Pharmaceutical compositions for the treatment or prevention of various inflammatory conditions

Abstract

Disclosed herein is a method for treating, preventing, and / or slowing the progression of various chronic inflammatory condition groups including: (1) the Type 2 Diabetes group (Metabolic Syndrome (MET), obesity, hyperglycemia); (2) ARDS (Acute Respiratory Distress Syndrome); (3) chronic autoimmune inflammatory conditions (Rheumatoid Arthritis (RA), Lupus, and Psoriasis); (4) inflammatory bowel diseases (IBD), such as Crohn's disease and ulcerative colitis; (5) metabolite group-mediated diseases (atherosclerosis, hypertension, and congestive heart failure); and (6) eating disorders, such as Prader-Willi Syndrome and other monogenic and symptomatic obesity disorders including leptin pathway defects, each method comprising orally administering a pharmaceutical composition comprising a denatonium salt. The present disclosure is based on the results of a series of studies that tracked biomarker cluster levels to track mediators of inflammatory conditions and mediators of gut signaling hormones in response to orally administered denatonium salts.

Description

TECHNICAL FIELD

[0001] The present disclosure provides a method for treating, preventing, and / or slowing the progression of various chronic inflammatory condition groups including (1) the Type 2 Diabetes group (Metabolic Syndrome (MET), obesity, hyperglycemia); (2) ARDS (Acute Respiratory Distress Syndrome); (3) chronic autoimmune inflammatory conditions (Rheumatoid Arthritis (RA), Lupus, and Psoriasis); (4) inflammatory bowel diseases (IBD), such as Crohn’s disease and ulcerative colitis; (5) metabolite group-mediated diseases (atherosclerosis, hypertension, and congestive heart failure); and (6) eating disorders, such as Prader-Willi Syndrome and other monogenic and symptomatic obesity disorders including leptin pathway defects, each method comprising oral administration of a pharmaceutical composition comprising a denatonium salt. The present disclosure is based on the results of a series of studies that tracked biomarker cluster levels to track mediators of inflammatory conditions and mediators of gut signaling hormones in response to oral administration of a denatonium salt. The present disclosure also provides a pharmaceutical composition for treating and preventing various inflammatory conditions that can be tracked by pro-inflammatory biomarkers, including administration of a pharmaceutical composition comprising a denatonium salt. Preferably, the pharmaceutical composition for daily oral administration comprises a denatonium salt that delivers a total daily dose from about 20 mg to about 5000 mg BID to an adult. Preferably, the denatonium salt is selected from the group consisting of denatonium acetate, denatonium citrate, denatonium maleate, and denatonium tartrate. BACKGROUND

[0002] Over the past 40 years, the level of obesity has increased more than twofold globally. Obesity is prone to cause metabolic syndrome and is associated with coronary heart disease, stroke, Type 2 diabetes, certain forms of cancer, and severe illness and higher risk of death even for the coronavirus pandemic, which represents one of the most important global health challenges currently. With the emergence of this problem, the understanding of the pathological mechanisms that link the obese state to disease development has also improved. At the core of these mechanisms is the exacerbation of a state of systemic inflammation due to obesity, which leads to multiple pathologies. Therefore, there is a significant need for therapeutic and prophylactic drugs that address the problem of appetite and inflammatory signaling. The present disclosure addresses this need.

[0003] Inflammatory diseases

[0004] Various inflammatory diseases are currently treated with anti-tumor necrosis factor (TNF) (and anti-interleukin (IL)-6) proteins and antibodies. Such therapeutic proteins are approved for rheumatoid arthritis, polyarticular juvenile idiopathic arthritis (JIA) in children, psoriatic arthritis, lupus, ankylosing spondylitis (AS), chronic plaque psoriasis (Ps), uveitis, IBD (including ulcerative colitis and Crohn's disease), and many others. These biologic drugs act by binding to and clearing circulating TNFa (and IL-6) with antibodies or fusion proteins such as etanercept ) However, these anti-TNFa drugs and other any biologic drugs that bind to and clear inflammatory cytokines have serious side effects. Side effects are caused by the inhibition of most TNF signaling. Since TNF has immune surveillance functions that are also inhibited by these biologic drugs, infections and reduced immune surveillance are more likely to occur, including various infectious diseases listed on black box warning labels and increased incidence of malignancies including leukemias and lymphomas. Thus, there is a need in the art for more cost-effective small molecule therapeutics that knock down (but not necessarily eliminate) circulating TNF. Since protein-based therapeutics cannot be administered orally, there is a need in the art for oral small molecule agents that are more subtle or more self-limiting in eliminating circulating TNF by preventing TNF production as a pro-inflammatory cytokine rather than any clearance of existing and already produced TNF.

[0005] For example, the US FDA approved label for adalimumab indicates the following side effects: serious infections (i.e., including TB and infections by viruses, fungi, or bacteria), worsening of hepatitis B infection in viral carriers, allergic reactions, and increased risk of various leukemias and lymphomas.

[0006] Metabolic syndrome

[0007] Metabolic Syndrome (METS) is a cluster of multiple factors that increase the risk of developing type 2 diabetes and cardiovascular disease. METS is a clustering of at least three of the following five medical conditions: (1) visceral obesity; (2) elevated blood pressure; (3) elevated blood glucose; (4) elevated serum triglycerides; and (5) low serum high-density lipoprotein (HDL).

[0008] According to the International Diabetes Foundation (IDF), metabolic syndrome presents with central obesity and any two of the following: (1) elevated triglycerides (TG) > 150 mg / dL (1.7 mmol / L) or specific treatment for elevated triglycerides; (2) reduced HDL, < 40 mg / dL (1.03 mmol / L) in men, < 50 mg / dL (1.29 mmol / L) in women; (3) elevated blood pressure (BP) with systolic > 130 or diastolic > 85 mm Hg or hypertension treatment and (4) elevated fasting plasma glucose (FPG) > 100 mg / dL (5.6 mmol / L) or previous diagnosis of type 2 diabetes.

[0009] Metabolic syndrome can also be defined as presenting with hyperinsulinemia and any two of the following: (1) abdominal obesity (waist-to-hip ratio > 0.90 or BMI 30 kg / m 2 ), (2) dyslipidemia (TG > 1.7 or HDL < 0.9 mmol / L) and (3) hypertension (BP > 140 / 90 mm Hg or use of antihypertensive medication). In one clinical study, carbohydrate restriction was considered a first-line dietary intervention for METS, which looked for significance in a panel of biomarkers from fasting participants, including the inflammatory biomarkers TNFα, IL-6 and MCP-1 (Al-Sarraj et al., J. Nutrition 139(9): 1667-1675, 2009). The study (n = 20) found significance for MPC-1, ICAM-1 and TNFα, but not for IL-6.

[0010] METS affects 20-25% of the global adult population, including 35% of the U.S. adult population. METS is present in about 60% of U.S. residents > 50 years of age. And METS is associated with a higher frequency of autoimmune diseases. Thus, there is a need in the art for providing safer and more effective METS therapeutics.

[0011] ARDS and viral respiratory infections

[0012] Acute respiratory distress syndrome (ARDS) is a life-threatening condition characterized by acute onset of hypoxia and pulmonary infiltrates and is caused by conditions such as sepsis, pneumonia, trauma, burns, pancreatitis, and blood transfusion. ARDS causes diffuse lung inflammation, which leads to increased lung vascular permeability, pulmonary edema, and alveolar epithelial injury. Diagnosis of ARDS is made according to the following criteria: (1) acute onset; (2) bilateral lung infiltrates of non-cardiogenic origin on chest x-ray or computed tomography (CT) scan; and (3) moderate to severe impairment of oxygenation. The mortality rate for severe ARDS is 45%. The severity of ARDS is defined by the degree of hypoxemia, which is calculated as the ratio of arterial oxygen tension to fraction of inspired oxygen (PaO2 / FiO2). As classified by the Berlin definition of ARDS, ARDS can be mild, moderate, or severe, with mild PaO2 / FiO2 being 200-300, moderate being 100-199, and severe being <100.

[0013] Generally, the development of ARDS can be divided into two phases: an initiation phase, followed by an effector phase. The initiation phase of ARDS involves the release of inflammatory mediators (i.e., cytokines; complement and coagulation factors; and arachidonic acid metabolites) that promote systemic inflammation, leading to lung neutrophil recruitment. The second phase, the effector phase, involves activation of neutrophils and subsequent release of toxic oxygen radicals and proteolytic enzymes, specifically neutrophil elastase (NE). NE has the ability to damage lung endothelial cells and degrade extracellular matrix products including elastin, collagen, and fibronectin of the lung basement membrane.

[0014] While the end-stage pathologies of these different forms are identical, there are many different forms of ARDS with vastly different etiologies and courses. Examples of clinical events that can lead to different forms of ARDS include trauma, hemorrhage, diffuse pneumonia, viral-induced pneumonia (including but not limited to COVID-19 and SARS), inhalation of toxic gases, and sepsis. In the case of the COVID-19 pandemic in 2020, it is a viral pneumonia that can be observed in many patients requiring intensive care driving ARDS. Regardless of the initiating cause, ARDS has the following in common: accumulation of fluid within the lung and exudate leads to diffuse alveolar damage and impaired gas exchange in the alveoli. Downstream, common to all (regardless of the initiating cause of ARDS) is exacerbation due to inflammation, fluid release, cell migration and proliferation, and increase in proinflammatory cytokines.

[0015] Viral respiratory infections are typically characterized by a latent period of usually 2-7 days, during which infected individuals typically exhibit high fever, sometimes accompanied by chills, headache, malaise, and myalgias. Viral infections of the lung account for approximately 10-15% of ICU admissions per year in the United States in the absence of a pandemic, and a significant percentage of deaths per year from influenza in the absence of a coronavirus pandemic. The 2020 pandemic from COVID-19 illustrates this course of disease. The illness progresses with the onset of nonproductive dry cough or dyspnea, with or without development of hypoxemia. A large number of cases require intubation and mechanical ventilation. In addition, during the peak of respiratory illness, approximately 50% of infected individuals develop leukopenia and thrombocytopenia (MMWR Morb Mortal Wkly Rep. 2003 Mar 28;52(12):255-6).

[0016] The mode of spread of viral load, such as coronavirus or influenza virus, indicates that the viral pathogen is droplet transmission or contact transmission (N. Engl. J. Med. 2003 May 15;348(20):1995-2005). SARS-1 and -2 are etiologically related to viruses, and the SARS-associated coronavirus (SARS-CoV) is a member of the coronavirus family of enveloped viruses that replicate in the cytoplasm of infected animal host cells. Coronaviruses are typically characterized as single-stranded RNA viruses with a genome of approximately 30,000 nucleotides (Science. 2003 May 30;300(5624):1394-9). Coronaviruses are divided into three known groups; the first two groups cause mammalian coronavirus infections, and the third group causes avian coronavirus infections (J. S. M. Peiris, in Medical Microbiology (Eighteenth Edition), 2012, 587-593). Coronaviruses are considered to be the causative agents of several serious diseases in many animals, such as infectious bronchitis virus, feline infectious peritonitis virus, and infectious gastroenteritis virus, are important veterinary pathogens (Viruses. 2019 Jan;11(1):59).

[0017] Thus, there is a need for effective treatment of patients diagnosed with SARS, patients infected with an infectious agent associated with SARS, such as patients infected with SARS-CoV, or patients at imminent risk of infection with SARS, such as individuals exposed to or likely to be exposed to an infectious agent associated with SARS in the near future.

[0018] Treatment of ARDS in the prior art is inadequate. Thus, there is an urgent need for effective treatment of ARDS.

[0019] Metabolome

[0020] The gut microbiota has attracted much attention and an imbalance in the gut microbiota system is associated with several diseases depending on which bacterial groups are increased or decreased. Atherosclerotic disease, with manifestations such as myocardial infarction and stroke, is a major cause of serious illness and death in subjects with metabolic syndrome. The disease is thought to be caused by accumulation of cholesterol and recruitment of macrophages to the arterial wall and can thus be seen as both a metabolic and an inflammatory disease. Since the upper half of the 19th century, it has been thought that atherosclerosis is caused or promoted by pro-atherogenic changes in vascular cells being strengthened. However, there is still a need for better ways of slowing down the atherosclerotic changes in vascular cells and related diseases in the early stage. The present invention provides a method for slowing down the atherosclerotic changes in vascular cells by reducing gut signals supporting the atherosclerotic changes in vascular cells.

[0021] Hyperphagia

[0022] Modulation of food behaviour, including both control of appetite for some food components and preference for less fat food or lower calorie content food, can provide a mechanism for preventing the development of metabolic disorders including cardiovascular disease (Langley-Evans et al., Matern Child Nutr., 1, 142-148, 2005), particularly as occurs in our developed societies when high calorie density or fat rich, particularly saturated fat, foods are widely used.

[0023] One of the more important signals acting in the maintenance of energy balance and body weight is leptin, a circulating protein encoded by the ob gene expressed mainly in adipose tissue. Leptin plays a central role in the regulation of energy balance, suppressing food intake and increasing energy expenditure (Zhang et al., Nature, 372, 425-432, 1994). This protein circulates in the blood at a concentration proportional to the size of the fat depots; it passes through the blood-brain barrier by means of saturable systems and exerts most of its effects on central levels of energy balance through the interaction of the protein with receptors located in hypothalamic neurons and other areas of the brain (Tartaglia et al., Cell. 83, 1263-1271, 1995).

[0024] Animals with defects in the leptin signaling axis (either because they are unable to produce functional proteins or because they express defective forms of the leptin receptor) are characterized by overeating and severe obesity with an early appearance, as well as diabetes, hypothermia, and infertility. In humans, congenital defects in leptin signaling (lack of leptin or its receptor) are also associated with morbid obesity with an early appearance (Clement et al., Nature, 392, 398-401, 1998; Montague et al., Nature, 387, 903-908, 1997; Strobel et al., Nat. Genet., 18, 213-215, 1998). In this sense, the use of leptin in the treatment or prevention of diabetes whose direct cause is obesity has been proposed (WO 97 / 02004).

[0025] Although the short-term appetite-suppressing effects of leptin are thought to help manage obesity and related disorders in obese individuals, unfortunately, leptin administration alone is ineffective as a practical treatment, partly due to tolerability and compensatory upregulation of other pathways mediating hunger and satiety. Long-term treatment outcomes remain unsatisfactory.

[0026] With age, circulating leptin levels increase (Matheny et al., Diabetes 1997, 46, 2035-9; Iossa et al., J Nutr. 1999, 129, 1593-6), and sensitivity to this hormone is impaired (Qian et al., Proc. Soc. Exp. Biol. Med. 1998, 219, 160-5; Scarpace et al., Neuropharmacology, 2000, 39, 1872-9). Furthermore, high levels of circulating leptin can promote tolerance to the appetite-suppressing effects of this hormone, thus contributing to the maintenance and development of obesity and / or its complications. In fact, there is evidence that leptin tolerance is a major determinant of weight gain and age-related hyperlipidemia in rats [Iossa et al., J. Nutr., 1999, 129, 1593-6]. However, although circulating leptin concentration is generally considered to be proportional to body fat mass and that this amount usually increases with age, there is evidence that the increase in hyperleptinemia and the development of leptin resistance occur with age and are at least partially unrelated to the increase in hyperlipidemia (Gabriely et al., Diabetes, 2002, 51, 1016-21).

[0027] High leptin circulating levels are also associated with increased risk of cardiovascular disease [Ren, J. Endocrinol., 2004, 181, 1-10] and the development of insulin resistance [Huang et al., Int. J. Obes. Relat. Metab. Disord., 2004, 28, 470-5] in humans, and this is independent of body mass index / adiposity. SUMMARY

[0028] The present disclosure provides a method for treating, preventing, and / or slowing the progression of various chronic inflammatory disorder groups including (1) the Type 2 Diabetes group (Metabolic Syndrome (MET), obesity, hyperglycemia); (2) ARDS (Acute Respiratory Distress Syndrome); (3) chronic autoimmune inflammatory disorders (Rheumatoid Arthritis (RA), Lupus, and Psoriasis); (4) inflammatory bowel diseases (IBD), such as Crohn's disease and ulcerative colitis; (5) metabolite group-mediated diseases (atherosclerosis, hypertension, and congestive heart failure); and (6) hyperphagia disorders, such as Prader-Willi Syndrome and other monogenic and symptomatic obesity disorders including leptin pathway defects, each method comprising oral administration of a pharmaceutical composition comprising a denatonium salt. The present disclosure is based on the results of a series of studies that tracked biomarker cluster levels to track mediators of inflammatory disorders and mediators of gut signaling hormones in response to oral administration of a denatonium salt. The present disclosure also provides a pharmaceutical composition for treating and preventing various inflammatory conditions that can be tracked by proinflammatory biomarkers, including administration of a pharmaceutical composition comprising a denatonium salt. Preferably, the pharmaceutical composition for daily oral administration comprises a denatonium salt that delivers from about 20 mg to about 5000 mg of total daily dose BID to adults. Preferably, the denatonium salt is selected from the group consisting of denatonium acetate, denatonium citrate, denatonium maleate, and denatonium tartrate.

[0029] The present disclosure provides a method for treating, preventing and slowing the worsening of acute pulmonary inflammatory conditions including ARDS, the method comprising orally administering 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 sugar and denatonium tartrate. Preferably, the pharmaceutical composition further comprises about 0.5 g to about 5 g acetic acid. More preferably, for an adult, the daily dose of acetic acid is from about 1.5 g to about 3 g. Preferably, for an adult, the daily dose of the denatonium salt is from about 20 mg to about 5000 mg or from about 5 mg / kg to about 150 mg / kg body weight per day. More preferably, for an adult, the daily dose of DA is from about 50 mg to about 1000 mg. Most preferably, for an adult, the daily dose of DA is from about 60 mg to about 500 mg, or achieves a concentration in the gastrointestinal tract of from about 10 ppb to about 50 ppm. The daily dose of the denatonium salt is administered once a day, twice a day or three times a day.

[0030] The present disclosure provides a method for treating, preventing and slowing the worsening of acute pulmonary inflammatory conditions including ARDS, the method comprising orally administering 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 sugar and denatonium tartrate. Preferably, the pharmaceutical composition further comprises about 0.5 g to about 5 g acetic acid. More preferably, for an adult, the daily dose of acetic acid is from about 1.5 g to about 3 g. Preferably, for an adult, the daily dose of the denatonium salt is from about 20 mg to about 5000 mg or from about 5 mg / kg to about 150 mg / kg body weight per day. More preferably, for an adult, the daily dose of DA is from about 50 mg to about 1000 mg. Most preferably, for an adult, the daily dose of DA is from about 60 mg to about 500 mg, or achieves a concentration in the gastrointestinal tract of from about 10 ppb to about 50 ppm. The daily dose of the denatonium salt is administered once a day, twice a day or three times a day.

[0031] The present disclosure provides a method for treating, preventing, and slowing the progression of indications selected from the group of chronic autoimmune inflammatory disorders consisting of rheumatoid arthritis (RA), lupus, and psoriasis, comprising orally administering 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 sugar, and denatonium tartrate. Preferably, the pharmaceutical composition further comprises about 0.5 g to about 5 g acetic acid. More preferably, the daily dose of acetic acid is from about 1.5 g to about 3 g for an adult human. Preferably, the daily dose of the denatonium salt is from about 20 mg to about 5000 mg or from about 5 mg / kg to about 150 mg / kg body weight per day for an adult human. More preferably, the daily dose of DA is from about 50 mg to about 1000 mg for an adult human. Most preferably, the daily dose of DA is from about 60 mg to about 500 mg for an adult human, or achieves a concentration of from about 10 ppb to about 50 ppm in the gastrointestinal tract. The daily dose of the denatonium salt is administered once per day, twice per day, or three times per day.

[0032] The present disclosure provides a method for treating, preventing, and slowing the progression of indications selected from the group of chronic IBD consisting of Crohn's disease and ulcerative colitis, comprising orally administering 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 sugar, and denatonium tartrate. Preferably, the pharmaceutical composition further comprises about 0.5 g to about 5 g acetic acid. More preferably, the daily dose of acetic acid is from about 1.5 g to about 3 g for an adult human. Preferably, the daily dose of the denatonium salt is from about 20 mg to about 5000 mg or from about 5 mg / kg to about 150 mg / kg body weight per day for an adult human. More preferably, the daily dose of DA is from about 50 mg to about 1000 mg for an adult human. Most preferably, the daily dose of DA is from about 60 mg to about 500 mg for an adult human, or achieves a concentration of from about 10 ppb to about 50 ppm in the gastrointestinal tract. The daily dose of the denatonium salt is administered once per day, twice per day, or three times per day.

[0033] The present disclosure provides a method for treating, preventing, or slowing the progression of an indication selected from the group of atherosclerosis, hypertension, and congestive heart failure (CHF), comprising orally administering 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 sugar, and denatonium tartrate. Preferably, the pharmaceutical composition further comprises about 0.5 g to about 5 g acetic acid. More preferably, the daily dose of acetic acid is from about 1.5 g to about 3 g for an adult human. Preferably, the daily dose of the denatonium salt is from about 20 mg to about 5000 mg or from about 5 mg / kg to about 150 mg / kg body weight per day for an adult human. More preferably, the daily dose of DA is from about 50 mg to about 1000 mg for an adult human. Most preferably, the daily dose of DA is from about 60 mg to about 500 mg for an adult human, or achieves a concentration in the gastrointestinal tract of from about 10 ppb to about 50 ppm. The daily dose of the denatonium salt is administered once per day, twice per day, or three times per day.

[0034] The present disclosure provides a method for treating or slowing the progression of an indication selected from the group of Prader-Willi Syndrome and leptin pathway defects, comprising orally administering 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 sugar, and denatonium tartrate. Preferably, the pharmaceutical composition further comprises about 0.5 g to about 5 g acetic acid. More preferably, the daily dose of acetic acid is from about 1.5 g to about 3 g for an adult human. Preferably, the daily dose of the denatonium salt is from about 20 mg to about 5000 mg or from about 5 mg / kg to about 150 mg / kg body weight per day for an adult human. More preferably, the daily dose of DA is from about 50 mg to about 1000 mg for an adult human. Most preferably, the daily dose of DA is from about 60 mg to about 500 mg for an adult human, or achieves a concentration in the gastrointestinal tract of from about 10 ppb to about 50 ppm. The daily dose of the denatonium salt is administered once per day, twice per day, or three times per day. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 Body weight change over time is shown with DA administration compared to vehicle control.

[0036] Figure 2 Body weight change over time is shown with DA administration compared to vehicle control.

[0037] Figure 3 Body weight change at day 28 is shown. There is no statistically significant difference in body weight change at day 28 between the two experimental groups.

[0038] Figure 4 Fasting blood glucose levels at day 28 are shown. There was no statistically significant difference in fasting blood glucose levels at day 28 between the two experimental groups.

[0039] Figure 5 HbAlc levels at day 28 are shown. There was no statistically significant difference in blood HbAlc levels at day 28 between the two experimental groups.

[0040] Figure 6 Blood HDL levels at day 28 are shown. Animals treated with 23.1 mg / kg of DA showed a statistically significant decrease in blood HDL levels at day 28 compared to vehicle-treated animals.

[0041] Figure 7 Blood LDL cholesterol levels at day 28 are shown. There was no statistically significant difference in blood LDL levels at day 28 between the two experimental groups.

[0042] Figure 8 Blood total cholesterol levels (LDL plus HDL) at day 28 are shown. Animals treated with 23.1 mg / kg of DA showed a nearly significant decrease in blood total cholesterol levels at day 28 compared to vehicle-treated animals.

[0043] Figure 9 Blood insulin levels at day 28 are shown. There was no statistically significant difference in blood insulin levels at day 28 between the two experimental groups.

[0044] Figure 10 Blood bile acid levels at day 28 are shown. There was no statistically significant difference in blood bile acid levels at day 28 between the two experimental groups.

[0045] Figure 11 Neutrophil number and percentage at pre-dose and day 28 are shown. Although there was no statistically significant difference, DA-treated animals showed a trend toward increased neutrophil number compared to vehicle-treated controls.

[0046] Figure 12 Monocyte number and percentage at pre-dose and day 28 are shown. Although there was no statistically significant difference, DA-treated animals showed a trend toward increased monocyte number and percentage compared to vehicle-treated controls.

[0047] Figure 13Changes in lymphocyte and white blood cell numbers pre-dose and at day 28 are shown. Although there were no statistically significant differences, DA-treated animals showed a trend toward increased numbers and percentages of lymphocytes and white blood cells compared to vehicle-treated controls.

[0048] Figure 14 Cumulative food consumption over 28 days is shown. There were no statistically significant differences in food consumption over 28 days between the two experimental groups.

[0049] Figure 15 Various cytokine analyses in blood at day 28 are shown. KC: Cytokine-induced neutrophil chemoattractant (CXCL1); MCP-1: Monocyte chemoattractant protein-1; MIP-1: Macrophage inflammatory protein 1; M-CSF, Macrophage colony-stimulating factor; MIP-2: Macrophage inflammatory protein 2 (CXCL2); VEGF: Vascular endothelial growth factor. KC or CXCL1 and M-CSF showed significant reductions with DA administration.

[0050] Figure 16 Various cytokine analyses in blood at day 28 are shown. IP-10: IFN-gamma-inducible protein 10 (CXCL10). IL-10 and IL-12 showed significant reductions with DA administration.

[0051] Figure 17 Various cytokine analyses in blood at day 28 are shown. G-CSF: Granulocyte colony-stimulating factor; GM-CSF: Granulocyte-macrophage colony-stimulating factor; IFNy: Interferon gamma; IL-1a, IL-1b, IL-2, and IL-5. GM-CSF, IFNy, and IL-5 showed significant reductions with DA administration.

[0052] Figure 18 A graph showing infiltrating cell counts in the pouch exudate is shown, where pre-treatment with DA reduced infiltrating cell counts in the pouch exudate after LPS induction in a dose-dependent manner. Between results, animals pre-treated with 96.4 mg / kg of DA showed significantly lower infiltrating cell counts compared to animals pre-treated with vehicle and lower doses of DA.

[0053] Figure 19 A graph showing IL-6 levels in the pouch exudate is shown, where pre-treatment with DA reduced infiltrating cell counts in the pouch exudate after LPS induction in a dose-dependent manner. Between results, animals pre-treated with 96.4 mg / kg of DA showed significantly lower IL-6 levels compared to animals pre-treated with vehicle and lower doses of DA.

[0054] Figures 20-27 The levels of cytokines G-CSF, eosinophil chemokine, GM-CSF, IFNγ, IL-1a, IL-1b, IL-2, and IL-3 are shown separately. Among these cytokines, IL-1b showed a significant decrease at higher doses of DA.

[0055] Figures 28-35 Cytokine levels of IL-4, IL-5, IL-7, IL-9, IL-10, IL-12p40, IL-12p70, and IL-13 are shown. Among these cytokines, IL-10 showed a significant decrease at higher doses of DA.

[0056] Figures 36-43 Cytokine levels of IL-15, IL-17, LIF, LIX, IP-10, KC, MCP-1, and MCP-1a are shown separately. Among these cytokines, IL-17 showed a significant decrease at higher doses of DA.

[0057] Figures 44-50 The cytokine levels of MIP-1b, MIP-2, M-CSF, MIG, RANTES, VEGF, and TNF-1a are shown separately. Among these cytokines, TNF-1a showed a significant decrease at higher doses of dopamine (DA).

[0058] Figure 51 A summary of higher doses (orange) and lower doses (blue) is shown, with asterisks indicating significance.

[0059] Figure 52 The changes in body weight during the study period are shown. Treatment with DA showed a significant major effect on body weight (P = 0.0052).

[0060] Figure 53 The body weight on day 10 is shown. Animals treated with 69.3 mg / kg DA BID showed a significant effect on DSS-induced weight loss compared to the medium.

[0061] Figure 54 Fecal occult blood scores during the study period are shown. Treatment with DA showed a significant major effect on the status of rectal bleeding.

[0062] Figure 55 The fecal consistency scores during the study period are shown. The DA treatment demonstrates a significant major effect on fecal consistency.

[0063] Figure 56 Combined fecal scores during the study period are shown. The DA treatment demonstrates a significant primary effect on combined fecal status.

[0064] Figure 57 and Figure 58 Colonic weight and length at day 10 are shown. Although no significant differences were observed, treatment with high dose of DA can counteract DSS-induced decrease in mice colonic weight and length.

[0065] Figure 59 Spleen weight at day 10 is shown. Although no significant effects were observed, treatment with high dose of DA showed a trend to counteract DSS-induced loss of mice spleen weight.

[0066] Figure 60 Changes at phylum level are shown, where week 4 shows >95% confidence change at phylum level for the following microbiota: treatment increased proteobacteria*, verrucomicrobia*, cyanobacteria*. Treatment decreased Bacteroidetes, firmicutes*, deferribacteres and spirochetes* (*significant difference relative to control or time 0).

[0067] Figure 61 Significant differences at family level for treatment vs control are shown.

[0068] Figure 62 Principal coordinate analysis plot is shown.

[0069] Figure 63 shows significant enrichment of the biosynthesis of unsaturated fatty acids pathway after 4 weeks of DA treatment (top: individual data; bottom: group data).

[0070] Figure 64 shows significant enrichment of the arachidonic acid metabolism pathway after 4 weeks of DA treatment (top: individual data; bottom: group data).

[0071] Figure 65 shows significant enrichment of the pathways of cofactor and vitamin metabolism after 4 weeks of DA treatment (top: individual data; bottom: group data).

[0072] Figure 66 shows significant enrichment of the lysine degradation pathway after 4 weeks of DA treatment (top: individual data; bottom: group data).

[0073] Figure 67 Significant enrichment of the glycolysis and gluconeogenesis pathways after 4 weeks of DA treatment is shown (group data).

[0074] Figure 68 Significant enrichment of the phosphatidylinositol signaling after 4 weeks of DA treatment is shown (group data).

[0075] Figure 69 shows that signaling of arginine and ornithine metabolism is significantly reduced after 4 weeks of DA treatment (top: individual data; bottom: group data).

[0076] Figures 70A-70C A plot showing biomarkers in many studies compared by family is shown, showing the average percent decrease.

[0077] In Figure 71 , it is noted that clusters of multiple biomarkers predicted effectiveness for each disease indication, and the grouping is shown as Figure 71 .

[0078] Figure 72 and Figure 73 show cytokine profiles in lung lavage fluid from the data of Example 7 and Example 8, respectively.

[0079] Figure 74 It is shown that DA treatment significantly reduced weight gain in DIO mice at day 57 compared to vehicle and CQL.

[0080] Figure 75A It is shown that treatment with DA significantly reduced daily food intake in DIO mice at day 14 compared to vehicle, and Figure 75B It is shown that treatment with DA significantly increased daily water intake at day 28 compared to vehicle, while treatment with CQL significantly reduced daily water intake, both from Example 9.

[0081] Figure 76 It is shown that treatment with DA and CQL significantly reduced serum HbAlc levels in DIO mice at day 28, but substantially increased HbAlc levels at day 56.

[0082] Figure 77 It is shown that treatment with DA significantly reduced serum insulin levels in DIO mice at day 28 compared to vehicle control.

[0083] In Figure 78 , although no significant differences were observed, treatment with DA resulted in a clear decrease in serum LDL levels at day 28 and day 56 compared to vehicle control.

[0084] Figure 79 It is shown that treatment with DA significantly increased serum GLP-1 levels in DIO mice at day 7 and day 56 compared to vehicle control.

[0085] Figure 80 It is shown that treatment with DA significantly increased serum GLP-2 levels in DIO mice at day 56 compared to vehicle control.

[0086] Figure 81 It is shown that treatment with DA significantly increased serum CCK levels in DIO mice at day 56 compared to vehicle control.

[0087] Figure 82 It is shown that treatment with DA significantly increased serum PYY levels in DIO mice at day 56 compared to vehicle control.

[0088] Figure 83 It is shown that treatment with DA significantly decreased serum glucose levels in ob / ob mice.

[0089] Figure 84 It is shown that treatment with DA significantly decreased serum triglyceride levels in ob / ob mice compared to vehicle control.

[0090] Figure 85 It is shown that treatment with DA significantly increased serum bile acid levels in ob / ob mice compared to vehicle control.

[0091] Figure 86 It is shown that treatment with DA significantly decreased serum LDL levels in ob / ob mice compared to vehicle control. DETAILED DESCRIPTION

[0092] The present disclosure provides a method for treating, preventing, and / or slowing the progression of each of the following groups of chronic inflammatory disorders: (1) the Type 2 Diabetes group (Metabolic Syndrome (MET), obesity, hyperglycemia); (2) ARDS (Acute Respiratory Distress Syndrome); (3) chronic autoimmune inflammatory disorders (Rheumatoid Arthritis (RA), Lupus, and Psoriasis); (4) inflammatory bowel diseases (IBD), such as Crohn’s disease and ulcerative colitis; (5) metabolite group-mediated diseases (atherosclerosis, hypertension, and congestive heart failure); and (6) hyperphagic disorders, such as Prader-Willi Syndrome and other monogenic and symptomatic obesity disorders including leptin pathway defects, each method comprising orally administering a pharmaceutical composition comprising a denatonium salt. The present disclosure is based on the results of a series of studies that tracked biomarker cluster levels to track mediators of inflammatory disorders and mediators of gut signaling hormones in response to orally administered denatonium salts. The present disclosure also provides a pharmaceutical composition for treating and preventing various inflammatory conditions that can be tracked by pro-inflammatory biomarkers, including administering a pharmaceutical composition comprising a denatonium salt. Preferably, the pharmaceutical composition for daily oral administration comprises a denatonium salt that delivers a total daily dose from about 20 mg to about 5000 mg BID to an adult. Preferably, the denatonium salt is selected from the group consisting of denatonium acetate, denatonium citrate, denatonium maleate, and denatonium tartrate.

[0093] The present disclosure is based on the following findings: (1) an unexpected set of results from a predictive ob / ob obesity mouse model with denatonium salts and placebo controls. Data from several studies in various in vivo models show that by first measuring inflammatory cytokines in blood and other fluids (e.g., bleb exudates and lung lavage fluids) as biomarkers, then measuring gut signaling peptides, orally administered denatonium salts with organic acid anions show therapeutic efficacy and show significant anti-inflammatory effects. The method of treatment by oral administration (but not intravenous administration) provides data showing efficacy of treatment, prevention indications (including metabolic syndrome (METS), obesity (inflammation-mediated), ARDS, rheumatoid arthritis (RA), lupus, and psoriasis), and slowing of disease progression of these indications (Examples 1 and 2); (2) in vivo studies of a dextran sodium sulfate (DSS)-induced mouse colitis model show therapeutic and prophylactic efficacy in indications including inflammatory bowel disease (IBD) (predominantly containing ulcerative colitis and Crohn’s disease) (Example 3); and (3) a four-week microbiome study in mice fed a high-fat diet shows therapeutic and prophylactic efficacy for atherosclerosis, hypertension, and congestive heart failure (Example 4 and following). The set of pro-inflammatory indicator cytokines measured achieved a significant difference between drug-administered mice and control mice. Body weight loss showed a strong trend for in vivo efficacy in the case of DA administration, but was not similarly statistically significant.

[0094] The cytokine data provided herein in the inflammatory bowel disease model (Example 3) and the air pouch model of inflammatory disease, in accordance with the microbiome data, indicate that the study drug DA does exhibit therapeutic activity in the following three areas: (1) treating or preventing METS, (2) treating or preventing general inflammatory diseases including autoimmune diseases; (3) treating inflammatory bowel diseases including Crohn's disease and ulcerative colitis; and (4) treating cardiovascular diseases such as atherosclerosis, hypertension, and congestive heart failure. Thus, the data obtained in these studies do have a story to tell, and that story is that the denatonium salt pharmaceutical compositions exhibit safety and efficacy in the following areas: (1) treating or preventing METS; (2) treating obesity and achieving weight loss; (3) treating autoimmune inflammatory conditions rheumatoid arthritis (RA), lupus, and psoriasis; (4) treating Crohn's disease and inflammatory bowel disease (IBD); and (5) treating or slowing disease progression of cardiovascular diseases atherosclerosis, hypertension, and congestive heart failure. Preferably, the denatonium salt is selected from the group consisting of denatonium acetate, denatonium citrate, denatonium maleate, and denatonium tartrate. More preferably, from about 25 mg to about 500 mg per day of the denatonium salt is administered orally to an adult BID for the treatment of the above-listed indications.

[0095] In addition, the Example 2 study provides surprising results with statistical significance in reducing IL-5 production, which indicates the effectiveness of the denatonium salt pharmaceutical compositions of the present invention, including DA, in treating ARDS.

[0096]

[0097] This example describes the synthesis of denatonium acetate (DA).

[0098] Step 1: Synthesis of denatonium hydroxide from lidocaine

[0099] To a reflux apparatus was added 25 g of lidocaine, 60 ml of water, and 17.5 g of benzyl chloride, with stirring and heating at 70-90 °C. The solution was heated and stirred for 24 h before the given value, the solution was cooled to 30 °C. The unreacted reagent was removed with 3 x 10 mL of toluene. 65 g of sodium hydroxide was dissolved in 65 mL of cold water, and it was added to the aqueous solution under stirring over the course of 3 hours. The mixture was filtered, washed with some water, and dried in the open air. Recrystallized in hot chloroform or hot ethanol.

[0100]

[0101] Step 2: Preparation of denatonium acetate from denatonium hydroxide.

[0102] To a fluid bed granulator equipped with a rotor insert (rotor granulator) was added 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. The mixture was stirred and heated to 35°C for 3 h. It was then evaporated to dryness and recrystallized in hot acetone.

[0103]

[0104] Formulation of DA tablets

[0105] This provides an immediate release 50 mg granular formulation of denatonium acetate monohydrate (DA) as a free base, as an immediate gastric release oral pharmaceutical formulation.

[0106] Table 1 shows the qualitative and quantitative formulation composition of DA.

[0107]

[0108] IID, inactive ingredient database; API, active pharmaceutical ingredient; USP, United States Pharmacopeia; NF, National Formulary

[0109] * Solvents such as ethanol USP 190 Proof (190 Proof pure ethanol) and purified water (USP) are used for the preparation of pharmaceutical solutions and seal coating dispersions, but are removed during the manufacturing process.

[0110] The detailed manufacturing steps are described below.

[0111] 1. Drug layering process - drug layered pellets

[0112] The drug layering process was performed in a fluid bed granulator equipped with a rotor insert (rotor granulator). A drug solution was prepared by dissolving povidone K30 (Kollidon 30) and denatonium acetate in ethanol. The drug solution was sprayed tangentially onto the bed of sugar spheres (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 ten (10) minutes and sieved through a #20 mesh.

[0113] 2. Seal coating process - seal coated pellets

[0114] The seal coat dispersion was prepared by dissolving hypromellose E5 individually in a mixture of ethanol and purified water (1 : 1) until a clear solution was obtained. The remaining quantity of ethanol was then added to the above solution followed by the addition of talc. The dispersion was mixed for 20 minutes to allow uniform dispersion of the talc. The seal coat dispersion was sprayed tangentially onto the drug loaded pellets to achieve a 5% weight gain. The seal coated pellets were then dried in a rotor granulator for five (5) minutes, removed and further dried in a tray drier / oven at 55°C for 2 hours. The seal coated pellets were then passed through a #20 mesh screen.

[0115] 3. Final Blend - Denagerelease immediate (IR) pellets

[0116] The seal coated pellets were blended with talc passed through a #60 mesh screen using a V-Blender for ten (10) minutes and removed. The blended seal coated beads, i.e., denagerelease immediate (IR) pellets were used for encapsulation.

[0117] 4. Encapsulation - Denagel capsules, 50 mg

[0118] The 50 mg denagel IR pellets were filled into size 1 white opaque hard gelatin capsules using an automatic capsule filling machine. The capsules were then passed through an in-line capsule polisher and metal detector. In-process controls for capsule weight and appearance were performed during the encapsulation process. During the encapsulation process, acceptance quality limit (AQL) sampling was performed by quality assurance (QA) on composite samples. The finished product composite samples were collected and analyzed according to the specifications for release testing.

[0119] 5. Packaging - Capsules, 50 mg - 30 count

[0120] The 50 mg capsules were packaged in 30 count into 50 / 60 cc white HDPE round S-line bottles with 33 mm white CRC cap. The bottles were twisted and sealed using an induction sealer.

[0121] Relationship of biomarkers to disease indications

[0122] Many of the examples provided herein show the effects of denageream salts on various in vivo and in vitro models of various disease indications. In addition, blood samples were collected from the test (and control) animals and various biomarkers were measured and compared. Figures 70A-70C Plots showing the biomarkers from many of the studies are shown. Table 2 groups the biomarkers by family, shows the average percent decrease, and shows which disease indication is affected and predicted by each biomarker. It should be noted that clusters of multiple biomarkers predict the effectiveness for each disease indication and the grouping is shown as Figure 71 indicated.

[0123] Table 2

[0124]

[0125]

[0126]

[0127] Microbiome

[0128] There were changes in the microbiome in a mouse model using a high fat diet after 4 weeks of treatment with or without oral administration of DA. The high fat diet itself caused extensive changes in the microbiota of all groups. However, importantly, there were clear differences between the DA treated groups and the control groups at week 4.

[0129] A classification of different organisms that changed in the control and treated groups at week 4 and that observed extensive changes in the major and minor phyla groups of bacteria and also changes at the family and genus level was performed. For example, the Firmicutes phylum was substantially reduced in the treated groups, while the Proteobacteria and Verrucomicrobia phyla were substantially increased. Due to the diet effect, there was a decrease in diversity at week 4 in the course of the study in both the control and treated groups. At week 4, the treated groups had a further significant decrease in overall diversity compared to the controls, which indicates an increase in specialized populations.

[0130] The genetic potential for changes induced by the treatment related to predicted physiological and metabolic pathways is consistent with the observed benefits of treatment with DA in reducing inflammation and metabolic syndrome. Most of the affected pathways are directly related to a reduction in inflammation and are known to be beneficial for cardiovascular health and other conditions related to metabolic syndrome in humans. Observations include:

[0131] Increased metabolism of unsaturated fatty acids

[0132] Increased metabolism of arachidonic acid

[0133] Increased metabolism of cofactors and vitamins

[0134] Increased lysine degradation

[0135] Increased glycolysis and gluconeogenesis

[0136] Increased phosphatidylinositol signaling

[0137] Decreased arginine and ornithine metabolism

[0138] Changes below from phyla, family, genus level

[0139] Genetic potential 1: increased metabolism of unsaturated fatty acids. There is a significant enrichment of the biosynthetic pathway of unsaturated fatty acids. Accumulating evidence supports the benefit of unsaturated fatty acids in the diet over saturated fatty acids for cardiovascular health (Front Pharmacol. 2018; 9: 1082; Circulation. 2017; 136(3):el- e23; Ann. Intern. Med. 2014; 160(6):398-406).

[0140] Genetic potential 2: increased metabolism of arachidonic acid. Arachidonic acid metabolites are important factors in the initiation and resolution of inflammation and are associated with obesity, diabetes, nonalcoholic fatty liver disease (NAFLD) / nonalcoholic steatohepatitis (NASH), and cardiovascular disease (Int. J. Mol. Sci. 2018; 19(11):3285).

[0141] Genetic potential 3: increased metabolism of cofactors and vitamins. There is an interaction in the increased production of cofactors and vitamins. Cofactors including l-carnitine, nicotinamide riboside (NR), l-serine, and N-acetyl-l-cysteine (NAC) have been shown in human clinical studies to improve altered biological functions associated with different human diseases (Nutrients. 2019; 11(7): 1578). Multiple vitamins and their derivatives have therapeutic potential for the prevention and treatment of metabolic syndrome diseases including diabetes (Can. J. Physiol. Pharmacol. 2015; 93(5):355-62; Endocr. Metab. Immune Disord. Drug Targets. 2015; 15(1):54-63).

[0142] Genetic potential 4: increased lysine degradation. The main end product of lysine degradation is bacterial butyrate (Annu. Rev. Biochem. 1981; 50:23-40), which has been shown to prevent atherosclerosis by maintaining gut barrier function (Nat. Microbiol. 2018; 3(12):1332-1333). Another end product, acetate, also has a similar role in reducing inflammation (J. Atheroscler. Thromb. 2017; 24(7):660-672).

[0143] Genetic potential 5: increased glycolysis and gluconeogenesis. The production of short-chain fatty acids (SCFA) in bacteria proceeds in sequence from glycolysis of glucose to pyruvate, to acetyl coenzyme A (CoA), and finally to acetate, propionate, and butyrate (J. Lipid Res. 2016; 57(6):943-54). This modulation is coordinated with the pathways previously noted, including lysine degradation.

[0144] Genetic potential 6: increased phosphatidylinositol signaling. There was a significant upregulation of the phosphatidylinositol pathway. It is documented that the phosphatidylinositol pathway (e.g., PI3K / AKT, MAPK, and AMPK pathways) is critical for glucose homeostasis. Also, dysregulation of these pathways is often associated with obesity and diabetes (Expert Rev. Mol. Med. 2012; 14:e1).

[0145] Genetic potential 7: decreased arginine and ornithine metabolism. We observed a significant decrease in the arginine and ornithine metabolic pathway. A randomized study showed that high arginine levels are associated with a higher risk of ischemic heart disease (Am. Heart J. 2016; 182:54-61), and accumulation of ornithine is also involved in the pathogenesis of several metabolic diseases (Biomed. Pharmacother. 2017; 86:185-194).

[0146] Figure 60 Changes in phyla levels are shown, with Week 4 showing >95% confidence change in phyla levels for the following microbiome: treatment increased proteobacteria*, verrucomicrobia*, cyanobacteria*. Treatment decreased Bacteroidetes, firmicutes*, deferribacteres, and spirochetes*.

[0147] *Significant difference relative to control or time 0

[0148] Figure 61 Significant differences in treatment versus control at the family level are shown.

[0149] Significant differences in treatment versus control at the genus level at 4 weeks.

[0150] Significant increase

[0151] Parabacteroides

[0152] Escherichia

[0153] Erysipelatoclostridium

[0154] Peptoclostridium-

[0155] Sutterella

[0156] Shigella

[0157] Brenneria

[0158] significantly reduced

[0159] Lachnoclostridium

[0160] Barnesiella

[0161] Clostridium

[0162] Oscillospira

[0163] Dorea

[0164] Candidatus soleaferrea

[0165] Dehalobacterium

[0166] Oscillibacter

[0167] Flavonifractor

[0168] Figure 62 Principal coordinate analysis plot is shown.

[0169] Figure 63 shows significant enrichment of the biosynthesis of unsaturated fatty acids pathway after 4 weeks of DA treatment (top: individual data; bottom: group data).

[0170] Figure 64 shows significant enrichment of the arachidonic acid metabolism pathway after 4 weeks of DA treatment (top: individual data; bottom: group data).

[0171] Figure 65 shows significant enrichment of the pathways of cofactor and vitamin metabolism after 4 weeks of DA treatment (top: individual data; bottom: group data).

[0172] Figure 66 shows significant enrichment of the lysine degradation pathway after 4 weeks of DA treatment (top: individual data; bottom: group data).

[0173] Figure 67 Significant enrichment of glycolysis and gluconeogenesis pathways after 4 weeks of DA treatment is shown (group data).

[0174] Figure 68 Significant enrichment of phosphatidylinositol signaling after 4 weeks of DA treatment is shown (group data).

[0175] Figure 69 shows significant decrease in arginine and ornithine metabolism signaling after 4 weeks of DA treatment (top: individual data; bottom: group data).

[0176] Example 1

[0177] This example describes an in vivo study of the effect of denatonium acetate on body weight in leptin-deficient (ob / ob) mice. Adult leptin-deficient mice (homozygotes, ob / ob mice) were fed a high-fat diet. There was a vehicle control group (15 mice) that was treated by gavage twice daily with distilled water. The DA group (15 mice) was treated with a 23.1 mg / kg BID dose of DA solution.

[0178] Body weight and body weight change were determined on days 1, 3, 7, 10, 14, 21, 24, and 28. Food intake was determined on days 3, 7, 10, 14, 17, 21, 14, and 28. On day 28, blood samples were collected for analysis of cytokines, HbAlc, HDL, LDL, insulin, and bile acids. Statistics were performed by two-way repeated measures ANOVA followed by Tukey’s multiple comparison post-test.

[0179] Table 3 and Figure 1 Body weight measurements from days 1-28 are shown.

[0180]

[0181] Drug treatment had no significant main effect on body weight of ob / ob mice [F(l,28) = 2.076, P = 0.163] is shown.

[0182] Table 3 and Figure 2 Body weight change from days 1-28 is shown.

[0183]

[0184] Drug treatment had no significant main effect on body weight change of ob / ob mice [F(l,28) = 3.849, P = 0.105] is shown.

[0185] Figure 3 Body weight change on day 28 is shown. There was no statistically significant difference in body weight change on day 28 between the two experimental groups.

[0186] Figure 4 Fasting blood glucose levels at day 28 are shown. There was no statistically significant difference in fasting blood glucose levels at day 28 between the two experimental groups.

[0187] Figure 5 HbAlc levels at day 28 are shown. There was no statistically significant difference in blood HbAlc levels at day 28 between the two experimental groups.

[0188] Figure 6 Blood HDL levels at day 28 are shown. Animals treated with 23.1 mg / kg of DA showed a statistically significant decrease in blood HDL levels at day 28 compared to vehicle treated animals.

[0189] Figure 7 Blood LDL cholesterol levels at day 28 are shown. There was no statistically significant difference in blood LDL levels at day 28 between the two experimental groups.

[0190] Figure 8 Blood total cholesterol levels (LDL plus HDL) at day 28 are shown. Animals treated with 23.1 mg / kg of DA showed a nearly significant decrease in blood total cholesterol levels at day 28 compared to vehicle treated animals.

[0191] Figure 9 Blood insulin levels at day 28 are shown. There was no statistically significant difference in blood insulin levels at day 28 between the two experimental groups.

[0192] Figure 10 Blood bile acid levels at day 28 are shown. There was no statistically significant difference in blood bile acid levels at day 28 between the two experimental groups.

[0193] Figure 11 Neutrophil number and percentage at pre-dose and day 28 are shown. Although there was no statistically significant difference, there was a trend for DA treated animals to show an increase in neutrophil number compared to vehicle treated controls.

[0194] Figure 12 Monocyte number and percentage at pre-dose and day 28 are shown. Although there was no statistically significant difference, there was a trend for DA treated animals to show an increase in monocyte number and percentage compared to vehicle treated controls.

[0195] Figure 13Changes in lymphocyte and white blood cell numbers pre-dose and at day 28 are shown. Although there were no statistically significant differences, there was a trend toward increased lymphocyte and white blood cell numbers and percentages in DA-treated animals compared to vehicle-treated controls.

[0196] Figure 14 Cumulative food consumption over 28 days is shown. There were no statistically significant differences in food consumption over 28 days between the two experimental groups.

[0197] Figure 15 Various cytokine analyses in blood at day 28 are shown. KC: Cytokine-induced neutrophil chemoattractant (CXCL1); MCP-1: Monocyte chemoattractant protein-1; MIP-1: Macrophage inflammatory protein 1; M-CSF, Macrophage colony-stimulating factor; MIP-2: Macrophage inflammatory protein 2 (CXCL2); VEGF: Vascular endothelial growth factor. KC / CXCL1 and M-CSF showed significant reductions with DA administration.

[0198] Figure 16 Various cytokine analyses in blood at day 28 are shown. IP-10: IFN-gamma-inducible protein 10 (CXCL10). IL-10 and IL-12 showed significant reductions with DA administration.

[0199] Figure 17 Various cytokine analyses in blood at day 28 are shown. G-CSF: Granulocyte colony-stimulating factor; GM-CSF: Granulocyte-macrophage colony-stimulating factor; IFNy: Interferon gamma; IL-1a, IL-1b, IL-2, and IL-5. GM-CSF, IFNy, and IL-5 showed significant reductions with DA administration.

[0200] There is a direct link between chronic inflammation and the development of metabolic syndrome and other metabolic disorders (McLaughlin et al. J. Clin. Invest. 2017; 127(1): 5-13). Adipose tissue is considered a metabolic risk factor for these medical conditions and contains a variety of immune cells, including macrophages, eosinophils, innate lymphoid cells (ILCs), T cells, and B cells. This accumulation of immune cells induces chronic low-grade inflammation, affecting the metabolism of adipose tissue, promoting systemic inflammation, and impairing insulin action to cause systemic deleterious effects (Wisse, J. Am. Soc. Nephrol. 2004: 15(11): 2792-800). The overproduction of proinflammatory factors by this accumulation of immune cells has been demonstrated to play a role in this pathogenesis context (Saltiel and Olefsky, J. Clin. Invest. 2017; 127(1): 1-4). A broad range of proinflammatory factors, including cytokines and chemokines, show elevated circulating levels in individuals with metabolic syndrome, obesity, diabetes, or other metabolic disorders (Tchernof and Després, Physiol. Rev. 2013; 93(1): 359-404). Some proinflammatory factors, like TNF-a or IL-6, have been found to impair insulin action or affect lipid metabolism, thus contributing to insulin resistance or functional disturbances of fat storage (McLaughlin et al. J. Clin. Invest. 2017; 127(1): 5-13).

[0201] Bitter taste receptors (TAS2Rs) are members of the G protein-coupled receptor (GPCR) family and are not only present on the tongue, but throughout the body (Lu et al. J. Gen. Physiol. 2017; 149(2): 181-197). In this study, we did indeed observe that ob / ob mice treated with DA for 28 days showed a significant body weight reduction compared to vehicle-treated controls; whereas there was no difference in the average daily average individual food intake between these two groups of animals. However, in case DA was administered, a panel of cytokines including GM-CSF, IFNy, IL-5, IL-10, IL-12, KC, and M-CSF showed a significant reduction in DA-treated mice. Thus, the body weight reduction of the DA-treated group can at least in part be attributed to the fact that DA-induced agonism of TAS2Rs on immune cells inhibited the production of these cytokines, subsequently improving the inflammatory state in adipose tissue and mitigating the functional abnormalities of lipid metabolism.

[0202] Example 2

[0203] This example provides results of a study of DA modulation of immune response in a murine air pouch model of inflammation. Eight C57BL / 6 mice were assigned to groups of control (distilled water), DA at a dose of 23.1 mg / kg BID (low dose DA), and DA at a dose of 96.4 mg / kg BID (high dose DA) with twice daily gavage (BID). Measured were infiltrating cell counts of the air pouch exudate, IL-6 levels in the air pouch exudate (assayed by ELISA (R&D Systems Cat# M6000B)), and a multiplex cytokine analysis (Mouse 32Plex Kit MilliporeSigma Cat# MCYTMAG70PMX32BK). Statistical analysis was by one-way ANOVA followed by Tukey’s multiple comparison post-test (for data with normal distribution); Kruskal-Wallis test followed by Dunn’s multiple comparison post-test (for data with skewed distribution); and the ROUT method (for identification of outliers).

[0204] Duarte et al., Current Protocols in Pharmacology, 5.6.1-5.6.8 March 2012 describe “Subcutaneous air pouches are an in vivo model that can be used to study acute and chronic inflammation, resolution of inflammatory responses, and oxidative stress responses. Injection of irritants into the air pouch of rats or mice induces an inflammatory response that can be quantified by the volume of exudate produced, the infiltration of cells, and the release of inflammatory mediators. The model presented in this unit has been widely used to identify potential anti-inflammatory drugs.” It can be used to study local inflammation without systemic effects. But in this case, drugs are administered orally by twice daily gavage. In an earlier study of this model, Romano et al. (1997) showed that dexamethasone (a potent anti-inflammatory steroid with serious side effects) reduced TNF levels by gavage.

[0205] Test administration was 5 ml / kg body weight, BID dosing with 8 hours between doses. Air pouches were created in each test BL6 mouse by sc injection of 1.5 ml / mouse of sterile air (on day 0) and 1.5 ml / mouse of sterile air (on day 3). Compounds (or control distilled water) were administered BID on day -2. LPS (0.75 mg / animal in 1 ml of endotoxin-free PBS) was administered at hour 0 or one hour after dosing with test compound. Plasma samples and air pouch exudate were collected at termination for all groups. Cell count analysis and IL-6 assay were performed at the animal facility and plasma and exudate samples were sent out for cytokine analysis. Each of the distilled water control, 23.1 mg / kg DA, and 92.4 mg / kg DA groups had 8 mice.

[0206] Figure 18 A graph showing infiltrating cell counts in the air pouch exudate, where pre-treatment with DA reduced infiltrating cell counts in the air pouch exudate after LPS induction in a dose-dependent manner. Among the results, animals pre-treated with 96.4 mg / kg of DA showed significantly lower infiltrating cell counts compared to animals pre-treated with vehicle and lower doses of DA.

[0207] Figure 19 A graph showing IL-6 levels in the air pouch exudate, where pre-treatment with DA reduced infiltrating cell counts in the air pouch exudate after LPS induction in a dose-dependent manner. Among the results, animals pre-treated with 96.4 mg / kg of DA showed significantly lower IL-6 levels compared to animals pre-treated with vehicle and lower doses of DA.

[0208] Figures 20-27 Cytokine levels for G-CSF, Eotaxin, GM-CSF, IFNg, IL-la, IL-1β, IL-2, and IL-3 are shown, respectively. Among this group of cytokines, IL-1β showed a significant reduction with higher doses of DA.

[0209] Figures 28-35 Cytokine levels for IL-4, IL-5, IL-7, IL-9, IL-10, IL-12p40, IL-12p70, and IL-13 are shown, respectively. Among this group of cytokines, IL-10 showed a significant reduction with higher doses of DA.

[0210] Figures 36-43 Cytokine levels for IL-15, IL-17, LIF, LIX, IP-10, KC, MCP-1, and MCP-1a are shown, respectively. Among this group of cytokines, IL-17 showed a significant reduction with higher doses of DA.

[0211] Figures 44-50 Cytokine levels for MIP-1β, MIP-2, M-CSF, MIG, RANTES, VEGF, and TNF-1a are shown, respectively. Among this group of cytokines, TNF-1a showed a significant reduction with higher doses of DA.

[0212] In summary, Figure 51 A summary is shown for higher (orange) and lower (blue) doses, indicating significance when denoted with an asterisk. Also, the pro-inflammatory biomarkers TNFa, IL-1β, IL-10, and IL-17 show a significant dose response reduction with higher dose DA administration.

[0213] Example 3

[0214] This example provides the results of an in vivo study of dextran sodium sulfate (DSS)-induced colitis in a mouse model. Inflammatory Bowel Disease (IBD), which includes primarily ulcerative colitis and Crohn's disease, is a complex and multifactorial disease with unknown etiology. To mechanistically study human IBD, a number of murine colitis models have been developed. These models are potential mechanisms to decipher the pathogenesis of IBD as well as to evaluate potential therapeutic agents. Among various chemically induced colitis models, the dextran sodium sulfate (DSS)-induced colitis model is widely used due to its many similarities to human ulcerative colitis. Also, a number of existing approved IBD drugs have been studied in this model to allow comparison of new potential drug compounds to the existing drugs approved for IBD indications.

[0215] C5BL / 6 mice were divided into 5 groups of 3-10 mice and provided with standard mouse chow ad libitum, housed up to 5 per cage. Dexamethasone 21-phosphate disodium salt (DMS; Alfa Aesar Catalog # J64083-1G, Lot # R02F035) was used as a positive control. Hemoccult kits were obtained from Beckman (Hemoccult SENSA kit). Dextran sodium sulfate (DSS) reagent grade (MPI Catalog # 160110, Lot # 6046H, MW 36,000-50,000, CAS 9011-18-1) was supplemented in the water of certain groups to induce IBD-like symptoms. Treatment started on day -3 prior to DSS delivery. On day 1, all mice were pre-weighed and given fresh 4-5% DSS in water daily for 5 days, then water was given for the remainder of the study to induce disease. Additional control groups were given water (no DSS) for the duration of the study (10 days). Body weight was measured daily, hemoccult was measured 3X weekly, stool consistency was measured 3X weekly, and general health was determined daily. Mice were sacrificed on day 10 and serum obtained was used for cytokine analysis and colon length and weight were determined. There were two control groups, water only and DSS with no drug treatment. There were two treatment groups, 69.3 mg / kg (n=10) bid and 23.1 mg / kg bid (n=10).

[0216] Figure 52 Body weight change during the study period is shown. Treatment with DA showed a significant main effect on body weight (P=0.0052).

[0217] Figure 53 Body weight on day 10 is shown. Animals treated with 69.3 mg / kg DA BID showed a significant effect on DSS-induced weight loss compared to vehicle.

[0218] Figure 54 Fecal occult blood scores during the study period are shown. Treatment with DA showed a significant major effect on the status of rectal bleeding.

[0219] Figure 55 The fecal consistency scores during the study period are shown. The DA treatment demonstrates a significant major effect on fecal consistency.

[0220] Figure 56 Combined fecal scores during the study period are shown. The DA treatment demonstrates a significant primary effect on combined fecal status.

[0221] Figure 57 and Figure 58 Colon weight and length on day 10 are shown separately. Although no significant differences were observed, treatment with high doses of DA could counteract the DSS-induced reduction in colon weight and length in mice.

[0222] Figure 59 Spleen weight on day 10 is shown. Although no significant effect was observed, treatment with high doses of DA showed a trend to counteract DSS-induced spleen weight loss in mice.

[0223] Example 4

[0224] In microbiome studies, low levels of *Pseudomonas* (protective commensal bacteria) are associated with atherosclerosis, higher levels of *Escherichia* lead to coronary heart disease (CHD), *Ruminococcus* is typically increased in patients with ACVD (atherosclerotic cardiovascular disease), and short-chain fatty acids (SCFAs) produced by microbes result in reduced atherosclerosis, inflammation, and moderate hypertension.

[0225] In a mouse model of nonalcoholic steatohepatitis (NASH), the effects of a small-molecule orally administered TAS2R agonist (DA) on the microbial community were investigated. Two groups of 4-week-old male C57BL / 6 mice (n=20 / group) were fed an islet amyloid-liver NASH (AMLN) diet and administered daily doses of ARD-101 (30 mg / mL in water) or a mediator (water) via intragastric force-feeding. DNA was isolated from fecal samples collected at weeks 0 and 4, and microbial ecology was evaluated using bTEFAP (FLX amplicon pyrosequencing encoding bacterial tags). Operational taxonomic units were classified using BLAST against a select NCBI database. Diversity within specific ecosystems and microbial community structures was analyzed using Qiime 2. Differences were determined using repeated measures ANOVA and post-hoc pairwise comparisons using the map base test. Taxonomic data were evaluated using a two-level phylogenetic tree.

[0226] The AMLN diet resulted in changes in the microbiota population in both groups at week 4. At week 4, significant increases / decreases were observed at the phylum, family, and genus levels in the DA group compared to the vehicle group. For example, at the phylum level, there were significant increases in the phyla Proteobacteria, Verrucomicrobia, and Cyanobacteria and significant decreases in the phyla Firmicutes, Deferribacteres, and Spirochaetes. Diversity within the ecosystem and microbial communities was significantly less at week 4 compared to week 0 in both treatment groups and DA compared to vehicle group at week 4 (p < 0.05 for all comparisons). Genetic analysis indicated that DA resulted in increased metabolism of unsaturated fatty acids and arachidonic acid, increased production of cofactors and vitamins; increased lysine degradation, glycolysis, gluconeogenesis, and phosphatidylinositol signaling; and decreased production of arginine and ornithine. DA treatment induced significant changes in physiological and metabolic pathways and attenuated the diet-induced decrease in SCFAs in feces. The overall findings are consistent with data indicating that DA attenuates inflammation and metabolic syndrome.

[0227] Example 5

[0228] This example provides an in vivo study to determine the effect of DA on mouse peritoneal macrophages. Peritoneal exudates were obtained from Balb / c female mice by lavage 4 days after intraperitoneal injection of 4 ml of sterile 4% thioglycolate broth. After washing with RPMI 1640 medium, cell suspensions were centrifuged at 800 g for 5 min at 4°C. Red blood cells were eliminated by ACK buffer, and cells were washed and resuspended in RPMI 1640 supplemented with 10% inactivated FBS, 10 mM HEPES, 2 mM glutamine, and 100 U / ml penicillin-100 mg / ml streptomycin. Peritoneal macrophages were plated in 24-well tissue culture plates (2 X 10 5 cells / mL / well) in a 5% CO2 humidified atmosphere at 37°C. Macrophages were pre-cultured in serum-free RPMI 1640 medium for 24 h to reduce mitogenic effects. Macrophages were pre-treated with various concentrations of DA for 1 h before LPS treatment and stimulated with LPS (100 ng / mL) for 24 h. Treatment groups were:

[0229] Group Number of wells Treatment 1 6 Vehicle 2 6 LPS 3 6 LPS + SB203580 (positive control) 4 6 LPS + ARD_101 (1 μΜ) 5 6 LPS + ARD_101 (10 μΜ) 6 6 LPS + ARD_101 (100 μΜ)

[0230] At the stimulation time points of 12 and 24 h, about 200 ul of supernatant was removed and stored (-80°C) for cytokine analysis (13 Plex). The cytokines analyzed were - GM-CSF, IFNy, IL-1a, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12 (p70), IL-13, IL-17A, KC / CXCL1, LIX, MCP-1, MIP-2, TNF-a.

[0231] Table 5 reports the mean ± SD for each cytokine:

[0232] Cytokines DA concentration (μΜ) Significance of LPS incubation for 12 hours Significance of LPS incubation for 24 hours GM-CSF 1 / 10 / 100 P = 0.029 / not significant / not significant Not significant / not significant / not significant IFNγ 1 / 10 / 100 P = 0.031 / 0.037 / not significant Not significant / not significant / not significant IL-1α 1 / 10 / 100 P = 0.021 / 0.036 / not significant Not significant / not significant / not significant IL-1β 1 / 10 / 100 P = 0.023 / 0.023 / not significant Not significant / not significant / not significant IL-2 1 / 10 / 100 P = 0.005 / 0.004 / not significant Not significant / not significant / not significant IL-4 1 / 10 / 100 P = 0.009 / 0.009 / not significant Not significant / not significant / not significant IL-6 1 / 10 / 100 P = 0.096 / 0.029 / not significant Not significant / not significant / not significant IL-7 1 / 10 / 100 P＝0.024 / 0.010 / 010 Not significant / not significant / not significant IL-10 1 / 10 / 100 P＝0.045 / 0.026 / 0.015 Not significant / not significant / not significant IL-12 (p70) 1 / 10 / 100 P＝0.017 / 0.007 / 0.008 Not significant / not significant / not significant IL-13 1 / 10 / 100 P＝0.038 / 0.019 / 0.021 Not significant / not significant / not significant IL-17A 1 / 10 / 100 P＝0.044 / 0.024 / 0.042 Not significant / not significant / not significant KC / CXCL1 1 / 10 / 100 Not significant / not significant / P = 0.022 Not significant / not significant / not significant LIX 1 / 10 / 100 Not significant / not significant / not significant Not significant / not significant / not significant MCP-1 1 / 10 / 100 Not significant / not significant / not significant Not significant / not significant / not significant MIP-2 1 / 10 / 100 P＝0.081 / 0.021 / 0.033 Not significant / not significant / not significant TNF-α 1 / 10 / 100 P＝0.059 / 0.024 / 0.033 Not significant / not significant / not significant

[0233] In summary, 24 h incubation with LPS did not cause significant differences from 12 h LPS incubation.

[0234] Example 6

[0235] This example provides the results of a study to evaluate the effects of denatonium acetate on healthy mice (as measured by cytokine profile and route of DA administration). The study groups were: (1) vehicle group, N=12, treated with distilled water, gavage, BID; (2) DA oral low dose group, N=12, treated with a 23.1 mg / kg (salt weight) dose of DA, gavage, BID; (3) DA oral high dose group, N=12, treated with a 92.4 mg / kg (salt weight) dose of DA, gavage, BID; (4) DA IV low dose group, N=12, treated with a 1 mg / kg (salt weight) dose of DA, iv bolus, QD; (5) DA IV high dose group, N=12, treated with a 3 mg / kg (salt weight) dose of ARD-101, iv bolus, QD.

[0236] First, no biomarker (cytokine) effect was observed with iv DA doses. It can be concluded with certainty that DA needs to be administered orally to show an effect. Also, only iv administration was toxic. Group 3 is the lower dose oral DA group, and Group 4 is the higher dose oral DA group, and the lower dose DA resulted in a significant decrease (compared to control) in cytokines G-CSF (p=0.003), IL-la (p=0.04), IL-13 (P=0.03), MCP-1 (p=0.005), MIP-2 (p=0.015), and VEGF (p=0.001). The higher dose DA resulted in a significant decrease (compared to control) in cytokines GM-CSF (p=0.03), IL-9 (p=0.003), KC (p=0.05), and VEGF (p=0.001). This study confirms biomarker effects in normal mice and confirms that oral dosing should be used rather than iv.

[0237] Example 7

[0238] This example provides the results of a study to evaluate the effects of denatonium acetate in a mouse model of acute lung injury plus hyperthermia. The procedure was to administer to three groups of CD-1 mice: (1) saline by gavage for oral administration BID, (2) DA at a dose of 92.4 mg / kg BID orally administered, and (3) DA at a dose of 3 mg / kg iv bolus QD iv administered. Lung lavage was measured and cytokine analysis was performed. Statistics were: one-way ANOVA followed by Tukey's multiple comparison post-test (for data with normal distribution); Kruskal-Wallis test followed by Dunn's multiple comparison post-test (for data with skewed distribution); and ROUT method (for identification of outliers). Control or drug was administered for 3 days, then 50 μL of 1 mg / ml LPS was delivered endotracheally with a Penn Century needle at a core temperature of 39C, 24 hours after LPS, then sacrificed to measure lung lavage protein concentration and serum cytokine levels.

[0239] DA showed a dramatic but not significant decrease in protein concentration in lung lavage for both oral and iv dosing. The cytokine profile in lung lavage is shown in Figure 72 where DA = ARD-101.

[0240] Example 8

[0241] This example provides the results of a second modified acute lung injury plus hyperthermia study to evaluate the effects of denatonium acetate. The same procedure as in Example 7 was used. Groups of six CD-I mice were each prophylactically treated with vehicle or 92.4 mg / kg denatonium acetate (DA) (administered by twice daily (BID) oral gavage (PO)) or with 3 mg / kg DA (administered by once daily (QD) intraperitoneal (IP) injection) beginning three days prior to induction of lung injury. On Day 0, lung injury was induced by intratracheal instillation of 50 μL of 1 mg / mL bacterial lipopolysaccharide (LPS) and hyperthermia was induced by placing the animals in a 39°C incubator. On Day 1 (i.e., 24 hours after induction), the animals were euthanized and bronchoalveolar lavage fluid (BALF) was collected. BALF samples were evaluated for cytokine concentrations (using a multiplex bead-based assay) and protein levels and neutrophil counts (by fluorescence-activated cell sorting (FACS)). In addition, lungs were collected, fixed, stained with Masson's trichrome, and evaluated histologically. Three days of dosing with 92.4 mg / kg DA (BID) PO or with 3 mg / kg DA (QD) IP were well tolerated in female CD-I mice. Although two mice [one dosed with vehicle and one dosed with DA (92.4 mg / kg)] were found dead on Day 1, the timing of these deaths (within 24 hours of LPS instillation) suggested that the deaths reflected the instillation procedure, hyperthermia, or associated inflammation (rather than the test article). This inference was consistent with the observation that deaths were observed with both vehicle and test article dosing. No other adverse clinical observations were noted during the three days of test article administration. Oral dosing with 92.4 mg / kg DA resulted in a significant decrease in BALF concentrations of 7 of 32 cytokines tested (including IL-2, IL-3, IL-10, IL-13, MIP-1 β, MCSF, and MIG) compared to vehicle. IP dosing with 3 mg / kg DA resulted in a significant decrease in BALF concentrations of 10 of 32 cytokines tested (including G-CSF, eotaxin, IL2, IL-3, IL-4, IL-13, IP-10, MCP-1, M-CSF, and MIG) compared to vehicle (see Table 1). Figure 73). Oral and IP dosing with the indicated levels of DA were associated with nominally (but not significantly) changes in BALF protein concentration; nominally decreased BALF neutrophil counts (as determined by FACS); and nominally decreased lung pathology severity (as scored histologically). Thus, BID PO treatment with 92.4 mg / kg DA or QD IP injection with 3 mg / kg DA provided nominally active in countering neutrophil infiltration and lung damage in this acute lung injury mouse model, as well as a significant attenuation of the accumulation of multiple cytokines in the lungs of these animals.

[0242] Example 9

[0243] This example provides results of a study of the effects of DA plus another compound (CQL) on body weight in diet-induced (DIO) mice. Adult C57BL / 6NTac mice were fed a high fat diet (60%). The vehicle group (N=15) was treated with distilled water twice daily by gavage, CQL (N=15) was treated with 50 mg / kg twice daily by gavage, and DA (N=15) was treated with a dose of 92.4 mg / kg twice daily by gavage. The study period was 56 days + a subsequent 2-3 day test period. Body weight change was measured 3X weekly, food and water consumption was measured on days 0, 12, 28, 42, and 56. Metabolic biomarkers were measured on days 28 and 56. Cytokine analysis was performed on days 28 and 56. Serum levels of GLP-1, GLP-2, and CCK were measured 1 h post-dose on day 1 and 2 h post-dose on day 7 (fasted >6 h prior to dosing until blood collection after); and serum levels of PPY were measured on day 56.

[0244] Figure 74 It is shown that DA treatment significantly reduced body weight gain in DIO mice on day 57 compared to vehicle and CQL. Figure 75A It is shown that DA treatment significantly reduced daily food intake in DIO mice on day 14 compared to vehicle, and Figure 75B It is shown that DA treatment significantly increased daily water intake on day 28 compared to vehicle, while CQL treatment significantly reduced daily water intake. DA treatment did not show a significant effect on serum glucose levels in DIO mice. Figure 76 It is shown that DA and CQL treatment significantly reduced serum HbAlc levels in DIO mice on day 28, but substantially increased HbAlc levels on day 56. Figure 77 It is shown that DA treatment significantly reduced serum insulin levels in DIO mice on day 28 compared to vehicle controls. On Figure 78In this case, although no significant differences were observed, treatment with DA resulted in a significant decrease in serum LDL levels at day 28 and day 56 compared to vehicle control. Figure 79 It was shown that treatment with DA significantly increased serum GLP-1 levels in DIO mice at day 7 and day 56 compared to vehicle control. Figure 80 It was shown that treatment with DA significantly increased serum GLP-2 levels in DIO mice at day 56 compared to vehicle control. Figure 81 It was shown that treatment with DA significantly increased serum CCK levels in DIO mice at day 56 compared to vehicle control. Figure 82 It was shown that treatment with DA significantly increased serum PYY levels in DIO mice at day 56 compared to vehicle control.

[0245] At day 28 and day 56 (28 / 56), serum cytokines were measured and showed a significant increase in G-CSR (p=0.063 / 0.039), Eotaxin (p=0.031 / not significant), IL-6 (p=0.041 / not significant), IP-10 (p=0.013 / not significant), and MIG (p=not significant / 0.028) for G-CSR. Many mice did not allow sufficient blood to be obtained to produce statistical significance.

[0246] Example 10

[0247] Leptin-deficient ob / ob mice exhibit hyperphagia and obesity, as well as hyperglycemia and hypertriglyceridemia, which are also found in patients with hyperphagic disorders such as Prader-Willi syndrome and other monogenic and symptomatic obesity disorders (Diabetes. 2006 Dec;55(12):3335-43; Clin Genet. 2005 Mar;67(3):230-9; Biochim Biophys Acta. 2012 May;1821(5):819-25). Thus, ob / ob mice are a predictive in vivo model for these indications. This example provides results of a study of the effect of DA plus another compound (CQL) on body weight in leptin-deficient (ob / ob) mice. Vehicle group (N=14) was treated with distilled water twice daily by gavage, and DA (N=14) was treated at a dose of 50 mg / kg twice daily by gavage. The study period was 56 days + subsequent 2-3 day test period. Body weight change was measured 3X weekly, food intake was measured twice weekly, metabolic biomarkers (blood glucose, blood insulin, blood HbAlc, HDL, LDL, triglycerides, and bile acids) were measured at the beginning and end of the study. Cytokine analysis was performed at the end of day 56.

[0248] Treatment with DA did not show a significant effect on body weight of ob / ob mice. Treatment with DA did not show a significant effect on daily food consumption of ob / ob mice. Figure 83 Treatment with DA was shown to significantly decrease serum glucose levels of ob / ob mice. Treatment with DA did not show a significant effect on serum HBA1c levels or insulin levels of ob / ob mice. Figure 84 Treatment with DA was shown to significantly decrease serum triglyceride levels of ob / ob mice compared to vehicle control. Figure 85 Treatment with DA was shown to significantly increase serum bile acid levels of ob / ob mice compared to vehicle control. Figure 86 Treatment with DA was shown to significantly decrease serum LDL levels of ob / ob mice compared to vehicle control. However, there was no significant effect on serum HDL levels.

[0249] The DA group allowed for a significant decrease to be observed in the levels of eosinophil chemotactic factor (p=0.047) and MIG (p=0.026) cytokines at day 56 compared to the control group. Furthermore, although no significant differences were observed, the DA group showed a decrease in the levels of the following cytokines at day 56 compared to the vehicle group: RANTES (1.7% decrease), IL-1 beta (19.1% decrease), IL-6 (61.4% decrease) and MCP-1 (20.9% decrease).

Claims

1. Use of a denatonium salt or a pharmaceutical composition comprising a denatonium salt for the manufacture of a medicament for the treatment, prevention and slowing of the worsening of an acute pulmonary inflammatory condition, wherein the denatonium salt is selected from the group consisting of denatonium acetate, denatonium citrate, denatonium maleate, denatonium sugar and denatonium tartrate.

2. Use according to claim 1, wherein the denatonium salt is denatonium acetate.

3. Use according to claim 1, wherein the pharmaceutical composition further comprises 0.5 g to 5 g acetic acid.

4. Use according to claim 1, wherein the daily dose of the denatonium salt is from 20 mg to 5000 mg for an adult human.

5. Use according to claim 4, wherein the daily dose of denatonium acetate is from 50 mg to 1000 mg for an adult human.

6. Use according to claim 5, wherein the daily dose of denatonium acetate is from 60 mg to 500 mg for an adult human.

7. Use according to claim 5, wherein the daily dose of denatonium acetate reaches a concentration in the gastrointestinal tract of from 10 ppb to 50 ppm for an adult human.

8. Use according to claim 1, wherein the daily dose of the denatonium salt is administered once a day, twice a day or three times a day.

9. Use according to any one of claims 1 to 8, wherein the acute pulmonary inflammatory condition is acute respiratory distress syndrome.

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