WO2022108870A1

WO2022108870A1 - Compositions and methods for treating pulmonary fibrosis

Info

Publication number: WO2022108870A1
Application number: PCT/US2021/059334
Authority: WO
Inventors: Patrick H. GRIFFIN; Nir Barak; Sireesh APPAJOSYULA
Original assignee: 9 Meters Biopharma, Inc.
Priority date: 2020-11-17
Filing date: 2021-11-15
Publication date: 2022-05-27

Abstract

The present disclosure, in the various aspects and embodiments, provides peptide compositions comprising larazotide or derivatives of larazotide and methods for treating or preventing pulmonary fibrosis in a patient in need.

Description

COMPOSITIONS AND METHODS FOR TREATING PULMONARY FIBROSIS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to, and the benefit of, U.S. Provisional Application No. 63/181,486, filed April 29, 2021, and U.S. Provisional Application No. 63/114,756, filed November 17, 2020, which are hereby incorporated by reference in their entireties.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: A computer readable format copy of the Sequence Listing (Filename: “NMT-032PC_ST25.txt”; Date recorded: November 17, 2021; File size: 4,971 bytes).

BACKGROUND

Pulmonary fibrosis is a condition in which the lung tissue becomes scarred over time. The tissue gets thick and stiff making it difficult to breath and for the blood to get sufficient oxygen. Causes of pulmonary fibrosis include genetic factors, gastrointestinal reflux, environmental pollutants, medications, connective tissue diseases, and interstitial lung disease. Interstitial lung disease is the term for a group of diseases that inflame or scar the lungs. Where the cause cannot be determined, the condition is referred to as idiopathic pulmonary fibrosis or IPF.

Treatment of pulmonary fibrosis is individualized depending on the patient’s disease and severity, and may include oxygen therapy, rehabilitation, symptom management, and medications such as anti-fibrotic agents and glucocorticoids. While anti-fibrotic drugs such as pirfenidone and nintedanib can reduce lung function decline for some patients, these agents are not effective for all patients, have limited efficacy, and can have substantial adverse effects. See G. Raghu, Pharmacotherapy for idiopathic pulmonary fibrosis: current landscape and future potential, Eur Respir Rev 2017 26(145): 170071.

Additional or alternative agents for treatment of pulmonary fibrosis are needed. DESCRIPTION OF THE FIGURES

FIG. 1 shows representative photomicrographs of Masson’s Trichome-stained lung sections from a bleomycin-induced pulmonary fibrosis model. Treatments include vehicle, larazotide-per os (po), larazotide-intratracheal (it), and dexamethasone.

FIG. 2 shows the Ashcroft score from the bleomycin (BLM)-induced pulmonary fibrosis model.

FIG. 3 shows the changes in body weight over a course of 21 days after BLM administration when the mice were given vehicle, larazotide, or all d-larazotide.

FIG. 4 depicts survival percentages over a course of 21 days after BLM administration when the mice were given vehicle, larazotide, or all d-larazotide.

FIG. 5 shows body weight of the pulmonary fibrosis model mice on the day of sacrifice (Day 21).

FIG. 6 depicts left lung weight of the pulmonary fibrosis model mice on the day of sacrifice (Day 21).

FIG. 7 depicts post-caval lobe weight of the pulmonary fibrosis model mice on the day of sacrifice (Day 21).

FIG. 8 shows lung hydroxyproline concentrations calculated from the hydroxyproline standard curve. Lung hydroxyproline contents are expressed as pg per left lung.

FIG. 9 depicts photographs of stained sections of right lung tissue of pulmonary fibrosis model mice that were administered vehicle, larazotide, and all d-larazotide.

FIG. 10 depicts a plot of the graded results of the stained right lung sections per the Ashcroft evaluation and grading criteria. DETAILED DESCRIPTION

The present disclosure in the various aspects and embodiments, provides compositions and methods for treating or preventing pulmonary fibrosis in a patient in need. The method comprises administering an effective amount of larazotide or a derivative of larazotide to the patient. Larazotide is a peptide agent that restores integrity of tight junctions in epithelial and endothelial tissues. In accordance with this disclosure, larazotide or a derivative, including larazotide or derivative administered locally or to the gastrointestinal tract, can reduce pulmonary fibrosis.

Pulmonary fibrosis is characterized by accumulation of excessive connective tissue in the lungs. Causes of pulmonary fibrosis include administration of drugs such as bleomycin and cyclophosphamide; exposure to certain environmental factors such as gases, asbestos and silica, and microbial infections. Some systemic inflammatory diseases such as rheumatoid arthritis and SLE may also predispose to pulmonary fibrosis. Symptoms of pulmonary fibrosis include dyspnea, non-productive cough, fever and damage to the lung cells. Pulmonary fibrosis can be diagnosed with the aid of chest radiography, high resolution computed tomographic scanning and the results of pulmonary function tests.

In some embodiments, the patient has or is at risk to develop an interstitial lung disease, such as an interstitial lung disease selected from sarcoidosis and idiopathic pulmonary fibrosis (IPF). In various embodiments, the patient has a chronic inflammatory lung disease or condition in addition to pulmonary fibrosis, or which may predispose to or exacerbate pulmonary fibrosis. Exemplary inflammatory diseases or conditions include cystic fibrosis, asthma, chronic obstructive pulmonary disease (COPD), emphysema, chronic bronchitis, and chronic pneumonia.

In some embodiments, a patient at risk of pulmonary fibrosis is at least 60 years of age, or at least 70 years of age. In some embodiments, a patient at risk is a tobacco smoker or a former tobacco smoker. In some embodiments, the patient is genetically predisposed to develop pulmonary fibrosis. In these or other embodiments, the patient has chronic gastrointestinal reflux. In some embodiments, the patient at risk has a chronic or recurring respiratory infection. Exemplary microbial infections that may predispose to pulmonary fibrosis include infections of Pseudomonas aeruginosa, Streptococcus pneumonia, and mycobacterium (tuberculosis or non-tuberculous mycobacterium). Other microbial infections include viral infections such as parainfluenza and coronavirus, such as Sars-CoV2.

In some embodiments, the patient has sarcoidosis, which is a disease characterized by granulomas, commonly in the lungs and lymph nodes. Sarcoidosis can affect other organs. Alternatively, the patient may have IPF. IPF is a condition in which the lungs become scarred without a clear etiology.

In still other embodiments, the patient has or is at risk of drug-induced pulmonary fibrosis. For example, the patient may be undergoing treatment with an antibiotic that induces pulmonary fibrosis (e.g., nitrofurantoin), methotrexate, amiodarone, and cancer chemotherapy (e.g., bleomycin or alkylating agent such as cyclophosphamide). Thus, in some embodiments, the patient is undergoing therapy with an agent that induces pulmonary fibrosis, and treatment with larazotide or derivative thereof is provided prophylactically.

In still other embodiments, the patient having or at risk of pulmonary fibrosis has significant occupational exposure to asbestos.

In various embodiments, the patient having pulmonary fibrosis has mild or moderate pulmonary fibrosis. In still other embodiments, the pulmonary fibrosis is severe. The severity of the disease can be determined using, for example, lung function tests (e.g., forced vital capacity, FVC), high resolution CT scan (HRCT), and/or severity of symptoms such as breathlessness and cough.

Larazotide has the amino acid sequence: Gly Gly Vai Leu Vai Gin Pro Gly (SEQ ID NO: 1). Derivatives of larazotide comprise from one to four amino acid modifications with respect to SEQ ID NO: 1, and the modifications may be independently selected from substitutions, deletions, insertions or additions with respect to SEQ ID NO: 1. In some embodiments, the larazotide derivative is a derivative having 1, 2, 3, or 4 amino acid deletions, insertions, and/or substitutions with respect to SEQ ID NO: 1. By way example, in some embodiments, the larazotide derivative is a derivative described in U.S. Patent Nos. 8,785,374, 8,957,032, and 9,279,807, which are hereby incorporated by reference in their entirety. In some embodiments, the derivative has one or more non-genetically encoded amino acids, or one or more (or all) (d)-amino acids. Exemplary derivatives of larazotide include:

Gly Arg Vai Cys Vai Gin Pro Gly (SEQ ID NO: 2);

Gly Arg Vai Cys Vai Gin Asp Gly (SEQ ID NO: 3);

Gly Arg Vai Leu Vai Gin Pro Gly (SEQ ID NO: 4);

Gly Arg Vai Leu Vai Gin Asp Gly (SEQ ID NO: 5);

Gly Arg Leu Cys Vai Gin Pro Gly (SEQ ID NO: 6);

Gly Arg Leu Cys Vai Gin Asp Gly (SEQ ID NO: 7);

Gly Arg Leu Leu Vai Gin Pro Gly (SEQ ID NO: 8);

Gly Arg Leu Leu Vai Gin Asp Gly (SEQ ID NO: 9);

Gly Arg Gly Cys Vai Gin Pro Gly (SEQ ID NO: 10);

Gly Arg Gly Cys Vai Gin Asp Gly (SEQ ID NO: 11);

Gly Arg Gly Leu Vai Gin Pro Gly (SEQ ID NO: 12);

Gly Arg Gly Leu Vai Gin Asp Gly (SEQ ID NO: 13);

Gly Gly Vai Cys Vai Gin Pro Gly (SEQ ID NO: 14);

Gly Gly Vai Cys Vai Gin Asp Gly (SEQ ID NO: 15);

Gly Gly Vai Leu Vai Gin Asp Gly (SEQ ID NO: 16);

Gly Gly Leu Cys Vai Gin Pro Gly (SEQ ID NO: 17);

Gly Gly Leu Cys Vai Gin Asp Gly (SEQ ID NO: 18);

Gly Gly Leu Leu Vai Gin Pro Gly (SEQ ID NO: 19);

Gly Gly Leu Leu Vai Gin Asp Gly (SEQ ID NO: 20);

Gly Gly Gly Cys Vai Gin Pro Gly (SEQ ID NO: 21);

Gly Gly Gly Cys Vai Gin Asp Gly (SEQ ID NO: 22);

Gly Gly Gly Leu Vai Gin Pro Gly (SEQ ID NO: 23); and

Gly Gly Gly Leu Vai Gin Asp Gly (SEQ ID NO: 24).

In some embodiments, a larazotide derivative is administered that exhibits resistance to exopeptidases, such as aminopeptidases, thus avoiding substantial accumulation of inactive peptide fragments. Exemplary modifications include amino acid substitutions at the N- and/or C- terminus to reduce exopeptidase digestion, extension of the N- and/or C- termini to delay exopeptidase digestion of the functional peptide, incorporation of d-amino acids, as well as cyclization. Exemplary larazotide derivatives are disclosed in WO 2019/165346, which is hereby incorporated by reference in its entirety.

In various embodiments, the peptide has at least one, at least two, at least three, at least four d-amino acids. In an embodiment, each amino acid of the larazotide derivative (other than Gly) is a d-amino acid, and is optionally a retro-inverso peptide. A retro-inverso peptide contains the inverse amino acid sequence (e.g., GPQVLVGG, SEQ ID NO: 25), with all non-glycine amino acids present as d-amino acids. See US Patent 10,526,372, which is hereby incorporated by reference in its entirety. Retro-inverso peptides maintain side chain topology similar to that of the original L-amino acid peptide, and render the peptide more resistant to proteolytic degradation. In some embodiments, the N-terminal Gly of the retro- inverso peptide is substituted with Ala, Leu, He, Vai, or Allylgly. In these or other embodiments, one or both of the C-terminal Gly residues of the retro inverso peptide is/are substituted with an amino acid independently selected from Ala, Leu, He, Vai, or Allylgly.

In other embodiments, the peptide having the amino acid sequence of SEQ ID NO: 1 has one or two d-amino acids at the N- and optionally the C-terminus, with all other amino acids in the L configuration. In these embodiments, the N- and/or C-terminus are substituted or extended such that the peptide does not have a glycine at the terminus (Gly does not have D- and L- configurations). In some embodiments, the terminal Gly residues are replaced with an amino acid independently selected from (d)-Ala, (d)-Leu, (d)-Ile, (d)-Val, or (d)- Allylgly.

In some embodiments, a larazotide derivative is administered having the amino acid sequence of SEQ ID NO: 1 with one or more (d) amino acids. In some embodiments, the treatment is with (d)-larazotide, that is, where all non-glycine residues are in the (d) form. (d)-larazotide may provide advantages in dosing, as compared to larazotide. Specifically, (d)-larazotide may display a substantially less inverse dose response at higher doses, as compared to larazotide.

In various embodiments, the larazotide or derivative thereof is administered by pulmonary delivery. For example, the larazotide or derivative thereof may be administered by solution or powder aerosol. See U.S. Patent 10,723,763, which is hereby incorporated by reference in its entirety.

In some embodiments, the larazotide or derivative thereof is administered intratracheally (e.g., endotracheal administration).

In some embodiments, the larazotide or derivative thereof is administered parenterally (e.g., by intravenous infusion). See Rittirsch et al., Zonulin prehaptoglobin2 Regulates Lung Permeability and Activates the Complement System, American Journal of Physiology - Lung Cellular and Molecular Physiology 302: 12 (2013).

In some embodiments, the larazotide or a derivative thereof (e.g., (d)-larazotide) is administered to the gastrointestinal tract. Larazotide and its derivatives are effective for improving integrity of tight junctions of epithelial cells (including epithelial cells if the gastrointestinal mucosa). In some embodiments, the larazotide or derivative thereof is administered to the small and/or large intestine. For example, the larazotide or derivative may be administered to one or more of the duodenum, jejunum, and/or the ileum. In these or other embodiments, the larazotide or derivative is administered to the large intestine.

In some embodiments, the larazotide or derivative is administered in any suitable form, including as a salt. By way of example, in some embodiments, the larazotide is administered as an acetate salt or a hydrochloride salt. Non-limiting examples of salts of larazotide are disclosed in US 2013/0281384, which is hereby incorporated by reference in its entirety. Alternative salts may be employed, including any pharmaceutically acceptable salt of the peptide, for example, such as those listed in Journal of Pharmaceutical Science, 66, 2-19 (1977) and The Handbook of Pharmaceutical Salts; Properties, Selection, and Use. P. H. Stahl and C. G. Wermuth (eds.), Verlag, Zurich (Switzerland) 2002, which are hereby incorporated by reference in their entirety.

In some embodiments, the larazotide or derivative thereof is administered as a pharmaceutical composition. Pharmaceutical compositions can take the form of tablets, pills, pellets, capsules, capsules containing liquids, capsules containing multiparticulates, powders, solutions, emulsion, drops, suppositories, suspensions, delayed-release formulations, sustained-release formulations, controlled-release formulations, or any other form suitable for use. The pharmaceutical compositions are formulated for oral administration (administration to the GI).

In some embodiments, the pharmaceutical composition comprising larazotide (or derivative such as (d)-larazotide) is formulated for targeted delivery to the gastrointestinal tract including the stomach, small intestine, and large intestine including a plurality of subsections thereof. In some embodiments, the pharmaceutical composition is formulated to release larazotide or a derivative thereof in the small intestine, for example, in one or more of the duodenum, jejunum, and the ileum. In some embodiments, the pharmaceutical composition is formulated to release larazotide or a derivative thereof in the large intestine, for example, in one or more of the cecum, the ascending colon, the transverse colon, the descending colon, and the sigmoid colon.

In some embodiments, the pharmaceutical composition is formulated so as to not substantially release or partially release larazotide or a derivative thereof in the stomach, but to release the peptide agent after entry into the small bowel. That is, the pharmaceutical composition is formulated to have a delayed-release profile, i.e., not immediately release the peptide agent upon ingestion; but rather, postponement of release until the pharmaceutical composition is lower in the gastrointestinal tract. By way of example, the pharmaceutical composition may be formulated for release of the peptide agent in the small intestine (e.g., one or more of duodenum, jejunum, and ileum) and/or the large intestine (e.g., one or more of cecum, ascending, transverse, descending and/or sigmoid portions of the colon). In some embodiments, the pharmaceutical composition is formulated to have a delayed-release profile as described in, for example, U.S. Patent No. 8,168,594, the entire contents of which are hereby incorporated by reference.

In some embodiments, the pharmaceutical composition has a coating that remains essentially intact, or may be essentially insoluble, in gastric fluid. In some embodiments, the stability of the delayed-release coating is pH dependent. Delayed-release coatings that are pH dependent are substantially stable in acidic environments (pH of about 5 or less), and substantially unstable in near neutral to alkaline environments (pH greater than about 5). For example, in some embodiments, the delayed-release coating essentially disintegrates or dissolves in near neutral to alkaline environments such as are found in the small intestine (e.g., one or more of the duodenumjejunum, and ileum) and/or large intestine (e.g., one or more of the cecum, ascending colon, transverse colon, descending colon, and sigmoid colon). Exemplary delayed-release coatings are described elsewhere herein.

In some embodiments, the pharmaceutical composition is formulated to have sustained-release profiles, i.e., slow release of the larazotide or a derivative thereof (e.g., (d)- larazotide) in the GI tract over an extended period of time. In various embodiments, the larazotide or derivative is administered in a sustained release or controlled release formulation. For example, the formulation may deliver and/or functionally release from 0.5 to about 5 mg of larazotide or derivative, or from about 0.5 to about 4 mg of larazotide or derivative, or from about 0.5 to about 3 mg of larazotide or derivative, or from about 0.5 to about 2 mg of larazotide or derivative, or from about 0.5 to about 1 mg of larazotide or derivative. In various embodiments, the sustained release or controlled release formulation contains at least 1 mg or at least 2 mg of larazotide or derivative. For example, the formulation may contain from about 1 mg to about 5 mg of larazotide or derivative, or about 1 mg to about 3 mg of larazotide or derivative.

In some embodiments, the larazotide or derivative is formulated for sustained or modified or controlled delivery in one or more locations of the GI. For Example, the present invention contemplates a sustained or controlled release formulation that may functionally release the peptide over the course of at least about 2 hours, or over the course of at least about 2.5 hours, or over the course of at least about 3 hours, or over the course of at least about 4 hours, or over the course of at least about 5 hours. In some embodiments, the sustained or controlled release composition begins to release peptide starting within about 10 to about 30 minutes of exposure to simulated intestinal fluid, with release of peptide continuing for at least about 180 minutes, or at least about 210 minutes, or at least about 240 minutes, or at least about 280 minutes of exposure to simulated intestinal fluid. Release profiles can be prepared, for example, using compositions with different enteric polymer coats and/or different thicknesses of the polymer coats. In some embodiments, the invention provides a composition comprising an effective amount of a peptide that is (d)-larazotide, or salt thereof, contained within a biodegradable or erodible polymer matrix, which further comprises an enteric coating. Formulations employing a biodegradable or erodible matrix are described in PCT/US2020/046272, which is hereby incorporated by reference in its entirety. Further, the erodible polymer matrix can comprise a polysaccharide matrix. In some embodiments, the matrix comprises one or more of cellulose, chitin, chitosan, alginate, amylose, pectin, callose, laminarin, chrysolaminarin, xylan, arabinoxylan, mannan, fucoidan, galactomannan, xanthan gum, dextran, welan gum, gellan gum, diutan gum, pullulan, hyaluronic acid, and derivatives thereof. In further embodiments, the matrix comprises microcrystalline cellulose.

By way of example, in some embodiments, the larazotide, and/or a derivative thereof is administered to the small intestine of the patient, as an oral dosage, delayed-release composition that contains larazotide (or a derivative thereof)-coated beads that are stable in gastric fluid and unstable in intestinal fluid so as to substantially release the peptide in the small intestine. In an exemplary oral dosage composition, an effective amount of larazotide (e.g., as the acetate salt) is provided in first delayed-release particles that are capable of releasing larazotide or derivative in the duodenum of a patient, and second delayed release particles that are capable of releasing larazotide or derivative in the jejunum of a patient, and optionally a third delayed release particle capable of releasing larazotide or derivative in the ileum of a patient. Each particle may have a core particle, a coat comprising larazotide or derivative over the core particle, and a delayed-release coating (e.g., a 1 : 1 co-polymer of acrylate and methacrylate) outside the coat comprising larazotide or derivative. The first delayed-release particles may release at least 70% of the larazotide or derivative in the first delayed-release particles by about 60 minutes of exposure to simulated intestinal fluid having a pH of greater than 5; the second delayed-release particles may release at least 70% of the larazotide or derivative by about 30 and about 90 minutes of exposure to simulated intestinal fluid having a pH of greater than 5. The third delayed-release particles may release at least 70% of the larazotide or derivative by about 120 minutes to about 240 minutes (e.g., about 120 minutes to about 180 minutes) of exposure to simulated intestinal fluid.

In some embodiments, the larazotide, or derivative thereof, is administered to the colon of a patient, which can be via the same or different composition for administration to the small intestine. Various colon-specific delivery approaches may be utilized. For example, in some embodiments, the modified release formulation is formulated using a colon-specific drug delivery system (CODES), as described for example, in Li et al., AAPS PharmSciTech (2002), 3(4): 1-9, the entire contents of which are incorporated herein by reference. Drug release in such a system is triggered by colonic microflora coupled with pH- sensitive polymer coatings.

In some embodiments, the formulation is designed as a core tablet with three layers of polymer. The first coating is an acid-soluble polymer (e.g., EUDRAGIT E), the outer coating is enteric, along with a hydroxypropyl methylcellulose barrier layer interposed in between. In some embodiments, colon delivery is achieved by formulating the larazotide or derivative with specific polymers that degrade in the colon such as, for example, pectin. The pectin may be further gelled or cross-linked with a cation such as a zinc cation. Additional colon specific formulations include, but are not limited to, pressure-controlled drug delivery systems (prepared with, for example, ethylcellulose) and osmotic controlled drug delivery systems (i.e., ORDS-CT).

In some embodiments, the delayed-release coating includes an enteric agent that is substantially stable in acidic environments and substantially unstable in near neutral to alkaline environments. In some embodiments, the delayed-release coating contains an enteric agent that is substantially stable in gastric fluid. By way of example, in some embodiments, the enteric agent is selected from: solutions or dispersions of methacrylic acid copolymers, cellulose acetate phthalate, hydroxypropylmethyl cellulose phthalate, polyvinyl acetate phthalate, carboxymethylethylcellulose, EUDRAGIT®-type polymer (poly(methacrylic acid, methylmethacrylate), hydroxypropyl methylcellulose acetate succinate, cellulose acetate trimellitate, shellac, or other suitable enteric coating polymers. In some embodiments, the EUDRAGIT®-type polymer is selected from, for example, EUDRAGIT® FS 30D, L 30 D-55, L 100-55, L 100, L 12,5, L 12,5 P, RL 30 D, RL PO, RL 100, RL 12,5, RS 30 D, RS PO, RS 100, RS 12,5, NE 30 D, NE 40 D, NM 30 D, S 100, S 12,5, and S 12,5 P. In some embodiments, one or more of EUDRAGIT® FS 30D, L 30 D- 55, L 100-55, L 100, L 12,5, L 12,5 P RL 30 D, RL PO, RL 100, RL 12,5, RS 30 D, RS PO, RS 100, RS 12,5, NE 30 D, NE 40 D, NM 30 D, S 100, S 12,5 and S 12,5 P is used. In some embodiments, the enteric agent is a combination of any of the foregoing solutions or dispersions.

The pharmaceutical compositions described herein for targeting the gastrointestinal tract (e.g., the small intestine) can contain less than about 0.5 mg of the larazotide or derivative (e.g., (d)-larazotide). For example, in some embodiments, the pharmaceutical composition contains about 0.4 mg of the peptide or less, or about 0.3 mg of the peptide or less, or about 0.25 mg of the peptide of less, or about 0.2 mg of the peptide or less, or about 0.15 mg of the peptide or less, or about 0.1 mg of the peptide or less, or about 50 pg of the peptide of less, or about 25 pg of the peptide or less. In some embodiments, the pharmaceutical composition contains from about 50 pg to about 400 pg of the peptide, or from about 50 pg to about 200 pg, or from about 50 pg to about 150 pg. In other embodiments, the present invention contemplates a pharmaceutical composition that contains more than about 0.5 mg of peptide. For example, in some embodiments, the pharmaceutical composition contains about 0.6 mg of the peptide or more, or about 0.75 mg of the peptide or more, or about 1.0 mg of the peptide or more, or about 1.25 mg of the peptide or more, or about 1.5 mg of the peptide or more, or about 2.0 mg of the peptide or more. In some embodiments, the peptide is administered at about 0.5 mg. For doses less than about 0.2 mg or above about 1 mg, (d)-larazotide can provide advantages.

In some embodiments, compositions comprising or releasing larazotide or a derivative thereof (e.g., (d)-larazotide) are administered in a regimen of at least once per day. In some embodiments, the compositions are administered in a regimen including administration from 1 to 5 times daily, such as from 1 to 3 times daily.

In some embodiments, the subject further receives a probiotic. Probiotics suitable for use in the present invention include, but are not limited to, Saccharomyces boulardii: Lactobacillus rhamnosus GG; Lactobacillus plantarum 299v; Clostridium butyricum M588; Clostridium difficile VP20621 (non-toxigenic C. difficile strain); combination of Lactobacillus casei. Lactobacillus acidophilus (Bio-K + CL1285); combination of Lactobacillus casei, Lactobacillus bulgaricus, Streptococcus thermophilus (Actimel); combination of Lactobacillus acidophilus, Bifidobacterium bifidum (Florajen3); combination of Lactobacillus acidophilus, Lactobacillus bulgaricus delbrueckii subsp. bulgaricus, Lactobacillus bulgaricus casei, Lactobacillus bulgaricus plantarum, Bifidobacterium longum, Bifidobacterium infantis, Bifidobacterium breve, and Streptococcus salivarius subsp. therm ophilus (VSL#3)).

In accordance with embodiments of the invention, larazotide or a derivative thereof may be delivered in a larazotide-producing probiotic strain, that is where a polynucleotide encoding larazotide or a derivative is heterologously expressed in a microorganism, such as a commensal microorganism of the human gastrointestinal tract, or a microbial species that finds conventional use as a probiotic, as described in International Application No. PCT/US19/19348, which is hereby incorporated by reference in its entirety. The larazotide can contain a secretion signal to direct secretion from the microbial strain. For example, the microorganism may be a bacterium or fungus, and exemplary microorganisms include those of the genus Saccharomyces, Lactobacillus, Clostridium, Streptococcus, Staphylococcus, or Bifidobacterium. For example, the microorganism may be a species selected from Saccharomyces boulardii, Lactobacillus rhamnosus, Lactobacillus plantarum, Clostridium butyricum, non-toxigenic Clostridium difficile, Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus bulgaricus, Streptococcus thermophilus, Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacterium lactis, Bifidobacterium infantis, Bifidobacterium breve, and Streptococcus salivarius. In some embodiments, a probiotic strain (bacterial or fungal) is engineered for expression and optionally secretion of larazotide or derivative thereof from the cell.

In various embodiments, the microorganism is derived from a commensal microorganism of the human gastrointestinal tract, such as those of the genera Bacteroides, Faecalibacterium, Corynebacterium, Eubacterium, Ruminococcus, Peptococcus, Peptostreptococcus, Escherichia, or Helicobacter. In some embodiments, the microbe is E. coli. In some embodiments, the microbe is selected from a Fungal genera of Candida, Saccharomyces, Aspergillus, Penicillium, Rhodotorula, Trametes, Pleospora, Sclerotinia, Bullera, and Galactomyces. In accordance with embodiments of the invention, larazotide or a derivative thereof may be delivered in a larazotide-encoding bacteriophage that infects microbes in the GI tract of the subject. In some embodiments, the present invention contemplates a bacteriophage comprising a polynucleotide encoding a peptide that comprises the amino acid sequence of larazotide (SEQ ID NO: 1) or a derivative thereof. The polynucleotide further comprises a promoter controlling expression of the polynucleotide in a host bacterium, as described. The host bacterium may be of the genus Lactobacillus, Clostridium, Streptococcus, or Bifidobacterium.

Generally, the host bacterium is a commensal microorganism of the human gastrointestinal tract, and may belong to the genera Bacteroides, Faecalibacterium, Eubacterium, Ruminococcus, Peptococcus, Peptostreptococcus, Escherichia, or Helicobacter. In some embodiments, the host bacterium is E. coli.

The bacteriophage may further encode a secretory signal at the N-terminus of the peptide (as described), so as to drive secretion of the peptide from the host cell. The secretory signal may be cleaved by the host bacteria upon export of the peptide out of the cell. Various types of bacteriophages may be engineered in accordance with these embodiments, including lytic and lysogenic bacteriophages. In some embodiments, the phage is a lytic phage, allowing release of peptide upon lysis of the host cell, rather than through use of a signal peptide.

Exemplary bacteriophages include those of the order Caudovirales, Siphoviridae, Myoviridae, or Podoviridae.

In some embodiments, the bacteriophage is a coliphage, such as lambda phage, M13, T7, T4, or T3 bacteriophage. In other embodiments, the bacteriophage is lactobacillus phage, such as phages infecting Lactobacillus delbrueckii subsp. bulgaricus, known as Ld3, Ldl7, and Ld25A. Casey E., Molecular Characterization

Lactobacillus delbrueckii subsp. bulgaricus Phages, Appl. Environ. Microbiol. 2014 vol. 80 no. 18 5623-5635. Phages can be engineered to optimize the spectrum of infection. Various other phages have been isolated from human feces, which can be used in accordance with this disclosure. Breitbart M, Metagenomic Analyses of an Uncultured Viral Community from Human Feces, J Bacteriol.

Vol. 185, No. 20 pages 6220-6223 (2003).

Where larazotide or derivative is delivered as a larazotide-producing probiotic, the probiotic may be delivered, for example, about once daily, about once weekly, or about once monthly.

In some embodiments, the patient further receives therapy with an anti-fibrotic agent such as pirfenidone or nintedanib. In some embodiments, the patient further receives therapy with a corticosteroid, such as cortisone, hydrocortisone, prednisone, dexamethasone, and betamethasone. In various embodiments, the combined treatment with larazotide can be synergistic.

In other aspects, this disclosure provides a pharmaceutical composition for the treatment of pulmonary fibrosis. The composition comprises larazotide or derivative thereof, and an anti-fibrotic agent and/or a glucocorticoid. In various embodiments, the composition is formulated for enteral delivery. The larazotide unit dose and enteric formulation can be as described herein.

EXAMPLES

Example 1 : Dexamethasone and Larazotide Reduces Fibrosis

Pathogen-free 6 weeks old female C57BL/6J mice were obtained. At Day 0, 48 mice were induced to develop pulmonary fibrosis by a single intratracheal administration of bleomycin hydrochloride (BLM) in saline at a dose of 3.0 mg/kg, in a volume of 50 pL per animal using Microsprayer® (Penn-Century, USA).

The 48 mice were randomized into 4 groups of 12 mice based on the body weight on the day before the BLM administration. Individual body weight was measured daily during the experimental period. Survival, clinical signs, and behavior of mice were monitored daily. If an animal shows >40% body weight loss compared to Day 0, and/or if it shows a moribundity sign such as prone position, the animal will be euthanized ahead of study termination. Samples will not be collected from euthanized animals. The mice were grouped for the study as follows:

Group 1 (Vehicle): Twelve BLM-induced pulmonary fibrosis model mice were orally administered vehicle [pure water] in a volume of 10 mL/kg once daily from Day 0 to 20;

Group 2 (Larazotide-po): Twelve BLM-induced pulmonary fibrosis model mice were orally administered vehicle supplemented with larazotide at a dose of 1 mg/kg once daily from Day 0 to 20;

Group 3 (Larazotide-it): Twelve BLM-induced pulmonary fibrosis model mice were intratracheally administered vehicle supplemented with larazotide at a dose of 25 pg/mouse in a volume of 50 pL/mouse once weekly from Day 0 to 20 (Day 0, 7 and 14); and

Group 4 (Dexamethasone): Twelve BLM-induced pulmonary fibrosis model mice were orally administered pure water supplemented with Dexamethasone at a dose of 0.25 mg/kg once daily from Day 0 to 20.

Mice in all groups were sacrificed at Day 21 for the following assays:

Survival analysis: Survival curves will be established using the Kaplan-Meier survival method and compared using the Log Rank test.

Measurement of lung weight: Individual lung weight (left and post-caval lobes) will be measured.

Biochemical analysis: Lung hydroxyproline will be quantified by a hydrolysis method.

Histological analyses for lung sections: Masson's Trichrome staining and estimation of Ashcroft score.

Sample collection: After completion of the in-life portion of the study, frozen lung samples were collected for further analyses. Statistical tests were performed using Bonferroni Multiple Comparison Test. P values <0.05 were considered statistically significant.

Results are shown in FIG. 1 and FIG. 2. FIG. 1 shows representative photomicrographs of Masson’s Trichome-stained lung sections. Both Dexamethasone and larazotide samples show substantially less fibrosis than vehicle. FIG. 2 shows the Ashcroft score from the groups. As shown, all treatment groups demonstrate a lower score than vehicle. Larazotide-po was somewhat more effective than larazotide-it, and at least as effective as dexamethasone.

Example 2: All D-Amino Acid Larazotide Analog Reduces Pulmonary Fibrosis

An experiment was conducted to evaluate the effect of larazotide and all d-amino acid larazotide on lung fibrosis in bleomycin (BLM)-induced pulmonary fibrosis model.

The experiment consisted of 3 study groups: (1) Group 1, which represented the vehicle group, consisted of 12 BLM-induced pulmonary fibrosis model mice that were orally administered vehicle (pure water) in a volume of 10 mL/kg once daily from Day 0 to 20; (2) Group 2, which represented the larazotide group, consisted of 12 BLM-induced pulmonary fibrosis model mice that were orally administered vehicle supplemented with larazotide at a dose of 1 mg/kg in a volume of 10 mL/kg once daily from Day 0 to 20; and Group 3, which represented the all d-larazotide group, consisted of 12 BLM-induced pulmonary fibrosis model mice that were orally administered vehicle supplemented with all d-larazotide at a dose of 1 mg/kg in a volume of 10 mL/kg once daily from Day 0 to 20.

The animals were monitored daily for viability, clinical signs, and behavior. Body weight was recorded daily, and mice were observed for significant clinical signs of toxicity, moribundity, and mortality. FIG. 3 depicts the changes in body weight over the course of the study in days after BLM administration when the mice were given vehicle, larazotide, or all d-larazotide. FIG. 4 shows survival over the course of the study.

The animals were sacrificed on Day 21 (FIG. 5 shows body weight of the mice on the day of sacrifice), and lung samples were collected. Specifically, left and post-caval lobe bronchus were ligated to avoid leakage of the fixative. FIG. 6 shows left lung weight, and FIG. 7 shows post-caval lobe weight of the mice on the day of sacrifice. An indwelling needle was inserted into the trachea and connected to instillation route of syringe. The syringe was then loaded 10% neutral buffered formalin and kept at the height of 20 cm. Then, superior (A), middle (B) and inferior lobes (C) were instilled 10% neutral buffered formalin and ligated after inflation. Three fixed lobes (for histological analyses), unfixed left lung (E) (for biochemistry) and unfixed post-caval lobe (D) were harvested. Two unfixed lobes were washed with cold saline and measured for wet weight. Three fixed lobes were fixed in 10% neutral buffered formalin for 24 hours. After fixation, these specimens proceeded to paraffin embedding for Masson's trichrome staining.

In order to quantify lung hydroxyproline content, frozen left lung samples were processed by an acid hydrolysis method as follows. Lung samples were acid-hydrolyzed with 300 pL of 6N HC1 at 121°C for 20 minutes, and neutralized with 300 pL of 4N NaOH containing 10 mg/mL activated carbon. AC buffer (2.2M acetic acid/0.48M citric acid, 300 pL) was then added to the samples, followed by centrifugation to collect the supernatant. A standard curve of hydroxyproline was constructed with 16, 8, 4, 2, 1 and 0.5 pg/mL of trans- 4-hydroxy-L-proline (Sigma-Aldrich Co. LLC., USA Code: 54409). The prepared samples and standards (each 400 pL) were mixed with 400 pL chloramine T solution (NACALAI TESQUE, INC., Japan, Code: 08005-52) and incubated for 25 minutes at room temperature. The samples were then mixed with Ehrlich's solution (400 pL) and heated at 65°C for 20 minutes to develop the color. After the samples were cooled on ice and centrifuged to remove precipitates, the optical density of each supernatant was measured at 560 nm. The concentrations of hydroxyproline were calculated from the hydroxyproline standard curve, as depicted in FIG. 8. Lung hydroxyproline contents are expressed as pg per left lung.

Histological analyses of the samples were also conducted. Right lung tissues prefixed in 10% neutral buffered formalin were embedded in paraffin and sectioned at 4pm. For Masson’s Tri chrome staining, the sections were deparaffinized and rehydrated, followed by re-fixation with Bouin’s solution for 15 minutes. The sections were stained in Weigert’s iron Hematoxylin working solution (Sigma-Aldrich), Biebrich scarlet-Acid fuchsin solution (SigmaAldrich), Phosphotungstic/phosphomolybdic Acid solution, Aniline blue solution, and 1% Acetic Acid solution (Sigma-Aldrich). For quantitative analysis of lung fibrosis area, bright field images of Masson’s Trichrome-stained sections were randomly captured using a digital camera (DFC295; Leica, Germany) at 100-fold magnification, and the subpleural regions in 20 fields/mouse were evaluated according to the criteria for grading lung fibrosis (Ashcroft, T., et al., J Clin Pathol, 1988; 41 :467-70), as shown below:

Table 1: Criteria for grading lung fibrosis

FIG. 9 shows sections of right lung tissue of mice that were administered vehicle, larazotide, and all d-larazotide, respectively, after being subjected to the staining process described above. FIG. 10 depicts the grading results of the stained right lung sections per the Ashcroft evaluation and grading criteria. The all d-larazotide right lung samples exhibit less fibrosis than either the vehicle or larazotide samples.

Claims

CLAIMS:

1. A method for treating or preventing pulmonary fibrosis in a patient, comprising: administering an effective amount of larazotide or a derivative thereof, to a patient in need.

2. The method of claim 1, wherein the patient has a chronic inflammatory lung disease.

3. The method of claim 2, wherein the patient has cystic fibrosis, asthma, chronic obstructive pulmonary disease (COPD), emphysema, chronic bronchitis, chronic pneumonia, or interstitial lung disease.

4. The method of claim 3, wherein the patient has an interstitial lung disease selected from sarcoidosis and idiopathic pulmonary fibrosis (TPF).

5. The method of claim 1, wherein the patient is a tobacco smoker or former tobacco smoker.

6. The method of any one of claims 1 to 3, wherein the patient is undergoing therapy with a drug that induces pulmonary fibrosis as a side effect, and which is optionally nitrofurantoin, methotrexate, amiodarone, bleomycin or cyclophosphamide.

7. The method of any one of claims 1 to 6, wherein a larazotide derivative is administered having the amino acid sequence of SEQ ID NO: 1 with one or more (d) amino acids.

8. The method of claim 7, wherein (d)-larazotide is administered.

9. The method of any one of claims 1 to 8, wherein the larazotide or derivative thereof is administered by pulmonary delivery.

10. The method of claim 9, wherein the larazotide or derivative thereof is administered by solution of powder aerosol.

11. The method of claim 9, wherein the larazotide or derivative thereof is administered endotracheally.

12. The method of any one of claims 1 to 8, wherein the larazotide or derivative thereof is administered parenterally.

13. The method of any one of claims 1 to 8, wherein the larazotide or derivative thereof is administered to the small and/or large intestine.

14. The method of claim 13, wherein the larazotide or derivative is administered to one or more of the duodenumjejunum, and/or the ileum.

15. The method of claim 13 or 14, wherein the larazotide or derivative is administered to the large intestine.

16. The method of any one of claims 13 to 15, wherein the larazotide or derivative is administered in a composition comprising an enteric coating.

17. The method of claim 16, wherein the composition provides for a sustained or controlled release of larazotide or derivative thereof.

18. The method of any one of claims 9 to 17, wherein the larazotide or derivative thereof is administered from 1 to 3 times per day.

19. The method of any one of claims 1 to 6, wherein larazotide is delivered by expression in a bacterial strain or bacteriophage that is administered to the gastrointestinal tract of the patient.

20. The method of claim 19, wherein the bacterial strain or bacteriophage is administered about once weekly or about once or twice monthly.

21. The method of any one of claims 1 to 20, wherein the patient further receives therapy with an anti-fibrotic agent or corticosteroid.

22. A pharmaceutical composition for the treatment of pulmonary fibrosis, the composition comprising larazotide or derivative thereof, and an anti-fibrotic agent and/or a glucocorticoid.

23. The pharmaceutical composition of claim 22, wherein the composition is formulated for enteral delivery.