Pharmaceutical compositions for treating or preventing various inflammatory disorders

Abstract

Methods are provided for treating, preventing, and / or slowing the progression of various chronic inflammatory disorders, including: (1) type 2 diabetes; (2) acute respiratory distress syndrome; (3) chronic autoimmune inflammatory disorders; (4) inflammatory bowel disease; (5) metabolome-mediated diseases; and (6) hyperphagia disorders. [Solution] A method is provided that includes orally administering a pharmaceutical composition containing a denatonium salt. Also provided is a pharmaceutical composition for treating and preventing various inflammatory conditions that can be tracked by pro-inflammatory biomarkers, the pharmaceutical composition comprising a denatonium salt delivering a total daily dose of about 20 mg to about 5000 mg BID to an adult human. Preferably, the denatonium salt is selected from the group consisting of denatonium acetate, denatonium citrate, denatonium maleate, denatonium saccharide, and denatonium tartrate.

Description

[Technical Field]

[0001] The present disclosure provides methods for treating, preventing, and / or slowing the progression of various chronic inflammatory disorders, including: (1) type 2 diabetes (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 disease (IBD), e.g., Crohn's disease and ulcerative colitis; (5) metabolome-mediated diseases (atherosclerosis, hypertension, and congestive heart failure); and (6) hyperphagic disorders, e.g., Prader-Willi syndrome, and other monogenic syndromic obesity disorders involving leptin pathway defects, each comprising orally administering a pharmaceutical composition comprising a denatonium salt. The present disclosure is based on readouts from a series of studies tracking clusters of biomarker levels to track mediators of inflammatory disorders and mediators of gut signaling hormones in response to orally administered denatonium salt. The present disclosure further provides pharmaceutical compositions for treating and preventing various inflammatory conditions that can be tracked by proinflammatory biomarkers, comprising administering a pharmaceutical composition containing a denatonium salt. Preferably, the pharmaceutical compositions of the present invention, administered orally daily, contain a denatonium salt that delivers a total daily dose of about 20 mg to about 5000 mg to an adult human BID. Preferably, the denatonium salt is selected from the group consisting of denatonium acetate, denatonium citrate, denatonium maleate, and denatonium tartrate. [Background technology]

[0002] Over the past 40 years, global obesity levels have more than doubled. This growing epidemic represents one of the most significant global health challenges today, as obesity predisposes to metabolic syndrome and is associated with increased risk of severe illness and death from coronary heart disease, stroke, type 2 diabetes, certain forms of cancer, and even the coronavirus pandemic. As this problem emerges, our understanding of the pathological mechanisms linking obese states to disease progression has improved. Central to these mechanisms is the state of increased systemic inflammation as a result of obesity, which results in many pathologies. Thus, there is a significant need for treatments and preventions that address appetite and inflammatory signaling. The present disclosure addresses this need.

[0003] inflammatory diseases Currently, various inflammatory diseases are treated with anti-tumor necrosis factor (TNF) (and anti-interleukin (IL)-6) proteins and antibodies. Such therapeutic proteins have been approved for rheumatoid arthritis, pediatric polyarticular juvenile idiopathic arthritis (JIA), psoriatic arthritis, lupus, ankylosing spondylitis (AS), chronic plaque psoriasis (Ps), panuveitis, IBD including ulcerative colitis and Crohn's disease, and many other diseases. These biologic drugs are antibodies or fusion proteins, such as etanercept (Embrel®), that act by binding and scavenging circulating TNFα (and IL-6). However, these anti-TNFα drugs, as well as other biologic drugs that indiscriminately bind and scavenge inflammatory cytokines, have severe side effects caused by their inhibition of most TNF signaling pathways. Because TNF has an immune surveillance function (which is also inhibited by these biologic drugs), it increases susceptibility to infection and impaired immune surveillance, including increased incidence of various infectious diseases and malignancies, including leukemia and lymphoma, which are listed on black box warning labels. Therefore, there is a need in the art for more cost-effective small molecule therapeutics that knock down (but do not necessarily eliminate) circulating TNF. Because protein-based therapeutics cannot be administered orally, there is a need in the art for oral small molecule agents that are more sensitive or self-limiting in eliminating circulating TNF by preventing the production of TNF as a pro-inflammatory cytokine, rather than indiscriminately clearing existing and produced TNF.

[0004] For example, the US FDA-approved label for adalimumab (Humira®) lists side effects such as an increased risk of serious infections (i.e., TB; viral, fungal, and bacterial infections), worsening of hepatitis B infection in carriers, allergic reactions, and various leukemias and lymphomas.

[0005] metabolic syndrome Metabolic syndrome (METS) is a complex set of factors that increase the risk of developing type 2 diabetes and cardiovascular disease. METS is the clustering of at least three of the following five conditions: (1) visceral obesity; (2) elevated blood pressure; (3) elevated blood glucose levels; (4) elevated serum triglycerides; and (5) low serum high-density lipoprotein (HDL) levels.

[0006] According to the International Diabetes Foundation (IDF), metabolic syndrome is defined as 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) (<50 mg / dL in men and 1.29 mmol / L in women); (3) elevated blood pressure (BP) (systolic >130 or diastolic >85 mm Hg) or treatment for hypertension; and (4) elevated fasting plasma glucose (FPG) >100 mg / dL (5.6 mmol / L) or a prior diagnosis of type 2 diabetes.

[0007] Metabolic syndrome can also be defined as the presence of 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 medications). A clinical study examining carbohydrate restriction as a first-line dietary intervention for METS examined whether there was significance in a panel of biomarkers, including the inflammatory biomarkers TNFα, IL-6, and MCP-1, in fasting participants (Al-Sarraj et al., J. Nutrition 139(9):1667-1675, 2009). This study (n=20) found significance in MPC-1, ICAM-1, and TNFα, but not IL-6.

[0008] METS affects 20-25% of the adult population worldwide, including 35% in the United States. METS is present in approximately 60% of U.S. residents aged >50 years. METS is also correlated with a high incidence of autoimmune diseases. Therefore, there is a need in the art to provide safer and more effective METS treatments.

[0009] ARDS and viral respiratory infections Acute respiratory distress syndrome (ARDS) is a life-threatening disease characterized by the acute onset of hypoxia and pulmonary infiltrates and triggered by conditions such as sepsis, pneumonia, trauma, burns, pancreatitis, and blood transfusion. ARDS causes generalized pulmonary inflammation leading to increased pulmonary vascular permeability, pulmonary edema, and alveolar epithelial damage. The diagnosis of ARDS is based on the following criteria: (1) acute onset; (2) bilateral pulmonary infiltrates of noncardiac origin on chest radiography or computed tomography (CT) scan; and (3) moderate to severe oxygenation impairment. Severe ARDS has a 45% mortality rate. The severity of ARDS is defined by the degree of hypoxemia, calculated as the ratio of arterial oxygen tension to fraction of inspired oxygen (PaO2 / FiO2). There are three types of ARDS: mild, moderate, or severe. The Berlin definition of ARDS is PaO2 / FiO2 of 200-300 (mild), 100-199 (moderate), and <100 (severe).

[0010] The progression of ARDS is generally divided into two phases: the initiator stage and the subsequent effector stage. The initiator stage 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 the sequestration of pulmonary neutrophils. The second, effector, stage involves neutrophil activation and the subsequent release of toxic oxygen radicals and proteolytic enzymes, particularly neutrophil elastase (NE). NE has the ability to injure pulmonary endothelial cells and degrade extracellular matrix products, such as elastin, collagen, and fibronectin, including those in the lung base membrane.

[0011] ARDS exists in many forms, each with different etiologies and progression, but the ultimate pathology is the same across these diverse forms. Examples of clinical events that can precipitate various forms of ARDS include trauma, hemorrhage, generalized pneumonia, viral-induced pneumonia (including but not limited to COVID-19 and SARS), inhalation of toxic gases, and sepsis. In the case of the 2020 COVID-19 pandemic, viral pneumonia was the driving force behind ARDS observed in many patients requiring critical care. Regardless of the initial cause, ARDS shares the following commonalities: intrapulmonary fluid accumulation and exudate lead to generalized alveolar damage and impaired alveolar gas exchange. Common factors (regardless of the initial cause of ARDS) include inflammation, fluid release, cell migration and proliferation, and increased proinflammatory cytokines, exacerbating the condition.

[0012] Viral respiratory infections generally have an incubation period of 2–7 days. Infected individuals typically present with a high fever, sometimes accompanied by chills, headache, fatigue, and muscle aches. Pulmonary viral infections account for approximately 10–15% of annual ICU admissions in the United States, even without a pandemic, and a significant proportion of influenza-related deaths annually, even without a coronavirus pandemic. The 2020 COVID-19 pandemic illustrates this trajectory of disease progression. The illness progresses with the onset of a dry cough or dyspnea, which may be accompanied by or progress to hypoxemia. A significant number of cases require intubation and mechanical ventilation. Furthermore, at 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).

[0013] The pattern of viral load spread (e.g., coronavirus or influenza virus) suggests droplet or contact transmission of viral pathogens (N. Engl. J. Med. 2003 May 15;348(20):1995-2005). SARS-1 and SARS-2 are members of the coronavirus family of enveloped viruses that replicate in the cytoplasm of infected animal host cells. Coronaviruses are generally characterized as single-stranded RNA viruses with genomes of approximately 30,000 nucleotides (Science. 2003 May 30;300(5624):1394-9). Coronaviruses are classified into three known groups; the first two groups cause mammalian coronavirus infections, and the third group causes avian coronavirus infections (JSM Peiris, in Medical Microbiology (Eighteenth Edition), 2012, 587-593). Coronaviruses are thought to be the causative agents of several severe diseases in many animals; for example, infectious bronchitis virus, feline infectious peritonitis virus, and gastroenteritis virus are important veterinary pathogens (Viruses. 2019 Jan; 11(1): 59).

[0014] Thus, there is a need for effective treatments for patients diagnosed with SARS, infected with an infectious agent associated with SARS, e.g., patients infected with SARS-CoV, or who are at imminent risk of contracting SARS, e.g., individuals who have been exposed to, or may be exposed in the near future to, an infectious agent associated with SARS.

[0015] Prior art treatments for ARDS are inadequate. Therefore, there is an urgent need for effective treatments for ARDS.

[0016] metabolome The gut microbiota has attracted much attention, and imbalances in the gut microbiota have been linked to several diseases, depending on which groups of bacteria are increased or decreased. Atherosclerotic disease, accompanied by symptoms such as myocardial infarction and stroke, is a major cause of severe illness and death in patients with metabolic syndrome. This disease is thought to be caused by cholesterol accumulation and macrophage recruitment to the arterial wall and can therefore be considered both a metabolic and inflammatory disease. Since the early 19th century, it has been suggested that infections cause or promote atherosclerosis by increasing pro-atherosclerotic changes in vascular cells. However, better methods for early delaying atherosclerotic changes in vascular cells and their associated diseases remain necessary. The present invention provides a method for delaying atherosclerotic changes in vascular cells by reducing intestinal signals that support atherosclerotic changes in vascular cells.

[0017] Hyperphagia Particularly in developed countries, where calorie-dense foods and foods high in fat, especially saturated fat, are widely available, modulation of eating behavior, including both appetite control for certain food components and dietary preferences for foods with less fat or lower calorie content, 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).

[0018] One of the key signals that plays a role in maintaining energy balance and thus body weight is leptin, a circulating protein encoded by the ob gene, which is expressed primarily in adipose tissue. Leptin plays a central role in regulating energy balance, which suppresses food intake and increases energy expenditure (Zhang et al., Nature, 372, 425–432, 1994). This protein circulates in the blood at concentrations proportional to the size of fat depots, crosses the blood-brain barrier by saturable pathways, and exerts most of its influence on energy balance at the central level through protein interactions with receptors located on hypothalamic neurons and other brain regions (Tartaglia et al., Cell, 83, 1263–1271, 1995).

[0019] Animals with defects in the leptin signaling axis, either because they do not produce functional proteins or because they express defective forms of their receptors, are characterized by early hyperphagia and severe obesity, diabetes, hypothermia, and infertility. In humans, congenic defects in leptin signaling (lack of leptin or its receptor) have also been associated with early-onset morbid obesity (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 context, the use of leptin for the treatment or prevention of diabetes, which is a direct result of obesity, has been proposed (WO97 / 02004).

[0020] The short-term anorectic effects of leptin were thought to contribute to the control of obesity and related disorders in obese individuals, but unfortunately, leptin administration alone was not effective as a practical treatment, partly due to tolerance and compensatory upregulation of other hunger- and satiety-mediated pathways, and long-term treatment outcomes were also unsatisfactory.

[0021] With aging, circulating levels of leptin 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 develop resistance to the appetite-suppressing effects of this hormone, which contributes to the development and maintenance of obesity and / or its complications. Indeed, evidence suggests that leptin resistance is a major determinant of weight gain and age-related obesity in rats [Iossa et al., J. Nutr., 1999, 129, 1593-6]. However, although circulating leptin levels are normally proportional to body fat mass, which is generally thought to increase with age, there is evidence that age-related increases in leptinemia and the development of leptin resistance occur, at least in part, independently of increases in fat mass (Gabriely et al., Diabetes, 2002, 51, 1016-21).

[0022] High circulating levels of leptin are associated with increased risk of cardiovascular disease in humans [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], independent of body mass index / obesity. Summary of the Invention

[0023] The present disclosure provides methods for treating, preventing, and / or slowing the progression of various chronic inflammatory disorders, including: (1) type 2 diabetes (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 disease (IBD), e.g., Crohn's disease and ulcerative colitis; (5) metabolome-mediated diseases (atherosclerosis, hypertension, and congestive heart failure); and (6) hyperphagic disorders, e.g., Prader-Willi syndrome, and other monogenic syndromic obesity disorders involving leptin pathway defects, each comprising orally administering a pharmaceutical composition comprising a denatonium salt. The present disclosure is based on readouts from a series of studies tracking clusters of biomarker levels to track mediators of inflammatory disorders and mediators of gut signaling hormones in response to orally administered denatonium salts. The present disclosure further provides pharmaceutical compositions for treating and preventing various inflammatory conditions that can be tracked by proinflammatory biomarkers, comprising administering a pharmaceutical composition containing a denatonium salt. Preferably, the pharmaceutical compositions of the present invention, administered orally daily, contain a denatonium salt that delivers a total daily dose of about 20 mg to about 5000 mg to an adult human BID. Preferably, the denatonium salt is selected from the group consisting of denatonium acetate, denatonium citrate, denatonium maleate, and denatonium tartrate.

[0024] The present disclosure provides methods for treating, preventing, and delaying the progression of metabolic syndrome (MET), obesity, and type 2 diabetes, including hyperglycemia, comprising orally administering a pharmaceutical composition containing a denatonium salt selected from the group consisting of denatonium acetate (DA), denatonium citrate, denatonium maleate, denatonium saccharide, and denatonium tartrate. Preferably, the pharmaceutical composition further contains 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. Preferably, the daily dose of denatonium salt for an adult is about 20 mg to about 5,000 mg, or about 5 mg / kg body weight to about 150 mg / kg body weight per day. More preferably, the daily dose of DA for an adult is about 50 mg to about 1,000 mg. Most preferably, the adult daily dose of DA is about 60 mg to about 500 mg, or a dose that achieves a GI tract concentration of about 10 ppb (parts per billion) to about 50 ppm. The daily dose of denatonium salt is administered once daily, twice daily, or three times daily.

[0025] The present disclosure provides methods for treating, preventing, and delaying the worsening of acute pulmonary inflammatory disorders, including ARDS, comprising orally administering a pharmaceutical composition containing a denatonium salt selected from the group consisting of denatonium acetate (DA), denatonium citrate, denatonium maleate, denatonium saccharide, and denatonium tartrate. Preferably, the pharmaceutical composition further contains 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. Preferably, the daily dose of denatonium salt for an adult is about 20 mg to about 5,000 mg, or about 5 mg / kg body weight to about 150 mg / kg body weight per day. More preferably, the daily dose of DA for an adult is about 50 mg to about 1,000 mg. More preferably, the daily dose of DA for an adult is a dose that achieves a GI tract concentration of about 60 mg to about 500 mg, or about 10 ppb to about 50 ppm. The daily dose of denatonium salt is administered once daily, twice daily, or three times daily.

[0026] The present disclosure provides methods for treating, preventing, and delaying the progression of chronic autoimmune inflammatory disorders, the symptoms of which are selected from the group consisting of rheumatoid arthritis (RA), lupus, and psoriasis, comprising orally administering a pharmaceutical composition containing a denatonium salt selected from the group consisting of denatonium acetate (DA), denatonium citrate, denatonium maleate, denatonium saccharide, and denatonium tartrate. Preferably, the pharmaceutical composition further contains 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. Preferably, the daily dose of denatonium salt for an adult is about 20 mg to about 5,000 mg, or about 5 mg / kg body weight to about 150 mg / kg body weight per day. More preferably, the daily dose of DA for an adult is about 50 mg to about 1,000 mg. Most preferably, the adult daily dose of DA is a dose that achieves a GI tract concentration of about 60 mg to about 500 mg, or about 10 ppb to about 50 ppm. The daily dose of denatonium salt is administered once daily, twice daily, or three times daily.

[0027] The present disclosure provides methods for treating, preventing, and delaying the worsening of chronic IBD symptoms selected from the group consisting of Crohn's disease and ulcerative colitis, comprising orally administering a pharmaceutical composition containing a denatonium salt selected from the group consisting of denatonium acetate (DA), denatonium citrate, denatonium maleate, denatonium saccharide, and denatonium tartrate. Preferably, the pharmaceutical composition further contains 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. Preferably, the daily dose of denatonium salt for an adult is about 20 mg to about 5000 mg, or about 5 mg / kg body weight to about 150 mg / kg body weight per day. More preferably, the daily dose of DA for an adult is about 50 mg to about 1000 mg. Most preferably, the adult daily dose of DA is a dose that achieves a GI tract concentration of about 60 mg to about 500 mg, or about 10 ppb to about 50 ppm. The daily dose of denatonium salt is administered once daily, twice daily, or three times daily.

[0028] The present disclosure provides methods for treating, preventing, and delaying the progression of metabolome-mediated conditions selected from the group consisting of atherosclerosis, hypertension, and congestive heart failure (CHF), comprising orally administering a pharmaceutical composition containing a denatonium salt selected from the group consisting of denatonium acetate (DA), denatonium citrate, denatonium maleate, denatonium saccharide, and denatonium tartrate. Preferably, the pharmaceutical composition further contains 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. Preferably, the daily dose of denatonium salt for an adult is about 20 mg to about 5000 mg, or about 5 mg / kg body weight to about 150 mg / kg body weight per day. More preferably, the daily dose of DA for an adult is about 50 mg to about 1000 mg. Most preferably, the adult daily dose of DA is a dose that achieves a GI tract concentration of about 60 mg to about 500 mg, or about 10 ppb to about 50 ppm. The daily dose of denatonium salt is administered once daily, twice daily, or three times daily.

[0029] The present disclosure provides a method for treating or delaying the progression of hyperphagia associated with symptoms selected from the group consisting of Prader-Willi syndrome and leptin pathway deficiency, comprising orally administering a pharmaceutical composition containing a denatonium salt selected from the group consisting of denatonium acetate (DA), denatonium citrate, denatonium maleate, denatonium saccharide, and denatonium tartrate. Preferably, the pharmaceutical composition further contains about 0.5 g to about 5 g of acetic acid. More preferably, the adult daily dose of acetic acid is about 1.5 g to about 3 g. Preferably, the adult daily dose of denatonium salt is about 20 mg to about 5000 mg, or about 5 mg / kg body weight to about 150 mg / kg body weight per day. More preferably, the adult daily dose of DA is about 50 mg to about 1000 mg. Most preferably, the adult daily dose of DA is a dose that achieves a GI tract concentration of about 60 mg to about 500 mg, or about 10 ppb to about 50 ppm. The daily dose of denatonium salt is administered once daily, twice daily, or three times daily. [Brief explanation of the drawings]

[0030] [Figure 1] FIG. 1 shows the body weight over time due to administration of DA compared to vehicle control.

[0031] [Figure 2] FIG. 2 shows the change in body weight over time due to administration of DA compared to vehicle control.

[0032] [Figure 3] Figure 3 shows the weight change on day 28. There was no statistically significant difference in weight change on day 28 between the two experimental groups.

[0033] [Figure 4] Figure 4 shows fasting blood glucose levels on day 28. There was no statistically significant difference in fasting blood glucose levels on day 28 between the two experimental groups.

[0034] [Figure 5] Figure 5 shows the HbA1c levels on day 28. There was no statistically significant difference in blood HbA1c levels on day 28 between the two experimental groups.

[0035] [Figure 6] Figure 6 shows blood HDL levels on day 28. Animals administered 23.1 mg / kg DA showed a statistically significant decrease in blood HDL levels on day 28 compared to vehicle-treated animals.

[0036] [Figure 7] Figure 7 shows blood LDL cholesterol levels on day 28. There was no statistically significant difference in blood LDL levels on day 28 between the two experimental groups.

[0037] [Figure 8]Figure 8 shows blood total cholesterol levels (LDL + HDL) on day 28. Animals administered 23.1 mg / kg DA showed a nearly significant reduction in blood total cholesterol levels on day 28 compared to vehicle-treated animals.

[0038] [Figure 9] Figure 9 shows the blood insulin levels on day 28. There was no statistically significant difference in blood insulin levels on day 28 between the two experimental groups.

[0039] [Figure 10] Figure 10 shows the blood bile acid levels on day 28. There was no statistically significant difference in blood bile acid levels on day 28 between the two experimental groups.

[0040] [Figure 11] Figure 11 shows the number and percentage of granulocytes before administration and on day 28. Although there was no statistically significant difference, the change in granulocyte number showed a trend toward an increase in DA-treated animals compared to vehicle-treated controls.

[0041] [Figure 12] Figure 12 shows the number and percentage of monocytes before administration and on day 28. Although there was no statistically significant difference, the changes in monocyte number and percentage showed a trend toward an increase in DA-administered animals compared to vehicle-treated controls.

[0042] [Figure 13] Figure 13 shows the changes in lymphocyte and leukocyte counts before administration and on day 28. Although there were no statistically significant differences, the changes in lymphocyte and leukocyte counts and percentages tended to increase in DA-treated animals compared to vehicle-treated controls.

[0043] [Figure 14] Figure 14 shows the cumulative food intake over 28 days. There was no statistically significant difference in food intake over 28 days between the two experimental groups.

[0044] [Figure 15] Figure 15 shows the analysis of various blood cytokines on day 28. KC: cytokine-induced neutrophil chemoattractant (CXCL1); MCP-1: monocyte chemotactic 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. DA administration significantly reduced KC or CXCL1 and M-CSF.

[0045] [Figure 16] Figure 16 shows the analysis of various blood cytokines on day 28. IP-10: IFN-γ-inducible protein 10 (CXCL10). IL-10 and IL-12 showed significant decreases with DA administration.

[0046] [Figure 17] Figure 17 shows the analysis of various blood cytokines on day 28. G-CSF: granulocyte colony-stimulating factor; GM-CSF: granulocyte-macrophage colony-stimulating factor; IFNγ: interferon gamma; IL-1α, IL-1β, IL-2, and IL-5. DA administration showed significant decreases in GM-CSF, IFNα, and IL-5.

[0047] [Figure 18] Figure 18 shows a graph of the number of infiltrating cells in the air pouch exudate, demonstrating that DA pretreatment dose-dependently reduced the number of infiltrating cells in the air pouch exudate after LPS challenge. Among the results, animals pretreated with 96.4 mg / kg DA showed significantly lower infiltrating cell numbers compared with animals pretreated with vehicle and low-dose DA.

[0048] [Figure 19]Figure 19 shows a graph of IL-6 levels in air pouch exudates, demonstrating that DA pretreatment dose-dependently reduced the number of infiltrating cells in the air pouch exudates after LPS induction. Among the results, animals pretreated with 96.4 mg / kg DA exhibited significantly lower IL-6 levels compared with animals pretreated with vehicle and low-dose DA.

[0049] [Figure 20-27] 20 to 27 show the cytokine levels of G-CSF, eotaxin, GM-CSF, IFNγ, IL-1a, IL-1b, IL-2, and IL-3, respectively. In this cytokine group, IL-1b showed a significant decrease with high-dose DA.

[0050] [Figure 28-35] Figures 28 to 35 show the cytokine levels of IL-4, IL-5, IL-7, IL-9, IL-10, IL-12p40, IL-12p70, and IL-13, respectively. In this cytokine group, IL-10 showed a significant decrease with high-dose DA.

[0051] [Figure 36-43] Figures 36 to 43 show the cytokine levels of IL-15, IL-17, LIF, LIX, IP-10, KC, MCP-1, and MCP-1a, respectively. Among these cytokine groups, IL-17 showed a significant decrease with high-dose DA.

[0052] [Figure 44-50] 44 to 50 show the cytokine levels of MIP-1b, MIP-2, M-CSF, MIG, RANTES, VEGF, and TNF-1a, respectively. Among these cytokine groups, TNF-1a showed a significant decrease with high-dose DA.

[0053] [Figure 51] Figure 51 shows a summary of the high dose (orange) and low dose (blue) with asterisks indicating significance.

[0054] [Figure 52] Figure 52 shows the changes in body weight during the study period. DA administration showed a significant main effect on body weight (P=0.0052).

[0055] [Figure 53] Figure 53 shows body weight on day 10. Animals treated with 69.3 mg / kg DA BID showed a significant effect on DSS-induced weight loss compared to vehicle.

[0056] [Figure 54] Figure 54 shows the fecal occult blood scores during the test period. DA administration showed a significant main effect on the fecal occult blood status.

[0057] [Figure 55] Figure 55 shows fecal consistency scores over the study period. DA administration showed a significant main effect on fecal consistency.

[0058] [Figure 56] Figure 56 shows the total fecal scores over the study period. DA administration showed a significant main effect on total fecal status.

[0059] [Figure 57-58] Figures 57 and 58 show the colon weight and length, respectively, on day 10. In mice, high-dose DA administration was able to counteract the DSS-induced decrease in colon weight and length, although no significant differences were observed.

[0060] [Figure 59] Figure 59 shows spleen weight on day 10. In mice, high dose DA administration showed a tendency to counteract the DSS-induced decrease in spleen weight, although no significant effect was observed.

[0061] [Figure 60]Figure 60 shows the phylum level changes and showed >95% confidence changes in the microbiome at the phylum level at week 4: Treatment increased Proteobacteria*, Verrucomicrobia*, and Cyanobacteria*. Treatment decreased Bacteroidetes, Firmicutes*, Deferibacter, and Firmicutes* (*significant difference from control or time 0).

[0062] [Figure 61] Figure 61 shows the significant differences between treated versus control groups at the family level.

[0063] [Figure 62] Figure 62 shows the principal coordinate analysis plot.

[0064] [Figure 63] FIG. 63 shows that 4 weeks of DA administration significantly enhanced the biosynthetic pathway of unsaturated fatty acids (upper panel: individual data, lower panel: group data).

[0065] [Figure 64] FIG. 64 shows that 4 weeks of DA administration significantly enhanced the pathway of arachidonic acid metabolism (upper panel: individual data; lower panel: group data).

[0066] [Figure 65] Figure 65 shows significant enrichment of pathways for cofactor and vitamin metabolism by 4 weeks of DA administration (upper panel: individual data; lower panel: group data).

[0067] [Figure 66] FIG. 66 shows that 4 weeks of DA administration significantly enhanced the pathway of lysine degradation (upper panel: individual data; lower panel: group data).

[0068] [Figure 67] FIG. 67 shows that 4 weeks of DA administration significantly enhanced the glycolytic and gluconeogenic pathways (group data).

[0069] [Figure 68] FIG. 68 shows significant enhancement of phosphatidylinositol signaling by 4 weeks of DA administration (group data).

[0070] [Figure 69] FIG. 69 shows that 4 weeks of DA administration significantly reduced signaling for arginine and ornithine metabolism (upper panel: individual data; lower panel: group data).

[0071] [Figure 70A-C] Figures 70A-C show graphs comparing biomarkers by department across multiple studies, showing the average percentage decline.

[0072] [Figure 71] It should be noted in Figure 71 that clusters of biomarkers predict efficacy for each disease indication and are presented in Figure 71 as groups.

[0073] [Figure 72] Figure 72 and Figure 72 show the cytokine profiles in lung lavage fluid from the data of Examples 7 and 8, respectively.

[0074] [Figure 74] FIG. 74 shows that DA administration significantly reduced weight gain on day 57 in DIO mice compared to vehicle and CQL.

[0075] [Figure 75] Figure 75A shows that in DIO mice, on day 14, DA administration significantly reduced daily food intake compared to vehicle, and Figure 75B shows that on day 28, DA administration significantly increased daily water intake compared to vehicle, while CQL administration significantly reduced daily water intake compared to vehicle, both from Example 9.

[0076] [Figure 76]FIG. 76 shows that administration of DA and CQL significantly reduced serum HbA1c levels on day 28, but significantly increased HbA1c levels on day 56 in DIO mice.

[0077] [Figure 77] FIG. 77 shows that serum insulin levels were significantly reduced on day 28 with DA administration compared to vehicle controls in DIO mice.

[0078] [Figure 78] In Figure 78, although no significant differences were observed, DA administration significantly reduced serum LDL levels on days 28 and 56 compared to vehicle controls.

[0079] [Figure 79] FIG. 79 shows that serum GLP-1 levels were significantly elevated in DIO mice on days 7 and 56 with DA administration compared to vehicle controls.

[0080] [Figure 80] FIG. 80 shows that serum GLP-2 levels were significantly elevated in DIO mice on day 56 with DA administration compared to vehicle controls.

[0081] [Figure 81] FIG. 81 shows that serum CCK levels were significantly elevated in DIO mice on day 56 with DA administration compared to vehicle controls.

[0082] [Figure 82] FIG. 82 shows that serum PYY levels were significantly elevated in DIO mice on day 56 with DA administration compared to vehicle controls.

[0083] [Figure 83] FIG. 83 shows that DA administration significantly reduced serum glucose levels in ob / ob mice.

[0084] [Figure 84] FIG. 84 shows that DA administration significantly reduced serum triglyceride levels in ob / ob mice compared to vehicle controls.

[0085] [Figure 85] FIG. 85 shows that DA administration significantly elevated serum bile acid levels in ob / ob mice compared to vehicle controls.

[0086] [Figure 86] FIG. 86 shows that DA administration significantly reduced serum LDL levels in ob / ob mice compared to vehicle controls.

[0087] (Detailed explanation) The present disclosure provides methods for treating, preventing, and / or delaying the progression of various chronic inflammatory disorders, including: (1) type 2 diabetes (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 disease (IBD), e.g., Crohn's disease and ulcerative colitis; (5) metabolome-mediated diseases (atherosclerosis, hypertension, and congestive heart failure); and (6) hyperphagic disorders, e.g., Prader-Willi syndrome, and other monogenic syndromic obesity disorders involving leptin pathway defects, each comprising orally administering a pharmaceutical composition comprising a denatonium salt. The present disclosure is based on readouts from a series of studies tracking clusters of biomarker levels to track mediators of inflammatory disorders and mediators of gut signaling hormones in response to orally administered denatonium salt. The present disclosure further provides pharmaceutical compositions for treating and preventing various inflammatory conditions that can be tracked by proinflammatory biomarkers, comprising administering a pharmaceutical composition containing a denatonium salt. Preferably, the pharmaceutical composition is orally administered daily and contains a denatonium salt that delivers a total daily dose of about 20 mg to about 5000 mg to an adult human BID. Preferably, the denatonium salt is selected from the group consisting of denatonium acetate, denatonium citrate, denatonium maleate, and denatonium tartrate.

[0088] This disclosure is based on the discovery of a cluster of surprising results from what began as (1) an in vivo study of weight loss in a prospective ob / ob obese mouse model using denatonium salts and a placebo control. Data from several studies in various in vivo models showed that oral administration of denatonium salts bearing organic acid anions demonstrated therapeutic efficacy, demonstrating significant anti-inflammatory effects, first measured as biomarkers of inflammatory cytokines in blood and other fluids (e.g., air pouch exudates and lung lavage fluids) and then as measured by gut signaling peptides. We have provided data demonstrating the efficacy of oral (but not intravenous) treatment methods for treating, preventing, and delaying disease progression in indications including metabolic syndrome (METS), obesity (inflammatory mediation), ARDS, rheumatoid arthritis (RA), lupus, and psoriasis (Examples 1 and 2); (2) an in vivo study of dextran sulfate sodium (DSS)-induced colitis in a mouse model demonstrating therapeutic and preventative effects in indications primarily including ulcerative colitis and inflammatory bowel disease (IBD), including Crohn's disease (Example 3); and (3) a 4-week microbiome study in mice fed a high-fat diet demonstrating therapeutic and preventative effects in atherosclerosis, hypertension, and congestive heart failure (Examples 4 and subsequent). Measurement of a cluster of proinflammatory cytokines achieved significant differences between drug-treated and control mice. Weight loss showed a strong trend toward an in vivo effect of DA administration, but similarly did not achieve statistical significance.

[0089] The cytokine data provided herein show that in an inflammatory bowel disease model (Example 3) and an air pouch model of inflammatory disease, the test drug, DA, demonstrated therapeutic activity in three areas: (1) treatment or prevention of METS; (2) treatment or prevention of general inflammatory diseases, including autoimmune diseases; (3) treatment of inflammatory bowel diseases, including Crohn's disease and ulcerative colitis; and (4) treatment of cardiovascular diseases, such as atherosclerosis, hypertension, and congestive heart failure, from the microbiome data. Thus, the data from these studies tell a story: denatonium salt pharmaceutical compositions demonstrate safety and efficacy for (1) treating or preventing METS; (2) treating obesity and promoting weight loss; (3) treating the autoimmune inflammatory conditions rheumatoid arthritis (RA), lupus, and psoriasis; (4) treating Crohn's disease and inflammatory bowel disease (IBD); and (5) treating or delaying disease progression in the cardiovascular diseases of 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, the denatonium salt for treating the above indications is administered orally to adults at a dose of about 25 mg to about 500 mg BID per day.

[0090] Furthermore, the test in Example 2 surprisingly yielded a statistically significant reduction in IL-5 production, demonstrating the effectiveness of the pharmaceutical composition of the present invention of a denatonium salt containing DA in treating ARDS. JPEG2026041895000001.jpg35151

[0091] This example describes the synthesis of denatonium acetate (DA). 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 with stirring and heat to 70-90°C. Heat and stir the solution at the above-specified temperatures for 24 hours, then cool the solution to 30°C. Remove unreacted reagents with 3 x 10 ml of toluene. Dissolve 65 g of sodium hydroxide in 65 ml of cold water with stirring and add the solution to the solution with stirring over a period of 3 hours. Filter the mixture, wash with water, and dry in the open air. Recrystallize in hot chloroform or hot ethanol. [ka] [ka]

[0092] Step 2: Preparation of denatonium acetate from denatonium hydroxide. A reflux apparatus was charged with 10 g of denatonium hydroxide (MW: 342.475 g / mol, 0.029 mol), 20 mL of acetone, and 2 g (0.033 mol) of glacial acetic acid dissolved in 15 mL of acetone, and the mixture was stirred and heated to 35° C. for 3 hours, then evaporated to dryness and recrystallized in hot acetone. [ka]

[0093] Formulation of DA tablets This provides an immediate release 50 mg granular formulation of denatonium acetate monohydrate (DA) as the free base as an immediate gastric release oral pharmaceutical formulation.

[0094] Table 1 shows the qualitative and quantitative formulation composition of DA. [Table 1]

[0095] The detailed manufacturing process is shown below. 1. Drug layering process - drug layered pellets The drug layering process was carried out in a fluidized-bed granulator (rotor granulator) equipped with rotor inserts. The drug solution was prepared by solubilizing povidone K30 (Kollidon 30) and denatonium acetate in ethyl alcohol. The drug solution was sprayed tangentially onto a bed of refined sucrose (35 / 45 mesh) moving in a circular motion within the rotor granulator. The final drug-loaded pellets were then dried in the rotor granulator for 10 minutes, discharged, and sieved through a #20 mesh sieve.

[0096] 2. Seal coating process - Seal coating pellets A seal coating dispersion was prepared by separately dissolving hypromellose E5 in a 1:1 mixture of ethyl alcohol and purified water until a clear solution was obtained. The remaining amount of ethyl alcohol was then added to the solution, followed by talc. The talc dispersion was mixed for 20 minutes until the dispersion was uniform. The drug-loaded pellets were tangentially sprayed with the seal coating dispersion to achieve a 5% weight gain. The seal-coated pellets were then dried in a rotor granulator for 5 minutes, discharged, and further dried in a tray dryer or oven at 55°C for 2 hours. The seal-coated pellets were then sieved through a #20 mesh screen.

[0097] 3. Final Mix – Denatonium Immediate Release (IR) Pellets The seal-coated pellets were mixed with the talc sieved through a #60 mesh in a V-blender for 10 minutes and then discharged. The mixed seal-coated beads and denatonium IR pellets were used for encapsulation.

[0098] 4. Encapsulation - Denatonium Capsules, 50mg Denatonium IR pellets, 50 mg, were filled into size 1, white, opaque, hard gelatin capsules using an automated capsule filling machine. The capsules then passed through a single-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) spot checks were performed by Quality Assurance (QA) on composite samples during the encapsulation process. Composite samples of the finished product were collected and analyzed as per the release test protocol.

[0099] 5. Packaging - Capsules, 50mg - 30 pieces Thirty 50 mg capsules were packaged in 50 / 60 cc white HDPE round S-line bottles with 33 mm white CRC caps. The bottles were rotated and sealed using an induction sealer.

[0100] Association of biomarkers with disease symptoms Many of the examples provided herein demonstrate the effects of denatonium salts on various in vivo and in vitro models of various disease indications. Additionally, blood samples were collected from test (and control) animals, and various biomarkers were measured and compared. Figures 70A-C are graphs comparing biomarkers across multiple studies. Table 2 groups the biomarkers by family, showing the average percentage reduction and indicating which disease indications are affected and predicted by each biomarker. It should be noted that clusters of multiple biomarkers predict efficacy for each disease indication, and the groupings are shown in Figure 71.

[0101] [Table 2]

[0102] Microbiome In a mouse model fed a high-fat diet, there were changes in the microbiome after 4 weeks in groups with and without oral DA administration. The high-fat diet itself induced widespread changes in the microbial population in all groups. However, importantly, there was a significant difference between the DA-treated and control groups at 4 weeks.

[0103] The various organisms that changed in the control and treatment groups at week 4 were classified, and extensive changes were observed at the major or dominant bacterial phylum, and at the family and genus levels. For example, the treatment group saw a dramatic decrease in the Firmicutes phylum, while Proteobacteria and Verrucomicrobia increased dramatically. Diversity at week 4 decreased in both the control and treatment groups over the study period due to the effects of diet. Overall diversity at week 4 in the treatment group was significantly reduced compared to the control group, indicating an increase in specialized populations.

[0104] The genetic potential of treatment-induced changes related to predicted physiological and metabolic pathways was aligned with the observed benefits of DA treatment in suppressing inflammation and metabolic syndrome. The majority of affected pathways are directly related to reduced inflammation, known to be beneficial for human cardiovascular health and other conditions related to metabolic syndrome. The following were observed: Increased metabolism of unsaturated fatty acids Increased metabolism of arachidonic acid Increased metabolism of cofactors and vitamins Increased lysine breakdown Increased glycolysis and gluconeogenesis Increased phosphatidylinositol signaling Decreased metabolism of arginine and ornithine Below, changes from the phylum, family, and genus levels

[0105] Genetic Possibility 1: Increased Unsaturated Fatty Acid Metabolism. The unsaturated fatty acid biosynthetic pathway was significantly enhanced. Accumulating evidence supports the cardiovascular benefits of dietary unsaturated fatty acids over saturated fatty acids (Front Pharmacol. 2018; 9:1082; Circulation. 2017; 136(3):e1-e23; Ann. Intern. Med. 2014; 160(6):398-406).

[0106] Genetic Possibility 2: Elevated Arachidonic Acid Metabolism. Arachidonic acid metabolites are key factors in the initiation and resolution of inflammation and are associated with the pathophysiology of obesity, diabetes, nonalcoholic fatty liver disease (NAFLD) / nonalcoholic steatohepatitis (NASH), and cardiovascular disease (Int. J. Mol. Sci. 2018;19(11):3285).

[0107] Genetic Possibility 3: Increased Metabolism of Cofactors and Vitamins. Increased production of cofactors and vitamins has synergistic effects. Cofactors, including l-carnitine, nicotinamide riboside (NR), l-serine, and N-acetyl-l-cysteine ​​(NAC), have been demonstrated in human clinical trials to improve altered biological functions associated with various human diseases (Nutrients. 2019;11(7):1578). Multiple vitamins and their derivatives have therapeutic potential for the prevention and treatment of metabolic syndrome disorders, including diabetes (Can. J. Physiol. Pharmacol. 2015;93(5):355-62; Endocr. Metab. Immune Disord. Drug Targets. 2015;15(1):54-63).

[0108] Genetic Possibility 4: Increased Lysine Degradation. The primary end product of lysine degradation is bacterial butyrate (Annu. Rev. Biochem. 1981; 50:23-40), which has been shown to prevent atherosclerosis by maintaining intestinal barrier function (Nat. Microbiol. 2018; 3(12):1332-1333). Another end product, acetate, also has a similar inflammation-reducing effect (J. Atheroscler. Thromb. 2017; 24(7):660-672).

[0109] Genetic Possibility 5: Increased Glycolysis and Gluconeogenesis. Short-chain fatty acid (SCFA) production in bacteria proceeds sequentially from glucose glycolysis to pyruvate, acetyl-coenzyme A (CoA), and ultimately acetate, propionate, and butyrate (J. Lipid Res. 2016;57(6):943-54). This regulation is linked to previously mentioned pathways, including lysine degradation.

[0110] Genetic Possibility 6: Increased Phosphatidylinositol Signaling. There was significant upregulation of the phosphatidylinositol pathway. It has been documented that phosphatidylinositol pathways (e.g., PI3K / AKT, MAPK, and AMPK) are essential for glucose homeostasis. Furthermore, deregulation of these pathways often results in obesity and diabetes (Expert Rev. Mol. Med. 2012;14:e1).

[0111] Genetic Possibility 7: Decreased arginine and ornithine metabolism. Significantly reduced arginine and ornithine metabolic pathways were observed. Randomized trials have shown that elevated arginine levels are associated with a higher risk of ischemic heart disease (Am. Heart J. 2016;182:54-61), and ornithine accumulation is also implicated in the pathogenesis of several metabolic diseases (Biomed. Pharmacother. 2017;86:185-194).

[0112] Figure 60 shows the phylum level changes and showed >95% confidence changes in the microbiome at the phylum level at week 4: treatment increased Proteobacteria*, Verrucomicrobia*, and Cyanobacteria*. Treatment decreased Bacteroidetes, Firmicutes*, Deferibacter, and Firmicutes*. **Significant difference from control or time 0

[0113] Figure 61 shows the significant differences between treated and control groups at the family level. Genus is significantly different between treated and baseline and control at week 4. Significantly increased Parabacteroides Escherichia Erysipelatoclostridium Peptoclostridium - Sutterella Shigella Brenneria Significantly decreased Lachnoclostridium Barnesiella Clostridium Oscillospira Dorea Candidatus soleaferrea Dehalobacterium Oscillibacter Flavonifractor

[0114] Figure 62 shows the principal coordinate analysis plot.

[0115] FIG. 63 shows that 4 weeks of DA administration significantly enhanced the biosynthetic pathway of unsaturated fatty acids (upper panel: individual data, lower panel: group data).

[0116] FIG. 64 shows that 4 weeks of DA administration significantly enhanced the pathway of arachidonic acid metabolism (upper panel: individual data; lower panel: group data).

[0117] Figure 65 shows significant enrichment of pathways for cofactor and vitamin metabolism by 4 weeks of DA administration (upper panel: individual data; lower panel: group data).

[0118] FIG. 66 shows that 4 weeks of DA administration significantly enhanced the pathway of lysine degradation (upper panel: individual data; lower panel: group data).

[0119] FIG. 67 shows that 4 weeks of DA administration significantly enhanced the glycolytic and gluconeogenic pathways (group data).

[0120] FIG. 68 shows significant enhancement of phosphatidylinositol signaling by 4 weeks of DA administration (group data).

[0121] FIG. 69 shows that 4 weeks of DA administration significantly reduced signaling for arginine and ornithine metabolism (upper panel: individual data; lower panel: group data). [Example]

[0122] Example 1 This example describes an in vivo study of denatonium acetate on the body weight of leptin-deficient (ob / ob) mice. Adult leptin-deficient mice (homozygous ob / ob mice) fed a high-fat diet were used. The vehicle control group (15 mice) was gavaged with distilled water BID. The DA group (15 mice) was administered DA solution BID at a dose of 23.1 mg / kg.

[0123] Body weight and weight change were measured on days 1, 3, 7, 10, 14, 21, 24, and 28. Food intake was measured on days 3, 7, 10, 14, 17, 21, 14, and 28. On day 28, blood samples were collected for cytokine analysis (HbA1c, HDL, LDL, insulin, and bile acids). Statistics were performed by two-way repeated measures ANOVA followed by Tukey's multiple comparison post hoc test.

[0124] Table 3 and Figure 1 show the body weight measurements on days 1-28. [Table 3]

[0125] Drug administration did not have a significant main effect on body weight in ob / ob mice [F(1, 28) = 2.076, P = 0.163].

[0126] Table 3 and Figure 2 show the changes in body weight from days 1 to 28. [Table 4]

[0127] Drug administration did not have a significant main effect on body weight change in ob / ob mice [F(1,28)=3.849, P=0.105].

[0128] Figure 3 shows the weight change on day 28. There was no statistically significant difference in weight change on day 28 between the two experimental groups.

[0129] Figure 4 shows fasting blood glucose levels on day 28. There was no statistically significant difference in fasting blood glucose levels on day 28 between the two experimental groups.

[0130] Figure 5 shows the HbA1c levels on day 28. There was no statistically significant difference in blood HbA1c levels on day 28 between the two experimental groups.

[0131] Figure 6 shows blood HDL levels on day 28. Animals administered 23.1 mg / kg DA showed a statistically significant decrease in blood HDL levels on day 28 compared to vehicle-treated animals.

[0132] Figure 7 shows blood LDL cholesterol levels on day 28. There was no statistically significant difference in blood LDL levels on day 28 between the two experimental groups.

[0133] Figure 8 shows the blood total cholesterol levels (LDL + HDL) on day 28. Animals administered 23.1 mg / kg of DA showed a nearly significant decrease in blood total cholesterol levels on day 28 compared to vehicle-administered animals.

[0134] Figure 9 shows the blood insulin levels on day 28. There was no statistically significant difference in blood insulin levels on day 28 between the two experimental groups.

[0135] Figure 10 shows the blood bile acid levels on day 28. There was no statistically significant difference in blood bile acid levels on day 28 between the two experimental groups.

[0136] Figure 11 shows the number and percentage of granulocytes before administration and on day 28. Although there was no statistically significant difference, the change in granulocyte number showed a trend toward an increase in DA-treated animals compared to vehicle-treated controls.

[0137] Figure 12 shows the number and percentage of monocytes before administration and on day 28. Although there was no statistically significant difference, the changes in monocyte number and percentage showed a trend toward an increase in DA-administered animals compared to vehicle-administered controls.

[0138] Figure 13 shows the changes in lymphocyte and leukocyte counts before administration and on day 28. Although there were no statistically significant differences, the changes in lymphocyte and leukocyte counts and percentages tended to increase in DA-treated animals compared to vehicle-treated controls.

[0139] Figure 14 shows the cumulative food intake over 28 days. There was no statistically significant difference in food intake over 28 days between the two experimental groups.

[0140] Figure 15 shows the analysis of various blood cytokines on day 28. KC: cytokine-induced neutrophil chemoattractant (CXCL1); MCP-1: monocyte chemotactic 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. DA administration significantly reduced KC / CXCL1 and M-CSF.

[0141] Figure 16 shows the analysis of various blood cytokines on day 28. IP-10: IFN-γ-inducible protein 10 (CXCL10). IL-10 and IL-12 showed significant decreases with DA administration.

[0142] Figure 17 shows the analysis of various blood cytokines on day 28. G-CSF: granulocyte colony-stimulating factor; GM-CSF: granulocyte-macrophage colony-stimulating factor; IFNγ: interferon gamma; IL-1α, IL-1β, IL-2, and IL-5. DA administration showed significant decreases in GM-CSF, IFNα, and IL-5.

[0143] 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 conditions and contains a variety of immune cells, including macrophages, eosinophils, innate lymphoid cells (ILCs), T cells, and B cells. The accumulation of these immune cells induces chronic low-grade inflammation, which affects adipose tissue metabolism, promotes systemic inflammation, impairs insulin action, and causes deleterious effects throughout the body (Wisse, J. Am. Soc. Nephrol. 2004: 15(11):2792-800). Overproduction of pro-inflammatory factors by this immune cell accumulation has been demonstrated to play a role in this pathogenic context (Saltiel and Olefsky, J. Clin. Invest. 2017; 127(1):1-4). A wide range of pro-inflammatory factors, including cytokines and chemokines, exhibit elevated circulating levels in individuals with metabolic syndrome, obesity, diabetes, or other metabolic disorders (Tchernof and Despres, Physiol. Rev. 2013; 93(1):359-404). Some pro-inflammatory factors, such as TNF-α or IL-6, have been found to inhibit insulin action or affect lipid metabolism, thereby contributing to insulin resistance or adiposity dysfunction (McLaughlin et al. J. Clin. Invest. 2017; 127(1):5-13).

[0144] The bitter taste receptor (TAS2R) is a member of the G protein-coupled receptor (GPCR) family and is present throughout the body, not just on the tongue (Lu et al. J. Gen. Physiol. 2017; 149(2): 181-197). In this study, ob / ob mice treated with DA for 28 days showed significant weight loss compared with vehicle-treated controls; there was no difference in individual average daily food intake between these two groups of animals. Nevertheless, a panel of cytokines, including GM-CSF, IFNγ, IL-5, IL-10, IL-12, KC, and M-CSF, showed significant decreases in DA-treated mice. Therefore, the weight loss in the DA-treated group may be at least partly attributable to the fact that DA-induced agonism at TAS2R on immune cells inhibits the production of these cytokines, subsequently ameliorating the inflammatory state in adipose tissue and improving lipid metabolic dysfunction.

[0145] Example 2 This example examines the role of DA in regulating immune responses in a mouse model of air pouch inflammation. Eight C57BL / 6 mice were assigned to three groups: control (distilled water) gavage (BID), 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). The number of infiltrating cells in the air pouch exudate was measured using an ELISA assay (R&D Systems Cat. No. M6000B) and multiple cytokine analysis (Mouse 32Plex Kit MilliporeSigma Cat. No. MCYTMAG70PMX32BK). IL-6 levels in the air pouch exudate were also measured. Statistical analysis was performed by one-way ANOVA followed by Tukey's multiple comparison post hoc test for normally distributed data and by Kruskal-Wallis test followed by Dunn's multiple comparison post hoc test for skewed distributed data, with the ROUT method used to identify outliers.

[0146] Duarte et al., Current Protocols in Pharmacology, 5.6.1-5.6.8, March 2012, states the following: "The subcutaneous air pouch is an in vivo model that can be used to study acute and chronic inflammation, resolution of inflammatory responses, and oxidative stress responses. Injection of an irritant into the air pouch of a rat or mouse induces an inflammatory response that can be quantified by the volume of exudate produced, cellular infiltration, and release of inflammatory mediators. The model presented in this unit has been used extensively to identify potential anti-inflammatory drugs." It can be used to study local inflammation without systemic effects. However, in this case, drugs were administered by gavage twice a day. In a previous study using this model, R. Romano et al. (1997) showed that gavage administration of dexamethasone (a potent anti-inflammatory steroid with severe side effects) reduced TNF levels.

[0147] Test administration was 5 ml / kg body weight, administered BID every 8 hours. Each test BL6 mouse received a subcutaneous injection of 1.5 ml / mouse of sterile air on day 0 and 1.5 ml / mouse of sterile air on day 3 to create an air pouch. 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 time 0 or 1 hour after administration of the test compound. Plasma samples were collected at the end of all groups and from the air pouch exudates. Cell count analysis and IL-6 assays were performed in the animal facility, and plasma and exudate samples were sent for cytokine analysis. Eight mice were included in each of the distilled water control, 23.1 mg / kg DA, and 92.4 mg / kg DA groups.

[0148] Figure 18 shows a graph of the number of infiltrating cells in the air pouch exudate, demonstrating that DA pretreatment dose-dependently reduced the number of infiltrating cells in the air pouch exudate after LPS induction. Among the results, animals pretreated with 96.4 mg / kg DA showed significantly lower infiltrating cell counts compared to animals pretreated with vehicle and low-dose DA.

[0149] Figure 19 shows a graph of IL-6 levels in air pouch exudates, demonstrating that DA pretreatment dose-dependently reduced the number of infiltrating cells in the air pouch exudates after LPS induction. Among the results, animals pretreated with 96.4 mg / kg DA exhibited significantly lower IL-6 levels compared with animals pretreated with vehicle and low-dose DA.

[0150] Figures 20-27 show the cytokine levels of G-CSF, eotaxin, GM-CSF, IFNg, IL-1a, IL-1β, IL-2, and IL-3, respectively. Among this cytokine group, IL-1b showed a significant decrease with high dose DA.

[0151] Figures 28-35 show the cytokine levels of IL-4, IL-5, IL-7, IL-9, IL-10, IL-12p40, IL-12p70, and IL-13, respectively. Among these cytokine groups, IL-10 showed a significant decrease with high-dose DA.

[0152] Figures 36-43 show the cytokine levels of IL-15, IL-17, LIF, LIX, IP-10, KC, MCP-1, and MCP-1α, respectively. Among these cytokine groups, IL-17 showed a significant decrease with high dose DA.

[0153] Figures 44-50 show the cytokine levels of MIP-1β, MIP-2, M-CSF, MIG, RANTES, VEGF, and TNF-1α, respectively. Among this cytokine group, TNF-1α showed a significant decrease with high-dose DA.

[0154] In summary, Figure 51 shows a summary of the high (orange) and low (blue) doses, with asterisks indicating significance. Additionally, the pro-inflammatory biomarkers TNFα, IL-1β, IL-10, and IL-17 showed a significant dose-response decrease with high-dose DA administration.

[0155] Example 3 This example provides the results of an in vivo study of dextran sulfate sodium (DSS)-induced colitis in a mouse model. Inflammatory bowel disease (IBD), primarily including ulcerative colitis and Crohn's disease, is a complex, multifactorial disease with unknown etiology. Numerous mouse models of colitis have been developed to mechanistically study human IBD. These models are tools for deciphering the underlying mechanisms of IBD pathogenesis and evaluating potential therapies. Among various chemically induced colitis models, the dextran sulfate sodium (DSS)-induced colitis model is widely used due to its many similarities to human ulcerative colitis. Furthermore, many existing approved IBD drugs have been studied in this model, allowing for the comparison of new potential drug compounds with existing drugs for approved IBD indications.

[0156] C5BL / 6 mice were divided into five groups of 3–10 mice and fed a standard mouse chow diet ad libitum, housed up to five per cage. Dexamethasone 21-phosphate disodium salt (DMS; Alfa Aesar Catalog #J64083-1G, Lot R02F035) was used as a positive control. A Hemoccult kit was obtained from Beckman (Hemoccult SENSA kit). Dextran sulfate sodium (DSS) reagent grade (MPI Catalog #160110, Lot #6046H, MW 36,000–50,000, CAS 0247-1000) was used. IBD-like symptoms were induced by supplementing the water of certain groups with 9011-18-1. Treatment began on day -3, prior to DSS delivery. All mice were pre-weighed on day 1 and given fresh 4-5% DSS in water daily for 5 days, followed by water for the remainder of the study to induce disease. An additional control group received water (without DSS) for the entire study period (10 days). Body weight was measured daily, fecal occult blood status (Hemoccult) was measured three times weekly, fecal viscosity was measured three times weekly, and general health status was assessed daily. Mice were sacrificed on day 10, and serum was collected for cytokine analysis, and colon length and weight were measured. Two control groups were water only and DSS without drug administration. The two treatment groups were 69.3 mg / kg bid (n = 10) and 23.1 mg / kg bid (n = 10).

[0157] Figure 52 shows the changes in body weight during the study period. DA administration showed a significant main effect on body weight (P=0.0052).

[0158] Figure 53 shows body weight on day 10. Animals treated with 69.3 mg / kg DA BID showed a significant effect on DSS-induced weight loss compared to vehicle.

[0159] Figure 54 shows the fecal occult blood scores during the test period. DA administration showed a significant main effect on the fecal occult blood status.

[0160] Figure 55 shows the fecal viscosity scores over the study period. DA administration showed a significant main effect on fecal viscosity.

[0161] Figure 56 shows the total fecal scores over the study period. DA administration showed a significant main effect on total fecal status.

[0162] Figures 57 and 58 show the colon weight and length, respectively, on day 10. In mice, high-dose DA administration was able to counteract the DSS-induced decrease in colon weight and length, although no significant differences were observed.

[0163] Figure 59 shows spleen weight on day 10. In mice, high-dose DA administration showed a tendency to counteract DSS-induced decrease in spleen weight, although no significant effect was observed.

[0164] Example 4 Microbiome studies have shown that low levels of Parabacteroides (protective commensal bacteria) correlate with atherosclerosis, high levels of Escherichia are linked to coronary heart disease (CHD), Ruminococcacea are often elevated in patients with ACVD (atherosclerotic cardiovascular disease), and microbially produced short-chain fatty acids (SCFAs) are linked to reduced atherosclerosis, inflammation, and moderate hypertension.

[0165] We investigated the effects of a small-molecule oral TAS2R agonist (DA) on microbial communities in a mouse model of nonalcoholic steatohepatitis (NASH). Two groups of 4-week-old male C57BL / 6 mice (20 / group) were fed an Amylin Liver NASH (AMLN) diet and administered a daily dose of ARD-101 (30 mg / mL in water) or vehicle (water) via intragastric gavage. DNA was isolated from fecal samples collected at weeks 0 and 4, and microbial ecology was assessed using bTEFAP (bacterial tag-encoded FLX amplicon pyrosequencing). Operational taxonomic units were classified using BLAST against a curated NCBI database. Diversity within specific ecosystems and microbial community structure was analyzed using Qiime2. Differences were determined by repeated-measures ANOVA and post-hoc pairwise comparisons using Tukey's test. Taxonomic classification data were evaluated using a dual hierarchical dendrogram.

[0166] The AMLN diet altered the microbial communities in both groups at week 4. Significant increases and decreases were observed at the phylum, family, and genus levels between the DA and vehicle groups at week 4. For example, at the phylum level, significant increases were observed in Proteobacteria, Verrucomicrobia, and Cyanobacteria, while significant decreases were observed in Firmicutes, Deferibacter, and Firmicutes. Ecosystem and microbial community diversity was significantly lower in both treatment groups at week 4 vs. week 0, and between DA and vehicle at week 4 (p<0.05 for all comparisons). Genetic analysis showed that DA increased unsaturated fatty acid and arachidonic acid metabolism, increased cofactor and vitamin production; increased lysine degradation, glycolysis, gluconeogenesis, and phosphatidylinositol signaling; and decreased arginine and ornithine production. DA administration induced significant changes in physiological and metabolic pathways, mitigating the decline in fecal SCFAs with the diet. The overall findings are consistent with data showing that DA attenuates inflammation and metabolic syndrome.

[0167] Example 5 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 with 4 ml of sterile 4% thioglycollate broth. After washing with RPMI 1640 medium, the cell suspension was centrifuged at 800 g for 5 minutes at 4°C. Red blood cells were removed with ACK buffer, and the 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 cultured in a 24-well tissue culture plate (2 x 10 5 Macrophages were plated at 1000 x g (cells / mL / well). Macrophages were pre-cultured in serum-free RPMI 1640 medium for 24 hours to reduce the mitogenic effect. Macrophages were pre-treated with various concentrations of DA for 1 hour before LPS administration and then stimulated with LPS (100 ng / mL) for 24 hours. Treatment groups are shown in Table 4: [Table 5]

[0168] Approximately 200 μl of supernatant was removed and stored at -80°C for cytokine analysis (13plex) at 12 and 24 h post-stimulation. The cytokines analyzed were: GM-CSF, IFNγ, IL-1α, IL-1β, 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, and TNF-α.

[0169] [Table 6]

[0170] In summary, 24-hour incubation with LPS did not induce significant differences as did 12-hour incubation with LPS.

[0171] Example 6 This example provides the results of a study evaluating the effect of denatonium acetate on healthy mice based on cytokine profiles and DA administration routes. The test groups are as follows: (1) vehicle group (N=12), gavage-administered with distilled water, BID; (2) oral low-dose DA group (N=12), gavage-administered with DA at a dose of 23.1 mg / kg (salt weight), BID; (3) oral high-dose DA group (N=12), gavage-administered with DA at a dose of 92.4 mg / kg (salt weight), BID; (4) IV low-dose DA group (N=12), IV bolus-administered with DA at a dose of 1 mg / kg (salt weight), QD; (5) IV high-dose DA group (N=12), IV bolus-administered with ARD-101 at a dose of 3 mg / kg (salt weight), QD.

[0172] First, intravenous administration of DA did not result in any biomarker (cytokine) effects. It is safe to conclude that oral administration of DA is necessary for its effectiveness. Furthermore, intravenous administration alone resulted in toxic side effects. Group #3 was the low-dose oral DA group, and Group #4 was the high-dose oral DA group. Low-dose DA significantly reduced the cytokines G-CSF (p = 0.003), IL-1α (p = 0.04), IL-13 (p = 0.03), MCP-1 (p = 0.005), MIP-2 (p = 0.015), and VEGF (p = 0.001) compared with controls. High-dose DA significantly reduced the cytokines GM-CSF (p = 0.03), IL-9 (p = 0.003), KC (p = 0.05), and VEGF (p = 0.001) compared with controls. This study confirmed the effects of the biomarker in normal mice and confirmed that oral administration should be used rather than IV administration.

[0173] Example 7 This example provides the results of a study evaluating the effects of denatonium acetate in a mouse model of acute lung injury and hyperthermia. Three groups of CD-1 mice were administered: (1) saline by gavage BID; (2) DA orally at a dose of 92.4 mg / kg BID; and (3) DA iv at a 3 mg / kg iv bolus QD. Lung lavage fluid was measured and cytokine analysis was performed. Statistics included one-way ANOVA followed by Tukey's multiple comparison post-hoc test for normally distributed data, Kruskal-Wallis test followed by Dunn's multiple comparison post-hoc test for skewed data, and the ROUT method for outlier identification. Control or drug treatment was administered for 3 days, followed by intratracheal administration of 1 mg / ml LPS using a 50L Penn Century needle. Mice were sacrificed 24 hours post-LPS when their core temperature reached 39°C, and lung lavage fluid protein concentrations and serum cytokine levels were measured.

[0174] DA dramatically reduced protein concentrations in lung lavage fluid both orally and intravenously, but not significantly. The cytokine profile in lung lavage fluid is shown in Figure 72 for DA = ARD-101.

[0175] Example 8 This example provides the results of a second modified acute lung injury plus hyperthermia study to evaluate the effects of denatonium acetate. The same procedures as in Example 7 were used. Starting 3 days before lung injury induction, groups of six CD-1 mice were prophylactically administered vehicle or 92.4 mg / kg denatonium acetate (DA) (administered by oral gavage twice daily (BID)) or 3 mg / kg DA (administered by intraperitoneal (IP) injection once daily (QD)). 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 specimens were evaluated for cytokine concentrations (using various bead-based assays), protein levels, and neutrophil counts (by fluorescence-activated cell sorting (FACS)). Additionally, lungs were harvested, fixed, stained with Masson's trichrome, and histologically evaluated. Female CD-1 mice received repeated PO (BID) administration of 92.4 mg / kg DA or IP (QD) administration of 3 mg / kg DA for 3 days and were well tolerated. Two mice (one vehicle-treated and one DA (92.4 mg / kg)-treated) died on day 1; however, the timing of these deaths (within 24 h after LPS injection) suggests that these deaths reflect the injection process (not the test article), hyperthermia, or associated inflammation. This is consistent with the observation that deaths occurred with both vehicle and test article administration. No other adverse clinical observations were noted during the 3-day test article administration. Oral administration of 92.4 mg / kg DA significantly reduced (compared to vehicle) the BALF concentrations of 7 of the 32 cytokines tested (including IL-2, IL-3, IL-10, MIP-1β, MCSF, and MIG). IP administration of 3 mg / kg DA significantly reduced (compared to vehicle) the BALF concentrations of 10 of the 32 cytokines tested (G-CSF, eotaxin, IL2, IL-3, IL-4, IL-13, IP-10, MCP-1, M-CSF, and MIG) (see Figure 73).Oral and IP administration of DA at the indicated levels was associated with nominal (but not significant) changes in BALF protein concentration, a nominal decrease in BALF neutrophil counts (by FACS assay), and a nominal reduction in the severity of lung pathology (by histological scoring). Thus, 92.4 mg / kg DA administered PO twice daily or 3 mg / kg DA administered IP once daily resulted in significant attenuation of the accumulation of multiple cytokines in the lungs of this mouse model of acute lung injury, along with nominal activity against neutrophil infiltration and lung injury in these animals.

[0176] Example 9 This example presents the results of a study on the effects of dietary induced DA plus another compound (CQL) on mouse body weight (DIO). Adult C57BL / 6NTac mice were fed a high-fat diet (60%). The vehicle group (N=15) received distilled water by gavage BID, the CQL group (N=15) received a 50 mg / kg dose by gavage BID, and the DA group (N=15) received a 92.4 mg / kg dose by gavage BID. The study period was 56 days followed by a 2-3 day test period. Body weight changes were measured three times weekly, and food and water intakes were 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 1 hour after dosing on days 1 and 56, and 2 hours after dosing on day 7 (dosing (until after blood sampling, >6 h fasting before dosing); serum levels of PPY on day 56).

[0177] Figure 74 shows that in DIO mice, DA administration significantly reduced weight gain on day 57 compared with vehicle and CQL. Figure 75A shows that in DIO mice, DA administration significantly reduced daily food intake compared with vehicle on day 14, and Figure 75B shows that DA administration significantly increased daily water intake compared with vehicle on day 28, while CQL administration significantly reduced daily water intake compared with vehicle. Figure 76 shows that in DIO mice, DA and CQL administration significantly reduced serum HbA1c levels on day 28, but significantly elevated HbA1c levels on day 56. Figure 77 shows that in DIO mice, DA administration significantly reduced serum insulin levels on day 28 compared with vehicle control. In Figure 78, no significant difference was observed, but DA administration significantly reduced serum LDL levels on days 28 and 56 compared with vehicle control. Figure 79 shows that serum GLP-1 levels were significantly elevated in DIO mice on days 7 and 56 with DA administration compared to vehicle controls. Figure 80 shows that serum GLP-2 levels were significantly elevated in DIO mice on day 56 with DA administration compared to vehicle controls. Figure 81 shows that serum CCK levels were significantly elevated in DIO mice on day 56 with DA administration compared to vehicle controls. Figure 82 shows that serum PYY levels were significantly elevated in DIO mice on day 56 with DA administration compared to vehicle controls.

[0178] Serum cytokines measured on days 28 and 56 (28 / 56) showed significant increases in G-CSR (p=0.063 / 0.039), eotaxin (p=0.031 / no sig), IL-6 (p=0.041 / no sig), IP-10 (p=0.013 / 0.028), and MIG (p=no sig). In many mice, sufficient blood was not available to achieve statistical significance.

[0179] Example 10 Leptin-deficient ob / ob mice exhibit hyperphagia and obesity, as well as hyperglycemia and hypertriglyceridemia, which are also observed in patients with hyperphagic disorders, such as Prader-Willi syndrome and monogenic syndromic 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). Therefore, ob / ob mice are a predictive in vivo model of these conditions. This example provides the results of a study of the effects of DA plus another compound (CQL) on the body weight of leptin-deficient (ob / ob) mice. The vehicle group (N = 14) received distilled water by force BID, and the DA group (N = 14) received a dose of 50 mg / kg by force BID. The study period was 56 days, followed by a 2-3 day test period. Body weight change was measured three times per week, food intake was measured twice per week, and metabolic biomarkers (blood glucose, blood insulin, blood HbA1c, blood HDL, blood LDL, blood triglycerides, and blood bile acids) were measured at the beginning and end of the study. Cytokine analysis was measured at the end of the 56th day.

[0180] DA administration did not significantly affect the body weight of ob / ob mice. DA administration did not significantly affect daily food intake in ob / ob mice. Figure 83 shows that DA administration significantly reduced serum glucose levels in ob / ob mice. DA administration did not significantly affect serum HBA1c or insulin levels in ob / ob mice. Figure 84 shows that DA administration significantly reduced serum triglyceride levels in ob / ob mice compared to vehicle controls. Figure 85 shows that DA administration significantly increased serum bile acid levels in ob / ob mice compared to vehicle controls. Figure 86 shows that DA administration significantly reduced serum LDL levels in ob / ob mice compared to vehicle controls. However, there was no significant effect on serum HDL levels.

[0181] The DA group had significantly reduced cytokines (vs. control) for eotaxin (p=0.047) and MIG (p=0.026) at day 56. Although not significantly different, the DA group also showed reduced levels of the following cytokines at day 56 compared to the vehicle group: RANTES (1.7% decrease), IL-1β (19.1% decrease), IL-6 (61.4% decrease), and MCP-1 (20.9% decrease).

Claims

1. A pharmaceutical composition for oral administration, comprising a denatonium salt selected from the group consisting of denatonium acetate (DA), denatonium citrate, denatonium maleate, denatonium saccharide, and denatonium tartrate, for treating, preventing, and delaying the exacerbation of acute inflammatory lung disease.

2. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition further comprises about 0.5 g to about 5 g of acetic acid.

3. The pharmaceutical composition according to claim 2, wherein the pharmaceutical composition further comprises about 1.5 g to about 3 g of acetic acid.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the daily dose of denatonium salt for adults is approximately 20 mg to approximately 5000 mg.

5. The pharmaceutical composition according to claim 4, wherein the daily dose of denatonium salt for adults is approximately 50 mg to approximately 1000 mg.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the daily dose of denatonium salt for adults is approximately 60 mg to approximately 500 mg, or a dose that achieves an intra-gastrointestinal concentration of approximately 10 ppb to approximately 50 ppm.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the daily dose of denatonium salt for adults is approximately 5 mg / kg (body weight) to approximately 150 mg / kg (body weight) per day.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein the denatonium salt is denatonium acetate (DA).

9. The pharmaceutical composition according to any one of claims 1 to 7, wherein the denatonium salt is denatonium citrate.

10. The pharmaceutical composition according to any one of claims 1 to 7, wherein the denatonium salt is denatonium maleate.

11. The pharmaceutical composition according to any one of claims 1 to 7, wherein the denatonium salt is denatonium tartrate.

12. The pharmaceutical composition according to any one of claims 1 to 8, wherein the daily dose of DA for adults is approximately 50 mg to approximately 1000 mg.

13. The pharmaceutical composition according to claim 12, wherein the daily dose of DA for adults is approximately 60 mg to approximately 500 mg.

14. The pharmaceutical composition according to claim 12 or 13, wherein the daily dose of DA for adults is a dose that achieves an intratube concentration of approximately 10 ppb to approximately 50 ppm.

15. The pharmaceutical composition according to any one of claims 1 to 14, wherein the daily dose of denatonium salt is administered once, twice, or three times a day.

16. The pharmaceutical composition according to any one of claims 1 to 15, wherein the acute inflammatory pneumonia is acute respiratory distress syndrome (ARDS).

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