Use of cyclosporine analogs to treat fibrosis

Abstract

Methods, compositions, and kits suitable for use in the prevention, treatment, or recovery of fibrosis are disclosed. [Solution] The above problems are solved by a method comprising administering a composition comprising a cyclosporine analog (e.g., CRV431) or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof to a subject requiring prevention, treatment, or recovery from fibrosis, and by compositions and kits for the prevention, treatment, or recovery from fibrosis comprising a cyclosporine analog (e.g., CRV431) or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 978,526, filed on February 19, 2020, and U.S. Provisional Patent Application No. 62 / 981,383, filed on February 25, 2020. The entire content of each of these related applications is incorporated herein by reference.

Background Art

[0002] Background Field The present disclosure generally relates to the fields of molecular biology and medicine. One aspect relates to preventing, treating, and reversing fibrosis with cyclophilin inhibitors.

[0003] Description of Related Art Fibrosis is a pathological condition characterized by the uncontrolled deposition of fibrous connective tissue proteins and reduced clearance, ultimately leading to fatal end - stage organ scarring. Tissue fibrosis can occur across all organs. Fibrotic disorders remain very difficult to treat clinically, and for example, treatment options for idiopathic pulmonary fibrosis (IPF) and scleroderma are extremely limited. There is a need to find anti - fibrotic drugs that are effective in preventing, treating, and reversing fibrosis affecting various organs, including non - hepatic fibrosis.

Summary of the Invention

[0004] Summary The disclosure herein includes a method of treating non - hepatic fibrosis. The method can include, for example, administering to a subject in need thereof a composition comprising a cyclosporine analog of formula L below, or a pharmaceutically acceptable salt, solvate, stereoisomer thereof.

[0005]

Chemical Formula

[0006] Where: a. R' is either H or acetyl; b. R1 is a saturated or unsaturated linear or branched aliphatic carbon chain with a length of 2 to 15 carbon atoms; c. R2 is as follows: i. H; ii. Unsubstituted, N-substituted, or N,N disubstituted amides; iii. N-substituted or unsubstituted acyl-protected amines; iv. N-substituted or unsubstituted amines; v. Carboxylic acid; vi. nitrile; vii. Esther; viii. Ketones; ix. Hydroxy, dihydroxy, trihydroxy, or polyhydroxyalkyl; and x. Substitutive or non-substitutive aryl groups; xi. A saturated or unsaturated linear or branched aliphatic chain that may optionally contain substituents selected from the group consisting of hydrogen, ketones, hydroxyl, nitrile, carboxylic acid, ester, 1,3-dioxolane, halogen, and oxo; xii. Aromatic groups containing substituents selected from the group consisting of halogens, esters, and nitros; and xiii. A combination of a saturated or unsaturated linear or branched aliphatic chain of (xi) and an aromatic group of (xii). Selected from the group consisting of; and d. R23 is a saturated or unsaturated linear or branched aliphatic carbon chain which may be optionally substituted.

[0007] The subjects requiring this treatment may be those suffering from non-hepatic fibrosis. The method may include reducing the amount of non-hepatic fibrosis in the subject. In some embodiments, the amount of non-hepatic fibrosis in the subject is reduced by 5%, 10%, 20%, or more. The method may include reducing the formation of non-hepatic fibrosis in the subject. In some embodiments, the formation of non-hepatic fibrosis in the subject is reduced by 5%, 10%, 20%, or more. Non-hepatic fibrosis is not limited to fibrosis of the lungs, liver, kidneys, heart, skin, eyes, gastrointestinal tract, peritoneum, bone marrow, muscle, blood vessels, vascular structures, or any combination thereof. In some embodiments, subjects requiring this are those suffering from fibrotic disorders selected from the group consisting of idiopathic pulmonary fibrosis (IPF), cardiac fibrosis, cutaneous fibrosis, renal fibrosis, or a combination thereof. This specification also includes methods for preventing or delaying the onset of fibrosis. Such methods may include, for example, administering to a subject in need a composition comprising a cyclosporine analog of formula L below, or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof.

[0008] [ka]

[0009] During the ceremony: a. R' is either H or acetyl; b. R1 is a saturated or unsaturated linear or branched aliphatic carbon chain with a length of 2 to 15 carbon atoms; c. R2 is as follows: i. H; ii. Unsubstituted, N-substituted, or N,N disubstituted amides; iii. N-substituted or unsubstituted acyl-protected amines; iv. N-substituted or unsubstituted amines; v. Carboxylic acid; vi. nitrile; vii. Esther; viii. Ketones; ix. Hydroxy, dihydroxy, trihydroxy, or polyhydroxyalkyl; and x. Substitutive or non-substitutive aryl groups; xi. A saturated or unsaturated linear or branched aliphatic chain that may optionally contain substituents selected from the group consisting of hydrogen, ketones, hydroxyl, nitrile, carboxylic acid, ester, 1,3-dioxolane, halogen, and oxo; xii. an aromatic group containing a substituent selected from the group consisting of halogen, ester, and nitro; and xiii. a combination of the saturated or unsaturated linear or branched aliphatic chain of (xi) and the aromatic group of (xii) selected from the group consisting of; and d. R23 is a saturated or unsaturated, straight-chain or branched, optionally substituted aliphatic carbon chain.

[0010] The subject in need thereof can be, for example, a subject at risk of developing fibrosis. In some embodiments, the risk of developing fibrosis in the subject is reduced by at least 5%, 10%, 20% or more compared to an untreated subject. In some embodiments, the onset of fibrosis is delayed by at least 1 month, 1 year, or more. In some embodiments, the method includes reducing fibrogenesis, which is reduced by at least 1 month, 1 year, or more compared to an untreated subject. This specification also includes a method of reducing or restoring fibrosis. This method can include, for example, administering to a subject in need thereof a composition comprising a cyclosporine analog of formula L below, or a pharmaceutically acceptable salt, solvate, stereoisomer thereof.

[0011]

Chemical formula

[0012] Where: a. R’ is H or acetyl; b. R1 is a saturated or unsaturated, straight-chain or branched aliphatic carbon chain having 2 to 15 carbon atoms in length; c. R2 is as follows: i. H; ii. an unsubstituted, N-substituted, or N,N-disubstituted amide; iii. an N-substituted or unsubstituted acyl-protected amine; iv. an N-substituted or unsubstituted amine; [[ID=​vi. nitrile; vii. Esther; viii. Ketones; ix. Hydroxy, dihydroxy, trihydroxy, or polyhydroxyalkyl; and x. Substitutive or non-substitutive aryl groups; xi. A saturated or unsaturated linear or branched aliphatic chain that may optionally contain substituents selected from the group consisting of hydrogen, ketones, hydroxyl, nitrile, carboxylic acid, ester, 1,3-dioxolane, halogen, and oxo; xii. Aromatic groups containing substituents selected from the group consisting of halogens, esters, and nitros; and xiii. A combination of a saturated or unsaturated linear or branched aliphatic chain of (xi) and an aromatic group of (xii). Selected from the group consisting of; and d. R23 is a saturated or unsaturated linear or branched aliphatic carbon chain which may be optionally substituted.

[0013] In some embodiments, the subjects requiring it are subjects suffering from fibrosis. In some embodiments, the method includes suppressing fibrosis formation in the subject. In some embodiments, the fibrosis is non-hepatic fibrosis. In some embodiments, non-hepatic fibrosis includes fibrosis of the lungs, kidneys, heart, skin, eyes, gastrointestinal tract, peritoneum, bone marrow, muscles, blood vessels, vascular structures, or any combination thereof. In some embodiments, the subjects requiring it are subjects suffering from a fibrotic disorder selected from the group consisting of pulmonary fibrosis, cardiac fibrosis, cutaneous fibrosis, renal fibrosis, hepatic fibrosis, or a combination thereof. In some embodiments, the subjects requiring this are those suffering from idiopathic pulmonary fibrosis (IPF). In some embodiments, the fibrosis is hepatic fibrosis, such as cirrhosis. In some embodiments, the cirrhosis is associated with viral hepatitis, schistosomiasis, and chronic alcoholism. In some embodiments, the cyclosporine analog of formula L is CRV431.

[0014] [ka]

[0015] In some embodiments, the composition comprises a therapeutic or prophylactic effective amount of a cyclosporine analog of formula L, or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof. In some embodiments, non-hepatic fibrosis or fibrosis is induced by therapeutic agents, injury, or a combination thereof. In some embodiments, non-hepatic fibrosis or fibrosis is associated with trauma, inflammation, tissue repair, immune responses, cell hyperplasia, tumors, or accumulation of extracellular matrix components occurring after a combination thereof. In some embodiments, non-hepatic fibrosis or fibrosis is associated with major organ disease, fibroproliferative disorders, trauma-related scarring, or a combination thereof. In some embodiments, fibrosis is associated with interstitial lung disease, cirrhosis, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, kidney disease, heart or vascular disease, eye disease, systemic and focal scleroderma, keloids, hypertrophic scarring, atherosclerosis, restenosis, Dupuytren's contracture, surgical complications, chemotherapy-induced fibrosis, radiation-induced fibrosis, accident-related injury and burns, retroperitoneal fibrosis, peritoneal fibrosis / peritoneal scarring, or a combination thereof. In some embodiments, fibrosis associated with interstitial lung disease includes sarcoidosis, silicosis, drug reaction, infection, collagen vascular disease, rheumatoid arthritis, systemic sclerosis, scleroderma, pulmonary fibrosis, idiopathic pulmonary fibrosis, typical interstitial pneumonia, interstitial lung disease, fibrotic alveolitis of unknown cause, bronchiolitis obstructive, bronchiectasis, or a combination thereof.

[0016] The method can reduce fibrosis formation in subjects by, for example, at least 5%, 10%, 20%, 50%, 70%, 90%, or more compared to untreated subjects. In some embodiments, the method includes delaying fibrosis development in a subject compared to an untreated subject. The subject may be, for example, a mammal (e.g., human). In some embodiments, the composition includes one or more pharmaceutically acceptable excipients. In some embodiments, the composition includes one or more additional therapeutic agents. In some embodiments, the method further comprises administering one or more additional therapeutic agents to subjects who require it. In some embodiments, the one or more additional therapeutic agents include additional antifibrotic agents. In some embodiments, the one or more additional therapeutic agents include type II interferon receptor agonists, pirfenidone and pirfenidone analogs, nintedanib and nintedanib analogs, anti-angiogenic agents, anti-inflammatory agents, IL-1 antagonists, angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers and aldosterone antagonists, mitomycin C (MMC), 5-fluorouracil (5-FU), adenylyl cyclase activators, β-adrenergic receptor (adenoreceptor) agonists, flavonoids, mast cell stabilizers, phosphodiesterase inhibitors, procyanidins, or combinations thereof.

[0017] In some embodiments, at least one of one or more additional therapeutic agents is administered to the subject concurrently with the composition. In some embodiments, at least one of one or more additional therapeutic agents is administered to the subject before, after, or before or after the administration of the composition. The composition can be administered to the subject, for example, intravenously, orally, or parenterally. The composition may be in the form of, for example, powder, pill, tablet, microtablet, pellet, micropellet, capsule, microtablet-containing capsule, liquid, aerosol, or nanoparticles. In some embodiments, the composition is administered to the subject in an effective daily dose of 10 mg to 250 mg of a cyclosporine analog or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof. This specification also provides pharmaceutical compositions comprising a cyclosporine analog of the following formula L, or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof, for use in the prevention or treatment of fibrosis, for use in the prevention or reduction of fibrosis formation, for use in the recovery from fibrosis, for reduction of the amount of fibrosis, or for delaying the onset of fibrosis.

[0018] [ka]

[0019] During the ceremony: a. R' is either H or acetyl; b. R1 is a saturated or unsaturated linear or branched aliphatic carbon chain with a length of 2 to 15 carbon atoms; c. R2 is as follows: i. H; ii. Unsubstituted, N-substituted, or N,N disubstituted amides; iii. N-substituted or unsubstituted acyl-protected amines; iv. N-substituted or unsubstituted amines; v. Carboxylic acid; vi. nitrile; vii. Esther; viii. Ketones; ix. Hydroxy, dihydroxy, trihydroxy, or polyhydroxyalkyl; and x. Substitutive or non-substitutive aryl groups; xi. A saturated or unsaturated linear or branched aliphatic chain that may optionally contain substituents selected from the group consisting of hydrogen, ketones, hydroxyl, nitrile, carboxylic acid, ester, 1,3-dioxolane, halogen, and oxo; xii. Aromatic groups containing substituents selected from the group consisting of halogens, esters, and nitros; and xiii. A combination of a saturated or unsaturated linear or branched aliphatic chain of (xi) and an aromatic group of (xii). Selected from the group consisting of; and d. R23 is a saturated or unsaturated linear or branched aliphatic carbon chain which may be optionally substituted. In some embodiments, the cyclosporine analog is CRV431.

[0020] [ka]

[0021] In some embodiments, the pharmaceutical composition is for intravenous, oral, or parenteral administration. In some embodiments, the pharmaceutical composition is in the form of a powder, pill, tablet, microtablet, pellet, micropellet, capsule, microtablet-containing capsule, liquid, aerosol, or nanoparticles. In this specification, Pharmaceutical compositions disclosed herein; and The kit includes a label, which is shown below: (a) The kit is for the prevention or treatment of fibrosis, (b) The kit is for reducing or suppressing fibrosis formation. (c) The kit is for the recovery of fibrosis. (d) The kit is for reducing the amount of fibrosis, and (e) The kit is for delaying the onset of fibrosis. A kit showing one or more of the following Disclose the following. In some embodiments, the kit further includes instructions for identifying subjects at risk of developing fibrosis, instructions for identifying subjects who have fibrosis, or both. [Brief explanation of the drawing]

[0022] [Figure 1A] This histogram shows the dose-dependent decrease in procollagen levels in human LX-2 hepatic stellate cell cultures due to CRV431 treatment. [Figure 1B] This histogram shows the dose-dependent decrease in fibronectin levels in human LX-2 hepatic stellate cell cultures due to CRV431 treatment. [Figure 2A] This histogram shows a dose-dependent decrease in procollagen levels in human IPF lung fibroblast cultures due to CRV431 treatment. [Figure 2B] This histogram shows a dose-dependent decrease in fibronectin levels due to CRV431 treatment in human IPF lung fibroblast cultures. [Figure 3A]This histogram shows a dose-dependent decrease in procollagen levels in human cardiac fibroblast cultures due to CRV431 treatment. [Figure 3B] This histogram shows a dose-dependent decrease in fibronectin levels in human cardiac fibroblast cultures due to CRV431 treatment. [Figure 4A] This histogram shows a dose-dependent decrease in procollagen levels in human renal mesangial cell cultures due to CRV431 treatment. [Figure 4B] This histogram shows a dose-dependent decrease in fibronectin levels due to CRV431 treatment in human renal mesangial cell cultures. [Figure 5A] This histogram shows a dose-dependent decrease in the amount of procollagen in human dermal fibroblast cultures due to CRV431 treatment. [Figure 5B] This histogram shows a dose-dependent decrease in fibronectin levels in human dermal fibroblast cultures due to CRV431 treatment. [Figure 6A] This plot shows the daily mean percentage change in secretion markers in human precision-sectioned IPF lung thin sections during culture. [Figure 6B] This plot shows gene expression in thin section cultures of human IPF lung tissue. [Figure 7A] This plot shows the anti-fibrotic activity of CRV431 in a mouse unilateral ureteral ligation (UUO) model of renal fibrosis. [Figure 7B] This plot shows the anti-fibrotic activity of CRV431 in a mouse unilateral ureteral ligation (UUO) model of renal fibrosis. [Figure 8A] This plot shows the antifibrotic activity of CRV431 in human precision-sectioned liver thin sections (PCLS). [Figure 8B] This plot shows the antifibrotic activity of CRV431 in human precision-sectioned liver thin sections (PCLS). [Figure 8C] This plot shows the antifibrotic activity of CRV431 in human precision-sectioned liver thin sections (PCLS). [Figure 9A] This is a PCA plot showing the distribution of samples and donors for comparing TGFb / PDGF+CRV431 versus TGFb / PDGF+vehicle by group. [Figure 9B] This is a PCA plot showing the distribution of samples and donors for comparing TGFb / PDGF+CRV431 versus TGFb / PDGF+vehicle by group. [Figure 9C] This is an MA plot for comparing TGFb / PDGF+CRV431 versus TGFb / PDGF+vehicle by group. [Figure 10A] This heatmap was generated to compare TGFb / PDGF+CRV431 versus TGFb / PDGF+vehicle in separate groups. [Figure 10B] This heatmap was generated to compare TGFb / PDGF+CRV431 versus TGFb / PDGF+vehicle in separate groups. [Figure 11] This is a volcano plot showing significantly differently expressed genes identified in a group comparison between TGFb / PDGF+CRV431 and TGFb / PDGF+vehicles. [Figure 12A] This is a Venn diagram showing significant genetic overlap among all three donors. [Figure 12B] This is a Venn diagram showing significant genetic overlap among all three donors. [Figure 13A] This is a PCA plot showing the distribution of samples and donors for comparing unstimulated + CRV431 versus unstimulated + vehicle groups. [Figure 13B] This is a PCA plot showing the distribution of samples and donors for comparing unstimulated + CRV431 versus unstimulated + vehicle groups. [Figure 13C] This is an MA plot for comparing the groups of unstimulated + CRV431 versus unstimulated + vehicle. [Figure 14A] This heatmap was generated to compare the groups of non-stimulated + CRV431 versus non-stimulated + vehicle. [Figure 14B]This heatmap was generated to compare the groups of non-stimulated + CRV431 versus non-stimulated + vehicle. [Figure 15] This is a volcano plot showing significantly differently expressed genes identified in a group comparison between unstimulated + CRV431 and unstimulated + vehicle. [Figure 16A] This Venn diagram shows significant gene overlaps among all three donors to compare unstimulated CRV431 versus unstimulated vehicle by donor. [Figure 16B] This Venn diagram shows significant gene overlaps among all three donors to compare unstimulated CRV431 versus unstimulated vehicle by donor. [Modes for carrying out the invention]

[0023] Detailed explanation The following detailed description refers to the accompanying drawings, which form part thereof. In the drawings, similar symbols are typically considered identical to similar components unless the context requires them to be interpreted otherwise. The exemplary embodiments described in the detailed description, drawings, and claims are not intended to be limiting. Other embodiments may be used, and other modifications may be made without departing from the spirit or scope of the subject matter presented herein. The aspects of this disclosure can be arranged, substituted, combined, separated, and designed in a variety of different configurations, as generally described herein and as shown in the drawings, all of which are expressly intended herein and will readily be seen as forming part of the disclosure herein. All patents, published patent applications, other published documents, and sequences from GenBank, as well as other databases referenced herein, are incorporated herein in their entirety with respect to the relevant technology.

[0024] definition Unless otherwise defined, the technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which this disclosure belongs. See, for example, Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press (Cold Spring Harbor, NY 1989). For the purposes of this disclosure, the following terms are defined below. As used herein, “subject” refers to an animal being used for treatment, observation, or experimentation. “Animal” includes cold-blooded and warm-blooded vertebrates and invertebrates, such as fish, crustaceans, reptiles, and especially mammals. Mammals include, but are not limited to, mice; rats; rabbits; guinea pigs; dogs; cats; sheep; goats; cattle; horses; primates, such as monkeys, chimpanzees, and apes, and especially humans. As used herein, “patient” means a subject being treated by a medical professional, such as a doctor of medicine (i.e., a physician of allopathic medicine or osteopathic medicine) or a physician of veterinary medicine, in an attempt to cure a particular disease or disorder, or at least mitigate its effects, or primarily to prevent the disease or disorder from occurring.

[0025] As used herein, “administer” or “to administer” refers to a method of giving a certain dose of a pharmaceutically active ingredient to a vertebrate. As used herein, “dosage” refers to the total amount of the active ingredient (e.g., cyclosporine analogs including CRV431). As used herein, "unit dose" refers to the amount of therapeutic agent administered to a patient in a single dose. As used herein, "daily dose" refers to the total amount of therapeutic agent administered to a patient on a daily basis. As used herein, “therapeutically effective dose” or “pharmaceutically effective dose” means the amount of a therapeutic agent that has a therapeutic effect. The dose of a pharmaceutically active ingredient that is effective in treatment when administered alone or in combination with one or more additional therapeutic agents is a therapeutically effective dose. Therefore, as used herein, a therapeutically effective dose means the amount of a therapeutic agent that produces the desired therapeutic effect as determined by clinical trial results and / or model animal studies.

[0026] As used herein, the terms “to treat,” “to treat,” or “to treat” refer to administering a therapeutic agent or pharmaceutical composition to a subject for preventive and / or therapeutic purposes. The term “preventive treatment” refers to treating a subject who does not yet show symptoms of a disease or illness but is susceptible to, or at least at risk of developing, a particular disease or illness, and this treatment reduces the likelihood that the patient will develop the disease or illness. The term “therapeutic treatment” refers to administering treatment to a subject who already has a disease or illness. As used herein, the term “therapeutic effect” refers to reducing, to some extent, one or more symptoms of a disease or disorder. For example, a therapeutic effect may be observed by a reduction in subjective discomfort reported by the subject (e.g., a reduction in discomfort described on a self-administering patient questionnaire). As used herein, the term “prophylaxis” or “prevention” refers to the preventive treatment of an asymptomatic disease state in a subject, such as a mammal (including humans), to reduce the probability of the development of a clinical disease state. Subjects are selected for preventive therapy based on factors known to increase their risk of developing a clinical disease state compared to the population. “Preventive” therapy can be classified into (a) primary prevention and (b) secondary prevention. Primary prevention is defined as the treatment of a subject who does not yet present with a clinical disease state, while secondary prevention is defined as preventing the secondary development of the same or similar clinical disease state.

[0027] Fibrosis Fibrosis is a pathological condition characterized by an excessive accumulation of fibrous connective tissue. In some cases, fibrosis is considered a result of acute or chronic stress on a tissue or organ, characterized by extracellular matrix deposition, decreased patency of blood vessels / tubules / tubules / airways, and ultimately organ failure. The formation of excess fibrous connective tissue within an organ or tissue can occur during reactive or repair processes. Reactive fibrosis is a reversible process that occurs in the absence of functional cell necrosis (i.e., diseased cells remain alive), while repair fibrosis involves scar formation after cell death. Fibrosis can affect all tissue and organ systems, including, but not limited to, the heart, liver, lungs, skeletal muscle, kidneys, eyes, blood vessels, skin, brain, bone marrow, gastrointestinal tract, peritoneum, and vascular structures. Fibrosis is the end stage of many chronic tissue diseases, accounting for nearly half of all deaths worldwide: skeletal muscle tissue (e.g., dystrophic muscle disease), cardiovascular tissue (e.g., myocardial infarction), liver tissue (e.g., non-alcoholic fatty liver disease / cirrhosis), lung tissue (e.g., idiopathic pulmonary fibrosis), and kidney tissue (e.g., chronic kidney disease / renal fibrosis). Fibrosis and fibrotic disorders may be associated with major organs, including, but are not limited to, the lungs, liver, kidneys, heart, skin, eyes, gastrointestinal tract, peritoneum, bone marrow, or combinations thereof. As described herein, fibrosis may be non-hepatic fibrosis, where fibrosis is present in non-hepatic tissues and organs other than the liver, and / or where fibrosis affects non-hepatic tissues and organs other than the liver. Non-limiting examples of non-hepatic fibrosis include fibrosis affecting the heart, lungs, skeletal muscle, kidneys, eyes, blood vessels, skin, brain, bone marrow, gastrointestinal tract, peritoneum, and vascular structures. Examples of non-hepatic fibrotic disorders include, but are not limited to, fibrotic conditions associated with the lungs, kidneys, heart, skin, eyes, gastrointestinal tract, peritoneum, bone marrow, muscle (e.g., skeletal muscle), blood vessels, vascular structures, or any combination thereof.

[0028] As used herein, a fibrous disease or condition is any disease or condition characterized by the formation of excessive fibrous connective tissue. A common feature of these diseases is the excessive proliferation of fibrotic cells, and tissue or organ fibrosis often includes pulmonary fibrosis, hepatic fibrosis, chronic pancreatitis, scleroderma, glomerular fibrosis, and multi-organ fibrosis resulting from radiotherapy and tissue transplantation. The formation of excessive fibrous connective tissue may be in response to repair or reaction processes. Examples of fibrosis, but not limited to, include pulmonary fibrosis, hepatic fibrosis, myelofibrosis, cutaneous fibrosis (e.g., nephrogenic systemic fibrosis and keloid fibrosis), mediastinal fibrosis, cardiac fibrosis, renal fibrosis, interstitial fibrosis, epidural fibrosis, epithelial fibrosis, idiopathic fibrosis, cirrhosis, and any combination thereof.

[0029] In some embodiments, fibrosis is a fibrous condition involving the accumulation of extracellular matrix components that occurs after trauma, inflammation, tissue repair, immune response, cell hyperplasia, and tumors. Non-limiting examples of fibrosis and fibrotic disorders include, but are not limited to, cystic fibrosis; but are not limited to, fibrosis and fibrotic disorders associated with major organ diseases, including interstitial lung disease (ILD), cirrhosis, non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) (hepatic fibrosis), kidney disease (renal fibrosis), heart or vascular disease (cardiac fibrosis), and eye diseases; fibroproliferative disorders, such as, but are not limited to, systemic and focal scleroderma, keloids and hypertrophic scars, atherosclerosis, restenosis, and Dupuytren's contracture; trauma-related scars, such as, but are not limited to, surgical complications, chemotherapy-induced fibrosis (e.g., bleomycin-induced fibrosis), radiation-induced fibrosis, accidental injuries, and burns; retroperitoneal fibrosis (Ormond's disease); and peritoneal fibrosis / peritoneal scars, usually in patients who have undergone peritoneal dialysis after kidney transplantation.

[0030] Fibrosis may be ocular fibrosis, but is not limited to ocular fibrosis. Ocular fibrosis may result from eye diseases or disorders, such as diabetic retinopathy, bacterial infections, viral infections, fungal infections, amoebic infections, eye trauma, chemical corneal burns, thermal corneal burns, pterygium, glaucoma, surgical trauma, Fuchs' corneal endothelial dystrophy (FECD), proliferative vitreoretinopathy (PVR, e.g., anterior proliferative vitreoretinopathy (anterior PVR)), or a combination thereof. Ocular fibrosis may include, for example, retinal fibrosis, corneal fibrosis, conjunctival fibrosis, trabecular meshwork fibrosis, or a combination thereof. Ocular fibrosis may include, for example, corneal opacity, corneal scarring, trabecular meshwork blebs, or a combination thereof. Ocular fibrosis may result from, for example, corneal burns, pterygium, FECD, glaucoma, or a combination thereof. In some embodiments, ocular fibrosis includes retinal fibrosis associated with anti-VEGF therapy.

[0031] Renal fibrosis is a fibrotic condition in which fibrosis occurs in scar tissue due to infection, causing parts of the kidney to harden and lose function. This hardening of the kidneys can lead to chronic renal failure and may be accompanied by anemia, coagulation disorders, hypertension, various cardiopulmonary and gastrointestinal complications, and infections. For example, when kidney function declines to less than 15 percent of normal, the kidneys produce less erythropoietin, resulting in reduced red blood cell production. Furthermore, uremia caused by inactive urine secretion shortens the lifespan of red blood cells and causes severe anemia. Additionally, the development of uremia increases the likelihood of systemic infection, a major cause of sepsis. In some cases, kidney disease (which may be associated with diabetes) can damage and scar the kidneys, leading to progressive loss of function and hypertension. Renal fibrosis can occur at any stage of kidney disease, from chronic kidney disease (CKD) to end-stage renal disease (ESRD). Renal fibrosis can develop as a result of cardiovascular diseases such as hypertension or diabetes, both of which place a tremendous strain on kidney function and promote fibrotic reactions. In some cases, renal fibrosis can be idiopathic (of unknown cause), and certain mitochondrial genetic disorders also present with signs and associated symptoms of renal fibrosis.

[0032] Examples of fibrotic disorders of the lung include pulmonary fibrosis, idiopathic pulmonary fibrosis (IPF), usual interstitial pneumonia (UIP), interstitial lung disease (ILD), fibrotic alveolitis of unknown cause (CFA), bronchiolitis obstructive, and bronchiectasis. Interstitial lung disease (ILD) includes disorders in which inflammation and fibrosis of the lung are the last common pathway of pathogenesis, such as sarcoidosis, silicosis, drug reactions, infections, and collagen vascular diseases, such as rheumatoid arthritis and systemic sclerosis (scleroderma). IPF is the most common type of ILD. IPF is known to result from abnormalities in the healing process of damaged sites caused by continuous stimulation of epithelial cells or goblet cells, although the stimulating factors are unknown. While inflammation of the lung may not directly cause pulmonary fibrosis, pulmonary fibrosis is known to arise from differences between patients with idiopathic pulmonary fibrosis and healthy individuals in the healing process of lung tissue after inflammation. Another mechanism of fibrosis involves the deposition of extracellular matrix and fibrosis through the activation and proliferation of fibroblasts by T helper 2 cytokines.

[0033] Hepatic fibrosis can be defined as the excessive deposition of extracellular matrix resulting from chronic inflammation of the liver. If chronic liver disease persists due to this excessive deposition of extracellular matrix, it eventually leads to cirrhosis due to distortion of the liver's internal structure and a decrease in hepatocytes. Representative cells involved in hepatic fibrosis include hepatic stellate cells, Kupffer cells, and endothelial cells. Activated hepatic stellate cells are the main source of extracellular matrix production and are involved in increasing the production of various extracellular matrix components, including collagen. Kupffer cells are located in the sinusoidal space of the liver, and substances produced by activated Kupffer cells act around hepatocytes, endothelial cells, and hepatic stellate cells, thereby promoting hepatic fibrosis. Endothelial cells not only play a crucial role in regulating blood flow within the liver but also participate in the production of growth factors and extracellular matrix components involved in the proliferation of hepatic stellate cells due to inflammation or hepatic fibrosis. Cytokines that affect hepatic fibrosis include transforming growth factor β (TGFβ) and platelet-derived growth factor (PDGF). TGFβ is the most potent cytokine promoting hepatic stellate cell fibrosis, with hepatic stellate cells themselves being the primary source of TGFβ. PDGF is the most potent cytokine promoting hepatic stellate cell division and proliferation. While the hepatic fibrosis process was traditionally considered irreversible, it has recently been reported to be reversibly altered. Therefore, it may be a dynamic process. Consequently, accurately measuring this alteration becomes clinically important. In some embodiments, hepatic fibrosis is cirrhosis, which includes, for example, cirrhosis associated with viral hepatitis, schistosomiasis, and chronic alcoholism.

[0034] In some embodiments, fibrosis is associated with heart disease, eye disease, or a combination thereof. Heart disease may result in scar tissue that impairs the heart's pumping ability. Non-limited examples of cardiac fibrosis include atrial fibrosis, endocardial fibrosis, and combinations thereof. Eye diseases, but not limited to, include macular degeneration and retinal and vitreoretinopathy, which may impair vision. Fibrosis can be muscle fibrosis, such as reactive muscle fibrosis. Muscle fibrosis can be cardiac fibrosis, which includes, but not limited to, interstitial cardiac fibrosis, perivascular cardiac fibrosis, skeletal muscle fibrosis, and combinations thereof. Cardiac fibrosis can play a significant role in the pathogenesis of cardiac disease in patients through various distinct mechanisms. Fibrotic myocardial remodeling can impair ventricular diastolic function, contributing to the development of heart failure with preserved ejection fraction. The development of atrial fibrosis can increase the likelihood of atrial tachycardia in subjects. Furthermore, collagen deposition in the ventricular myocardium can contribute to an increased incidence of ventricular arrhythmias and sudden death observed in individuals with metabolic abnormalities. In addition, in some patients, the development of fibrotic changes in the right ventricle may disrupt right ventricular function. Moreover, metabolically abnormal-related modulation of the post-infarction repair fibrosis response may increase the likelihood of developing post-infarction heart failure in these subjects. Reducing fibrosis may be advantageous in lowering the incidence and mortality of cardiac disease in subjects with metabolic abnormalities.

[0035] As used herein, fibrotic disorders may include, for example, fibrovascular disease, cystic fibrosis, pulmonary fibrosis, idiopathic pulmonary fibrosis, musculoskeletal fibrosis, renal fibrosis, HIV-associated lymphadenopathy, inflammatory pulmonary fibrosis, pancreatic fibrosis, hepatic fibrosis, myocardial fibrosis, or a combination thereof. Fibrosis can be age-related (e.g., as a result of tissue injury or cardiovascular disease), induced by injury, or induced by stress. Age-related fibrosis can be of the heart, kidneys, blood vessels, liver, brain, intestines, skin, pancreas, and lungs, or any combination thereof. Fibrosis can be, for example, cardiac fibrosis, hepatic fibrosis, cerebral fibrosis, renal fibrosis, vascular fibrosis, pulmonary fibrosis, cutaneous fibrosis, or any combination thereof. Non-limited examples of cutaneous fibrotic disorders include abnormal wound healing, wrinkles, cellulite and cutaneous neoplastic fibrosis, vascular disorders, vasculitis, hyperproliferative burn wound healing, diabetic foot disease syndrome, arthral fibrosis, Peyronie's disease, and any combination thereof. Non-limited examples of cerebral fibrotic conditions include glial scarring. In some embodiments, the fibrous condition is arterial rigidity, articular fibrosis, Crohn's disease, Dupuytren's contracture, keloids, mediastinal fibrosis, myelofibrosis, Peyronie's disease (PD), nephrogenic systemic fibrosis, progressive nodular fibrosis, retroperitoneal fibrosis, burn scars, postoperative fibrosis of any organ, urethral fibrosis, diabetic nephropathy, scleroderma / systemic sclerosis, or any combination thereof. The fibrous condition may be associated with organs of the gastrointestinal system, e.g., liver, small intestine, large intestine, or pancreas; organs of the respiratory system, e.g., lungs; organs of the cardiovascular system, e.g., heart or blood vessels; skin; organs of the nervous system, e.g., brain; organs of the urinary system, e.g., kidneys; and / or organs of the musculoskeletal system, e.g., muscle tissue. In some embodiments, the fibrosis or fibrous condition has a known cause and / or trigger, e.g., skin burns, postoperative fibrosis, or a combination thereof. In some embodiments, the fibrosis or fibrous condition has no known cause and / or trigger.

[0036] Prevention and treatment methods This specification discloses methods, compositions, and kits for treating fibrosis (e.g., one or more fibrous diseases / disorders). These methods, compositions, and kits, for example, alleviate or improve one or more symptoms of fibrosis in a subject who needs them. This specification also discloses methods, compositions, and kits for preventing (including primary and secondary prevention) fibrosis (e.g., one or more fibrous diseases / disorders). Furthermore, methods, compositions, and kits are provided for preventing or delaying the onset of fibrosis. In some embodiments, the methods, compositions, and kits disclosed herein may restore fibrosis in a subject, reduce or suppress fibrosis formation, and / or reduce the amount of fibrosis. In some embodiments, the fibrosis is non-hepatic fibrosis.

[0037] In some embodiments, the method includes identifying subjects who have fibrosis. In some embodiments, the method includes identifying subjects who are at risk of developing fibrosis. The kit may include instructions for identifying subjects who have fibrosis, instructions for identifying subjects who are at risk of developing fibrosis, or both. The method may include, for example, administering to a subject in need a composition comprising a cyclosporine analog (e.g., CRV431) or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof. In some embodiments, the subject in need is a subject at risk of developing fibrosis. In some embodiments, the subject does not have fibrosis. For example, the subject is an individual who may or will develop fibrosis in the absence of preventive measures (e.g., treatment with a fibrosis prevention method or composition disclosed herein). In some embodiments, the subject in need is a subject suffering from fibrosis. In some embodiments, the subject in need is a subject having one or more symptoms of fibrosis. In some embodiments, the subject in need is a subject who has recently suffered from fibrosis.

[0038] For example, the methods, compositions, and kits disclosed herein can be used to prevent the onset of fibrosis in subjects at risk of developing fibrosis (e.g., subjects who are likely to develop or will develop fibrosis in the absence of fibrosis-preventive treatment). Furthermore, the methods, compositions, and kits disclosed herein can be used to delay the onset of fibrosis in subjects at risk of developing fibrosis (e.g., subjects who would have previously developed fibrosis in the absence of fibrosis-preventive treatment). As described herein, subjects at risk of developing fibrosis are subjects who do not yet have fibrosis, and therefore fibrosis has not progressed in them. In some embodiments, the onset of fibrosis is delayed. The delay may be, for example, one second or more, one minute or more, one hour or more, one day or more, one week or more, one month or more, or one year or more. In some embodiments, the delay in treated subjects is that of the same subjects who did not receive treatment. In some embodiments, the delay in treated subjects is that of untreated subjects. In some embodiments, the onset of fibrosis is delayed by 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 250, 300, 350, or approximately 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 250, 300, 350, or a range of days between any of these values. In some embodiments, the onset of fibrosis is delayed by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or a range of months or years between any of these values. In some embodiments, the onset of fibrosis is delayed by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 months or year, or by at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 months or year. In some embodiments, the onset of fibrosis is delayed by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 hours or longer, or by at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 hours or longer.

[0039] In some embodiments, fibrosis (e.g., histological fibrosis) recovers in the subject. Recovery may be 1%, 2%, 5%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% of the fibrosis present in the subject, or approximately 1%, 2%, 5%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or a range between any two of these values. In some embodiments, recovery may be at least 1%, 2%, 5%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more of the fibrosis present in the subject, or at least about 1%, 2%, 5%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more. In some embodiments, the fibrosis is non-hepatic fibrosis.

[0040] In some embodiments, the amount of fibrosis (e.g., histological fibrosis) is reduced in the subject. In the subject, the reduction in the amount of fibrosis may be 1%, 2%, 5%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or approximately 1%, 2%, 5%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or a range between any two of these values. In some embodiments, the reduction in the amount of fibrosis in a subject may be at least 1%, 2%, 5%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more, or at least about 1%, 2%, 5%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more. In some embodiments, the fibrosis is non-hepatic fibrosis.

[0041] In some embodiments, the formation of fibrosis (e.g., histological fibrosis) is reduced in subjects. The reduction in fibrosis formation may be 1%, 2%, 5%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or approximately 1%, 2%, 5%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or a range between any two of these values. In some embodiments, the reduction in fibrosis formation may be at least 1%, 2%, 5%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more in subjects, or at least about 1%, 2%, 5%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more. In some embodiments, the fibrosis is non-hepatic fibrosis. The reduction in fibrosis formation in treated subjects is compared to the same subjects who did not receive treatment. In some embodiments, the reduction in fibrosis formation in treated subjects is compared to untreated subjects.

[0042] The therapeutic efficacy of one or more cyclosporine analogs (e.g., CRV431) or their pharmaceutically acceptable salts, solvates, or stereoisomers disclosed herein can be determined using methods known to measure the amount of fibrosis (e.g., fibrosis in an affected organ, tissue, or area) in a subject. A subject may be, for example, a patient with fibrosis or a patient who has recently had fibrosis. The amount of fibrosis in a subject can be measured by methods known to those skilled in the art to measure the amount of fibrosis. For example, but not limited to, the amount of fibrosis can be determined by taking a muscle biopsy from a subject, thinning the muscle on a slide, and evaluating the amount of fibrosis revealed by staining techniques known in the art (e.g., hematoxylin-eosin (H&E) staining and / or Masson's trichrome staining). Alternatively, the amount of fibrosis can be determined in vivo by utilizing magnetic resonance imaging (MRI).

[0043] Exemplary therapeutic endpoints achievable by the compositions, methods, or kits disclosed herein may be a reduction in the amount of fibrosis in a subject administered one or more cyclosporine analogs disclosed herein (e.g., CRV431) or their pharmaceutically acceptable salts, solvates, stereoisomers, and optionally one or more additional therapeutic agents (e.g., antifibrotic agents). The relative amount of fibrosis in a subject may be quantified, for example, by tissue biopsy and subsequent histological examination, by quantifying Evans blue dye uptake as a measure of muscle fiber or cell damage (e.g., as described in Heydemann et al., Neuromuscular Disorders 15(9-10): 601-9 (2005)), or by quantifying hydroxyproline content as described in Swaggart et al., Physiol Genomics 43: 24-31 (2011), or both. In some embodiments, the amount of fibrosis in subjects administered one or more cyclosporine analogs disclosed herein (e.g., CRV431), or pharmaceutically acceptable salts, solvates, or stereoisomers thereof, and optionally one or more additional therapeutic agents (e.g., antifibrotic agents) is 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, and 6% lower than in patients not treated in this way. Decrease by 5%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or approximately 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or any value between 1% and 100%, or any range between any two of these values.In some embodiments, the amount of fibrosis in subjects administered one or more cyclosporine analogs disclosed herein (e.g., CRV431), or pharmaceutically acceptable salts, solvates, or stereoisomers thereof, and optionally one or more additional therapeutic agents (e.g., antifibrotic agents) is at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40% lower than in patients not treated in this manner. It decreases by 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, or decreases by at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%.

[0044] Functional assays can be used to determine the efficacy of the compounds disclosed herein in, for example, the treatment of fibrosis, prevention of fibrosis, recovery from fibrosis, reduction of the amount of fibrosis, delay of the onset of fibrosis, reduction or suppression of fibrosis formation, or any combination thereof. For example, a compound can be tested in relation to inhibiting collagen gel contraction as a measure of myofibroblast contractility, a characteristic function of myofibroblasts that distinguishes them from fibroblasts. Fibroblasts were packed into a collagen gel, and contraction was measured after stimulation with TGFβ1. Effective compounds are expected to inhibit TGFβ1-induced contraction of collagen. The efficacy of a compound can also be tested in relation to inhibiting ECM protein production, one of the important characteristic functions of myofibroblasts.

[0045] The methods disclosed herein can be applied, for example, to the treatment of subjects having organ failure, scarring, alteration of the normal extracellular matrix balance, increased collagen deposition, increased collagen volume fraction, differentiation of fibroblasts into myofibroblasts, decreased matrix metalloproteinase levels and increased tissue inhibitor levels of matrix metalloproteinases, increased levels of either the N-terminal propeptide or C-terminal propeptide (PINP or PICP) of type I procollagen, decreased levels of the C-terminal telopeptide (CTP or CITP) of type I collagen, increased collagen deposition and cardiac dysfunction as measured by various non-invasive imaging techniques, as well as renal dysfunction as measured by increased proteinuria and albuminuria, decreased glomerular filtration rate or doubling of creatinine levels.

[0046] Treatment drugs As used herein, the term “antifibrotic agent” refers to an agent (e.g., a chemical or biochemical agent) that has antifibrotic activity in mammals (e.g., preventing, reducing, or reversing fibrosis). Antifibrotic agents can have a variety of mechanisms of action, for example, some that reduce the formation of collagen or other proteins, or that promote the catabolism or removal of collagen in the affected area of ​​the body. Antifibrotic agents that are active in reducing the presence of fibrous tissue are included herein, regardless of the specific mechanism of action by which the agent functions. Some non-limiting examples of antifibrotic agents are described in U.S. Patent No. 5,720,950, which is incorporated herein by reference. Additional antifibrotic agents, though not limited to them, include type II interferon receptor agonists (e.g., interferon-γ); pirfenidone and pirfenidone analogs; anti-angiogenic agents, such as VEGF antagonists, VEGF receptor antagonists, bFGF antagonists, bFGF receptor antagonists, TGFβ antagonists; anti-inflammatory agents, IL-1 antagonists, such as IL-1Ra, angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers, and aldosterone antagonists.

[0047] Antifibrotic agents, including cyclosporine analogs (e.g., CRV431) or their pharmaceutically acceptable salts, solvates, and stereoisomers, disclosed herein can be used to treat fibrosis (e.g., fibrotic disorders) in subjects, or for primary or secondary prevention of fibrosis (e.g., fibrotic disorders). Cyclosporine analogs, or their pharmaceutically acceptable salts, solvates, and stereoisomers, can, for example, delay the onset of fibrosis in subjects (e.g., subjects at risk of developing fibrosis). Cyclosporine analogs, or their pharmaceutically acceptable salts, solvates, and stereoisomers, can, for example, reverse fibrosis in subjects. Cyclosporine analogs, or their pharmaceutically acceptable salts, solvates, and stereoisomers, can be used to reduce or suppress fibrosis formation in subjects. Cyclosporine analogs, or their pharmaceutically acceptable salts, solvates, and stereoisomers, can be used to reduce the amount of fibrosis in subjects. In some embodiments, the fibrosis is non-hepatic fibrosis.

[0048] Cyclosporine analogs disclosed herein (e.g., CRV431), or their pharmaceutically acceptable salts, solvates, and stereoisomers, can prevent the development of fibrosis (e.g., histofibrosis). For example, these antifibrotic agents can prevent fibrosis formation in subjects, delay the onset of fibrosis in subjects, or both. The delay may be, for example, more than one second, more than one minute, more than one hour, more than one day, more than one week, more than one month, or more than one year. The delay in treated subjects may be that of the same subjects who did not receive treatment, or that of untreated subjects. In some embodiments, the onset of fibrosis is delayed by a number of days ranging from 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 250, 300, 350, or approximately 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 250, 300, 350, or any of these values. In some embodiments, the onset of fibrosis is delayed by a number of months or years ranging from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or any of these values. In some embodiments, the onset of fibrosis is delayed by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 months or year, or by at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 months or year. In some embodiments, the onset of fibrosis is delayed by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 hours or more, or by at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 hours or more.

[0049] Cyclosporine analogs disclosed herein (e.g., CRV431), or their pharmaceutically acceptable salts, solvates, and stereoisomers, can restore fibrosis (e.g., histofibrosis) in a subject. The restoration may be 1%, 2%, 5%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% of the fibrosis present in the subject, or about 1%, 2%, 5%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or a range between any two of these values. In some embodiments, recovery may be at least 1%, 2%, 5%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more of the fibrosis present in the subject, or at least about 1%, 2%, 5%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more.

[0050] Cyclosporine analogs disclosed herein (e.g., CRV431), or their pharmaceutically acceptable salts, solvates, and stereoisomers, can reduce the amount of fibrosis (e.g., histofibrosis) in a subject. The reduction in the amount of fibrosis in a subject may be 1%, 2%, 5%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 1%, 2%, 5%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or in the range between any two of these values. In some embodiments, the reduction in the amount of fibrosis may be at least 1%, 2%, 5%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more in subjects, or at least about 1%, 2%, 5%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more. In some embodiments, the fibrosis is non-hepatic fibrosis.

[0051] Cyclosporine analogs disclosed herein (e.g., CRV431), or their pharmaceutically acceptable salts, solvates, and stereoisomers, can reduce the formation of fibrosis (e.g., histological fibrosis) in subjects. The reduction in fibrosis formation may be 1%, 2%, 5%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 1%, 2%, 5%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or a range between any two of these values. In some embodiments, the reduction in fibrosis formation may be at least 1%, 2%, 5%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more in subjects, or at least about 1%, 2%, 5%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more. In some embodiments, the fibrosis is non-hepatic fibrosis. The reduction in fibrosis formation in treated subjects is compared to the same subjects who did not receive treatment, or to untreated subjects. In some embodiments, the cyclosporine analog is a compound of the following formula L.

[0052] [ka]

[0053] During the ceremony: a. R' is either H or acetyl; b. R1 is a saturated or unsaturated linear or branched aliphatic carbon chain with a length of 2 to 15 carbon atoms; c. R2 is as follows: i. H; ii. Unsubstituted, N-substituted, or N,N disubstituted amides; iii. N-substituted or unsubstituted acyl-protected amines; iv. N-substituted or unsubstituted amines; v. Carboxylic acid; vi. nitrile; vii. Esther; viii. Ketones; ix. Hydroxy, dihydroxy, trihydroxy, or polyhydroxyalkyl; and x. Substitutive or non-substitutive aryl groups; xi. A saturated or unsaturated linear or branched aliphatic chain that may optionally contain substituents selected from the group consisting of hydrogen, ketones, hydroxyl, nitrile, carboxylic acid, ester, 1,3-dioxolane, halogen, and oxo; xii. Aromatic groups containing substituents selected from the group consisting of halogens, esters, and nitros; and xiii. A combination of a saturated or unsaturated linear or branched aliphatic chain of (xi) and an aromatic group of (xii). Selected from the group consisting of; and d. R23 is a saturated or unsaturated linear or branched aliphatic carbon chain which may be optionally substituted. In some embodiments, R1-R2 are selected from the group consisting of the following groups.

[0054] [ka]

[0055] In some embodiments, R1-R2 may optionally be substituted with substituents selected from the group consisting of hydrogen, ketones, hydroxyl, nitrile, halogen, oxo, carboxylic acid, ester, and 1,3-dioxolane, comprising a saturated or unsaturated linear or branched aliphatic chain with carbon atoms between 2 and 5. In some embodiments, R2 is based on the following:

[0056] [ka]

[0057] Selected from the group consisting of: R5 is a saturated or unsaturated linear or branched aliphatic carbon chain with carbon lengths between 1 and 10; and R6 is a monohydroxylated, dihydroxylated, trihydroxylated or polyhydroxylated saturated or unsaturated linear or branched aliphatic carbon chain with carbon lengths between 1 and 10. In some embodiments, R23 is selected from the group consisting of the following groups.

[0058] [ka]

[0059] In some embodiments, R23 is an optionally substituted alkyl, for example, an optionally substituted C1-C3 alkyl. The alkyl may be substituted with an amino and may contain C1-C3-Ala, in which case the compound contains a D-epimer of amino acid 3, to which R23 is attached. In some embodiments, R23 may be MeAla. In some embodiments, R23 is a linear or branched aliphatic carbon chain with a length of 1-6, 1-5, 1-4, 1-3, or 2 carbon atoms. In some embodiments, formula L

[0060] [ka]

[0061] The group is selected from the group consisting of the following groups.

[0062] [ka]

[0063] In some embodiments, the cyclosporine analog is given by the following formula L:

[0064] [ka]

[0065] (In the formula: a. R' is either H or acetyl; b. R1 is a saturated or unsaturated linear or branched aliphatic carbon chain with a length of 2 to 15 carbon atoms; c. R2 is as follows: i. Unsubstituted, N-substituted, or N,N disubstituted amides; ii. Carboxylic acid; iii. Nitrile; iv. Esther; v. Ketones; vi. Hydroxy, dihydroxy, trihydroxy, or polyhydroxyalkyl; and vii. Substitutive or non-substitutive aryl groups; viii. Saturated or unsaturated linear or branched aliphatic carbon chains substituted with substituents selected from the group consisting of ketones, hydroxyls, nitriles, carboxylic acids, esters, 1,3-dioxolanes, and oxos; ix. Aromatic groups substituted with substituents selected from the group consisting of halogens, esters, and nitros; and x. viii) Saturated or unsaturated linear or branched aliphatic carbon chains and ix) Combinations of aromatic groups Selected from the group consisting of; and d. R23 is an unsubstituted C1-C3 alkyl group. It is a compound of [the compound]. In some embodiments, R' is H. In some embodiments, R1 is a saturated or unsaturated linear or branched aliphatic carbon chain with a length of 5 to 8 carbon atoms. In some embodiments, R2 is based on the following:

[0066] [ka]

[0067] Selected from the group consisting of; R5 is a saturated or unsaturated linear or branched aliphatic carbon chain with carbon lengths between 1 and 10; and R6 is a monohydroxylated, dihydroxylated, trihydroxylated, or polyhydroxylated saturated or unsaturated linear or branched aliphatic carbon chain with carbon lengths between 1 and 10. In some embodiments, R1-R2 are selected from the group consisting of the following groups.

[0068] [ka]

[0069] In some embodiments, R1-R2 are substituted with substituents selected from the group consisting of ketones, hydroxyl, nitriles, oxo, carboxylic acids, esters, and 1,3-dioxolanes. In some embodiments, R1-R2 have a length of at least 6 carbon atoms. In some embodiments, formula L

[0070] [ka]

[0071] The group is selected from the group consisting of the following groups.

[0072] [ka]

[0073] In some embodiments, R23 is selected from the group consisting of the following groups.

[0074] [ka]

[0075] In some embodiments, R23 is methyl. In some embodiments, the compound comprises a D-epimer of amino acid 3, to which R23 is attached. In some embodiments, the cyclosporine analog is a compound selected from the group consisting of the following compounds.

[0076] [Table 1] TIFF2026069500000021.tif216158

[0077] During the ceremony: R is

[0078] [ka]

[0079] and; R' is either H or acetyl; and Isomers are isomorphic forms of amino acid 3, which is the amino acid to which R23 is attached. In some embodiments, the cyclosporine analog is given by the following formula L:

[0080] [ka]

[0081] (In the formula: a. R' is either H or acetyl; b. R1 is a saturated or unsaturated linear or branched aliphatic carbon chain with a length of 2 to 15 carbon atoms; c. R2 is as follows: i. Unsubstituted, N-substituted, or N,N disubstituted amides; ii. Carboxylic acid; iii. Nitrile; iv. Esther; v. Ketones; vi. Hydroxy, dihydroxy, trihydroxy, or polyhydroxyalkyl; vii. Substitutive or non-substitutive aryl groups; viii. Saturated or unsaturated linear or branched aliphatic carbon chains substituted with substituents selected from the group consisting of ketones, hydroxyls, nitriles, carboxylic acids, esters, 1,3-dioxolanes, and oxos; ix. Aromatic groups substituted with substituents selected from the group consisting of halogens, esters, and nitros; and x. A combination of a saturated or unsaturated linear or branched aliphatic carbon chain of (viii) and an aromatic group of (ix). Selected from the group consisting of; and d. R23 is a saturated or unsaturated linear or branched aliphatic carbon chain which may be optionally substituted. R1-R2 has a length of at least 6 carbon atoms. It is a compound of [the compound]. In some embodiments, R' is H. In some embodiments, R1 is a saturated or unsaturated linear or branched aliphatic carbon chain with a length of 5 to 8 carbon atoms. In some embodiments, R1-R2 are selected from the group consisting of the following groups.

[0082] [ka]

[0083] In some embodiments, R1-R2 are substituted with substituents selected from the group consisting of ketones, hydroxylomers, nitriles, oxoomers, carboxylic acids, esters, and 1,3-dioxolanes. In some embodiments, R23 is selected from the group consisting of the following groups.

[0084] [ka]

[0085] In some embodiments, R23 comprises an optionally substituted C1-C3 alkyl group. In some embodiments, R23 is substituted with an amino group. In some embodiments, R23 is a C1-C3 alkyl group, and the compound comprises a D-epimer of amino acid 3, to which R23 is attached. In some embodiments, R23 is methyl. In some embodiments, R23 is a linear or branched aliphatic carbon chain with a length of 1 to 6 carbon atoms. In some embodiments, formula L

[0086] [ka]

[0087] The group is selected from the group consisting of the following groups.

[0088] [ka]

[0089] In some embodiments, R1-R2 are

[0090] [ka]

[0091] Therefore, R23 is methyl, and the compound is the D-epimer of amino acid 3, which is the amino acid to which R23 is attached. In some embodiments, the cyclosporine analog is given by the following formula L:

[0092] [ka]

[0093] (In the formula: R' is either H or acetyl; R1 is a saturated or unsaturated linear or branched aliphatic carbon chain with a length of 2 to 15 carbon atoms; R2 is an N-substituted or unsubstituted acyl-protected amine; and R23 is either methyl or ethyl. It is a compound of [the compound]. In some embodiments, R' is H. In some embodiments, R1 is a saturated or unsaturated linear or branched aliphatic carbon chain with a length of 5 to 8 carbon atoms. In some embodiments, R2 is

[0094] [ka]

[0095] R5 is a saturated or unsaturated linear or branched aliphatic carbon chain with carbon lengths between 1 and 10. In some embodiments, R1-R2 are selected from the group consisting of the following groups.

[0096] [ka]

[0097] In some embodiments, R23 is methyl. In some embodiments, formula L

[0098] [ka]

[0099] The basis is as follows:

[0100] [ka]

[0101] In some embodiments, R', R1-R2, and R23, as well as isomers of the compound, are selected from the following.

[0102] [ka]

[0103] Here, the isomer is an isomorph of amino acid 3, which is the amino acid to which R23 is attached. In some embodiments, the cyclosporine analog is given by the following formula L:

[0104] [ka]

[0105] (In the formula: R' is either H or acetyl; R1-R2 are based on the following:

[0106] [ka]

[0107] Selected from the group consisting of; and R23 is a saturated or unsaturated linear or branched aliphatic carbon chain that may be optionally substituted. It is a compound of [the compound]. In some embodiments, R' is H. In some embodiments, R1-R2 are the following groups.

[0108] [ka]

[0109] In some embodiments, formula L

[0110] [ka]

[0111] The basis is as follows:

[0112] [ka]

[0113] In some embodiments, R23 is selected from the group consisting of the following groups.

[0114] [ka]

[0115] In some embodiments, R23 is the following group.

[0116] [ka]

[0117] In some embodiments, R23 is the following group.

[0118] [ka]

[0119] In some embodiments, R23 is (a) comprising an optionally substituted C1-C3 alkyl group; (b) substituted with an amino group; (c) C1-C3-Ala, wherein the compound comprises a D-epimer; (d) MeAla; and / or (e) a linear or branched aliphatic carbon chain with lengths of 1-6, 1-5, 1-4, 1-3, or 2 carbon atoms. In some embodiments, R', R1-R2, and R23, as well as isomers of the compound, are selected from the following.

[0120] [ka]

[0121] In some embodiments, the cyclosporine analog is a small molecule cyclophylline inhibitor CRV431 (shown below), which is a derivative of cyclosporine A (CsA), and is a neutral cyclic peptide consisting of 11 amino acids, with amino acids 1 and 3 being chemically modified.

[0122] [ka]

[0123] CRV431 is a small molecule cyclophylline inhibitor being clinically developed for the treatment of liver diseases, including hepatic fibrosis and hepatocellular carcinoma. In preclinical studies, CRV431 has shown antiviral activity against numerous viruses, including hepatitis B virus, hepatitis C virus, and HIV, and has shown antifibrotic activity in the liver in numerous in vivo models. CRV431 can reduce hepatic fibrosis arising from non-alcoholic steatohepatitis ("NASH") and hepatocellular carcinoma tumor burden in experimental models of NASH. As disclosed herein, including in vitro studies, CRV431 can reduce the production of extracellular matrix (ECM) molecules, collagen, and fibronectin, from fibroblasts derived from various different organs. Overproduction of collagen and fibronectin from these cell types leads to fibrous scarring in damaged organs, and CRV431 can reduce collagen and fibronectin production from multiple cell types. These results demonstrate that CsA derivatives such as CRV431 can exert anti-fibrotic activity across a variety of diseases and various cells, tissues, and organs.

[0124] For example, as described herein, CRV431 can reduce the production of extracellular matrix (ECM) molecules, collagen, and fibronectin from fibroblasts derived from five cell types, including pulmonary fibroblasts, cardiac fibroblasts, cutaneous fibroblasts, renal mesangial cells, and the LX2 hepatic stellate cell line, from patients with idiopathic pulmonary fibrosis ("IPF"). IPF is known to be an invasive fibrotic disease in great need of new treatments. As described herein, CRV431 dose-dependently reduced the secretion of procollagen and fibronectin from all cell types of comparable size, as measured by enzyme-linked immunosorbent assay (ELISA). The degree of suppression was similar regardless of whether the cells were stimulated with transforming growth factor β (TGFβ), a profibrotic factor consistent with its direct effect on ECM synthesis. CRV431 dose-dependently reduced ECM production by up to 55% at clinically significant concentrations without causing any reduction in cell viability. As disclosed herein, ECM production can be reduced by inhibiting cyclophyllin B using CRV431, and consistent with this finding, downregulation of cyclophyllin B by small interfering RNA (siRNA) similarly reduced procollagen and fibronectin secretion.

[0125] Fibrous scarring is a major pathological feature and driver of organ failure in many diseases, including cirrhosis, IPF, chronic kidney disease, and severe heart disease. Nevertheless, there are few treatments available to reduce scarring. Many treatments attempt to reduce fibrosis by targeting fibroblast stimulation, but these signaling phenomena can vary depending on the patient, the type of fibrotic disease, or the stage of the disease. Without being limited to any particular theory, in some embodiments, the effect of CRV431 is independent of the type of stimulating signal, making it advantageous to use CRV431 for the treatment of fibrotic diseases (not only in the liver, but also in organs other than the liver).

[0126] In some embodiments, the method includes administering one or more additional therapeutic agents to subjects who require it. For example, the composition may contain one or more additional antifibrotic agents. Non-limiting examples of antifibrotic agents include type II interferon receptor agonists (e.g., interferon-γ); pirfenidone and pirfenidone analogs; nintedanib and nintedanib analogs; anti-angiogenic agents, such as VEGF antagonists, VEGF receptor antagonists, bFGF antagonists, bFGF receptor antagonists, TGFβ antagonists; anti-inflammatory agents; IL-1 antagonists, such as IL-1Ra; angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers and aldosterone antagonists; mitomycin C (MMC); 5-fluorouracil (5-FU); adenylyl cyclase activators; β-adrenergic receptor (adenoreceptor) agonists; flavonoids; mast cell stabilizers; phosphodiesterase inhibitors and procyanidins; and any combination thereof.

[0127] In some embodiments, an additional therapeutic agent (e.g., an additional antifibrotic agent) is administered to the subject concurrently with a composition comprising one or more cyclosporine analogs (e.g., CRV431) disclosed herein, or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof. In some embodiments, the additional therapeutic agent is administered to the subject before administration of a composition comprising one or more cyclosporine analogs (e.g., CRV431) disclosed herein, or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof, and / or after administration of a composition comprising one or more cyclosporine analogs (e.g., CRV431) disclosed herein, or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof.

[0128] Kit, composition, and administration method In some embodiments, a kit is provided comprising a cyclosporine analog (e.g., CRV431) or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof, and a label indicating the use of the kit. In some embodiments, the label indicates that the kit is for the prevention of fibrosis, e.g., fibrotic disorders, in a subject. In some embodiments, the label indicates that the kit is for the treatment of fibrosis, e.g., fibrotic disorders, in a subject. In some embodiments, the label indicates that the kit is for reducing the amount of fibrosis in a subject. In some embodiments, the label indicates that the kit is for delaying the onset of fibrosis in a subject. In some embodiments, the label indicates that the kit is for reducing or suppressing fibrosis formation in a subject. In some embodiments, the label indicates that the kit is for the recovery of fibrosis in a subject. In some embodiments, this specification also provides compositions comprising one or more cyclosporine analogs disclosed herein (e.g., CRV431), or pharmaceutically acceptable salts, solvates, or stereoisomers thereof, for use in the prevention of fibrosis (e.g., fibrotic disorders), treatment of fibrosis (e.g., fibrotic disorders), reduction of the amount of fibrosis, delay of the onset of fibrosis, reduction or suppression of fibrosis formation, recovery from fibrosis, or any combination thereof.

[0129] Fibrosis can affect the heart, liver, lungs, skeletal muscle, kidneys, eyes, blood vessels, skin, brain, bone marrow, gastrointestinal tract, peritoneum, vascular structures, or any combination thereof. In some embodiments, fibrosis is non-hepatic fibrosis. Fibrous disorders may include, but are not limited to, any of the fibrotic disorders disclosed herein, including, retinal fibrosis, corneal fibrosis, conjunctival fibrosis, trabecular fibrosis, renal fibrosis, pulmonary fibrosis, idiopathic pulmonary fibrosis (IPF), usual interstitial pneumonia (UIP), interstitial lung disease (ILD), fibrotic alveolitis of unknown cause (CFA), bronchiolitis obliterans, bronchiectasis, cirrhosis, hepatic fibrosis, fibrous vascular disease, cystic fibrosis, pulmonary fibrosis, idiopathic pulmonary fibrosis, musculoskeletal fibrosis, renal fibrosis, HIV-associated lymphadenopathy, inflammatory pulmonary fibrosis, pancreatic fibrosis, cardiac fibrosis, vascular fibrosis, myocardial fibrosis, or any combination thereof.

[0130] In some embodiments, the composition is a stable self-microemulsifying drug delivery system ("SMEDDS") formulation comprising a derivative or analog of cyclosporine A (e.g., CRV431), or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof. The composition enables good solubility of, for example, a derivative of cyclosporine A (e.g., CRV431), or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof, and enables significant blood exposure in humans. In some embodiments, the composition further comprises polyoxyl castor oil (polyoxyl 40 hydrogenated castor oil, macrogol glycerol hydroxysteart, and PEG-40 hydrogenated castor oil, also known as Cremophor® RH40 and Kolliphor® RH40). In some embodiments, the composition comprises ethanol. In some embodiments, the composition comprises diethylene glycol monoethyl ether (2-(2-ethoxyethoxy)ethanol, also known as Transcutol®). In some embodiments, the composition comprises propylene glycol (PG). In some embodiments, the composition comprises glyceryl monolinoleate, e.g., Maisine® CC. In some embodiments, the composition comprises vitamin E. Various pharmaceutical compositions / drug delivery systems (e.g., SMEDDS formulations) comprising cyclosporine analogs (e.g., CRV431) or pharmaceutically acceptable salts thereof are described in a PCT patent application published as WO 2020 / 112562, which is incorporated herein by reference to its entirety.

[0131] In some embodiments, the system contains vitamin E, Maisine® CC, propylene glycol, Transcutol®, ethanol, and Cremophor® RH40 in 1 / 1 / 5 / 5 / 2.4 / 4 or 1 / 1.5 / 2.5 / 5 / 2.4 / 5. The system may contain, for example, a cyclosporine analog (e.g., CRV431) at concentrations ranging from about 10 mg / mL to about 90 mg / mL, such as 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, a range between any two of these values, or any value within 10 mg / mL to 90 mg / mL. In some embodiments, the system contains a cyclosporine analog (e.g., CRV431) at a concentration of about 90 mg / mL. In some embodiments, the system contains a cyclosporine analog (e.g., CRV431) at a concentration of 70 mg / mL or about 70 mg / mL.

[0132] The composition may be a pharmaceutical composition comprising, for example, a cyclosporine analog (e.g., CRV431), or a pharmaceutically acceptable salt, solvate, stereoisomer thereof, and one or more pharmaceutically acceptable excipients. In some embodiments, the composition is administered to a subject by intravenous, nasal, intrapulmonary, oral, or parenteral administration. In some embodiments, the composition is in the form of a powder, pill, tablet, microtablet, pellet, micropellet, capsule, microtablet-containing capsule, liquid, aerosol, suspension, or nanoparticles. In some embodiments, the composition is administered to a subject once, twice, or three times daily. In some embodiments, the composition is administered to a subject once or twice in an emergency (e.g., during surgery). In some embodiments, the composition is administered to a subject over a period of at least one day, at least two days, at least three days, at least one week, or longer. In some embodiments, the composition is administered to subjects in an effective daily dose of 10 mg to 250 mg of a cyclosporine analog (e.g., CRV431) or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof.

[0133] The therapeutically effective dose and frequency of administration of cyclosporine analogs (e.g., CRV431), as well as the duration of treatment with cyclosporine analogs (e.g., CRV431), are determined by the treating physician and depend on various factors, including the nature and severity of proliferative disorders such as cancer, the potency and mode of administration of the cyclosporine analog (e.g., CRV431), the subject's age, weight, overall health, sex, and dietary restrictions, and the subject's response to treatment. In some embodiments, the therapeutically effective dose of a cyclosporine analog (e.g., CRV431) for treating or preventing proliferative disorders such as cancer, or for reducing or suppressing one or more symptoms of the proliferative disorders described herein, is about 0.1 to 200 mg, 0.1 to 150 mg, 0.1 to 100 mg, 0.1 to 50 mg, 0.1 to 30 mg, 0.5 to 20 mg, 0.5 to 10 mg, or 1 to 10 mg (e.g., per day or per dose), or, if the treating physician deems it appropriate, it may be administered as a single dose or in divided doses. In some embodiments, the therapeutically effective dose (e.g., per day or per dose) of a cyclosporine analog (e.g., CRV431) for treating or preventing proliferative disorders such as cancer, or for reducing or suppressing one or more symptoms of the proliferative disorders described herein, is approximately 0.1 to 1 mg (e.g., approximately 0.1 mg, 0.5 mg, or 1 mg), approximately 1 to 5 mg (e.g., approximately 1 mg, 2 mg, 3 mg, 4 mg, or 5 mg), approximately 5 to 10 mg (e.g., approximately 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, or 10 mg), approximately 10 to 20 mg ( For example, approximately 10 mg, 15 mg, or 20 mg; approximately 20-30 mg (for example, approximately 20 mg, 25 mg, or 30 mg); approximately 30-40 mg (for example, approximately 30 mg, 35 mg, or 40 mg); approximately 40-50 mg (for example, approximately 40 mg, 45 mg, or 50 mg); approximately 50-100 mg (for example, approximately 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, or 100 mg); approximately 100-150 mg (for example, approximately 100 mg, 125 mg, or 150 mg); or approximately 150-200 mg (for example, approximately 150 mg, 175 mg, or 200 mg).In some embodiments, a therapeutically effective dose of a cyclosporine analog (e.g., CRV431) is administered once or more times daily (e.g., twice, three or four times or more times), or once every two or three days, or once, two or three times a week, or as the treating physician deems appropriate. In some embodiments, the composition comprises a therapeutically or prophylactically effective amount of a cyclosporine analog (e.g., CRV431) or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof.

[0134] Cyclosporine analogs (e.g., CRV431) can also be administered in an irregular pattern. For example, cyclosporine analogs (e.g., CRV431) can be administered in an irregular pattern, once, twice, or three times, at intervals of 30 minutes, one hour, two hours, or more. Furthermore, cyclosporine analogs (e.g., CRV431) can be taken as needed. For example, cyclosporine analogs (e.g., CRV431) can be administered one, two, three, four, five times, or more times, whether in a regular or irregular pattern, until the proliferative disorder / condition (e.g., cancer) improves. Once relief from the proliferative disorder / condition (e.g., cancer) is achieved, administration of cyclosporine analogs (e.g., CRV431) may be discontinued if necessary. If the disease impairment / condition returns, administration of cyclosporine analogs (e.g., CRV431) can be resumed, whether in a regular or irregular pattern. The appropriate dosage, frequency of administration, and duration of treatment with cyclosporine analogs (e.g., CRV431) can be determined by the treating physician.

[0135] Cyclosporine analogs (e.g., CRV431) can be used prophylactically to treat or prevent proliferative disorders such as cancer, or to prevent or reduce one or more symptoms of proliferative disorders (e.g., fibrosis), or to reduce or suppress the occurrence of one or more symptoms of proliferative disorders (e.g., fibrosis). The prophylactically effective amount of cyclosporine analog (e.g., CRV431) may be any therapeutically effective amount of cyclosporine analog (e.g., CRV431) described herein. Cyclosporine analogs (e.g., CRV431) can be administered via any suitable route. Possible routes of administration for cyclosporine analogs (e.g., CRV431) include, but are not limited to, oral, parenteral (including intramuscular, subcutaneous, intradermal, intravascular, intravenous, intraarterial, intramedullary, and subarachnoid), intracavitary, intraperitoneal, and topical (including skin / supercutaneous, percutaneous, mucous membrane, transmucosal, intranasal [e.g., by nasal spray or nasal drop], intraocular [e.g., by eye drop], lung [e.g., by oral or nasal inhalation], buccal, sublingual, rectal, and vaginal). In some embodiments, cyclosporine analogs (e.g., CRV431) are administered orally (e.g., as capsules or tablets, possibly with enteric coating). In other embodiments, cyclosporine analogs (e.g., CRV431) are administered parenterally (e.g., intravenous, subcutaneous, or intradermal). In further embodiments, a cyclosporine analog (e.g., CRV431) is administered topically (e.g., skin, intracutaneous, transdermal, mucous membrane, transmucosal, buccal, or sublingually).

[0136] In some embodiments, the cyclosporine analog (e.g., CRV431) is administered without food. In some embodiments, the cyclosporine analog (e.g., CRV431) is administered at least about 1 or 2 hours before or after a meal. In some embodiments, the cyclosporine analog (e.g., CRV431) is administered at least about 2 hours after dinner. The cyclosporine analog (e.g., CRV431) can also be taken substantially simultaneously with a meal (e.g., within about 0.5, 1 or 2 hours before or after a meal, or with a meal). In some embodiments where a more rapid establishment of therapeutic levels with a cyclosporine analog (e.g., CRV431) is desired, the cyclosporine analog (e.g., CRV431) is administered under a dosing schedule in which, after the administration of a loading dose, (i) one or more additional loading doses are administered, followed by one or more therapeutically effective maintenance doses, or (ii) one or more therapeutically effective maintenance doses are administered without additional loading doses, if the treating physician deems it appropriate. The loading dose of the drug is typically greater than the subsequent maintenance doses (e.g., about 1.5, 2, 3, 4, or 5 times greater) and is designed to establish therapeutic levels of the drug more rapidly. One or more therapeutically effective maintenance doses may be any therapeutically effective dose described herein. In some embodiments, the loading dose is about three times greater than the maintenance doses. In some embodiments, after administering a loading dose of a cyclosporine analog (e.g., CRV431), maintenance doses of the cyclosporine analog (e.g., CRV431) are administered at an appropriate time interval (e.g., about 12 or 24 hours) and thereafter for the duration of treatment. For example, a loading dose of the cyclosporine analog (e.g., CRV431) is administered on day 1, and maintenance doses are administered on day 2 and thereafter for the duration of treatment. In some embodiments, a cyclosporine analog (e.g., CRV431) is administered orally (e.g., as a tablet) on day 1 at a loading dose of approximately 1.5, 3, 15, or 30 mg (e.g., 3 × approximately 0.5, 1, 5, or 10 mg), followed by a maintenance dose of approximately 0.5, 1, 5, or 10 mg orally (e.g., as a tablet) once daily, possibly before bedtime, for at least approximately 2 weeks, 1 month (4 weeks), 6 weeks, 2 months, 10 weeks, 3 months, 4 months, 5 months, 6 months, 1 year, 1.5 years, 2 years, 3 years or longer (e.g., at least approximately 6 weeks, 2 months, 3 months, or 6 months). In some embodiments, a cyclosporine analog (e.g., CRV431) is administered orally (e.g., as a tablet) at a loading dose of approximately 15 mg (e.g., 3 × approximately 5 mg) on ​​day 1, followed by a maintenance dose of approximately 5 mg orally (e.g., as a tablet) once daily, possibly before bedtime, for at least approximately 2 weeks, 1 month, 6 weeks, 2 months, 3 months, 6 months, 1 year, 1.5 years, 2 years, 3 years or longer (e.g., at least approximately 6 weeks, 2 months, 3 months or 6 months). In some embodiments, the initial loading dose of a cyclosporine analog (e.g., CRV431) is administered on day 1, the second loading dose on day 2, and the maintenance dose on day 3 and thereafter for the duration of treatment. In some embodiments, the initial loading dose is approximately three times greater than the maintenance dose, and the second loading dose is approximately twice greater than the maintenance dose.

[0137] As disclosed herein, therapeutic agents (e.g., cyclosporine analogs (e.g., CRV431)) may be formulated for administration in pharmaceutical compositions comprising physiologically acceptable surfactants, carriers, diluents, excipients, lubricants, suspensions, film-forming agents, coating aids, or combinations thereof. In some embodiments, therapeutic agents (e.g., cyclosporine analogs (e.g., CRV431)) are formulated for administration with pharmaceutically acceptable carriers or diluents. The therapeutic agents (e.g., cyclosporine analogs (e.g., CRV431)) may be formulated as drugs comprising standard pharmaceutically acceptable carriers and / or excipients that are routine in the pharmaceutical field. The exact nature of the formulation depends on several factors, including the desired route of administration. In some embodiments, cyclosporine analogs (e.g., CRV431) are formulated for oral, intravenous, intragastric, intravascular, or intraperitoneal administration. Standard pharmaceutical formulation techniques can utilize the techniques disclosed in Remington's *The Science and Practice of Pharmacy*, 21st Ed., Lippincott Williams & Wilkins (2005), which are incorporated herein by reference in their entirety.

[0138] The terms “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” include all kinds of solvents, dispersion media, coatings, antibacterial agents, antifungal agents, isotonic agents, and absorption retarders. The use of such media and agents for pharmaceutically active substances is technically well known. Any common media or agent is intended for use in a therapeutic composition, provided it is not incompatible with the active ingredient. Furthermore, various adjuvants commonly used in the art may be included. Considerations for including various components in a pharmaceutical composition are, for example, described in Gilman et al. (Eds.) (1990); Goodman and Gilman's: The Pharmacological Basis of Therapeutics, 8th Ed., Pergamon Press, which is incorporated herein by reference in its entirety. Some examples of substances that can serve as pharmaceutically acceptable carriers or components thereof include sugars, e.g., lactose, glucose, and sucrose; starches, e.g., corn starch and potato starch; cellulose and its derivatives, e.g., sodium carboxymethylcellulose, tragacanth powder; malt; gelatin; talc; solid lubricants, e.g., stearic acid and magnesium stearate; calcium sulfate; vegetable oils, e.g., peanut oil, cottonseed oil, sesame oil, olive oil, com oil, and cocoa butter; polyols, e.g., propylene glycol, glycerin, sorbitol, mannitol, and polyethylen glycol; alginic acid; emulsifiers, e.g., TWEENS; humectants, e.g., sodium lauryl sulfate; colorants; flavoring agents; tableting agents, stabilizers; antioxidants; preservatives; pyrogenic substance removal water; isotonic saline; and phosphate buffer. The selection of a pharmaceutically acceptable carrier to be used in combination with the target therapeutic agent is basically determined by the method by which the composition is administered.

[0139] The compositions described herein are preferably provided in unit dosage forms. As used herein, “unit dosage form” is a composition containing a quantity of therapeutic agent (e.g., a cyclosporine analog (e.g., CRV431)) suitable for administration in a single dose to an animal, preferably a mammalian subject, in accordance with good medical practice. However, the preparation of a single or unit dosage form does not mean that the dosage form is administered once daily or once per course of treatment. The dosage form is intended to be administered once, twice, three or more times daily, and may be administered as an infusion over a period of time (e.g., from about 30 minutes to about 2 to 6 hours), or as a continuous infusion, and may be given multiple times during a course of treatment, but a single dose is not explicitly excluded. A skilled practitioner will recognize that the formulation is not explicitly intended for an entire course of treatment, and that judgment is left to those skilled in the art of treatment rather than the formulation.

[0140] As described above, useful compositions may be in any form suitable for various routes of administration, e.g., oral, nasal, rectal, topical (including percutaneous), ocular, intracerebral, intracranial, subarachnoid, intra-arterial, intravenous, intramuscular, or other parental routes of administration. A skilled craftsman will understand that oral and nasal compositions include compositions administered by inhalation and are prepared using available methodologies. Depending on the desired specific route of administration, various technically known and pharmaceutically acceptable carriers may be used. Examples of pharmaceutically acceptable carriers include solid or liquid fillers, diluents, hydrotropes, surfactants, and encapsulating materials. Optional pharmaceutically active materials that do not substantially interfere with the inhibitory activity of the therapeutic agent (e.g., cyclosporine analogs (e.g., CRV431)) may also be included. The amount of carrier used in combination with a therapeutic agent (e.g., a cyclosporine analog (e.g., CRV431)) is sufficient to provide a practical amount of material for each unit dose of the therapeutic agent (e.g., a cyclosporine analog (e.g., CRV431)). Techniques and compositions for producing dosage forms useful for the methods described herein are described in the following references, which are incorporated herein by reference: Modern Pharmaceutics, 4th Ed., Chapters 9 and 10 (Banker & Rhodes, editors, 2002); Lieberman et al, Pharmaceutical Dosage Forms: Tablets (1989); and Ansel, Introduction to Pharmaceutical Dosage Forms 8th Edition (2004).

[0141] Various oral dosage forms can be used, including solid forms such as tablets, capsules, and granules. Tablets may be compressed powder tablets, enteric-coated tablets, sugar-coated tablets, film-coated tablets, or multi-compressed tablets containing a suitable binder, lubricant, diluent, disintegrant, colorant, flavoring agent, flow inducer, and melting agent. Liquid oral dosage forms include aqueous solutions, emulsions, suspensions, solutions and / or suspensions reconstituted from non-foaming granules, and effervescent formulations reconstituted from effervescent granules, containing a suitable solvent, preservative, emulsifier, suspending agent, diluent, sweetener, melting agent, colorant, and flavoring agent. Pharmacovigilant carriers suitable for the preparation of oral dosage forms are technically well known. Tablets typically contain a conventional pharmaceutically acceptable adjuvant as an inert diluent, e.g., calcium carbonate, sodium carbonate, mannitol, lactose, and cellulose; a binder, e.g., starch, gelatin, and sucrose; a disintegrant, e.g., starch, alginic acid, and croscarmellose; and a lubricant, e.g., magnesium stearate, stearic acid, and talc. Flow facilitators such as silicon dioxide can be used to improve the flow properties of the powder mixture. Colorants such as FD&C dyes can be added for appearance. Sweeteners and flavorings, e.g., aspartame, saccharin, menthol, peppermint, and fruit flavors, are useful adjuvants for chewable tablets. Capsules typically contain one or more of the above-mentioned solid diluents. The selection of carrier components is determined by secondary considerations such as taste, cost, and storability, which are not critical and can be easily done by those skilled in the art.

[0142] Oral compositions also include solutions, emulsions, suspensions, etc. pharmaceutically acceptable carriers suitable for the preparation of such compositions are technically well known. Typical components of carriers for syrups, elixirs, emulsions, and suspensions include ethanol, glycerol, propylene glycol, polyethylene glycol, liquid sucrose, sorbitol, and water. Typical suspension agents include sodium carboxymethylcellulose, AVICEL RC-591, tragacanth, and sodium alginate; typical humectants include lecithin and polysorbate 80; and typical preservatives include methylparaben and sodium benzoate. Oral liquid compositions may contain one or more of the above-mentioned sweeteners, flavorings, and colorants.

[0143] Other compositions useful for achieving the delivery system of the target therapeutic agent include sublingual, buccal, and nasal formulations. These compositions typically contain soluble filler substances, such as sucrose, sorbitol, and mannitol; and one or more binders, such as gum arabic, microcrystalline cellulose, carboxymethylcellulose, and hydroxypropylmethylcellulose. They may also contain the flow enhancers, lubricants, sweeteners, colorants, antioxidants, and flavorings. For topical use, creams, ointments, gels, solutions, or suspensions containing the therapeutic agents disclosed herein (e.g., cyclosporine analogs (e.g., CRV431)) are used. Topical formulations may generally contain a pharmaceutical carrier, a co-solvent, an emulsifier, a penetration enhancer, a preservative, and a emollient.

[0144] For intravenous administration, the therapeutic agents described herein (e.g., cyclosporine analogs (e.g., CRV431)) and compositions may be dissolved or dispersed in a pharmaceutically acceptable diluent, such as physiological saline or dextrose solution. The desired pH can be achieved by including, but is not limited to, suitable excipients such as NaOH, sodium carbonate, sodium acetate, HC1, and citric acid. In various embodiments, the pH of the final composition is in the range of 2 to 8, preferably 4 to 7. Examples of antioxidant excipients include sodium bisulfite, acetone bisulfite, sodium formaldehyde, sulfoxylates, thiourea, and EDTA. Other non-limiting examples of suitable excipients found in the final intravenous composition include sodium phosphate or potassium phosphate, citric acid, tartaric acid, gelatin, and carbohydrates such as dextrose, mannitol, and dextran. Further acceptable excipients are described in Powell, et al., Compendium of Excipients for Parenteral Formulations, PDA J Pharm Sci and Tech 1998, 52 238-31 1 and Nema et al., Excipients and Their Role in Approved Injectable Products: Current Usage and Future Directions, PDA J Pharm Sci and Tech 2011, 65 287-332. Both documents are incorporated herein by reference in their entirety. Bacteriostatic or fungal solutions may be obtained by including, but not limited to, antimicrobial agents such as phenylmercury nitrate, thimerosal, benzethonium chloride, benzalkonium chloride, phenol, cresol, and chlorobutanol.

[0145] One or more solid forms that are reconstituted with a suitable diluent such as sterile water, saline, or dextrose in water shortly before administration may be provided to the caregiver as a composition for intravenous administration. In other embodiments, the composition is provided as a solution that is immediately suitable for parenteral administration. In still other embodiments, the composition is provided as a solution that is further diluted before administration. In embodiments that include administering a combination of a therapeutic agent described herein (e.g., a cyclosporine analog such as CRV431) and another agent, the combination may be provided to the caregiver as a mixture, or the caregiver may mix the two agents before administration, or the two agents may be administered separately. In non-human animal studies, the application of a potential product is started at a higher dosage level and the dosage is reduced until the desired effect is no longer achieved or the adverse side effects disappear. The dosage can be in a wide range depending on the desired effect and the therapeutic index. Typically, the dosage can be between about 0.1 mg / kg (body weight) and 4000 mg / kg (body weight), preferably between about 80 mg / kg (body weight) and 1600 mg / kg (body weight). Alternatively, as would be understood by one of ordinary skill in the art, the dosage can be calculated based on the patient's surface area.

[0146] Depending on the severity and responsiveness of the condition to be treated, the medication may consist of a single dose of a sustained-release composition over a course of treatment lasting several days to several weeks, or until a cure is achieved or a reduction in the disease state is achieved. The amount of composition to be administered will, of course, be determined by many factors, including the patient being treated, the severity of the pain, the method of administration, and the judgment of the prescribing physician. The therapeutic agents disclosed herein (e.g., cyclosporine analogs (e.g., CRV431)) or combinations of therapeutic agents may be administered orally or by injection at doses of 0.1 mg to 4000 mg per kg of body weight per day. The dose range for adults is generally 1 g to 100 g / day. Other forms of presentation, provided as tablets or individual units, contain, for convenience, an amount of the therapeutic agent disclosed herein (e.g., a cyclosporine analog (e.g., CRV431)) or a combination of therapeutic agents in an amount effective as a unit containing multiple identical, for example, 1g to 60g (e.g., about 5g to 20g, about 10g to 50g, about 20g to 40g, or about 25g to 35g). The exact amount of therapeutic agent administered to a patient is the responsibility of the attending physician. However, the dosage will depend on a number of factors, including the patient's age and sex, the exact disorder being treated, and its severity. Furthermore, the route of administration may vary depending on the condition and its severity. Typical doses of the therapeutic agent (e.g., a cyclosporine analog (e.g., CRV431)) may be 0.02g to 1.25g per kg of body weight, for example, 0.1g to 0.5g per kg of body weight, depending on the parameters. In some embodiments, the dosage of the therapeutic agent (e.g., a cyclosporine analog (e.g., CRV431)) may range from 1 g to 100 g, for example, 10 g to 80 g, 15 g to 60 g, 20 g to 40 g, or 25 g to 35 g. A physician can determine the required dosage of the therapeutic agent (e.g., a cyclosporine analog (e.g., CRV431)) for any particular subject.

[0147] For the pharmaceutical compositions of the therapeutic agents (e.g., cyclosporine analogs (e.g., CRV431)) or combinations of therapeutic agents disclosed herein, the exact formulation, route of administration, and dosage will be selected by the individual physician taking into account the patient's condition. Typically, the dosage range of the composition administered to a patient can be from about 0.1 to about 4000 mg per kg of the patient's body weight. The dosage can be a single dosage or a series of two or more dosages given over a period of one or more days, as needed by the patient. Where the human dosage of a therapeutic agent has been established for at least some conditions, the present disclosure will use those same dosages, or dosages between about 0.1% and about 5000%, more preferably between about 25% and about 1000% of the established human dosage. In cases of newly discovered pharmaceutical compounds, where the human dosage has not been established, an appropriate human dosage can be inferred from the ED 50 or ID 50 value, or from other appropriate values derived from in vitro or in vivo studies identified by animal toxicity and efficacy studies.

[0148] The attending physician should be aware of how and when to discontinue, interrupt, or adjust administration due to toxicity or organ failure. Conversely, the attending physician also knows to adjust the treatment to a higher level if the clinical response is not appropriate (toxicity not excluded). The magnitude of the dosage administered in the management of the disorder of interest will vary depending on the severity of the condition to be treated and the route of administration. The severity of the condition can be evaluated, in part, by standard prognostic assessment methods, for example. Further, the dosage and perhaps the dosing frequency will also vary depending on the age, weight, and response of the individual patient. Programs comparable to the above program may be used in veterinary medicine. The appropriate dosage is determined for each drug, but in most cases, the dosage can be generalized to some extent. In the case of pharmaceutically acceptable salt administration, the dosage may be calculated as free base. In some embodiments, the composition is administered 1 to 4 times a day. Alternatively, the compositions disclosed herein may be administered by continuous intravenous infusion, for example, in doses of up to 100 g of each active ingredient per day. As will be apparent to those skilled in the art, in certain circumstances, particularly to effectively and aggressively treat invasive diseases or infections, it may be necessary to administer the compositions disclosed herein in amounts exceeding, or even far exceeding, the preferred dosage range described above. In some embodiments, the therapeutic agents disclosed herein (e.g., cyclosporine analogs (e.g., CRV431)) or combinations of therapeutic agents will be administered over a period of continuous treatment, for example, over a week or more, or over several months or years.

[0149] In some embodiments, the administration schedule of the therapeutic agents (e.g., cyclosporine analogs (e.g., CRV431)) or combinations of therapeutic agents disclosed herein is carried out over a period that may be, for example, at least about 1 week to at least about 4 weeks, at least about 4 weeks to at least about 8 weeks, at least about 4 weeks to at least about 12 weeks, at least about 4 weeks to at least about 16 weeks, or longer. The administration schedule of the therapeutic agents (e.g., cyclosporine analogs (e.g., CRV431)) or combinations of therapeutic agents disclosed herein may be carried out three times a day, twice a day, daily, every other day, three times a week, every other week, three times a month, once a month, substantially continuously or continuously. Cyclosporine analogs (e.g., CRV431) may be administered alone or in the form of a composition (e.g., a pharmaceutical composition). In some embodiments, a pharmaceutical composition comprises a cyclosporine analog (e.g., CRV431) or a pharmaceutically acceptable salt, solvate, hydrate, clathrate, polymorph, prodrug or metabolite thereof, and one or more pharmaceutically acceptable carriers or excipients. The composition may optionally contain one or more additional therapeutic agents as described herein. A pharmaceutical composition comprising a therapeutically effective amount of a therapeutic agent (e.g., a cyclosporine analog (e.g., CRV431)) and one or more pharmaceutically acceptable carriers or excipients is formulated for administration to a subject for therapeutic use. In the sense of the content of a pharmaceutical composition, the terms “therapeutic agent,” “active ingredient,” “active drug,” and “drug” include prodrugs.

[0150] The pharmaceutical composition contains a therapeutic agent (e.g., a cyclosporine analog (e.g., CRV431)) in a substantially pure form. In some embodiments, the purity of the therapeutic agent is at least about 95%, 96%, 97%, 98%, or 99%. In some embodiments, the purity of the therapeutic agent is at least about 98% or 99%. Furthermore, the pharmaceutical composition is substantially free of contaminants or impurities. In some embodiments, the level of contaminants or impurities other than residual solvents in the pharmaceutical composition is about 5%, 4%, 3%, 2%, or 1% or less relative to the total mass of the intended active and inactive components. In some embodiments, the level of contaminants or impurities other than residual solvents in the pharmaceutical composition is about 2% or 1% or less relative to the total mass of the intended active and inactive components. The pharmaceutical composition is generally prepared in accordance with current Good Manufacturing Practices (GMP), as recommended and required by, for example, the Federal Food, Drug, and Cosmetic Act) §501(a)(2)(B) and the International Conference on Harmonisation Q7 Guideline.

[0151] Medicinally acceptable carriers and excipients include medicinal materials, vehicles, and substances. Non-limiting examples of excipients include liquid and solid fillers, diluents, binders, lubricants, flow enhancers, solubilizers, surfactants, dispersants, disintegrants, emulsifiers, wetting agents, suspending agents, thickeners, solvents, isotonic agents, buffers, pH adjusters, stabilizers, preservatives, antioxidants, antibacterial agents, antifungal agents, absorption retarders, sweeteners, flavoring agents, colorants, adjuvants, encapsulating materials, and coating materials. The use of such excipients in pharmaceutical formulations is technically well known. For example, common vehicles and carriers include, but are not limited to, oils (e.g., vegetable oils, e.g., sesame oil), aqueous solvents (e.g., physiological saline, phosphate-buffered saline [PBS] and isotonic solutions [e.g., Ringer's solution]), and solvents (e.g., dimethyl sulfoxide [DMSO] and alcohols [e.g., ethanol, glycerol, and propylene glycol]). Unless any common carrier or excipient is incompatible with the active ingredient, this disclosure encompasses the use of common carriers and excipients in formulations containing therapeutic agents (e.g., cyclosporine analogs (e.g., CRV431)). For example, see Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott Williams & Wilkins (Philadelphia, Pennsylvania

[2005] ); Handbook of Pharmaceutical Excipients, 5th Ed., Rowe et al., Eds., The Pharmaceutical Press and the American Pharmaceutical Association (2005); Handbook of Pharmaceutical Additives, 3rd Ed., Ash and Ash, Eds., Gower Publishing Co. (2007); and Pharmaceutical Preformulation and Formulation, Gibson, Ed., CRC Press (Boca Raton, Florida, 2004).

[0152] The appropriate formulation may depend on various factors, such as the chosen mode of administration. Possible modes of administration of a pharmaceutical composition containing a cyclosporine analog (e.g., CRV431) include, but are not limited to, oral, parenteral (including intramuscular, subcutaneous, intradermal, intravascular, intravenous, intraarterial, intraperitoneal, intramedullary, subarachnoid, and topical), intracavitary, and topical (skin / supercutaneous, percutaneous, mucous membrane, transmucosal, intranasal [e.g., by nasal spray or nasal drop], intrapulmonary [e.g., by oral or nasal inhalation], buccal, sublingual, rectal [e.g., by suppositories], and vaginal [e.g., by suppositories]). For example, formulations of cyclosporine analogs suitable for oral administration (e.g., CRV431) may be provided, for example, as boluses; as tablets, capsules, pills, cachets or lorangeries; as powders or granules; as semi-solids, licks, pastes or gels; as solutions or suspensions in aqueous and / or non-aqueous liquids; or as oil-in-water or water-in-oil emulsions.

[0153] The tablets may contain a cyclosporine analog (e.g., CRV431) in a mixture of, for example, a filler or inert diluent (e.g., calcium carbonate, calcium phosphate, lactose, mannitol, or microcrystalline cellulose), a binder (e.g., starch, gelatin, gum arabic, alginic acid or its salts, or microcrystalline cellulose), a lubricant (e.g., stearic acid, magnesium stearate, talc, or silicon dioxide), a disintegrant (e.g., crospovidone, croscarmellose sodium, or colloidal silica), and optionally a surfactant (e.g., sodium lauryl sulfate). The tablets may be uncoated or coated, for example, with an enteric coating to protect the active ingredient from the acidic environment of the stomach, or with a material that delays the disintegration and absorption of the active ingredient in the gastrointestinal tract, thereby providing a longer-lasting effect. In some embodiments, the tablets comprise a cyclosporine analog (e.g., CRV431), mannitol, microcrystalline cellulose, magnesium stearate, silicon dioxide, croscarmellose sodium, and sodium lauryl sulfate, and optionally lactose monohydrate, and the tablets may optionally be film-coated (e.g., with Opadry®).

[0154] Push-fit capsules or two-piece hard gelatin capsules may contain a cyclosporine analog (e.g., CRV431) in a mixture with, for example, a filler or an inert solid diluent (e.g., calcium carbonate, calcium phosphate, kaolin, or lactose), a binder (e.g., starch), a flow enhancer or lubricant (e.g., talc or magnesium stearate), and a disintegrant (e.g., crospovidone), and optionally a stabilizer and / or preservative. For soft capsules or single-piece gelatin capsules, a cyclosporine analog (e.g., CRV431) can be dissolved or suspended in a suitable liquid (e.g., liquid polyethylene glycol or an oil medium, e.g., fatty oil, peanut oil, olive oil, or liquid paraffin), and liquid-filled capsules may contain one or more other liquid excipients and / or semi-solid excipients, e.g., stabilizers and / or amphiphilic agents (e.g., fatty acid esters of glycerol, propylene glycol, or sorbitol).

[0155] Orally administered compositions can also be formulated as solutions or suspensions in aqueous and / or non-aqueous liquids, or as oil-in-water or water-in-oil emulsions. Dispersible powders or granules of cyclosporine analogs (e.g., CRV431) can be mixed with aqueous liquids, organic solvents and / or oils and any suitable combination of suitable excipients (e.g., any combination of dispersants, wetting agents, suspending agents, emulsifiers, and / or preservatives) to form solutions, suspensions, or emulsions. Cyclosporine analogs (e.g., CRV431) can also be formulated for parenteral administration by injection or infusion to avoid gastrointestinal absorption and first-pass metabolism. A typical route of parenteral administration is intravenous.

[0156] Further advantages of intravenous administration include the direct delivery of therapeutic agents into the systemic circulation to achieve rapid systemic effects, as well as the ability to administer drugs continuously and / or in large quantities as needed. Injectable or infusion formulations may be, for example, solutions, suspensions, or emulsions in oily or aqueous vehicles, and may contain excipients such as suspending agents, dispersants, and / or stabilizers. For example, an aqueous or non-aqueous (e.g., oily) sterile injection solution may contain a cyclosporine analog (e.g., CRV431) along with excipients such as antioxidants, buffers, bacteriostatic agents, and solutes that give the formulation isotonicity with the subject's blood. An aqueous or non-aqueous sterile suspension may contain a cyclosporine analog (e.g., CRV431) along with suspending agents and thickeners, and optionally with excipients such as stabilizers and agents that increase the solubility of the cyclosporine analog (e.g., CRV431) to enable the preparation of more concentrated solutions or suspensions. As another example, a sterile aqueous solution for injection or infusion (e.g., subcutaneous or intravenous) may contain a cyclosporine analog (e.g., CRV431), NaCl, a buffer (e.g., sodium citrate), a preservative (e.g., meta-cresol), and optionally a base (e.g., NaOH) or / and an acid (e.g., HC1) to adjust the pH.

[0157] For local administration, cyclosporine analogs (e.g., CRV431) can be formulated, for example, as buccal tablets, sublingual tablets, or pills. Buccal tablets, sublingual tablets, or pills can bypass first-pass metabolism and gastrointestinal absorption. Buccal tablets, sublingual tablets, or pills can be designed to achieve faster release of cyclosporine analogs (e.g., CRV431) for more rapid uptake into systemic circulation. In addition to a therapeutically effective amount of cyclosporine analog (e.g., CRV431), buccal tablets, sublingual tablets, or pills may contain, but are not limited to, appropriate excipients including any combination of fillers and diluents (e.g., mannitol and sorbitol), binders (e.g., sodium carbonate), humectants (e.g., sodium carbonate), disintegrants (e.g., crospovidone and croscarmellose sodium), lubricants (e.g., silicon dioxide [including colloidal silicon dioxide] and sodium stearyl fumarate), stabilizers (e.g., sodium bicarbonate), flavoring agents (e.g., spearmint flavor), sweeteners (e.g., sucralose), and colorants (e.g., yellow iron oxide).

[0158] For local administration, cyclosporine analogs (e.g., CRV431) can also be formulated for intranasal administration. The nasal mucosa provides a large surface area, porous endothelium, highly vascularized subepithelial layer, and high absorption rate, enabling high bioavailability. Furthermore, intranasal administration avoids first-pass metabolism, allowing a considerable concentration of cyclosporine analog (e.g., CRV431) to be introduced into the central nervous system, enabling the cyclosporine analog (e.g., CRV431) to block the central cough reflex by the nucleus tractus solitarius in the cough center of the medulla oblongata where vagal afferent nerves terminate. Intranasal solutions or suspension formulations may contain a cyclosporine analog (e.g., CRV431) together with excipients such as a solubility enhancer (e.g., propylene glycol), a wetting agent (e.g., mannitol or sorbitol), a buffer, and water, and optionally an excipient such as a preservative (e.g., benzalkonium chloride), a mucosal adhesion agent (e.g., hydroxyethylcellulose), and / or an osmotic enhancer. In some embodiments, nasal spray formulations contain a cyclosporine analog (e.g., CRV431), microcrystalline cellulose, sodium carboxymethylcellulose, dextrose, and water, and optionally an acid (e.g., HC1) to adjust the pH. Intranasal solutions or suspension formulations may be administered into the nasal cavity by any suitable means, including, but not limited to, a dropper, pipette, or spray using, for example, a metered-dose spray pump. Further modes of local administration include intrapulmonary administration, including oral inhalation and nasal inhalation.

[0159] In some embodiments, a cyclosporine analog (e.g., CRV431) is delivered from a sustained-release composition. As used herein, the term “sustained-release composition” encompasses sustained-release, prolonged-release, extended-release, slow-release, and controlled-release compositions, systems, and devices. The use of sustained-release compositions may have benefits such as improved profile of the amount of drug or its active metabolite delivered to a target site over a period of time, including the delivery of therapeutically effective amounts of the drug or its active metabolite over a long period of time. In some embodiments, a sustained-release composition delivers a cyclosporine analog (e.g., CRV431) over a period of at least about 1 day, 2 days, 3 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, or longer. In some embodiments, the sustained-release composition is a drug encapsulation system, such as nanoparticles, microparticles, or capsules made of a biodegradable polymer and / or hydrogel. In some embodiments, the sustained-release composition includes a hydrogel. Non-limiting examples of polymers that can constitute a hydrogel include polyvinyl alcohol, acrylate polymers (e.g., sodium polyacrylate), and other homopolymers and copolymers having a relatively large number of hydrophilic groups (e.g., hydroxyl and / or carboxylate groups). In some embodiments, the sustained-release drug encapsulation system includes a membrane-enclosed reservoir containing a drug, and the membrane is permeable to the drug. The drug delivery system may be in the form of, for example, a transdermal patch.

[0160] In some embodiments, the sustained-release composition is in oral dosage form, such as tablets or capsules. For example, the drug can be embedded in an insoluble porous matrix such that it must create a pathway for the dissolved drug to exit the matrix before it can be absorbed through the gastrointestinal tract. Alternatively, the drug can be embedded in a matrix that swells to form a gel through which the drug can exit. Sustained release can also be achieved by means of a monolayer or multilayer osmotically controlled-release oral delivery system (OROS). An OROS is a tablet having a semipermeable outer membrane and one or more small laser-drilled holes inside. As the tablet passes through the body, water is absorbed through the semipermeable membrane by osmosis, and the resulting osmotic pressure pushes the drug out of the holes in the tablet into the gastrointestinal tract, where it can be absorbed.

[0161] The sustained-release composition can be formulated as polymer nanoparticles or microparticles, which can be delivered, for example, by inhalation, injection, or from an implant. In some embodiments, the polymer implant or polymer nanoparticles or microparticles are composed of a biodegradable polymer. In some embodiments, the biodegradable polymer includes lactic acid and / or glycolic acid [e.g., L-lactic acid copolymers, e.g., poly(L-lactide-co-glycolide) or poly(L-lactic acid-co-D,L-2-hydroxyoctanoic acid)]. For example, biodegradable polymer microspheres composed of polylactic acid and / or polyglycolic acid can function as a sustained-release lung drug delivery system. The biodegradable polymer of the polymer implant or polymer nanoparticles or microparticles can be selected such that the polymer is substantially completely degraded by the time the end of the treatment period is expected, and that the by-products of polymer degradation, such as the polymer being biocompatible.

[0162] For delayed or sustained release of a cyclosporine analog (e.g., CRV431), the composition can also be formulated as a depot that can be implanted or injected into a subject, for example, intramuscularly or subcutaneously. Depot formulations can be designed to deliver the cyclosporine analog (e.g., CRV431) over longer periods, for example, over periods of at least about 1 week, 2 weeks, 3 weeks, 1 month, 6 weeks, 2 months, 3 months or longer. For example, the cyclosporine analog (e.g., CRV431) can be formulated with polymer materials (e.g., polyethylene glycol (PEG), polylactic acid (PLA), or polyglycolic acid (PGA), or their copolymers (e.g., PLGA)), hydrophobic materials (e.g., as an emulsion in oil) and / or ion exchange resins, or poorly soluble derivatives (e.g., poorly soluble salts). As a helpful example, the cyclosporine analog (e.g., CRV431) can be incorporated or embedded in sustained-release microparticles composed of PLGA and formulated as a depot lasting 1 month.

[0163] A cyclosporine analog (e.g., CRV431) may also be contained in or dispersed in the matrix material. The matrix material may contain a polymer (e.g., ethylene-vinyl acetate) that can control the release of the compound by controlling its dissolution and / or, for example, its diffusion from the reservoir, thereby increasing the stability of the compound while it is contained in the reservoir. The release system can be designed as a sustained-release system, for example, formed as a transdermal or transmucosal patch, and may contain excipients that can accelerate the release of the compound, such as a water-swellable material (e.g., hydrogel) that helps to expel the compound from the reservoir. The release system can achieve a temporally regulated release profile (e.g., pulsed release) when a temporal variation in plasma levels is desired, or a more continuous or consistent release when a constant plasma level is desired. Pulsed release can be achieved from individual reservoirs or from multiple reservoirs. For example, if each reservoir provides a single pulse, multiple pulses ("pulsed" release) can be achieved by temporally staggering the single pulse releases from each of the multiple reservoirs so as not to overlap.

[0164] Furthermore, pharmaceutical compositions containing cyclosporine analogs (e.g., CRV431) can be formulated, for example, as liposomes, micelles (e.g., composed of biodegradable natural polymers and / or biodegradable synthetic polymers, e.g., lactosomes), microspheres, microparticles, or nanoparticles, whether or not they are designed for sustained release. Pharmaceutical compositions can be manufactured using any suitable method that is technically well known, for example, by conventional mixing, dissolving, suspending, granulating, sugar-coating, levigating, emulsifying, encapsulating, or compressing processes. All active and inactive components can be mixed in a suitable system, and pharmaceutical compositions can be provided in unit dosage forms as single doses without the need to mix components to form a composition to be administered. The unit dosage form may contain an effective dose, or an appropriate fraction thereof, of a therapeutic agent (e.g., a cyclosporine analog (e.g., CRV431)). Typical examples of unit dosage forms include tablets, capsules, or pills for oral administration, and powders in vials or ampoules for oral or nasal inhalation.

[0165] A pharmaceutical composition disclosed herein can be provided as a kit in which the active ingredient, excipients, and carrier (e.g., solvent) are provided in two or more separate containers (e.g., ampoules, vials, tubes, bottles, or syringes) and need to be mixed to form a composition to be administered. The kit may contain instructions for storing, preparing, and administering the composition (e.g., a solution to be administered intravenously). The kit can contain all the active and inactive ingredients in a unit dosage form or the active and inactive ingredients can be contained in two or more separate containers, and may contain instructions for using the pharmaceutical composition. In some embodiments, the kit includes a cyclosporine analog (e.g., CRV431) or a pharmaceutically acceptable salt, solvate, hydrate, clathrate, polymorph, prodrug or metabolite thereof, and instructions for administering the compound.

Example

[0166] Example Some aspects of the above embodiments are further disclosed in detail in the following examples, which are not intended to limit the scope of the present disclosure in any way. Example 1 Determination of the antifibrotic activity of CRV431 in fibroblasts from multiple different organs In this example, the ability of CRV431 to reduce the production of ECM molecules, collagen and fibronectin was determined using five different types of fibroblasts, including (1) lung fibroblasts from patients with idiopathic pulmonary fibrosis ("IPF"), (2) cardiac fibroblasts, (C) skin fibroblasts, (4) renal mesangial cells, and (5) LX2 hepatic stellate cell line. As shown in this example, CRV431 dose-dependently reduced the secretion of procollagen and fibronectin from all five types of fibroblasts of similar size, measured by enzyme-linked immunosorbent assay (ELISA). The degree of inhibition was similar regardless of the presence or absence of cell stimulation by the fibrogenic factor, transforming growth factor β (TGFβ), and was consistent with a direct effect on ECM synthesis. Furthermore, CRV decreased ECM production by up to 55% dose-dependently without causing any decrease in cell viability at clinically significant concentrations. As disclosed herein, ECM production can be reduced by inhibiting cyclophilin B using CRV431, and in line with this hypothesis, downregulation of cyclophilin B by small interfering RNA (siRNA) similarly decreased the secretion of procollagen and fibronectin.

[0167] Human LX-2 hepatic stellate cells (Millipore-Sigma, Cat. # SCC064) were cultured in 96-well plates. Three wells were treated for 24 hours (100 μl treatment per well). Cells were nearly confluenced at the start of treatment. Cell viability analysis at the end of treatment showed that CRV431 treatment did not affect cell viability. Used media collected at the end of treatment were assayed for procollagen and fibronectin using enzyme-linked immunosorbent assay (ELISA) (procollagen ELISA antibody pair from Abcam (ab216064) and fibronectin ELISA antibody pair (ab222262)). Procollagen and fibronectin were not detected in media not applied to cells. The levels of procollagen and fibronectin in the media are expressed as the mean ± SD (bar) from the three replicated wells (white circles). As shown in Figures 1A and 1B, CRV431 dose-dependently reduced the levels of procollagen and fibronectin in the culture medium, both in the absence and in the presence of 0.1 ng / ml TGFβ.

[0168] Human lung fibroblasts (Lonza Bioscience, Cat. # CC-7231) from patients with idiopathic pulmonary fibrosis (IPF) were cultured in 96-well plates. Three wells were treated for 6 days (100 μl treatment per well). Cells were nearly confluenced at the start of treatment. Cell viability analysis at the end of treatment showed that CRV431 treatment did not affect cell viability. Used media collected at the end of treatment were assayed for procollagen and fibronectin by enzyme-linked immunosorbent assay (ELISA) (procollagen ELISA antibody pair from Abcam (ab216064) and fibronectin ELISA antibody pair (ab222262)). Procollagen and fibronectin were not detected in media not applied to cells. The levels of procollagen and fibronectin in the media are expressed as mean ± SD (bar) from the three replicated wells (white circles). As shown in Figures 2A and 2B, CRV431 dose-dependently reduced the levels of procollagen and fibronectin in the culture medium, both in the absence and in the presence of 0.4 ng / ml TGFβ.

[0169] Human cardiac fibroblasts (Lonza Bioscience, Cat. #CC-2903) were cultured in 96-well plates. Three wells were treated for 6 days (100 μl treatment per well). Cells were nearly confluenced at the start of treatment. Cell viability analysis at the end of treatment showed that CRV431 treatment did not affect cell viability. Used media collected at the end of treatment were assayed for procollagen and fibronectin using enzyme-linked immunosorbent assay (ELISA) (procollagen ELISA antibody pair from Abcam (ab216064) and fibronectin ELISA antibody pair (ab222262)). Procollagen and fibronectin were not detected in media not applied to cells. The levels of procollagen and fibronectin in the media are expressed as mean ± SD (bar) from the three replicated wells (white circles). As shown in Figures 3A and 3B, CRV431 dose-dependently reduced the levels of procollagen and fibronectin in the culture medium, both in the absence and in the presence of 0.4 ng / ml TGFβ.

[0170] Human renal mesangial cells (ScienCell, Cat. # 4200(SC)) were cultured in 96-well plates. Three wells were treated for 2 days (100 μl treatment per well), followed by a repeat treatment for another 2 days. Cells were nearly confluenced at the start of treatment. Cell viability analysis at the end of treatment (4 days) showed that CRV431 treatment did not affect cell viability. Used media collected from days 2 to 4 of treatment were assayed for procollagen and fibronectin using enzyme-linked immunosorbent assay (ELISA) (procollagen ELISA antibody pair from Abcam (ab216064) and fibronectin ELISA antibody pair (ab222262)). Procollagen and fibronectin were not detected in media not applied to cells. The levels of procollagen and fibronectin in the media are expressed as the mean ± SD (bar) from three replicated wells (white circles). As shown in Figures 4A and 4B, CRV431 dose-dependently reduced the levels of procollagen and fibronectin in the culture medium, both in the absence and in the presence of 0.4 ng / ml TGFβ.

[0171] Human adult dermal fibroblasts (Lonza Bioscience, Cat. # CC-2511) were cultured in 96-well plates. Three wells were treated for 1 day (100 μl treatment per well), followed by a repeat treatment for another day. Cells were nearly confluenced at the start of treatment. Cell viability analysis at the end of treatment (4 days) showed that CRV431 treatment did not affect cell viability. Used media collected on days 1 and 2 of treatment were assayed for procollagen and fibronectin using enzyme-linked immunosorbent assay (ELISA) (procollagen ELISA antibody pair from Abcam (ab216064) and fibronectin ELISA antibody pair (ab222262)). Procollagen and fibronectin were not detected in media not applied to cells. The levels of procollagen and fibronectin in the media are expressed as the mean ± SD (bar) from the three replicated wells (white circles). As shown in Figures 5A and 5B, CRV431 dose-dependently reduced the levels of procollagen and fibronectin in the culture medium, both in the absence and in the presence of 10 ng / ml epidermal growth factor (EGF) or 0.4 ng / ml TGFβ.

[0172] Example 2 Treatment of human IPF lung thin sections in culture with CRV431 This example describes a study conducted using precision sectioned lung tissue. Precision sectioned lung tissue from biopsy-confirmed idiopathic pulmonary fibrosis (IPF) patients was cultured for 6 days. Six replicated sections were treated with each treatment on day 2 of the culture, and were replaced daily on days 3, 4, and 5 of the culture. To create the secretion marker results (daily mean change %) shown in Figure 6A, used culture media (at 24-hour intervals) were collected on days 3, 4, 5, and 6, and the secretion of six markers of inflammation and fibrosis—monocyte chemotactic protein (MCP-1), interleukin-6 (IL-6), matrix metalloproteinase-7 (MMP-7), tissue inhibitor of metalloproteinase-1 (TIMP1), hyaluronic acid, and collagen 1α1—was measured by ELISA. For each drug treatment, the mean secretion levels from six replicate sections were expressed as a percentage change relative to DMSO vehicle treatment for each daily treatment, and then the mean percentage change from four 1-day treatment intervals was calculated.

[0173] To create the gene expression results shown in Figure 6B, RNA was isolated from six lung thin sections pooled for each treatment on day 6 of culture, four days after drug or vehicle treatment. Six markers of inflammation and fibrosis—MCP-1, IL-6, TGFβ, TIMP1, collagen 1α1, and α-smooth muscle actin (αSMA)—were evaluated by reverse transcription-polymerase chain reaction (RT-PCR). The relative levels of each target gene RNA were calculated using β-actin as the reference gene. CRV431, applied at a concentration of 5 μM, reduced the mean daily secretion and gene expression of all markers, similar to Alk5i (a TGFβ receptor kinase inhibitor), pirfenidone (a treatment approved for IPF), and nintedanib (a treatment approved for IPF).

[0174] Example 3 Treatment of renal fibrosis using CRV431 in a mouse unilateral ureteral ligation (UUO) model This example describes a study conducted using a mouse unilateral ureteral ligation (UUO) model of renal fibrosis. This study used 7-week-old female C57BL / 6 mice (n=8 mice / group). Pseudogroup mice did not undergo UUO. The remaining mice underwent complete surgical ligation of the left ureter. Starting on the day of surgery, vehicles and CRV431 were administered orally daily at a dose of 50 mg / kg / day for 13 days. Mice were sacrificed on day 14, and the left kidneys were histologically processed and fibrous collagen stained with Sirius Red. Morphometrically, the percentage of tissue sections with Sirius Red staining was measured in 5 sections from each kidney. The results are shown in Figure 7A (showing Sirius Red in individual tissue sections) and Figure 7B (showing Sirius Red in individual mice (5 tissue sections / mouse)). One-way ANOVA and Tukey's multiple comparisons, presenting data both section-wise and animal-wise, demonstrated that CRV431 treatment resulted in a statistically significant reduction in Sirius Red staining.

[0175] Example 4 Antifibrotic activity of CRV431 in human precision-sectioned liver thin sections (PCLS) This example describes a study conducted in human PCLS to determine the anti-fibrotic activity of CRV431. PCLS from all five human donors were observed to have some pre-existing fibrosis, increased to 7–11% near the fraction area by TGFβ+PDGF-BB stimulation. CRV431 was the most effective of the five NASH drug candidates in the prevention of TGFβ+PDGF-BB-induced histofibrosis. Most CRV431-treated thin sections also showed less fibrosis than vehicle-treated thin sections after 6 days of culture in the absence of exogenous TGFβ+PDGF-BB. The reduction in histofibrosis was accompanied by decreased secretion of collagen 1α1, fibronectin, hyaluronic acid, IL-6, and MCP-1, as well as decreased RNA levels of collagen 1α1, αSMA, TIMP1, IL-6, and MCP-1 as demonstrated by qRT-PCR. RNA-Seq analysis similarly showed that CRV431 reduces the expression of many fibrosis-related genes, such as more than 10 collagen genes, collagen hydroxylase and oxidase, ACTA2, VEGF, and TIMPs. Gene expression varied considerably among donors, with less than 200 genes altered by CRV431 in all donors without exception. Many of these pan-donor transcriptional changes were consistent with the anti-NASH, anti-fibrotic, and anti-cancer activities described in the literature for these genes. Notable genes affected by CRV431 without exception in the absence of TGFβ+PDGF-BB were ESM1 (RNA decreased 2.2 times; -2.2), NCOA3 (-2.7), IFI44L (-4.8), mIR-194-2 (-7.9), and DKK1 (RNA increased 3.8 times; +3.8). In the presence of exogenous TGFβ+PDGF-BB, the notable genes affected by CRV431 without exception were LOXL2 (-1.9), UBD / FAT10 (-2.0), ESM1 (-2.6), STRA6 (-3.1), RCCD1 (-3.6), and DUOX2 (-4.5). While not bound by any particular theory, the results described herein suggest that CRV431 has the ability to prevent fibrosis formation and reverse fibrosis.

[0176] PCLS was obtained from five human donors who underwent resection for liver cancer. Replicate sections were collected from healthy resection margins and cultured for 4 or 6 days according to the experimental protocol. In the non-stimulation protocol, sections were cultured for 6 days and treated with DMSO vehicle or 5 μM CRV431 throughout the period. In the stimulation protocol, sections were rested for 1 day, and then administered cytokines, TGFβ, and PDGF-BB for 3 days to stimulate inflammation and fibrosis. In the stimulation protocol, DMSO vehicle, CRV431 (1 and 5 μM), Alk5i (10 μM), obeticholic acid (5 μM), ellafibranol (5 μM), resmetirom (5 μM), and aramchol (5 μM) were administered individually as drug treatments simultaneously with TGFβ+PDGF-BB. The culture medium and treatment agents were changed daily. The following four types of evaluations were performed on PCLS: (a) secretion of inflammatory and fibrotic biomarkers into the culture medium, (b) histological staining and quantification of Sirius Red as measures of histological fibrosis, (c) gene expression of inflammatory and fibrotic biomarkers by RT-PCR, and (d) RNA sequencing of the complete transcriptome (RNA-Seq). Each of the four types of evaluations is described below.

[0177] (a) Biomarker secretion. Used culture media was collected daily from paired tissue sections for each treatment. The secretion of monocyte chemotactic protein (MCP-1), interleukin-6 (IL-6), tissue inhibitor of metalloproteinase 1 (TIMP1), hyaluronic acid, fibronectin, and collagen 1α1 was quantified using ELISA. For each donor, the average level of biomarker secretion in each treatment group was expressed as a percentage change relative to the DMSO vehicle. The average percentage was calculated for all donors, and finally, the average daily change percentage was calculated from all evaluation days. The results are shown in Figure 8A (secretion marker - average daily change %). (b) Sirius Red Histological Staining. At the end of the experiment, PCLS were histologically treated and stained with Sirius Red to demonstrate histological fibrosis. In the histological sections, Sirius Red was quantified morphometrically as the percentage of area stained with Sirius Red in 10 sections from each treatment. In the non-stimulation protocol, the amount of Sirius Red staining was expressed relative to the vehicle group. In the stimulation protocol, the amount of Sirius Red staining was expressed relative to non-stimulated PCLS (0%) and TGFβ+PDGF-BB stimulated+vehicle PCLS (100%). The results are shown in Figure 8B (Sirius Red Staining for Histological Fibrosis).

[0178] (c) Gene expression by RT-PCR. RNA was isolated from paired liver thin sections after each treatment at the end of the experiment, and five markers of inflammation and fibrosis: MCP-1, IL-6, TIMP1, αSMA, and collagen 1α1 were evaluated by RT-PCR. The relative levels of each target gene RNA were calculated using β-actin as the reference gene. CRV431, applied at a concentration of 5 μM, reduced the average daily secretion and gene expression of all markers, similar to Alk5i (a TGFβ receptor kinase inhibitor), pirfenidone (a treatment approved for IPF), and nintedanib (a treatment approved for IPF). The results are shown in Figure 8C (genes by RT-PCR). (d) Gene expression by RNA-Seq. Full transcriptome RNA sequencing (30 million reads per sample) was performed on vehicle and 5 μM CRV431 treated groups from three donors using both non-stimulating and stimulated protocols. The data were analyzed using bioinformatics software to identify genes that were differentially expressed between vehicle treatment and CRV431 treatment. For the evaluation of (d), 12 samples corresponding to 3 different donor slides that received 4 different treatments were examined. In this analysis, the following comparisons were made: (1) TGFb / PDGF + CRV vs. TGFb / PDGF + Vehicle(V), and (2) unstimulated + CRV vs. unstimulated + vehicle. Table 1 below shows the sample ID and corresponding treatment.

[0179] [Table 2]

[0180] Quality control checks were performed on all samples. All samples had over 30 million reads and a mean quality score of >35 for almost all bases in the sequence. In the realities of RNA sequencing, a level of duplication is expected, and it is anticipated that different copies of the same RNA may be present in the samples. Since all samples had a good quantity and quality of reads, all samples passed the checked QC and were included in the analysis. TGFb / PDGF+CRV431 versus TGFb / PDGF+vehicle were compared by group. Principal component analysis (PCA) plots were created to analyze the distribution of samples (Figure 9A). This shows a strong donor influence, as they cluster based on donor, but there are also differences between treatments, as they separate in the PCA plot as shown in Figure 9B. In the MA plot in Figure 9C, the mean count is plotted against the log fold change. Significant genes that were expressed differently in the comparison between TGFb / PDGF+CRV and TGFb / PDGF+vehicle (p-adjusted <0.05) are shown in red.

[0181] Heatmaps were also created to compare TGFb / PDGF+CRV and TGFb / PDGF+vehicle, plotting significantly differently expressed genes (p-value adjustment <0.05, logarithmic scaling >0.5). To observe the different expression patterns within the groups, heatmaps of samples grouped by group are shown in Figure 10A (blue = vehicle; red = CRV). The same significant genes were also plotted in a separate heatmap (Figure 10B), but this time the samples were grouped by donor to observe differences between donors (blue = vehicle; red = CRV). Furthermore, significant hits were plotted on a volcano plot (Figure 11), significant genes were plotted in red, and their corresponding symbols (IDs) were displayed. Downward and upward control were relative to CRV treatment. Table 2 below shows the genes that were significantly expressed differently in a comparison between TGFb / PDGF+CRV and TGFb / PDGF+vehicle (p-adjusted >0.05, logarithmic scaling >0.5). The scaling factor is relative to the treatment (CRV).

[0182] [Table 3] TIFF2026069500000047.tif178162

[0183] As PCA analysis shows, there is a strong influence from the donor. Therefore, each donor was analyzed individually. Count comparisons were performed donor-specific for TGFb / PDGF+CRV341 versus TGFb / PDGF+vehicle. First, genes with a difference in count (TGFb / PDGF+vehicle - TGFb / PDGF+CRV) <-300 were selected. Therefore, the vehicle group had at least 300 fewer copies than the CRV-treated group. In other words, this treatment had at least 300 more copies than the vehicle-treated group. After performing this selection individually for each donor, Venn diagrams were plotted to show the overlap among all three donors. As shown in Figure 12A, 117 genes had at least 300 more copies in the CRV-treated samples than in the vehicle-treated samples. Next, we selected genes with a difference in count (TGFb / PDGF + vehicle - TGFb / PDGF + CRV) > 300, and therefore genes that had at least 300 more copies in the vehicle sample compared to the CRV-treated sample. In other words, these genes had at least 300 fewer copies in the CRV-treated sample compared to the vehicle sample. After performing this selection individually for each donor, we plotted Venn diagrams to show the overlaps among all three donors. As shown in Figure 12B, 279 genes had at least 300 fewer copies in the CRV-treated sample compared to the vehicle sample.

[0184] Next, we compared the unstimulated + CRV431 group with the unstimulated + vehicle group. Principal component analysis (PCA) plots were created to analyze the distribution of the samples (Figure 13A). As expected, a strong influence of the donor was observed, as they clustered based on the donor rather than the treatment. However, they separated in the PCA plot (Figure 13B), indicating differences between the treatments. For the MA plot shown in Figure 13C, the mean count was plotted against the logarithmic scaling change. Significant genes (enes) that were expressed differently in the unstimulated + CRV and unstimulated + vehicle comparison are shown in red (p-adjusted <0.05). Heatmaps were created by plotting significantly differently expressed genes to compare unstimulated + CRV and unstimulated + vehicle (p-value adjustment <0.05, logarithmic scaling change >0.5). First, to observe the different expression patterns present within the groups, heatmaps of samples grouped by group are shown (Figure 14A) (blue = vehicle; red = CRV). Next, the same significant genes were plotted on the heatmap (Figure 14B), but this time the samples were grouped by donor to observe differences between donors (blue = vehicle; red = CRV). Significant hits were plotted on a volcano plot (Figure 15). Significant genes are plotted in red, and their corresponding symbols (IDs) are displayed. Downregulatory and upregulatory genes are for treated CRV. Table 3 below shows the genes that were significantly expressed differently in a comparison between unstimulated CRV and unstimulated vehicle (p-adjusted >0.05, logarithmic scaling >0.5). The scaling changes shown are relative to treated CRV.

[0185] [Table 4] TIFF2026069500000049.tif249153 TIFF2026069500000050.tif86162

[0186] Next, we compared unstimulated + CRV431 versus unstimulated + vehicle, donor by donor. As shown in the PCA, there was a strong influence from the donor. Therefore, we repeated the analysis as in the previous comparison. First, we selected genes where there was a difference in count (unstimulated + vehicle - unstimulated + CRV) < -300, and therefore the vehicle group had at least 300 fewer copies than the CRV-treated group. In other words, the treated CRV had at least 300 more copies than the vehicle. After performing this selection individually for each donor, we plotted Venn diagrams to show the overlap among all three donors. As shown in Figure 16A, 210 genes had at least 300 more copies in the CRV-treated group than in the vehicle. Next, we selected genes where there was a difference in count (unstimulated + vehicle - unstimulated + CRV) > 300, and therefore the vehicle sample had at least 300 more copies compared to the CRV-treated group. That is, the treated group had at least 300 fewer copies than the vehicle group. After making this selection individually for each donor, a Venn diagram was plotted to show the overlap among all three donors. As shown in Figure 16B, 255 genes had fewer than 300 copies in the vehicle after CRV treatment.

[0187] In at least some of the embodiments described above, one or more elements used in one embodiment may be used interchangeably in another embodiment, provided that such substitution is not technically impossible. As will be apparent to those skilled in the art, various other omissions, additions, and modifications may be made to the above methods and structures, provided that they do not depart from the scope of the subject matter of the claims. All such modifications and changes are intended to fall within the scope of the subject matter as defined by the appended claims. In substantially any use of plural and / or singular terms herein, those skilled in the art can substitute plurals for singular and / or singulars for plural as appropriate to the context and / or this application. Various singular / plural substitutions are explicitly shown herein for clarity. As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise. Any reference to "or" herein is intended to include "and / or" unless otherwise specified.

[0188] Generally, those skilled in the art will understand that the terms used herein, and in particular in the appended claims (e.g., the main part of the appended claims), are intended to be generally "open" terms (for example, the term "including" should be interpreted as "including, but not limited to," the term "having" should be interpreted as "having at least," and the term "include" should be interpreted as "including, but not limited to," etc.). Furthermore, where a specific number of introductory claims are intended, that intention will be explicitly enumerated in the claims, and in the absence of such enumeration, that intention does not exist. For example, for the sake of understanding, the following appended claims may introduce a list of claims, including the introductory phrases "at least one" and "one or more." However, the use of the phrase should not be interpreted as meaning that the introduction of a claim enumeration by the indefinite article "a" or "an" limits any particular claim containing such an introductory claim enumeration to embodiments containing only one such enumeration, even when the claim includes the introductory phrase "one or more" or "at least one" and the indefinite article, e.g., "a" or "an" (for example, "a" and / or "an" should be interpreted as meaning "at least one" or "one or more"); the same applies to the use of the definite article to introduce a claim enumeration. Furthermore, even if a specific number of introductory claim enumerations are explicitly stated, a person skilled in the art will recognize that such enumerations should be interpreted as meaning at least the number enumerated (for example, the enumeration "two enumerations" without other modifiers means at least two enumerations, or two or more enumerations). Furthermore, when using a similar convention to "at least one of A, B, and C, etc.," the configuration is generally intended to mean that a person skilled in the art will understand this convention (for example, "a system having at least one of A, B, and C" would include, but not be limited to, a system having only A, only B, only C, A and B together, A and C together, B and C together, and / or a system having A, B, and C together).Where a similar convention is used to "at least one of A, B, or C, etc.," it is generally intended that a person skilled in the art will understand this convention (for example, "a system having at least one of A, B, or C" would include, but not be limited to, a system having only A, only B, only C, A and B together, A and C together, B and C together, and / or a system having A, B, and C together). Furthermore, a person skilled in the art will understand that any disjunctive word and / or phrase that presents effectively two or more alternative terms, whether in this description, claims, or drawings, should be understood as intended to include one of those terms, either of those terms, or both of those terms.

[0189] Furthermore, if any feature or aspect of the present disclosure is described from the perspective of the Markush group, a person skilled in the art will recognize that the present disclosure is also described from the perspective of any individual member or subgroup member of the Markush group. As will be obvious to those skilled in the art, for all purposes, from the perspective of such written descriptions, all scopes disclosed herein also encompass all possible subscopes and combinations thereof. Any described scope can be readily considered sufficiently described and can be divided into at least equal 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 10, etc. As a non-limiting example, each scope described herein can be readily divided into a lower third, a middle third, and an upper third, etc. Similarly, as will be obvious to those skilled in the art, all words such as “up to,” “at least,” “greater than,” and “less than” include the listed numbers and refer to scopes that can then be divided into subgroups as described above. Finally, as will be obvious to those skilled in the art, a scope includes each individual member. Thus, for example, a group having 1 to 3 items refers to a group having 1, 2, or 3 items. Similarly, a group having 1 to 5 items refers to a group having 1, 2, 3, 4, or 5 items, and so on.

[0190] While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be obvious to those skilled in the art. The various aspects and embodiments disclosed herein are for illustrative purposes only and are not intended to limit the scope and spirit, and the true scope and spirit are shown in the following claims.

Claims

1. A method for treating non-liver fibrosis, wherein the subject requiring it is given the following formula L: 【Chemistry 1】 (In the formula: a. R' is either H or acetyl; b. R1 is a saturated or unsaturated linear or branched aliphatic carbon chain with a length of 2 to 15 carbon atoms; c. R2 is as follows: i. H; ii. Unsubstituted, N-substituted, or N,N disubstituted amides; iii. N-substituted or unsubstituted acyl-protected amines; iv. N-substituted or unsubstituted amines; v. Carboxylic acid; vi. Nitrile; vii. Esther; viii. Ketones; ix. Hydroxy, dihydroxy, trihydroxy, or polyhydroxyalkyl; and x. Substitutable or non-substitutable aryl; xi. A saturated or unsaturated linear or branched aliphatic chain that may optionally contain substituents selected from the group consisting of hydrogen, ketones, hydroxyl, nitrile, carboxylic acid, ester, 1,3-dioxolane, halogen, and oxo; xii. Aromatic groups containing substituents selected from the group consisting of halogens, esters, and nitros; and xiii. A combination of a saturated or unsaturated linear or branched aliphatic chain of (xi) and an aromatic group of (xii). Selected from the group consisting of; and d. R23 is a saturated or unsaturated linear or branched aliphatic carbon chain that may be optionally substituted. The method comprising administering a composition comprising a cyclosporine analog or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof.

2. The method according to claim 1, wherein the subject requiring the same is a subject suffering from non-liver fibrosis.

3. The method according to any one of claims 1 to 2, comprising reducing the amount of non-liver fibrosis in the subject.

4. The method according to claim 3, wherein the amount of non-liver fibrosis in the subject is reduced by 5%, 10%, 20%, or more.

5. The method according to any one of claims 1 to 2, comprising reducing the formation of non-liver fibrosis in the subject.

6. The method according to claim 5, wherein the formation of non-liver fibrosis in the subject is reduced by 5%, 10%, 20%, or more.

7. The method according to any one of claims 1 to 6, wherein the non-hepatic fibrosis includes fibrosis of the lungs, kidneys, heart, skin, eyes, gastrointestinal tract, peritoneum, bone marrow, muscle, blood vessels, vascular structures, or any combination thereof.

8. The method according to any one of claims 1 to 6, wherein the subject requiring the same is suffering from a fibrotic disorder selected from the group consisting of idiopathic pulmonary fibrosis (IPF), cardiac fibrosis, cutaneous fibrosis, renal fibrosis, or a combination thereof.

9. A method for preventing or delaying the onset of fibrosis, wherein the subject in need is given the following formula L: 【Chemistry 2】 (In the formula: a. R' is either H or acetyl; b. R1 is a saturated or unsaturated linear or branched aliphatic carbon chain with a length of 2 to 15 carbon atoms; c. R2 is as follows: i. H; ii. Unsubstituted, N-substituted, or N,N disubstituted amides; iii. N-substituted or unsubstituted acyl-protected amines; iv. N-substituted or unsubstituted amines; v. Carboxylic acid; vi. Nitrile; vii. Esther; viii. Ketones; ix. Hydroxy, dihydroxy, trihydroxy, or polyhydroxyalkyl; and x. Substitutable or non-substitutable aryl; xi. A saturated or unsaturated linear or branched aliphatic chain that may optionally contain substituents selected from the group consisting of hydrogen, ketones, hydroxyl, nitrile, carboxylic acid, ester, 1,3-dioxolane, halogen, and oxo; xii. Aromatic groups containing substituents selected from the group consisting of halogens, esters, and nitros; and xiii. A combination of a saturated or unsaturated linear or branched aliphatic chain of (xi) and an aromatic group of (xii). Selected from the group consisting of; and d. R23 is a saturated or unsaturated linear or branched aliphatic carbon chain that may be optionally substituted. The method comprising administering a composition comprising a cyclosporine analog or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof.

10. The method according to claim 9, wherein the subject requiring the same is a subject at risk of developing fibrosis.

11. The method according to any one of claims 9 to 10, wherein the subject requiring the same is a subject without progression of fibrosis.

12. The method according to any one of claims 10 to 11, wherein the risk of the subject developing fibrosis is reduced by at least 5%, 10%, 20%, or more compared to an untreated subject.

13. The method according to any one of claims 9 to 12, wherein the onset of fibrosis is delayed by at least one month, one year, or more in the subject.

14. The method according to any one of claims 9 to 13, comprising reducing fibrosis formation, wherein the fibrosis formation is reduced in a subject by at least one month, one year, or more compared to an untreated subject.

15. A method for reducing or reversing fibrosis, wherein the subject in need is given the following formula L: 【Transformation 3】 (In the formula: a. R' is either H or acetyl; b. R1 is a saturated or unsaturated linear or branched aliphatic carbon chain with a length of 2 to 15 carbon atoms; c. R2 is as follows: i. H; ii. Unsubstituted, N-substituted, or N,N disubstituted amides; iii. N-substituted or unsubstituted acyl-protected amines; iv. N-substituted or unsubstituted amines; v. Carboxylic acid; vi. Nitrile; vii. Esther; viii. Ketones; ix. Hydroxy, dihydroxy, trihydroxy, or polyhydroxyalkyl; and x. Substitutable or non-substitutable aryl; xi. A saturated or unsaturated linear or branched aliphatic chain that may optionally contain substituents selected from the group consisting of hydrogen, ketones, hydroxyl, nitrile, carboxylic acid, ester, 1,3-dioxolane, halogen, and oxo; xii. Aromatic groups containing substituents selected from the group consisting of halogens, esters, and nitros; and xiii. A combination of a saturated or unsaturated linear or branched aliphatic chain of (xi) and an aromatic group of (xii). Selected from the group consisting of; and d. R23 is a saturated or unsaturated linear or branched aliphatic carbon chain that may be optionally substituted. The method comprising administering a composition comprising a cyclosporine analog or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof.

16. The method according to claim 15, wherein the subject requiring it is a subject suffering from fibrosis.

17. The method according to any one of claims 9 to 16, comprising suppressing the formation of fibrosis in the subject.

18. The method according to any one of claims 9 to 17, wherein the fibrosis is non-hepatic fibrosis.

19. The method according to claim 18, wherein non-hepatic fibrosis includes fibrosis of the lungs, kidneys, heart, skin, eyes, gastrointestinal tract, peritoneum, bone marrow, muscle, blood vessels, vascular structures, or any combination thereof.

20. The method according to any one of claims 9 to 17, wherein the subject requiring the same is suffering from a fibrotic disorder selected from the group consisting of pulmonary fibrosis, cardiac fibrosis, cutaneous fibrosis, renal fibrosis, hepatic fibrosis, or a combination thereof.

21. The method according to any one of claims 9 to 17, wherein the subject requiring the same is a subject suffering from idiopathic pulmonary fibrosis (IPF).

22. The method according to any one of claims 9 to 17, wherein the fibrosis is hepatic fibrosis.

23. The method according to claim 22, wherein the liver fibrosis is cirrhosis.

24. The method according to claim 23, wherein the cirrhosis is associated with viral hepatitis, schistosomiasis, and chronic alcoholism.

25. The method according to any one of claims 1 to 24, wherein the cyclosporine analog of formula L is CRV431 as described below. 【Chemistry 4】

26. The method according to any one of claims 1 to 25, wherein the composition comprises a therapeutically or prophylactically effective amount of a cyclosporine analog of formula L, or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof.

27. The method according to any one of claims 1 to 26, wherein the non-hepatic fibrosis or fibrosis is induced by a therapeutic agent, injury, or a combination thereof.

28. The method according to any one of claims 1 to 26, wherein the non-hepatic fibrosis or fibrosis is associated with the accumulation of extracellular matrix components occurring after trauma, inflammation, tissue repair, immune response, cell hyperplasia, tumor, or a combination thereof.

29. The method according to any one of claims 1 to 26, wherein the non-hepatic fibrosis or fibrosis is associated with major organ disease, fibroproliferative disorder, trauma-related scarring, or a combination thereof.

30. The method according to any one of claims 9 to 26, wherein the fibrosis is associated with interstitial lung disease, cirrhosis, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, kidney disease, heart disease or vascular disease, eye disease, systemic and focal scleroderma, keloids, hypertrophic scars, atherosclerosis, restenosis, Dupuytren's contracture, surgical complications, chemotherapy-induced fibrosis, radiation-induced fibrosis, accidental injuries and burns, retroperitoneal fibrosis, peritoneal fibrosis / peritoneal scarring, or a combination thereof.

31. The method according to claim 30, wherein the fibrosis associated with the interstitial lung disease is sarcoidosis, silicosis, drug reaction, infection, collagen vascular disease, rheumatoid arthritis, systemic sclerosis, scleroderma, pulmonary fibrosis, idiopathic pulmonary fibrosis, typical interstitial pneumonia, interstitial lung disease, fibrotic alveolitis of unknown cause, bronchiolitis obstructive, bronchiectasis, or a combination thereof.

32. The method according to any one of claims 9 to 31, comprising reducing the fibrosis formation in a subject by at least 5%, 10%, 20%, 50%, 70%, 90%, or more compared to an untreated subject.

33. The method according to any one of claims 9 to 31, comprising delaying the formation of fibrosis in the subject compared to an untreated subject.

34. The method according to any one of claims 1 to 33, wherein the subject is a mammal.

35. The method according to any one of claims 1 to 33, wherein the subject is a human.

36. The method according to any one of claims 1 to 35, wherein the composition comprises one or more pharmaceutically acceptable excipients.

37. The method according to any one of claims 1 to 36, wherein the composition comprises one or more additional therapeutic agents.

38. The method according to any one of claims 1 to 36, further comprising administering one or more additional therapeutic agents to the subject in need thereof.

39. The method according to claim 37 or 38, wherein the one or more additional therapeutic agents include additional antifibrotic agents.

40. The method according to claim 37 or 38, wherein the one or more additional therapeutic agents include a type II interferon receptor agonist, pirfenidone and pirfenidone analogs, nintedanib and nintedanib analogs, anti-angiogenic agents, anti-inflammatory agents, IL-1 antagonists, angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers and aldosterone antagonists, mitomycin C (MMC), 5-fluorouracil (5-FU), adenylyl cyclase activators, β-adrenergic receptor (adenoreceptor) agonists, flavonoids, mast cell stabilizers, phosphodiesterase inhibitors, procyanidins, or a combination thereof.

41. The method according to any one of claims 37 to 40, wherein at least one of the one or more additional therapeutic agents is administered to the subject simultaneously with the composition.

42. The method according to any one of claims 37 to 40, wherein at least one of the one or more additional therapeutic agents is administered to the subject before administration of the composition, after administration of the composition, or before and after administration of the composition.

43. The method according to any one of claims 1 to 42, wherein the composition is administered to the subject by intravenous, oral, or parenteral administration.

44. The method according to any one of claims 1 to 43, wherein the composition is in the form of a powder, pill, tablet, microtablet, pellet, micropellet, capsule, microtablet-containing capsule, liquid, aerosol, or nanoparticles.

45. The method according to any one of claims 1 to 44, wherein the composition is administered to the subject in an effective daily dose of 10 mg to 250 mg of the cyclosporine analog or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof.

46. For use in the prevention or treatment of fibrosis, or for use in the prevention or reduction of fibrosis formation, or for use in the recovery from fibrosis, or for reduction of the amount of fibrosis, or for delaying the onset of fibrosis, the following formula L: 【Transformation 5】 (In the formula: a. R' is either H or acetyl; b. R1 is a saturated or unsaturated linear or branched aliphatic carbon chain with a length of 2 to 15 carbon atoms; c. R2 is as follows: i. H; ii. Unsubstituted, N-substituted, or N,N disubstituted amides; iii. N-substituted or unsubstituted acyl-protected amines; iv. N-substituted or unsubstituted amines; v. Carboxylic acid; vi. Nitrile; vii. Esther; viii. Ketones; ix. Hydroxy, dihydroxy, trihydroxy, or polyhydroxyalkyl; and x. Substitutable or non-substitutable aryl; xi. A saturated or unsaturated linear or branched aliphatic chain that may optionally contain substituents selected from the group consisting of hydrogen, ketones, hydroxyl, nitrile, carboxylic acid, ester, 1,3-dioxolane, halogen, and oxo; xii. Aromatic groups containing substituents selected from the group consisting of halogens, esters, and nitros; and xiii. A combination of a saturated or unsaturated linear or branched aliphatic chain of (xi) and an aromatic group of (xii). Selected from the group consisting of; and d. R23 is a saturated or unsaturated linear or branched aliphatic carbon chain that may be optionally substituted. A pharmaceutical composition comprising a cyclosporine analog, or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof.

47. The pharmaceutical composition according to claim 46, wherein the cyclosporine analog is CRV431 as described below. 【Transformation 6】

48. The pharmaceutical composition according to claim 46, wherein the pharmaceutical composition is for intravenous, oral, or parenteral administration.

49. The pharmaceutical composition according to any one of claims 46 to 48, wherein the pharmaceutical composition is in the form of a powder, pill, tablet, microtablet, pellet, micropellet, capsule, microtablet-containing capsule, liquid, aerosol, or nanoparticles.

50. The pharmaceutical composition according to any one of claims 46 to 49; and A kit including a label, wherein the label is as follows: (a) The kit is for the prevention or treatment of fibrosis, (b) The kit is for reducing or suppressing fibrosis formation. (c) The kit is for the recovery of fibrosis, (d) The kit is for reducing the amount of fibrosis, and (e) The kit is for delaying the onset of fibrosis. The kit, which shows one or more of the following.

51. The kit according to claim 50, further comprising instructions for identifying subjects at risk of developing fibrosis, instructions for identifying subjects who have fibrosis, or both.

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