Oral pharmaceutical agents and methods for preventing, inhibiting progression and treating fatty liver disease

Abstract

Disclosed is a pharmaceutical composition for use in a method for preventing, preventing progression and / or treating fatty liver disease, comprising a bitter taste receptor agonist, comprising a denatonium salt selected from the group consisting of denatonium acetate (DA), denatonium citrate, denatonium maleate, denatonium saccharide and denatonium tartrate. In some embodiments, the daily dose of the denatonium salt for preventing or preventing progression of fatty liver disease is 25 mg / kg to about 45 mg / kg QD, and the daily dose of the denatonium salt for treating existing fatty liver disease is 70 mg / kg to 200 mg / kg per day administered QD or BID.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 144,386, filed February 1, 2021.

[0002] FIELD OF THEINVENTION The present disclosure provides a method for preventing, preventing the progression of, and treating fatty liver disease, comprising administering an effective amount of a pharmaceutical composition comprising a bitter taste receptor agonist comprising a denatonium salt, wherein the denatonium salt is selected from the group consisting of denatonium acetate (DA), denatonium citrate, denatonium maleate, denatonium saccharide, and denatonium tartrate; the daily dose of the denatonium salt for preventing or preventing the progression of fatty liver disease is about 0.1 mg / kg / day to about 8 mg / kg / day, and the daily dose of the denatonium salt for treating existing fatty liver disease is about 1 mg / kg / day to about 8 mg / kg / day, administered QD, BID, or TID, respectively. [Background technology]

[0003] Fatty liver disease is a term used to describe a group of liver diseases that includes nonalcoholic steatohepatitis (NASH), alcoholic steatohepatitis (ASH), nonalcoholic fatty liver disease (NAFLD) and HIV-associated steatohepatitis, with or without liver fibrosis. Specifically, nonalcoholic steatohepatitis (NASH) is a common liver disease associated with increased morbidity and mortality. However, despite many compounds being tested in purported NASH treatment models, there are no FDA-approved treatment options. Nonalcoholic fatty liver disease (NAFLD) is a disorder that affects one in three to five adults and one in ten children in the United States. These are conditions in which excess fat accumulates in the liver of people who drink little or no alcohol. The most common form of NAFLD is a non-serious condition called hepatic steatosis (fatty liver), in which fat accumulates in liver cells (which is not normal, but is probably not damaging to the liver in itself). NAFLD most often presents in individuals with a set of risk factors called metabolic syndrome, characterized by elevated fasting plasma glucose (FPG) with or without intolerance to postprandial sugars, overweight or obesity, high blood lipids, such as cholesterol and triglycerides (TG) and low high-density lipoprotein cholesterol (HDL-C) levels, and high blood pressure, although not all patients have all the symptoms of metabolic syndrome. Obesity is thought to be the most common cause of NAFLD, and some experts estimate that about two-thirds of obese adults and about one-half of obese children may have fatty liver. The majority of patients with NAFLD have no symptoms and a normal physical exam (although they may have a slightly enlarged liver). Children may present with symptoms such as abdominal pain and fatigue, and may show patchy dark skin discoloration (acanthosis nigricans). The diagnosis of NAFLD is usually first suspected in overweight or obese people who have mildly elevated liver blood tests during routine examinations, but NAFLD can also be present with normal liver blood tests or be detected incidentally on imaging tests, e.g., ultrasound or CT scan.This is confirmed by imaging tests, most commonly liver ultrasound or magnetic resonance imaging (MRI), to rule out other causes.

[0004] Some patients with NAFLD may develop a more severe condition called nonalcoholic steatohepatitis (NASH), which affects approximately 2-5% of adult Americans and up to 20% of obese adult Americans. In NASH, fat accumulation in the liver is associated with inflammation and varying degrees of scarring. NASH is a potentially serious condition with significant risk of progression to end-stage liver disease, cirrhosis, and hepatocellular carcinoma. Some patients who develop cirrhosis are at risk of liver failure and may eventually require a liver transplant. Thus, weight loss is a recommended measure to prevent or slow the progression of NASH. However, weight loss has not been shown to treat NASH once liver fibrotic damage has occurred.

[0005] NAFLD can be differentiated from NASH by the NAFLD activity score (NAS), which is the sum of the histopathological scores of liver biopsies for steatosis (0–3), lobular inflammation (0–2), and hepatocellular ballooning (0–2). An NAS of less than 3 corresponds to NAFLD, 3–4 to borderline NASH, and ≥5 to NASH. Biopsies are also scored for fibrosis (0–4).

[0006] NASH is the leading cause of end-stage liver disease.

[0007] Treatment of NAFLD and NASH No drugs are currently approved to prevent or treat NAFLD or NASH. Many pharmacological interventions have been attempted in NAFLD / NASH with limited overall benefit. Antioxidants can prevent lipid peroxidation and cytoprotectants can stabilize phospholipid membranes, but drugs that have been tried without success or with only modest benefit include ursodeoxycholic acid, vitamin E (α-tocopherol) and vitamin C, and pentoxifylline. Weight loss agents such as orlistat offer no significant advantage over weight loss achieved through diet and exercise alone ("weight loss alone"). Most weight loss studies in NAFLD / NASH have been short-term pilot studies with limited success, reporting only modest improvements in necroinflammation or fibrosis. A randomized, double-blind, placebo-controlled, 6-month trial of pioglitazone, a thiazolidinedione peroxisome proliferator-activated receptor-γ (PPARγ) agonist and insulin sensitizer, with weight loss alone (Belfort, "A placebo-controlled trial of pioglitazone in subjects with nonalcoholic steatohepatitis", N. Engl. J. Med., 355, 2297-2307 (2006)) showed no improvement with weight loss alone, but treatment with pioglitazone improved glycemic control, insulin sensitivity, indices of systemic inflammation (including hsCRP, tumor necrosis factor α, and transforming growth factor β), and liver histology in patients with NASH and IGT or T2DM. Treatment with pioglitazone also improved fat, liver, and muscle IR and was associated with an approximately 50% reduction in necroinflammation (p<0.002) and a 37% reduction in fibrosis (p=0.08). Amelioration of hepatocellular injury and fibrosis was reported in another controlled trial using pioglitazone for 12 months.In contrast, the first randomized clinical study with rosiglitazone, another thiazolidinedione approved for diabetes treatment, showed a reduction in IR, plasma alanine aminotransferase (ALT) levels, and steatosis in NASH, while rosiglitazone treatment had no significant effect on necrosis, inflammation, or fibrosis. It is important to note that in these results, liver fibrosis, an important indicator of NASH, was not reduced even though ALT, insulin resistance, and other diabetes indicators were reduced. Thus, diabetes control is not sufficient to treat or prevent NASH. In addition, both pioglitazone and rosiglitazone have strict safety limitations. The preliminary report of the two-year open-label follow-up of this trial was also disappointing, with no significant benefit from rosiglitazone treatment. One drug with some efficacy in NASH is pioglitazone. Unfortunately, pioglitazone is also associated with a significant increase in the risk of weight gain, edema, congestive heart failure, and osteoporotic fractures in both women and men.

[0008] A phase 2 trial in patients with NASH showed that treatment with daily subcutaneous semaglutide (a GLP-1 agonist) resulted in a higher proportion of patients with NASH resolution than placebo. However, the trial did not show a significant difference between groups in the proportion of patients with improved fibrosis stage (Newsome et al., N. Engl. J. Med. "A Placebo-Controlled Trial of Subcutaneous Semaglutide in Nonalcoholic Steatohepatitis" November 13, 2020). Unfortunately, "the percentage of patients who achieved resolution of NASH without worsening fibrosis was 40% in the 0.1 mg group, 36% in the 0.2 mg group, 59% in the 0.4 mg group, 17% in the 0.4 mg group, and 17% in the placebo group (P = .48). The mean percentage of weight loss was 13% in the 0.4 mg group and 1% in the placebo group. The incidence of nausea, constipation, and vomiting was higher in the 0.4 mg group than in the placebo group (nausea 42% vs. 11%, constipation 22% vs. 12%, vomiting 15% vs. 2%). Malignant neoplasms were not associated with semaglutinin. "Neoplasms were reported in three patients (1%) receiving semaglutide and none in those receiving placebo. Overall, neoplasms (benign, malignant or indeterminate) were reported in 15% of patients in the semaglutide group and 8% in the placebo group. No specific organ occurrence patterns were observed." Thus, even GLP-1 agonists such as semaglutide are not a mild treatment for the prevention or treatment of NASH that justifies the risks of long-term treatment required to treat, prevent or slow the progression of NASH.

[0009] A summary of the clinical data obtained showed that the treatment of NASH appears to be independent of weight loss as a treatment measure, whatever the weight loss technique. However, weight loss may be an effective means of preventing NASH, which may indicate NASH progression. Therefore, there is a need for a better accepted translational model to predict the prevention, progression prevention and treatment of fatty liver disease, including NASH. Therefore, there is a need for effective and safe NASH treatment options, especially if the treatment can be delivered orally rather than by injection. There is also a need for safe agents to prevent the development of complete NASH liver disease and damage and to indicate NASH progression. Summary of the Invention

[0010] The present disclosure provides a method for preventing, preventing the progression of, and / or treating fatty liver disease comprising administering an effective amount of a pharmaceutical composition comprising a bitter taste receptor agonist comprising a denatonium salt, wherein the denatonium salt is selected from the group consisting of denatonium acetate (DA), denatonium citrate, denatonium maleate, denatonium saccharide, and denatonium tartrate; and wherein the daily dose of the denatonium salt for preventing or preventing the progression of fatty liver disease is about 0.1 mg / kg / day to about 8 mg / kg / day, and the daily dose of the denatonium salt for treating existing fatty liver disease is about 1 mg / kg / day to about 8 mg / kg / day, administered QD, BID, or TID, respectively.

[0011] More specifically, the present disclosure provides a method for preventing and preventing the progression of fatty liver disease selected from the group consisting of non-alcoholic steatohepatitis (NASH), alcoholic steatohepatitis (ASH), non-alcoholic fatty liver disease (NAFLD) and HIV-associated steatohepatitis, with or without liver fibrosis, comprising administering an effective amount of a pharmaceutical composition comprising a denatonium salt, wherein the denatonium salt is selected from the group consisting of denatonium acetate (DA), denatonium citrate, denatonium maleate, denatonium saccharide and denatonium tartrate, and the daily dose of the denatonium salt for preventing or preventing the progression of fatty liver disease is about 0.1 mg / kg / day to about 8 mg / kg / day QD, BID, or TID. Preferably, the pharmaceutical composition further comprises about 0.5 g to about 5 g of acetic acid. More preferably, the daily dose of acetic acid for an adult is about 1.5 g to about 3 g.

[0012] The present disclosure provides a method for treating a liver disease selected from the group consisting of non-alcoholic steatohepatitis (NASH), alcoholic steatohepatitis (ASH), non-alcoholic fatty liver disease (NAFLD) and HIV-associated steatohepatitis, with or without liver fibrosis, comprising administering an effective amount of a pharmaceutical composition comprising a denatonium salt, wherein the denatonium salt is selected from the group consisting of denatonium acetate (DA), denatonium citrate, denatonium maleate, denatonium saccharide and denatonium tartrate, and the daily dose of the denatonium salt for the treatment of the existing fatty liver disease is about 1.0 mg / kg / day to about 8 mg / kg / day administered QD, BID or TID. Preferably, the pharmaceutical composition further comprises about 0.5 g to about 5 g of acetic acid. More preferably, the daily dose of acetic acid for an adult is about 1.5 g to about 3 g. [Brief description of the drawings]

[0013] [Figure 1] 1A-B show group mean absolute body weights (FIG. 1A) and relative body weights (% baseline, FIG. 1B) of mice fed the AMLN diet during 1 year of daily treatment with vehicle or 30 mg / kg DA. [Diagram 2] Figure 2 shows that fasting blood glucose levels in DA-treated mice were significantly lower than those in vehicle-treated mice at week 24 only. Comparison with vehicle values ​​on each day by unpaired two-tailed t-test. Differences were considered statistically significant at p<0.05. *p<0.05. NS, not significant. [Diagram 3] Figure 3 shows that insulin levels in DA-treated mice were significantly elevated at weeks 4 and 12, but not thereafter (compared to those in vehicle-treated mice). Comparisons with vehicle values ​​on each day by unpaired two-tailed t-test. Differences were considered statistically significant at p<0.05. *p<0.05. **p<0.01. NS, not significant. [Figure 4] FIG. 4 shows that HbA1c levels in DA-treated mice were significantly reduced at weeks 12 and 48, but not at week 24 (compared to those in vehicle-treated mice). (HbA1c levels were not assayed pre-dosing or at week 4.) Comparison with vehicle values ​​on each day by unpaired two-tailed t-test. Differences were considered statistically significant at p<0.05. *p<0.05. **p<0.01. ***p<0.001. NS, not significant. HbA1c was not assayed pre-dosing or at week 4. [Diagram 5] FIG. 5A shows that GLP-1 levels in DA-treated mice were significantly reduced only at week 24 (compared to those in vehicle-treated mice). The magnitude of the difference was similar to the other time points (not statistically significant), but the variation at week 24 was smaller than the other weeks, suggesting that the statistical significance of this effect is not meaningful. Comparisons with vehicle values ​​on each day by unpaired two-tailed t-test. Differences were considered statistically significant at p<0.05. ***p<0.001. NS, not significant. FIG. 5B shows GLP-2 levels. Note that GLP-2 was assayed only at week 48. GLP-2 levels in DA-treated mice were not significantly different from those in vehicle-treated mice at week 48, the only time point at which this parameter was assessed. [Figure 6]Figure 6 shows that serum ALT activity levels in DA-treated mice were significantly decreased at 24 and 48 weeks, but not earlier (compared to those in vehicle-treated mice). Comparison with vehicle values ​​on each day by unpaired two-tailed t-test. Differences were considered statistically significant at p<0.05. **p<0.01. ***p<0.001. NS, not significant. [Figure 7] Figure 7 shows that serum AST activity levels in DA-treated mice were significantly reduced only at week 24 (compared to those in vehicle-treated mice). Comparison with vehicle values ​​on each day by unpaired two-tailed t-test. Differences were considered statistically significant at p<0.05. **p<0.01. NS, not significant. [Figure 8] Figure 8 shows that serum ALB levels in DA-treated mice were not significantly different from those in vehicle-treated mice at any assessment time point. Comparison with vehicle values ​​on each day by unpaired two-tailed t-test. Differences were considered statistically significant at p<0.05. NS, not significant. [Figure 9] Figure 9 shows that serum BA levels in DA-treated mice were significantly reduced only at week 24 (compared to those in vehicle-treated mice). Comparison with vehicle values ​​on each day by unpaired two-tailed t-test. Differences were considered statistically significant at p<0.05. *p<0.05. NS, not significant. [Figure 10]FIG. 10 shows blood levels of cytokines that showed statistically significant differences (n=10) in mice fed the AMLN diet during one year of daily treatment with vehicle or 30 mg / kg for IL-6 (FIG. 10A), IL-9 (FIG. 10B), IL-13 (FIG. 10C), IP-10 (FIG. 10D), KC (FIG. 10E), MCP-1 (FIG. 10F), MIP-1a (FIG. 10G), MIP-1B (FIG. 10H), MIG (FIG. 10I) and TNFα-6 (FIG. 10J). Integers in parentheses in the cytokine names / graph titles correspond to the assay numbers used as part of the multiplex assay. Comparisons with vehicle values ​​on each day by unpaired two-tailed t-test. Differences were considered statistically significant at p<0.05. *p<0.05. **p<0.01. NS, not significant. [Figure 11] Figure 11A (absolute values) and B (relative values, normalized to body weight) show autopsy liver weights after one year of daily treatment with vehicle or 30 mg / kg DA. Comparisons with vehicle values ​​on each day by unpaired two-tailed t-test. Differences were considered statistically significant at p<0.05. *p<0.05. NS, not significant. [Figure 12] Figure 12 shows serum HDL, LDL and TGA levels after one year of daily treatment with vehicle or 30 mg / kg DA. Serum TGA levels were significantly reduced in DA-treated mice after one year of daily treatment, but not HDL or LDL (compared to vehicle-treated animals). DA, denatonium acetate. HDL, high-density lipoprotein. LDL, low-density lipoprotein. TGA, triglycerides. Comparison with vehicle values ​​on each day by unpaired two-tailed t-test. Differences were considered statistically significant at p<0.05. *p<0.05. NS, not significant. [Figure 13] Figure 13 shows liver TGA (Figure 13A), TC (Figure 13B) and FFA (Figure 13C) after one year of daily treatment with vehicle or 30 mg / kg DA. After one year of daily treatment, liver TGA levels were significantly reduced in DA-treated mice, but not TC or FFA levels (compared to vehicle-treated mice). [Figure 14]FIG. 14 shows that treatment with DA (ARD-101) significantly improved NAFLD activity scores based on blinded histopathological examination. [Figure 15] Figures 15A and 15B show that treatment with ARD-101(DA) showed a significant effect on body weight (Figure 15A) and body weight change (Figure 15B) of AMLN diet-induced NASH mice. Data are expressed as mean values. Statistical analysis was performed by one-tailed t-test. ***p<0.001 compared to vehicle; $$P<0.01 and $$$P<0.001 compared to the combination. [Figure 16] Figures 16A and 16B show liver weight (Figure 16A) and liver / body weight ratio (Figure 16B), demonstrating that DA (ARD-101) significantly reduced liver weight and liver / body weight ratio compared to vehicle. [Figure 17] Figure 17A shows ALT levels and Figure 17B shows AST levels. At the end of the study, DA (ARD-101) significantly reduced ALT and AST levels compared to the vehicle control. [Figure 18] Figures 18A (triglycerides (TG)), 18B (LDL) and 18C (HDL) show that DA (ARD-101) significantly reduced triglycerides (TG), low density lipoprotein (LDL) and high density lipoprotein (HDL), respectively, at the end of the study in Example 2. [Figure 19] FIG. 19 shows the change in glucose levels at the end of the study of Example 2 for the indicated treatment groups. [Figure 20] Figure 20 shows the HbA1c levels for the indicated treatment groups at the end of the study in Example 2. Baseline HbA1c levels were 5.0%. [Figure 21] Figure 21 shows insulin levels for the indicated treatment groups at the end of the study in Example 2. Baseline insulin levels were 1.5 ng / ml. [Figure 22] Figure 22 shows that the two treatments did not significantly affect bile acid levels compared to the vehicle control. Baseline bile acid levels were 30 μmol / L. [Figure 23] Figure 23A (CK-18) shows that DA (ARD-101) significantly reduced CK-18 levels compared to vehicle controls. Figure 23B shows TGF-β1 levels for the indicated treatment groups. [Figure 24] 24A and 24B show that at the end of the study in Example 2, the two treatments did not significantly affect IL-6 and TNF-α levels compared to vehicle. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] This disclosure is based on a first in vivo study (shown in Example 1) focused on the prevention of fatty liver disease, followed by a second in vivo study (shown in Example 2) focused on the treatment of fatty liver disease. These data from both studies provide surprising evidence that treatment with denatonium salts at various dosing frequencies and dose ranges can provide both prevention of fatty liver disease in susceptible individuals (such as type 2 diabetes patients who are at extremely high risk of developing NASH) and prevention of disease progression with a lower oral dose of denatonium salts once a day. Also, a higher dose of denatonium salts twice a day is required to treat NASH in an in vivo model, with similar efficacy to GLP-1 agonists, but without the known (in humans) severe side effects of semaglutide, which is sold as a prototype GLP-1 agonist.

[0015] Definition: "Fatty liver disease" means any of a group of diseases characterized by the accumulation of unwanted fat in the liver, including nonalcoholic steatohepatitis (NASH), alcoholic steatohepatitis (ASH), nonalcoholic fatty liver disease (NAFLD), and HIV-associated steatohepatitis, with or without liver fibrosis.

[0016] A "therapeutically effective amount" of a denatonium salt means an amount that, when administered to a human for treating fatty liver disease, such as NAFLD or NASH, is sufficient to effect treatment of the fatty liver disease. "Treating" or "treatment" of NAFLD or NASH in a human includes one or more of the following: (1) preventing or reducing the risk of developing NAFLD or NASH, i.e., preventing the development of clinical symptoms of NAFLD or NASH in subjects who may be predisposed to NAFLD or NASH but have not yet experienced or exhibited symptoms of NAFLD or NASH (i.e., prevention); (2) inhibiting NAFLD or NASH, i.e., arresting or reducing the onset of NAFLD or NASH or its clinical symptoms; and (3) alleviating NAFLD or NASH, i.e., causing regression, reversal, or improvement of NAFLD or NASH, or reducing the number, frequency, duration, or severity of its clinical symptoms. The therapeutically effective amount for a particular subject varies according to the health and physical condition of the subject being treated, the degree of NAFLD or NASH, evaluation of medical condition, and other relevant factors.It is expected that the therapeutically effective amount will fall in a relatively broad range that can be determined through routine testing.

[0017] "Or" is used in its inclusive sense (equivalent to "and / or") unless the context requires otherwise.

[0018] Ranges and amounts used herein may be expressed as "about" a particular value or range. "About" includes the exact amount. Thus, "about 5 mg" means "about 5 mg" and also "5 mg". In general, the term "about" includes amounts that are expected to be within experimental error, for example, within 15%, 10%, or 5%.

[0019] The section headings are provided for the reader's convenience only and do not limit the disclosure.

[0020] (Dosage) The results of the two in vivo mouse studies in Examples 1 and 2, and the first results in a human Phase 1 clinical safety study, provided a safe and effective dosage range for the denatonium salt of the present disclosure. Based on the mouse-to-human conversion formula, the human dose equivalent to 50 mg / kg, BID in mice (the dose used for treatment in the in vivo study in Example 2) is 4 mg / kg, BID (or 8 mg / kg / day). Considering that the average body weight of an adult is 60 kg, the corresponding dose is 240 mg, BID (or 480 mg / day). In the first-in-human clinical trial, 240 mg, BID (480 mg / day) was well tolerated. Thus, for treatment purposes, a daily dose upper limit (adult) of 600 mg to 1000 mg is safe and tolerable, and efficacy has been shown at lower daily doses in the preclinical mouse model provided herein.

[0021] Specifically, for fatty liver disease, the daily adult dose of denatonium salt is about 10 mg to about 600 mg or about 0.2 mg / kg body weight to about 10 mg / kg body weight per day. Most preferably, the denatonium salt for adults is about 40 mg to about 400 mg or about 1 mg / kg body weight to about 8 mg / kg body weight per day. The daily dose of denatonium salt is administered once a day, twice a day, or three times a day. Most preferably, the daily dose of denatonium salt is administered twice a day.

[0022] Specifically, for the prevention of fatty liver disease, the daily adult dose of denatonium salt is about 5 mg to about 400 mg or about 0.1 mg / kg body weight to about 8 mg / kg body weight per day. Most preferably, the denatonium salt for adults is about 20 mg to about 200 mg or about 0.5 mg / kg body weight to about 4 mg / kg body weight per day. The daily dose of denatonium salt is administered once a day, twice a day or three times a day. Most preferably, the daily dose of denatonium salt is administered once a day.

[0023] To the extent that the material incorporated by reference conflicts with the explicit content of this disclosure, the explicit content controls.

[0024] [ka] Denatonium acetate anhydrous, or DA, is an anhydrous salt in which for every 100 mg of DA there are 83 mg of denatonium cation and 17 mg of acetate anion.

[0025] Scheme A illustrates the synthesis of denatonium acetate (DA).

[0026] Step 1: Synthesis of denatonium hydroxide from lidocaine Add 25 g of lidocaine, 60 ml of water and 17.5 g of benzyl chloride to a reflux apparatus and heat at 70-90 ° C with stirring. The solution needs to be heated and stirred at the previously indicated values ​​for 24 hours, and then the solution needs to be cooled to 30 ° C. Remove the unreacted reagent with 3 × 10 mL of toluene. Dissolve 65 g of sodium hydroxide in 65 mL of cold water with stirring and add it to the aqueous solution over 3 hours with stirring. Filter the mixture, wash with some water and dry in the open air. Recrystallize in hot chloroform or hot ethanol. [ka]

[0027] Step 2: Preparation of denatonium acetate anhydride from denatonium hydroxide To a reflux apparatus, 10 g of denatonium hydroxide (MW: 342.475 g / mol, 0.029 mol), 20 mL of acetone, and 2 g of glacial acetic acid (0.033 mol) dissolved in 15 mL of acetone were added, and the mixture was stirred and heated at 35° C. for 3 h, then evaporated to dryness and recrystallized in hot acetone. [ka]

[0028] Prescription of DA tablets This provides an immediate release 50 mg granular formulation of denatonium acetate (DA) as the free base in an intragastric immediate release oral pharmaceutical formulation.

[0029] Table 1 shows the qualitative and quantitative formulation composition of DA. [Table 1]

[0030] The manufacturing process will be described in detail below.

[0031] 1. Drug Layering Process – Drug Layered Pellets The drug layering process was carried out in a fluid bed granulator equipped with a rotor insert (rotor granulator). The drug solution was prepared by dissolving povidone K30 (Kollidon 30) and denatonium acetate in ethyl alcohol. The drug solution was sprayed tangentially onto a bed of white sugar spherical granules (35 / 45 mesh) moving in a circular motion in the rotor granulator. The final drug-loaded pellets were then dried in the rotor granulator for 10 minutes, removed, and sieved through a #20 mesh.

[0032] 2. Seal Coating Process – Seal Coating Pellets The seal coating dispersion was prepared by separately dissolving hypromellose E5 in a mixture of ethyl alcohol and purified water (1:1) until a clear solution was obtained. The remaining amount of ethyl alcohol was then added to the above solution followed by talc. The dispersion was mixed for 20 minutes to uniformly disperse the talc. The seal coating dispersion was sprayed tangentially onto the drug loaded pellets to achieve a weight gain of 5%. The seal coated pellets were then dried in a rotor granulator for 5 minutes, removed and further dried in a tray dryer / oven at 55°C for 2 hours. The seal coated pellets were then sieved through a #20 mesh.

[0033] 3. Final Mix – Denatonium Immediate Release (IR) Pellets The seal coating pellets were mixed with talc sieved through mesh #60 using a V-blender and discharged. The mixed seal coating beads, denatonium IR pellets were used for encapsulation.

[0034] 4. Encapsulation – Denatonium Capsules 50mg 50 mg of denatonium IR pellets were filled into size 1 white opaque hard gelatin capsules using an automated capsule filling machine. The capsules were then passed through an in-line capsule polisher and metal detector. In-process control of capsule weight and appearance was performed during the encapsulation process. Acceptable Quality Level (AQL) sampling and inspection was performed by Quality Assurance (QA) on composite samples during the encapsulation process. Composite samples of the finished product were taken and analyzed per release test specifications.

[0035] 5. Packaging - Capsules 50mg - 30 pieces The 50 mg capsules were packaged in packs of 30 in 50 / 60 cc white HDPE round S-line bottles with 33 mm white CRC caps. The bottles were clamped and sealed using an induction sealer. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5]

[0036] Table 2 shows various results in widely different in vivo models of NASH. This makes direct comparison of the data difficult. The study corresponding to the first row (called Aardvark Therapeutics) is provided in Example 1 below, and a figure is provided with this disclosure. It should be noted that unlike many prior art NASH models, this disclosure involved an extremely long 48 weeks of daily dosing, since it is estimated that at least 30 weeks of AMLN diet is required to induce fully fibrotic NASH in mouse models. Many of the other studies were of shorter duration. Thus, the study in Example 1 is more predictive of the efficacy of methods to prevent or delay the progression of NASH and similar liver diseases characterized by liver fibrosis.

[0037] Studies that emphasize weight loss as a model for NASH treatment appear to be directed toward treating existing NASH conditions. Thus, this disclosure further incorporates by reference U.S. Patent Application No. 17 / 132,580, filed December 23, 2020. In particular, Example 9 and the accompanying figures show the results of a weight loss study using a higher dose of DA (23.1 mg / kg BID, equivalent to 46.2 mg / kg / day of DA or 37.4 mg / kg / day of dry denatonium), which shows a significant reduction in weight gain. In contrast to the shorter-term weight loss study of DIO using approximately twice the daily dose of DA, this NASH prevention study using a lower dose of DA showed no difference in weight gain over 48 weeks with vehicle controls (Figure 1 herein). Thus, this comparison implies that either a higher dose of DA per day for prevention is required for treatment, or more frequent administration is required for treatment, or both aspects are required for treatment. However, the study of Example 1 clearly shows that a similar dose, but only once a day (rather than twice a day), is sufficient to prevent and delay progression of NASH, but not to reduce body weight, which may be a marker for the treatment of NASH. Thus, the dosage range of denatonium salts for the method of treating NASH and related liver diseases is about 1.0 mg / kg / day to about 12 mg / kg / day; preferably about 2 mg / kg / day to about 8 mg / kg / day; most preferably about 3 mg / kg / day to about 6 mg / kg / day. Thus, the dosage range of denatonium salts in the method of preventing and delaying progression of NASH and related liver diseases is about 0.1 mg / kg / day to about 6 mg / kg / day, preferably about 0.25 mg / kg / day to about 4 mg / kg / day; most preferably about 0.5 mg / kg / day to about 3 mg / kg / day.

[0038] (Implementation) In a first aspect, the present disclosure provides a method for preventing or preventing the progression of fatty liver disease with or without liver fibrosis (e.g., nonalcoholic steatohepatitis (NASH), alcoholic steatohepatitis (ASH), nonalcoholic fatty liver disease (NAFLD) and HIV-associated steatohepatitis), comprising administering an effective amount of a pharmaceutical composition comprising a denatonium salt, wherein the denatonium salt is selected from the group consisting of denatonium acetate (DA), denatonium citrate, denatonium maleate, denatonium saccharide and denatonium tartrate, and the daily dose of the denatonium salt for preventing or preventing the progression of fatty liver disease is about 0.1 mg / kg / day to about 8 mg / kg / day QD, BID or TID (Method 1). Preferably, the pharmaceutical composition further comprises about 0.5 g to about 5 g of acetic acid. More preferably, the daily dose of acetic acid for an adult is about 1.5 g to about 3 g.

[0039] The present disclosure further provides the following embodiments of methods for preventing or preventing the progression of fatty liver disease: 1.1 Method 1, wherein the fatty liver disease is NASH. 1.2 Method 1 or 1.1, wherein the fatty liver disease is NASH or NAFLD or ASH. 1.3 Any of the preceding processes wherein the denatonium salt is denatonium acetate. 1.4 Any of the preceding processes wherein the denatonium salt is denatonium citrate. 1.5 Any of the preceding processes wherein the denatonium salt is denatonium maleate. 1.6 Any of the preceding methods, wherein the daily dose of denatonium salt is 0.2 mg / kg / day QD. 1.7 Any of the preceding methods, wherein the daily dose of denatonium salt is 1.0 mg / kg / day QD. 1.8 Any of the preceding methods, wherein the daily dose of denatonium salt is 4.0 mg / kg / day QD.

[0040] In a second aspect, the present disclosure provides a method for treating fatty liver disease with or without liver fibrosis (e.g., nonalcoholic steatohepatitis (NASH), alcoholic steatohepatitis (ASH), nonalcoholic fatty liver disease (NAFLD) or HIV-associated steatohepatitis) comprising administering an effective amount of a pharmaceutical composition comprising a denatonium salt, wherein the denatonium salt is selected from the group consisting of denatonium acetate (DA), denatonium citrate, denatonium maleate, denatonium saccharide and denatonium tartrate, and the daily dose of the denatonium salt for the treatment of existing fatty liver disease is about 1.0 mg / kg / day to about 10 mg / kg / day administered QD, BID or TID (Method 2). Preferably, the pharmaceutical composition further comprises about 0.5 g to about 5 g of acetic acid. More preferably, the daily dose of acetic acid for an adult is about 1.5 g to about 3 g.

[0041] The present disclosure further provides the following embodiments of methods for the treatment of fatty liver disease: 2.1 Method 2, wherein the fatty liver disease is NASH. 2.2 Method 2 or 2.1, wherein the fatty liver disease is NASH or NAFLD or ASH. 2.3 Any of the preceding processes wherein the denatonium salt is denatonium acetate. 2.4 Any of the preceding processes wherein the denatonium salt is denatonium citrate. 2.5 Any of the preceding processes wherein the denatonium salt is denatonium maleate. 2.6 Any of the preceding methods wherein the daily dose of denatonium salt is 2.5 mg / kg / day BID. 2.7 Any of the preceding methods wherein the daily dose of denatonium salt is 5 mg / kg / day BID. 2.8 Any of the preceding methods wherein the daily dose of denatonium salt is 7.5 mg / kg / day BID.

[0042] (FDA Guidance on Clinical Trials for the Treatment of NASH) On January 29, 2021, the U.S. Food and Drug Administration (FDA) held a seminar on how potential treatments can show efficacy in animal models and clinical trials for NASH with fibrosis. The FDA confirmed that NASH (with fibrosis, hereafter NASH) is a serious condition and that the clinical use of surrogate endpoints can predict clinical benefit. In animal studies (such as those provided in Example 1 herein), histopathological examination is better evidence of the benefit of treatment, prevention, or disease progression (depending on the length of the animal study). Therefore, in clinical trials, the FDA accepts surrogate endpoints and liver biopsies as a means of showing clinical benefit (or lack thereof). The FDA recognized that the challenge of developing NASH drugs is due to the gradual and slow progression of chronic inflammatory changes in the liver, and that a NASH drug that completely prevents or treats or delays progression of NASH (progressive liver fibrosis) may be a lifelong treatment. Regarding surrogate endpoints, the FDA suggests that histopathology is "substantially likely to predict clinical benefit." The FDA has shown that NASH-driven liver "fibrosis, but not other histologic features of steatohepatitis, is independently associated with increased mortality, transplantation, and liver-related events" (citing Angulo et al. Gastroenterology, 149:389-397, 2015).

[0043] When conducting clinical trials, the FDA suggests that early-phase trials should assess liver stiffness using non-invasive disease-specific biomarkers (e.g., aminotransferases), total bilirubin, and radiographic methods (e.g., elastography, MRI-PDFF). For approval, the FDA will accept improvement in liver histology. "Liver biopsy is a surrogate based on studies that have demonstrated that histological improvement is likely to predict improved clinical outcomes in patients with NASH." Liver fibrosis is graded as stage 0 (none), stage 1, stage 2, stage 3, and stage 4 (cirrhosis). The recommended points for NASH are (1) resolution of steatohepatitis and no worsening of liver fibrosis; or (2) improvement of liver fibrosis and no worsening of steatohepatitis; or (3) both resolution of steatohepatitis and improvement of fibrosis. EXAMPLES

[0044] Example 1 This example provides the results of a 52-week study of low-dose DA (30 mg / kg / day QD by oral gavage) in mice in which the NASH state was dietarily induced. Such long-term (48-52 weeks) animal-induced NASH data can be interpreted as supporting methods to prevent and slow the progression of fatty liver disease, including NASH. Male C57BL / 6 mice (4 weeks old at the start of the study) were maintained on a high-fat "Western diet" (AMLN diet) (DIO-NASH) (D09100301, Research Diet, USA) (40% fat (18% trans fat), 40% carbohydrate (20% fructose), 2% cholesterol, 40 kcal% fat, 20 kcal% fructose, 2% cholesterol) upon arrival to induce nonalcoholic steatohepatitis (NASH). After acclimation, groups 1 and 2 (n=22 each) received vehicle (distilled water) or denatonium acetate (DA) at 30 mg / kg (respectively) by intragastric gavage (PO). Doses were administered once daily (QD) for a full year (up to day 366) at a dose of 1 mL / kg. Body weight and clinical findings were monitored throughout the study. Blood was collected from fasted animals at baseline (day 1) and periodically throughout the study (weeks 4, 12, 24, and 48) and processed to serum or plasma to assess metabolic parameters [glucose, insulin, hemoglobin A1c (HbA1c), glucagon-like peptides 1 and 2 (GLP-1 and -2)], selected serum chemistry parameters [Ala aminotransferase (ALT), Asp aminotransferase (AST), albumin (ALB), and total bile acids (BA)], and multiple cytokines (32 in total). After 52 weeks of QD treatment, fasted animals were bled and euthanized, and livers were harvested and weighed. Final serum samples were assessed for low- and high-density cholesterol (LDL and HDL, respectively) and triglycerides (TGA); livers were assessed for total cholesterol (TC), TGA, and free fatty acids (FFA).

[0045] Three animals were found to have died during the study, including one vehicle-treated mouse (at week 51) and two DA-treated mice (at weeks 12 and 49, respectively). None of these three animals had shown clinical signs prior to their deaths, but one of the DA-treated deceased animals showed weight loss beginning at week 44, and the other DA-treated deceased was significantly heavier than the other animals from the start of the study. It was unclear whether any of the deaths in Group 2 (DA-treated) were related to the test article. No other clinical findings were noted during the study.

[0046] For both groups, body weight increased by an average of approximately 200% from baseline (i.e., a tripling of body weight) during the 1-year chronic study. Of note, with the sole exception of relative body weight at the first time point post-treatment (day 4), when relative body weight was lower in DA-treated mice in group 2, body weight (expressed as absolute or relative values) was not statistically significantly different between the two groups during the study.

[0047] For in-life blood samples, DA-treated mice showed the following significant differences (compared to vehicle): Metabolic parameters: fasting plasma glucose and GLP-1 levels at week 24, and reduction in HbA1c at weeks 12 and 48; and insulin levels at weeks 4 and 12. No significant differences were found in GLP-2 levels (assessed only at week 48). · Serum chemistry: Decreased ALT at weeks 24 and 48, and AST and BA at week 24. No significant differences were seen in serum ALB levels. Cytokines: IP-10 and MIP-1B at week 4; IL-13 at week 12; MIP-1a at week 24; IL-6, IL-9 and KC at week 48; and reduced MIG at weeks 4 and 48. No significant treatment-related differences were observed in the levels of other evaluated cytokines (n=24).

[0048] At necropsy after 1 year of QD treatment, relative (normalized to body weight) but not absolute liver weight was reduced in DA-treated mice (compared to vehicle-treated animals).

[0049] Final blood samples revealed that after one year of daily dosing, serum TGA levels were significantly reduced in DA-treated mice (compared to vehicle-treated animals). Postmortem liver samples showed significantly reduced TGA levels, but not TC or FFA levels, in DA-treated mice (compared to vehicle-treated animals).

[0050] Thus, DA administration (30 mg / kg, PO, QD) for one year to AMLN diet-induced NASH mice did not appear to adversely affect survival, body weight, or clinical signs. Compared to vehicle, DA led to significant changes in many metabolic parameters (fasting glucose, GLP-1, HbA1c, and insulin), serum chemistry (ALT, AST, and BA), and a subset of cytokines (IL-6, IL-9, IL-13, IP-10, KC, MIG, MIP-1a, and MIP-1B) at selected time points during life. After one year of QD administration, DA-treated mice showed reduced relative liver weight and TGA levels in both serum and liver (compared to vehicle).

[0051] Male C57BL / 6 mice (n=44) were purchased as 4-week-old animals from Taconic Biosciences, Inc. (Rensselaer, NY). Upon arrival, animals were weighed using an electronic balance (Ohaus SCOUT® PRO, Parsippany, NJ), underwent clinical examination to ensure the mice were in good condition, and were group-housed (up to 4 per cage). Animals were maintained in HEPA-filtered static cages with SaniChip bedding 7090A (Harlan Teklad, Hayward, CA). Animal room controls were set to maintain temperature and relative humidity at 22°C ± 4°C and 50% ± 20%, respectively. The animal room had a 12:12 light / dark cycle. Animals were allowed to acclimate in situ for at least 3 days before participating in the study. Upon arrival and throughout the study period, mice had free access to water (via water bottle) and (except where noted for fasting) a high-fat "Western diet" containing 40 kcal% fat, 20 kcal% fructose, and 2% cholesterol (AMLN diet, No. D09100301; Research Diets, New Brunswick, NJ). DA was formulated at 30 mg / mL in DW. Solid DA was weighed and added to the appropriate amount of DW; the solution was mixed well and visually inspected to ensure no precipitation was present and the test article was fully solubilized. DA dosing solutions were made up fresh every week and kept refrigerated at 4°C between uses for daily dosing.

[0052] Blood and fecal samples were collected from fasted animals on day -1 (i.e., 1 day before dosing began); and at weeks 4, 12, 24 and 48 (i.e., 1, 3, 6, and 12 months after dosing). Fecal samples (4 pellets per cage of 3–4 mice) were collected directly from the floor of each cage, divided into two aliquots, and stored at -80 °C for future microbiota and microbiome analyses. The results of these analyses are not included in this report. Blood (100 μL / animal) was collected from each animal via the submandibular route. All blood samples were evaluated for glucose levels and processed to both serum and plasma. Depending on the time point and assay, either all or half of the samples (i.e., 10 or 11) per group were evaluated for the following parameters: - Serum: selected serum chemistry parameters [Ala aminotransferase (ALT), Asp aminotransferase (AST), albumin (ALB), and total bile acids (BA)], insulin, and glucagon-like peptides 1 and 2 (GLP-1 and -2) - Plasma: Hemoglobin A1c (HbA1c) and cytokines.

[0053] On day 366 (week 52), fasted mice were weighed, subjected to terminal cardiac puncture, and euthanized. Blood was processed to serum. Livers were removed, weighed, flash frozen, and stored at -80°C; remaining tissue was discarded. Serum was assessed for low- and high-density cholesterol (LDL and HDL, respectively) and triglycerides (TGA). Livers were assessed for total cholesterol (TC), TGA, and free fatty acids (FFA).

[0054] Blood and liver parameters were measured using the kits and devices shown in Table 3. [Table 3]

[0055] result: For both groups, body weight increased by an average of approximately 200% from baseline (i.e., body weight tripled) during the 1-year chronic study, with the majority of this increase (approximately 150%) occurring during the first 5 months. Of note, body weight (expressed as absolute or relative values) was not statistically significantly different between the two groups during the study, with the only exception of relative weight at the first time point after treatment initiation (day 4), where the relative weight of DA-treated mice in group 2 was lower.

[0056] FIG. 1 shows group mean absolute body weights (FIG. 1A) and relative body weights (% baseline, FIG. 1B) of mice fed the AMLN diet during one year of daily treatment with vehicle or 30 mg / kg DA.

[0057] Figure 2 shows that fasting blood glucose levels in DA-treated mice were significantly lower than those in vehicle-treated mice at week 24 only. Comparison with vehicle values ​​on each day by unpaired two-tailed t-test. Differences were considered statistically significant at p<0.05. *p<0.05. NS, not significant.

[0058] Figure 3 shows that insulin levels in DA-treated mice were significantly elevated at weeks 4 and 12, but not thereafter (compared to those in vehicle-treated mice). Comparisons with vehicle values ​​on each day by unpaired two-tailed t-test. Differences were considered statistically significant at p<0.05. *p<0.05. **p<0.01. NS, not significant.

[0059] Figure 4 shows that HbA1c levels in DA-treated mice were significantly reduced at weeks 12 and 48, but not at week 24 (compared to those in vehicle-treated mice). (HbA1c levels were not assayed pre-dose or at week 4.) Comparison with vehicle values ​​on each day by unpaired two-tailed t-test. Differences were considered statistically significant at p<0.05. *p<0.05. **p<0.01. ***p<0.001. NS, not significant. HbA1c was not assayed pre-dose or at week 4.

[0060] Figure 5A-B shows that GLP-1 levels in DA-treated mice were significantly reduced only at week 24 (compared to those in vehicle-treated mice). The magnitude of the difference was similar to other time points (not statistically significant), but the variation at week 24 was smaller than at other weeks, suggesting that the statistical significance of this effect is not meaningful. Comparisons with vehicle values ​​at each day by unpaired two-tailed t-test. Differences were considered statistically significant at p<0.05. ***p<0.001. NS, not significant. Note that GLP-2 was only assayed at week 48.

[0061] Serum chemistry: Figure 6 shows that serum ALT activity levels in DA-treated mice were significantly decreased at 24 and 48 weeks, but not earlier (compared to those in vehicle-treated mice). Comparison with vehicle values ​​on each day by unpaired two-tailed t-test. Differences were considered statistically significant at p<0.05. **p<0.01. ***p<0.001. NS, not significant.

[0062] Figure 7 shows that serum AST activity levels in DA-treated mice were significantly reduced only at week 24 (compared to those in vehicle-treated mice). Comparison with vehicle values ​​on each day by unpaired two-tailed t-test. Differences were considered statistically significant at p<0.05. **p<0.01. NS, not significant.

[0063] Figure 8 shows that serum ALB levels in DA-treated mice were not significantly different from those in vehicle-treated mice at any assessment time point. Comparison with vehicle values ​​on each day by unpaired two-tailed t-test. Differences were considered statistically significant at p<0.05. NS, not significant.

[0064] Figure 9 shows that serum BA levels in DA-treated mice were significantly reduced only at week 24 (compared to those in vehicle-treated mice). Comparison with vehicle values ​​on each day by unpaired two-tailed t-test. Differences were considered statistically significant at p<0.05. *p<0.05. NS, not significant.

[0065] Blood cytokine levels The mean values ​​(n=10) of cytokines with statistically significant differences between groups are plotted in Figure 10. Of these 10 cytokines, 8 showed significant group differences after treatment initiation. The other two cytokines [MCP-1 (monocyte chemoattractant protein 1, also known as CCL2) and TNFα (tumor necrosis factor alpha)] showed significant group differences only at the pre-treatment time point, an effect apparently unrelated to treatment. Statistically significant reductions in cytokine levels in DA-treated mice (compared to vehicle-treated mice) were observed as follows: Week 4 only: IP-10 (interferon-gamma-inducible protein 10, aka CXCL10); MIP-1B (macrophage inflammatory protein 1 beta, aka CCL4). Week 12 only: IL-13 (interleukin 13). Week 24 only: MIP-1a (macrophage inflammatory protein 1 alpha). Week 48 only: IL-6 (Interleukin 6); IL-9 (Interleukin 9); KC (Keratinocyte-inducing chemokine, also known as CXCL1 or CINC-1). Weeks 4 and 48 only: MIG (monokine-inducible gamma interferon, also known as CXCL9).

[0066] FIG. 10 shows blood levels of cytokines that showed statistically significant differences (n=10) in mice fed the AMLN diet during one year of daily treatment with vehicle or 30 mg / kg for IL-6 (FIG. 10A), IL-9 (FIG. 10B), IL-13 (FIG. 10C), IP-10 (FIG. 10D), KC (FIG. 10E), MCP-1 (FIG. 10F), MIP-1a (FIG. 10G), MIP-1B (FIG. 10H), MIG (FIG. 10I) and TNFα-6 (FIG. 10J). Integers in parentheses in the cytokine names / graph titles correspond to the assay numbers used as part of the multiplex assay. Comparisons with vehicle values ​​on each day by unpaired two-tailed t-test. Differences were considered statistically significant at p<0.05. *p<0.05. **p<0.01. NS, not significant.

[0067] Final liver parameters Figure 11A (absolute values) and B (relative values, normalized to body weight) show autopsy liver weights after one year of daily treatment with vehicle or 30 mg / kg DA. Comparisons with vehicle values ​​on each day by unpaired two-tailed t-test. Differences were considered statistically significant at p<0.05. *p<0.05. NS, not significant.

[0068] Figure 12 shows serum HDL, LDL and TGA levels after one year of daily treatment with vehicle or 30 mg / kg DA. Serum TGA levels were significantly reduced in DA-treated mice after one year of daily treatment, but not HDL or LDL (compared to vehicle-treated animals). DA, denatonium acetate. HDL, high-density lipoprotein. LDL, low-density lipoprotein. TGA, triglycerides. Comparison with vehicle values ​​on each day by unpaired two-tailed t-test. Differences were considered statistically significant at p<0.05. *p<0.05. NS, not significant.

[0069] Figures 13A-C show liver TGA (Figure 13A), TC (Figure 13B) and FFA (Figure 13C) after one year of daily treatment with vehicle or 30 mg / kg DA. After one year of daily treatment, liver TGA levels, but not TC or FFA levels, were significantly decreased in DA-treated mice (compared to vehicle-treated mice).

[0070] The conclusion of this study was that DA administration (30 mg / kg, PO, QD) for 1 year in AMLN diet-induced NASH mice did not appear to adversely affect survival, body weight, or clinical signs. Compared to vehicle, DA caused significant changes in a small number of metabolic parameters (fasting glucose, GLP-1, HbA1c, and insulin), serum chemistry (ALT, AST, and BA), and a subset of cytokines (IL-6, IL-9, IL-13, IP-10, KC, MIG, MIP-1a, and MIP-1B) at selected time points during life. After 1 year of QD administration, DA-treated mice showed reduced relative liver weight and TGA levels in both serum and liver (compared to vehicle).

[0071] In conclusion, the histopathology data from this 1-year NASH study in Figures 12-14 showed that treatment with DA significantly improved steatosis and improved / inhibited fibrosis based on blinded histopathology examination (as an example, total fibrosis score 1.52 vs. 0.58, p-value <0.0001). Based on the new FDA NASH / fibrosis criteria, histopathology data not seen in many other NASH animal studies (often very short term studies, see Table 2 herein) support the conclusion that a method for preventing or slowing the progression of fatty liver disease selected from the group consisting of nonalcoholic steatohepatitis (NASH), alcoholic steatohepatitis (ASH), nonalcoholic fatty liver disease (NAFLD) and HIV-associated steatohepatitis, with or without liver fibrosis, can be accomplished by administering an oral dose of a denatonium salt selected from the group consisting of denatonium acetate (DA), denatonium citrate, denatonium maleate, denatonium saccharide, and denatonium tartrate.

[0072] Example 2 This example provides results from a second in vivo mouse model of fatty liver disease treatment to investigate the therapeutic effect of DA on the treatment of NASH compared to the positive control semaglutide (a commercially available GLP-1 agonist drug for lowering HbA1c).The differences of this study from the prevention model of Example 1 are that a higher dose of DA (75mg / kg) was used compared to 30mg / kg, administration was twice a day (BID compared to QD in the study of Example 1), the positive control semaglutide was used, a different mouse strain (B6 mice) was used, and the animals were already adults at the start of the study (4 weeks old compared to 23 weeks old in Example 2 compared to Example 1) because they had already been fed the AMLN diet for 17 weeks before the start of the study.The test dose started at 75mg / kg BID. However, after 2 weeks of treatment, this DA dose was found to be poorly tolerated, so the DA dose was reduced to 50 mg / kg BID for the remaining 10 weeks of treatment (total of 12 weeks).

[0073] The study included three groups of 10 mice each: (A) vehicle control with distilled water by gavage BID, (B) DA by gavage BID, and (C) semaglutide 10 mmol / kg subcutaneous QD. Body weight and changes were measured three times a week. Serum metabolic markers (blood glucose, blood insulin, HbA1c, HDL, LDL triglycerides and bile acids) were measured at the beginning (baseline) and end of the study. At the end of the study, histopathology of liver samples and serum levels of inflammatory biomarkers (IL-6, TNFα, CK-18 and TGF-β) were evaluated. Histopathology was performed blinded using score scales according to the NAFLD activity score and fibrosis score according to Table 4. [Table 4]

[0074] Figure 14 shows that treatment with DA (ARD-101) significantly improved NAFLD activity scores based on blinded histopathological examination. Figures 15A-B show that treatment with ARD-101 (DA) showed a significant effect on body weight and body weight change (Figures 16A-B) in AMLN diet-induced NASH mice.

[0075] Figures 16A and 16B show liver weight (Figure 16A) and liver / body weight ratio (Figure 16B), demonstrating that DA (ARD-101) significantly reduced liver weight and liver / body weight ratio compared to vehicle.

[0076] Figure 17A shows ALT levels and Figure 17B shows AST levels. At the end of the study, DA (ARD-101) significantly reduced ALT and AST levels compared to the vehicle control.

[0077] Figures 18A (triglycerides), 18B (LDL) and 18C (HDL) show that DA (ARD-101) significantly reduced triglycerides (TG), low density lipoprotein (LDL) and high density lipoprotein (HDL) at the end of the study, respectively.

[0078] Figure 19 shows fasting blood glucose levels at the end of the study. Figure 20 shows HbA1c levels at the end of the study. Baseline HbA1c levels were 5.0%. Figure 21 shows insulin levels at the end of the study. Baseline insulin levels were 1.5ng / ml. Figure 22 shows that the two treatments did not significantly affect bile acid levels compared to vehicle controls. Baseline bile acid levels were 30 μmol / L.

[0079] Figures 23A (CK-18) and 23B (TGF-β) show that DA (ARD-101) significantly reduced CK-18 levels compared to vehicle controls (Figure 24A).

[0080] Figures 24A and 24B show that at the end of the study, the two treatments did not significantly affect IL-6 and TNF-α levels compared to vehicle.

Claims

1. A pharmaceutical composition for preventing, inhibiting the progression of and / or treating fatty liver disease with or without liver fibrosis, comprising a denatonium salt, wherein the denatonium salt is selected from the group consisting of denatonium acetate (DA), denatonium citrate, denatonium maleate, denatonium saccharide and denatonium tartrate.

2. The pharmaceutical composition of claim 1, wherein the fatty liver disease is non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH) or alcoholic steatohepatitis (ASH), and the denatonium salt is a racemic mixture or an enantiomer or diastereoisomer.

3. 3. The pharmaceutical composition of claim 1, wherein the daily dosage of the denatonium salt for an adult is from about 1.0 mg / kg / day to about 12 mg / kg / day.

4. 4. The pharmaceutical composition of claim 3, wherein the daily dosage of the denatonium salt for an adult is from about 2 mg / kg / day to about 8 mg / kg / day.

5. The pharmaceutical composition of claim 4, wherein the daily dosage of DA for an adult is about 3 mg / kg / day to about 6 mg / kg / day.

6. The pharmaceutical composition of claim 1 or 2, wherein the dosage range of the denatonium salt for preventing or preventing the progression of fatty liver disease in adults is from about 0.1 mg / kg / day to about 8 mg / kg / day.

7. 7. The pharmaceutical composition of claim 6, wherein the daily dosage of the denatonium salt for an adult is from about 0.25 mg / kg / day to about 4 mg / kg / day.

8. The pharmaceutical composition of claim 1 or 2, wherein the dosage range of the denatonium salt for preventing or preventing the progression of fatty liver disease in adults is from about 0.1 mg / kg / day to about 6 mg / kg / day.

9. 9. The pharmaceutical composition of any one of claims 1 to 8, wherein the daily dose of the denatonium salt is administered once daily (QD), twice daily (BID) or three times daily (TID).

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the fatty liver disease comprises NASH.

11. The pharmaceutical composition according to any one of claims 1 to 9, wherein the fatty liver disease comprises ASH.

12. The pharmaceutical composition according to any one of claims 1 to 9, wherein the fatty liver disease comprises NAFLD.

13. The pharmaceutical composition according to any one of claims 1 to 9, wherein the fatty liver disease comprises HIV-associated steatohepatitis.

14. The pharmaceutical composition according to any one of claims 1 to 13, wherein the fatty liver disease is accompanied by liver fibrosis.

15. The pharmaceutical composition according to any one of claims 1 to 13, wherein the fatty liver disease is not accompanied by liver fibrosis.

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