Treatment of liver damage by THR-β agonists

Abstract

Provided herein are methods for treating liver damage, including non-alcoholic steatohepatitis and its symptoms and signs, in a patient utilizing treatment with a THR-β agonist.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to PCT / CN2021 / 130083, filed November 11, 2021, and PCT / CN2022 / 097426, filed June 7, 2022. The contents of the aforementioned patent applications are incorporated herein by reference in their entirety for all purposes.

[0002] The present invention relates to methods and compositions for treating liver damage in a patient. [Background technology]

[0003] Fatty liver disease (FLD) encompasses a variety of disease states characterized by excess accumulation of fat in the liver, often accompanied by inflammation. FLD can lead to nonalcoholic fatty liver disease (NAFLD), which can be characterized by insulin resistance. If untreated, NAFLD can progress to persistent inflammatory responses or nonalcoholic steatohepatitis (NASH), progressive liver fibrosis, and ultimately cirrhosis. In Europe and the United States, NAFLD is the second most common reason for liver transplantation. Thus, the need for treatment is urgent, but due to the lack of obvious symptoms in patients, patients may lack motivation to maintain treatment regimens, especially burdensome treatment regimens (such as medicines that are injected, medicines that are administered multiple times a day, or any that produce dangerous or irritating side effects). Currently, there is no approved treatment for NASH.

[0004] Thyroid hormone receptor-β (THR-β) agonists have recently been investigated in the treatment of liver diseases, including NASH. THR-β is the major form of THR in the liver and plays an important role in energy balance and fatty acid and lipid metabolism, whereas THR-α predominates in the heart and is responsible for the majority of the undesirable cardiovascular effects of thyroid hormone stimulation. A significant problem to overcome involves developing a THR-β agonist for treating NASH that does not produce the undesirable side effects associated with THR-α agonism. Summary of the Invention

[0005] Provided herein are methods and compositions for treating liver damage in a patient in need thereof. The method includes administering to the patient a thyroid hormone receptor beta (THR-β) agonist, referred to herein as Compound 1, or a pharma- ceutically acceptable salt thereof. [ka] Compound 1 (having the chemical name 2-(3,5-dichloro-4-((4-oxo-3,4-dihydrophthalazin-1-yl)oxy)phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-nitrile) was described in U.S. Pat. No. 11,084,802, which is incorporated herein by reference in its entirety.

[0006] The present inventors have found that compound 1 or its pharmacologic equivalent can be administered to patients suffering from liver damage at surprisingly low doses and still maintain a desired level of efficacy.As a result, compound 1 can be used to treat liver damage without the undesirable side effects generally associated with THR agonism.

[0007] In some embodiments of the present disclosure, compound 1 or a pharma- ceutically acceptable salt thereof may be orally administered once a day to a patient at a dose as low as 1 mg or less and still sufficiently reduce amine oxidase activity and reduce lymphocyte adhesion and migration. For example, compound 1 or a pharma- ceutically acceptable salt thereof may be orally administered once a day to a patient with liver damage (e.g., NASH) at a dose of about 1 mg to about 60 mg. In some embodiments, compound 1 or a pharma- ceutically acceptable salt thereof may be orally administered once a day to a patient with liver damage (e.g., NASH) at a dose of about 0.5 mg to about 25 mg. In some embodiments, compound 1 or a pharma- ceutically acceptable salt thereof may be orally administered once a day to a patient with liver damage (e.g., NASH) at a dose of about 1 mg to about 15 mg. In some embodiments, compound 1 or a pharma- ceutically acceptable salt thereof may be orally administered once a day to a patient with liver damage (e.g., NASH) at a dose of about 3 mg to about 10 mg. In some embodiments, Compound 1 or a pharma- ceutically acceptable salt thereof may be orally administered once a day to a patient with hepatic impairment (e.g., NASH) at a dose of about 1 mg. In some embodiments, Compound 1 or a pharma- ceutically acceptable salt thereof may be orally administered once a day to a patient with hepatic impairment (e.g., NASH) at a dose of about 3 mg. In some embodiments, Compound 1 or a pharma- ceutically acceptable salt thereof may be orally administered once a day to a patient with hepatic impairment (e.g., NASH) at a dose of about 4 mg. In some embodiments, Compound 1 or a pharma- ceutically acceptable salt thereof may be orally administered once a day to a patient with hepatic impairment (e.g., NASH) at a dose of about 5 mg. In some embodiments, Compound 1 or a pharma- ceutically acceptable salt thereof may be orally administered once a day to a patient with hepatic impairment (e.g., NASH) at a dose of about 6 mg. In some embodiments, Compound 1 or a pharma- ceutically acceptable salt thereof may be orally administered once a day to a patient with hepatic impairment (e.g., NASH) at a dose of about 10 mg. In some embodiments, Compound 1 or a pharma- ceutically acceptable salt thereof may be orally administered once daily to a patient with liver damage (e.g., NASH) at a dose of about 15 mg. In some embodiments, Compound 1 or a pharma- ceutically acceptable salt thereof may be orally administered once daily to a patient with liver damage (e.g., NASH) at a dose of about 20 mg.In some embodiments, Compound 1 or a pharma- ceutically acceptable salt thereof may be orally administered once daily to a patient with liver damage (e.g., NASH) at a dose of about 30 mg. In some embodiments, Compound 1 or a pharma- ceutically acceptable salt thereof may be orally administered once daily to a patient with liver damage (e.g., NASH) at a dose of about 50 mg. In some embodiments, Compound 1 or a pharma- ceutically acceptable salt thereof may be orally administered once daily to a patient with liver damage (e.g., NASH) at a dose of about 60 mg.

[0008] In another aspect, the present disclosure provides a method for treating or preventing NASH in a patient in need of such treatment or prevention, comprising administering to the patient a therapeutically effective amount of compound 1 or its pharmacologic acceptable salt.In one embodiment, the patient in need of such treatment or prevention is a patient suffering from fatty liver disease, such as NAFLD.In another embodiment, the patient in need of such treatment or prevention is a patient suffering from metabolic syndrome.

[0009] In one aspect, the present disclosure provides a method for reducing hepatic inflammation in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of Compound 1 or a pharma- ceutically acceptable salt thereof. The reduced hepatic inflammation is characterized by reduced expression of inflammatory genes in the liver and markers of leukocyte activation. In some embodiments, the reduced hepatic inflammation is achieved without increasing the low-density lipoprotein cholesterol (LDL-C) level in the patient's blood.

[0010] In another aspect, the present disclosure provides a method for treating a disease or condition characterized by liver fibrosis, comprising administering a therapeutically effective amount of Compound 1 or a pharmaceutically acceptable salt thereof to a patient. The reduction in fibrosis is characterized by histological improvement in the liver and reduced expression of profibrotic genes. In some embodiments, liver fibrosis is reduced without increasing low-density lipoprotein cholesterol (LDL-C) levels in the patient's blood. In some embodiments, administration of Compound 1 or a pharmaceutically acceptable salt thereof results in reduced liver fibrosis and liver inflammation.

[0011] In some embodiments, the patient has liver disease and diabetes. In some embodiments, the patient has liver disease and cardiovascular disease. In some embodiments, the treatment period is the remaining lifespan of the patient. In some embodiments, the method does not include administering an antihistamine, an immunosuppressant, a steroid, rifampicin, an opioid antagonist, or a selective serotonin reuptake inhibitor (SSRI).

[0012] In some embodiments, Compound 1 is administered to a patient as a pharma- ceutically acceptable salt. In some embodiments, the pharma- ceutically acceptable salt is a potassium salt. In some embodiments, the pharma- ceutically acceptable salt is a sodium salt.

[0013] The present disclosure also provides novel compositions comprising Compound 1 or a pharma- ceutically acceptable salt thereof. Compound 1 has very low aqueous solubility even when administered in salt form. It has been found that certain ionic surfactants can effectively solubilize Compound 1 and its pharmaceutical salts with minimal or no degradation of the compound. In some embodiments, the ionic surfactant is sodium lauryl sulfate. In some such embodiments, the amount of SLS in the composition is about 1% to about 8% by weight. In other such embodiments, the amount of SLS in the composition is about 5% by weight. In some embodiments, the pharmaceutical composition comprises a potassium salt of Compound 1 and SLS. [Brief description of the drawings]

[0014] [Figure 1] 1 shows the plasma concentrations of Compound 1 in patients up to 72 hours after administration of a single dose of Compound 1. [Diagram 2] Shown is the area under the curve (AUCinf) and maximum plasma concentration (Cmax) of Compound 1 in patients plotted against the administered dose. [Diagram 3] 1 shows the mean percent change in sex hormone binding globulin (SHBG) four days after administration of a single dose of Compound 1. [Figure 4] 1 shows the mean percent change in apolipoprotein B (Apo B) four days after administration of a single dose of Compound 1. [Diagram 5] FIG. 1 shows free T3, T4, and TSH on day 15 after 14 days of daily administration of Compound 1 or placebo in humans. [Figure 6] 1 shows the percent change from baseline in free testosterone, total testosterone, and sex hormone binding globulin (SHBG) on day 15 following 14 days of daily administration of Compound 1 or placebo in humans. [Figure 7] FIG. 1 shows plasma concentrations of Compound 1 over time on days 1 and 14 of a multiple ascending dose study in which Compound 1 was dosed once daily. [Figure 8] Figure 1 shows the percent change from baseline in pharmacodynamic markers (sex hormone binding globulin, ApoB, total cholesterol, LDL-c, HDL-c, and triglycerides) on day 15 following 14 days of daily administration of Compound 1 or placebo in humans. [Figure 9] 1 shows the effect of Compound 1 on body and organ weights in a mouse NASH model. [Figure 10] 1 shows the effects of Compound 1 on hepatic steatosis, inflammation, and fibrosis in a mouse NASH model. [Figure 11] 1 shows the effect of Compound 1 on lipids and an indicator of liver damage (ALT) in a mouse NASH model. [Figure 12] FIG. 1 shows the effect of Compound 1 on the expression of genes associated with collagen extracellular matrix and hepatic stellate cell activation. [Figure 13] FIG. 1 shows plasma concentrations of Compound 1 in beagle dogs following administration of a single 50 mg dose provided in one of two oral formulations (PO1 or PO2). [Figure 14] 1 shows the plasma concentrations of Compound 1 in two groups of beagle dogs following administration of a single 10 mg dose provided in oral formulation PO3. One group was pretreated with pentagastrin, while the other group was pretreated with famotidine. [Figure 15] FIG. 1 shows plasma concentrations of Compound 1 in fasted and fed beagle dogs following administration of a single 10 mg dose provided in oral formulation PO3. [Figure 16] 1 shows the XRPD spectrum of compound 1 (potassium salt form, Form A). [Figure 17] 1 shows the XRPD spectrum of Compound 1 (free acid form, Form A). [Figure 18] 1 shows the XRPD spectrum of compound 1 (sodium salt form, Form A). [Figure 19] 1 shows the XRPD spectrum of Compound 1 (L-arginine salt form, Form A). [Figure 20] 1 shows the XRPD spectrum of compound 1 (magnesium salt form, Form A). [Figure 21] The overall study design for Compound 1 is shown. [Figure 22] Demographic and baseline characteristics for Compound 1 are shown. [Diagram 23] Plasma concentration-time profile for Compound 1 (day 14) is shown. [Figure 24] PK parameters of Compound 1 on Day 14 are shown. [Diagram 25] Sex hormone binding globulin (SHBG) is shown (percent change from baseline to day 15). [Figure 26] LDL-c is shown (percent change from baseline to day 15). [Figure 27] Percent change from baseline at the end of treatment (day 15) for SHBG and LDL-c per compound dose is shown. [Figure 28] Reduction in total cholesterol (TC), Apo B, and triglycerides (TG) per compound dose is shown. [Figure 29] Treatment-emergent adverse events were mild and often not associated with significant changes in vital signs. [Diagram 30] Figure 1 shows body weight change in male DIO-NASH mice confirmed by biopsy. Compound 2 reduced body weight alone and in combination with Compound 1. Body weight was measured daily during the study. Body weight change during the study is shown in A, with dots representing the mean body weight change compared to baseline (n=10-16 mice per group). In B, bars represent the mean (standard deviation) body weight measured at week 11 of the study. Low fat vehicle control, white; DIO-GAN vehicle control, grey; Compound 2, blue; Compound 1-low, light orange; Compound 1-medium, orange; Compound 1-high, dark orange; Combination-low, light purple; Combination-medium, purple; Combination-high, dark purple. Statistical comparisons to DIO-GAN vehicle control were determined by ANOVA followed by Tukey correction for multiple comparisons. *p<0.05;**p<0.01;***p<0.001;****p<0.0001. [Diagram 31] Weekly food intake during the study is shown. Dots represent average weekly food intake in grams (n=10-16 mice per group). Low fat vehicle control, white; DIO-GAN vehicle control, grey; Compound 2, blue; Compound 1-low, light orange; Compound 1-medium, orange; Compound 1-high, dark orange; Combination-low, light purple; Combination-medium, purple; Combination-high, dark purple. [Diagram 32]Liver and spleen weights are shown. Compound 2 and Compound 1, alone and in combination, significantly reduced hepatomegaly without changes in spleen weight. Bars represent mean (standard deviation) liver (A) and spleen (B) organ weights determined at the end of the study (n=10-16 mice per group). Statistical comparisons to DIO-GAN vehicle control were determined by ANOVA followed by Tukey correction for multiple comparisons. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. [Diagram 33] Body weight composition at baseline is shown. Body weight composition was well balanced across treatment groups at baseline (week -1). Bars represent mean (standard deviation) adipose tissue (A) and lean tissue (B) mass as a percentage of body weight (%BW, n=10-16 mice per group) as determined by whole-body EchoMRI at week -1 of the study. Statistical comparisons to DIO-GAN vehicle control were determined by ANOVA followed by Tukey correction for multiple comparisons. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. [Diagram 34] Body weight composition at week 11 is shown. Compound 2, alone and in combination with Compound 1, significantly reduced adipose tissue mass. Bars represent mean (standard deviation) adipose tissue (A) and lean tissue (B) mass as a percentage of body weight (%BW, n=10-16 mice per group) determined by whole-body EchoMRI at week 11 of the study. Statistical comparisons to DIO-GAN vehicle control were determined by ANOVA followed by Tukey correction for multiple comparisons. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. [Diagram 35]Plasma and liver total cholesterol are shown. Compound 2 and Compound 1, alone and in combination, significantly reduced total cholesterol. Bars represent mean (standard deviation) total cholesterol levels measured at the end of the study in plasma (A) and liver (B) (n=10-16 mice per group). Statistical comparisons to DIO-GAN vehicle control were determined by ANOVA followed by Tukey correction for multiple comparisons. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. [Diagram 36] Plasma and liver triglycerides are shown. Compound 2, alone and in combination with compound 1, significantly reduced plasma triglycerides. Bars represent mean (standard deviation) triglyceride levels measured at the end of the study in plasma (A) and liver (B) (n=10-16 mice per group). Statistical comparisons to DIO-GAN vehicle control were determined by ANOVA followed by Tukey correction for multiple comparisons. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. [Figure 37] Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels are shown. Compound 1 alone significantly reduced alanine aminotransferase (ALT) levels. Bars represent mean (standard deviation) liver (A) and aspartate aminotransferase (AST) (B) levels determined at the end of the study (n=10-16 mice per group). Statistical comparisons to DIO-GAN vehicle control were determined by ANOVA followed by Tukey correction for multiple comparisons. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. [Figure 38]Alkaline phosphatase levels are shown. Alkaline phosphatase (ALP) levels were not significantly altered by treatment. Bars represent mean (standard deviation) ALP levels determined at the end of the study (n=10-16 mice per group). Statistical comparisons to DIO-GAN vehicle control were determined by ANOVA followed by Tukey correction for multiple comparisons. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. [Figure 39] NAFLD activity scores (NAS) at baseline and end of treatment are shown. NAFLD activity scores (NAS) were well balanced at baseline and significantly improved in the combination treatment group. NAS (defined as the composite unweighted sum of the histological scores of ballooning, steatosis, and lobular inflammation) was determined at baseline (A) and after 12 weeks of treatment (B). Dots represent individual mice in each treatment group (n=14-16 mice). Statistical comparisons to DIO-GAN vehicle control were determined by ANOVA followed by Tukey correction for multiple comparisons. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Diagram 40] Shows hepatic steatosis by histological morphometric analysis. The combination of compound 2 and compound 1 resulted in greater reduction of hepatic steatosis as determined by histological morphometric analysis. Hepatocyte steatosis, including the percentage of hepatocytes with lipid droplets and hepatic lipid content as percent fractional area (FA), was determined by morphometric analysis of liver histological samples at the end of the study. Statistical comparisons to DIO-GAN vehicle control were determined by ANOVA followed by Tukey correction for multiple comparisons. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Diagram 41]Figure 1 shows hepatocyte lipid droplet size. Combination treatment significantly reduces hepatocyte lipid droplet size. Lipid droplet size was determined by morphometric analysis of liver histological samples at the end of the study. Statistical comparisons to DIO-GAN vehicle control were determined by ANOVA followed by Tukey correction for multiple comparisons. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Diagram 42] Plasma CK18 M30 is shown. The apoptosis biomarker cytokeratin 18 M30 (CK18 M30) levels were not significantly altered by treatment. CK18 M30, a biomarker of apoptosis, was measured in plasma samples at the end of the study. Statistical comparisons to DIO-GAN vehicle control were determined by ANOVA followed by Tukey correction for multiple comparisons. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Diagram 43] Figure 1 shows hepatic protein expression of galectin-3 and smooth muscle actin proteins. Compound 2 treatment reduces expression of galectin-3 (Gal-3). Expression of Gal-3 (A) and α-smooth muscle actin (α-SMA) (B) was assessed by immunohistochemical (IHC) staining of livers of treated mice at the end of the study. Statistical comparisons to DIO-GAN vehicle control were determined by ANOVA followed by Tukey's correction for multiple comparisons. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Diagram 44]Figure 1 shows the expression of energy and lipid metabolism-related genes in liver. Comparison of gene expression values ​​between Compound 1-High and Combination-High treatment groups for selected genes involved in energy and lipid metabolism. Liver samples were processed for transcriptomics analysis by RNAseq at the end of the study. Dots represent the average fold change expression values ​​compared to DIO-GAN vehicle control (n=10 mice per group). Red and blue coloring indicates the trend of change (red=increased expression; blue=decreased expression) between DIO-GAN vehicle and low fat control groups. Dotted lines indicate lines that are identical. Solid lines were generated by linear regression analysis of fold change expression values ​​for selected genes in Compound 1-High and Combination-High treatment groups. Selected genes are involved in energy and lipid metabolism. Squalene epoxidase (Sqle), 7-dehydrocholesterol (Dhcr7), hydroxymethylgluteraryl-CoA synthase (Hmgcs1), and stearoyl-CoA desaturase (Scd1). [Diagram 45]Expression of selected genes involved in energy and lipid metabolism is shown. Hepatic expression of genes involved in energy and lipid metabolism. Liver samples were processed for transcriptomics analysis by RNAseq at termination. Bars represent mean (standard deviation) expression (FPKM) values ​​for selected genes involved in energy and lipid metabolism as indicated. Squalene epoxidase (Sqle, A), 7-dehydrocholesterol (Dhcr7, B), hydroxymethylglutararyl-CoA synthase (Hmgcs1, C), and stearoyl-CoA desaturase (Scd1, D). Low fat, white (leftmost bar); DIO-GAN vehicle control, gray (second from left); Compound 2, blue (third from left); Compound 1-low, light orange (fourth from left); Compound 1-medium, orange (fifth from left); Compound 1-high, dark orange (fourth from right); Combination-low, light purple (third from right); Combination-medium, purple (second from right); Combination-high, dark purple (rightmost bar). Statistical comparisons for individual treatment groups are shown against DIO-GAN vehicle control. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. The combination treatment groups were additionally compared to the Compound 2 treatment groups (+p<0.05, ++p<0.01, +++p<0.0001, ++++p<0.00001) or the respective Compound 1 monotherapy groups (i.e., Compound 1-low vs. combination-low); #p<0.05, ##p<0.01, ###p<0.0001, ####p<0.00001). [Diagram 46]Expression of genes involved in fibrosis and inflammation is shown. Liver expression of genes involved in fibrosis and inflammation. Liver samples were processed for transcriptomics analysis by RNAseq at termination. Bars represent mean (standard deviation) expression (FPKM) values ​​for selected genes involved in fibrosis and inflammation. Collagen type I alpha 1 (Col1a1), actin alpha 2, smooth actin (Acta2), galectin 3 (Lgals3), and melanoma cell adhesion molecule (CD146). Low, white (left-most bar); DIO-GAN vehicle control, gray (second from left); Compound 2, blue (third from left); Compound 1-low, light orange (fourth from left); Compound 1-medium, orange (fifth from left); Compound 1-high, dark orange (fourth from right); Combination-low, light purple (third from right); Combination-medium, purple (second from right); Combination-high, dark purple (right-most bar). Statistical comparisons for individual treatment groups are shown versus DIO-GAN vehicle control. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] definition As used herein, the following definitions shall apply unless otherwise indicated. Furthermore, any term or symbol used herein shall have its ordinary meaning in the art, unless defined as set forth below.

[0016] "Comprising" is intended to mean that the compositions and methods include the recited elements but do not exclude others. "Consisting essentially of," when used to define compositions and methods, is intended to mean excluding other elements of any essential importance to the combination. For example, a composition consisting essentially of the elements defined herein does not exclude other elements that do not materially affect the basic and novel feature(s) of the claimed invention. "Consisting of" is intended to mean excluding more than trace amounts, e.g., of other ingredients and more than substantial method steps recited. Embodiments defined by each of these transition terms are within the scope of the present invention.

[0017] "Combination therapy" or "combination treatment" refers to the use of two or more drugs or agents in treatment (e.g., the use of compound 1 utilized herein together with another agent useful for treating liver damage (NAFLD, NASH, etc.)), the symptoms and signs of each of which are treated in combination. "Combined" administration refers to the administration of two agents (e.g., compound 1 utilized herein and another agent) in any manner in which both pharmacological effects are simultaneously manifested in the patient. Thus, combined administration does not require that a single pharmaceutical composition, the same dosage form, or even the same route of administration be used for the administration of both agents, or that the two agents be administered at exactly the same time. Both agents may also be formulated in a single pharmaceutically acceptable composition. A non-limiting example of such a single composition is an oral composition or oral dosage form. For example, and without limitation, it is contemplated that compound 1 may be administered in combination therapy with another agent according to the present invention.

[0018] The term "excipient" as used herein means an inert or inactive substance that may be used in the production of a drug or pharmaceutical product, such as a tablet containing a compound of the invention as an active ingredient. A variety of substances may be encompassed by the term excipient, including, but not limited to, binding substances, disintegration substances, coatings, compression aids / encapsulation aids, creams or lotions, lubricating substances, solutions for parenteral administration, materials for chewable tablets, sweeteners or flavoring substances, suspending / gelling agents, or any substance used as a wet granulation agent. Binding substances include, for example, carbomer, povidone, xanthan gum, etc.; coatings include, for example, cellulose acetate phthalate, ethylcellulose, gellan gum, maltodextrin, enteric coatings, etc.; compression aids / encapsulation aids include, for example, calcium carbonate, dextrose, fructose dc (dc = "directly compressible"), honey dc, lactose (anhydrous or monohydrate; optionally in combination with aspartame, cellulose, or microcrystalline cellulose), starch dc, sucrose, etc.; disintegrants include, for example, croscarmellose sodium, gellan gum, sodium starch glycolate, etc.; creams or lotions include, for example, Examples of suitable agents include maltodextrin, carrageenan, etc.; lubricating agents include magnesium stearate, stearic acid, sodium stearyl fumarate, etc.; materials for chewable tablets include dextrose, fructose dc, lactose (monohydrate, optionally in combination with aspartame or cellulose), etc.; suspending / gelling agents include carrageenan, sodium starch glycolate, xanthan gum, etc.; sweetening agents include aspartame, dextrose, fructose dc, sorbitol, sucrose dc, etc.; wet granulating agents include calcium carbonate, maltodextrin, microcrystalline cellulose, etc.

[0019] "Patient" refers to a mammal, including humans and non-human mammals. Examples of patients include, but are not limited to, mice, rats, hamsters, guinea pigs, pigs, rabbits, cats, dogs, goats, sheep, cows, and humans. In some embodiments, a patient refers to a human.

[0020] "Pharmaceutically acceptable" refers to safe and non-toxic, preferably for in vivo, and more preferably for human administration.

[0021] "Pharmaceutically acceptable salt" refers to a salt that is pharma- ceutically acceptable. The compounds described herein may be administered as a pharma- ceutically acceptable salt.

[0022] "Salt" refers to an ionic compound formed between an acid and a base. When the compounds provided herein contain an acidic functional group, such salts include, but are not limited to, alkali metal salts, alkaline earth metal salts, and ammonium salts. As used herein, ammonium salts include salts containing protonated nitrogen bases and alkylated nitrogen bases. Exemplary, non-limiting cations useful in pharma-ceutically acceptable salts include Na, K, Rb, Cs, NH4, Ca, Ba, imidazolium, and ammonium cations based on naturally occurring amino acids. When the compounds utilized herein contain a basic functional group, such salts include, but are not limited to, salts of organic acids (such as carboxylic and sulfonic acids) and mineral acids (such as hydrogen halides, sulfuric acids, phosphoric acids, and the like). Exemplary, non-limiting anions useful in pharma- ceutically acceptable salts include oxalate, maleate, acetate, propionate, succinate, tartrate, chloride, sulfate, bisulfate, monobasic, dibasic, and tribasic phosphates, mesylate, tosylate, and the like.

[0023] A "therapeutically effective amount" or dose of a compound or composition refers to that amount of the compound or composition that results in a reduction or inhibition of symptoms or prolonged survival in a patient. Results may require multiple doses of the compound or composition.

[0024] "Treatment" or "treating" refers to an approach to obtain beneficial or desired results, including clinical results. For the purposes of the present invention, beneficial or desired results include, but are not limited to, one or more of the following: reduction of one or more symptoms resulting from a disease or disorder, diminishing the extent of a disease or disorder, stabilizing a disease or disorder (e.g., preventing or delaying the worsening of a disease or disorder), delaying the appearance or recurrence of a disease or disorder, delaying or slowing the progression of a disease or disorder, relieving the disease or disorder state, providing remission (whether partial or complete) of a disease or disorder, reducing the dosage of one or more other pharmaceuticals required to treat a disease or disorder, enhancing the effect of another pharmaceutical used to treat a disease or disorder, delaying the progression of a disease or disorder, increasing the quality of life, and / or prolonging the survival of a patient. Reduction of the pathological consequences of a disease or disorder is also encompassed by "treatment". The method of the present invention contemplates any one or more of these aspects of treatment.

[0025] As used herein, "delaying" the onset of a disease means suspending, preventing, slowing, retarding, stabilizing, and / or postponing the onset of the disease and / or slowing down the progression, or altering the process and / or course of the underlying disease once it has started. This delay can be of variable length depending on the history of the disease and / or the individual being treated. As will be apparent to those skilled in the art, a sufficient or significant delay can in effect encompass prevention, in that the individual does not develop clinical symptoms associated with the disease. A method of "delaying" the onset of a disease is one that reduces the probability of the onset of the disease in a given time frame and / or reduces the extent of the disease in a given time frame when compared to not using the method, and includes stabilizing one or more symptoms resulting from the disease.

[0026] An individual "at risk" of developing a disease may or may not have detectable disease, and may or may not present detectable disease prior to the treatment methods described herein. "At risk" indicates that the individual has one or more so-called risk factors, which are measurable parameters that correlate with the development of the disease. Individuals with one or more of these risk factors have a higher probability of developing the disease than individuals without these risk factor(s). These risk factors may include, but are not limited to, age, sex, race, diet, history of previous disease, presence of precursor disease, and genetic (i.e., hereditary) considerations. The compounds may, in some embodiments, be administered to subjects (including humans) at risk or with a family history of a disease or condition.

[0027] The term "optional" or "optionally," as used throughout this specification, means that the subsequently described event or circumstance can, but need not, occur, and that the description encompasses instances in which the event or circumstance occurs as well as instances in which it does not occur. For example, "a nitrogen atom is optionally oxidized to provide an N-oxide (N→O) moiety" means that the nitrogen atom can, but does not need to be oxidized, and that the description encompasses situations in which the nitrogen atom is not oxidized as well as situations in which the nitrogen atom is oxidized.

[0028] As used herein, the term "substantially as shown in," when referring to, for example, an XRPD pattern, encompasses a pattern or graph that is not necessarily identical to that depicted herein, but that falls within the limits of experimental error or deviation as determined by one of ordinary skill in the art.

[0029] Pharmaceutically acceptable compositions and formulations Compound 1 (Thyroid hormone receptor β (THR-β) agonist) [ka] Pharmaceutically acceptable compositions of, or simply "pharmaceutical compositions" are encompassed by the present invention. Thus, the present invention encompasses pharmaceutical compositions comprising Compound 1 or a pharmaceutical acceptable salt thereof, and a pharmaceutical acceptable carrier or excipient. In some embodiments, the pharmaceutical acceptable salt is a base addition salt (such as a salt formed with an inorganic or organic base). In some embodiments, the pharmaceutical acceptable salt is a potassium salt of Compound 1. In some embodiments, the pharmaceutical acceptable salt is a sodium salt of Compound 1. Pharmaceutical compositions according to the present invention may be in a form suitable for oral, buccal, parenteral, nasal, topical, or rectal administration, or in a form suitable for administration by inhalation.

[0030] Compound 1 as detailed herein may be in a purified form in one aspect, and compositions comprising compound 1 in purified form are detailed herein. Compositions (such as compositions of substantially pure compounds) comprising compound 1 as detailed herein or a salt thereof are provided. In some embodiments, compositions as detailed herein containing compound 1 or a salt thereof are in substantially pure form. In one variation, "substantially pure" contemplates a composition containing 35% or less impurities, where impurities refer to compounds other than the compound or a salt thereof that constitutes the majority of the composition. For example, a composition of a substantially pure compound contemplates a composition containing 35% or less impurities, where impurities refer to compounds other than the compound or a salt thereof. In one variation, a composition of a substantially pure compound or a salt thereof is provided, where the composition contains 25% or less impurities. In another variation, a composition of a substantially pure compound or a salt thereof is provided, where the composition contains 20% or less impurities. In yet another variation, a composition of a substantially pure compound or a salt thereof is provided, where the composition contains 10% or less impurities. In a further variation, a composition of a substantially pure compound or a salt thereof is provided, where the composition contains 5% or less impurities. In another variation, a composition of a substantially pure compound or a salt thereof is provided, wherein the composition contains 3% or less of impurities. In yet another variation, a composition of a substantially pure compound or a salt thereof is provided, wherein the composition contains 1% or less of impurities. In a further variation, a composition of a substantially pure compound or a salt thereof is provided, wherein the composition contains 0.5% or less of impurities. In yet another variation, a composition of a substantially pure compound means that the composition contains 15% or less of impurities, or preferably 10% or less, or more preferably 5% or less, or even more preferably 3% or less, and most preferably 1% or less of impurities.

[0031] In one variation, Compound 1 is a synthetic compound prepared for administration to an individual, such as a human. In another variation, compositions containing Compound 1 in substantially pure form are provided. In another variation, the invention encompasses pharmaceutical compositions comprising Compound 1 and a pharma- ceutically acceptable carrier or excipient. In another variation, methods of administering Compound 1 are provided. The purified forms, pharmaceutical compositions, and methods of administering the compounds are suitable for any of the compounds or forms thereof detailed herein.

[0032] Compound 1 may be formulated for any available delivery route, including oral, mucosal (e.g., nasal, sublingual, vaginal, buccal, rectal), parenteral (e.g., intramuscular, subcutaneous, intravenous), topical, or transdermal delivery forms. Compound 1 may be formulated with a suitable carrier to provide a delivery form that includes, but is not limited to, tablets, caplets, capsules (such as hard gelatin capsules or soft elastic gelatin capsules), cachets, troches, lozenges, gums, dispersions, suppositories, ointments, poultices, pastes, powders, dressings, creams, solutions, patches, aerosols (e.g., nasal sprays or inhalers), gels, suspensions (e.g., aqueous or non-aqueous liquid suspensions, oil-in-water or water-in-oil liquid emulsions), solutions, and elixirs.

[0033] Compound 1 can be used in the preparation of formulations (such as pharmaceutical formulations) by combining Compound 1 as an active ingredient with a pharma- ceutically acceptable carrier (such as those mentioned above). Depending on the form of systemic therapy (e.g., transdermal patch vs. oral tablet), the carrier can be in various forms. In addition, pharmaceutical formulations can contain preservatives, solubilizing substances, stabilizing substances, rewetting agents, emulsifying substances (emulgators), sweetening substances, dyes, adjusting substances, and salts for adjusting osmotic pressure, buffers, coating agents, or antioxidants. Formulations containing Compound 1 can also contain other substances with beneficial therapeutic properties. Pharmaceutical formulations can be prepared by known pharmaceutical methods. Suitable formulations are described, for example, in Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins, 21 st ed. (2005), incorporated herein by reference.

[0034] The compounds described herein can be administered to an individual (e.g., a human) in the form of a generally acceptable oral composition (such as tablets, coated tablets, and hard-shell or soft-shell gel capsules), emulsion, or suspension. Examples of carriers that can be used to prepare such compositions include microcrystalline cellulose, mannitol, lactose, corn starch or its derivatives, talc, stearates or their salts, etc. Acceptable carriers for soft-shell gel capsules include, for example, vegetable oils, waxes, fats, semi-solid and liquid polyols, etc. In addition, pharmaceutical preparations can contain preservatives, solubilizing agents, stabilizing agents, rewetting agents, emulsifying agents, sweetening agents, dyes, adjusting agents, and salts for adjusting osmotic pressure, buffers, coating agents, or antioxidants.

[0035] Compound 1 has very low aqueous solubility even when administered in salt form. The potassium salt of Compound 1 has a solubility of approximately 2.2 μg / mL in a pH 6.0 buffer and approximately 8.0 μg / mL in a pH 8.0 buffer. Solubilizing agents such as poloxamer 188 result in significant degradation of Compound 1. It has been found that certain ionic surfactants (such as sodium lauryl sulfate (SLS)) are compatible with Compound 1 and can be co-formulated with Compound 1 and its pharma- ceutically acceptable salts with minimal or no degradation of the compound. Co-formulation of Compound 1 pharmaceutical salts thereof with ionic surfactants (e.g., SLS) results in dramatic enhancement of solubility at all pH levels.

[0036] In some embodiments, a formulation is provided that includes Compound 1 or a pharma- ceutically acceptable salt thereof and about 1% to about 10% by weight of SLS. In some embodiments, a formulation is provided that includes Compound 1 or a pharma- ceutically acceptable salt thereof and about 1% to about 8% by weight of SLS. In some embodiments, a formulation is provided that includes Compound 1 or a pharma- ceutically acceptable salt thereof and about 1.5% to about 8% by weight of SLS. In some embodiments, a formulation is provided that includes Compound 1 or a pharma- ceutically acceptable salt thereof and about 1.5% to about 5% by weight of SLS. In some embodiments, a formulation is provided that includes Compound 1 or a pharma- ceutically acceptable salt thereof and about 5% by weight of SLS. In some embodiments, a formulation is provided that includes the potassium salt of Compound 1 and about 1.5% by weight of sodium lauryl sulfate (SLS).

[0037] Other potential solubilizing agents that may be used in combination with Compound 1 or a pharma- ceutically acceptable salt thereof include docusate sodium, polysorbates, phospholipids, or D-α-tocopheryl polyethylene glycol succinate (vitamin E TPGS).

[0038] In some embodiments, a formulation is provided that includes Compound 1 or a pharma- ceutically acceptable salt thereof and sodium lauryl sulfate. In some embodiments, a formulation is provided that includes Compound 1 or a pharma- ceutically acceptable salt thereof, sodium lauryl sulfate, and croscarmellose sodium. In some embodiments, a formulation is provided that includes Compound 1 or a pharma- ceutically acceptable salt thereof, sodium lauryl sulfate, croscarmellose sodium, colloidal silicaon dioxide, and magnesium stearate. In some embodiments, a formulation is provided that includes Compound 1 or a pharma- ceutically acceptable salt thereof, sodium lauryl sulfate, croscarmellose sodium, colloidal silicaon dioxide, magnesium stearate, hypromellose (HPMC) capsule, mannitol, and microcrystalline cellulose.

[0039] In some embodiments, compound 1 is provided in a capsule formulation of any dosage form described herein. In some embodiments, a capsule formulation containing about 0.5 mg to about 50 mg of compound 1 or a pharma- ceutically acceptable salt thereof is provided. In some embodiments, a capsule formulation containing about 0.5 mg, about 1 mg, about 2 mg, about 3 mg, about 5 mg, about 6 mg, about 10 mg, about 15 mg, about 25 mg, or about 50 mg of compound 1 or a pharma- ceutically acceptable salt thereof is provided.

[0040] In some embodiments, Compound 1 is provided formulated in a capsule with one or more of a variety of other ingredients as listed below in Table A. In some embodiments, Compound 1 is provided formulated in a capsule with one or more of a variety of other ingredients in the amounts as listed below in Table A. [Table 1]

[0041] In some embodiments, compound 1 is provided in a capsule formulation of any dosage form described herein. In some embodiments, a tablet formulation containing about 0.5 mg to about 50 mg of compound 1 is provided. In all embodiments, it will be understood that the weight of compound 1 refers to the active portion (free acid) of the molecule. In embodiments where a salt form of compound 1 is used, the weight of the salt will be adjusted to ensure that there is an appropriate amount of active compound in the composition. In some embodiments, a tablet formulation containing about 0.5 mg, about 1 mg, about 2 mg, about 3 mg, about 5 mg, about 6 mg, about 10 mg, about 15 mg, about 25 mg, or about 50 mg of compound 1 is provided.

[0042] The present disclosure further encompasses kits (e.g., pharmaceutical packages). The kits provided may include a pharmaceutical composition or compound described herein and a container (e.g., a drug bottle, an ampoule, a bottle, a syringe, and / or a subpackage, or other suitable container).

[0043] Method and use The compounds and compositions described herein can be used in some aspects in the treatment or prevention of liver damage.In some embodiments, the method for treating or preventing liver damage in a patient in need of such treatment or prevention comprises administering compound 1 or a pharma- ceutically acceptable salt thereof to the patient.

[0044] Liver disorders include, but are not limited to, liver inflammation, fibrosis, and steatohepatitis. In some embodiments, liver disorders are selected from liver inflammation, liver fibrosis, alcohol-induced fibrosis, steatosis, alcoholic steatosis, primary sclerosing cholangitis (PSC), primary biliary cirrhosis (PBC), non-alcoholic fatty liver disease (NAFLD), and non-alcoholic steatohepatitis (NASH). In certain embodiments, liver disorders are selected from liver fibrosis, alcohol-induced fibrosis, steatosis, alcoholic steatosis, NAFLD, and NASH. In one embodiment, liver disorders are NASH. In another embodiment, liver disorders are liver inflammation. In another embodiment, liver disorders are liver fibrosis. In another embodiment, liver disorders are alcohol-induced fibrosis. In another embodiment, liver disorders are steatosis. In another embodiment, liver disorders are alcoholic steatosis. In another embodiment, liver disorders are NAFLD. In one embodiment, the therapeutic method provided herein prevents or slows down the progression of NAFLD to NASH.In one embodiment, the therapeutic method provided herein prevents or slows down the progression of NASH.NASH can progress to one or more of, for example, cirrhosis, liver cancer, etc.In some embodiments, the liver disorder is NASH.In some embodiments, the patient has undergone liver biopsy.In some embodiments, the method further comprises obtaining the results of liver biopsy.

[0045] In some embodiments, a method of treating liver damage in a patient in need thereof is provided, wherein the liver damage is selected from the group consisting of liver inflammation, liver fibrosis, alcohol-induced fibrosis, steatosis, alcoholic steatosis, primary sclerosing cholangitis (PSC), primary biliary cirrhosis (PBC), non-alcoholic fatty liver disease (NAFLD), and non-alcoholic steatohepatitis (NASH).

[0046] Provided herein is a method for treating or preventing liver damage in a patient (e.g., a human patient) in need thereof with Compound 1 or a pharma- ceutically acceptable salt thereof, comprising administering a therapeutically effective amount of Compound 1 or a pharma- ceutically acceptable salt thereof, wherein the liver damage is selected from hepatic inflammation, hepatic fibrosis, alcohol-induced fibrosis, steatosis, alcoholic steatosis, primary sclerosing cholangitis (PSC), primary biliary cirrhosis (PBC), nonalcoholic fatty liver disease (NAFLD), and nonalcoholic steatohepatitis (NASH).

[0047] Also provided herein is a method for preventing or slowing the progression of non-alcoholic fatty liver disease (NAFLD) to non-alcoholic steatohepatitis (NASH) in a patient (e.g., a human patient) in need of such a method, comprising administering Compound 1 or a pharmaceutically acceptable salt thereof. In some embodiments, the method comprises administering a therapeutically effective amount of Compound 1 or a pharmaceutically acceptable salt thereof. Also provided herein is a method for preventing or slowing the progression of NASH in a patient (e.g., a human patient) in need of such a method, comprising administering Compound 1 or a pharmaceutically acceptable salt thereof. In some embodiments, the method comprises administering a therapeutically effective amount of Compound 1 or a pharmaceutically acceptable salt thereof.

[0048] In some embodiments, a method of reducing liver damage is provided herein, comprising administering compound 1 or a pharma- ceutically acceptable salt thereof to an individual in need of reducing liver damage, whereby fibrosis is reduced. In some embodiments, the level of expression of one or more markers for fibrosis is reduced. In some embodiments, the level of expression of Ccr2, Col1a1, Col1a2, Col1a3, Cxcr3, Dcn, Hgf, Il1a, Inhbe, Lox, Loxl1, Loxl2, Loxl3, Mmp2, Pdgfb, Plau, Serpine1, Perpinh1, Snai, Tgfb1, Tgfb3, Thbs1, Thbs2, Timp2, and / or Timp3 is reduced. In some embodiments, the level of collagen is reduced. In some embodiments, the level of collagen fragment is reduced. In some embodiments, the level of expression of fibrosis marker is reduced by at least 2, at least 3, at least 4, or at least 5 times. In some embodiments, the level of expression of the fibrosis marker is reduced by about 2-fold, about 3-fold, about 4-fold, or about 5-fold.

[0049] In some embodiments, a method of reducing liver damage is provided herein, comprising administering Compound 1 or a pharma- ceutically acceptable salt thereof to an individual in need of reducing liver damage, whereby inflammation is reduced. In some embodiments, one or more markers of inflammation are reduced. In some embodiments, the expression levels of Adgre1, Ccr2, Ccr5, Il1A, and / or Tlr4 are reduced. In some embodiments, the expression levels of inflammation markers are reduced by at least 2, at least 3, at least 4, or at least 5 times. In some embodiments, the expression levels of fibrosis markers are reduced by about 2, about 3, about 4, or about 5 times.

[0050] In a patient, the levels of alkaline phosphatase, gamma-glutamyltransferase (GGT), alanine aminotransferase (ALT), and / or aspartate aminotransferase (AST) may be elevated. In some embodiments, provided herein is a method of reducing liver damage comprising administering Compound 1 or a pharma- ceutically acceptable salt thereof, wherein the levels of GGT, ALT, and / or AST are elevated prior to treatment. In some embodiments, the patient's ALT level is about 2-4 times greater than the upper limit of normal. In some embodiments, the patient's AST level is about 2-4 times greater than the upper limit of normal. In some embodiments, the patient's GGT level is about 1.5-3 times greater than the upper limit of normal. In some embodiments, the patient's alkaline phosphatase level is about 1.5-3 times greater than the upper limit of normal. Methods for determining the levels of these molecules are well known. Normal levels of ALT in blood range from about 7-56 units / liter. Normal levels of AST in blood range from about 10-40 units / liter. Normal levels of GGT in blood range from about 9 to 48 units / liter. Normal levels of alkaline phosphatase in blood range from about 53 to 128 units / liter for men aged 20 to 50 years and about 42 to 98 units / liter for women aged 20 to 50 years.

[0051] Thyroid hormone deficiency is more common in patients with NAFLD and NASH (Pagadala MR, Zein CO, Dasarathy S, Yerian LM, Lopez R, McCullough AJ. Prevalence of hypothyroidism in nonalcoholic fatty liver disease. Dig Dis Sci. 2012;57:528-34.). The thyroid gland produces triiodothyronine (T3) and thyroxine (T4) under the control of thyrotropin (thyroid-stimulating hormone [TSH]) from the anterior pituitary gland in response to thyrotropin-releasing hormone (TRH) from the hypothalamus. Without being bound by any particular theory, THR-β agonism in the liver is responsible for lowering free T4 without changes in T3 or TSH, which may be due to peripheral thyroid hormone regulation (Taub R, Chiang E, Chabot-Blanchet M, Kelly MJ, Reeves RA, Guertin MC, et al. Lipid lowering in healthy volunteers treated with multiple doses of MGL-3196, a liver-targeted thyroid hormone receptor-β agonist. Atherosclerosis. 2013; 230: 373-80.; Berry MJ, Kates AL, Larsen PR. Thyroid hormone regulates type I deiodinase messenger RNA in rat liver. Mol Endocrinol 1990; 4: 743-748.). Evidence of an effect on the central thyroid axis may indicate undesirable effects. Thus, in some embodiments, administration of Compound 1, or a pharma- ceutically acceptable salt thereof, does not result in changes in a patient's serum TSH, free T3, or T4 levels outside the normal range.

[0052] In some embodiments, administration of Compound 1, or a pharma- ceutically acceptable salt thereof, does not result in changes in a patient's cardiac biomarkers, including CK, CK-MB, and troponin I, to levels outside the normal range.

[0053] Sex hormone binding globulin (SHBG) is produced in the liver and binds and stabilizes sex hormones, including androgens and estrogens, affecting the total and free fractions of circulating hormones (Hammond 2016). THR-β agonism upregulates SHBG expression, making SHBG a useful PD marker. In some embodiments, administration of Compound 1 or a pharmaceutically acceptable salt thereof results in an increase in serum SHBG in the patient. In some embodiments, the increase in serum SHBG in the patient occurs within 4 days of administration of Compound 1 or a pharmaceutically acceptable salt thereof. In some embodiments, the serum SHBG in the patient increases by at least 5% compared to baseline after administration of Compound 1 or a pharmaceutically acceptable salt thereof. In some embodiments, the serum SHBG in the patient increases by at least 10% compared to baseline after administration of Compound 1 or a pharmaceutically acceptable salt thereof. In some embodiments, the serum SHBG in the patient increases by at least 25% compared to baseline after administration of Compound 1 or a pharmaceutically acceptable salt thereof.

[0054] In some embodiments, the patient is a human. Obesity is highly correlated with NAFLD and NASH, but lean people can also be affected by NAFLD and NASH. Thus, in some embodiments, the patient is obese. In some embodiments, the patient is not obese. Obesity can be correlated with or can cause other diseases, such as diabetes or cardiovascular disorders. Thus, in some embodiments, the patient also has diabetes and / or cardiovascular disorders. Without being bound by theory, it is believed that comorbidities (such as obesity, diabetes, and cardiovascular disorders) can make NAFLD and NASH more difficult to treat. Conversely, the only currently recognized way to address NAFLD and NASH is weight loss, which may have little or no effect on lean patients.

[0055] Although risk for NAFLD and NASH increases with age, there are literature reports of children as young as 2 years old, and children can also suffer from NAFLD and NASH (Schwimmer, et al., Pediatrics, 2006, 118:1388-1393). In some embodiments, the patient is 2-17 years old (e.g., 2-10, 2-6, 2-4, 4-15, 4-8, 6-15, 6-10, 8-17, 8-15, 8-12, 10-17, or 13-17 years old). In some embodiments, the patient is 18-64 years old (e.g., 18-55, 18-40, 18-30, 18-26, 18-21, 21-64, 21-55, 21-40, 21-30, 21-26, 26-64, 26-55, 26-40, 26-30, 30-64, 30-55, 30-40, 40-64, 40-55, or 55-64 years old). In some embodiments, the patient is 65 years old or older (e.g., 70 or older, 80 or older, or 90 or older).

[0056] Although NAFLD and NASH are common causes of liver transplantation, patients who have already undergone one liver transplant often develop NAFLD and / or NASH again. Thus, in some embodiments, the patient undergoes a liver transplant.

[0057] In some embodiments, the treatment according to the methods provided herein results in a reduction in NAFLD activity (NAS) score in the patient. For example, in some embodiments, steatosis, inflammation, and / or ballooning are reduced upon treatment. In some embodiments, the methods of treatment provided herein reduce liver fibrosis. In some embodiments, the methods reduce serum triglycerides. In some embodiments, the methods reduce liver triglycerides.

[0058] Dyslipidemia, characterized by elevated low-density lipoprotein (LDL), cholesterol, and triglycerides, is an important risk factor for cardiovascular disease (Nelson RH. Hyperlipidemia as a risk factor for cardiovascular disease. Prim Care. 2013 March;40(1):195-211.) and is commonly observed in patients with NASH (Loomba R. Nonalcoholic fatty liver disease progression rates to cirrhosis and progression of cirrhosis to decompensation and mortality: a real world analysis of Medicare data. Aliment Pharmacol Ther. 2020;51:1149-59.). Furthermore, dyslipidemia is a possible etiological driver of the liver inflammation underlying NASH (Walenbergh S. Cholesterol is a significant risk factor for nonalcoholic steatohepatitis. Expert Review of Gastroenterology & Hepatology. 2015;9:11:1343-46.). Apolipoprotein B (Apo B) is associated with LDL cholesterol in the blood and can be useful in assessing a patient's lipid profile. Thus, in some embodiments, administration of Compound 1 or a pharma- ceutically acceptable salt thereof reduces serum Apo B in the patient. In some embodiments, administration of Compound 1 or a pharma- ceutically acceptable salt thereof results in a reduction in serum Apo B in the patient within 4 days of administration. In some such embodiments, the reduction in serum Apo B in the patient is at least 5% compared to baseline. In some such embodiments, the reduction in serum Apo B in the patient is at least 10% compared to baseline. In some such embodiments, the reduction in serum Apo B in the patient is at least 15% compared to baseline.

[0059] In some embodiments, the patient is at risk of developing a side effect prior to administration in accordance with the methods provided herein. In some embodiments, the side effect is a side effect affecting the kidneys, lungs, heart, and / or skin. In some embodiments, the side effect is itching.

[0060] In some embodiments, the patient has undergone one or more prior therapies. In some embodiments, the liver damage progresses during the treatment. In some embodiments, the patient suffers from pruritus during at least one of the one or more prior therapies.

[0061] Preclinical animal models with Compound 1 demonstrated a steady-state plasma area under the curve (AUC) from time 0 to infinity of approximately 3,320 ng*hg / mL, as described in the Examples below. 0-∞ ) was established as sufficient exposure to ensure efficacy based on various pharmacodynamic markers. Compound 1 exposures of 3,320 ng*hg / mL or greater do not result in undesirable thyroid hormone effects often associated with THR agonism. For example, in the single-dose escalation study described in the Examples, the steady-state plasma AUC of Compound 1 was approximately 50,000 ng*hg / mL. 0-∞ did not result in any significant side effects.

[0062] In some embodiments, Compound 1, or a pharma- ceutical acceptable salt thereof, has a steady-state plasma area under the curve (AUC) from time 0 to infinity of about 2,500 ng*hr / mL to about 50,000 ng*hr / mL. 0-∞ In some embodiments, Compound 1 or a pharma- ceutical acceptable salt thereof is administered daily to an individual in need thereof (e.g., a patient with NASH) at a dose such that the individual achieves a steady-state plasma area under the curve (AUC) from time 0 to infinity of about 3,000 ng*hr / mL to about 50,000 ng*hr / mL. 0-∞In some embodiments, Compound 1 or a pharma- ceutical acceptable salt thereof is administered daily to an individual in need thereof (e.g., a patient with NASH) at a dose such that the individual achieves a steady-state plasma area under the curve (AUC) from time 0 to infinity of about 5,000 ng*hr / mL to about 50,000 ng*hr / mL. 0-∞ In some embodiments, Compound 1 or a pharma- ceutical acceptable salt thereof is administered daily to an individual in need thereof (e.g., a patient with NASH) at a dose such that the individual achieves a steady-state plasma area under the curve (AUC) from time 0 to infinity of about 5,000 ng*hr / mL to about 30,000 ng*hr / mL. 0-∞ In some embodiments, Compound 1 or a pharma- ceutical acceptable salt thereof is administered daily to an individual in need thereof (e.g., a patient with NASH) at a dose such that the individual achieves a steady-state plasma area under the curve (AUC) from time 0 to infinity of about 5,000 ng*hr / mL to about 25,000 ng*hr / mL. 0-∞ In some embodiments, Compound 1 or a pharma- ceutical acceptable salt thereof is administered daily to an individual in need thereof (e.g., a patient with NASH) at a dose such that the individual achieves a steady-state plasma area under the curve (AUC) from time 0 to infinity of about 5,000 ng*hr / mL to about 20,000 ng*hr / mL. 0-∞ In some embodiments, Compound 1 or a pharma- ceutical acceptable salt thereof is administered daily to an individual in need thereof (e.g., a patient with NASH) at a dose such that the individual achieves a steady-state plasma area under the curve (AUC) from time 0 to infinity of about 3,000 ng*hr / mL to about 10,000 ng*hr / mL. 0-∞ In some embodiments, Compound 1 or a pharma- ceutical acceptable salt thereof is administered daily to an individual in need thereof (e.g., a patient with NASH) at a dose such that the individual achieves a steady-state plasma area under the curve (AUC) from time 0 to infinity of about 5,000 ng*hr / mL to about 10,000 ng*hr / mL. 0-∞Compound 1 or a pharma- ceutically acceptable salt thereof is administered daily to an individual in need thereof (e.g., a patient with NASH) at a dose such that a therapeutically effective dose of Compound 1 or a pharma- ceutically acceptable salt thereof is achieved.

[0063] Surprisingly, it has been found that Compound 1 or a pharma- ceutically acceptable salt thereof can achieve the desired exposure at very low doses. In some embodiments of the present disclosure, Compound 1 or a pharma- ceutically acceptable salt thereof can be orally administered once a day to a patient at a dose as low as 1 mg or less and still sufficiently reduce amine oxidase activity and reduce lymphocyte adhesion and migration. For example, Compound 1 or a pharma- ceutically acceptable salt thereof can be orally administered once a day to a patient with liver damage (e.g., NASH) at a dose of about 1 mg to about 60 mg. In some embodiments, Compound 1 or a pharma- ceutically acceptable salt thereof can be orally administered once a day to a patient with liver damage (e.g., NASH) at a dose of about 0.5 mg to about 25 mg. In some embodiments, Compound 1 or a pharma- ceutically acceptable salt thereof can be orally administered once a day to a patient with liver damage (e.g., NASH) at a dose of about 1 mg to about 15 mg. In some embodiments, Compound 1 or a pharma- ceutically acceptable salt thereof may be orally administered once a day to a patient with liver damage (e.g., NASH) at a dose of about 2 mg to about 10 mg. In some embodiments, Compound 1 or a pharma- ceutically acceptable salt thereof may be orally administered once a day to a patient with liver damage (e.g., NASH) at a dose of about 1 mg. In some embodiments, Compound 1 or a pharma- ceutically acceptable salt thereof may be orally administered once a day to a patient with liver damage (e.g., NASH) at a dose of about 3 mg. In some embodiments, Compound 1 or a pharma- ceutically acceptable salt thereof may be orally administered once a day to a patient with liver damage (e.g., NASH) at a dose of about 4 mg. In some embodiments, Compound 1 or a pharma- ceutically acceptable salt thereof may be orally administered once a day to a patient with liver damage (e.g., NASH) at a dose of about 5 mg. In some embodiments, Compound 1 or a pharma- ceutically acceptable salt thereof may be orally administered once a day to a patient with liver damage (e.g., NASH) at a dose of about 6 mg. In some embodiments, Compound 1 or a pharma- ceutically acceptable salt thereof may be orally administered once daily to a patient with liver damage (e.g., NASH) at a dose of about 10 mg. In some embodiments, Compound 1 or a pharma- ceutically acceptable salt thereof may be orally administered once daily to a patient with liver damage (e.g., NASH) at a dose of about 15 mg.In some embodiments, Compound 1 or a pharma- ceutically acceptable salt thereof may be orally administered once daily to a patient with hepatic impairment (e.g., NASH) at a dose of about 20 mg. In all embodiments, it will be understood that the weight of Compound 1 refers to the active portion (free acid) of the molecule. In some embodiments, Compound 1 or a pharma- ceutically acceptable salt thereof may be orally administered once daily to a patient with hepatic impairment (e.g., NASH) at a dose of about 30 mg. In some embodiments, Compound 1 or a pharma- ceutically acceptable salt thereof may be orally administered once daily to a patient with hepatic impairment (e.g., NASH) at a dose of about 50 mg. In some embodiments, Compound 1 or a pharma- ceutically acceptable salt thereof may be orally administered once daily to a patient with hepatic impairment (e.g., NASH) at a dose of about 60 mg. In embodiments in which a salt form of the compound is used, the weight of the salt will be adjusted to ensure that there is an appropriate amount of active compound in the composition.

[0064] The treatment period may be generally one week or longer. In some embodiments, the treatment period is at least one week, two weeks, three weeks, four weeks, five weeks, one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, one year, two years, three years, four years, or more. In some embodiments, the treatment period is about one week to about one month, about one month to about one year, about one year to about several years. In some embodiments, the treatment period is at least about any of one week, two weeks, three weeks, four weeks, five weeks, one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, one year, two years, three years, four years, or more. In some embodiments, the treatment period is the remaining lifespan of the patient.

[0065] The administration of Compound 1 or a pharma- ceutically acceptable salt thereof may be once a day, twice a day, or every other day for a treatment period of one week or more. In some embodiments, the administration comprises administering the compound daily for a treatment period of one week or more. In some embodiments, the administration comprises administering the compound twice a day for a treatment period of one week or more. In some embodiments, the administration comprises administering the compound every other day for a treatment period of one week or more.

[0066] In some embodiments, the amount of Compound 1 or a pharma- ceutically acceptable salt thereof administered on day 1 of the treatment period is equal to or greater than the amount administered on all subsequent days of the treatment period. In some embodiments, the amount administered on day 1 of the treatment period is equal to the amount administered on all subsequent days of the treatment period.

[0067] In some embodiments, administering compound 1 or its pharma- ceutically acceptable salt reduces steatosis in an individual. Methods for assessing steatosis are known to those skilled in the art and may include histological analysis and assigning a histological score. It is therefore understood that the methods of treatment detailed herein in some embodiments include treating liver disorders in an individual in need of treatment, such as liver inflammation, liver fibrosis, alcohol-induced fibrosis, steatosis, alcoholic steatosis, primary sclerosing cholangitis (PSC), primary biliary cirrhosis (PBC), nonalcoholic fatty liver disease (NAFLD), and nonalcoholic steatohepatitis (NASH), and the like, and the treatment includes reducing the histological markers associated with steatosis.

[0068] In some embodiments, administering compound 1 or its pharma- ceutically acceptable salt reduces liver inflammation in an individual. Methods for assessing liver inflammation are known to those skilled in the art and may include histological analysis of lobular inflammation and assigning a histological score. Thus, it is understood that the method of treatment detailed herein in some embodiments includes treating liver disorders (such as liver inflammation, liver fibrosis, alcohol-induced fibrosis, steatosis, alcoholic steatosis, primary sclerosing cholangitis (PSC), primary biliary cirrhosis (PBC), nonalcoholic fatty liver disease (NAFLD), and nonalcoholic steatohepatitis (NASH)) in an individual in need of treatment, and the treatment includes reducing lobular inflammation or the histological markers associated with lobular inflammation.

[0069] In some embodiments, administering compound 1 or its pharma- ceutically acceptable salt reduces liver fibrosis in an individual.Methods for assessing liver fibrosis are known to those skilled in the art and may include histological analysis.Therefore, it is understood that the method of treatment detailed herein includes, in some embodiments, treating liver injury in an individual in need of treatment (such as liver inflammation, liver fibrosis, alcohol-induced fibrosis, steatosis, alcoholic steatosis, primary sclerosing cholangitis (PSC), primary biliary cirrhosis (PBC), nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH)), and the treatment includes reducing fibrosis or the histological markers associated with fibrosis.

[0070] In some embodiments, administering compound 1 or its pharmacologic acceptable salt reduces at least one or at least two of hepatic steatosis, inflammation, and fibrosis in an individual.Therefore, it is understood that the method of treatment detailed herein in some embodiments includes treating liver disorders (such as hepatic inflammation, hepatic fibrosis, alcohol-induced fibrosis, steatosis, alcoholic steatosis, primary sclerosing cholangitis (PSC), primary biliary cirrhosis (PBC), nonalcoholic fatty liver disease (NAFLD), and nonalcoholic steatohepatitis (NASH)) in an individual in need of treatment, and the treatment includes reducing at least one or at least two of steatosis, lobular inflammation, fibrosis, or any histological markers of the above.

[0071] In some embodiments, administering compound 1 or its pharma- ceutically acceptable salt reduces serum triglycerides in an individual.Thus, it is understood that the methods of treatment detailed herein in some embodiments include treating liver disorders in an individual in need of treatment, such as liver inflammation, liver fibrosis, alcohol-induced fibrosis, steatosis, alcoholic steatosis, primary sclerosing cholangitis (PSC), primary biliary cirrhosis (PBC), nonalcoholic fatty liver disease (NAFLD), and nonalcoholic steatohepatitis (NASH), and the like, and the treatment includes reducing serum triglycerides.

[0072] In some embodiments, administering compound 1 or its pharma- ceutically acceptable salt reduces serum total cholesterol in an individual.Thus, it is understood that the method of treatment detailed herein in some embodiments includes treating liver disorders in an individual in need of treatment (such as liver inflammation, liver fibrosis, alcohol-induced fibrosis, steatosis, alcoholic steatosis, primary sclerosing cholangitis (PSC), primary biliary cirrhosis (PBC), nonalcoholic fatty liver disease (NAFLD), and nonalcoholic steatohepatitis (NASH)), and the treatment includes reducing serum cholesterol.

[0073] In some embodiments, administration of compound 1 or a pharma- ceutically acceptable salt thereof reduces serum alanine aminotransferase in an individual.Thus, it is understood that the methods of treatment detailed herein in some embodiments include treating liver disorders in an individual in need of treatment, such as liver inflammation, liver fibrosis, alcohol-induced fibrosis, steatosis, alcoholic steatosis, primary sclerosing cholangitis (PSC), primary biliary cirrhosis (PBC), nonalcoholic fatty liver disease (NAFLD), and nonalcoholic steatohepatitis (NASH), and the like, and the treatment includes reducing serum alanine aminotransferase.

[0074] In some embodiments, administration of compound 1 or a pharmaceutically acceptable salt thereof reduces at least one or at least two of serum triglycerides, total cholesterol, and alanine aminotransferase in an individual. In some embodiments, administration of compound 1 or a pharmaceutically acceptable salt thereof reduces serum triglycerides, total cholesterol, and alanine aminotransferase in an individual. Thus, it is understood that the method of treatment detailed herein in some embodiments includes treating liver disorders (such as liver inflammation, liver fibrosis, alcohol-induced fibrosis, steatosis, alcoholic steatosis, primary sclerosing cholangitis (PSC), primary biliary cirrhosis (PBC), non-alcoholic fatty liver disease (NAFLD), and non-alcoholic steatohepatitis (NASH)) in an individual in need of treatment, and the treatment includes reducing at least one or at least two of serum triglycerides, total cholesterol, and alanine aminotransferase.

[0075] In some of the above embodiments, Compound 1 or a pharma- ceutically acceptable salt thereof is administered to a patient who has not eaten for at least 10 hours prior to dosing. In some embodiments, Compound 1 or a pharma- ceutically acceptable salt thereof is administered to a patient who has consumed a high-fat, high-calorie meal less than 30 minutes prior to dosing.

[0076] polymorph In one aspect, provided herein is a polymorph of Compound 1, or a pharma- ceutically acceptable salt thereof. Polymorphs may have properties, such as bioavailability and stability under certain conditions, that are suitable for medical or pharmaceutical use.

[0077] In some embodiments, provided herein is polymorph Form A of the potassium salt of Compound 1. In some embodiments, Form A of the potassium salt of Compound 1 has an XRPD pattern substantially as shown in Figure 16. The angles 2θ and relative peak intensities that can be observed for Form A of the potassium salt of Compound 1 using XRPD are shown in Table B below. [Table 2]

[0078] In some embodiments, polymorph Form A of the potassium salt of Compound 1 has an XRPD pattern that exhibits at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 peaks at the angle 2θ with maximum intensity in an XRPD pattern as shown in FIG. 16 or as provided in Table B.

[0079] In some embodiments, polymorph Form A of the potassium salt of Compound 1 has an XRPD pattern including peaks at 6.78±0.20, 11.35±0.20, and 20.51±0.20 degrees 2θ. In some embodiments, polymorph Form A of the potassium salt of Compound 1 has an XRPD pattern including peaks at 6.78±0.20, 11.35±0.20, 14.44±0.20, 20.51±0.20, and 29.13±0.20 degrees 2θ. In some embodiments, polymorph Form A of the potassium salt of Compound 1 has an XRPD pattern including peaks at 6.16±0.20, 6.78±0.20, 11.35±0.20, 13.51±0.20, 14.44±0.20, 15.76±0.20, 20.51±0.20, 24.63±0.20, 25.97±0.20, and 29.13±0.20 degrees 2θ.

[0080] In some embodiments, provided herein is polymorph Form A of the sodium salt of Compound 1. In some embodiments, Form A of the sodium salt of Compound 1 has an XRPD pattern substantially as shown in Figure 18. The angles 2θ and relative peak intensities that can be observed for Form A of the sodium salt of Compound 1 using XRPD are shown in Table C below. [Table 3]

[0081] In some embodiments, polymorph Form A of the sodium salt of Compound 1 has an XRPD pattern that exhibits at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 peaks at the angle 2θ with maximum intensity in an XRPD pattern as shown in FIG. 18 or as provided in Table C.

[0082] In some embodiments, polymorph Form A of the sodium salt of Compound 1 has an XRPD pattern including peaks at 5.51±0.20, 8.47±0.20, and 16.57±0.20 degrees 2θ. In some embodiments, polymorph Form A of the sodium salt of Compound 1 has an XRPD pattern including peaks at 5.51±0.20, 6.99±0.20, 8.47±0.20, 15.24±0.20, and 16.57±0.20 degrees 2θ. In some embodiments, polymorph Form A of the sodium salt of Compound 1 has an XRPD pattern including peaks at 5.51±0.20, 6.99±0.20, 8.47±0.20, 13.12±0.20, and 15.24±0.20, 16.57±0.20, 20.42±0.20, 21.02±0.20, 28.55±0.20, and 31.33±0.20 degrees 2θ.

[0083] It should be understood that the relative intensities may vary depending on many factors, including sample preparation, mounting, and the instrumentation and analytical procedures and settings used to obtain the spectrum. Relative peak intensities and peak assignments may vary within experimental error. In some embodiments, the XRPD peak assignments listed herein (including for polymorph Form A of the potassium salt of Compound 1 or polymorph Form A of the sodium salt of Compound 1) may vary by ±0.6 degrees, ±0.4 degrees, ±0.2 degrees, or ±0.1 degrees 2θ. In some embodiments, the peak assignments listed herein may vary by ±0.6 degrees 2θ. In some embodiments, the peak assignments listed herein may vary by ±0.4 degrees 2θ. In some embodiments, the peak assignments listed herein may vary by ±0.2 degrees 2θ. In some embodiments, the peak assignments listed herein may vary by ±0.1 degrees 2θ.

[0084] Use in combination The present disclosure further provides the combination of compound 1 or its pharmaceutically acceptable salt with other therapeutic agents used to treat liver disease.In particular, the present disclosure provides the combination of compound 1 or its pharmaceutically acceptable salt with other therapeutic agents used in the treatment of NASH.As disclosed herein, due to its low clinical dose, compound 1 or its pharmaceutically acceptable salt is an attractive candidate for use in fixed dose combination for the treatment of NASH.

[0085] In some embodiments, Compound 1 or a pharma- ceutically acceptable salt thereof is administered in combination with a farnesoid X receptor (FXR) agonist. In some embodiments, the FXR agonist is obeticholic acid. In some embodiments, the FXR agonist is cilofexor. In some embodiments, the FXR agonist is tropifexor. In some embodiments, the FXR agonist is EYP001 (bonafexor, INN proposal). In some embodiments, the FXR agonist is MET409 (Metacrine). In some embodiments, the FXR agonist is EDP-305 (by Enanta). In some embodiments, the FXR agonist is [ka] ("or Compound 2"), or a pharma- ceutically acceptable salt thereof.

[0086] In some embodiments, Compound 1 or a pharma- ceutically acceptable salt thereof is administered in combination with a peroxisome proliferator-activated receptor (PPAR) agonist. In some embodiments, the PPAR agonist is pioglitazone. In some embodiments, the PPAR agonist is rosiglitazone. In some embodiments, the PPAR agonist is elalafibranor. In some embodiments, the PPAR agonist is saroglitazar. In some embodiments, the PPAR agonist is lanifibranor. In some embodiments, the PPAR agonist is elafibranor. In some embodiments, the PPAR agonist is seladelphar.

[0087] In some embodiments, Compound 1, or a pharma- ceutically acceptable salt thereof, is administered in combination with a pan-caspase inhibitor. In some embodiments, the pan-caspase inhibitor is emricasan.

[0088] In some embodiments, Compound 1, or a pharma- ceutically acceptable salt thereof, is administered in combination with a galectin-3 inhibitor. In some embodiments, the galectin-3 inhibitor is belapectin.

[0089] In some embodiments, Compound 1, or a pharma- ceutically acceptable salt thereof, is administered in combination with an inhibitor of stearoyl Co-A desaturase 1. In some embodiments, the inhibitor of stearoyl Co-A desaturase 1 is armachol.

[0090] In some embodiments, Compound 1, or a pharma- ceutically acceptable salt thereof, is administered in combination with a chemokine receptor type 2 and chemokine receptor type 5 (CCR2 / CCR5 chemokine) antagonist. In some embodiments, the CCR2 / CCR5 chemokine agonist is cenicriviroc.

[0091] In some embodiments, Compound 1, or a pharma- ceutically acceptable salt thereof, is administered in combination with an antioxidant. In some embodiments, the antioxidant is vitamin E.

[0092] In some embodiments, Compound 1 is co-administered with a cholesterol-lowering drug. In some embodiments, the cholesterol-lowering drug is a statin. In some such embodiments, the statin is atorvastatin, simvastatin, or rosuvastatin.

[0093] Manufactured Products and Kits The present disclosure further provides an article of manufacture comprising the compound or its salt described herein, the composition described herein, or one or more unit doses described herein in suitable packaging.In certain embodiments, the article of manufacture is for use in any of the methods described herein.Suitable packaging (e.g., containers) are known in the art, and include, for example, vials, containers, ampoules, bottles, jars, flexible packaging, and the like.The article of manufacture can be further sterilized and / or sealed.

[0094] The present disclosure further provides kits for carrying out the methods of the present disclosure, the kits comprising Compound 1 or a pharma- ceutically acceptable salt thereof, or a composition comprising Compound 1 or a pharma- ceutically acceptable salt thereof. The kits may be used for any one or more of the uses described herein, and thus may contain instructions for the treatments described herein.

[0095] The kit generally includes suitable packaging. The kit may include one or more containers containing Compound 1 or a pharma- ceutically acceptable salt thereof.

[0096] The kit may be in unit dosage form, bulk package (e.g., multiple dose package), or sub-unit dose. For example, a kit containing sufficient dosage of the compound of formula (I) or its pharmaceutically acceptable salt, and / or additional pharmaceutically active compound useful for the diseases detailed herein may be provided to provide effective treatment to an individual for an extended period of time (any of 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 4 months, 5 months, 7 months, 8 months, 9 months, or more, etc.). The kit may also include multiple unit doses of the compound and instructions for use, packaged in sufficient quantities for storage and use in pharmacies (e.g., hospital pharmacies and compounding pharmacies).

[0097] The kit may optionally include a set of instructions (generally written instructions), although instructions containing an electronic storage medium (e.g., a magnetic diskette or optical disk) relating to the use of the component(s) of the disclosed method are also acceptable. The instructions included in the kit generally include information regarding the components and their administration to an individual. EXAMPLES

[0098] Example 1: Single Ascending Dose Study of Compound 1 in Healthy Human Subjects method A single ascending dose clinical trial of Compound 1 (potassium salt) was conducted. Four groups of eight healthy participants were randomized in a 3:1 ratio (n=6 active and n=2 placebo) to receive Compound 1 (3 mg, 10 mg, 30 mg, or 60 mg capsules) or matching placebo, administered during the fasting state on day 1 of the study. Plasma levels of Compound 1 and PD biomarkers were determined at various pre- and post-dose time points.

[0099] Adverse event (AE) monitoring, routine clinical laboratory tests (including thyroid axis tests [free and total thyroid hormone triiodothyronine (T3), free and total thyroid hormone thyroxine (T4), thyroid stimulating hormone (TSH)], cardiac biomarkers [CK-MB, troponin I], and liver biochemistry), aggregate vital signs, electrocardiogram telemetry, and electrocardiograms were assessed throughout the study. Plasma and urinary concentrations of Compound 1 were determined using a validated liquid chromatography-tandem mass spectrometry assay.

[0100] Plasma samples for Compound 1 concentration and PK sampling were collected pre-dose and 0.5, 1, 2, 3, 4, 6, 8, 12, 24, 48, and 72 hours after administration of a single dose of test medication (placebo or compound). Urine samples for Compound 1 concentration and PK sampling were collected pre-dose and at the following time points: 0-6 hours, 6-12 hours, 12-24 hours, and 24-48 hours. PK parameters were estimated by noncompartmental methods using Phoenix® WinNonlin® (Certara, LP, Princeton, NJ). Serum pharmacodynamic (PD) biomarkers apolipoprotein B (Apo B) and sex hormone binding globulin (SHBG) concentrations were measured using immunoassays, and serum lipids were determined using spectrophotometry.

[0101] PD sampling was performed pre-dose and 48 and 72 hours post-dose. Percent change from baseline for PD markers was calculated using an ANCOVA model with percent change from baseline as the dependent variable, treatment group as a fixed effect, and baseline as a covariate. Analyses used only observed data without imputation for missing data.

[0102] result Monitoring for treatment-emergent adverse events All adverse events were mild or moderate and most were unrelated to study drug. No cardiac-related AEs (e.g., tachycardia, arrhythmia) were reported, and no notable changes in vital signs or ECG parameters were observed (see Table 1). [Table 4]

[0103] Safety and Thyroid Axis Monitoring Heart rate remained stable and within normal range for 24 hours after Compound 1 administration, following a similar pattern to that observed in the placebo group. TSH, free T3, and free T4 levels remained within normal range. No subject experienced an increase in ALT more than 2 times the upper limit of normal (ULN); no subject showed bilirubin levels above normal range. In addition, no significant changes in cardiac biomarkers (troponin I, CK-MB) or other clinical safety tests were observed.

[0104] Pharmacokinetics Compound 1 was absorbed with low variability (%CV≦33%) under fasting conditions. Exposure (AUC, C max ) was approximately dose proportional. The median half-life of Compound 1 ranged from 13.8 to 17.3 hours, supporting once-daily dosing. Minimal renal excretion was determined at all doses. See Table 2, Figures 1 and 2. [Table 5]

[0105] Pharmacodynamics The mean percent changes in sex hormone binding globulin (SHBG) and apolipoprotein B (Apo B) on day 3 after a single dose of Compound 1 on day 1 of the study are shown in Figure 3 and Figure 4, respectively. Mean percent changes refer to least squares mean (LSM) and standard error (SE) from ANCOVA model. P value vs. placebo: *<0.05; **<0.01; ***<0.001; ****<0.0001. A significant increase in SHBG was observed after a single dose of ≧10 mg Compound 1 compared to placebo. A dose-dependent decrease in LDL-c, total cholesterol, and Apo B was observed on day 3 after a single dose of Compound 1, with similar results on day 4. No significant changes in triglyceride levels were observed after a single dose of Compound 1 (see Table 3). [Table 6]

[0106] conclusion Single dose escalation of Compound 1 up to 60 mg was generally safe and well tolerated, with linear dose-proportional plasma exposure with low variability. Half-lives were >13 hours at all single dose levels, supporting once-daily oral dosing. Renal excretion of unchanged Compound 1 was minimal, indicating that renal excretion is a minor route.

[0107] Significant dose-dependent effects on SHBG, Apo B, and LDL-c were observed after a single dose of Compound 1, pointing to potential efficacy. Sufficient exposure levels were achieved in human subjects across all Compound 1 doses, relative to efficacious doses of Compound 1 in preclinical models (a threshold AUC of 3,320 ng*hr / mL achieved significant histological improvement in a mouse model of NASH).

[0108] Safety, PK, and PD results support the continued development of compound 1 and indicate that compound 1 is well suited for co-formulation with other oral small molecule NASH agents as an oral, once-daily, fixed-dose combination.

[0109] Example 2: Multiple Ascending Dose Study of Compound 1 in Healthy Human Subjects A multiple-dose ascending clinical trial of Compound 1 was conducted. Four groups of eight healthy participants were randomized in a 3:1 ratio (n=6 active and n=2 placebo) to receive Compound 1 (1 mg, 3 mg, 6 mg, or 10 mg capsules) or matching placebo, administered once daily during a 14-day fasting state. Plasma levels of Compound 1 and PD biomarkers were determined at various pre- and post-dose time points.

[0110] Adverse event (AE) monitoring (Table 4), routine clinical laboratory tests (including thyroid axis tests [free and total thyroid hormone triiodothyronine (T3), free and total thyroid hormone thyroxine (T4), thyroid stimulating hormone (TSH)], cardiac biomarkers [CK-MB, troponin I], and liver biochemistry) (Figure 5, Figure 6), aggregate vital signs, electrocardiogram telemetry, and electrocardiograms were assessed throughout the study. Compound 1 concentrations were determined using a validated liquid chromatography-tandem mass spectrometry assay.

[0111] Plasma samples for Compound 1 concentration and PK sampling were collected pre-dose and 0.5, 1, 2, 3, 4, 6, 8, 12, and 24 hours after the first dose of test medication (placebo or compound), pre-dose on days 3, 4, 5, 8, 11, and 13 of dosing, and pre-dose and 0.5, 1, 2, 3, 4, 6, 8, 12, 24, 48, and 72 hours after 14 days of once-daily administration (see Table 5, Figure 7). PK parameters were estimated by non-compartmental methods using Phoenix® WinNonlin® (Certara, LP, Princeton, NJ). Serum pharmacodynamic (PD) biomarkers apolipoprotein B (Apo B) and sex hormone binding globulin (SHBG) concentrations were measured using immunoassays, and serum lipids were determined using spectrophotometry.

[0112] PD sampling was also performed. Percent change from baseline for PD markers was calculated using an ANCOVA model with percent change from baseline as the dependent variable, treatment group as a fixed effect, and baseline as a covariate. Analyses used only observed data without imputation for missing data. PD data for day 15 of the study are shown in Figure 8.

[0113] Results and Conclusions safety [Table 7]

[0114] Daily dosing of placebo or Compound 1 up to 10 mg for 14 days was generally well tolerated. No study or dose escalation discontinuation criteria were met. All AEs were grade 1 and were determined to be largely unrelated to study drug. All subjects randomized to Compound 1 completed the study without interrupting study drug. Heart rate and blood pressure remained stable throughout the study. Free T4 declined in a dose-dependent manner without apparent changes in TSH or free T3. T4 changes were asymptomatic and not determined to be clinically significant, suggesting that there is peripheral thyroid hormone regulation leading to the decline in free T4 without changes in T3 or TSH.

[0115] Mean ALT values ​​were similar across groups and were not significantly different from placebo. No treated subjects had an increase in ALT ≥ 2x ULN. A dose-dependent increase in GGT was observed, with values ​​remaining below the ULN for treated patients. A dose-dependent increase in total testosterone was observed, but no significant changes in free levels were identified.

[0116] No notable changes in ECGs, cardiac biomarkers, or other clinical laboratory tests were identified.

[0117] Pharmacokinetics and Pharmacodynamics [Table 8]

[0118] Pharmacokinetic data showed good oral bioavailability with low PK variability. Multiple doses of Compound 1 led to significant and dose-dependent increases in SHBG, even at the low dose of 3 mg once daily. Reductions in total cholesterol, LDL-c, Apo B, and triglycerides were observed at all dose levels of Compound 1, with significant reductions observed at day 15 in the 10 mg dose cohort. HDL-c did not change significantly by day 15. These results support the efficacy of low doses of Compound 1 once daily.

[0119] Example 3: Drug-drug interaction studies with Compound 1 in healthy humans In vitro studies have shown that Compound 1 has the potential for first-pass inhibition of organic anion transporting polypeptide (OATP) 1B1 / 1B3 and intestinal inhibition of breast cancer resistance protein (BCRP) transporter (OATP1B1 IC50=2.01 micromolar; OATP1B3 IC50=0.71 micromolar; BCRP IC50=9.37 micromolar). The effect of Compound 1 on the pharmacokinetics of co-administered rosuvastatin (ROS), an antihyperlipidemic drug and substrate of OATP and BCRP, in healthy participants is determined by administration of Compound 1 and ROS as described in Table 6, in conjunction with PK sampling. [Table 9]

[0120] In addition, Compound 2 is an inhibitor of the intestinal expressed transporters P-glycoprotein (P-gp) and BCRP (P-gp IC50=3.92 micromolar; BCRP IC50=4.39 micromolar). Based on in vitro studies, inhibition of these transporters by Compound 2 has the potential to increase the absorption of co-administered Compound 1. Therefore, as described in Table 7, a drug-drug interaction (DDI) study is performed to assess the potential of Compound 2 to enhance the absorption of Compound 1 through inhibition of intestinal P-gp and BCRP. [Table 10]

[0121] Example 4: Effect of food on Compound 1 in healthy humans The effect of food on the uptake and pharmacokinetics of Compound 1 in healthy participants is determined by administering Compound 1 during fed and fasted states as described in Table 8, in conjunction with PK sampling. [Table 11]

[0122] Participants will fast overnight (no food or liquids other than water for at least 10 hours prior to dosing). On day 9 of sequence A and day 1 of sequence B, a high-fat / high-calorie breakfast containing approximately 1000 kcal and 45%-55% fat will be provided 30 minutes prior to administration of study medication. Study medication will be administered when participants have completed breakfast (100% consumption) or within 5 minutes of completion.

[0123] Thyroid axis safety monitoring and cardiovascular safety monitoring will be performed as described in the preceding examples.

[0124] Example 5: Effect of Compound 1 on mouse NASH model C57BL / 6J mice were fed a high-fat diet for 10 weeks to induce obesity (>38 g body weight). Obese mice were injected intraperitoneally (ip) with 0.5 μl / g of 25% CCl4 (formulated in olive oil) twice a week for 4 weeks to induce fibrosis, and one group of normal weight mice was injected ip with olive oil twice a week for 4 weeks to serve as healthy controls. During the same dosing period, obese mice were orally given vehicle or varying doses of Compound 1 once a day for 28 days. On the dosing day of CCl4, CCl4 was administered 4 hours after compound or vehicle dosing. On day 27, all animals were fasted for approximately 16 hours before terminal euthanasia. On day 28, all animals were sacrificed and various biological parameters were analyzed. Total body weight, liver weight, heart weight, and brain weight were measured, and changes in liver and heart weight were normalized using brain weight.

[0125] Compound 1 significantly reduced liver / brain weights and had no effect on total body weight or heart / brain weights (Figure 9). Liver tissue histology was analyzed for the effect of Compound 1 on steatosis, inflammation, and fibrosis. Compound 1 significantly reduced steatosis at all doses tested, showed a trend toward reduced inflammation, and significantly reduced liver fibrosis at 3 and 10 mpk (Figure 10). Compound 1 also significantly reduced serum total cholesterol, triglycerides, and ALT at all doses tested (Figure 11).

[0126] Liver samples were collected for whole transcriptome analysis by RNA sequencing (RNAseq). Preparation and sequencing of RNAseq libraries (n=5 per group) was performed using Illumina's standard protocol. Alignment of sequencing reads was performed using STAR aligner software, and read counts were estimated using RSEM. Differentially expressed genes (compared to vehicle-treated NASH control mice) were determined using EdgeR software. Gene ontology analysis was performed using Advaita software with fold change and adjusted p-value cutoffs of >1.5 and <0.05, respectively. Gene ontology was derived from the Gene Ontology Consortium database (April 26, 2019) (Ashburner et al., Gene ontology: Tool for the unification of biology. Nature Genetics 25(1):25-9(2000); Gene Ontology Consortium, Creating the Gene Ontology Resource: Design and Implementation. Genome Research 11:1425-1433(2001)). Compound 1 had a significant effect on the expression of genes associated with collagen extracellular matrix and hepatic stellate cell activation, primarily by reducing their expression levels compared to NASH control mice (Figure 12).

[0127] Example 6: Effect of pH and solubilizing agents on the solubility of Compound 1 The solubility of Compound 1 (potassium salt) was evaluated at various pH levels. The solubility of Compound 1 in aqueous solution was pH dependent and increased with pH, ​​as shown in Table 9. In the presence of a solubilizing agent (sodium lauryl sulfate, SLS), the solubility of Compound 1 further improved to 308 μg / mL in pH 10.0 buffer + 2 wt% SLS after 24 hours at 25° C. [Table 12]

[0128] Example 7: Formulation, Pharmacokinetics, and Food Effect of Compound 1 in Beagle Dogs To determine the effect of solubilizing agent (SLS) on the uptake of Compound 1, 50 mg (based on the free acid) of Compound 1 was formulated in capsules with or without 5% by weight SLS (see Table 10) and administered to fasted beagle dogs pretreated with pentagastrin (6 μg / kg, administered by intramuscular injection 30±2 minutes prior to administration of Compound 1). Plasma concentrations of Compound 1 were measured over a 24 hour period (see FIG. 13). Formulations containing 5% by weight SLS significantly reduced Compound 1 exposure (C max , AUC) by more than 70% (see Table 11). [Table 13] [Table 14]

[0129] To determine pH effects on PK performance, beagle dogs were divided into two groups (n=3 per group). For group 1, dogs were pretreated with pentagastrin (6 μg / kg, intramuscularly injected 30±2 minutes prior to administration of Compound 1). For group 2, dogs were pretreated with famotidine (2 tablets, 20 mg / tablet, orally administered 180±10 minutes prior to administration of Compound 1). Compound 1 (10 mg capsule, 5% SLS) was administered and plasma concentrations of Compound 1 were monitored for 24 hours and are illustrated in FIG. 14. Minimal pH effects occurred in dogs under study conditions.

[0130] To determine the effect of food on PK performance, beagle dogs were divided into two groups (n=3 per group). For the fasted group, dogs were fasted overnight through 4 hours after dosing. For the fed group, dogs were fed a high fat meal 30 minutes prior to dosing. Plasma concentrations of Compound 1 were monitored for 24 hours. Compound 1 (10 mg capsule, 5% SLS) was administered and plasma concentrations of Compound 1 were monitored for 24 hours and are illustrated in FIG. 15. Food intake was associated with the increase in the concentration of Compound 1 t max The effect of 10-fold increased the plasma concentration of 10-fold increased creatinine, but had no effect on plasma exposure.

[0131] Example 8. Preparation of the potassium salt of compound 1 Ethyl (E)-(2-cyano-2-(2-(3,5-dichloro-4-((4-oxo-3,4-dihydrophthalazin-1-yl)oxy)phenyl)hydrazinylidene)acetyl)carbamate (7.4 kg, 0.99-1.01×), potassium acetate (7.4 kg, 0.95-1.00×), and DMAc (41 kg, 5.6-6.0×) were charged into a 500 L GL reactor. The resulting mixture was maintained at 80-90° C. for 12-16 hours. The mixture was adjusted to 20-30° C. A solution of KOH (0.85 kg, 0.11-0.17×) in process water (8 kg, 1.0-1.5×) was added at 20-30° C. for 1-2 hours. The mixture was stirred at 20-30° C. for 1-2 hours. Process water (39 kg, 5.0-5.5x) was added at 20-30°C for 4-6 hours. The mixture was stirred at 20-30°C for 2-3 hours. The resulting mixture was centrifuged through a stainless steel centrifuge. The wet cake was rinsed with process water twice (18+20 kg, 2-3x). The wet cake and process water (38 kg, 5.0-6.0x) were charged into a 500L GL reactor. The mixture was stirred at 20-30°C for 2-3 hours. The resulting mixture was centrifuged through a stainless steel centrifuge. The wet cake was rinsed with process water twice (18+22 kg, 2-3x) and dried through a stainless steel dryer under reduced pressure at 55-65°C to obtain crude potassium salt of compound 1 (5.45 kg, purity: 98.4%; assay: 95.9%; yield: 72%).

[0132] Crude potassium salt of compound 1 (5.3 kg, 0.99-1.01x) and DMSO (43 kg, 6.0-8.0x) were charged into a 250 GL reactor (R1). The resulting mixture was maintained at 40-50°C for 0.5-1 hour to obtain a clear solution. Ethyl acetate (EA) (22 kg, 4.0-4.5x) was added at 40-50°C over 1-2 hours. The resulting mixture was filtered through an in-line filter and transferred into a 250 GL reactor (R2). R1 ​​was rinsed with DMSO (1.8 kg, 0.2-0.5x) and the material was filtered through an in-line filter and transferred into R2. The temperature was adjusted to 40-50°C and EA (22 kg, 4.0-4.5x) was charged into R2 over 1-2 hours at 40-50°C. Compound 1 seed (0.010 kg, 0.001-0.002x) was charged into R2. The resulting mixture was stirred at 40-50°C for 1-2 hours. EA (67 kg, 12.0-13.0x) was charged into R2 over 12-15 hours at 40-50°C and stirred at 40-50°C for 2-3 hours. Sampled for analysis (Spec: XRPD of wet cake: consistent with reference Form A). EA (27 kg, 1.0-5.0x) was charged into R2 over 2 hours at 40-50°C and stirred at 40-50°C for 2-3 hours. R2 was adjusted to 20-30°C over 2 hours and stirred at 20-30°C for 4-6 hours. The resulting mixture was filtered in a stainless steel filter drier. The wet cake was rinsed twice with EA (16+14 kg, 2-3x). EA (36 kg, 6-7x) was charged into a stainless steel filter drier. Temperature was adjusted to 20-30°C and stirred at 20-30°C for 2-3 hours. The resulting mixture was filtered in a stainless steel filter drier. The wet cake was rinsed with EA (16 kg, 2-3x) and dried at 60-70°C under reduced pressure in a stainless steel filter drier. The material was sieved to obtain the purified potassium salt of compound 1 (4.16 kg, purity: 99.81%; assay: 97.6%; yield: 80%). See Figure 16.

[0133] Example 9. Salt Screening Studies for Compound 1 Several salt forms of Compound 1 were evaluated based on their properties, including solubility, stability, and hygroscopicity, in comparison to the potassium salt form prepared in Example 8 (see Figure 16, Table 12). [Table 15]

[0134] Using free acid form A (FIG. 17) as the starting material, a total of 50 salt screening experiments were performed with 10 salt formers and 5 solvents. Characterization results showed that a total of 14 salts were obtained in the salt screening and reformulation experiments, including ammonium salt form B / C, L-lysine salt form A / B / C, magnesium salt form A, choline salt form A, calcium salt form A, L-arginine salt form A / B, sodium salt form A, tris salt form A / B, diethyl amine salt form A, and four forms of the free acid (including free acid form A / B / C / D). According to XRPD comparison, the sample named ammonium salt form A was inferred to be a mixture of free form form D and ammonium salt.

[0135] Based on the characterization data (good crystallinity, clean DSC signal, integer molar ratio, high safety class, and simple polymorphism used as criteria), sodium salt form A (Figure 18, Table 13), L-arginine salt form A (Figure 19), and magnesium salt form A (Figure 20) were selected for reformulation and kinetic solubility evaluation, along with the potassium salt as a reference for comparison. The results of the solubility evaluation showed that the sodium salt demonstrated the highest solubility in water at room temperature for 24 hours (approximately 0.4 mg / mL, similar to the solubility of the potassium salt in water at 37°C). In simulated gastric fluid (SGF) at 37°C, the potassium salt showed the highest solubility (0.11 mg / mL), followed by the sodium salt. In fasted simulated intestinal fluid (FaSSIF) at 37°C, the magnesium salt showed the highest solubility (approximately 0.04 mg / mL), followed by the free acid. Similar solubilities (approximately 0.02 mg / mL) were observed for the four salts and the free acid in fed-state simulated intestinal fluid (FeSSIF) at 37° C. XRPD results showed that morphological changes were observed for all salts in the different test media, except for the free acid. [Table 16]

[0136] According to the results of the kinetic solubility evaluation, with higher solubility (compared to the free acid) and simple polymorphism as a criterion, the anhydrous sodium salt Form A was selected as a candidate for physicochemical stability evaluation, hygroscopicity, and PLM studies and compared with the potassium salt Form A (Table 14). The results of these studies showed that: 1) Physicochemical stability evaluation: After 1 and 2 weeks of storage at 60°C / closed and 60°C / 75% relative humidity, a minor decrease in HPLC purity (<0.1% area) was observed for sodium salt form A, but no morphological change was observed. 2) Hygroscopicity: Dynamic vapor sorption (DVS) results showed that sodium salt form A was slightly hygroscopic with no morphological changes after DVS testing. 3) PLM: Sodium salt Type A sample consisted of irregular snow crystals with particle size <20 μm. A comparison of the characterization and evaluation data for the sodium and potassium salts is shown in Table 14. [Table 17]

[0137] Example 10. Formulation of Compound 1 for use in clinical trials The compound is an orally bioavailable thyroid hormone receptor beta (THR-β) selective agonist for the treatment of adults with moderate to severe non-cirrhotic non-alcoholic steatohepatitis (NASH). Compound 1 is classified as a BCS class IV compound and was formulated as an immediate release capsule. To promote the intrinsic solubility of the active substance, compound 1 is isolated as a potassium salt form of the drug substance and sodium lauryl sulfate is used as a solubilizer in the immediate release formulation. The formulated product is a dry blend containing 0.5-50 mg of compound 1 (free acid equivalent) in a size 0 HPMC (hydroxypropyl methylcellulose) capsule. In addition to solubilizing substances, other classes of excipients such as diluents (e.g., microcrystalline cellulose (MCC), mannitol), disintegrants (e.g., croscarmellose sodium), flow agents (e.g., colloidal silicon dioxide), and lubricants (e.g., magnesium stearate) were also investigated and used in the compound 1 formulation. The quantitative unit dose formulation composition of the active capsules is set forth below in Table 15. [Table 18]

[0138] Example 11. Study of direct encapsulation formulations of Compound 1 A study of the direct encapsulation (DE) formulation of Compound 1 was performed. This work encompassed the development of a DE process for the preparation of 1 mg, 3 mg, 10 mg, 50 mg, and placebo capsules on semi-automatic or automatic capsule filling machines. The formulation ingredients, ratios, fill weight, flowability, and uniformity were all evaluated. The developed process is suitable for manufacturing Compound 1 drug product for future clinical trials. Preparation of prototype confirmation batches, short-term stability studies, GMP certification, and manufacturing of demonstration stability batches were completed to ensure successful manufacturing of GMP batches and quality for intended clinical use.

[0139] During initial formulation studies of Compound 1, types of solubilizers were investigated, including solubilizers such as poloxamer 188 and the anionic surfactant sodium lauryl sulfate (SLS). SLS showed good compatibility with Compound 1 and increased the aqueous solubility of Compound 1. Levels of SLS from 1.5% to 5.0% weight / weight were investigated. Pharmacokinetic data showed that using certain amounts of SLS in the formulation significantly improved in vivo bioavailability. Therefore, 1.5% to 5.0% SLS was suitable for further formulation development with Compound 1.

[0140] An efficient blending process was developed to address the challenge of dose uniformity at different dose levels. An equal amount blending process was developed for the 0.5mg-10mg dose levels. Compound 1 was first blended in equal amounts with the entire amount of SLS and a portion of the amount of MCC. The mixture was then blended with the remaining excipients, passed through a sieve, and then blended again (blend-sieve-blend process). By combining Compound 1 with excipients instead of alone, the yield loss of Compound 1 during the blending process was efficiently reduced. The blend-sieve-blend process effectively incorporated low doses of Compound 1 uniformly with excipients in higher yields into the formulation, allowing for downstream direct capsule filling or direct tableting. For the 10mg-50mg formulations, a similar blending and sieving procedure was used in addition to the equal amount blending process developed and described above. Demonstration batches and GMP batches of each dose strength showed that the blend uniformity results of the final blends using this blending process met the desired product specifications.

[0141] The capsule filling weight of the formulation containing different doses of Compound 1 and placebo is designed to be 300mg, because this material volume can meet the need for semi-automatic filling of No. 0 capsules based on the bulk density of the final blend.Semi-automatic filling can be used in early clinical stage where only small batch size of clinical drug product is required.In addition, fully automated capsule filling process is also developed to meet the large scale need of late stage clinical trials.

[0142] Short-term stability studies under stress conditions of 60°C / 75% relative humidity revealed the formation of degradation impurities at a titer of 3 mg after 2 weeks. Silica gel desiccant was subsequently suggested for packaging and proved effective in reducing the formation of impurities.

[0143] Demonstration stability and subsequent manufacturing of GMP batches of 1 mg, 3 mg, 10 mg, and 50 mg strengths using GMP Compound 1 were performed according to the final formulation and process. The effectiveness of silica gel desiccant in minimizing degradation was evaluated through comparative stability studies with and without desiccant included in the primary packaging. The stability data clearly demonstrated that packaging with desiccant significantly retarded the formation of degradation impurities at 40°C / 75% relative humidity conditions. Therefore, it was determined that packaging with 1 g desiccant improved product quality and increased the shelf life of the product.

[0144] Example 12. Clinical evaluation of Compound 1 in healthy subjects with elevated LDL-c

[0145] Objective: To assess the overall safety and tolerability of multiple ascending doses of Compound 1 in healthy subjects with elevated LDL-c.

[0146] Secondary Objective: To evaluate the PK and PD of Compound 1 in healthy subjects with elevated LDL-c following multiple ascending doses of Compound 1.

[0147] Primary endpoints: treatment-emergent adverse events (TEAEs), vital signs, clinical laboratory parameters, and electrocardiogram (ECG) monitoring.

[0148] Secondary endpoints: plasma PK parameters for Compound 1, PD markers of THR-β agonist target engagement including LDL-c and other lipid parameters, and sex hormone binding globulin (SHBG).

[0149] Healthy volunteers with mildly elevated LDL-c were randomized 3:1 to Compound 1 (n=6) or placebo (n=2). Volunteers randomized to Compound 1 received multiple doses of 1, 3, 6, or 10 mg of Compound 1 once daily for 14 days in the MAD cohort of the study.

[0150] result Compound 1 was generally safe and well tolerated with similar incidence of AEs across all Compound 1 treatment arms and placebo. All AEs were mild to moderate in severity and had no apparent dose-related association. One placebo subject (1 mg cohort) discontinued the study early due to withdrawal of consent; all Compound 1 subjects completed the study without premature discontinuation. No study or dose-escalation discontinuation criteria were met.

[0151] Liver biochemistry: ALT, AST, ALP, and total bilirubin values ​​were similar overall across treatment groups. Subjects receiving Compound 1 did not experience increases in ALT to > 2xULN. No evidence of DILI. Thyroid hormones: No symptoms of hyper / hypothyroidism. Mean TSH and free T3 values ​​were highly variable but generally similar across groups. A dose-dependent decline in free T4 was observed in the Compound 1 group, consistent with peripheral thyroid hormone regulation observed with other THR-agonists. Other laboratory assessments (e.g., clinical chemistry, hematology) showed no clear trends.

[0152] Compound 1 was administered once daily for 14 days at 1, 3, 6, and 10 mg, and was generally safe and well tolerated, with no clinical signs or symptoms of hypo / hyperthyroidism or THR-α agonism. Compound 1 exhibited dose-proportional PK with low variability and half-life suitable for once-daily dosing. Compound 1 increased SHBG, an important marker of hepatic THR-β engagement, in a dose-dependent manner. Compound 1 led to significant reductions in circulating atherogenic lipid levels, including LDL-c, Apo B, total cholesterol, and triglycerides. Taken together, the PD data indicate that administration of Compound 1 led to robust THR-β target engagement in the liver.

[0153] Example 13: Effects of 12-week monotherapy and combination treatment with Compound 2 and Compound 1 on NAFLD activity scores including metabolic parameters, liver pathology, and fibrosis stage in male biopsy-confirmed DIO-NASH mice [Table 19-1] [Table 19-2]

[0154] introduction Nonalcoholic steatohepatitis (NASH) is a disease in which liver inflammation and damage occur in the context of hepatic steatosis and will likely require combination therapy targeting multiple aspects of the disease to achieve a high level of disease cure. Small molecule agonists of the nuclear hormone receptors Farnesoid X Receptor (FXR), which maintains metabolic pathway homeostasis, and Thyroid Hormone Receptor β (THR-β), which regulates metabolic pathways complementary to FXR, are in development for the treatment of NASH. Compound 2, a nonsteroidal agonist of FXR, and Compound 1, a liver-distributed, selective agonist of THR-β, were evaluated alone and in combination in a diet-induced mouse model of NASH.

[0155] Materials and Methods Animal handling and study design C57BL / 6JRj male mice (n=138) were fed a Gubra Amylin NASH (GAN) diet (40% fat, 22% fructose, 2% cholesterol [D09100310, Research Diets]) or a low-fat chow diet for 35 weeks prior to the start of treatment. Prior to treatment, all animals underwent liver biopsy for histological confirmation (steatosis score ≧2 and fibrosis stage ≧1) and stratification using the Nonalcoholic Fatty Liver Disease (NAFLD) Activity Scoring (NAS) and Fibrosis Staging System. Diet-induced obese mice on the GAN diet (DIO-GAN) were randomized into eight treatment groups (Table 16) based on the percent area of ​​picrosirius red (PSR) staining. DIO-GAN mice (n=16 per group) were treated (PO, QD) with vehicle (0.5% HPMC in Tris buffer + 0.2% Tween 80 [50 mM, pH 8]), Compound 2 (10 mg / kg), Compound 1 (0.3 mg / kg [low], 2 mg / kg [medium], or 10 mg / kg [high]), or combination treatment of Compound 2 with Compound 1 (combined-low, combined-medium, or combined-high) for 12 weeks. Vehicle-dosed low-fat chow-fed controls served as healthy controls (n=10). Mice were maintained on their respective diets (GAN or low-fat chow) for the duration of the study. Intrasubject comparisons (pre- vs. post-treatment) were performed for liver biopsy histopathology scores. Final quantitative endpoints included plasma / liver biochemistry, liver histomorphometry, and liver transcriptome analysis by RNAseq. [Table 20]

[0156] Liver biopsy processing and scoring Formalin-fixed paraffin-embedded (FFPE) liver biopsies were prepared by placing liver samples in 10% neutral buffered formalin for approximately 24 hours, then transferring to 70% ethanol, followed by storage at 4C. FFPE were placed in a Histokinette, infiltrated, and then embedded in a block. Biopsy tissues were then cut at 3 μm using a microtome, and sections were mounted on slides. Liver sections were stained with hematoxylin and eosin (H&E) to assess steatosis, inflammation, and ballooning, and with PSR to assess fibrosis. Additionally, slides were processed to detect type I collagen (Col1a1), galectin-3 (Gal-3), and smooth muscle actin (α-SMA) protein expression by immunohistochemistry (IHC). For H&E staining, slides were incubated in Mayer's hematoxylin (Dako), washed in tap water, stained in Eosin Y solution (Sigma-Aldrich), dehydrated, and coverslipped. For PSR, slides were incubated in Weigert's iron hematoxylin (Sigma-Aldrich), washed in tap water, stained in Picro-Sirius Red (Sigma-Aldrich), and washed twice in acid water. Excess water was removed by shaking the slides, and then the slides were dehydrated in ethanol, cleared in xylene, and coverslipped. NAS and fibrosis stage were scored as previously described (Kleiner et al. 2005). NAS represents the unweighted sum of steatosis, inflammation, and ballooning scores and ranges from 0 to 8 (Table 17); fibrosis stage ranges from 0 (no fibrosis) to 4 (cirrhosis). For detection of Col1a1, Gal-3, and α-SMA, IHC was performed by standard procedures. Briefly, after antigen retrieval and blocking of endogenous peroxidase activity, slides were incubated with primary antibodies (Col1a1: Southern Biotech, Cat. 1310-01; Gal-3: Biolegend, Cat. 125402; α-SMA: Abcam, Cat. Ab124964). Primary antibodies were detected using polymeric HRP-linker antibody conjugates. Primary antibodies were visualized with DAB as chromogen.Finally, sections were counterstained in hematoxylin and coverslipped. [Table 21]

[0157] Analysis of liver enzymes, plasma lipids, and CK18 M30 Terminal blood was collected by cardiac puncture from mice anesthetized with isoflurane (2-3%), mixed with anticoagulant, placed at 4C, and then centrifuged at 3000×g for 10 min. Plasma supernatants were transferred to new tubes, immediately frozen on dry ice, and stored at -80C. Alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), triglycerides (TG), total cholesterol (TC), high-density lipoprotein (HDL-c), and low-density lipoprotein (LDL-c) were measured using commercial kits (Roche Diagnostics) on a Cobas c 501 automated analyzer according to the manufacturer's instructions. Cytokeratin 18 M30 (CK18 M30) was measured from plasma using a commercial ELISA kit (Cusabio) according to the manufacturer's instructions.

[0158] Liver lipid analysis Liver samples were homogenized and TGs and TCs were extracted in 5% NP-40 by heating (2x) at 90 C. Samples were centrifuged and the TG and TC contents in the supernatants were measured using commercial kits (Roche Diagnostics) on a Cobas c 501 automated analyzer according to the manufacturer's instructions.

[0159] Liver transcriptome analysis by RNAseq Tissues were collected, snap frozen in liquid nitrogen, and stored at -80C until processing. RNA was isolated using the NucleoSpin kit (MACHEREY-NAGEL). A total of 10ng-1μg of purified RNA from each sample was used to generate cDNA libraries using the NEBNext Utra II Directional RNA Library Prep Kit for Illumina (New England Biolabs). The cDNA libraries were then sequenced on a NextSeq 500 using the NextSeq 500 / 550 High Output Kit V2 (Illumina). Sequencing data were aligned to the mouse genome using Spliced ​​Transcripts Alignment to a Reference (STAR) software. Differentially expressed genes were identified using the R package DESeq2.

[0160] Analysis of trough plasma compound levels Terminal plasma samples were collected by cardiac puncture approximately 21-24 hours after the last dose of compound(s). Terminal plasma samples were analyzed by high-resolution LC-MS / MS using a Triple Quad 6500+ instrument. 20 μL of plasma sample was mixed with 200 μL of internal standard (100 ng / mL labetalol + 100 ng / mL tolbutamide in acetonitrile), vortexed, and centrifuged at 4,000 rpm for 15 min at 4 °C. The supernatant (100 μL) was transferred to a sample plate, mixed with water (100 μL), shaken (800 rpm) for 10 min, and then injected (2 μL) onto a 1.7 μm, 2.1×50 mm ACQUITY UPLC BEH C18 column (Waters) at a flow rate of 0.6 mL / min using a gradient (Table 18) mobile phase of 0.1% formic acid in water (mobile phase A) and 0.1% formic acid in acetonitrile (mobile phase B). Two internal standards (Standard 1 (retention time: 0.87 min) and Standard 2 (retention time: 0.96 min)) were used for the quantification of Compound 2 (retention time: 0.87 min) and Compound 1 (retention time: 0.97 min), respectively. An internal standard (retention time: 0.76 min) was used for the quantification of the glucuronide metabolite of Compound 2 (retention time: 0.77 min). Calibration curves (1-3000 ng / mL) were generated in a matrix of mouse plasma (pooled vehicle control) for each analyte. [Table 22]

[0161] result Study design overview The diet-induced obesity Gubra Amylin NASH model (DIO-GAN) recapitulates many of the histopathological features of human NASH (Hansen 2020). Using the DIO-GAN model, the efficacy of an FXR agonist (compound 2) and a THR-β agonist (compound 1) was assessed as single agents and in combination. To induce NASH disease, C57BL / 6JRj mice were maintained on a diet high in fat, cholesterol, and fructose (GAN diet) for over 35 weeks. Prior to therapeutic intervention, mice were biopsied to assess NASH disease and fibrosis severity, and mice with a steatosis score <2 and fibrosis stage <1 were excluded from the study. DIO-GAN mice were then randomized into eight treatment groups (n=16 per group) based on the percent area of ​​picrosirius red (PSR) staining of pretreatment biopsies and body fat tissue mass determined by whole body echo-magnetic resonance imaging (EchoMRI). In the monotherapy group, compound 2 was administered by oral gavage once a day at a dose of 10 mg / kg, while compound 1 was administered by oral gavage once a day at dose levels of 0.3 (compound 1-low), 2 (compound 1-medium) or 10 (compound 1-high) mg / kg. In the combination treatment group, a fixed dose level of compound 2 (10 mg / kg) was combined with low, medium, and high doses of compound 1 (i.e., combination-low, combination-medium, and combination-high, respectively). DIO-GAN mice administered vehicle by oral gavage once a day served as control. Mice were treated for a total of 12 weeks and maintained on the GAN diet throughout the study. Lean mice (n=10) maintained on a normal diet throughout the study served as healthy controls.

[0162] Effects of treatment on body weight, food intake, and liver weight Treatment with Compound 2 alone and in combination with Compound 1 (combination-low, combination-medium, and combination-high) reduced body weight during the study (Figure 30A). At the end of the study, Compound 2, combination-medium, and combination-high treatment groups were significantly lower than DIO-GAN vehicle control (Figure 30B). The reduction in body weight did not seem to be associated with a reduction in food intake (Figure 31). All treatment groups significantly improved hepatomegaly (Figure 32A), with the greatest reduction in liver weight observed in the combination treatment groups (combination-medium and combination-high), and the change in spleen weight was not significant (Figure 32B).

[0163] Effect of treatment on body weight composition Body composition was determined by whole body EchoMRI at baseline (week -1) and week 11 of the study to determine the relative levels of lean and adipose tissue as a percentage of body weight. Baseline levels of lean and adipose tissue were well balanced across treatment groups (Figures 33A and 33B). Compound 2 treatment and combination with Compound 1 treatment reduced the levels of adipose tissue at week 11 (Figure 34A). A significant increase in relative lean tissue mass was observed in the Compound 2 and combination treatment groups (Figure 34B).

[0164] Effect of treatment on plasma and liver lipid levels All treatment groups significantly reduced plasma total cholesterol (TC) (Figure 35AA), with the greatest reduction observed in the combination treatment groups (combination-medium and combination-high), and a similar trend was observed for TC in liver (Figure 35B). Reductions in plasma low-density lipoprotein cholesterol and high-density lipoprotein cholesterol (HDL-c and LDL-c, respectively) were consistent with the effects observed for TC. Compound 2 and Compound 1-high significantly reduced plasma triglycerides (TG), as did combination treatment (Figure 36A). Liver TG levels were significantly reduced only in the combination-high group (Figure 36B).

[0165] Effect of treatment on liver enzymes Single-agent treatment with Compound 1 (low, medium, and high) significantly reduced ALT levels compared to the DIO-GAN vehicle control (Figure 37A). ALT levels were not significantly reduced by combination treatment. AST levels showed a similar trend to ALT, but none of the treatment groups were significantly different from the DIO-GAN vehicle control (Figure 37B). None of the treatment groups were significantly different from the DIO-GAN vehicle control, but ALP was numerically lower in the single-agent treatment groups (Compound 2, Compound 1-low, and Compound 1-medium) and higher in the combination-medium and combination-high treatment groups compared to the DIO-GAN vehicle control (Figure 38).

[0166] Liver histology NAFLD Activity Score (NAS) The histological effect of treatment was assessed using the NAFLD activity score (NAS). NAS is defined as the unweighted sum of the histological scores of steatosis, inflammation, and ballooning, and can range from 0 to 8. NAS was determined for each animal before treatment (baseline) and at the end of the study after 12 weeks of treatment. NAS was well balanced across treatment groups, with a NAS range of 5 to 6 at baseline in most mice (Figure 39A). After 12 weeks of treatment, NAS was significantly improved in most treatment groups, with the most significant improvement observed with combination treatment (Figure 39B). In the Compound 2 treatment group, 56% of animals showed a NAS improvement of ≥ 1 point, compared to 31%, 27%, and 62% in the Compound 1-Low, Compound 1-Mid, and Compound 1-High treatment groups, respectively (Table 19). Combination treatment was more effective, with 69%, 81%, and 100% of mice showing ≥1 point NAS improvement in the combination-low, combination-medium, and combination-high combination treatment groups, respectively. In addition, the magnitude of NAS improvement was greater in the combination treatment groups. 0% of mice in the Compound 2 treatment group had a >1 point NAS improvement, while 19%, 25%, and 43% of mice in the combination-low, combination-medium, and combination-high combination groups, respectively, achieved a ≥2 point NAS improvement. These results were superior to the Compound 1 monotherapy groups, with 0%, 7%, and 25% of mice in the Compound 1-low, Compound 1-medium, and Compound 1-high groups, respectively, achieving a ≥2 point NAS improvement. [Table 23]

[0167] Fatty degeneration NAS improvement was mostly driven by a greater reduction in steatosis (Table 20). In the compound 2 group, 81% showed improvement in steatosis at the end of treatment, with the maximum improvement being 1 point. In the compound 1 monotherapy groups, a dose-dependent increase in the percentage of mice showing improvement in steatosis was observed, corresponding to 31%, 47%, and 81% of mice in the compound 1-low, compound 1-medium, and compound 1-high dose groups, respectively. In each compound 1 monotherapy group, one mouse (i.e., about 6%) showed an improvement in steatosis of ≧2 points. In contrast, in the combination-low, combination-medium, and combination-high treatment groups, 25%, 31%, and 71% of mice showed an improvement in steatosis of ≧2 points, respectively. These effects were supported by quantitative liver histomorphometry, which showed a reduction in the percentage of liver cells containing lipid droplets (Figure 40A) and lower levels of liver lipids (Figure 40B) and smaller lipid droplet size (Figure 41). [Table 24]

[0168] The combination of Compound 2 and Compound 1 resulted in greater improvement in hepatic steatosis.Hepatic steatosis was determined by histology at baseline and at the end of treatment for each individual mouse.Table 29 shows the percentage of mice in each treatment group with unchanged or improved steatosis scores (1 point and ≧2 point reduction from baseline).The total represents the percentage of mice in each treatment group with at least 1 point improvement in steatosis from baseline.

[0169] Ballooning, inflammation, and fibrosis Hepatocyte ballooning (an indicator of apoptosis) was observed occasionally and was not significantly altered by any of the treatments. CK18 M30 (a plasma biomarker associated with apoptosis) also did not differ significantly between treatment groups and the DIO-GAN vehicle control (Figure 42). Lobular inflammation was not significantly improved by treatment, but improvements in inflammation scores were observed, albeit infrequently, in the Compound 1-Low, Compound 1-Medium, and Compound 1-High treatment groups and the Combination-Medium combination group (Table 21). To further assess inflammation, protein expression of Galectin-3 (Gal-3), a marker of inflammatory lymphocyte infiltration, was determined by immunohistochemistry (IHC) staining of liver. Treatment with Compound 2 alone and in combination with Compound 1 (Combination-Low) resulted in lower levels of Gal-3 expression in the liver compared to the DIO-GAN vehicle control (Figure 43A). [Table 25]

[0170] Inflammation was not significantly improved by treatment. Lobular inflammation was determined by histology at baseline and at the end of treatment. Table 30 shows the percentage of mice in each treatment group with worsening (≧1 point increase from baseline), no change, or improvement (≧1 point decrease from baseline) lobular inflammation scores.

[0171] Improvement in fibrosis stage was observed more frequently in the combination drug treatment group compared to the single drug group, but the difference did not reach significance (Table 22). Changes in Col1a1 protein expression, as determined by IHC staining of the liver, did not differ significantly between treatment groups. Reductions in the values ​​of α-SMA (a marker of hepatic stellate cell activation) were observed in the Compound 2 treatment group, the Compound 1-low treatment group, and the Compound 1-medium treatment group. Significant reductions were observed only in the combination-low treatment group (Figure 43B). [Table 26]

[0172] Improvement in fibrosis was observed more frequently with combination treatment. Liver fibrosis stage was determined by histology at baseline and end of treatment. Table 31 shows the percentage of mice in each treatment group with worsening (increase of ≥ 1 stage from baseline), no change, or improvement in fibrosis (decrease of ≥ 1 stage from baseline).

[0173] Liver transcriptomic analysis by RNAseq Final liver samples (n=10) from treatment groups were processed for transcriptomics analysis by RNAseq. Differentially expressed genes (DEGs) were identified compared to the DIO-GAN vehicle control. DEGs were identified in all treatment groups, with the lowest number of DEGs in compound 1-low (987) and the highest number in the combination-high (3533) treatment group. Comparing compound 1-high to combination-high showed that genes involved in energy and lipid metabolism were differentially expressed to a greater extent with the combination treatment (Figure 44). This is shown by comparing the slope for the discrimination line of fold change values ​​(dotted line, slope=1) to the linear regression line (solid line, slope=0.55). Squalene epoxidase (Sqle) and 7-dehydrocholesterol reductase (Dhcr7), enzymes involved in cholesterol metabolism, were expressed at significantly higher levels in the combination group compared to single agent treatments (Figures 45A and 45B). Hydroxymethylglutaryl-CoA synthetase (Hmgcs1), a key enzyme in energy metabolism, showed a similar pattern of expression (Figure 45C). Stearoyl-CoA desaturase (Scd1), an enzyme involved in fatty acid metabolism, was reduced by Compound 2 treatment and further reduced by combination treatment (Figure 45D).

[0174] Finally, we examined the expression of select genes associated with fibrosis and inflammation, including type I collagen alpha 1 (Col1a1), actin alpha 2 smooth actin (Acta2), galectin 3 (Lgals3), and melanoma cell adhesion molecule (CD146). Overall, compound 2, alone and in combination with compound 1, significantly reduced the expression of these genes compared to the DIO-GAN vehicle control (Figure 46). The combination treatment groups were not significantly different from compound 2 treatment alone.

[0175] summary The mouse diet-induced obese Gubra-Amylin NASH (DIO-GAN) model was used to evaluate the efficacy of Compound 2 and Compound 1 as single agents and in combination on metabolic and histopathological indices of NASH and fibrosis. This model has been extensively characterized and recapitulates many aspects of human NASH (Hansen 2020) without the use of hepatotoxic drugs to induce the disease. In this model, mice were maintained on a diet high in fat, cholesterol, and fructose (GAN diet) for over 35 weeks. Prior to therapeutic intervention, mice were biopsied to assess NAFLD activity score (NAS) and fibrosis severity by histology, and mice with baseline steatosis score >2 and fibrosis stage >1 were used in the study. Importantly, this preselection step ensured that only mice with significant NAFLD activity were used in the study. In addition, baseline NAS information allows the evaluated treatment response to be compared not only to DIO-GAN vehicle control, but also to individual baseline values. DIO GAN mice were treated with Compound 2 and Compound 1, alone and in combination, for 12 weeks and maintained on a GAN diet throughout the study. To maximize the ability to identify potential additive therapeutic effects, mice were treated with a single dose level of Compound 2, alone or in combination with three dose levels of Compound 1 (low [0.3 mg / kg], medium [2 mg / kg], and high [10 mg / kg]).

[0176] The combination of Compound 2 and Compound 1 showed a greater reduction in NAS compared to single-agent treatment, both in terms of the percentage of mice showing a reduction in NAS and the magnitude of NAS improvement. The improvement in NAS was driven in large part by a greater reduction in steatosis, which was associated with a greater reduction in plasma and liver total cholesterol and triglycerides. The greater overall effect observed with combination treatment did not appear to be driven by a higher exposure of the individual drugs in the combination treatment group (Table 23). In addition, although changes in body weight may contribute to NAS improvement, weight loss was similar between the Compound 2 treatment group and the combination treatment group (combination-low and combination-medium), suggesting that weight loss alone does not fully explain the greater anti-steatotic activity of the combination treatment. Instead, the combination treatment had a greater effect on the expression of genes related to energy and lipid metabolism. These results suggest that the combination of Compound 2 and Compound 1 appears to have at least additive effects on these pathways, likely responsible for the greater anti-steatotic activity observed.

[0177] Histological improvement of inflammation and fibrosis was not significantly improved by treatment. However, evidence of fibrosis improvement was observed, including a higher number of mice with fibrosis improvement by combination. In addition, transcriptomics analysis identified key markers of fibrosis and inflammation that were reduced by combination treatment. The expression of these genes was also reduced by compound 2 treatment, suggesting that FXR agonism may be the main driver for the effect on fibrosis and inflammation at the gene expression level. In this case, FXR agonism may be considered to be complementary to the more anti-steatotic mechanism of THR-β agonism. Combined with the higher anti-steatotic effect observed by the combination of compound 2 and compound 1, these results suggest that this combination may address multiple aspects of NASH disease.

[0178] References Kleiner DE, Brunt EM, Van Natta M, Behling C, Contos MJ, Cummings OW, Ferrell LD, Liu YC, Torbenson MS, Unalp-Arida A, Yeh M, McCullough AJ, Sanyal AJ; Nonalcoholic Steatohepatitis Clinical Research Network. Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology.2005 Jun;41(6):1313-21. Hansen,HH,Agidius,HM,Oro,D.et al.Human translatability of the GAN diet-induced obese mouse model of non-alcoholic steatohepatitis.BMC Gastroenterol 20,210(2020).

[0179] supplement [Table 27]

[0180] All publications, including patents, patent applications, and scientific articles, mentioned in this specification are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, including patents, patent applications, or scientific articles, was specifically and individually indicated to be incorporated by reference.

[0181] Although the foregoing invention has been described in some detail by way of illustrations and examples for purposes of clarity of understanding, it will be apparent to those skilled in the art that certain minor changes and modifications may be practiced in light of the above teachings. Therefore, the descriptions and examples should not be construed as limitations on the scope of the invention.

Claims

1. having an XRPD spectrum containing peaks at angles 2θ of 6.78±0.20, 11.35±0.20, and 20.51±0.20 degrees, respectively, of the formula: 【Transformation 3】 A crystalline potassium salt of the compound represented by the formula:

2. 2. The crystalline potassium salt of claim 1 having an XRPD spectrum substantially as shown in the figure below. [Figure 1]

3. having an XRPD spectrum containing peaks at angles 2θ of 5.51±0.20, 8.47±0.20, and 16.57±0.20 degrees, respectively, of the formula: 【Transformation 8】 A crystalline sodium salt of the compound represented by the formula:

4. 4. The crystalline sodium salt of claim 3, having an XRPD spectrum substantially as shown in the figure below. [Figure 2]

5. A pharmaceutical composition comprising, as an active ingredient, the crystalline potassium salt of claim 1 or 2, or the crystalline sodium salt of claim 3 or 4.

6. 6. The pharmaceutical composition according to claim 5, wherein the amount of the compound as an active ingredient in the pharmaceutical composition is from about 1 mg to about 15 mg.

7. 7. The pharmaceutical composition according to claim 6, wherein the amount of the compound as an active ingredient in the pharmaceutical composition is from about 2 mg to about 10 mg.

8. The pharmaceutical composition of claim 5 further comprising an ionic surfactant.

9. 9. The pharmaceutical composition of claim 8, wherein the ionic surfactant is sodium lauryl sulfate.

10. 1. A pharmaceutical composition for the treatment of non-alcoholic steatohepatitis (NASH), comprising a compound of the formula: 【Chemistry 2】 or a pharmaceutically acceptable salt thereof, wherein said compound or pharmaceutically acceptable salt thereof is orally administered once daily at a dose of about 0.5 mg to about 25 mg.

11. 11. The pharmaceutical composition of claim 10, wherein the compound or a pharmaceutically acceptable salt thereof is administered once daily at a dose of about 1 mg to about 15 mg.

12. 11. The pharmaceutical composition of claim 10, wherein the compound or a pharmaceutically acceptable salt thereof is administered once daily at a dose of about 2 mg to about 10 mg.

13. 11. The pharmaceutical composition of claim 10, wherein the compound is administered as a potassium salt or a sodium salt.

14. The pharmaceutical composition according to any one of claims 10 to 13, wherein the compound or a pharmaceutically acceptable salt thereof is administered in a pharmaceutical composition comprising an ionic surfactant.

15. 15. The pharmaceutical composition of claim 14, wherein the ionic surfactant is sodium lauryl sulfate.

16. 16. The pharmaceutical composition of claim 15, wherein sodium lauryl sulfate is present in the composition at about 1% to about 8% by weight.

17. formula: 【Transformation 3】 or a pharmaceutically acceptable salt thereof, and sodium lauryl sulfate.

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