Method of treating liver diseases or disorders with ask1 inhibitors

Abstract

Provided herein are methods of treating liver diseases and disorders comprising administering to the subject a therapeutically effective amount of Compound A, or a pharmaceutically acceptable salt or solvate thereof.

Description

WSGR Docket No. 51760-717.601METHOD OF TREATING LIVER DISEASES OR DISORDERS WITH ASK1INHIBITORSCROSS REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 708,139 filed October 16, 2024 which application is incorporated herein by reference in their entirety.BACKGROUND

[0002] There is an unmet need to treat a variety of liver diseases and disorders, which can progress to fibrosis, cirrhosis, liver failure, or death if left untreated, or if the treatment is not effective.SUMMARY

[0003] There remains a need for novel compounds to treat these liver diseases and disorders. Apoptosis signal-regulating kinase 1 (ASK1) inhibitors may be useful for treating these liver diseases and disorders. Orally dosed ASK1 inhibitors described herein show preferential distribution to the liver and achieve organ concentration several fold above the plasma concentration. Intravenous (IV) administration of ASK1 inhibitors described herein also show preferential distribution to liver and thus are suitable for treatment of patients in whom the IV route of administration is preferred over the oral route, including patients who are unconscious, have swallowing difficulties or require a rapid onset of pharmacological activity. As such, in some embodiments ASK1 inhibitors described herein are useful for treating liver diseases and disorders.

[0004] In one aspect, described herein is a method for treating cholestatic liver disease in a subject in need thereof comprising administering to the subject a therapeutically effective amount of compounds described herein, or pharmaceutically acceptable salts or solvates thereof.

[0005] In some embodiments described herein is a method for treating cholestatic liver disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of Compound A, or a pharmaceutically acceptable salt or solvate thereof, wherein Compound A has the following structure:(Compound A).WSGR Docket No. 51760-717.601

[0006] In some embodiments, the cholestatic liver disease is acute cholestasis liver disease or chronic cholestasis liver disease. In some embodiments, the cholestatic liver disease is biliary obstruction, biliary atresia, cholangitis, total parenteral nutrition (TPN)-associated cholestasis, primary sclerosing cholangitis, secondary sclerosing cholangitis, or intrahepatic cholestasis of pregnancy. In some embodiments, the cholestatic liver disease is biliary atresia.

[0007] In some embodiments, Compound A is administered in a dose between about 1 mg / kg to about 100 mg / kg. In some embodiments, the dose is between about 5 mg / kg to about 80 mg / kg. In some embodiments, the dose is between about 10 mg / kg and about 60 mg / kg. In some embodiments, the dose is between about 15 mg / kg to about 50 mg / kg.

[0008] In some embodiments, the subject is a pediatric subject. In some embodiments, the subject is less than 5 years old. In some embodiments, the subject is less than 3 years old. In some embodiments, the subject is less than 1 year old.

[0009] In some embodiments, Compound A is administered orally or by intravenous delivery. In some embodiments, Compound A is administered by intravenous delivery. In some embodiments, Compound A is administered orally.

[0010] In an aspect, described herein is a method for treating primary liver cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of Compound A, or a pharmaceutically acceptable salt or solvate thereof, wherein Compound A has the following structure:

[0011] In some embodiments, the primary liver cancer is hepatocellular carcinoma (HCC) or bile duct cancer. In some embodiments, the primary liver cancer is hepatocellular carcinoma (HCC). In some embodiments, the subject previously had chronic hepatitis B or chronic hepatitis C.

[0012] In some embodiments, Compound A is administered in a dose between about 1 mg / kg to about 100 mg / kg. in some embodiments, the dose is between about 5 mg / kg to about 80 mg / kg. In some embodiments, the dose is between about 10 mg / kg and about 60 mg / kg. In some embodiments, the dose is between about 15 mg / kg to about 50 mg / kg.

[0013] In some embodiments, the subject is an adult subject. In some embodiments, the subject is at least 18 years old.WSGR Docket No. 51760-717.601

[0014] In some embodiments, Compound A is administered orally, by intravenous delivery, or by intraperitoneal injection. In some embodiments, Compound A is administered by intravenous delivery. In some embodiments, Compound A is administered orally.

[0015] In an aspect described herein is a method for treating advanced fibrosis or cirrhosis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of Compound A, or a pharmaceutically acceptable salt or solvate thereof, wherein Compound A has the following structure:

[0016] In some embodiments, the advanced fibrosis or cirrhosis is caused by a fibrotic liver disease. In some embodiments, the fibrotic liver disease is chronic hepatitis B, chronic hepatitis C, chronic hepatitis D, metabolic dysfunction-associated steatotic liver disease (MASLD) (formerly known as non-alcoholic fatty liver disease (NAFLD)), metabolic dysfunction- associated steatohepatitis (MASH) (formerly knowns as non-alcoholic steatohepatitis (NASH)), alpha- 1 -antitrypsin deficiency, biliary atresia, primary sclerosing cholangitis, secondary sclerosing cholangitis, autoimmune hepatitis, alcohol-related liver disease (ALD), hemochromatosis, schistosomiasis, or Wilson’s disease.

[0017] In some embodiments, the administration of Compound A inhibits stellate cell activation. In some embodiments, the administration of Compound A causes an antiinflammatory effect. In some embodiments, the anti-inflammatory effect comprises inhibition of reactive oxygen species-induced cytokine release.

[0018] In some embodiments, the method comprises treating advanced fibrosis. In some embodiments, the method comprises treating cirrhosis.

[0019] In some embodiments, Compound A is administered in a dose between about 1 mg / kg to about 100 mg / kg. in some embodiments, the dose is between about 5 mg / kg to about 80 mg / kg. In some embodiments, the dose is between about 10 mg / kg and about 60 mg / kg. In some embodiments, the dose is between about 15 mg / kg to about 50 mg / kg.

[0020] In some embodiments, Compound A is administered orally or by intravenous delivery. In some embodiments, Compound A is administered by intravenous delivery. In some embodiments, Compound A is administered orally.WSGR Docket No. 51760-717.601BRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIG. 1 illustrates the schematic of ASK1 cascade in the liver. Stress-inducers increase ROS resulting in the activation of ASK1 and the resulting pathological cascade. Compound A inhibits activated ASK1 resulting in decreased apoptosis, inflammation, and fibrosis in the liver. ASKl=apoptosis signal regulating kinase 1; IL=interleukin; JNK=C-Jun N-terminal kinase; LPS=lipopolysaccharide; MKK=mitogen-activated protein kinase kinase; ROS=reactive oxygen species; TLR=toll-like receptor; TNF=tumor necrosis factor; TRX=thi oredoxin .

[0022] FIGs. 2A-2F illustrates representative graphs demonstrating Compound A mechanisms of action in vitro. Increasing concentrations of Compound A were analyzed in an enzymatic assay (0.51-10,000 nM, 10 pt dose response format), or incubated with human PBMCs primary fibroblasts or HepG2 cells ((0.001 - 30 uM, dose response format) with appropriate stress to induce ASK1. Results show that Compound A dose dependently inhibits the activated ASK1 kinase (FIG. 2A), demonstrates ASK1 downstream target engagement cascade in vitro (FIG. 2B) inhibits LPS-induced cytokine secretion demonstrating an antiinflammatory mechanism (FIG. 2C) inhibits TNF-induced activation of myofibroblasts demonstrating antifibrotic mechanism (FIG. 2D) inhibits H2O2-induced apoptosis (FIG. 2E) and lacks off-target cytotoxicity (FIG. 2F). FIG. 2A shows that Compound A results in a dose dependent inhibition of the human ASK1 enzymatic assay demonstrating potent activity on the target kinase. FIG. 2B shows that Compound A inhibits LPS-induced p-p38 in human primary PBMCs demonstrating ASK1 downstream target engagement in vitro. FIG. 2C shows that Compound A dose dependently inhibits LPS-induced cytokine TNFa secretion in human PBMC demonstrating an anti-inflammatory mechanism. FIG. 2D shows that Compound A dose dependently inhibits TGFP-induced alpha-smooth muscle actin (aSMA) a biomarker of activated myofibroblasts in human primary fibroblasts demonstrating a direct anti-fibrotic mechanism. FIG. 2E shows that Compound A dose dependently inhibits H2O2 induced caspase 3 / 7 biomarker of early apoptosis demonstrating a direct effect on apoptosis. FIG. 2F shows that Compound A has no effect on HepG2 viability with no stress inducer present over 72 hours or three population doublings demonstrating no off-target cytotoxicity.

[0023] FIG. 3 illustrates Compound A reduced APAP-induced Plasma ALT, and Liver p- ASK1, p-JNK, and p-p38. In a short-term liver injury model, APAP treatment induces oxidative stress that significantly activates the ASK1-JNK cascade. Dose-responsive efficacy was demonstrated with Compound A treatment by a significant reduction of plasma ALT (markerWSGR Docket No. 51760-717.601 of liver injury), and reduced p-JNK, p-ASKl, and p-p38 by Western blot analysis. *** p<0.001 vs vehicle control, ### p<0.001 vs APAP -vehicle. ALT=alanine aminotransferase; APAP=acetaminophen; ASKl=apoptosis signal regulating kinase 1; JNK=C-Jun N-terminal kinase; Veh=vehicle.

[0024] FIG. 4 illustrates Compound A treatment reduced liver injury, fibrosis, inflammation, and cell death in DIO-MASH model in mice with biopsy-verified fibrosis and steatosis at treatment initiation. ALT = alanine aminotransferase, Collal = collagen 1A1, CASP1 = caspase 1.

[0025] FIG. 5 illustrates blood chemistries at 14 days in the BDL study. ALT = alanine aminotransferase, AST = aspartate aminotransferase, TBIL = total bilirubin, ALP = alkaline phosphatase, TBA = total bile acid.

[0026] FIG. 6 illustrates representative picrosirius red staining (PSR) images and quantitation demonstrating decreased fibrosis with Compound A treatment.

[0027] FIG. 7 illustrates Compound A treatment significantly decreased BDL-induced fibrosis as assed by hydroxyproline incorporation into the collagen matrix.

[0028] FIG. 8 illustrates Compound A significantly and dose-dependently decreased BDL- induced stellate cell activation.

[0029] FIG. 9 illustrates predicted human liver concentrations using PBPK model and liver concentrations from BDL model.DETAILED DESCRIPTION

[0030] Cholestatic liver diseases are diseases which can affect the bile flow from the liver by slowing or stalling the bile flow. The liver produces bile which is delivered through the digestive system via a network of vessels, the bile ducts. The bile ducts run through the liver as well as outside of it, connecting the liver, gallbladder, pancreas, and small intestine. These organs and bile ducts make up the biliary system. When bile can’t flow properly through the biliary system, the organs are affected, which can irritate the tissues and interfere with normal function. Cholestatic liver diseases can lead to permanent liver failure.

[0031] Cholestatic liver diseases include, but are not limited to, biliary obstruction, biliary atresia, cholangitis, total parenteral nutrition (TPN)-associated cholestasis, primary sclerosing cholangitis, secondary sclerosing cholangitis, intrahepatic cholestasis of pregnancy, congestive hepatopathy, sepsis, progressive familial intrahepatic cholestasis, and drug-induced liver injury.WSGR Docket No. 51760-717.601

[0032] Biliary atresia is a severe neonatal disease caused by an inflammatory and fibrotic obliteration of the extrahepatic biliary tree resulting in cholestasis and progressive hepatic failure. Neonates with biliary atresia develop scleral icterus, clay-colored acholic stools, and jaundice that persists beyond the first 2 weeks of life. Biliary atresia prevalence at birth is 1 / 15,000-19,000 in Europe and North America.

[0033] Biliary atresia can be classified into 3 categories, all of which are dependent on the level most proximal to the biliary obstruction. Type I involves obstruction of the common bile duct, characterized with luminal patency down to the common bile duct, and accounts for approximately 5% of cases. Type II, characterized with patency to the level of the common hepatic duct, accounts for approximately 2% of cases. In both types, there is some preservation of the intrahepatic ducts, although they may morphologically still be abnormal and irregular. Given this abnormality in structure, they typically do not dilate, despite the presence of an obstruction. Type III involves obstruction at the level of the porta hepatis. In most populations, type III accounts for more than 90% of cases and is characterized by most of the proximal part of the extrahepatic biliary tract within the porta hepatis entirely solid.

[0034] Susceptible infants with possible genetic predisposition experience an in-utero insult, leading to stimulation of innate and adaptive immunity, bile duct injury with impaired bile flow, accumulation of bile acids, and epithelial damage with alteration of intercellular junctions. While the exact triggering event remains unknown, evidence supports an in-utero onset of biliary atresia including identification of prenatal gallbladder anomalies, abnormally low gamma-glutamyl transferase (GGT) levels in amniotic fluid, and the presence of elevated direct / conjugated bilirubin levels within 24 to 48 hours of birth. Ongoing research suggests that there is variable contribution among the dominant contributing factors of disease pathogenesis thereby leading to different biliary atresia phenotypes despite a common clinical presentation.

[0035] A viral trigger causing biliary obstruction in-utero has long been considered a possible etiological factor for the development of biliary atresia. Viruses implicated in biliary atresia include cytomegalovirus (CMV), reovirus, rotavirus, Epstein-Barr virus, and human papillomavirus. An in vivo study of mice injected with biliatresone (an isoflavonoid found in Dysphania plant) confirmed biliary obstruction with associated inflammation and fibrosis.

[0036] Biliary atresia occurs in newborns, suggesting that the fetus and young neonate, by virtue of developmental stage, are particularly susceptible to injury and dysregulated repair (fibrosis) after an insult to the biliary tree. Extrahepatic bile ducts have an increased susceptibility to injury and increased potential for a fibrotic response.WSGR Docket No. 51760-717.601

[0037] Various immune mechanisms have been proposed including immune dysregulation, autoimmunity, and susceptibility of the neonate’s immature immune system. Innate immunity plays a key role in the early immune response to pathogens through pathogen recognition receptors (PRR), in particular toll-like receptors (TLRs). TLRs are upregulated in biliary atresia and recognize pathogen-associated molecular patterns (PAMPs) or stimuli released from apoptotic / necrotic cells, i.e., damage associated molecular patterns (DAMPs). Upon TLR activation type 1 interferons are released and initiate a complex cascade of immune signaling by tumor necrosis factor alpha (TNF-a), IL-1, IL-6, IL-8, and IL-15. Cholangiocytes, macrophages, and dendritic cells play a primary role in this early immune response that results in neutrophil recruitment and activation of adaptive immunity.

[0038] Within the adaptive immune arm, oligoclonal expansion of both T cells and B cells has been observed and various autoantibodies have been identified in infants with biliary atresia supporting a possible role for antigen-driven immune stimulation. Most infants with biliary atresia exhibit a dominant Thl immune response early in disease although Thl7 T cell immunity has been demonstrated in various studies and Th2 responses may be involved in cystic biliary atresia. Human and murine studies have also demonstrated a decrease in frequency and function of T regulatory cells (Tregs), further supporting the premise for immune dysregulation in the setting of immature neonatal immunity. Despite evidence of oligoclonality and the presence of various autoantibodies, a critical antigen independent role for B cells has also been shown.

[0039] The hepatic immune response at later stages of biliary atresia commonly exhibits a Th2 immune phenotype characterized by low-level inflammation and oxidative injury, bile duct proliferation, and progressive fibrosis. Important at this stage of disease is the hepatocyte derived alarmin IL-33 that triggers IL-13 driven activation of hepatic stellate cells and progressive hepatic fibrosis. Macrophages mediate both the pro restorative and maladaptive responses in cholestatic liver disease, and distinct transcriptional subsets have been identified in pediatric cholestatic liver disease at the time of transplant.

[0040] Studies suggest that while there is no established causal gene in biliary atresia, various susceptibility genes and specific gene pathways may be involved in pathogenesis and outcome. Of interest in syndromic biliary atresia is identification of variants of PKD1L1, a gene associated with ciliary development and laterality determination.

[0041] Biliary atresia is a disease that, if diagnosed and treated in time, can have a good outcome. Currently, kasai portoenterostomy (KPE) is the primary treatment of biliary atresia to promote biliary drainage. For those who do not have successful drainage after operation,WSGR Docket No. 51760-717.601 liver transplant remains the only option. Without surgical intervention, biliary atresia may progress to hepatic fibrosis, liver cirrhosis, liver failure, or death within 2 years. Biliary atresia is a lifelong illness although palliative surgery can be done to alleviate the severity of the illness and to deter mortality. Early diagnosis is important to achieve successful biliary drainage. Over time, biliary occlusion progresses. If not diagnosed and treated in a timely manner, sclerosis of the extrahepatic biliary tree progresses to cirrhosis, liver failure, and then death. Biliary atresia remains the most common reason for liver transplant in the pediatric population as there are no effective medical therapies to prevent or slow disease progression. Thus, there remains a need for novel new compound therapeutics for the treatment of these diseases.

[0042] Overall, about half of patients with biliary atresia require a liver transplant by 2 years of age, and most are transplanted by early adulthood. Rates of graft and patient survival in biliary atresia after liver transplantation are excellent with a recent report from Society of Pediatric Liver Transplantation (SPLIT) of graft and patient survival at 90% and 97% respectively. At 10 years post-transplant in the SPLIT registry, more than 80% of patients had normal liver and kidney function, 89% had height z-scores greater than the 3rd percentile, and 94% had weight z-scores for age greater than the 3rd percentile. In addition, there is growing recognition of the need to improve outcomes in patients that survive with their native liver beyond the age of 2 years. Patients surviving without transplant continue to experience complications of chronic liver disease including clinical manifestations of cirrhosis, portal hypertension, cholangitis, and pruritus. Prognostic models have recently been developed to risk stratify poor outcomes in patients with their native liver after the age of 2 years. Cumulative incidence of liver transplant or death was 23.7% and predicted by increased total bilirubin, low albumin, decreased platelet count, and a history of either cholangitis or ascites.

[0043] Children with biliary atresia are also at risk for impairments in neurodevelopmental outcomes and decreased quality of life. Multiple studies have shown variable levels of impairment in motor and / or language skills in infants with biliary atresia at diagnosis up to the time of transplant as well as school-age children with or without transplant. Importantly, a 2013 cross-sectional study from the Childhood Liver Disease Research Network found that biliary atresia patients had poorer health-related quality of life compared to healthy children across all domains. Similar findings have been validated by other groups and highlight the need for ongoing multidisciplinary support for this vulnerable patient population.

[0044] Generalized immune modulating therapies previously tested in biliary atresia have included steroids and intravenous immunoglobulin (IVIG). Theoretical benefits of using steroids include improving biliary inflammation to promote choleresis, however, their benefitWSGR Docket No. 51760-717.601 in biliary atresia remains unclear. Previous studies of the effect of oral prednisolone after KPE and found that while there was improvement in jaundice in the early postoperative period, particularly for infants less than 70 days old, there was no significant reduction in the need for liver transplantation. The utility of steroids was most extensively examined in the multicenter, double-blind Steroids in Biliary Atresia Randomized Trial (START), in which infants received a 13 -week course of either high-dose steroids or placebo starting within 72 hours after KPE. Results from START showed no difference in total bilirubin at 6 months post-KPE or 2-year survival with native liver in those that received steroids. Furthermore, the treatment group experienced impaired growth and a shorter time to first serious adverse event. While a small clinical benefit could not be excluded, the results did not establish a role for routine use of high-dose steroids after KPE. A prospective, multicenter, open-label clinical trial (PRIME) found no improvement in bilirubin levels at 90 days post-KPE or 1-year survival with native liver in participants that received IVIG after KPE compared to a placebo-arm group.

[0045] More recent studies include use of immune cell subset specific therapies in biliary atresia. A limited study examining the effect of B-cell depleting agents in biliary atresia showed that one dose of rituximab was safe and well-tolerated, however, long term clinical outcomes were not reported. A possible beneficial role for hematopoietic stem cell recruitment via granulocyte colony stimulating factor (G-CSF) in liver disease has been supported by various studies in adults. Based on this experience, a clinical trial using G-CSF in patients with biliary atresia is ongoing with initial Phase 1 data demonstrating safety and perhaps some improvement in early biliary drainage and frequency of cholangitis.

[0046] Additional therapeutic targets in BA include interrupting ongoing hepatic injury induced through oxidative injury and bile-acid toxicity. N-acetylcysteine (NAC) is an antioxidant that has been shown to improve hepatic injury and fibrosis, reduce biliary obstruction, and increase survival in murine BA. Ursodiol has various proposed mechanisms to reduce cholestatic liver injury and is standard supportive medical therapy after KPE. Newer agents that inhibit the ileal apical sodium-dependent bile acid transporter to interrupt enterohepatic bile acid recirculation include maralixibat and odevixibat, both with ongoing clinical trials in biliary atresia. Lastly, farnesoid x receptor agonists control metabolic homeostasis and inhibit bile acid synthesis and may be considered as future therapeutic options for pediatric cholestatic liver disease.

[0047] Biliary atresia remains the leading cause of neonatal obstructive jaundice and is the most common indication for pediatric liver transplantation. While outcomes after liver transplantation for biliary atresia are excellent, there remains an unmet need to develop medicalWSGR Docket No. 51760-717.601 therapies to prolong transplant-free survival and reduce the incidence of adverse events in patients surviving with their native liver.

[0048] Liver cancer is the third leading cause of cancer-related death and its incidence is increasing globally. The most common type is hepatocellular carcinoma (HCC), which accounts for 80-90% of all primary liver cancers.

[0049] Liver fibrosis is a substantial risk factor for the development and progression of liver cancer. Chronic liver injury of any etiology leads to hepatic fibrosis, which can then progress to cirrhosis, and, in one third of cases, to HCC. Stellate cells are responsible for making the extracellular matrix resulting in hepatic fibrosis. While excessive fibrosis is a common feature in multiple liver diseases it is most clearly defined in MASH, where fibrosis is the only histologic feature that correlates with clinical outcomes including HCC.

[0050] Pathogenesis of HCC has been associated with chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) infections, as well as cirrhosis-inducing conditions of liver. The five- year survival rate of those with HCC is about less than 20%, so there remains an unmet need to find effective treatment for this cancer.Definitions

[0051] Unless otherwise stated, the following terms used in this application, including the specification and claims, have the definitions given below. It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Unless otherwise indicated, conventional methods of mass spectroscopy, NMR, HPLC, protein chemistry, biochemistry, recombinant DNA techniques and pharmacology are employed. In this application, the use of “or” or “and” means “and / or” unless stated otherwise. Furthermore, use of the term “including” as well as other forms, such as “include”, “includes,” and “included,” is not limiting. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0052] The term “acceptable” with respect to a formulation, composition or ingredient, as used herein, means having no persistent detrimental effect on the general health of the subject being treated.

[0053] The term “subject” or “patient” encompasses mammals. Examples of mammals include, but are not limited to, any member of the Mammalian class: humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratoryWSGR Docket No. 51760-717.601 animals including rodents, such as rats, mice and guinea pigs, and the like. In one aspect, the mammal is a human.

[0054] The terms “treat,” “treating” or “treatment,” as used herein, include alleviating, abating or ameliorating at least one symptom of a disease or condition, preventing additional symptoms, inhibiting the disease or condition, e.g., arresting the development of the disease or condition, relieving the disease or condition, causing regression of the disease or condition, relieving a condition caused by the disease or condition, or stopping the symptoms of the disease or condition either prophylactically and / or therapeutically.Methods

[0055] ASK1 inhibitors that are useful for treating liver diseases and disorders described herein include Compound A:

[0056] Compound A is a small molecule inhibitor of apoptosis signal regulating kinase 1 (ASK1, FIG. 1). In enzymatic and binding assays, Compound A has been demonstrated to be a selective, potent ASK1 inhibitor. In cellular studies, Compound A treatment demonstrates anti-apoptotic, anti-inflammatory, and anti-fibrotic mechanism of action (MOA) in human primary cells and human cell lines with no off-target proliferation / cytotoxicity effects. In vivo studies with Compound A has been demonstrated efficacy in a short-term therapeutic (6 hours) acetaminophen liver toxicity model, a 3 -day model of alcoholic hepatitis and a chronic (1 year) therapeutic diet induced obesity mouse model of metabolic dysfunction-associated steatohepatitis (MASH). Human safety and PK profile was established in a phase 1 clinical trial.

[0057] In a rat bile duct ligation (BDL) study, a model of biliary atresia, Compound A treatment significantly, and dose-dependently, decreased BDL-induced fibrosis after 14 days of treatment in this severe model. Fibrosis was measured by both imaging analysis and biochemical hydroxyproline content. A significant and dose-dependent decrease of stellate cell activation (fibrosis generating cells) was also observed.

[0058] These antifibrotic data suggests that Compound A is a promising therapeutic for human cholestatic liver diseases with an established safe clinical dosing regimen. The data provided herein additional suggests that Compound A is a promising therapeutic for theWSGR Docket No. 51760-717.601 treatment of advanced fibrosis and cirrhosis, particularly within the context of fibrotic liver disease.

[0059] In some aspects described herein is a method for treating cholestatic liver disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of Compound A, or a pharmaceutically acceptable salt or solvate thereof, wherein Compound A has the following structure:

[0060] In some embodiments, the cholestatic liver disease is acute cholestasis liver disease or chronic cholestasis liver disease. Causes of acute and chronic cholestasis include but are not limited to cholangitis, drug-induced liver injury, sepsis, alcoholic hepatitis, biliary obstruction, biliary atresia, cholangitis, total parenteral nutrition (TPN)-associated cholestasis, primary sclerosing cholangitis, secondary sclerosing cholangitis, primary biliary cholangitis, intrahepatic cholestasis of pregnancy, congestive hepatopathy, progressive familial intrahepatic cholestasis, benign recurrent intrahepatic cholestasis, and alagille syndrome.

[0061] In some embodiments, the cholestatic liver disease is primary biliary cholangitis, biliary obstruction, biliary atresia, cholangitis, total parenteral nutrition (TPN)-associated cholestasis, primary sclerosing cholangitis, secondary sclerosing cholangitis, or intrahepatic cholestasis of pregnancy. In some embodiments, the cholestatic liver disease is biliary obstruction, biliary atresia, cholangitis, total parenteral nutrition (TPN)-associated cholestasis, primary sclerosing cholangitis, secondary sclerosing cholangitis, or intrahepatic cholestasis of pregnancy. In some embodiments, the cholestatic liver disease is biliary obstruction, biliary atresia, cholangitis, or total parenteral nutrition (TPN)-associated cholestasis. In some embodiments, the cholestatic liver disease is biliary obstruction. In some embodiments, the cholestatic liver disease is biliary atresia.

[0062] The doses can be modified as suitable for the subject being treated. In some embodiments, Compound A is administered in a dose between about 1 mg / kg to about 100 mg / kg. In some embodiments, the dose is between about 5 mg / kg to about 90 mg / kg. In some embodiments, the dose is between about 5 mg / kg to about 80 mg / kg. In some embodiments, the dose is between about 5 mg / kg to about 70 mg / kg. In some embodiments, the dose is between about 10 mg / kg to about 70 mg / kg. In some embodiments, the dose is between about 15 mg / kg to about 70 mg / kg. In some embodiments, the dose is between about 5 mg / kg toWSGR Docket No. 51760-717.601 about 60 mg / kg. In some embodiments, the dose is between about 10 mg / kg to about 60 mg / kg. In some embodiments, the dose is between about 15 mg / kg to about 60 mg / kg. In some embodiments, the dose is between about 5 mg / kg to about 50 mg / kg. In some embodiments, the dose is between about 10 mg / kg to about 50 mg / kg. In some embodiments, the dose is between about 15 mg / kg to about 50 mg / kg.

[0063] In some embodiments, the subject is a pediatric subject. A pediatric subject ranges from a newborn until about 18 years of age. In some embodiments, the subject is less than 5 years old. In some embodiments, the subject is less than 4 years old. In some embodiments, the subject is less than 3 years old. In some embodiments, the subject is less than 2 years old. In some embodiments, the subject is less than 1 year old. In some embodiments, the subject is less than 9 months old. In some embodiments, the subject is less than 6 months old.

[0064] In some embodiments, Compound A is administered orally, by intravenous delivery, or by intraperitoneal injection. In some embodiments, Compound A is administered orally or by intravenous delivery. In some embodiments, Compound A is administered orally. In some embodiments, Compound A is administered by intravenous delivery. In some embodiments, Compound A is administered by intraperitoneal injection.

[0065] In some embodiments, therapeutic intervention with Compound A in treating the cholestatic liver disease is effective to prevent or reduce the risk of developing primary liver cancer.

[0066] In some aspects is a method for treating primary liver cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of Compound A, or a pharmaceutically acceptable salt or solvate thereof, wherein Compound A has the following structure:(Compound A).

[0067] In some embodiments, the primary liver cancer is hepatocellular carcinoma(HCC) or bile duct cancer. In some embodiments, the primary liver cancer is hepatocellular carcinoma. In some embodiments, the primary liver cancer is bile duct cancer.

[0068] HCC has been associated with chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) infections, as well as cirrhosis-inducing conditions of liver. In some embodiments, the subject previously had chronic hepatitis B or chronic hepatitis C. In some embodiments, the subject has chronic hepatitis B. In some embodiments, the subject has chronic hepatitis C.WSGR Docket No. 51760-717.601

[0069] The doses of Compound A administered for the treatment of primary liver cancer, including but not limited to hepatocellular carcinoma or bile duct cancer can be modified as suitable for the subject being treated. In some embodiments, Compound A is administered in a dose between about 1 mg / kg to about 100 mg / kg. In some embodiments, the dose is between about 5 mg / kg to about 90 mg / kg. In some embodiments, the dose is between about 5 mg / kg to about 80 mg / kg. In some embodiments, the dose is between about 5 mg / kg to about 70 mg / kg. In some embodiments, the dose is between about 10 mg / kg to about 70 mg / kg. In some embodiments, the dose is between about 15 mg / kg to about 70 mg / kg. In some embodiments, the dose is between about 5 mg / kg to about 60 mg / kg. In some embodiments, the dose is between about 10 mg / kg to about 60 mg / kg. In some embodiments, the dose is between about 15 mg / kg to about 60 mg / kg. In some embodiments, the dose is between about 5 mg / kg to about 50 mg / kg. In some embodiments, the dose is between about 10 mg / kg to about 50 mg / kg. In some embodiments, the dose is between about 15 mg / kg to about 50 mg / kg.

[0070] In some embodiments, the subject is an adult subject. An adult subject is someone who is at least 18 years old. In some embodiments, the subject is at least 18 years old. In some embodiments, the subject is at least 21 years old. In some embodiments, the subject is at least 25 years old.

[0071] The compound can be administered by any suitable means for a therapeutic effect. In some embodiments, Compound A is administered orally, by intravenous delivery, or by intraperitoneal injection. In some embodiments Compound A is administered orally or by intravenous delivery. In some embodiments, compound A is administered orally. In some embodiments Compound A is administered by intravenous delivery. In some embodiments, Compound A is administered by intraperitoneal injection.

[0072] Also provided herein in an aspect a method for treating advanced fibrosis or cirrhosis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of Compound A, or a pharmaceutically acceptable salt or solvate thereof, wherein Compound A has the following structure:

[0073] In some embodiments, the advanced fibrosis or cirrhosis is caused by a fibrotic liver disease. In some embodiments, the fibrotic liver disease is chronic hepatitis B, chronic hepatitis C, chronic hepatitis D, metabolic dysfunction-associated steatotic liver disease (MASLD)WSGR Docket No. 51760-717.601(formerly known as non-alcoholic fatty liver disease (NAFLD)), metabolic dysfunction- associated steatohepatitis (MASH) (formerly knowns as non-alcoholic steatohepatitis (NASH)), alpha- 1 -antitrypsin deficiency, biliary atresia, primary sclerosing cholangitis, secondary sclerosing cholangitis, autoimmune hepatitis, alcohol-related liver disease (ALD) hemochromatosis, schistosomiasis, or Wilson’s disease. In some embodiments, the fibrotic liver disease is In some embodiments, the fibrotic liver disease is chronic hepatitis B. In some embodiments, the fibrotic liver disease is chronic hepatitis C. In some embodiments, the fibrotic liver disease is chronic hepatitis D. In some embodiments, the fibrotic liver disease is metabolic dysfunction-associated steatotic liver disease (MASLD) (formerly known as nonalcoholic fatty liver disease (NAFLD)). In some embodiments, the fibrotic liver disease is metabolic dysfunction-associated steatohepatitis (MASH) (formerly knowns as non-alcoholic steatohepatitis (NASH)). In some embodiments, the fibrotic liver disease is alpha- 1 -antitrypsin deficiency. In some embodiments, the fibrotic liver disease is biliary atresia. In some embodiments, the fibrotic liver disease is primary sclerosing cholangitis. In some embodiments, the fibrotic liver disease is secondary sclerosing cholangitis. In some embodiments, the fibrotic liver disease is autoimmune hepatitis. In some embodiments, the fibrotic liver disease is alcohol-related liver disease (ALD). In some embodiments, the fibrotic liver disease is hemochromatosis. In some embodiments, the fibrotic liver disease is schistosomiasis. In some embodiments, the fibrotic liver disease is Wilson’s disease.

[0074] In some embodiments, the administration of Compound A inhibits stellate cell activation. In some embodiments, the administration of Compound A causes an antiinflammatory effect. In some embodiments, the anti-inflammatory effect comprises inhibition of reactive oxygen species-induced cytokine release. In some embodiments, the cytokines for which release is inhibited include one or both of p38 and TNFa.

[0075] In some embodiments, the method comprises treating advanced fibrosis. In some embodiments, the advanced fibrosis is a liver fibrosis level of F3 or higher based on the METAVIR scoring system. In some embodiments, the method comprises treating cirrhosis. In some embodiments, administration of Compound A is effective to reduce fibrosis in the subject. In some embodiments, administration of Compound A is effective to slow the rate of growth of fibrosis in the subject. The ability of Compound A to treat advanced fibrosis and / or cirrhosis (e.g., to reduce or slow the growth of fibrosis in suitable subjects) can be assessed by a variety of methods known in the art. For example, the thioacetamide-induced pan-lobularWSGR Docket No. 51760-717.601 liver fibrosis mouse model or the BALBc.Mdr2(- / -) can be used to assess the effect of Compound A on liver fibrosis levels in model organisms.

[0076] The doses of Compound A administered for the treatment of advanced fibrosis and / or cirrhosis can be modified as suitable for the subject being treated. In some embodiments, Compound A is administered in a dose between about 1 mg / kg to about 100 mg / kg. In some embodiments, Compound A is administered in a dose between about 1 mg / kg to about 100 mg / kg. In some embodiments, the dose is between about 5 mg / kg to about 90 mg / kg. In some embodiments, the dose is between about 5 mg / kg to about 80 mg / kg. In some embodiments, the dose is between about 5 mg / kg to about 70 mg / kg. In some embodiments, the dose is between about 10 mg / kg to about 70 mg / kg. In some embodiments, the dose is between about 15 mg / kg to about 70 mg / kg. In some embodiments, the dose is between about 5 mg / kg to about 60 mg / kg. In some embodiments, the dose is between about 10 mg / kg to about 60 mg / kg. In some embodiments, the dose is between about 15 mg / kg to about 60 mg / kg. In some embodiments, the dose is between about 5 mg / kg to about 50 mg / kg. In some embodiments, the dose is between about 10 mg / kg to about 50 mg / kg. In some embodiments, the dose is between about 15 mg / kg to about 50 mg / kg.

[0077] The compound can be administered by any suitable means for a therapeutic effect. In some embodiments, Compound A is administered orally, by intravenous delivery, or by intraperitoneal injection. In some embodiments Compound A is administered orally or by intravenous delivery. In some embodiments, compound A is administered orally. In some embodiments Compound A is administered by intravenous delivery. In some embodiments, Compound A is administered by intraperitoneal injection.Further Forms of Compounds

[0078] In one aspect, compounds described herein possess one or more stereocenters and each stereocenter exists independently in either the R or S configuration. The compounds presented herein include all diastereomeric, enantiomeric, and epimeric forms as well as the appropriate mixtures thereof. The compounds and methods provided herein include all cis-, trans-, syn-, anti-, entgegen (E), and zusammen (Z) isomers as well as the appropriate mixtures thereof. In certain embodiments, compounds described herein are prepared as their individual stereoisomers by reacting a racemic mixture of the compound with an optically active resolving agent to form a pair of diastereoisomeric compounds / salts, separating the diastereomers and recovering the optically pure enantiomers. In some embodiments, resolution of enantiomers is carried out using covalent diastereomeric derivatives of the compounds described herein. In another embodiment, diastereomers are separated by separation / resolution techniques basedWSGR Docket No. 51760-717.601 upon differences in solubility. In other embodiments, separation of stereoisomers is performed by chromatography or by the forming diastereomeric salts and separation by recrystallization, or chromatography, or any combination thereof. Jean Jacques, Andre Collet, Samuel H. Wilen, “Enantiomers, Racemates and Resolutions”, John Wiley And Sons, Inc., 1981. In some embodiments, stereoisomers are obtained by stereoselective synthesis.

[0079] “Pharmaceutically acceptable,” as used herein, refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively nontoxic, i.e., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

[0080] The term “pharmaceutically acceptable salt” refers to a formulation of a compound that does not cause significant irritation to an organism to which it is administered and does not abrogate the biological activity and properties of the compound. In some embodiments, pharmaceutically acceptable salts are obtained by reacting a compound described herein with acids. Pharmaceutically acceptable salts are also obtained by reacting a compound described herein with a base to form a salt.

[0081] Compounds described herein may be formed as, and / or used as, pharmaceutically acceptable salts. The type of pharmaceutical acceptable salts, include, but are not limited to: (1) acid addition salts, formed by reacting the free base form of the compound with a pharmaceutically acceptable: inorganic acid to form a salt such as, for example, a hydrochloric acid salt, a hydrobromic acid salt, a sulfuric acid salt, a phosphoric acid salt, a metaphosphoric acid salt, and the like; or with an organic acid to form a salt such as, for example, an acetic acid salt, a propionic acid salt, a hexanoic acid salt, a cyclopentanepropionic acid salt, a glycolic acid salt, a pyruvic acid salt, a lactic acid salt, a malonic acid salt, a succinic acid salt, a malic acid salt, a maleic acid salt, a fumaric acid salt, a trifluoroacetic acid salt, a tartaric acid salt, a citric acid salt, a benzoic acid salt, a 3-(4-hydroxybenzoyl)benzoic acid salt, a cinnamic acid salt, a mandelic acid salt, a methanesulfonic acid salt, an ethanesulfonic acid salt, a 1,2- ethanedi sulfonic acid salt, a 2-hydroxyethanesulfonic acid salt, a benzenesulfonic acid salt, a toluenesulfonic acid salt, a 2-naphthalenesulfonic acid salt, a 4-methylbicyclo-[2.2.2]oct-2- ene-1 -carboxylic acid salt, a glucoheptonic acid salt, a 4,4’-methylenebis-(3-hydroxy-2-ene-l- carboxylic acid) salt, a 3 -phenylpropionic acid salt, a trimethylacetic acid salt, a tertiary butylacetic acid salt, a lauryl sulfuric acid salt, a gluconic acid salt, a glutamic acid salt, a hydroxynaphthoic acid salt, a salicylic acid salt, a stearic acid salt, a muconic acid salt, a butyric acid salt, a phenylacetic acid salt, a phenylbutyric acid salt, a valproic acid salt, and the like;WSGR Docket No. 51760-717.601(2) salts formed when an acidic proton present in the parent compound is replaced by a metal ion, e.g., an alkali metal ion (e.g. a lithium salt, a sodium salt, or a potassium salt), an alkaline earth ion (e.g. a magnesium salt, or a calcium salt), or an aluminum ion (e.g. an aluminum salt). In some cases, compounds described herein may coordinate with an organic base to form a salt, such as, but not limited to, an ethanolamine salt, a diethanolamine salt, a triethanolamine salt, a tromethamine salt, a N-methylglucamine salt, a dicyclohexylamine salt, or a tris(hydroxymethyl)methylamine salt. In other cases, compounds described herein may form salts with amino acids such as, but not limited to, an arginine salt, a lysine salt, and the like. Acceptable inorganic bases used to form salts with compounds that include an acidic proton, include, but are not limited to, aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, and the like.

[0082] It should be understood that a reference to a pharmaceutically acceptable salt includes the solvent addition forms. Solvates contain either stoichiometric or non- stoichiometric amounts of a solvent, and may be formed during the process of crystallization with pharmaceutically acceptable solvents such as water, ethanol, and the like. Hydrates are formed when the solvent is water, or alcoholates are formed when the solvent is alcohol. Solvates of compounds described herein can be conveniently prepared or formed during the processes described herein. In addition, the compounds provided herein can exist in unsolvated as well as solvated forms.Routes of Administration

[0083] Suitable routes of administration include, but are not limited to, oral, intravenous, rectal, aerosol, parenteral, ophthalmic, pulmonary, transmucosal, transdermal, vaginal, otic, nasal, and topical administration. In addition, by way of example only, parenteral delivery includes intramuscular, subcutaneous, intravenous, intramedullary injections, as well as intrathecal, direct intraventricular, intraperitoneal, intralymphatic, and intranasal injections.

[0084] In certain embodiments, a compound as described herein is administered in a local rather than systemic manner, for example, via injection of the compound directly into an organ, often in a depot preparation or sustained release formulation. In specific embodiments, long acting formulations are administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Furthermore, in other embodiments, the drug is delivered in a targeted drug delivery system, for example, in a liposome coated with organ specific antibody. In such embodiments, the liposomes are targeted to and taken up selectively by the organ. In yet other embodiments, the compound as described herein is provided in the form of a rapid release formulation, in the form of an extended release formulation, or in theWSGR Docket No. 51760-717.601 form of an intermediate release formulation. In yet other embodiments, the compound described herein is administered topically.

[0085] In some embodiments described herein, a therapeutically effective amount of a compound of Compound A, or a pharmaceutically acceptable salt or solvate thereof, is administered by intravenous delivery to a subject in need thereof. In some embodiments described herein, a therapeutically effective amount of a compound of Compound A, or a pharmaceutically acceptable salt or solvate thereof, is administered by intravenous delivery to a subject in need thereof, wherein the intravenous delivery is selected from bolus injection, intravenous drip, and infusion pump.Pharmaceutical Compositions / F ormulations

[0086] In some embodiments, the compounds described herein are formulated into pharmaceutical compositions. Pharmaceutical compositions are formulated in a conventional manner using one or more pharmaceutically acceptable inactive ingredients that facilitate processing of the active compounds into preparations that can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. A summary of pharmaceutical compositions described herein can be found, for example, in Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999), herein incorporated by reference for such disclosure.

[0087] Provided herein are pharmaceutical compositions that include a compound of Compound A, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable inactive ingredient. In some embodiments is a pharmaceutical composition that includes a compound of Compound A, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient.

[0088] In other embodiments, the pharmaceutical compositions include other medicinal or pharmaceutical agents, carriers, adjuvants, preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure, and / or buffers. In yet other embodiments, the pharmaceutical compositions include other therapeutically valuable substances.

[0089] A pharmaceutical composition, as used herein, refers to a mixture of a compound of Compound A or a pharmaceutically acceptable salt thereof, with other chemical componentsWSGR Docket No. 51760-717.601(i.e. pharmaceutically acceptable inactive ingredients), such as carriers, excipients, binders, filling agents, suspending agents, flavoring agents, sweetening agents, disintegrating agents, dispersing agents, surfactants, lubricants, colorants, diluents, solubilizers, moistening agents, plasticizers, stabilizers, penetration enhancers, wetting agents, anti-foaming agents, antioxidants, preservatives, or one or more combination thereof. The pharmaceutical composition facilitates administration of the compound to a mammal.

[0090] A therapeutically effective amount can vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the compound used and other factors. The compounds can be used singly or in combination with one or more therapeutic agents as components of mixtures.

[0091] The pharmaceutical formulations described herein are administered to a subject by appropriate administration routes, including but not limited to, oral, parenteral (e.g., intravenous, intravitreal, subcutaneous, intramuscular), intranasal, buccal, topical, rectal, or transdermal administration routes. The pharmaceutical formulations described herein include, but are not limited to, aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release formulations, fast melt formulations, tablets, capsules, pills, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations, and mixed immediate and controlled release formulations.

[0092] Pharmaceutical compositions including a compound of Compound A, or a pharmaceutically acceptable salt thereof, are manufactured in a conventional manner, such as, by way of example only, by means of conventional mixing, dissolving, granulating, drageemaking, levigating, emulsifying, encapsulating, entrapping or compression processes.

[0093] The pharmaceutical compositions will include at least one compound of Compound A, as an active ingredient in free-acid or free-base form, or in a pharmaceutically acceptable salt form. In addition, the methods and pharmaceutical compositions described herein include the use of N-oxides (if appropriate), crystalline forms, amorphous phases, as well as active metabolites of these compounds having the same type of activity. In some embodiments, compounds described herein exist in unsolvated form or in solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like. The solvated forms of the compounds presented herein are also considered to be disclosed herein.

[0094] In some embodiments, solid oral dosage forms are prepared by mixing a compound of Compound A, or a pharmaceutically acceptable salt thereof, with one or more of the following: antioxidants, flavoring agents, and carrier materials such as binders, suspendingWSGR Docket No. 51760-717.601 agents, disintegration agents, filling agents, surfactants, solubilizers, stabilizers, lubricants, wetting agents, and diluents.

[0095] In some embodiments, the solid dosage forms disclosed herein are in the form of a tablet, (including a suspension tablet, a fast-melt tablet, a bite-disintegration tablet, a rapiddisintegration tablet, an effervescent tablet, or a caplet), a pill, a powder, a capsule, solid dispersion, solid solution, bioerodible dosage form, controlled release formulations, pulsatile release dosage forms, multiparticulate dosage forms, beads, pellets, granules. In other embodiments, the pharmaceutical formulation is in the form of a powder. In still other embodiments, the pharmaceutical formulation is in the form of a tablet. In other embodiments, pharmaceutical formulation is in the form of a capsule.

[0096] In some embodiments, solid dosage forms, e.g., tablets, effervescent tablets, and capsules, are prepared by mixing particles of a compound of Formula I, Formula II, Formula Ila, or Formula III, or a pharmaceutically acceptable salt thereof, with one or more pharmaceutical excipients to form a bulk blend composition. The bulk blend is readily subdivided into equally effective unit dosage forms, such as tablets, pills, and capsules. In some embodiments, the individual unit dosages include film coatings. These formulations are manufactured by conventional formulation techniques.

[0097] Conventional formulation techniques include, e.g., one or a combination of methods: (1) dry mixing, (2) direct compression, (3) milling, (4) dry or non-aqueous granulation, (5) wet granulation, or (6) fusion. Other methods include, e.g., spray drying, pan coating, melt granulation, granulation, fluidized bed spray drying or coating (e.g., wurster coating), tangential coating, top spraying, tableting, extruding and the like.

[0098] In some embodiments, a compound of Compound A, or a pharmaceutically acceptable salt thereof, is formulated into a pharmaceutical composition suitable for intravitreal, intramuscular, subcutaneous, or intravenous injection. In one aspect, formulations suitable for intravitreal, intramuscular, subcutaneous, or intravenous injection include physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. In some embodiments, formulations suitable for subcutaneous injection contain additives such as preserving, wetting, emulsifying, and dispensing agents.

[0099] In some embodiments, a compound of Compound A, or a pharmaceutically acceptable salt thereof, is formulated into a pharmaceutical composition suitable for intravenous injection. Examples of intravenous injections include but are not limited to bolus injection, intravenous drip, and infusion pump.WSGR Docket No. 51760-717.601

[0100] For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

[0101] In certain embodiments, delivery systems for pharmaceutical compounds may be employed, such as, for example, nanoparticles, implants, mucoadhesives, liposomes and emulsions. In certain embodiments, compositions provided herein can also include an mucoadhesive polymer, selected from among, for example, carboxymethylcellulose, carbomer (acrylic acid polymer), poly(methylmethacrylate), polyacrylamide, polycarbophil, acrylic acid / butyl acrylate copolymer, sodium alginate and dextran.

[0102] In some embodiments, the compounds described herein may be administered topically and can be formulated into a variety of topically administrable compositions, such as solutions, suspensions, sprays, lotions, gels, pastes, medicated sticks, foams, balms, creams or ointments. Such pharmaceutical compounds can contain solubilizers, stabilizers, tonicity enhancing agents, buffers and preservatives.EXAMPLES

[0103] The following illustrative examples are representative of embodiments of the stimulation, systems, and methods described herein and are not meant to be limiting in any way.Example 1. Compound A ASK1 Kinase Inhibition, Kinase Selectivity, and Off-Target Receptor Panel

[0104] Inhibition: Compound A inhibition (IC50) of ASK1 catalytic domain enzymatic activity was determined to be 22.5 nM (n=22; FIG. 2A) using a 10-point curve and reference standard. The inhibitor binding constants (Kd) evaluated with 11 -point dose response curve in duplicate was 5.8 nM.

[0105] Selectivity: Due to evolutionary conservation of the ATP -binding site, most kinase inhibitors that target the ATP -binding site can inhibit multiple kinases. To determine kinase selectivity, Compound A was evaluated at 1 pM in a full kinome scan (489 kinases) resulting in the inhibition at 90% or greater of 17 non-mutated kinases. The follow-up full Kd or IC50 determinations showed that ASK1, DRAK2, ASK3, and LRRK2 are the key kinases inhibited by Compound A with an ASKl / kinase ratio of <10. Compound A binding of ASK1 was most potent at 5.8 nM. Additionally, in safety pharmacology studies of cardiovascular, respiratory, central nervous system endpoints, and GLP toxicology studies, no off-target effects of these kinases were observed with Compound A treatment in rats or non-human primates (NHP) after 4 weeks of treatment. The kinase screening results also demonstrate that Compound A doesWSGR Docket No. 51760-717.601 not directly bind the downstream kinases p38 and JNK thus indicating the decreases in these kinases observed in cells or in vivo are due to the inhibition of the upstream ASK1 kinase.

[0106] Off-Target Receptor Panel: No off-target binding (>50% inhibition at 10 pM) was observed with Compound A using a primary molecular target panel consisting of 13 enzymatic and 74 binding assays is designed to identity potential off-target liabilities.Example 2. Compound A Mechanism of Action in Cellular Studies

[0107] ASK1 activation is known to result in inflammation, apoptosis, and fibrosis in vitro and in vivo in multiple systems. To demonstrate the direct inhibition of these cellular mechanisms with Compound A, human primary cells and human cell lines challenged with ROS inducing factors were used to activate ASK1.

[0108] Human primary PBMCs were obtained from ALLCELLS and cell lines (HepG2 and human dermal fibroblasts [HDF]) were obtained from ATCC. As these cells are not engineered to have activated ASK1, cells were challenged using protocols to induce ROS and activate ASK1 (LPS, TGFbeta, H2O2). Compound A was evaluated in a dose-responsive manner to determine the IC50. Representative dose responses are shown in FIGs. 2B-D.

[0109] Compound A anti-inflammatory mechanism of action (MOA): In the innate immune system, ASK1 plays an important role in the intracellular signaling downstream of the Toll-like receptor 4 (TLR4). ASK1 is specifically activated upon stimulation with bacterial wall-derived lipopolysaccharide (LPS), a bona fide ligand of TLR4. The anti-inflammatory MOA of Compound A was demonstrated by inhibiting ROS-induced cytokine release. In human peripheral blood mononuclear cells (PBMCs) up to 9 different donors were first challenged with 100 ng / mL LPS to induce p38 and TNFa secretion via the TLR4-ASK1 pathway. Incubation with Compound A (0.001 - 30 pM) inhibited p-p38 at 30 minutes, and TNFa levels at 6 hours were 2.8±0.4 and 9.1±1.4, in Compound A exposed and unexposed cells, respectively (IC50 pM; Mean±SEM).

[0110] Compound A antiapoptotic mechanism of action: HepG2 cells, a human liver cell line, were challenged with H2O2 to result in ROS-induced apoptosis. The dose-dependent antiapoptotic MOA of Compound A was evaluated by assessing Compound A effects (0.94-30 pM) on H2O2-induced caspase3 / 7 activity. Apoptosis was induced in HepG2 cells by 1 mM H2O2 treatment for 20 hours in the presence Compound A. Cell viability was determined in parallel and caspase activity normalized to cell viability at each concentration. Decreased caspase3 / 7 was demonstrated with Compound A treatment in 2 independent experiments with no significant change in viability on H2O2 stressed cells. Compound A maximum inhibition of H2C>2-induced apoptosis was 34% at 7.5 pM (experimental n=2; FIG. 2E).WSGR Docket No. 51760-717.601

[0111] Compound A antifibrotic mechanism of action: Following liver injury, hepatic stellate cells undergo an activation response that induces a transition of quiescent cells into proliferative, fibrogenic, and contractile myofibroblasts. Liver fibrosis is characterized by the accumulation of excessive extracellular matrix, which is secreted by these myofibroblasts. The myofibroblast marker of cellular fibrosis, alpha-SMA (a-SMA), was induced in HDF cells by TGFbeta with and without Compound A co-treatment for 48 hours. Alpha-SMA was quantified by in-Cell ELISA using mouse anti -alpha smooth muscle Actin antibody (1 A4; ABCAM). The IC50 of Compound A inhibition of a SMA cellular fibrosis was 13.7±2.7 pM (Mean±SEM, n=4).

[0112] Compound A lacks off-target cellular proliferation / cytotoxicity: ASK1 is activated by many pathological and physiological stimuli and stresses, including inflammatory innate immunity signals, oxidative stress, ER-stress, and lipotoxicity. In cells, when no ROS inducing stimulus is added, ASK1 is not activated and consequently there should be no changes in proliferation or cytotoxicity with Compound A treatment unless there is off-target effect like that observed with certain ASK1 inhibitors. To evaluate off-target cytotoxicity, cytostasis or proliferation effects, viability of HepG2 and HDF cells was determined after 72 hours (3 population doublings) exposure to 0.3-30 pM Compound A. No changes in viability were observed (IC50 >30 pM, n=3) with either cell line indicating no changes in cytotoxicity, cytostasis or proliferation with Compound A treatment. Representative dose response in HepG2 cells is shown in FIG. 2F.Example 3. Compound A in Therapeutic Short-Term Acetaminophen (APAP) Liver Injury Model

[0113] High doses of APAP induce liver injury due to metabolism by the cytochrome P450 enzyme system producing excessive N-acetyl-p-benzoquinone imine (NAPQI), leading to peroxidatic reaction of hepatic cellular mitochondria inducing oxidative stress that activates the mitochondrial oxidative-stress ASK1-JNK pathway. Mitochondrial JNK translocation amplifies the oxidant stress and leads to collapse of mitochondrial membrane potential and hepatic cell death.

[0114] In the APAP therapeutic mouse model, APAP (300 mg / kg; intraperitoneal [IP]) was administered and 1 hour later Compound A (0.3-10 mg / kg), reference compound GS-4997 (selonsertib; 0.3 and 1 mg / kg) or vehicle (5% NMP + 10% Solutol + 55% PEG400 + 30% water) was administered PO (n=6 / group). Six hours after APAP administration, terminal plasma was measured for alanine aminotransferase (ALT). In a separate experiment, livers from treated mice were isolated at 6 hours post treatment of APAP and equal amount of pooledWSGR Docket No. 51760-717.601 liver protein (50 pg) from each group was subjected to SDS PAGE and western blotting for JNK, p-JNK, ASK1, p-ASKl, p38, and p-p38; same blots were stripped and re-probed with anti-P- Actin Ab. Immunocomplexes were detected using an enhanced chemiluminescence system.

[0115] As shown in FIG. 3, a single IP injection of APAP (300 mg / kg) markedly increased plasma ALT and liver p-JNK, with modest increases in p-ASKl and p-p38 as compared to the vehicle treated mice. Phosphorylation of ASK1, JNK, and p38 was reduced dose dependently after treatment of mice with Compound A.Example 4. Compound A in chronic DIO-MASH model

[0116] Compound A treatment was also effective in a high fat diet-induced obesity (DIO) MASH mouse model (FIG. 4). After 38 weeks on MASH diet (40% fat, 20% fructose, 2% cholesterol) a liver biopsy was performed on all animals. Only animals with biopsy confirmed liver fibrosis and steatosis advanced to study treatment. Twelve-week Compound A treatment (0.5% in chow) significantly decreased plasma ALT (liver injury marker), fibrosis, inflammation, and apoptosis compared to MASH controls (FIG. 4). RNAseq analysis confirmed these mechanisms and also demonstrated that Compound A had significant effects on the inflammasome pathway.Example 5. Compound A in BDL Model of Biliary Atresia

[0117] Male Sprague Dawletay rats (7 - 8 weeks) were randomized into four treatment groups as shown in Table 1.Table 1: BDL Protocol and EndpointsSham orBDL surgery TerminationVehicle or Compound A dosing (po, QD)Day 1 Day 14Endpoints:BW, LW, liver enzymesFibrosis: Hydroxyproline, PSRStellate ceil activation: alpha- SMA IHC

[0118] BDL surgery included ligation of the common bile duct below the junction of the hepatic ducts and above the pancreatic duct entrance. Sham animals underwent laparotomyWSGR Docket No. 51760-717.601 without ligation. Compound A or vehicle was dosed po (80:20 Capryol90: Labrafil M 1944 CS, % v / v) starting at Dayl, 1 h before surgery, then daily for 14 days.

[0119] On terminal Day 14, 2 h after the last dose, whole blood was collected for blood chemistry evaluation. Livers were evaluated for Compound A concentrations, fibrosis by biochemical hydroxyproline (HP) content, picrosirius red staining (PSR), and stellate cell activation (alpha-smooth muscle actin staining) by histomorphometry (Aperio ImageScope analysis software). Results are presented as mean ± S.E.M. Statistical analysis was performed by One-way ANOVA followed by Dunnett’s multiple comparison test using GraphPad Prism.compared to BDL + Vehicle group.

[0120] The BDL-elevated blood chemistries were unchanged with Compound A treatment (FIG. 5). The body weight (BW) of all BDL groups was decreased, and liver weight increased, compared to sham group and unchanged with Compound A treatment (data not shown).

[0121] Compound A treatment significantly and dose-dependently decreased BDL- induced fibrosis as demonstrated by PSR staining and HP analysis in FIGs. 6 and 7.

[0122] Using an orthogonal biochemical assessement of fibrosis, treatment with Compound A also decreased BDL-induced hydroxyproline 33% in the 50 mg / kg dose.

[0123] Compound A treatment also significantly and dose-dependently decreased BDL- induced stellate cell activation. Stellate cells are responsible for the formation of the collagen matrix. In FIG. 8 representative immunohistochemical (IHC) alpha-SMA images (marker of activated stellate cells) and alpha-SMA quantitation demonstrating decreased stellate cell activation with Compound A treatment.

[0124] The Compound A liver concentrations at terminal day 14 were 1,232 + / - 168 and 2,757 + / - 680 ng / g (Mean + / - SEM) at 15 mg / kg and 50 mg / kg, respectively. Using an established physiologically based pharmacokinetic (PBPK) model based on preclinical plasma and liver exposures and clinical data, the Compound A liver exposure levels from this BDL study were modeled to predict human liver exposures (FIG. 9). The Compound A liver concentrations from this study are demonstrated clinically achievable in this simulated model.

[0125] These antifibrotic BDL data suggests Compound A is a promising therapeutic for human biliary atresia with an established safe clinical dosing regimen.Example 6. Compound A is Dual ASK1 / DRAK2 Inhibitor of HCC and Fibrosis

[0126] Liver cancer is the third leading cause of cancer-related death and its incidence is increasing globally. The most common type is hepatocellular carcinoma (HCC), which accounts for 80-90% all primary liver cancers.WSGR Docket No. 51760-717.601

[0127] Liver fibrosis is a substantial risk factor for the development and progression of liver cancer. Chronic liver injury of any etiology leads to hepatic fibrosis, which can then progress to cirrhosis, and, in one third of cases, to HCC. Stellate cells are responsible for making the extracellular matrix resulting in hepatic fibrosis. While excessive fibrosis is a common feature in multiple liver diseases it is most clearly defined in MASH, where fibrosis is the only histologic feature that correlates with clinical outcomes including HCC.

[0128] Death-associated apoptosis-inducing protein kinase 2 (DRAK2), also known as STK17B, is a serine / threonine protein kinase and a member of the death-associated protein kinase (DAPK) family. It has been shown, in some systems, to be pro-apoptotic, related to lymphoid T-cell activation, and linked to fatty liver disease development.

[0129] DRAK2 is observed to be upregulated in HCC tumors and DRAK2 predicts poor clinicopathological features. DRAK2 was upregulated in 60 HCC samples paired to tumor adjacent tissue, and this upregulation was associated with increased tumor size, NTM stage, and venous invasion. DRAK2 is also significantly unregulated in HCC cell lines and overexpression promotes HCC cell proliferation and tumorigenesis in vitro and in vivo. Further, in preclinical studies, DRAK2 promotes HCC cell migration and invasion in vitro and in vivo.Kinome data

[0130] The KINOMEscan™ screening platform (DiscoverX, San Diego) was used to evaluate the kinome profile of Compound A. This platform employs an active site directed competition binding assay to quantitatively measure interactions between Compound A and 489 human kinases. These assays do not require ATP and thereby report true thermodynamic interaction affinities, as opposed to IC50 values, which can depend on the ATP concentration.

[0131] Compound A was evaluated at 1 pM in a full kinome scan (489 kinases) resulting in the inhibition at 90% or greater of 17 non-mutated kinases. Full Kds (binding constant) were determined using the KdElect platform at DiscoverX (San Diego). An 11 -point 3-fold serial dilution was prepared in 100% DMSO at lOOx final test concentration and subsequently diluted to lx in the assay (final DMSO concentration = 1%). Kds were determined using a compound top concentration of 60pM.

[0132] The follow-up full Kd determinations showed that ASK1, DRAK2, ASK3, and LRRK2 are the key kinases inhibited by Compound A with an ASKl / kinase ratio of <10. ASK1 and ASK3 are the targets of interest while inhibition of DRAK2 (STK17B) may be beneficial for liver diseases with fibrosis due to its role in hepatocellular carcinoma (HCC) progression.WSGR Docket No. 51760-717.601

[0133] Compound A, with dual inhibition of ASK1 and DRAK2, would decrease HCC formation initially by limiting fibrosis by the direct inhibition of activated stellate cells by ASK1 inhibition (FIGs. 4 and 8) and also inhibiting HCC development and migration by DRAK2 inhibition.Example 7: Compound A Has Low Risk of Drug-Drug Interaction

[0134] Compound A was tested in human microsomes to assess inhibitory potential on major drug metabolizing human cytochrome P450 (CYP) enzymes (CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4). The selective drug substrates used were phenacetin (CYP1A2), bupropion (CYP2B6), paclitaxel (CYP2C8), tolbutamide (CYP2C9), S-Mephenytoin (CYP2C19), bufuralol (CYP2D6), midazolam (CYP3A4) and testosterone (CYP3A4). Compound A showed marginal inhibitory effect on major human CYPs with IC50 values above 20 pM.

[0135] Compound A was incubated at 2 and 10 pM with cryopreserved human hepatocytes to assess the induction of major isoforms of human cytochrome P450. Compound A did not cause any significant changes in mRNA expression of CYP2B6 or CYP3 A4 under the study conditions. Compound A did not cause any significant changes in mRNA expression of CYP1 A2 at 2 pM, but weakly increased CYP1 A2 levels about 2-fold at 10 pM (less than 10% of the positive control omeprazole).

[0136] Compound A has a low potential for being an object (“victim”) of drug-drug interaction as suggested by a low metabolic turnover mediated by CYP3 A4. Having potential to be a substrate of p-glycoprotein (P-gp) efflux and organic anion transporter 3 (OAT3)- mediated uptake, Compound A distribution could also be affected by inhibitors or inducers of these drug transporters.

[0137] This data indicates that Compound A has limited risk of drug-drug interaction and can be used in combination with other drugs to treat cholestatic diseases.Example 8: Compound A Has Limited Effect on Bile Transport

[0138] Compound A was observed to have a limited effect on BCRP, P-gp and BSEP mediated transport as evidenced by 42%, 24%, and 8% inhibition, respectively, at 10 pM. Compound A did not inhibit NTCP, MRP2, and MRP3 vesicular transport, whereas MRP4 transport was inhibited by 28% at 10 pM. This data suggests that Compound A has a low potential to affect bile transport and is suitable for treatment of cholestatic diseases.

[0139] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments areWSGR Docket No. 51760-717.601 provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

WSGR Docket No. 51760-717.601CLAIMSWHAT IS CLAIMED IS:

1. A method for treating cholestatic liver disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of Compound A, or a pharmaceutically acceptable salt or solvate thereof, wherein Compound A has the following structure:

2. The method of claim 1, wherein the cholestatic liver disease is acute cholestasis liver disease or chronic cholestasis liver disease.

3. The method of claim 2, wherein the cholestatic liver disease is biliary obstruction, biliary atresia, cholangitis, total parenteral nutrition (TPN)-associated cholestasis, primary sclerosing cholangitis, secondary sclerosing cholangitis, or intrahepatic cholestasis of pregnancy.

4. The method of claim 2, wherein the cholestatic liver disease is biliary atresia.

5. The method of any one of claims 1 to 4, wherein Compound A is administered in a dose between about 1 mg / kg to about 100 mg / kg.

6. The method of claim 5, wherein the dose is between about 5 mg / kg to about 80 mg / kg.

7. The method of claim 6, wherein the dose is between about 10 mg / kg and about 60 mg / kg.

8. The method of claim 7, wherein the dose is between about 15 mg / kg to about 50 mg / kg.

9. The method of claim any one of claims 1 to 8, wherein the subject is a pediatric subject.

10. The method of claim 9, wherein the subject is less than 5 years old.

11. The method of claim 10, wherein the subject is less than 3 years old.

12. The method of claim 11, wherein the subject is less than 1 year old.

13. The method of any one of claims 1 to 12, wherein Compound A is administered orally or by intravenous delivery.WSGR Docket No. 51760-717.60114. The method of claim 13, wherein Compound A is administered by intravenous delivery.

15. The method of claim 13, wherein Compound A is administered orally.

16. A method for treating primary liver cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of Compound A, or a pharmaceutically acceptable salt or solvate thereof, wherein Compound A has the following structure:

17. The method of claim 16, wherein the primary liver cancer is hepatocellular carcinoma (HCC) or bile duct cancer.

18. The method of claim 17, wherein the primary liver cancer is hepatocellular carcinoma (HCC).

19. The method of any one of claims 16 to 18, wherein the subject previously had chronic hepatitis B or chronic hepatitis C.

20. The method of any one of claims 16 to 19, wherein Compound A is administered in a dose between about 1 mg / kg to about 100 mg / kg.

21. The method of claim 20, wherein the dose is between about 5 mg / kg to about 80 mg / kg.

22. The method of claim 21, wherein the dose is between about 10 mg / kg and about 60 mg / kg.

23. The method of claim 22, wherein the dose is between about 15 mg / kg to about 50 mg / kg.

24. The method of any one of claims 16 to 23, wherein the subject is an adult subject.

25. The method of claim 24, wherein the subject is at least 18 years old.

26. The method of any one of claims 16 to 25, wherein Compound A is administered orally, by intravenous delivery, or by intraperitoneal injection.

27. The method of claim 26, wherein Compound A is administered by intravenous delivery.

28. The method of claim 26, wherein Compound A is administered orally.WSGR Docket No. 51760-717.60129. A method for treating advanced fibrosis or cirrhosis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of Compound A, or a pharmaceutically acceptable salt or solvate thereof, wherein Compound A has the following structure:(Compound A).

30. The method of claim 29, wherein the advanced fibrosis or cirrhosis is caused by a fibrotic liver disease.

31. The method of claim 30, wherein the fibrotic liver disease is chronic hepatitis B, chronic hepatitis C, chronic hepatitis D, metabolic dysfunction-associated steatotic liver disease (MASLD) (formerly known as non-alcoholic fatty liver disease (NAFLD)), metabolic dysfunction-associated steatohepatitis (MASH) (formerly knowns as non-alcoholic steatohepatitis (NASH)), alpha- 1 -antitrypsin deficiency, biliary atresia, primary sclerosing cholangitis, secondary sclerosing cholangitis, autoimmune hepatitis, alcohol-related liver disease (ALD), hemochromatosis, schistosomiasis, or Wilson’s disease.

32. The method of any one of claims 29 to 31, wherein the administration of Compound A inhibits stellate cell activation.

33. The method of any one of claims 29 to 32, wherein the administration of Compound A causes an anti-inflammatory effect.

34. The method of claim 33, wherein the anti-inflammatory effect comprises inhibition of reactive oxygen species-induced cytokine release.

35. The method of any one of claims 29 to 34, wherein the method comprises treating advanced fibrosis.

36. The method of any one of claims 29 to 35, wherein the method comprises treating cirrhosis.

37. The method of any one of claims 29 to 36, wherein Compound A is administered in a dose between about 1 mg / kg to about 100 mg / kg.

38. The method of claim 37, wherein the dose is between about 5 mg / kg to about 80 mg / kg.

39. The method of claim 38, wherein the dose is between about 10 mg / kg and about 60 mg / kg.WSGR Docket No. 51760-717.60140. The method of claim 39, wherein the dose is between about 15 mg / kg to about 50 mg / kg.

41. The method of any one of claims 29 to 40, wherein Compound A is administered orally, by intravenous delivery, or by intraperitoneal injection.

42. The method of claim 41, wherein Compound A is administered by intravenous delivery.

43. The method of claim 41, wherein Compound A is administered orally.

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