodiments, an altered biomarker is elevated relative to a threshold level (e.g., elevated by at least about 0.5, about 1.0, about 1.5, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, or about 5.0, or more standard deviations). In some embodiments, an altered biomarker is reduced relative to a threshold level (e.g., reduced by at least about 0.5, about 1.0, about 1.5, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, or about 5.0, or more standard deviations). 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).

[0049] 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). 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).

[0050] In some embodiments, 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. 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).

[0051] In some embodiments, a level of a biomarker corresponds to a level of protein, including 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 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”).

[0052] In some embodiments, a biomarker useful in methods provided herein is a biomarker selected from Table 1 (e.g., gene expression of and/or level of protein produced by a gene selected from Table 1), or a human analog thereof (e.g., a human analog selected from Table 1 A). In some embodiments, a level of a biomarker selected from Table l is a level of gene expression of a gene selected from Table 1 or a human analog thereof (e.g., a human analog selected from Table 1 A). Table 1.

[0053] In some embodiments, patients are selected and/or characterized based on an elevated level of one or more biomarkers selected from Table 2 or a human analog thereof (e.g., a human analog selected from Table 1 A) and/or a reduced level of one or more biomarkers selected from Table 3 or a human analog thereof (e.g., a human analog selected from Table 1 A). Table 2.

Table 3.

[0054] In some embodiments, a biomarker useful in methods provided herein is selected from Table 5 or a human analog thereof (e.g., a human analog selected from Table 1 A) (see Example 2 below). In some embodiments, a biomarker useful in methods provided herein is a biomarker selected from Table 5, or a human analog thereof, with a change in mean expression for sham animals relative to PANX animals of at least about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, or about 20-fold. In some embodiments, a biomarker useful in methods provided herein is a biomarker selected from Table 5, or a human analog thereof, with a change in mean expression for sham animals relative to PANX animals of at least about 0.5, about 1.0, about 1.5, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, or about 5.0 standard deviations. In some embodiments, a biomarker useful in methods provided herein is a biomarker selected from Table 5, or a human analog thereof, with a change in mean expression for PANX animals relative to PANX+Compound 1 animals of at least about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, or about 20-fold. In some embodiments, a biomarker useful in methods provided herein is a biomarker selected from Table 5, or a human analog thereof, with a change in mean expression for PANX animals relative to PANX+Compound 1 animals of at least about 0.5, about 1.0, about 1.5, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, or about 5.0 standard deviations. In some embodiments, a biomarker useful in methods provided herein is a biomarker selected from Table 5, or a human analog thereof, with a change in mean expression for sham animals relative to PANX+Compound 1 animals of less than about 100%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, or about 10%. In some embodiments, a biomarker useful in methods provided herein is a biomarker selected from Table 5, or a human analog thereof, with a change in mean expression for sham animals relative to PANX+Compound 1 animals of less than about 0.5, about 1.0, about 1.5, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, or about 5.0 standard deviations.

[0055] In some embodiments, a biomarker useful in methods provided herein is a biomarker selected from Table 5, or a human analog thereof, with: a change in mean expression for sham animals relative to PANX animals of at least about 1.5- fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, or about 20-fold; and/or a change in mean expression for PANX animals relative to PANX+Compound 1 animals of at least about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, or about 20-fold; and/or a change in mean expression for sham animals relative to PANX+Compound 1 animals of less than about 100%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, or about 10%.

[0056] In some embodiments, a biomarker useful in methods provided herein is a biomarker selected from Table 5, or a human analog thereof, with: a change in mean expression for sham animals relative to PANX animals of at least about 0.5, about 1.0, about 1.5, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, or about 5.0 standard deviations; and/or a change in mean expression for PANX animals relative to PANX+Compound 1 animals of at least about 0.5, about 1.0, about 1.5, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, or about 5.0 standard deviations; and/or a change in mean expression for sham animals relative to PANX+Compound 1 animals of less than about 0.5, about 1.0, about 1.5, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, or about 5.0 standard deviations.

[0057] In some embodiments, the present disclosure provides insights that altered levels of one or more biomarkers selected from Table 1 and/or Table 2 and/or Table 3 and/or Table 5, or a human analog thereof, may be useful in selecting and/or characterizing patients for Compound 1 therapy.

[0058] In some embodiments, patients are selected and/or characterized based on the percentage of biomarkers with altered levels observed in a biological sample obtained from the patient. For example, in some embodiments, a patient has been determined to have an altered level of at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% of the biomarkers in Table 1, or a human analog thereof. In some embodiments, a patient has been determined to have an elevated level of at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% of the biomarkers in Table 2, or a human analog thereof. In some embodiments, a patient has been determined to have a reduced level of at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% of the biomarkers in Table 3, or a human analog thereof. In some embodiments, a patient has been determined to have an elevated level of at least one biomarker in Table 2, or a human analog thereof, and a reduced level of at least one biomarker in Table 3, or a human analog thereof. In some embodiments, a patient has been determined to have an elevated level of at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% of the biomarkers in Table 2, or a human analog thereof, and a reduced level of at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% of the biomarkers in Table 3, or a human analog thereof.

[0059] In some embodiments, a patient has been determined to have an altered level of at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% of the biomarkers in Table 5 or a human analog thereof. In some embodiments, a patient has been determined to have an altered level of at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% of the biomarkers in Table 5, or a human analog thereof, with a change in mean expression for sham animals relative to PANX animals of at least about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, or about 20-fold. In some embodiments, a patient has been determined to have an altered level of at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% of the biomarkers in Table 5, or a human analog thereof, with a change in mean expression for sham animals relative to PANX animals of at least about 0.5, about 1.0, about 1.5, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, or about 5.0 standard deviations. In some embodiments, a patient has been determined to have an altered level of at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% of the biomarkers in Table 5, or a human analog thereof, with a change in mean expression for PANX animals relative to PANX+Compound 1 animals of at least about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, or about 20-fold. In some embodiments, a patient has been determined to have an altered level of at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% of the biomarkers in Table 5, or a human analog thereof, with a change in mean expression for PANX animals relative to PANX+Compound 1 animals of at least about 0.5, about 1.0, about 1.5, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, or about 5.0 standard deviations. In some embodiments, a patient has been determined to have an altered level of at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% of the biomarkers in Table 5, or a human analog thereof, with a change in mean expression for sham animals relative to PANX+Compound 1 animals of less than about 100%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, or about 10%. In some embodiments, a patient has been determined to have an altered level of at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% of the biomarkers in Table 5, or a human analog thereof, with a change in mean expression for sham animals relative to PANX+Compound 1 animals of less than about 0.5, about 1.0, about 1.5, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, or about 5.0 standard deviations.

[0060] In some embodiments, a patient has been determined to have an altered level of at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% of the biomarkers in Table 5, or a human analog thereof, with: a change in mean expression for sham animals relative to PANX animals of at least about 1.5- fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, or about 20-fold; and/or a change in mean expression for PANX animals relative to PANX+Compound 1 animals of at least about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, or about 20-fold; and/or a change in mean expression for sham animals relative to PANX+Compound 1 animals of less than about 100%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, or about 10%.

[0061] In some embodiments, a patient has been determined to have an altered level of at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% of the biomarkers in Table 5, or a human analog thereof, with: a change in mean expression for sham animals relative to PANX animals of at least about 0.5, about 1.0, about 1.5, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, or about 5.0 standard deviations; and/or a change in mean expression for PANX animals relative to PANX+Compound 1 animals of at least about 0.5, about 1.0, about 1.5, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, or about 5.0 standard deviations; and/or a change in mean expression for sham animals relative to PANX+Compound 1 animals of less than about 0.5, about 1.0, about 1.5, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, or about 5.0 standard deviations.

[0062] In some embodiments, a patient has been determined to have an altered level of at least one biomarker selected from Table 5, or a human analog thereof, with a change in mean expression for sham animals relative to PANX animals of at least about 1.5-fold, about 2-fold, or about 3-fold and at least one biomarker selected from Table 5, or a human analog thereof, with a change in mean expression for sham animals relative to PANX animals of at least about 4-fold, about 5-fold, about 10-fold, or about 20-fold. In some embodiments, a patient has been determined to have an altered level of at least one biomarker selected from Table 5, or a human analog thereof, with a change in mean expression for sham animals relative to PANX animals of at least about 0.5, about 1.0, about 1.5, about 2.0, or about 2.5 standard deviations and at least one biomarker selected from Table 5, or a human analog thereof, with a change in mean expression for sham animals relative to PANX animals of at least about 3.0, about 3.5, about 4.0, about 4.5, or about 5.0 standard deviations.

[0063] In some embodiments, a patient has been determined to have an altered level of at least one biomarker selected from Table 5, or a human analog thereof, with a change in mean expression for PANX animals relative to PANX+Compound 1 animals of at least about 1.5-fold, about 2-fold, or about 3-fold and at least one biomarker in Table 5, or a human analog thereof, with a change in mean expression for PANX animals relative to PANX+Compound 1 animals of at least about 4-fold, about 5-fold, about 10-fold, or about 20-fold. In some embodiments, a patient has been determined to have an altered level of at least one biomarker selected from Table 5, or a human analog thereof, with a change in mean expression for PANX animals relative to PANX+Compound 1 animals of at least about 0.5, about 1.0, about 1.5, about 2.0, or about 2.5 standard deviations and at least one biomarker selected from Table 5, or a human analog thereof, with a change in mean expression for PANX animals relative to PANX+Compound 1 animals of at least about 3.0, about 3.5, about 4.0, about 4.5, or about 5.0 standard deviations.

[0064] In some embodiments, a patient has been determined to have an altered level of at least one biomarker selected from Table 5, or a human analog thereof, with a change in mean expression for sham animals relative to PANX+Compound 1 animals of less than about 100%, about 90%, about 80%, about 70%, or about 60% and at least one biomarker selected from Table 5, or a human analog thereof, with a change in mean expression for sham animals relative to PANX+Compound 1 animals of less than about 50%, about 40%, about 30%, about 20%, or about 10%. In some embodiments, a patient has been determined to have an altered level of at least one biomarker selected from Table 5, or a human analog thereof, a change in mean expression for sham animals relative to PANX+Compound 1 animals of less than about 0.5, about 1.0, about 1.5, about 2.0, or about 2.5 standard deviations and at least one biomarker selected from Table 5, or a human analog thereof, with a change in mean expression for sham animals relative to PANX+Compound 1 animals of less than about 3.0, about 3.5, about 4.0, about 4.5, or about 5.0 standard deviations.

[0065] It will be appreciated that a “change in mean expression” between, e.g., sham animals relative to PANX animals or PANX animals relative to PANX+Compound 1 animals or sham animals relative to PANX+Compound 1 animals, in Table 5 refers to a comparison of a mean expression value in Table 5 with another mean expression value in Table 5. For example, a biomarker with a change in mean expression for sham animals relative to PANX animals of at least about 2-fold refers to a biomarker with mean expression for PANX animals that is at least about 2-fold higher or lower than mean expression for sham animals. As another example, a biomarker with a change in mean expression for sham animals relative to PANX+Compound 1 animals of less than about 50% refers to a biomarker with a mean expression for PANX+Compound 1 animals that is less than 50% higher or lower than (i.e., within 50% of) mean expression for sham animals.

[0066] In some embodiments, a patient is determined to have an altered level of at least two biomarkers (e.g., those selected from Table 1 and/or Table 2 and/or Table 3 and/or Table 5) which are part of the same biochemical pathway. Non-limiting examples of biochemical pathways common to at least two biomarkers listed in Table 1 include: glycosphingolipid metabolic process, cytokine biosynthetic process, lysosome organization, regulation of transcription, protein localization, negative regulation of cell migration, JAK/STAT cascade, regulation of protein secretion, keratinocyte differentiation, embryonic morphogenesis, cell development, actin filament polymerization, positive regulation of lymphocyte activation, organ development, intrinsic apoptotic signaling, carboxylic acid metabolic process, growth, chromosome segregation, catabolic process, hemostasis, regulation of cytoskeleton organization, mitotic spindle organization, regulated secretory pathway, regulation of synapse structure and activity, viral infectious cycle, positive regulation of sequence specific DNA binding transcription factor activity, regulation of cellular protein metabolic process, cell cycle checkpoint, tissue remodeling, inflammatory response, tube development, microtubule polymerization or depolymerization, meiosis I, chromatin assembly, embryo implantation, regulation of cell migration, synapse organization, cytoskeleton organization, apoptotic mitochondrial changes, histone modification, acute inflammatory response, actin filament-based movement, protein processing, positive regulation of protein phosphorylation, monocarboxylic acid metabolic process, signal transduction in response to DNA damage, interleukin 2 production, negative regulation of hydrolase activity, body fluid secretion, regulation of cell proliferation, regulation of developmental process, negative regulation of secretion, defense response, microtubule cytoskeleton organization, cell-cell junction assembly, regulation of DNA replication, neuron projection development, monovalent inorganic cation transport, cell division, regulation of transcription from RNA polymerase II promoter, DNA-dependent DNA replication, heme metabolic process, amyloid precursor protein metabolic process, negative regulation of apoptotic process, protein O-linked glycosylation, regulation of protein stability, protein catabolic process, and cellular response to stress.

[0067] In some embodiments, the present disclosure encompasses the recognition that levels of one or more urinary and/or circulating biomarkers may be indicative of and/or correlated with levels of one or more biomarkers described herein, e.g., those selected from Table 1 and/or Table 2 and/or Table 3 and/or Table 5. In some embodiments, such urinary and/or circulating biomarkers may be used in provided methods, e.g., to select and/or characterize patients for Compound 1 therapy.

[0068] In some embodiments, the present disclosure encompasses the recognition that a collagen 6 biomarker is useful in methods provided herein. Collagen 6 is a member of the collagen family, primarily present in the extracellular matrix and reported to perform both mechanical and cytoprotective roles. See Cescon, M., et al., J. Cell Sci. (2015) 128 (19):3525- 3531. A collagen 6 biomarker can be a level of gene expression (e.g., RNA, e.g., mRNA) or a level of protein expression (e.g., a level of collagen 6 protein and/or a corresponding procollagen polypeptide, collagen protein, preprocollagen polypeptide, amino-terminal procollagen polypeptide, collagen fibril, collagen fiber, and/or any fragment or degradation product of any thereof). For example, in some embodiments, a collagen 6 biomarker can be a level of collagen 6, pro-collagen 6, and/or endotrophin, as well as fragments and/or degradation products thereof. In particular, the pro-peptide of the alpha-3 chain of collagen 6 has a sequence overlap with endotrophin (see Rasmussen, D. G. K., et al., Scientific Reports, 7, 2017, 17328); as such, in some embodiments, a collagen 6 biomarker may be or comprise an endotrophin level.

[0069] In some embodiments, the present disclosure encompasses the recognition that a collagen 1 and/or collagen 3 biomarker is useful in methods provided herein, e.g., in combination with one or more other biomarkers described herein. A collagen 1 biomarker can include a level of gene expression (e.g., COL1 Al gene expression) or a level of protein expression (e.g., a level of collagen 1 protein and/or a corresponding procollagen polypeptide, collagen protein, preprocollagen polypeptide, amino-terminal procollagen polypeptide, collagen fibril, collagen fiber, and/or any fragment or degradation product of any thereof). A collagen 3 biomarker can include a level of gene expression (e.g., COL3A1 gene expression) or a level of protein expression (e.g., a level of collagen 3 protein and/or a corresponding procollagen polypeptide, collagen protein, preprocollagen polypeptide, amino-terminal procollagen polypeptide, collagen fibril, collagen fiber, and/or any fragment or degradation product of any thereof). For example, in some embodiments, the present disclosure encompasses the recognition that a combination of a collagen 6 biomarker with a collagen 1 and/or collagen 3 biomarker may be useful in selecting and/or characterizing patients for Compound 1 therapy. In some embodiments, a combination of a collagen 6 biomarker with one or more biomarkers selected from Table 1, Table 2, Table 3, and/or Table 5 may be useful in selecting and/or characterizing patients for Compound 1 therapy. [0070] In some embodiments, a patient is determined to have an altered level of a biomarker when the level of the biomarker is above or below a threshold level (e.g., a predetermined median or mean level). In some embodiments, a patient is determined to have an altered level of a biomarker when the level of the biomarker is different from a threshold level by at least about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, or about 20-fold, or more. In some embodiments, a patient is determined to have an altered level of a biomarker when the level of the biomarker is different from a threshold level by at least about 0.5, about 1.0, about 1.5, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, or about 5.0, or more standard deviations. [0071] In some embodiments, a patient is determined to have an elevated level of a biomarker when the level of the biomarker is above a threshold level (e.g., a predetermined median or mean level). In some embodiments, a patient is determined to have an elevated level of a biomarker when the level of the biomarker is above a threshold level by at least about 1.5- fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, or about 20-fold, or more. In some embodiments, a patient is determined to have an elevated level of a biomarker when the level of the biomarker is above a threshold level by at least about 0.5, about 1.0, about 1.5, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, or about 5.0, or more standard deviations.

[0072] In some embodiments, a patient is determined to have a reduced level of a biomarker when the level of the biomarker is below a threshold level (e.g., a predetermined median or mean level). In some embodiments, a patient is determined to have a reduced level of a biomarker when the level of the biomarker is below a threshold level by at least about 1.5-fold, about 2- fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, or about 20-fold, or more. In some embodiments, a patient is determined to have a reduced level of a biomarker when the level of the biomarker is below a threshold level by at least about 0.5, about 1.0, about 1.5, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, or about 5.0, or more standard deviations. [0073] In some embodiments, the present disclosure provides methods of identifying biomarkers useful for selecting, identifying, and/or characterizing patients likely to benefit from a treatment with Compound 1. For example, in some embodiments, biomarkers are identified based on a mean change across a population of subjects administered Compound 1 relative to a comparable reference population (e.g., as described in Example 2 or 17). In some embodiments, biomarkers useful in methods provided herein are biomarkers that have been established to have a mean increase or decrease of at least about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, or about 20-fold, or more in a population of subjects administered Compound 1 relative to a comparable reference population. In some embodiments, biomarkers useful in methods provided herein are biomarkers that have been established to have a mean increase or decrease of at least about 0.5, about 1.0, about 1.5, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, or about 5.0, or more standard deviations in a population of subjects administered Compound 1 relative to a comparable reference population. In some embodiments, a population of subjects is a population of human subjects. In some embodiments, a population of subjects is a population of non-human animal subjects (e.g., rodent subjects). In some embodiments, a reference population has not received Compound 1. In some embodiments, a reference population has received an otherwise comparable composition that does not provide Compound 1 (e.g., a placebo).

[0074] Alternatively or additionally, in some embodiments, biomarkers are identified based on a mean change across a population of subjects with confirmed fibrotic disease (e.g., confirmed fibrotic disease of the kidney) relative to a population of healthy volunteers (e.g., as described in Example 2 or 17). In some embodiments, biomarkers useful in methods provided herein are biomarkers that have been established to have a mean increase or decrease of at least about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, or about 20- fold, or more in a population of subjects with confirmed fibrotic disease (e.g., confirmed fibrotic disease of the kidney) relative to a population of healthy volunteers. In some embodiments, biomarkers useful in methods provided herein are biomarkers that have been established to have a mean increase or decrease of at least about 0.5, about 1.0, about 1.5, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, or about 5.0, or more standard deviations in a population of subjects with confirmed fibrotic disease (e.g., confirmed fibrotic disease of the kidney) relative to a population of healthy volunteers. In some embodiments, a population of subjects is a population of human subjects. In some embodiments, a population of subjects is a population of non-human animal subjects (e.g., rodent subjects).

Provided Methods

[0075] 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.

[0076] 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.). [0077] In some embodiments, provided methods relate to treatment of fibrotic disease(s) (e.g., fibrotic diseases of the kidney) and selecting, identifying, and/or characterizing patients likely to benefit from a treatment with Compound 1.

[0078] The present disclosure is based in part on the recognition 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. In some embodiments, drivers of their kidney disease correspond with the mechanism of action of Compound 1. 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 proteins that are part of the mechanism of action of Compound 1.

[0079] In some embodiments, the present disclosure provides a method of treating a patient diagnosed with, suspected of having, or at risk of a fibrotic disease (e.g., a fibrotic disease described herein, such as a fibrotic disease of the kidney), comprising administering an effective amount of Compound 1 to a patient that has been determined to have an altered level of one or more biomarkers described herein.

[0080] In some embodiments, the present disclosure provides a method of treating a fibrotic disease (e.g., a fibrotic disease described herein, such as a fibrotic disease of the kidney) in a patient characterized by an altered level of one or more biomarkers described herein, comprising administering an effective amount of Compound 1 to the patient.

[0081] In some embodiments, the present disclosure provides a method comprising administering an effective amount of Compound 1 to a patient that has been determined to have (i) at least one symptom selected from proteinuria and/or hypoalbuminemia and/or hyperlipidemia and/or edema; and (ii) an altered level of one or more biomarkers described herein.

[0082] In some embodiments, the present disclosure provides a method comprising administering an effective amount of Compound 1 to a patient that has been determined to have (i) nephrotic syndrome; and (ii) an altered level of one or more biomarkers described herein. [0083] In some embodiments, the present disclosure provides a method comprising administering an effective amount of Compound 1 to a patient in need thereof, wherein the patient has been determined to have an altered level of one or more biomarkers described herein. [0084] In some embodiments, one or more biomarkers are selected from Table 1 and/or Table 2 and/or Table 3 and/or Table 5, or a human analog thereof (e.g., a human analog selected from Table 1 A), including any classes and subclasses thereof as described herein, both singly and in combination. In some embodiments, one or more biomarkers comprise a collagen 6 biomarker. In some embodiments, one or more biomarkers comprise a collagen 1 biomarker or a collagen 3 biomarker. In some embodiments, one or more biomarkers are selected from a collagen 6 biomarker, a collagen 1 biomarker, a collagen 3 biomarker, and a biomarker in Table 1 and/or Table 2 and/or Table 3 and/or Table 5, or a human analog thereof (e.g., a human analog selected from Table 1 A), including any classes and subclasses thereof as described herein, both singly and in combination. In some embodiments, a patient has been determined to have an altered level of at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% of the biomarkers in Table 1 and/or Table 2 and/or Table 3 and/or Table 5, or a human analog thereof (e.g., a human analog selected from Table 1 A), including any classes and subclasses thereof as described herein, both singly and in combination. In some embodiments, a patient has been determined to have an altered level of a collagen 6 biomarker. In some embodiments, a patient has been determined to have an altered level of a collagen 1 and/or collagen 3 biomarker. In some embodiments, an altered level of a biomarker is a level that is different from (e.g., at least about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, or about 20-fold, or more, above or below) a corresponding threshold level.

[0085] In some embodiments, one or more biomarkers are selected from biomarkers identified using a method described herein. In some embodiments, one or more biomarkers are selected from biomarkers whose levels have been established to have a mean increase or decrease of at least about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, or about 20-fold, or more in a population of subjects administered Compound 1 relative to a comparable reference population. In some embodiments, one or more biomarkers are selected from biomarkers whose levels have been established to have a mean increase or decrease of at least about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, or about 20-fold, or more in a population of subjects with confirmed fibrotic disease (e.g., fibrotic disease of the kidney) relative to a population of healthy volunteers. [0086] In some embodiments, the present disclosure provides a method of 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), comprising (i) obtaining or determining a level of one or more biomarkers described herein in a biological sample obtained from the patient; and (ii) comparing the determined level(s) to a corresponding threshold level. In some embodiments, a method further comprises performing an assay on a biological sample obtained from the patient to determine level(s) of one or more biomarkers. In some embodiments, if the level of one or more biomarkers is different from the corresponding threshold level, then Compound 1 is administered to the patient. In some embodiments, if the level of one or more biomarkers is comparable to (e.g., is not different from) the corresponding threshold level, then Compound 1 is not administered to the subject.

[0087] 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 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 increases or decreases a level of one or more biomarkers (i.e., such that the level of the one or more biomarkers is less different from a threshold level than prior to treatment with Compound 1). In some embodiments, treatment of a patient with Compound 1 decreases a level of one or more biomarkers that was elevated prior to treatment with Compound 1 (e.g., one or more biomarkers selected from Table 2 or a human analog thereof, and/or a collagen 6, collagen 1, or collagen 3 biomarker). In some embodiments, treatment of a patient with Compound 1 increases a level of one or more biomarkers that was reduced prior to treatment with Compound 1 (e.g., one or more biomarkers selected from Table 3 or a human analog thereof).

[0088] 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). 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 the 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.

[0089] In some embodiments, provided methods 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 (i) administering an effective amount of Compound 1 to a patient characterized by an altered level of one or more biomarkers described herein; and (ii) monitoring levels of the one or more biomarkers, e.g., over a period of time. In some embodiments, if the level of one or more biomarkers remains altered in the patient, then therapy with Compound 1 is discontinued. In some embodiments, if the level of one or more biomarkers remains altered in the patient, then the dose and/or dosing frequency of Compound 1 is increased.

[0090] In some embodiments, the present disclosure provides a method comprising determining levels of one or more biomarkers described herein in each of a plurality of biological samples obtained at different time points from a single patient; and comparing the determined levels from a first time point with those from at least one later time point. In some embodiments, the present disclosure provides a method comprising determining levels of one or more biomarkers described herein from a biological sample obtained from a subject for whom levels of the one or more biomarkers have previously been obtained at least once; and comparing the determined levels with the previously obtained levels. In some embodiments, a first time point and one or more later time points are separated from one another by a reasonably consistent interval. 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).

[0091] In some embodiments, a meaningful change in a determined level over time indicates a change in the subject’s status. In some embodiments, a meaningful change in a determined level over time is a change (e.g., an increase or a decrease) of at least about 1.5-fold, about 2- fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, or about 20-fold, or more compared to a threshold level. In some embodiments, a meaningful 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.

[0092] 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 is or was obtained from a subject prior to administration of Compound 1, and a sample from a second time point is or was obtained from the subject after administration of Compound 1. In some such embodiments, if the levels of one or more biomarkers are no longer altered and/or are altered to a lesser degree in a later sample compared to a first sample, then Compound 1 therapy is continued. In some such embodiments, if the levels of one or more biomarkers remain altered in a later sample compared to a first sample, then Compound 1 therapy is discontinued, or dosage amount and/or dosing frequency of Compound 1 therapy is increased. [0093] In some embodiments, the present disclosure provides a method for treatment with Compound 1 that includes: (i) receiving a report listing the level of one or more biomarkers (e.g., one or more biomarkers described herein) for a patient with a fibrotic disease (e.g., a fibrotic kidney disease) and/or a symptom of a fibrotic disease (e.g., proteinuria); (ii) receiving a request for reimbursement of the screening and/or of a particular therapeutic regimen; and (iii) approving payment and/or reimbursement for treatment with Compound 1 therapy if the report indicates the level of one or more biomarkers is above a threshold level.

[0094] 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.

[0095] 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). 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.

Pulmonary Diseases, Disorders, and Conditions

[0096] In some embodiments, provided methods are useful for treating pulmonary diseases, disorders, and 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).

[0097] 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 a 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.

[0098] 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, et al. The Lancet. 2020 Jan 24).

[0099] 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.

[0100] 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).

[0101] 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-β1), 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

[0102] 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).

[0103] 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, endstage 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).

[0104] 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 nonalcoholic fatty liver disease have no effective treatment other than liver transplantation.

[0105] 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

[0106] In some embodiments, provided methods are useful for treating renal diseases, disorders, and conditions. In some embodiments, provided methods are useful for reducing fibrosis of the kidney in a subject in need thereof. In some embodiments, provided methods are useful for treating a kidney 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 kidney therapies. It will be appreciated that provided methods may be suitable for treating kidney 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).

[0107] In some embodiments, provided methods are useful for treating acute injuries (e.g., acute organ injuries, such as acute kidney injury), as well as for treating chronic injuries (e.g., 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. 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.

[0108] 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 glomerulonephritides (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., autosomal dominant 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), 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. 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).

[0109] 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.

[0110] Currently, there is a need for 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 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(l l):2535-42).

[OHl] 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.

[0112] 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 mmHg), and HMG-CoA reductase inhibitors (e.g., statins) in patients with hyperlipidemia. The inhibitors of the mineralocorticoid receptor and sodium glucose cotransporter-2 (SGLT-2) are increasingly being used in these patients as well.

[0113] In some embodiments, provided methods are useful for treating primary glomerular diseases (e.g., FSGS, membranous nephropathy, or IgA nephropathy) and persistent proteinuria. [0114] 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. Pediatr. 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).

[0115] 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 endstage 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).

[0116] 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. [0117] In some embodiments, a kidney disease to be treated by methods of the present disclosure is membranous glomerulonephritis (MG or MGN). MG 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.

[0118] In some embodiments, a kidney disease to be treated by methods of the present disclosure is anti -neutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis. 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.

[0119] 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 an 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.

[0120] In some embodiments, a kidney disease to be treated by methods of the present disclosure is anti-globular basement membrane (anti-GBM) nephropathy. 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.

[0121] 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. [0122] 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.

[0123] 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). Caroli’s disease 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 often driven by both progressive organ dysfunction and by significant enlargement of the diseased organ(s), and is accompanied by severe pain (www.arpkdchf.org). [0124] In some embodiments, a kidney disease to be treated by methods of the present disclosure is or comprises 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).

[0125] 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.

[0126] 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.

[0127] 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, H., 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 1); 1358-66.

Dermal Diseases, Disorders, and Conditions

[0128] 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).

[0129] 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).

[0130] 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.

[0131] 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).

[0132] 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). TGFβ1, 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 TGFβ1 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).

[0133] 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.

[0134] 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 Furst D.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

[0135] 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.

[0136] 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 to any part of 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).

[0137] 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). [0138] 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 cure.

[0139] 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 TNFα, and activate fibroblasts via TGFβ1. 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).

[0140] 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; 11(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; 1 l(6):e0158017; Cosnes, J., et al. Gut 2005;54:237-241). [0141] 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 l;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).

[0142] Studies indicate that angiogenesis as 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

[0143] 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.

[0144] 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.

[0145] 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.

[0146] 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.

[0147] 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

[0148] 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 an altered level of one or more biomarkers, etc.

[0149] 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 an assessment of one or more biomarkers described herein). 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). [0150] 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.

[0151] 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.

[0152] 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.

[0153] In some embodiments, a subject or population thereof is suffering from or is susceptible to a pulmonary disease as described herein. 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.

[0154] In some embodiments, a subject or population thereof is suffering from or is susceptible to a hepatic disease as described herein. 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). [0155] In some embodiments, a subject or population thereof is suffering from or is susceptible to a kidney disease as described herein. In some embodiments, a subject or population thereof is suffering from or is susceptible to fibrotic disease of the kidney as described herein. 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 disease of the kidney as described herein.

[0156] In some embodiments, a subject or population thereof is suffering from or is susceptible to an acute kidney injury. In some embodiments, a subject or population thereof is suffering from or is susceptible to a 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.

[0157] 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 glomerulonephritides (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), 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 (FSGS). In some embodiments, a subject or population thereof is suffering from or is susceptible to collagen type III glomerulopathy. In some embodiments, a subject or population thereof is suffering from or is susceptible to nailpatella syndrome. 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). In some embodiments, a subject or population thereof is suffering from or is susceptible to renal fibrosis and has an altered level of one or more biomarkers (e.g., an altered level of one or more biomarkers described herein).

[0158] 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.

[0159] In some embodiments, a subject or population thereof has one or more symptoms selected from proteinuria, hypoalbuminemia, hyperlipidemia, and edema. In some embodiments, a subject or population thereof has proteinuria. In some embodiments, a subject or population thereof has hypoalbuminemia. In some embodiments, a subject or population thereof has hyperlipidemia. In some embodiments, a subject or population thereof has edema.

[0160] In some embodiments, a subject or population thereof is suffering from or is susceptible to a dermal disease as described herein. 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). [0161] In some embodiments, a subject or population thereof is suffering from or is susceptible to a gastrointestinal disease as described herein. 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).

[0162] 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.

[0163] 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. [0164] 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.

[0165] 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.

Administration

[0166] A composition providing Compound 1, as described herein, can be administered to subjects in accordance with methods provided herein.

[0167] 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 a 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.

[0168] 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.

[0169] 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, Compound 1 is administered as one or more oral unit dosage forms. In some embodiments, a composition providing Compound 1 is an immediate release solid unit dosage form. [0170] In some embodiments, a composition providing Compound 1 is a capsule. In some embodiments, a composition providing Compound l is a tablet.

[0171] 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).

[0172] 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.

[0173] 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 (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 (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.

[0174] 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.

[0175] 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).

[0176] 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.

[0177] 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 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.

[0178] 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.

[0179] 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.

[0180] 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.

[0181] 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). Exemplary Embodiments

[0182] The following numbered embodiments, while non-limiting, are exemplary of certain aspects of the disclosure:

1. A method comprising administering an effective amount of Compound 1 :

to a patient that has been determined to have an altered level of one or more biomarkers selected from a collagen 6 biomarker and/or Table 1 or a human analog thereof.

2. A method of treating a fibrotic disease of the kidney, the method comprising administering an effective amount of Compound 1 :

3. A method comprising administering an effective amount of Compound 1 :

to a patient that has been determined to have (i) proteinuria and/or hypoalbuminemia and/or hyperlipidemia and/or edema; and (ii) an altered level of one or more biomarkers selected from a collagen 6 biomarker and/or Table 1 or a human analog thereof.

4. A method comprising administering an effective amount of Compound 1 :

to a patient that has been determined to have (i) nephrotic syndrome; and (ii) an altered level of one or more biomarkers selected from a collagen 6 biomarker and/or Table 1 or a human analog thereof.

5. A method comprising administering an effective amount of Compound 1 :

to a patient that has been determined to have an altered level of one or more biomarkers selected from Table 1 or a human analog thereof.

6. A method of treating a fibrotic disease, the method comprising administering an effective amount of Compound 1 :

7. A method comprising administering an effective amount of Compound 1 :

to a patient that has been determined to have (i) proteinuria and/or hypoalbuminemia and/or hyperlipidemia and/or edema; and (ii) an altered level of one or more biomarkers selected from Table 1 or a human analog thereof.

8. A method comprising administering an effective amount of Compound 1 :

to a patient that has been determined to have (i) nephrotic syndrome; and (ii) an altered level of one or more biomarkers selected from Table 1 or a human analog thereof.

9. The method of any one of embodiments 1-8, wherein the patient has been determined to have an elevated level of one or more biomarkers selected from Table 2 or a human analog thereof.

10. The method of any one of embodiments 1-9, wherein the patient has been determined to have a reduced level of one or more biomarkers selected from Table 3 or a human analog thereof.

11. The method of any one of embodiments 1-10, wherein the patient has been determined to have an altered level of one or more biomarkers selected from those in Table 5, or a human analog thereof, with: a change in mean expression for sham animals relative to PANX animals of at least about 1.5- fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, or about 20-fold; and/or a change in mean expression for PANX animals relative to PANX+Compound 1 animals of at least about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, or about 20-fold; and/or a change in mean expression for sham animals relative to PANX+Compound 1 animals of less than about 100%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, or about 10%.

12. The method of any one of embodiments 1-11, wherein the patient has been determined to have an altered level of at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% of the biomarkers. 13. The method of any one of embodiments 1-12, wherein the level of the one or more biomarkers is at least about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, or about 20-fold different from that of a corresponding threshold level.

14. The method of embodiment 13, wherein the threshold level corresponds to a predetermined mean or median level of the biomarker in a population of healthy subjects.

15. The method of any one of embodiments 1-14, wherein the altered level of one or more biomarkers was obtained from or determined in a biological sample obtained from the patient.

16. The method of any one of embodiments 1-15, wherein the method further comprises obtaining or determining a level of one or more biomarkers in a biological sample obtained from the patient.

17. The method of any one of embodiments 1-16, wherein the patient has been determined to have an altered level of a collagen 6 biomarker.

18. The method of any one of embodiments 1-17, wherein the patient has further been determined to have an altered level of a collagen 1 and/or collagen 3 biomarker.

19. A method comprising:

(i) obtaining or determining a level of one or more biomarkers in a biological sample obtained from the patient, wherein the one or more biomarkers are selected from a collagen 6 biomarker and/or Table 1 or a human analog thereof; and

(ii) comparing the level of the one or more biomarkers with that of a corresponding threshold level.

20. A method comprising:

(i) obtaining or determining a level of one or more biomarkers in a biological sample obtained from the patient, wherein the one or more biomarkers are selected from Table 1 or a human analog thereof; and

21. The method of embodiment 19 or 20, wherein if the level of one or more of the biomarkers is different from the corresponding threshold level, then administering to the patient an effective amount of Compound 1. 22. The method of any one of embodiments 19-21, wherein if the level of one or more of the biomarkers is comparable to the corresponding threshold level, then the patient is not administered Compound 1.

23. The method of any one of embodiments 19-22, wherein the one or more biomarkers are selected from Table 2 or a human analog thereof.

24. The method of any one of embodiments 19-23, wherein the one or more biomarkers are selected from Table 3 or a human analog thereof.

25. The method of any one of embodiments 19-24, wherein the one or more biomarkers are selected from those in Table 5, or a human analog thereof, with: a change in mean expression for sham animals relative to PANX animals of at least about 1.5- fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, or about 20-fold; and/or a change in mean expression for PANX animals relative to PANX+Compound 1 animals of at least about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, or about 20-fold; and/or a change in mean expression for sham animals relative to PANX+Compound 1 animals of less than about 100%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, or about 10%.

26. The method of any one of embodiments 19-25, wherein if the level of at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% of the biomarkers is different from the corresponding threshold level, then administering to the patient an effective amount of Compound 1.

27. The method of any one of embodiments 19-26, wherein if the level of the one or more biomarkers is at least about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, or about 20-fold different from the corresponding threshold level, then administering to the patient an effective amount of Compound 1.

28. The method of any one of embodiments 19-27, wherein the one or more biomarkers is or comprises a collagen 6 biomarker.

29. The method of any one of embodiments 19-28, further comprising obtaining or determining a level of a collagen 1 and/or collagen 3 biomarker in a biological sample obtained from the patient; and comparing the level of the collagen 1 and/or collagen 3 biomarker with that of a corresponding threshold level.

30. A method of treating a patient diagnosed with, suspected of having, or at risk of a fibrotic disease, wherein the patient is characterized by an altered level of one or more biomarkers selected from a collagen 6 biomarker and/or Table 1 or a human analog thereof, the method comprising:

(i) administering an effective amount of Compound 1 :

(ii) monitoring levels of the one or more biomarkers.

31. A method of treating a patient diagnosed with, suspected of having, or at risk of a fibrotic disease, wherein the patient is characterized by an altered level of one or more biomarkers selected from Table 1 or a human analog thereof, the method comprising:

(i) administering an effective amount of Compound 1 :

(ii) monitoring levels of the one or more biomarkers.

32. The method of embodiment 30 or 31, wherein if the levels of one or more biomarkers remain altered in the patient, discontinuing further therapy with Compound 1. 33. The method of any one of embodiments 30-32, wherein if the levels of at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% of the one or more biomarkers remain altered in the patient, discontinuing further therapy with Compound 1.

34. The method of any one of embodiments 30-33, wherein if the levels of the one or more biomarkers remain altered in the patient, increasing the dose and/or dosing frequency of Compound 1 administered to the patient.

35. The method of embodiment 34, wherein if the levels of at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% of the one or more biomarkers remain altered in the patient, increasing the dose and/or dosing frequency of Compound 1 administered to the patient.

36. The method of any one of embodiments 30-35, wherein the patient is characterized by an elevated level of one or more biomarkers selected from Table 2 or a human analog thereof.

37. The method of any one of embodiments 30-36, wherein the patient is characterized by a reduced level of one or more biomarkers selected from Table 3 or a human analog thereof.

38. The method of any one of embodiments 30-37, wherein the one or more biomarkers are selected from those in Table 5, or a human analog thereof, with: a change in mean expression for sham animals relative to PANX animals of at least about 1.5- fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, or about 20-fold; and/or a change in mean expression for PANX animals relative to PANX+Compound 1 animals of at least about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, or about 20-fold; and/or a change in mean expression for sham animals relative to PANX+Compound 1 animals of less than about 100%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, or about 10%.

39. The method of any one of embodiments 30-38, wherein the one or more biomarkers is or comprises a collagen 6 biomarker.

40. The method of any one of embodiments 30-39, wherein the patient is further characterized by an altered level of a collagen 1 and/or collagen 3 biomarker. 41. A method of treating a fibrotic disease, the method comprising administering an effective amount of Compound 1 :

to a patient that has been determined to have an altered level of one or more biomarkers, wherein the one or more biomarkers are selected from biomarkers or human analogs thereof whose levels have been established to have: a mean change in a population of subjects administered Compound 1 relative to a comparable reference population; and/or a mean change in a population of subjects with confirmed fibrotic disease relative to a population of healthy volunteers.

42. The method of embodiment 41, wherein the one or more biomarkers are selected from biomarkers or human analogs thereof whose levels have been established to have: a mean change of at least about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, or about 20-fold in a population of subjects administered Compound 1 relative to a comparable reference population; and/or a mean change of at least about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, or about 20-fold in a population of subjects with confirmed fibrotic disease relative to a population of healthy volunteers.

43. The method of embodiment 41 or 42, wherein the population of subjects is a population of rodent subjects.

44. The method of embodiment 41 or 42, wherein the population of subjects is a population of human subjects.

45. The method of any one of embodiments 41-44, wherein the confirmed fibrotic disease is a fibrotic disease of the kidney. 46. The method of any one of embodiments 41-45, wherein the patient has been determined to have a level of the one or more biomarkers that is at least about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, or about 20-fold different from that of a corresponding threshold level.

47. The method of embodiment 46, wherein the threshold level corresponds to a predetermined mean or median level of the biomarker in a population of healthy subjects.

48. The method of any one of embodiments 41-47, wherein the patient has been determined to have an altered level of at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% of the biomarkers.

49. The method of any one of embodiments 41-47, wherein the altered level of one or more biomarkers was obtained from or determined in a biological sample obtained from the patient.

50. The method of any one of embodiments 41-48, wherein the method further comprises obtaining or determining a level of one or more biomarkers in a biological sample obtained from the patient.

51. The method of any one of embodiments 1-50, wherein the level of one or more biomarkers is a level of expression of one or more gene products or proteins.

52. The method of any one of embodiments 1-51, wherein the biological sample is a renal biopsy sample.

53. The method of any one of embodiments 1-51, wherein the biological sample is a urine sample.

54. The method of any one of embodiments 1-51, wherein the biological sample is a blood sample.

55. The method of any one of embodiments 1-54, wherein the patient is diagnosed with, suspected of having, or at risk of fibrosis of gastrointestinal tract, heart, kidney, lung, liver, muscle, pancreas, or skin.

56. The method of any one of embodiments 1-55, wherein the patient is diagnosed with, suspected of having, or at risk of pulmonary fibrosis.

57. The method of any one of embodiments 1-56, wherein the patient is diagnosed with, suspected of having, or at risk of idiopathic pulmonary fibrosis.

58. The method of any one of embodiments 1-57, wherein the patient is diagnosed with, suspected of having, or at risk of renal fibrosis. 59. The method of any one of embodiments 1-58, wherein the patient is diagnosed with, suspected of having, or at risk of focal segmental glomerulosclerosis (FSGS), steroid resistant nephrotic syndrome (SRNS), proteinuria, lupus nephritis, minimal change disease, an antineutrophil 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, nailpatella syndrome, Alport syndrome, or chronic kidney disease.

60. The method of embodiment 59, wherein the anti-neutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis is Wegener’s granulomatosis, microscopic polyangiitis (MPA), or renal limited vasculitis.

61. The method of any one of embodiments 1-60, wherein the patient has proteinuria.

62. The method of any one of embodiments 1-61, wherein the patient is diagnosed with, suspected of having, or at risk of primary proteinuric kidney disease.

63. The method of any one of embodiments 1-62, wherein the patient is diagnosed with, suspected of having, or at risk of primary glomerular disease.

64. The method of any one of embodiments 1-63, wherein the patient is diagnosed with, suspected of having, or at risk of dermal fibrosis.

65. The method of any one of embodiments 1-64, wherein the patient is diagnosed with, suspected of having, or at risk of scleroderma or systemic sclerosis.

66. The method of any one of embodiments 1-65, wherein the patient is diagnosed with, suspected of having, or at risk of inflammatory bowel disease.

67. The method of any one of embodiments 1-66, wherein Compound 1 is administered as a pharmaceutical composition.

68. The method of any one of embodiments 1-67, wherein Compound 1 is administered orally.

69. The method of any one of embodiments 1-68, wherein Compound 1 is administered as a pharmaceutically acceptable salt form.

70. The method of any one of embodiments 1-69, wherein the human analog is selected from Table 1A.

71. The method of any one of embodiments 1-70, wherein the one or more biomarkers are human analog(s) selected from Table 1 A. EXAMPLES

[0183] The following examples are provided to illustrate, but not limit, the claimed invention.

Example 1. Preparation of a Composition that Provides Compound 1

Synthesis o f Compound 1

[0184] 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.

[0185] 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. [0186] 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-(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.

[0187] 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 hydrochloride salt channel hydrate (e.g., with approx, three equivalents of water).

[0188] The XRPD data of the material obtained from this Example are summarized below:

Capsule Formulation o f Compound 1

[0189] 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.

[0190] 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).

Example 2. Identification of Biomarkers for Treatment of Kidney Fibrotic Diseases with Compound 1

Materials and Methods

[0191] Whole kidney tissue samples for mRNA analysis were obtained as follows: Male Sprague-Dawley rats were subjected to uninephrectomy (UNX) and a week later (Study Day 1) administered a single 50 mg/kg IP dose of puromycin aminonucleoside (PAN, Treatment Day 1). Rats were randomized three days later to Compound 1 (n = 15) or vehicle (n = 15) groups, and treated twice daily for two weeks. Compound 1 (50 mg/kg) and vehicle (equivalent volume of tap water) were administered orally. Sham treated rats (n = 15) were not uninephrectomized nor administered PAN or Compound 1 or vehicle. In life, 24-hour urine samples were collected on Study Day 11 (Treatment Day 8) and Study Day 17 (Treatment Day 14). Kidneys were harvested, and apportioned to formalin, liquid nitrogen, or put in RNAlater buffer. Formalin samples were processed for histopathology. Kidney samples for RNA analysis were processed according to the following: Total RNA was extracted from fresh frozen tissue samples using Qiagen RNeasy Plus Universal mini kit following the manufacturer’s instructions (Qiagen, Hilden, Germany).

Urinalysis

[0192] FIG. l is a graph showing results of Compound 1 treatment on urine protein. Rats treated with Compound 1 displayed reduced urine protein on Study Day 17 (Treatment Day 14) compared to vehicle-treated animals.

[0193] FIG. 2 A and FIG. 2B are graphs showing change from Treatment Day 8 to Treatment

Day 14 in urine protein per animal. Rats treated with Compound 1 displayed reduced urine protein on Study Day 17 (Treatment Day 14) compared to vehicle-treated animals.

Histopathology

[0194] Kidney samples preserved with formalin were analyzed and scored blindly according to the following renal damage scale: 0 (no damage); 1 (>25% damage); 2 (26-50% damage); 3 (51-75% damage); or 4 (>75% damage). As shown in Table 4, the average renal damage score in the group treated with Compound 1 was significantly reduced compared to the vehicle-treated group.

Table 4.

Transcriptome Analysis

[0195] RNA samples were quantified using Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and RNA integrity was checked using Agilent TapeStation 4200 (Agilent Technologies, Palo Alto, CA, USA).

[0196] RNA sequencing libraries were prepared using the NEBNext Ultra RNA Library Prep Kit for Illumina using manufacturer’s instructions (NEB, Ipswich, MA, USA). Briefly, mRNAs were initially enriched with Oligod(T) beads. Enriched mRNAs were fragmented for 15 minutes at 94 °C. First strand and second strand cDNA were subsequently synthesized. cDNA fragments were end repaired and adenylated at 3 ’ends, and universal adapters were ligated to cDNA fragments, followed by index addition and library enrichment by PCR with limited cycles. The sequencing library was validated on the Agilent TapeStation (Agilent Technologies, Palo Alto, CA, USA), and quantified by using Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA), as well as by quantitative PCR (KAPA Biosystems, Wilmington, MA, USA).

[0197] The sequencing libraries were clustered on a single lane of a flowcell. After clustering, the flowcell was loaded on the Illumina HiSeq instrument (4000 or equivalent) according to manufacturer’s instructions. The samples were sequenced using a 2xl50bp Paired End (PE) configuration. Image analysis and base calling were conducted by the HiSeq Control Software (HCS). Raw sequence data (.bcl files) generated from Illumina HiSeq were converted into fastq files and de-multiplexed using Illumina's bcl2fastq 2.17 software. One mismatch was allowed for index sequence identification.

[0198] After investigating the quality of the raw data, sequence reads were trimmed to remove possible adapter sequences and nucleotides with poor quality using Trimmomatic v.0.36. The trimmed reads were mapped to the reference genome available on ENSEMBL using the STAR aligner v.2.5.2b. The STAR aligner uses a splice aligner that detects splice junctions and incorporates them to help align the entire read sequences. BAM files were generated as a result of this step. Unique gene hit counts were calculated by using feature Counts from the Subread package v.1.5.2. Only unique reads that fell within exon regions were counted.

[0199] After extraction of gene hit counts, the gene hit counts table was used for downstream differential expression analysis. Using DESeq2, a comparison of gene expression between the groups of samples was performed. The Wald test was used to generate p-values and Log2 fold changes. Genes with adjusted p-values < 0.05 and absolute log2 fold changes > 1 were called as differentially expressed genes for each comparison. A gene ontology analysis was performed on the statistically significant set of genes by implementing the software GeneSCF. The mgi GO list was used to cluster the set of genes based on their biological process and determine their statistical significance. A PCA analysis was performed using the "plotPCA" function within the DESeq2 R package.

[0200] A total of 14,618 mRNA were analyzed. The disease model (rats treated with PANX) displayed 2,667 mRNA that were modulated relative to sham animals. Treatment with Compound 1 was determined to significantly restore 218 mRNA toward sham levels (see Table 5 below). Data analysis (p<0.05, Wald’s test; p adjusted <0.05, Benjamini -Hochberg test) indicates that treatment with Compound 1 is associated with partial restitution of the PANX renal transcriptome. Results are summarized in Table 5.

Table 5.

^a Sham rats were not uninephrectromized or administered PAN. ^b PANX rats were uninephrectomized and administered PAN. ^c PANX+Cmpd 1 (or “PANX+Compound 1”) rats were uninephrectomized and administered PAN, and then treated with Compound 1 per the protocol described above. Example 3. Therapeutic Treatment of Patients Expressing Biomarkers for Compound 1 [0201] A nephropathy patient with suspected or confirmed glomerular disease (e.g., suspected of having FSGS or MCD) is subjected to a renal biopsy. For example, mRNA is extracted from single glomeruli (see Menon et al., JCI Insight. 2020, 5(6):el 33267), and sequenced to quantify levels of certain biomarkers (e.g., those described herein). If the subject has a level of one or more biomarkers that is altered compared to a threshold level, then the subject is to be treated with Compound 1. If the subject does not have a level of one or more biomarkers that is altered compared to a threshold level, then alternate treatment is determined. In some embodiments, in a patient to be treated with Compound 1, the kidney disease is stabilized and/or ameliorated.

Example 4. Identification of Biomarkers for Treatment of Pulmonary Fibrotic Diseases with Compound 1

[0202] The present disclosure encompasses a recognition that in different patients, diseasedriving 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.

[0203] 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 SiO₂ 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 staining 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 5. Therapeutic Treatment of Fibrotic Lung Disease Patients Expressing Biomarkers for Compound 1

[0204] 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 (e.g., 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 4 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. 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 6. 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

[0205] Primary: To assess the safety and tolerability of single and multiple ascending doses of Compound 1 in healthy adult participants.

[0206] 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

[0207] 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.

[0208] Secondary Endpoints:

[0209] Plasma PK endpoints include:

• Maximum concentration (C_max )

• Time to maximum concentration (Tmax)

• Area under drug concentration-time curve, from time zero to the last measurable concentration (AUC_0-last )

• 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 (t_1/2)

• Apparent terminal elimination rate constant (K_el)

• 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

[0210] Urine PK endpoints include:

• Amount of drug excreted in urine over time (Aet1-t2), 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 (Feti-t2), including cumulative Fe(o-72 h)

Methodology

[0211] 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)

[0212] Up to 48 participants were enrolled into one of up to six cohorts (Cohorts Al 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.

[0213] 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.

[0214] 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.

[0215] Cohorts Al, 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.

[0216] 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).

[0217] 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. [0218] 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. [0219] 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).

[0220] 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 C_max > 600 ng/mL or mean whole blood AUC_0-last > 2000 ng*h/mL. These C_max and AUC_0-last values were extrapolated from the mean plasma C_max 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.

[0221] For Part A (SAD), single oral dosing was carried out according to Table 6.

Table 6.

* Compound 1 dose did not exceed 1200 mg or a dose level that leads to mean whole blood C_max >600 ng/mL or mean whole blood AUC_0-last > 2000 ng*h/mL. These C_max and AUC_last values were extrapolated from the mean plasma C_max of 800 ng/mL and the mean plasma AUC_last 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)

[0222] 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 postdose.

[0223] 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.

[0224] 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 C_max > 600 ng/mL or mean whole blood AUC_0-last > 2000 ng*h/mL. These C_max and AUC_0-last values were extrapolated from the mean plasma C_max 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.

[0225] For Part B (MAD), once daily oral dosing was carried out according to Table 7.

* Compound 1 dose did 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 AUC_0-last > 2000 ng*h/mL. These C_max and AUC_last values were extrapolated from the mean plasma C_max of 800 ng/mL and the mean plasma AUC_last 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)

[0226] 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.

[0227] For Part C (MAD QD), once daily oral dosing was carried out according to Table 8.

* Compound 1 dose did not exceed dose level that leads to mean whole blood C_max >600 ng/mL or mean whole blood AUC_0-last > 2000 ng*h/mL. These C_max and AUC_last values were extrapolated from the mean plasma C_max of 800 ng/mL and the mean plasma AUC_last 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

[0228] Eight participants were enrolled into 1 cohort (Cohort DI). 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.

[0229] For Part D (Single Dose Food Effect), once daily oral dosing was carried out according to Table 9.

Table 9.

[0230] 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:

[0231] 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. 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. 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. 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 lU/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-ovulationmethods) 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.

[0232] 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. 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. 20. 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).

21. 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.

22. 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.

23. Use of any prescription drugs (other than hormonal contraception: OCPs, long-acting implantable hormones, injectable hormones, a vaginal ring or an IUD), 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

[0233] 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). [0234] 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

[0235] 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 10.

Table 10.

[0236] 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.

[0237] Part B (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.

[0238] 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).

[0239] 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

[0240] 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 (MAD QD) and Part D (Single Dose Food Effect

[0241] 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.

[0242] 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 DI).

Statistical Methods

[0243] 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. [0244] 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.

[0245] 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.

[0246] No formal hypothesis testing was performed for this study.

[0247] Analysis Populations: Participant inclusion into each population was determined prior to the final analysis.

[0248] 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.

[0249] 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.

[0250] 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.

[0251] 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.

[0252] 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.

[0253] 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.

[0254] Physical examinations at each visit were listed for each participant and summarized using descriptive statistics at each visit by treatment arm.

[0255] Medical history, pregnancy test/FSH, urine drug screen/alcohol breath test, physical examinations and serology (HIV, Hepatitis B & C screen) were listed by participant.

[0256] 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.

[0257] The following plasma Compound 1 non-compartmental PK parameters were estimated, as appropriate: C_max , Tmax, AUC_0-last , AUC_0-inf, AUC_tau (calculated for MAD cohorts only), K_e1, t_1/2, CL/F (SAD cohorts), CL/Fss (MAD cohorts), Vz/F (SAD cohorts, Vz/Fss (MAD cohorts), RA (calculated for MAD cohorts only).

[0258] Value for K_e1, t_1/2, 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.

[0259] A food effect assessment was conducted on Compound 1 PK parameters in Cohort A3. Analyses of variance (ANOVA) were performed on the In-transformed AUC_0-last , AUCo-inf and C_max (fasting vs fed). Ratios of the geometric means were calculated using the exponentiation of the least squares mean (LSM) from the analyses on the In-transformed AUCo- iast, AUCo-inf and C_max . Ratios were expressed as a percentage relative to the fasting regimen. 90% CI for the ratios were derived by exponentiation of the Cis obtained for the difference between regimen LSM resulting from the analyses on the In-transformed AUC_0-last , AUC_0-inf, and C_max . [0260] The analysis of dose proportionality was conducted for AUC and C_max 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 (CI) of the slope for each exposure parameter was computed from the model and presented in a summary table.

[0261] Urine collection time, volume collected, and Compound 1 concentration (Aeti-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 were calculated, as appropriate: Aeti-t2, CLR, CL/F, and Feti-t2.

Example 7. Compound 1 Ameliorates Fibrosis in Bleomycin-induced Injury Model of Idiopathic Pulmonary Fibrosis

[0262] 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 TGFβ1, 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. [0263] 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. 3 A is a graph showing lung to body weight ratio (mg/g).

[0264] 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. 3B).

[0265] 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. 3C).

[0266] 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. 3D).

[0267] Compound 1 significantly reduced transforming growth factor beta (TGFβ1) staining. Lung sections were immunohistologically stained for TGFβ1. Two weeks after bleomycin exposure, animals treated with bleomycin had increased TGFβ1 staining (albeit not statistically significant) compared to sham-treated animals. Animals then treated with Compound 1 for three weeks had reduced TGFβ1 staining compared to vehicle-treated animals (FIG. 3E).

Example 8. Compound 1 Reduces Fibrosis in Inducible TGFβ1 Mouse Model of Lung Fibrosis

[0268] A line of mice were used which expresses TGFβ1 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 TGFβ1 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 TGFβ1 transgene was under the control of a dox-inducible promoter. In addition, age and gender matched TGFβ1 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 TGFβ1 overexpression was evidenced after 4 weeks of dox compared to sham mice using micro-CT images.

[0269] 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.

[0270] Compound 1 reduced lung fibrosis score (Ashcroft) in dox-fed TGFβ1 transgenic mice. Dox feeding of TGFβ1 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. 4A). 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 TGFβ1 transgenic mice significantly (p=0.005) decreased lung fibrotic score compared to vehicle treatment.

[0271] 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. 4B). A reduction in hydroxyproline, a component of collagen is indicative of a reduction in fibrosis.

[0272] 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. 4C); 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. [0273] Compound 1 significantly decreases aSMA, an early lung fibrosis marker. Compound 1 treatment for 4 weeks following 4-week dox feeding of TGFβ1 transgenic mice significantly (p=0.026) decreased aSMA staining signals compared to vehicle treatment (FIG. 4D). aSMA is a marker of myofibroblast activation, an early step in fibrosis development.

Example 9. Compound 1 Has Anti-Fibrotic Effects Across Multiple Organ Systems in Bleomycin Systemic Sclerosis Mouse Model

[0274] 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. [0275] 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. 5A summarizes these results.

[0276] 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. 5B summarizes these results.

[0277] 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. 5C summarizes these results.

[0278] 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. 5D summarizes these results.

[0279] 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. 5E summarizes these results

[0280] 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. 5F summarizes these results.

Example 10. Compound 1 Reduces Proteinuria and Sclerosis in FSGS-Relevant Rat Model of PAN-induced Proteinuria

[0281] 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.

[0282] 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).

[0283] 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 H4C. 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.

[0284] 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.

[0285] 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 t_1/2 obtained from this curve was used to calculate GFR using the formula: GFR (pL/min/100 g b wt) = 21.33 [pL/100 g b wt]/t_1/2(FITc- sini strin) [min]; b wt=body weight. Glomerular Filtration Rate determination by the FITC- sinistrin method was determined for three animals per experimental group.

[0286] 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. [0287] 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.

[0288] 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.

[0289] Overnight (24-hour) urines were collected on days 10-11, immediately prior to sacrifice. Animals in the PAN Vehicle and PAN Compound 1 groups produced similar amounts of urine as those in the Sham exposed animals.

[0290] 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. 6A). This shows a significant anti-proteinuric effect of Compound 1 treatment.

[0291] 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. 6B). [0292] 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. 6C, 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. 6C).

[0293] 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. 6D).

[0294] 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. 6E). The COL-3 level was significantly reduced with Compound 1 treatment (FIG. 6E) compared to PAN Vehicle treatment.

Example 11. Compound 1 Reduces Proteinuria and Fibrosis in DOCA/Salt Model of Renal Injury and Fibrosis

[0295] Compound 1 was tested in the uninephrectomy (UNX) + DOCA + 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.

[0296] 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). [0297] 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.

[0298] 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).

[0299] 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).

[0300] 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).

[0301] 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).

[0302] 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.

[0303] 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. 7A). 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.

[0304] 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. 7B). 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.

[0305] Urines were also analyzed for creatinine levels and the albumin-to-creatinine-ratio (ACR) was calculated (FIG. 7C). 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.

[0306] Kidney Injury Molecule- 1 (KIMI) (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 KIMI levels are found in the urine (Zhang, Q., et al. Am. J. Med. Sci. 2018 Apr;355(4):314-21). Urines were analyzed for KIMI and the total amount of KIMI produced over 24 hours was calculated (FIG. 7D). In urines collected from animals prerandomization, elevated KIMI 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 KIMI 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 KIMI production compared to DOCA + Vehicle treated animals (Group 4). This is consistent with the notion that Compound 1 treatment reduces renal damage.

[0307] 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. 7E). 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.

[0308] The H&E stained slides were scored for renal damage by two blinded observers and the scores were averaged. As shown in FIG. 7F, 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).

[0309] Picrosirius red-staining in the slides was quantified using Bioquant Image Analysis software. The data were 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. 7G, 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.

[0310] 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. 7H, 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 12. Unilateral Ureteral Obstruction (UUO) Model of Renal Fibrosis in Mice [0311] 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.

[0312] 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 11. 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 11

a 100 mg/kg Compound 1 Hydrochloride trihydrate.

[0313] 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.

[0314] 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. [0315] 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.

[0316] 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.

[0317] 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.

[0318] All H&E stained slides were scored for renal damage by two blinded observers and the scores were averaged. As shown in FIG. 8A, 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.

[0319] 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. 8B, 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).

[0320] All a-Smooth Muscle Actin-stained slides were quantified using Bioquant Image Analysis software. The data were 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. 8C, 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).

[0321] 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 12, Compound 1 had statistically significant beneficial effects on renal damage, picrosirius red staining and alpha-SMA staining.

Table 12

A = log-rank (Mantel-Cox) test; B = one-way ANOVA followed by Tukey’s test

Example 13. PCK Rat Model for Polycystic Kidney Disease

[0322] 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. J., 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).

[0323] 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 13. Table 13

[0324] 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.

[0325] 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.

[0326] Serum creatinine (SCr) and BUN levels were determined in sera of 14-week old animals (FIG. 9A and FIG. 9B, 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.

[0327] Kidneys were weighed at sacrifice and kidney weights are given in absolute weight (g) and as a percentage of body weight (FIG. 9C and FIG. 9D, 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.

[0328] 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. 9E). 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. [0329] 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. 9F). Compound 1 treatment markedly reduced cystic index compared to PCK Vehicle animals, indicating a significant improvement in renal pathology due to Compound 1 treatment.

[0330] 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. 9G). Compound 1 treatment in PCK rats reduced urine production to levels similar to the Sprague Dawley WT control animals.

[0331] 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. 9H). 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.

[0332] 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. 91). 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.

[0333] 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. 9J). 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. [0334] Hepatitis A virus cellular receptor 1 (HAVcr-1) also known as KIM-1 (KIMI, 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. 9K). 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. 9K), which indicates mitigation of renal damage, and in particular tubular damage.

[0335] Interleukin 18 (IL- 18 or IL 18) 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 al., 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. 9L). 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. [0336] 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. 9M). 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 14. Model of Inflammatory Bowel Disease (IBD)/Acute Colitis Induced by TNBS in Mice

[0337] 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-TNFα, 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.

[0338] 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.

[0339] 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.

[0340] 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. 10 A), colons were collected and colon weight (FIG. 10B) was recorded. Colon length was determined (FIG. 10C) and macroscopic colon damage was scored (FIG. 10D), 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.

[0341] 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. 10E and the composite (total) Colon Histopathological Score (the sum of individual component scores; 0-12) is shown in FIG. 10F. 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.

[0342] 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. 10G, 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.

[0343] 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. 10H (group averages shown). Similarly, F4/80 was performed, with quantified staining intensity shown in FIG. 101 (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.

[0344] Compound 1 treatment at doses of 15 mg/kg and higher, BID, significantly attenuates inflammatory bowel disease (IBD) in a mouse model.

Example 15. Compound 1 Decreases Acetic Acid (AA) Induced Colitis in Mice

[0345] Male CD-I mice were infused with 4% AA in saline in 150 pL 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. 11 A) and reduced colon length (FIG. 1 IB), increased colon weight (FIG. 11C) and increased gross morphological colon damage (FIG. 1 ID).

[0346] 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. 1 IE), gross morphological colon damage (FIG. 1 IF) and histopathological colon damage (FIG. 11G) by decreasing mucosal wall thickness, and preserving and crypt/villi architecture compared to vehicle cohort (FIG. 11H).

Example 16. Compound 1 Decreases Chronic Colitis in IBD Mouse Model

[0347] 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 (100 mg) from all groups was subjected to hydroxyproline assay for collagen estimation as a fibrotic end point.

[0348] DSS-Vehicle cohort had the shortest colon length; DSS-Compound 1 cohort had significantly increased colon length compared to the DSS-vehicle cohort (FIG. 12A). Compound 1 treatment decreased macroscopic colon damage score in terms of appearance, inflammation and rectal bleeding (FIG. 12B), decreased colonic hydroxyproline (FIG. 12C) and histopathological injury score based on crypt damage, epithelial erosion and inflammatory cell infiltration as seen in H&E stained colon sections (FIG. 12D). These results indicate that Compound 1 could prevent progression of inflammation-induced fibrosis.

Example 17. Identification of Col6 as a Serum Biomarker for Treatment of Fibrotic Kidney Diseases with Compound 1

[0349] Compound 1 was evaluated in three rodent models of renal dysfunction: PANX model, DOCA/salt model, and PHN model.

[0350] PANX model: Male Sprague-Dawley rats were subjected to uninephrectomy (UNX) and approx. 4 months later (Study Day 1) administered a single 50 mg/kg IP dose of puromycin aminonucleoside (PAN, Treatment Day 1). Rats were randomized three days later to Compound 1 (n = 10) or vehicle (n = 14) groups, and treated twice daily for two weeks. Compound 1 (50 mg/kg) and vehicle (equivalent volume of tap water) were administered orally. Sham treated rats (n = 6) were not uninephrectomized nor administered PAN or Compound 1 or vehicle. Animals were sacrificed after approx. 2 weeks, and kidney tissue, urine, and serum samples were collected for analysis.

[0351] DOCA/salt model: Animals were uninephrectomized and implanted with a DOCA pellet (21 day release, 25 mg) and provided drinking water containing NaCl (1%). Two weeks later animals were randomized to Compound 1 in water, BID, PO, 50 mg/kg (n = 7) or vehicle (water, BID, PO) (n = 6) treatment groups. Animals were treated for approx. 4 weeks with Compound 1. DOCA pellets were reinstalled after 21 days. Sham treated rats (n = 9) were not uninephrectomized nor administered DOCA or Compound 1 or vehicle. At sacrifice kidneys, urine and serum were collected.

[0352] PHN model: 65 CD® rats from Charles River Laboratory were obtained and acclimated. 60 of them were administered anti-FXl A serum (tail vein, IV, 600 pL), and 5 were administered saline (tail vein, IV, 600 pL, sham), on two consecutive days, when the average mass of the animals was 300 g. Twenty-four hour urine samples were collected 7 days later (on Day 9) and proteinuria was determined. Animals were randomized based on proteinuria levels, ensuring equivalent average protein to creatinine ratio (PCR) and standard deviation in each group. Experimental groups were (1) 15 mg/kg Compound 1, (2) Vehicle (water), and (3) Sham. Dosing of animals in the Compound 1 and Vehicle groups began on day 10 and continued 5 days a week for 12 weeks (PO, BID). At the end of the study (day 93), animals were sacrificed, and kidney tissue and blood samples were collected.

[0353] Serum samples obtained from the models were analyzed for pro-collagen 6 levels using quantitative competitive ELISA.

[0354] FIG. 13 A is a graph showing serum pro-collagen 6 levels in animals treated with Compound 1 compared to sham and control animals.

[0355] FIG. 13B is a graph showing a correlation between serum pro-collagen 6 and TGF-P levels in animals evaluated in this Example.

[0356] FIG. 13C is a graph showing serum pro-collagen 6 levels in animals treated with Compound 1 compared to sham and control animals in the PANX model.

[0357] FIG. 13D is graph showing serum pro-collagen 6 levels in animals treated with Compound 1 compared to sham and control animals in the DOC A model.

[0358] Table 14 summarizes the results from the PHN model:

Table 14.

[0359] 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 an effective amount of Compound 1 :

to a patient that has been determined to have an altered level of one or more biomarkers selected from the group consisting of a collagen 6 biomarker and biomarkers in Table 1 or human analogs thereof.

2. A method of treating a fibrotic disease, the method comprising administering an effective amount of Compound 1 :

3. A method comprising administering an effective amount of Compound 1 :

to a patient that has been determined to have (i) proteinuria and/or hypoalbuminemia and/or hyperlipidemia and/or edema; and (ii) an altered level of one or more biomarkers selected from the group consisting of a collagen 6 biomarker and biomarkers in Table 1 or human analogs thereof.

4. A method comprising administering an effective amount of Compound 1 :

to a patient that has been determined to have (i) nephrotic syndrome; and (ii) an altered level of one or more biomarkers selected from the group consisting of a collagen 6 biomarker and biomarkers in Table 1 or human analogs thereof.

5. The method of any one of claims 1-4, wherein the patient has been determined to have an altered level of one or more biomarkers selected from Table 1 or human analogs thereof.

6. The method of any one of claims 1-4, wherein the patient has been determined to have an elevated level of one or more biomarkers selected from Table 2 or human analogs thereof.

7. The method of any one of claims 1-4, wherein the patient has been determined to have a reduced level of one or more biomarkers selected from Table 3 or human analogs thereof.

8. The method of any one of claims 1-4, wherein the patient has been determined to have an altered level of one or more biomarkers selected from those in Table 5, or human analogs thereof, with: a change in mean expression for sham animals relative to PANX animals of at least about 1.5- fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, or about 20-fold; and/or a change in mean expression for PANX animals relative to PANX+Compound 1 animals of at least about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, or about 20-fold; and/or a change in mean expression for sham animals relative to PANX+Compound 1 animals of less than about 100%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, or about 10%.

9. The method of any one of claims 1-4, wherein the patient has been determined to have an altered level of at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% of the biomarkers in Table 1.

10. The method of any one of claims 1-4, wherein the level of the one or more biomarkers is at least about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, or about 20-fold different from that of a corresponding threshold level.

11. The method of claim 10, wherein the threshold level corresponds to a predetermined mean or median level of the biomarker in a population of healthy subjects.

12. The method of any one of claims 1-4, wherein the altered level of the one or more biomarkers was obtained from or determined in a biological sample obtained from the patient.

13. The method of any one of claims 1-4, wherein the method further comprises obtaining or determining a level of one or more biomarkers in a biological sample obtained from the patient.

14. The method of any one of claim 1-4, wherein the patient has been determined to have an altered level of a collagen 6 biomarker.

15. The method of any one of claims 1-4, wherein the patient has further been determined to have an altered level of a collagen 1 and/or collagen 3 biomarker.

16. A method comprising:

(i) obtaining or determining a level of one or more biomarkers in a biological sample obtained from the patient, wherein the one or more biomarkers are selected from the group consisting of a collagen 6 biomarker and biomarkers in Table 1 or human analogs thereof; and

17. The method of claim 16, wherein if the level of one or more of the biomarkers is different from the corresponding threshold level, then administering to the patient an effective amount of Compound 1.

18. The method of claim 16, wherein if the level of one or more of the biomarkers is comparable to the corresponding threshold level, then the patient is not administered Compound 1.

19. The method of any one of claims 16-18, wherein the one or more biomarkers are selected from Table 1 or human analogs thereof.

20. The method of any one of claims 16-18, wherein the one or more biomarkers are selected from Table 2 or human analogs thereof.

21. The method of any one of claims 16-18, wherein the one or more biomarkers are selected from Table 3 or human analogs thereof.

22. The method of any one of claims 16-18, wherein the one or more biomarkers are selected from those in Table 5, or human analogs thereof, with: a change in mean expression for sham animals relative to PANX animals of at least about 1.5- fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, or about 20-fold; and/or a change in mean expression for PANX animals relative to PANX+Compound 1 animals of at least about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, or about 20-fold; and/or a change in mean expression for sham animals relative to PANX+Compound 1 animals of less than about 100%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, or about 10%.

23. The method of any one of claims 16-18, wherein the one or more biomarkers is or comprises a collagen 6 biomarker.

24. The method of claim 16, further comprising obtaining or determining a level of a collagen 1 and/or collagen 3 biomarker in a biological sample obtained from the patient; and comparing the level of the collagen 1 and/or collagen 3 biomarker with that of a corresponding threshold level.

25. A method of treating a patient diagnosed with, suspected of having, or at risk of a fibrotic disease, wherein the patient is characterized by an altered level of one or more biomarkers selected from the group consisting of a collagen 6 biomarker and biomarkers in Table 1 or human analogs thereof, the method comprising:

(i) administering an effective amount of Compound 1 :

(ii) monitoring levels of the one or more biomarkers.

26. The method of claim 25, wherein if the levels of one or more biomarkers remain altered in the patient, discontinuing further therapy with Compound 1.

27. The method of claim 25, wherein if the levels of the one or more biomarkers remain altered in the patient, increasing the dose and/or dosing frequency of Compound 1 administered to the patient.

28. The method of any one of claims 25-27, wherein the patient is characterized by an altered level of one or more biomarkers selected from Table 1 or human analogs thereof.

29. The method of any one of claims 25-27, wherein the patient is characterized by an elevated level of one or more biomarkers selected from Table 2 or human analogs thereof.

30. The method of any one of claims 25-27, wherein the patient is characterized by a reduced level of one or more biomarkers selected from Table 3 or human analogs thereof.

31. The method of any one of claims 25-27, wherein the one or more biomarkers are selected from those in Table 5, or human analogs thereof, with: a change in mean expression for sham animals relative to PANX animals of at least about 1.5- fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, or about 20-fold; and/or a change in mean expression for PANX animals relative to PANX+Compound 1 animals of at least about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, or about 20-fold; and/or a change in mean expression for sham animals relative to PANX+Compound 1 animals of less than about 100%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, or about 10%.

32. The method of any one of claims 25-27, wherein the one or more biomarkers is or comprises a collagen 6 biomarker.

33. The method of any one of claims 25-27, wherein the patient is further characterized by an altered level of a collagen 1 and/or collagen 3 biomarker.

34. A method of treating a fibrotic disease, the method comprising administering an effective amount of Compound 1 :

35. The method of claim 34, wherein the one or more biomarkers are selected from biomarkers or human analogs thereof whose levels have been established to have: a mean change of at least about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, or about 20-fold in a population of subjects administered Compound 1 relative to a comparable reference population; and/or a mean change of at least about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, or about 20-fold in a population of subjects with confirmed fibrotic disease relative to a population of healthy volunteers.

36. The method of any one of claims 1-35, wherein the level of one or more biomarkers is a level of expression of one or more gene products or proteins.

37. The method of any one of claims 1-36, wherein the biological sample is a renal biopsy sample.

38. The method of any one of claims 1-36, wherein the biological sample is a urine sample.

39. The method of any one of claims 1-36, wherein the biological sample is a blood sample.

40. The method of any one of claims 1-39, wherein the patient is diagnosed with, suspected of having, or at risk of fibrosis of gastrointestinal tract, heart, kidney, lung, liver, muscle, pancreas, or skin.

41. The method of any one of claims 1-40, wherein the patient is diagnosed with, suspected of having, or at risk of renal fibrosis.

42. The method of any one of claims 1-41, wherein the patient is diagnosed with, suspected of having, or at risk of 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, Alport syndrome, or chronic kidney disease.

43. The method of claim 42, wherein the anti -neutrophil cytoplasmic antibody (ANCA)- associated glomerulonephritis is Wegener’s granulomatosis, microscopic polyangiitis (MPA), or renal limited vasculitis.

44. The method of any one of claims 1-43, wherein the patient has proteinuria.

45. The method of any one of claims 1-44, wherein the patient is diagnosed with, suspected of having, or at risk of primary proteinuric kidney disease.

46. The method of any one of claims 1-45, wherein the patient is diagnosed with, suspected of having, or at risk of primary glomerular disease.

47. The method of any one of claims 1-46, wherein Compound 1 is administered as a pharmaceutical composition.

48. The method of any one of claims 1-47, wherein Compound 1 is administered orally.

49. The method of any one of claims 1-48, wherein Compound 1 is administered as a pharmaceutically acceptable salt form.