WO2022069701A1 - Methods of treating pulmonary fibrosis - Google Patents

Abstract

The present disclosure relates to methods of preventing the onset of or treating pulmonary fibrosis, e.g., pulmonary fibrosis associated with a coronavirus infection or an aberrant level of ACE2. The methods generally include administering to a patient in need thereof a therapeutically effective amount of a peroxisome proliferator-activated receptor gamma (PPAR-γ) modulator or a pharmaceutically acceptable salt thereof, such as the compound of Formula (I).

Description

METHODS OF TREATING PULMONARY FIBROSIS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of, and priority to, U.S. Provisional Patent Application No. 63/086,189, filed on October 1, 2020; the disclosure of which is hereby incorporated by reference in its entirety for all purposes.

BACKGROUND

[0002] Pulmonary fibrosis is a condition in which the lungs become scarred over time. Pulmonary fibrosis is also commonly observed in and associated with coronavirus infectious diseases. For example, in fatal cases of COVID-19, pulmonary fibrosis is generally present at the autopsy, with anecdotal reports of severe fibrotic organizing pneumonia. While recent studies shed light on antifibrotic therapies in preventing or treating coronavirus infection in patients with pulmonary fibrosis, there is also a need to develop antifibrotic therapies, such as small molecules, for preventing the onset of or treating fibrosis after a coronavirus infection.

[0003] Accordingly, there remains the need for identifying new therapeutic agents for stopping or reversing pulmonary fibrosis, as well as in-depth understanding of their molecular mechanisms. The present disclosure provides methods for preventing or treating pulmonary fibrosis, such as pulmonary fibrosis associated with a coronavirus infection.

SUMMARY

[0004] In various embodiments, the present disclosure provides methods for preventing the onset of or treating pulmonary fibrosis, such as pulmonary fibrosis associated with a coronavirus infection or an aberrant ACE2 level. In some embodiments, the present disclosure provides a method of treating pulmonary fibrosis in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a peroxisome proliferator-activated receptor gamma (PPAR-y) modulator or a pharmaceutically acceptable salt thereof, wherein the patient is, or was previously, infected with a coronavirus, and the PPAR-y modulator is a compound of Formula (I):

[0005] In some embodiments, the therapeutic effect of a method may be determined by one or more of: a) reduced level of myofibroblasts in lung tissue of the patient as compared to a control value; b) reduction of myofibroblast differentiation in lung tissue of the patient as compared to a control value; c) increased level of one or more genes, or one or more proteins encoded by the one or more genes, selected from the group consisting of CDH1 and OCLN or decreased level of one or more genes, or one or more proteins encoded by the one or more genes, selected from the group consisting of TGFB1, ACTA2, COL1A1, FN1, MUC5B, W MAPK3 in lung tissue of the patient as compared to a control value; and d) increased level of one or more genes, or one or more proteins encoded by the one or more genes, selected from the group consisting of A CE2 w MASR or decreased level of gene TACE, or one or more proteins encoded by TACE, in lung tissue of the patient as compared to a control value.

[0006] In some embodiments, the therapeutic effect of a method may be determined by reduction of myofibroblast differentiation by at least about 50% in lung tissue of the patient as compared to a control value.

[0007] In some embodiments, the therapeutic effect of a method may be determined by reduction of myofibroblast differentiation in lung tissue of the patient as compared to a control value determined using nintedanib or pirfenidone.

[0008] In some embodiments, the therapeutic effect of a method may be determined by decreased level of gene COL1A1, or one or more proteins encoded by COL1A1, by at least about 70% in lung tissue of the patient as compared to a control value.

[0009] In some embodiments, the therapeutic effect of a method may be determined by decreased level of gene .4 ( 7 2. or one or more proteins encoded by ACTA2, by at least about 80% in lung tissue of the patient as compared to a control value.

[0010] In some embodiments, the therapeutic effect of a method may be determined by decreased level of gene MAPK3, or one or more proteins encoded by MAPK3, in lung tissue of the patient as compared to a control value. [0011] In various embodiments, the present disclosure provides a method of preventing the onset of pulmonary fibrosis in a patient having a coronavirus infection, and in need thereof, comprising administering to the patient a therapeutically effective amount of a compound of Formula (I):

or a pharmaceutically acceptable salt thereof.

[0012] In some embodiments, the therapeutic effect of a method may be determined by one or more of: a) inhibition of aberrant myofibroblast differentiation in lung tissue of the patient as compared to a control value; b) inhibition of aberrant level of one or more genes, or one or more proteins encoded by the one or more genes, selected from the group consisting of TGFB1, ACTA2, COL1A1, FN1, CDH1, OCLN, MUC5B, and MAPK3 in lung tissue of the patient as compared to a control value; and c) inhibition of aberrant decrease in level of one or more genes, or one or more proteins encoded by the one or more genes, selected from the group consisting of ACE2 and MASR or aberrant increase in level of gene TACE, or one or more proteins encoded by TACE, in lung tissue of the patient as compared to a control value.

[0013] In various embodiments, the present disclosure provides a method of preventing or treating pulmonary fibrosis in a patient having an aberrant level of ACE2, and in need thereof, comprising administering to the patient a therapeutically effective amount of a peroxisome proliferator-activated receptor gamma (PPAR-y) modulator or a pharmaceutically acceptable salt thereofln some embodiments, the PPAR-y modulator is a compound of Formula (I):

[0014] In some embodiments, the aberrant level of ACE2 of a method is a decreased level of ACE2 in lung tissue of the patient as compared to a control value. In some embodiments, the level of ACE2 is gene level or protein level.

[0015] In some embodiments, the pulmonary fibrosis associated with aberrant level of ACE2 may be a pulmonary fibrosis associated with a viral infection. In some embodiments, the viral infection may be a severe acute respiratory syndrome coronavirus (SARS-CoV), SARS- CoV-2, or Middle East respiratory syndrome coronavirus (MERS-CoV).

[0016] In various embodiments, the present disclosure provides a method of preventing or treating pulmonary fibrosis in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a peroxisome proliferator-activated receptor gamma (PPAR- y) modulator or a pharmaceutically acceptable salt thereof, wherein administering the PPAR-y modulator or a pharmaceutically acceptable salt thereof results in inhibition or reduction of myofibroblast differentiation in lung tissue of the patient, and the PPAR-y modulator is a compound of Formula (I):

H₂N ^^T (I)

[0017] In some embodiments, the inhibition or reduction of myofibroblast differentiation of a method may be determined by measuring one or more genes, or one or more proteins encoded by the one or more genes, selected from the group consisting of TGFB1, ACTA2, COL1A1, FN1, CDH1, OCLN, MUC5B, and MAPK3 in lung tissue of the patient, wherein: a) inhibition of aberrant level of one or more of TGFB1, ACTA2, COL1A1, FN1, CDH1, OCLN, MUC5B, and MAPK3 as compared to a control value indicates the inhibition of myofibroblast differentiation, or b) reduced level of one or more of TGFB1, ACTA2, COL1A1, FN1, MUC5B, and MAPK3 as compared to a control value or increased level of one or more of CDH1 and OCLN as compared to a control value indicates the reduction of myofibroblast differentiation.

[0018] In some embodiments, the control value of any method may be determined by measuring myofibroblasts or one or more genes, or one or more proteins encoded by the one or more genes, in a corresponding healthy tissue or in a sample from the patient collected prior to administering the PPAR-y modulator. In some embodiments, the one or more genes may be selected from the group consisting of TGFB1, ACTA2, COL1A1, FN1, CDH1, OCLN, MUC5B, MAPK3,ACE2 an&MASR. In some embodiments, the control value may be determined by measuring ACE2 in a corresponding healthy tissue.

[0019] In some embodiments, a coronavirus of the present disclosure may be SARS-CoV, SARS-CoV -2, or MERS-CoV. [0020] In some embodiments, a PPAR-y modulator or a compound of Formula (I), or a pharmaceutically acceptable salt thereof, of the present disclosure may be administered at a dose of about 5 mg/kg/day to about 100 mg/kg/day.

[0021] In some embodiments, a PPAR-y modulator or a compound of Formula (I), or a pharmaceutically acceptable salt thereof, of the present disclosure may be administered orally.

[0022] In some embodiments, administering a PPAR-y modulator or a compound of Formula (I), or a pharmaceutically acceptable salt thereof, of the present disclosure does not alter expression of TMPRSS2 in the patient.

[0023] In some embodiments, a PPAR-y modulator or a compound of Formula (I) of the disclosure is (S)-2-methoxy-3-(4’-aminophenyl)propionic acid.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to understand the invention and to demonstrate how it may be carried out in practice, embodiments are now described, by way of non-limiting example only, with reference to the accompanying drawings in which:

[0025] FIG. 1 shows the mRNA expression levels of TGFB1 (a), ACTA (b), COL1A1 (c), and FN1 (d) and the protein expression levels of a-SMA (e and f) and collagen (g) in MRC-5 cells induced for myofibroblast activation with TGFpi. Data represent the fold change compared to control cells (CNRL) and are expressed as mean ± SEM; *= p< 0.05; **= p< 0.01; ***= p< 0.001; ns= not significant.

[0026] FIG. 2 shows the mRNA expression levels of TGFB1 (a), ACTA (b), COL1A1 (c), FN1 (d), CDH1 (e), and OCLN (f) and the protein expression levels of a-SMA (g and h) and collagen (i) in A549 cells induced for myofibroblast activation with TGFp. Data represent the fold change compared to control cells (CNRL) and are expressed as mean ± SEM; *= p< 0.05; **= p< 0.01; ***= p< 0.001; ns= not significant.

[0027] FIG. 3 shows the mRNA expression levels of TGFB1 (a), ACTA (b), COL1A1 (c), and FN1 (d) and the protein expression levels of a-SMA (e), collagen (f), and fibronectin (g) in human fibroblast cells induced for myofibroblast activation with TGFpi . Data represent the fold change compared to control cells (CNRL) and are expressed as mean ± SEM; *= p< 0.05; **= p< 0.01; ***= p< 0.001; ns= not significant. [0028] FIG. 4 shows the mRNA expression levels of TNF (a and c) and IL1B (b and d) in preventive mode (a and b) and curative mode (c and d) in frozen lungs of survived mice. Data represent the fold change compared to PBS mice and are expressed as mean ± SEM; *= p< 0.05; **= p< 0.01; ***= p< 0.001; ns= not significant.

[0029] FIG. 5 shows the mRNA expression levels of TGFB1 (a), ACTA (b), COL1A1 (c), FN1 (d), CDH1 (e), OCLN (I), and MUC5B (g) and the protein expression levels of a-SMA (h and i) and collagen (j and k) in preventive mode in male mice induced for lung fibrosis with bleomycin. Data represent the fold change compared to PBS mice and are expressed as mean ± SEM; *= p< 0.05; **= p< 0.01; ***= p< 0.001; ns= not significant.

[0030] FIG. 6 shows the mRNA expression levels of TGFB1 (a), ACTA (b), COL1A1 (c), FN1 (d), CDH1 (e), OCLN (I), and MUC5B (g) and the protein expression levels of a-SMA (h and i) and collagen (j and k) in curative mode in male mice induced for lung fibrosis with bleomycin. Data represent the fold change compared to PBS mice and are expressed as mean ± SEM; *= p< 0.05; **= p< 0.01; ***= p< 0.001; ns= not significant.

[0031] FIG. 7 shows the mRNA expression levels oiMAPK3 (a), ACE2 (b), TACE (c), and MASR (d) in male mice induced for lung fibrosis with bleomycin. Data represent the fold change compared to PBS mice and are expressed as mean ± SEM; *= p< 0.05; **= p< 0.01; ***= p< 0.001; ns= not significant.

DETAILED DESCRIPTION

[0032] It has now been discovered that a PPAR-y modulator (e.g. , a PPAR-y agonist), such as a compound of Formula (I), or a pharmaceutically acceptable salt thereof, can be used in methods of preventing the onset of or treating pulmonary fibrosis, e.g, pulmonary fibrosis associated with a coronavirus infection or an aberrant level of ACE2.

[0033] As used herein, the term “pharmaceutically acceptable” refers to a material that is not biologically or otherwise undesirable, i.e. the material can be administered to an individual along with the relevant active compound without causing clinically unacceptable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.

[0034] The term “pharmaceutically acceptable salts,” as used herein, shall be given its ordinary meaning and refers to the relatively non-toxic, inorganic and organic acid or base addition salts of compounds, including, for example, a compound of Formula (I). [0035] The term “pharmaceutically acceptable carrier,” as used herein, shall be given its ordinary meaning and refers to a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting any subject composition or component thereof from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the subject composition and its components and not injurious to the patient. Examples of materials which may serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as com starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer’s solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.

[0036] As used herein, the term “pharmaceutical composition” means, for example, a mixture containing a specified amount of a therapeutic compound, e g., a therapeutically effective amount of a therapeutic compound, in a pharmaceutically acceptable carrier to be administered to a mammal, e.g. , a human, in order to treat or prevent fibrosis, and/or symptoms thereof. In embodiments herein are pharmaceutical compositions including a compound, such as a PPAR-y modulator, and a pharmaceutically acceptable carrier. In another aspect, the disclosure discloses the use of a PPAR-y modulator in the manufacture of a medicament for treating or preventing fibrosis. “Medicament,” as used herein, has essentially the same meaning as the term “pharmaceutical composition.”

[0037] The terms “disease,” “disorder,” and “condition” are used interchangeably herein.

[0038] A “patient,” as described herein, refers to any animal suffering from or diagnosed with a disease or disorder, such as pulmonary fibrosis, e.g, pulmonary fibrosis associated with a coronavirus infection or an aberrant level of ACE2, including, but not limited to, mammals, primates, and humans. In certain embodiments, the patient may be a non-human mammal such as, for example, a cat, a dog, or a horse. In a preferred embodiment, the patient is a human or a human subject. [0039] As used herein, a “subject” refers to an individual. Thus, the “subject” can include domesticated animals (e.g., cats, dogs, etc.), livestock (e.g, cattle, horses, pigs, sheep, goats, etc.), laboratory animals (e.g, mouse, rabbit, rat, guinea pig, etc.), and birds. “Subject” can also include a mammal, such as a primate or a human. The terms “subject” and “patient” may be used interchangeably, however, in some embodiments a subject may not be diagnosed with or is suffering from a disease or disorder, though may be in need of therapy.

[0040] Patients or subjects in need of treatment by the methods described herein include patients or subjects having pulmonary fibrosis, a viral infection (e.g, a coronavirus (e.g, SAR- CoV, SARS-CoV2, MERS-CoV) infection), or an aberrant ACE2 level.

[0041] “Treating,” includes any effect, e.g., lessening, reducing, modulating, preventing, eliminating, or reversing, that results in the improvement of a condition, disease, disorder, symptom, and the like. “Treating” or “treatment” of a disease state includes: (1) inhibiting the disease state, i.e., arresting the development of the disease state or its clinical symptoms; (2) relieving the disease state, i.e., causing temporary or permanent reversion or regression of the disease state or its clinical symptoms; (3) reducing or lessening the symptoms of the disease state; or (4) preventing the disease state, e.g., causing the clinical symptoms of the disease state not to develop in a subject that may be exposed to or predisposed to the disease state, but does not yet experience or display symptoms of the disease state.

[0042] As used herein, “preventing” or “prevent” describe reducing or eliminating the onset of the symptoms or complications of the disease, condition or disorder. The term “preventing,” when used in relation to a condition, such as fibrosis, is art-recognized, and relates to the ability of a formulation, composition, and/or device to reduce the frequency of, or delay the onset of, signs and/or symptoms of a medical condition in a subject relative to a subject which does not receive the composition. Insofar as the methods of the present disclosure are directed to preventing disorders, it is understood that the term “prevent” does not require that the disease state be completely thwarted.

[0043] By “reduce” or other forms of the word, such as “reducing” or “reduction,” is meant lowering of an event or characteristic (e.g, fibrosis). It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary for the standard or relative value to be referred to.

[0044] “Effective amount,” as used herein, refers to an amount sufficient to elicit the desired biological response. Desired biological responses include, but are not limited to, prevention of a disease onset, a decrease in the rate of disease progression, amelioration or palliation of the disease state, alleviation of symptoms, stabilizing or not worsening the disease state, remission or improved prognosis, and prolonged survival. As will be appreciated by those of ordinary skill in this art, the effective amount of a compound of the invention may vary depending on such factors as the desired biological endpoint, the pharmacokinetics of the compound, the disease being treated, the mode of administration, and the age, health, and condition of the subject. In some embodiments, an effective amount of a disclosed compound (e.g, a PPAR-y modulator) is the amount to treat pulmonary fibrosis in a patient such that the administration of the agent elicits a desired effect, such as remission, sustained remission, and/or improvement of quality of life of patients.

[0045] As used herein, a “therapeutically effective amount” of an agent is an amount sufficient to provide a therapeutic benefit in the treatment of a disease, disorder, or condition, or to delay or minimize one or more symptoms associated with the disease, disorder, or condition. A therapeutically effective amount of an agent means an amount of therapeutic agent, alone or in combination with other therapies, which provides a therapeutic benefit in the treatment of the disease, disorder, or condition. The term “therapeutically effective amount” can encompass an amount that improves overall therapy, reduces or avoids symptoms or causes of disease or condition, or enhances the therapeutic efficacy of another therapeutic agent.

[0046] As used herein, “upregulation” or “upregulated” refers to the increased level, i.e., increase of quantity, of a cellular component, such as a gene or a protein. “Downregulation” or “downregulated” refers to the decreased level, i.e., decrease of quantity, of a cellular component, such as a gene or a protein. Upregulation (or increased level) of a protein refers to expression of the protein that is elevated compared to a selected control. Downregulation (or decreased level) of a protein refers to expression of the protein that is reduced compared to a selected control. As an illustrative example, certain genes (e.g., ACE2) in a pulmonary fibrosis patient may be downregulated as compared to a healthy subject.

[0047] The term “pulmonary fibrosis” (“PF”), as used herein, shall be given its ordinary meaning, as a lung disease in which the lung tissue becomes damaged and scarred over time. “Idiopathic pulmonary fibrosis” (“IPF”) is a type of pulmonary fibrosis of unknown cause.

[0048] The term “viral infection,” as used herein, shall be given its ordinary meaning, as a general term for diseases in which harmful virus(es) proliferate in the subject. Exemplary viral infections may be caused by, for example, coronaviruses. Coronaviruses are a large family of viruses, including SARS coronavirus (SARS-CoV) which may cause severe acute respiratory syndrome (SARS), MERS coronavirus (MERS-CoV) which may cause Middle East respiratory syndrome (MERS), and SARS-CoV-2 which may cause coronavirus disease 2019 (COVID-19).

[0049] Throughout the description and claims of this specification the word “comprise” and other forms of the word, such as “comprising” and “comprises,” means including but not limited to, and is not intended to exclude, for example, other additives, components, integers, or steps.

[0050] Throughout the description, where formulations are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are formulations of the present disclosure that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present disclosure that consist essentially of, or consist of, the recited processing steps.

[0051] “Optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0052] The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present disclosure. As used throughout this disclosure, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to “a composition” includes a plurality of such compositions, as well as a single composition, and a reference to “a therapeutic agent” is a reference to one or more therapeutic and/or pharmaceutical agents and equivalents thereof known to those skilled in the art, and so forth.

[0053] In the application, where an element or component is said to be included in and/or selected from a list of recited elements or components, it should be understood that the element or component can be any one of the recited elements or components, or the element or component can be selected from a group consisting of two or more of the recited elements or components.

[0054] Further, it should be understood that elements and/or features of a composition or a method described herein can be combined in a variety of ways without departing from the spirit and scope of the present invention, whether explicit or implicit herein. For example, where reference is made to a particular compound, that compound can be used in various embodiments of compositions of the present invention and/or in methods of the present invention, unless otherwise understood from the context. In other words, within this application, embodiments have been described and depicted in a way that enables a clear and concise application to be written and drawn, but it is intended and will be appreciated that embodiments may be variously combined or separated without parting from the present teachings and invention(s). For example, it will be appreciated that all features described and depicted herein can be applicable to all aspects of the invention(s) described and depicted herein.

[0055] It should be understood that the expression “at least one of’ includes individually each of the recited objects after the expression and the various combinations of two or more of the recited objects unless otherwise understood from the context and use. The expression “and/or” in connection with three or more recited objects should be understood to have the same meaning unless otherwise understood from the context.

[0056] The use of the term “include,” “includes,” “including,” “have,” “has,” “having,” “contain,” “contains,” or “containing,” including grammatical equivalents thereof, should be understood generally as open-ended and non-limiting, for example, not excluding additional unrecited elements or steps, unless otherwise specifically stated or understood from the context.

[0057] Where the use of the term “about” is before a quantitative value, the present invention also includes the specific quantitative value itself, unless specifically stated otherwise. As used herein, the term “about” refers to a ±10% variation from the nominal value unless otherwise indicated or inferred from the context.

[0058] The use of any and all examples, or exemplary language herein, for example, “such as” or “including,” is intended merely to illustrate better the present invention and does not pose a limitation on the scope of the invention unless claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the present invention.

[0059] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The abbreviations used herein have their conventional meaning within the chemical and biological arts. The chemical structures and formulae set forth herein are constructed according to the standard rules of chemical valency known in the chemical arts. In the case of conflict, the present specification will control. [0060] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents and other references mentioned herein are incorporated by reference. The references cited herein are not admitted being prior art to the claimed disclosure. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods and examples are illustrative only and are not intended to be limiting.

PPAR-y Modulators

[0061] Provided herein are peroxisome proliferator-activated receptor gamma (PPAR-y or PPARG) modulators, generally useful for treatment of pulmonary fibrosis, e.g., pulmonary fibrosis associated with a coronavirus infection or an aberrant level of ACE2. A PPAR-y modulator of the present disclosure may be any agent that can modulate and/or regulate biological function of PPAR-y. In some embodiments, a PPAR-y modulator of the present disclosure may induce anti-inflammatory effects. A PPAR-y modulator may be endogenous or synthetic ligands of PPAR-y. In some embodiments, PPAR-y modulators of the present disclosure may be agonists of PPAR-y.

[0062] In some embodiments, the PPAR-y modulator is a compound of Formula (I):

or a pharmaceutically acceptable salt thereof. The compound of Formula (I) may also be referred to herein as (S)-2-methoxy-3-(4’-aminophenyl)propionic acid or as GED-0507.

[0063] The compound of Formula (I), or a pharmaceutically acceptable salt thereof, can be made by methods known in the art, for example, as described in International Publication No. WO 2020/161362.

Pharmaceutical Compositions and Routes of Administration

[0064] In various embodiments, provided herein are pharmaceutical compositions comprising a PPAR-y modulator, such as a compound of Formula (I):

or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients and/or carriers. Pharmaceutically acceptable excipients and carriers include inert solid diluents and fillers, diluents, including sterile aqueous solution and various organic solvents, permeation enhancers, solubilizers and adjuvants, for the treatment of pulmonary fibrosis (e.g., pulmonary fibrosis associated with a coronavirus infection or an aberrant level of ACE2).

[0065] Examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, sterile water, syrup, and methyl cellulose. The pharmaceutical compositions can additionally include lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as methyl and propylhydroxy-benzoates; sweetening agents; and flavoring agents.

[0066] In some embodiments, a pharmaceutical composition of the disclosure may further comprise a second therapeutic agent, for example another antifibrotic agent such as pirfenidone or nintedanib.

[0067] The pharmaceutical compositions provided herein may be administered alone or in combination with other therapeutic agents, such as the agents or therapies described herein.

[0068] Pharmaceutical compositions containing comprising a PPAR-y modulator, such as a compound of Formula (I), or a pharmaceutically acceptable salt thereof, can be presented in a dosage unit form and can be prepared by any suitable method. A pharmaceutical composition should be formulated to be compatible with its intended route of administration. Useful formulations can be prepared by methods well known in the pharmaceutical art. For example, see Remington's Pharmaceutical Sciences, 18th ed. (Mack Publishing Company, 1990).

[0069] Pharmaceutical compositions provided herein may be administered in either single or multiple doses by any of the accepted modes of administration of agents having similar utilities, including rectal, buccal, intranasal and transdermal routes, by intra-arterial injection, intravenously, intraperitoneally, parenterally, intramuscularly, subcutaneously, orally, topically, as an inhalant, or via an impregnated or coated device such as a stent, for example, or an artery- inserted cylindrical polymer. In some embodiments, a pharmaceutical composition may be administered orally. [0070] Oral administration may be the route for administration of compounds or pharmaceutical compositions of the disclosure. Administration may be via capsules, enteric coated tablets, solutions, or the like. In making the pharmaceutical compositions that include at least one compound described herein, the active ingredient is usually diluted by an excipient and/or enclosed within such a carrier that can be in the form of a capsule, sachet, paper or other container. When the excipient serves as a diluent, it can be in the form of a solid, semi-solid, or liquid material, which may act as a vehicle, carrier, or medium for the active ingredient. Thus, the pharmaceutical compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments, soft and hard gelatin capsules, sterile injectable solutions, or sterile packaged powders.

[0071] In some embodiments, compounds or pharmaceutical compositions provided herein may be administered orally in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, boluses, electuaries, or pastes, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in- water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia).

[0072] In solid dosage forms for oral administration (capsules, tablets, pills, film-coated tablets, sugar-coated tablets, powders, granules, and the like), pharmaceutical compositions provided herein may comprise one or more pharmaceutically acceptable excipients such as sodium citrate or dicalcium phosphate, and/or one or more of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and/or sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as acetyl alcohol and/or glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets, and pills, pharmaceutical compositions provided herein may also comprise buffering agents. Compounds provided herein may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

[0073] A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxy propylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surfaceactive or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the subject composition moistened with an inert liquid diluent. Tablets, and other solid dosage forms, such as film coated tablets or sugar coated tablets, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art.

[0074] In liquid dosage forms (e.g., emulsions, microemulsions, solutions, suspensions, syrups, or elixirs) for oral administration, pharmaceutical compositions provided herein may comprise inert diluents commonly used in the art, such as water or other solvents, solubilizing agents, and/or emulsifiers, e.g, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, com, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols, fatty acid esters of sorbitan, cyclodextrins, and mixtures thereof. In the case of suspensions, the pharmaceutical compositions may comprise suspending agents, such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol, sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, tragacanth, and mixtures thereof.

[0075] Some examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, sterile water, syrup, and methyl cellulose. The formulations can additionally include lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as methyl and propylhydroxy-benzoates; sweetening agents; and flavoring agents.

[0076] Another mode for administration is parenteral, particularly by injection or intravenous infusion. The forms in which the compounds of the disclosure may be incorporated for administration by injection include aqueous or oil suspensions, or emulsions, with sesame oil, com oil, cottonseed oil, or peanut oil, as well as elixirs, mannitol, dextrose, or a sterile aqueous solution, and similar pharmaceutical vehicles. Aqueous solutions in saline are also conventionally used for injection, but less preferred in the context of the present invention. Ethanol, glycerol, propylene glycol, liquid polyethylene glycol, and the like (and suitable mixtures thereol), cyclodextrin derivatives, and vegetable oils may also be employed. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.

[0077] Sterile injectable solutions or intravenous fluid may be prepared by incorporating a compound of the disclosure in the required amount in the appropriate solvent with various other ingredients as enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0078] Pharmaceutical compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders. The liquid or solid pharmaceutical compositions may contain suitable pharmaceutically acceptable excipients as described supra. Preferably, the pharmaceutical compositions are administered by the oral or nasal respiratory route for local or systemic effect. Pharmaceutical compositions in preferably pharmaceutically acceptable solvents may be nebulized by use of inert gases. Nebulized solutions may be inhaled directly from the nebulizing device or the nebulizing device may be attached to a facemask tent, or intermittent positive pressure breathing machine. Solution, suspension, or powder pharmaceutical compositions may be administered, preferably orally or nasally, from devices that deliver the formulation in an appropriate manner.

[0079] Pharmaceutical compositions described herein may provide quick, sustained, or delayed release of the active ingredient (e.g, PPAR-y modulator) after administration to the patient by employing procedures known in the art. Controlled release drug delivery systems for oral administration include osmotic pump systems and dissolutional systems containing polymer-coated reservoirs or drug-polymer matrix formulations. Examples of controlled release systems are given in, e.g, U.S. Patent Nos. 3,845,770; 4,326,525; 4,902,514; and 5,616,345. Pharmaceutical compositions for use in the methods of the present disclosure may employ transdermal delivery devices (“patches”). Such transdermal patches may be used to provide continuous or discontinuous infusion of the active ingredient (e.g, PPAR-y modulator) in controlled amounts. The construction and use of transdermal patches for the delivery of pharmaceutical agents is well known in the art. See, e.g, U.S. Patent Nos. 5,023,252;

4,992,445; and 5,001,139. Such patches may be constructed for continuous, pulsatile, or on demand delivery of pharmaceutical agents.

[0080] Pharmaceutical compositions of the disclosure may be presented in unit dosage forms to facilitate accurate dosing. The term “unit dosage forms” refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient (e.g, a tablet, capsule, ampoule). The active ingredient (e.g, PPAR-y modulator) comprised in the pharmaceutical compositions are generally administered in a pharmaceutically effective amount. It will be understood, however, that the amount of the active ingredient actually administered usually will be determined by a physician, in light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound administered and its relative activity, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the like. Typical unit dosage forms include prefilled, premeasured ampules or syringes of the liquid compositions or pills, tablets, capsules or the like in the case of solid compositions.

Methods of Treating Pulmonary Fibrosis

[0081] Described herein are methods of preventing the onset of or treating pulmonary fibrosis, such as pulmonary fibrosis associated with a coronavirus infection or an aberrant ACE2 level, in a patient in need thereof. In some embodiments, a pulmonary fibrosis may be idiopathic pulmonary fibrosis (IPF).

[0082] In some embodiments, the methods may reduce level of myofibroblasts in lung tissue of the patient as compared to a control value. In some embodiments, the methods may inhibit or reduce myofibroblast differentiation in lung tissue of the patient as compared to a control value. In some embodiments, the methods may inhibit aberrant myofibroblast differentiation in lung tissue of the patient as compared to a control value. In some embodiments, the methods may reduce myofibroblast differentiation by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, or at least about 80% in lung tissue of the patient as compared to a control value. In some embodiments, the methods may reduce myofibroblast differentiation by at least about 50% in lung tissue of the patient as compared to a control value.

[0083] In some embodiments, the inhibition or reduction of myofibroblast differentiation may be determined by measuring one or more genes, or one or more proteins encoded by the one or more genes. The one or more genes may be selected from the group consisting of TGFB1,ACTA2, COL1A1, FN1, CDH1, OCLN,MUC5B, and MAPK3 in lung tissue of the patient. For example, inhibition of aberrant level of one or more of TGFB1, ACTA2, COL1A1, FN1, CDH1, OCLN, MUC5B, and MAPK3 as compared to a control value indicates the inhibition of myofibroblast differentiation. As another example, reduced level of one or more of TGFB1, ACTA2, COL1A1, FN1, MUC5B, and MAPK3 as compared to a control value or increased level of one or more of CDH1 and OCLN as compared to a control value indicates the reduction of myofibroblast differentiation.

[0084] In various embodiments, the methods may increase the level of one or more genes, or one or more proteins encoded by the one or more genes, in lung tissue of the patient as compared to a control value. In some embodiments, the one or more genes may be selected from the group consisting of CDH1 and OCLN. In some embodiments, the one or more genes may be selected from the group consisting of ACE2 w MASR. In some embodiments, the one or more genes may be ACE2. In some embodiments, the one or more genes may be MASR.

[0085] In various embodiments, the methods may decrease the level of one or more genes, or one or more proteins encoded by the one or more genes, in lung tissue of the patient as compared to a control value. In some embodiments, the one or more genes may be selected from the group consisting of TGFB1, ACTA2, COL1A1, FN1, MUC5B, and MAPK3. In some embodiments, the one or more genes may be MAPK3. In some embodiments, the one or more genes may be TACE.

[0086] In some embodiments, the methods may decrease the level of gene ACTA2, or one or more proteins encoded by ACTA2, by at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, or at least about 90% in lung tissue of the patient as compared to a control value. In some embodiments, the methods may decrease the level of gene ACTA2, or one or more proteins encoded by ACTA2, by at least about 80% in lung tissue of the patient as compared to a control value.

[0087] In some embodiments, the methods may decrease the level of gene COL1A1, or one or more proteins encoded by COL1A1, by at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, or at least about 90% in lung tissue of the patient as compared to a control value. In some embodiments, the methods may decrease the level of gene COL1A1, or one or more proteins encoded by COL1A1, by at least about 70% in lung tissue of the patient as compared to a control value.

[0088] In some embodiments, the methods may inhibit aberrant level of one or more genes, or one or more proteins encoded by the one or more genes, selected from the group consisting of TGFB1,ACTA2, COL1A1, FN1, CDH1, OCLN,MUC5B, and MARKS in lung tissue of the patient as compared to a control value. In some embodiments, the methods may inhibit aberrant decrease in the level of gene A CE2, or one or more proteins encoded by A CE2, in lung tissue of the patient as compared to a control value. In some embodiments, the methods may inhibit aberrant decrease in the level of gene MASR, or one or more proteins encoded by MASR, in lung tissue of the patient as compared to a control value. In some embodiments, the methods may inhibit aberrant increase in the level of gene TACE, or one or more proteins encoded by TACE, in lung tissue of the patient as compared to a control value.

[0089] In some embodiments, the methods do not alter expression of TMPRSS2 in the patient.

[0090] In some embodiments of the methods, the patient has, or is at risk of developing, pulmonary fibrosis. The pulmonary fibrosis may be associated with a coronavirus infection or an aberrant level of ACE2. In some embodiments, the patient is, or was previously, infected with a coronavirus. In some embodiments, the patient has an aberrant level of ACE2. The aberrant level of ACE2 may be associated with a viral infection, such as a coronavirus infection. A coronavirus may be SARS-CoV, SARS-CoV-2, or MERS-CoV.

[0091] In some embodiments, the control value of a method may be determined by measuring myofibroblasts or one or more genes, or one or more proteins encoded by the one or more of genes, in a corresponding healthy tissue or in a sample from the patient. In some embodiments, the one or more genes may be selected from the group consisting of TGFB1, ACTA2, COL1A1, FN1, CDH1, OCLN,MUC5B,MAPK3, ACE2, TACE, and MASR. In some embodiments, the one or more genes may be ACE2.

[0092] In some embodiments, the control value of a method may be determined using a reference therapeutic agent. In some particular embodiments, the control value may be determined in a patient being treated with a reference therapeutic agent. The reference therapeutic agent may be, for example, an antifibrotic agent. The antifibrotic agent may be, for example, nintedanib or pirfenidone. As an illustrative example, a control value may be the level of myofibroblast differentiation in lung tissue of a patient being treated with nintedanib or pirfenidone.

[0093] In some embodiments, a sample from the patient may be a tissue sample, such as a lung tissue sample. In some embodiments, a tissue sample from the patient (e.g., a tissue sample from the lung of the patient) may be used as a source of cells, a source of RNA, a source of protein, or a source of thin sections, for measuring the amount of, e.g, myofibroblasts, gene expression, or protein expression, in the sample. The tissue sample may be obtained using conventional biopsy instruments and procedures, such as needle biopsy, open biopsy, transbronchial biopsy (bronchoscopy), or thoracoscopic biopsy. The tissue sample may be in any form sufficient for cell sorting, RNA extraction, protein extraction, or preparation of thin sections. Accordingly, the tissue sample may be fresh, preserved through suitable cryogenic techniques, or preserved through non-cryogenic techniques. An exemplary standard process for handling clinical biopsy specimens is to fix the tissue sample in formalin and then embed it in paraffin. Samples in this form are commonly known as formalin-fixed, paraffin-embedded (FFPE) tissue. Suitable techniques of tissue preparation for subsequent analysis are well-known to those of skill in the art. In some embodiments, a sample from the patient may be collected at predetermined time, prior to and/or after being treated by a method described herein. A sample may be obtained from a healthy subject following the methods and procedures described herein. Such a sample obtained from a healthy subject may be a healthy tissue sample, such as a healthy lung tissue sample.

[0094] In certain embodiments, administering a therapeutically effective amount of a PPAR- y modulator or a compound of Formula (I), or a pharmaceutically acceptable salt thereof, comprises administering to the patient a dose of about 1 mg/kg/day to about 200 mg/kg/day, about 1 mg/kg/day to about 150 mg/kg/day, about 1 mg/kg/day to about 140 mg/kg/day, about 1 mg/kg/day to about 130 mg/kg/day, about 1 mg/kg/day to about 120 mg/kg/day, about 1 mg/kg/day to about 110 mg/kg/day, about 1 mg/kg/day to about 100 mg/kg/day, about 2 mg/kg/day to about 100 mg/kg/day, about 3 mg/kg/day to about 100 mg/kg/day, about 4 mg/kg/day to about 100 mg/kg/day, about 5 mg/kg/day to about 100 mg/kg/day, about 10 mg/kg/day to about 100 mg/kg/day, about 20 mg/kg/day to about 100 mg/kg/day, or about 30 mg/kg/day to about 100 mg/kg/day. In certain embodiments, administering a therapeutically effective amount of a PPAR-y modulator or a compound of Formula (I), or a pharmaceutically acceptable salt thereof, comprises administering to the patient a dose of about 5 mg/kg/day to about 100 mg/kg/day. In certain embodiments, administering a therapeutically effective amount of a PPAR-y modulator or a compound of Formula (I), or a pharmaceutically acceptable salt thereof, comprises administering to the patient a dose of about 1 mg/kg/day, a dose of about 2 mg/kg/day, a dose of about 3 mg/kg/day, a dose of about 4 mg/kg/day, a dose of about 5 mg/kg/day, a dose of about 6 mg/kg/day, a dose of about 7 mg/kg/day, a dose of about 8 mg/kg/day, a dose of about 9 mg/kg/day, a dose of about 10 mg/kg/day, about 15 mg/kg/day, about 20 mg/kg/day, about 25 mg/kg/day, about 30 mg/kg/day, about 35 mg/kg/day, about 40 mg/kg/day, about 45 mg/kg/day, about 50 mg/kg/day, about 55 mg/kg/day, about 60 mg/kg/day, about 65 mg/kg/day, about 70 mg/kg/day, about 75 mg/kg/day, about 80 mg/kg/day, about 85 mg/kg/day, about 90 mg/kg/day, about 95 mg/kg/day, about 100 mg/kg/day, about 105 mg/kg/day, about 110 mg/kg/day, about 115 mg/kg/day, about 120 mg/kg/day, about 125 mg/kg/day, about 130 mg/kg/day, about 135 mg/kg/day, about 140 mg/kg/day, about 145 mg/kg/day, about 150 mg/kg/day, about 155 mg/kg/day, about 160 mg/kg/day, about 165 mg/kg/day, about 170 mg/kg/day, about 175 mg/kg/day, about 180 mg/kg/day, about 185 mg/kg/day, about 190 mg/kg/day, about 195 mg/kg/day, or about 200 mg/kg/day. In some embodiments, administering a therapeutically effective amount of a PPAR-y modulator or a compound of Formula (I), or a pharmaceutically acceptable salt thereof, comprises administering to the patient a dose of about 5 mg/kg/day. In some embodiments, administering a therapeutically effective amount of a PPAR-y modulator or a compound of Formula (I), or a pharmaceutically acceptable salt thereof, comprises administering to the patient a dose of about 30 mg/kg/day. In some embodiments, administering a therapeutically effective amount of a PPAR-y modulator or a compound of Formula (I), or a pharmaceutically acceptable salt thereof, comprises administering to the patient a dose of about 100 mg/kg/day.

[0095] In some embodiments, a PPAR-y modulator or a compound of Formula (I), or a pharmaceutically acceptable salt thereof, is administered to the patient once every week, once every six days, once every five days, once every four days, once every three days, once every two days, once daily, twice daily, three times daily, four times daily, five times daily, six times daily, or seven times daily. [0096] In some embodiments, a PPAR-y modulator or a compound of Formula (I), or a pharmaceutically acceptable salt thereof, is administered orally.

[0097] In certain embodiments, methods or uses of the disclosure may further comprise administering to the subject an additional therapy. Accordingly, administering a therapeutically effective amount of a PPAR-y modulator or a compound of Formula (I), or a pharmaceutically acceptable salt thereof, may be combined with administering an additional therapy such as another antifibrotic agent, for example, pirfenidone or nintedanib. Such additional therapy may be in the form of adjuvant or neoadjuvant therapy.

EXAMPLES

[0098] The disclosure is further illustrated by the following examples. The examples are provided for illustrative purposes only, and should not to be construed as limiting the scope or content of the disclosure in any way.

[0099] Example 1: In vitro activity of PPAR-y modulator

[00100] To evaluate the ability of a PPAR-y modulator to inhibit or reduce fibroblast-to- myofibroblast transition (FMT) and epithelial-to-mesenchymal transition (EMT), A549 (ATCC® CCL-185™) and MRC-5 (ATCC® CCL171™) cell lines stimulated with TGFpi were treated with GED-0507, pirfenidone (Pirf), or nintedanib (Nint), the latter two of which are FDA-approved drugs for treating IPF. Profibrotic markers were than evaluated.

[00101] A549 and MRC-5 cell lines were grown in Dulbecco’s modified Eagle's medium

(DMEM) supplemented with 100 U/mL penicillin, 100 pg/mL streptomycin, and 10% fetal bovine serum (FBS).

[00102] A549 cells and MRC-5 cells were treated with recombinant human TGFpi (Sigma

Aldrich), at 20 ng/mL and at 10 ng/mL, respectively, to induce myofibroblast differentiation and with GED-0507 (1-30 mM), Pirf (0.1-0.8 mM in DMSO), or Nint (0.1-1 pM in DMSO). After 2 days (MRC-5 cells) or 4 days (A549 cells), the expression of makers of fibrosis and epithelial cells at the mRNA level and/or protein level was determined as described below. Statistical analysis for Pirf and Nint treatment was performed in comparison with cells treated with TGFpi and DMSO.

[00103] mRNA expression levels of TGF (TGFB1), a-SMA (ACTA), collagen (COL1A1), and fibronectin (FNI) were assessed by quantitative RT-PCR using the Light Cycler FastStart DNA Master SYBR Green I (Roche Diagnostics) according to the manufacturer’s protocol. Relative gene expression (E) was calculated as: E=2'^ACt, ACt being the difference between the critical threshold cycle (Ct) values of a gene of interest and the Ct of GAPDH (glyceraldehyde- 3-phosphate dehydrogenase). Data were expressed as fold change of the mean relative gene expression values ± SEM between treatment and control groups.

[00104] a-SMA protein expression was determined by immunofluorescence staining. Fixed cells were incubated with a polyclonal anti-a-SMA primary antibody (ab5694, Abeam) at 4°C overnight, after which an Alexa-Fluor conjugated secondary antibody (Al exaFluor 488, Thermo Fisher Scientific) was added and incubated for Ih at room temperature. Nuclei were stained with DAPI (4’,6-diamidino-2-phenylindole) (Thermo Fisher Scientific). Quantitative analysis was performed on three different microscope fields (20//40/ magnification) analyzed by threshold detection method for grayscale image using the Image J software. Data were expressed as fold change between treatment and control groups of the mean staining intensity/total area ± SEM.

[00105] Collagen deposition was determined by Picrosirius red staining on paraffin embedded cells. Briefly, monolayers of cells were fixed in 2% paraformaldehyde (PFA) stained with 0.1% Direct Red stain (Sigma Aldrich)/0.5% Picric Acid (Sigma Aldrich) for Ih at room temperature. Bound dye was eluted by incubation with 0.1 N NaOH at 37°C for Ih. Solubilized dye was then recovered and measured for absorbance at 550 nm. Results were normalized to cell density, which was determined by staining the remaining cell layers with DNA binding Crystal Violet dye for 30 min at room temperature and measuring the absorbance of Crystal Violet eluted with methanol at 540 nm.

[00106] Exemplary results in MRC-5 cells are shown in FIG. 1. The fibroblast-to- myofi broblast transition induced by TGFpi led to significantly increased expression of TGFP, a-SMA, collagen, and fibronectin at both mRNA level (FIGS, la-d) and protein level (FIGS, le-g). Treatment of the cells with GED-0507 reduced the upregulation of mRNA of TGFP (TGFB1), a-SMA (ACTA2), collagen (COL1A1), and fibronectin (FN ), at the tested doses of 1 and 30 mM (FIGS. la-d). GED-0507 treatment also inhibited a-SMA and collagen expression at the protein level (FIGS. le-g). Treatment with Pirf reduced the upregulation of TGFB1 but not ACTA2, COL1A1, or FNF, Pirf inhibited TGF i-induced collagen deposition (FIG. 1g) but not a-SMA protein expression (FIGS. le-f). Treatment with Nint only reduced the upregulation of TGFB1, while no significant effect on ACTA2, COL1A1, or FN1 expression, collagen deposition, or a-SMA protein expression was observed. [00107] Exemplary results in A549 cells are shown in FIG. 2. Treatment with GED-0507 inhibited the TGF[3-induced mRNA upregulation of TGFB1, ACTA2, COL1A1, and FN1 and the mRNA downregulation of CDH1 (encoding E-cadherin), and OCLN (encoding occludin), at the tested doses of 1 and 30 mM (FIGS. 2a-f); treatment with Pirf only inhibited the downregulation of CDH1 and OCLN,' and treatment with Nint only reduced the upregulation of CDH1 at one tested dose (0.1 pM). Moreover, GED-0507 inhibited a-SMA and collagen expression at the protein level (FIGS. 2g-i) more significantly than Pirf or Nint.

[00108] Example 2: In vitro activity of PPAR-y modulator in human lung fibroblasts

[00109] To evaluate the ability of a PPAR-y modulator to inhibit or reduce fibroblast-to- myofibroblast transition, human lung fibroblast (HLF) (ATCC® PCS-201-013™) cell line stimulated with TGFJ31 was treated with GED-0507, Pirf, or Nint. Profibrotic markers were than evaluated.

[00110] HLF cell line was grown in Fibroblast Basal Medium (ATCC® PCS-201-030™) supplemented with 100 U/mL penicillin, 100 pg/mL streptomycin, and a Fibroblast Growth Kit, Low serum (ATCC® PCS-201-041™) according to the manufacturer’s protocol. Cells were maintained in a humidified atmosphere of 95% air and 5% CO2 at 37°C.

[00111] HLF cells were treated with 5 ng/mL TGFJ31 to induce fibroblast-to-myofibroblast transition and with GED-0507 (1-30 mM), Pirf (0.1 -0.4 mM in DMSO), or Nint (0.1 -0.5 pM in DMSO) at the same time. After 2 days, the expression of profibrotic makers were evaluated as described below. Statistical analysis for Pirf and Nint treatment was performed in comparison with cells receiving TGFpi and DMSO

[00112] mRNA expression levels of TGF (TGFB1), a-SMA (ACTA), collagen (COL1A1), fibronectin (FNI), E-cadherin (CDH1), and occludin OCLN) were assessed by quantitative RT- PCR as described in Example 1.

[00113] Protein expression levels of a-SMA, collagen, and fibronectin were determined by Western Blotting. Briefly, total proteins were extracted from HLF cells using a RIPA Buffer containing 50mM Tris HC1 pH 7.6, 150mM NaCl, 5mM EDTA, 1%-TritonX supplemented with 100 mM sodium fluoride (NaF), 2mM sodium orthovanadate (NasVCL), 2 mM sodium pyrophosphate (NaPPi), 1 mM phenylmethanesulphone (PMSF), and a classical proteaseinhibitor cocktail. 50 pg of total protein of each sample were separated by SDS-PAGE electrophoresis and transferred onto nitrocellulose membranes. The membranes were then immunobloted with specific rabbit monoclonal antibodies against a-SMA (1:1000, Proteintech), against collagen (1:1000, Proteintech), against fibronectin (1:1000, Proteintech), or against P- Actin (1:10000, Sigma Aldrich) at 4°C overnight, followed by incubation with an anti-rabbit secondary horseradish peroxi das e-conjugated antibody (1:10000, Sigma Aldrich) for Ih at room temperature. The membranes were then incubated with a chemiluminescent HRP substrate, from which the light signal was measured according to the manufacturer’s protocol (ECL; Millipore Corporation). Optical density of target bands was determined using a computer- assisted densitometer and the ImageJ public domain software. Protein levels were expressed as units of Optical Density (OD) per quantity of total protein, relative to the quantity of the internal control -Actin in the sample.

[00114] Exemplary results are shown in FIG. 3. GED-0507 significantly decreased the level of TGFB1, ACTA2, COL1A1, w FNl genes compared to cells only stimulated with TGF i, at the concentration of 1 mM and higher (FIGS. 3a-3d). GED-0507 also inhibited a-SMA, collagen, and fibronectin protein expressions at 30 mM (FIGS. 3e-3g). Pirf showed no or limited inhibitory effects on markers of fibrosis. Nint decreased the mRNA level of TGFB1 and that of COL1A1 at only one tested dose (0.1 pM) and the expression of a-SMA and fibronectin proteins at only one tested dose (0.5 pM).

[00115] Example 3: In vivo activity of PPAR-y modulator

[00116] To evaluate the antifibrotic effect of a PPAR-y modulator in vivo, a murine model of lung fibrosis was used.

[00117] Lung injury was induced in 8-week-old male C57BL/6 mice by intratracheal instillation of 0.8 mg/kg (0.025 U/kg) bleomycin (BLM) on Day 0. GED-0507 (100 mg/kg), Pirf (400 mg/kg/day), or Nint (60 mg/kg/day) in 0.5% carboxymethyl cellulose (CMC) supplemented with 1% v/v Tween 80 was administered via oral gavage to BLM-challenged mice daily from Day 1 to Day 28 (preventive mode) or from Day 14 to Day 28 (curative mode). Lungs of all mice were excised at the end of the experiment. The left lungs were fixed in 4% buffered formaldehyde and embedded in paraffin and the right lungs were dissected and stored at -80°C until further analyses.

[00118] mRNA expressions levels of pro-inflammatory cytokines (TNF-a and IL-1 ), pro- fibrotic and EMT markers (TGF , a-SMA, collagen, fibronectin, E-Cadherin, occludin, and mucin 5B), and components of EMT-related signaling pathway (MAPK/ERK and ACE2/TACE/MasR) were assessed by quantitative RT-PCR as described in Example 1. [00119] a-SMA protein expression was determined by immunofluorescence staining as described in Example 1, with the exception that permeabilized lung sections instead of fixed cells were used for experimentation.

[00120] Collagen deposition was determined by Picrosirius red staining on paraffin embedded lung sections. The slides were examined and analyzed using the Image J software. The mean staining intensity was measured by threshold detection method for the grayscale image on three different microscope fields (20* magnification).

[00121] FIG. 4 depicts the anti-inflammatory properties of GED-0507. BLM challenge resulted in the upregulation of TNF (encoding TNF-a) and IL1B (encoding IL-1|3) in the mice lungs. Treatment with GED-0507 prevented the upregulation of TNF and IL1B mRNA in both preventive mode (FIGS. 4a, b) and curative mode (FIGS. 4c, d). Treatment with Pirf prevented the upregulation of TNF and IL1B mRNA in preventive mode; however, when administrated in curative mode, Pirf induced 100% of mortality rate at Day 24. Nint treatment in curative mode also prevented the upregulation of TNF and IL1B mRNA.

[00122] FIGS. 5 and 6 exemplifies the ability of GED-507 to modulate pro-fibrotic and EMT markers in BLM-challenged mice. Lungs of mice receiving BLM showed increased mRNA expression of pro-fibrotic markers: TGFB1 (encoding TGF|3), ACTA2 (encoding a-SMA), COL1A1 (encoding collagen), and FN J (encoding fibronectin) and EMT marker: MUC5B (encoding mucin 5B), and decreased mRNA expression of EMT markers: CDH1 (encoding E- Cadherin) and OCLN (encoding occludin). In preventative mode (FIG. 5), GED-0507 prevented aberrant regulation of all tested pro-fibrotic and EMT markers induced by BLM treatment at both mRNA and protein levels. Namely, GED-0507 prevented the upregulation of TGFB1, ACTA2, COL1A1, FN1, and MUC5B, and prevented the downregulation of CDH1 and OCLN (FIGS. 5a-g). GED-0507 also inhibited a-SMA and collagen expression at the protein level (FIGS. 5h-k). On the other hand, Pirf prevented the aberrant regulation of tested pro- fibrotic markers TGFB1,ACTA2, COL1A1, and FN1 at the mRNA level; however, Pirf treatment only showed limited reduction of collagen deposition and no significant effect on a- SMA expression. Furthermore, Pirf treatment did not regulate the tested EMT markers. I.e., Pirf did not significantly prevent the downregulation of CDH1 or OCLN or the upregulation of MUC5B. In the curative mode (FIG 6), treatment with GED-0507 and Nint significantly decreased gene expressions of TGFB1,ACTA2, COL1A1, and FN11 to a similar level, and considerably reduced protein expressions of a-SMA and collagen, compared to untreated animals with BLM-induced lung fibrosis (FIGS. 6a-d). With respect to EMT markers, GED-0507 attenuated the downregulation of CDH1 (FIG. 6e) and significantly prevented the upregulation of MUC5B (FIG. 6g) compared to untreated animals with BLM- induced lung fibrosis; Nint reduced the upregulation of MUC5B but did not modify CDH1 or OCLN gene expression.

[00123] FIG. 7 shows exemplary results of GED-0507 modulation on EMT-related signaling pathways MAPK/ERK and ACE2/TACE/MasR. MAPK3 gene (encoding ERK1) was upregulated in lungs of BLM-challenged mice. In preventative mode, GED-0507 reduced the upregulation of MAPK3,' Pirf did not significantly affect MAPK3 expression; and Nint further elevated the expression level (AMAPK3 (FIG. 7a). With respect to ACE2/TACE/MasR axis, BLM treatment resulted in the downregulation of ACE2 and MasR genes and the upregulation of TACE gene in mice lungs. In both preventive mode and curative mode, GED-0507 restored gene expression levels of ACE2 and MasR and significantly reduced the upregulation of TACE (FIGS. 7b-d). Similar effects on ACE2, TACE, and MasR were observed for Nint in the curative mode. Pirf did not have significant effects on the ACE2, TACE, o MasR expression.

INCORPORATION BY REFERENCE

[00124] The entire disclosure of each of the patent documents and scientific articles cited herein is incorporated by reference for all purposes.

EQUIVALENTS

[00125] The disclosure can be embodied in other specific forms with departing from the essential characteristics thereof. The foregoing embodiments therefore are to be considered illustrative rather than limiting on the disclosure described herein. The scope of the disclosure is indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

CLAIMS WHAT IS CLAIMED IS:

1. A method of treating pulmonary fibrosis in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a peroxisome proliferator- activated receptor gamma (PPAR-y) modulator or a pharmaceutically acceptable salt thereof, wherein the patient is, or was previously, infected with a coronavirus, and the PPAR-y modulator is a compound of Formula (I):

2. The method of claim 1, wherein the therapeutic effect of the method is determined by one or more of: a) reduced level of myofibroblasts in lung tissue of the patient as compared to a control value; b) reduction of myofibroblast differentiation in lung tissue of the patient as compared to a control value; c) increased level of one or more genes, or one or more proteins encoded by the one or more genes, selected from the group consisting of CDH1 and OCLN or decreased level of one or more genes, or one or more proteins encoded by the one or more genes, selected from the group consisting of TGFB1, ACTA2, COL1A1, FN1, MUC5B, and MAP K3 in lung tissue of the patient as compared to a control value; and d) increased level of one or more genes, or one or more proteins encoded by the one or more genes, selected from the group consisting of A CE2 and M APR or decreased level of gene TACE, or one or more proteins encoded by TACE, in lung tissue of the patient as compared to a control value.

3. The method of claim 2, wherein the therapeutic effect of the method is determined by reduction of myofibroblast differentiation by at least about 50% in lung tissue of the patient as compared to a control value.

28 The method of claim 2, wherein the therapeutic effect of the method is determined by reduction of myofibroblast differentiation in lung tissue of the patient as compared to a control value determined using nintedanib or pirfenidone. The method of claim 2, wherein the therapeutic effect of the method is determined by decreased level of gene COL1A1, or one or more proteins encoded by COL1A1, by at least about 70% in lung tissue of the patient as compared to a control value. The method of claim 2, wherein the therapeutic effect of the method is determined by decreased level of gene 4( 742. or one or more proteins encoded by ACTA2, by at least about 80% in lung tissue of the patient as compared to a control value. The method of claim 2, wherein the therapeutic effect of the method is determined by decreased level of gene MAPK3, or one or more proteins encoded by MAPK3, in lung tissue of the patient as compared to a control value. The method of any one of claims 2-7, wherein the control value is determined by measuring myofibroblasts or the one or more genes, or one or more proteins encoded by the one or more of genes, in a sample from the patient collected prior to administering the PPAR-y modulator. The method of any one of claims 1-8, wherein the coronavirus is severe acute respiratory syndrome coronavirus (SARS-CoV), SARS-CoV-2, or Middle East respiratory syndrome coronavirus (MERS-CoV). The method of any one of claims 1-9, wherein the PPAR-y modulator or a pharmaceutically acceptable salt thereof is administered at a dose of about 5 mg/kg/day to about 100 mg/kg/day. The method of any one of claims 1-10, wherein the PPAR-y modulator or a pharmaceutically acceptable salt thereof is administered orally. The method of any one of claims 1-11, wherein administering the PPAR-y modulator or a pharmaceutically acceptable salt thereof does not alter expression of TMPRSS2 in the patient. A method of preventing the onset of pulmonary fibrosis in a patient having a coronavirus infection, and in need thereof, comprising administering to the patient a therapeutically effective amount of a compound of Formula (I):

or a pharmaceutically acceptable salt thereof. The method of claim 13, wherein the therapeutic effect of the method is determined by one or more of: a) inhibition of aberrant myofibroblast differentiation in lung tissue of the patient as compared to a control value; b) inhibition of aberrant level of one or more genes, or one or more proteins encoded by the one or more genes, selected from the group consisting of TGFB1, ACTA2, COL1A1, FN1, CDH1, OCLN,MUC5B, and MAPK3 in lung tissue of the patient as compared to a control value; and c) inhibition of aberrant decrease in level of one or more genes, or one or more proteins encoded by the one or more genes, selected from the group consisting of ACE2 and MASR or aberrant increase in level of gene TACE, or one or more proteins encoded by TACE, in lung tissue of the patient as compared to a control value. The method of claim 14, wherein the control value is determined by measuring myofibroblasts or the one or more genes, or one or more proteins encoded by the one or more genes, in a corresponding healthy tissue or in a sample from the patient collected prior to administering the PPAR-y modulator. The method of any one of claims 13-15, wherein the coronavirus is SARS-CoV, SARS- CoV-2, or MERS-CoV. The method of any one of claims 13-16, wherein the compound of Formula (I) or a pharmaceutically acceptable salt thereof is administered at a dose of about 5 mg/kg/day to about 100 mg/kg/day. The method of any one of claims 13-17, wherein the compound of Formula (I) or a pharmaceutically acceptable salt thereof is administered orally. The method of any one of claims 13-18, wherein administering the compound of Formula (I) or a pharmaceutically acceptable salt thereof does not alter expression of TMPRSS2 in the patient. A method of preventing or treating pulmonary fibrosis in a patient having an aberrant level of ACE2, and in need thereof, comprising administering to the patient a therapeutically effective amount of a peroxisome proliferator-activated receptor gamma (PPAR-y) modulator or a pharmaceutically acceptable salt thereof. The method of claim 20, wherein the PPAR-y modulator is a compound of Formula (I):

The method of claim 20 or 21, wherein the aberrant level of ACE2 is a decreased level of ACE2 in lung tissue of the patient as compared to a control value. The method of any one of claims 20-22, wherein the level of ACE2 is gene level or protein level. The method of claim 22 or 23, wherein the control value is determined by measuring ACE2 in a corresponding healthy tissue. The method of any one of claims 20-24, wherein the pulmonary fibrosis associated with aberrant level of ACE2 is a pulmonary fibrosis associated with a viral infection. The claim of claim 25, wherein the viral infection is a SARS-CoV viral infection, a SARS- CoV2 viral infection, or a MERS-CoV viral infection. The method of any one of claims 20-26, wherein the PPAR-y modulator or a pharmaceutically acceptable salt thereof is administered at a dose of about 5 mg/kg/day to about 100 mg/kg/day. The method of any one of claims 20-27, wherein the PPAR-y modulator or a pharmaceutically acceptable salt thereof is administered orally. The method of any one of claims 20-28, wherein administering the PPAR-y modulator or a pharmaceutically acceptable salt thereof does not alter expression of TMPRSS2 in the patient. A method of preventing or treating pulmonary fibrosis in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a peroxisome proliferator-activated receptor gamma (PPAR-y) modulator or a pharmaceutically acceptable salt thereof, wherein administering the PPAR-y modulator or a pharmaceutically acceptable salt thereof results in inhibition or reduction of myofibroblast differentiation in lung tissue of the patient, and the PPAR-y modulator is a compound of Formula (I):

The method of claim 30, wherein the inhibition or reduction of myofibroblast differentiation is determined by measuring one or more genes, or one or more proteins encoded by the one or more genes, selected from the group consisting of TGFB1, ACTA2, COL1A1, FN1, CDH1, OCLN, MUC5B, and MAPK3 in lung tissue of the patient, wherein: a) inhibition of aberrant level of one or more of TGFB1, ACTA2, COL1A1, FN1, CDH1, OCLN,MUC5B, and MAPK3 as compared to a control value indicates the inhibition of myofibroblast differentiation, or b) reduced level of one or more of TGFB1, ACTA2, COL1A1, FN1, MUC5B, and MAPK3 as compared to a control value or increased level of one or more of CDH1 and OCLN as compared to a control value indicates the reduction of myofibroblast differentiation. The method of claim 31, wherein the control value is determined by measuring the one or more genes, or one or more proteins encoded by the one or more genes, in a corresponding

32 healthy tissue or in a sample from the patient collected prior to administering the PPAR-y modulator. The method of any one of claims 30-32, wherein the compound of Formula (I) or a pharmaceutically acceptable salt is administered at a dose of about 5 mg/kg/day to about 100 mg/kg/day. The method of any one of claims 30-33, wherein the compound of Formula (I) or a pharmaceutically acceptable salt is administered orally. The method of any one of claims 30-34, wherein administering the compound of Formula (I) or a pharmaceutically acceptable salt thereof does not alter expression of TMPRSS2 in the patient. The method of any one of claims 1-35, wherein the PPAR-y modulator or the compound of Formula (I) is (S)-2-methoxy-3-(4’-aminophenyl)propionic acid.

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