Pharmaceutical compositions for treating or preventing various inflammatory disorders

Abstract

The present invention discloses 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 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 invention 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.

Description

Technical Field

[0001] The present disclosure provides a method for treating, preventing and / or delaying the progression of various chronic inflammatory disorder groups, including (1) 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 disease (IBD), such as Crohn's disease and ulcerative colitis; (5) metabolome-mediated diseases (atherosclerosis, hypertension and congestive heart failure); and (6) hyperphagia disorders, such as Prader-Willi syndrome, and other monogenic symptomatic obesity disorders including leptin pathway deficiency, which comprises orally administering a pharmaceutical composition containing a sodium salt. The present disclosure is based on the readouts from a series of studies tracking clusters of biomarker levels for tracking mediators of inflammatory disorders and mediators of intestinal signaling hormones responsive to orally administered sodium salts. The present disclosure further provides a pharmaceutical composition for treating and preventing various inflammatory conditions that can be tracked by inflammatory biomarkers, which comprises administering a pharmaceutical composition containing a sodium salt. Preferably, the pharmaceutical composition of the present invention for oral administration daily contains a sodium salt that delivers a total daily dose of about 20 mg to about 5000 mg to adult humans BID. Preferably, the sodium salt is selected from the group consisting of sodium acetate, sodium citrate, sodium maleate and sodium tartrate.

Background Art

[0002] Over the past 40 years, global obesity levels have more than doubled. Obesity increases the risk of metabolic syndrome and is associated with an increased risk of coronary heart disease, stroke, type 2 diabetes, certain forms of cancer, and ultimately, serious illness and death due to the coronavirus pandemic. This expanding epidemic has become one of the most critical challenges to global health today. With the emergence of this problem, our understanding of the pathological mechanisms linking obesity to disease progression has deepened. At the heart of these mechanisms lies the increased systemic inflammation resulting from obesity, which ultimately leads to numerous pathological conditions. Therefore, there is a great need for treatment and prevention to address appetite and inflammatory signaling. This disclosure addresses this need.

[0003] inflammatory disease Currently, various inflammatory diseases are 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), panuveitis, IBD including ulcerative colitis and Crohn's disease, and many other diseases. These biologics act by binding and scavenging circulating TNFα (and IL-6) with antibodies or fusion proteins, such as etanercept (Embrel®). However, these anti-TNFα drugs, and other biologics that indiscriminately bind and scavenge inflammatory cytokines, have severe side effects. These side effects are caused by the inhibition of a large portion of TNF signaling. Because TNF possesses immune surveillance capabilities (which are also inhibited by these biological drugs), it increases susceptibility to infection and impaired immune surveillance, including an increased incidence of various infections and malignancies, such as leukemia and lymphoma, listed on the black-bordered warning label. Therefore, there is a need in the art for more cost-effective small molecule therapies that knock down (but not necessarily eliminate) circulating TNF. Since protein-based therapies cannot be administered orally, there is a need in the art for more sensitive or self-limiting oral small molecule agents 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 label on adalimumab (Humira®), approved by the US FDA, lists side effects such as an increased risk of serious infections (i.e., TB; infections caused by viruses, fungi, or bacteria), exacerbation of hepatitis B infection in virus carriers, allergic reactions, and various leukemias and lymphomas.

[0005] Metabolic syndrome Metabolic syndrome (METS) is a combination of 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 conditions: (1) visceral obesity; (2) elevated blood pressure; (3) elevated blood glucose levels; (4) high serum triglycerides; and (5) low serum high-density lipoprotein (HDL).

[0006] According to the International Diabetes Foundation (IDF), metabolic syndrome is characterized by 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) decreased HDL <40 mg / dL (1.03 mmol / L) (<50 mg / dL in men, 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 having hyperinsulinemia and any two of the following: (1) abdominal obesity (waist / hip ratio > 0.90 or BMI 30 kg / m²) 2 (2) dyslipidemia (TG > 1.7 or HDL < 0.9 mmol / L), and (3) hypertension (BP > 140 / 90 mm Hg or use of antihypertensive drugs). In a clinical study that examined carbohydrate restriction as a first-line dietary intervention for METS, the significance of biomarkers including the inflammatory biomarkers TNFα, IL-6, and MCP-1 was investigated in fasting participants (Al-Sarraj et al., J. Nutrition 139(9):1667-1675, 2009). In this study (n=20), significance was found for MPC-1, ICAM-1, and TNFα, but not for IL-6.

[0008] METS affects 20-25% of the global adult population, with 35% of that in the United States. METS is present in approximately 60% of US residents aged >50. Furthermore, METS is correlated with a high frequency of autoimmune diseases. Therefore, there is a need in this field to provide safer and more effective treatments for METS.

[0009] ARDS and viral respiratory infections Acute respiratory distress syndrome (ARDS) is a life-threatening illness characterized by an acute onset of hypoxia and pulmonary infiltration, triggered by conditions such as sepsis, pneumonia, trauma, burns, pancreatitis, and blood transfusion. ARDS leads to generalized pneumonia resulting in increased pulmonary vascular permeability, pulmonary edema, and alveolar epithelial damage. Diagnosis of ARDS is based on the following criteria: (1) acute onset; (2) bilateral pulmonary infiltration of non-cardiac origin on chest X-ray or tomography (CT) scan; and (3) moderate to severe oxygenation impairment. Severe ARDS has a mortality rate of 45%. The severity of ARDS is defined by the degree of hypoxemia, calculated as the ratio of arterial oxygen partial pressure to inspired oxygen fraction (PaO2 / FiO2). ARDS is classified into three types: mild, moderate, and severe. According to the Berlin definition of ARDS, a PaO2 / FiO2 ratio of 200-300 is considered mild, 100-199 is moderate, and <100 is severe.

[0010] Generally, the progression of ARDS is divided into two stages: the initiator stage and the subsequent effector stage. The initiator stage of ARDS involves the release of inflammatory mediators (i.e., cytokines; complement and aggregation factors; and arachidonic acid metabolites) that promote systemic inflammation leading to the sequestration of pulmonary neutrophils. The second stage, the effector stage, involves the activation of neutrophils and the subsequent release of toxic oxygen radicals and proteolytic enzymes, particularly neutrophil elastase (NE). NE has the ability to damage pulmonary endothelial cells and degrade extracellular matrix products such as elastin, collagen, and fibronectin, including those in the basal membrane.

[0011] ARDS exists in many diverse forms, each with different etiologies and courses, but the ultimate pathogenesis is the same across these diverse forms. Examples of clinical events that can induce various forms of ARDS include trauma, bleeding, generalized pneumonia, virus-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 life-saving emergency care. Regardless of the initial cause, ARDS has the following commonalities: fluid accumulation and exudate in the lungs lead to generalized alveolar damage and impaired alveolar gas exchange. Common (regardless of the initial cause of ARDS) is exacerbation due to inflammation, fluid release, cell migration and proliferation, and increased pro-inflammatory cytokines.

[0012] Viral respiratory infections typically have an incubation period of 2–7 days, and infected individuals usually present with a high fever, sometimes accompanied by chills, headache, fatigue, and muscle aches. Viral lung infections account for approximately 10–15% of annual ICU admissions in the United States, even without a pandemic, and account for a significant proportion of annual influenza deaths, even without a coronavirus pandemic. The 2020 COVID-19 pandemic demonstrates the progression of this disease. The illness progresses with the onset of a dry cough or dyspnea, often accompanied by or progressing to hypoxemia. A significant number of cases require intubation and mechanical ventilation. Furthermore, at the peak of respiratory illness, approximately 50% of infected individuals progress to leukopenia or thrombocytopenia (MMWR Morb Mortal Wkly Rep. 2003 Mar 28;52(12):255-6).

[0013] The patterns of viral loading spread (such as coronaviruses or influenza viruses) suggest droplet or contact transmission of the viral pathogen (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 by single-stranded RNA viruses with a genome of approximately 30,000 nucleotides (Science. 2003 May 30;300(5624):1394-9). Coronaviruses are classified into three known groups, the first two causing coronavirus infections in mammals, and the third causing coronavirus infections in birds (JSM Peiris, in Medical Microbiology (Eighteenth Edition), 2012, 587-593). Coronaviruses are thought to be causative agents of several serious 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] Therefore, effective measures are needed for patients diagnosed with SARS, patients infected with SARS-related infectious agents, such as SARS-CoV, or patients at imminent risk of contracting SARS, such as individuals who have been exposed to or may be exposed to SARS-related infectious agents in the near future.

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

[0016] Metabolome The gut microbiota has attracted considerable attention, and imbalances in the gut microbiota are associated with several diseases, depending on which groups of bacteria increase or decrease. Atherosclerotic diseases, accompanied by signs such as myocardial infarction and stroke, are a major cause of severe illness and death in patients with metabolic syndrome. This disease is thought to be caused by the accumulation of cholesterol and the recruitment of macrophages to the arterial wall, and can therefore be considered both a metabolic and inflammatory disease. Since the first half of the 19th century, it has been suggested that infections cause or accelerate atherosclerosis by increasing pro-atherosclerotic changes in vascular cells. However, there is still a need for better methods to delay pro-atherosclerotic changes in vascular cells and the associated diseases at an early stage. This invention provides a method for delaying pro-atherosclerotic changes in vascular cells by reducing gut signals that support pro-atherosclerotic changes in vascular cells.

[0017] Increased appetite In particular, when high-calorie-density foods and foods high in fat, especially saturated fat, are widely available, as is often the case in developed countries, the regulation of eating behavior, including both controlling appetite for certain food compositions and developing a preference for low-fat or low-calorie foods, can provide a mechanism to prevent the progression 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, i.e., body weight, is leptin, a circulating protein encoded by the ob gene, which is primarily expressed in adipose tissue. Leptin plays a central role in regulating energy balance, suppressing food intake and increasing energy expenditure (Zhang et al., Nature, 372, 425-432, 1994). This protein circulates in the blood at concentrations proportional to the size of the fat depot, crosses the blood-brain barrier via a saturation system, and exerts most of its influence on energy balance at the central level through protein-receptor interactions with receptors located in hypothalamic neurons and other areas of the brain (Tartaglia et al., Cell. 83, 1263-1271, 1995).

[0019] Animals with defects in the leptin signaling axis either fail to produce functional proteins or express defective forms of their receptors, resulting in early hyperfeeding and severe obesity, as well as diabetes, hypothermia, and infertility. In humans, genetic deficiencies in leptin signaling (absence of leptin or its receptor) are also associated with early-onset pathological 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 sense, the use of leptin for the treatment or prevention of diabetes directly caused by obesity has been proposed (WO97 / 02004).

[0020] While leptin's short-term appetite-suppressing effect was thought to contribute to controlling obesity and related disorders in obese individuals, unfortunately, leptin administration alone was not a practical treatment due to partial tolerability and compensatory upregulation of other hunger and satiety-mediated pathways. Furthermore, long-term treatment outcomes were unsatisfactory.

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

[0022] High leptin circulation levels increase the risk of cardiovascular disease in humans [Ren, J. Endocrinol., 2004, 181, 1-10] and also lead to the progression of insulin resistance [Huang et al., Int. J. Obes. Relat. Metab. Disord., 2004, 28, 470-5], but this is unrelated to the body mass index / obesity. [Overview of the project]

[0023] This 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 disorders (atherosclerosis, hypertension, and congestive heart failure); and (6) hyperphagia, e.g., Prader-Willi syndrome, and other monogenic symptomatic obesity disorders, including leptin pathway deficiency, each comprising orally administering a pharmaceutical composition comprising a denatonium salt. This disclosure is based on readouts from a series of studies tracking biomarker-level clusters to track mediators of inflammatory disorders and mediators of intestinal signaling hormones in response to orally administered denatonium salts. This disclosure further provides pharmaceutical compositions for treating and preventing various inflammatory conditions that can be tracked by pro-inflammatory biomarkers, comprising administering a pharmaceutical composition comprising a denatonium salt. Preferably, the pharmaceutical composition of the present invention, administered orally daily, comprises a denatonium salt delivered to a human adult in a total daily dose of about 20 mg to about 5000 mg by 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 a method for treating, preventing, and delaying the progression of type 2 diabetes including metabolic syndrome (MET), obesity, and hyperglycemia, which comprises orally administering a pharmaceutical composition comprising a sodium salt selected from the group consisting of sodium acetate (DA), sodium citrate, sodium maleate, sodium saccharide, and sodium tartrate. Preferably, the pharmaceutical composition further comprises about 0.5 g to about 5 g of acetic acid. More preferably, the daily dose of acetic acid for an adult is about 1.5 g to about 3 g. Preferably, the daily dose of the sodium 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 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 (parts per billion) to about 50 ppm. The daily dose of the sodium salt is administered once a day, twice a day, or three times a day.

[0025] The present disclosure provides a method for treating, preventing, and delaying the progression of acute pulmonary inflammatory disorders including ARDS, which comprises orally administering a pharmaceutical composition comprising a sodium salt selected from the group consisting of sodium acetate (DA), sodium citrate, sodium maleate, sodium saccharide, and sodium tartrate. Preferably, the pharmaceutical composition further comprises about 0.5 g to about 5 g of acetic acid. More preferably, the daily dose of acetic acid for an adult is about 1.5 g to about 3 g. Preferably, the daily dose of the sodium 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. 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 the sodium salt is administered once a day, twice a day, or three times a day.

[0026] The present disclosure provides a method for treating, preventing, and delaying the exacerbation of a group of chronic autoimmune inflammatory disorders with symptoms selected from the group consisting of rheumatoid arthritis (RA), lupus, and psoriasis, which comprises orally administering a pharmaceutical composition comprising a sodium salt selected from the group consisting of sodium acetate (DA), sodium citrate, sodium maleate, sodium saccharide, and sodium tartrate. Preferably, the pharmaceutical composition further comprises about 0.5 g to about 5 g of acetic acid. More preferably, the daily dose of acetic acid for an adult is about 1.5 g to about 3 g. Preferably, the daily dose of the sodium 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 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 the sodium salt is administered once, twice, or three times a day.

[0027] The present disclosure provides a method for treating, preventing, and delaying the exacerbation of a group of chronic IBD with symptoms selected from the group consisting of Crohn's disease and ulcerative colitis, which comprises orally administering a pharmaceutical composition comprising a sodium salt selected from the group consisting of sodium acetate (DA), sodium citrate, sodium maleate, sodium saccharide, and sodium tartrate. Preferably, the pharmaceutical composition further comprises about 0.5 g to about 5 g of acetic acid. More preferably, the daily dose of acetic acid for an adult is about 1.5 g to about 3 g. Preferably, the daily dose of the sodium 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 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 the sodium salt is administered once, twice, or three times a day.

[0028] This disclosure provides a method for treating, preventing, and delaying the exacerbation of metabolome-mediated symptoms selected from the group consisting of atherosclerosis, hypertension, and congestive heart failure (CHF), comprising orally administering a pharmaceutical composition comprising 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 comprises about 0.5 g to about 5 g of acetic acid. More preferably, the daily dose of acetic acid for adults is about 1.5 g to about 3 g. Preferably, the daily dose of denatonium salt for adults 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 adults is about 50 mg to about 1000 mg. Most preferably, the daily dose of DA for adults is about 60 mg to about 500 mg, or a dose that achieves an intravascular concentration of about 10 ppb to about 50 ppm. The daily dose of the denatonium salt is administered once, twice, or three times a day.

[0029] This disclosure provides a method for treating or delaying the exacerbation of hyperfeeding in a symptom selected from the group consisting of Prader-Willi syndrome and leptin pathway deficiency, comprising orally administering a pharmaceutical composition comprising 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 comprises about 0.5 g to about 5 g of acetic acid. More preferably, the daily dose of acetic acid for adults is about 1.5 g to about 3 g. Preferably, the daily dose of denatonium salt for adults 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 adults is about 50 mg to about 1000 mg. Most preferably, the daily dose of DA for adults is about 60 mg to about 500 mg, or a dose that achieves an intravascular concentration of about 10 ppb to about 50 ppm. The daily dose of the denatonium salt is administered once, twice, or three times a day. [Brief explanation of the drawing]

[0030] [Figure 1] Figure 1 shows the time course of body weight with DA administration compared to the vehicle control.

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

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

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

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

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

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

[0037] [Figure 8]Figure 8 shows the total blood cholesterol levels (LDL + HDL) on day 28. In animals administered 23.1 mg / kg of DA, the total blood cholesterol levels on day 28 showed an almost significant decrease compared to vehicles-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 between the two experimental groups on day 28.

[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 between the two experimental groups on day 28.

[0040] [Figure 11] Figure 11 shows the number and percentage of granulocytes before administration and on day 28. Although there were no statistically significant differences, the change in granulocyte number tended to 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 at day 28. Although there were no statistically significant differences, the DA-treated animals showed a tendency for increased changes in the number and percentage of monocytes compared to vehicle-treated controls.

[0042] [Figure 13] Figure 13 shows the changes in lymphocyte and white blood cell counts before administration and at day 28. Although there were no statistically significant differences, DA-treated animals showed a tendency for increased changes in the number and percentage of lymphocytes and white blood cells 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 between the two experimental groups over the 28 days.

[0044] [Figure 15] Figure 15 shows the analysis of various serum 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. KC or CXCL1 and M-CSF showed a significant decrease with DA administration.

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

[0046] [Figure 17] Figure 17 shows the analysis of various serum 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. GM-CSF, IFNα, and IL-5 showed a significant decrease with DA administration.

[0047] [Figure 18] Figure 18 shows the number of infiltrating cells in the air sac exudate, demonstrating that pre-administration of DA dose-dependently reduced the number of infiltrating cells in the air sac exudate after LPS induction. Among the results, animals pre-administered with 96.4 mg / kg of DA showed significantly lower infiltrating cell counts compared to animals pre-administered with a vehicle and animals pre-administered with a low dose of DA.

[0048] [Figure 19]Figure 19 shows the IL-6 levels in the air sac exudate, demonstrating that pre-administration of DA resulted in a dose-dependent decrease in the number of infiltrating cells in the air sac exudate after LPS induction. Among the results, animals pre-administered with 96.4 mg / kg of DA showed significantly lower IL-6 levels compared to animals pre-administered with a vehicle and animals pre-administered with a low dose of DA.

[0049] [Figure 20-27] Figures 20-27 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 28-35 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 36-43 show the cytokine levels of IL-15, IL-17, LIF, LIX, IP-10, KC, MCP-1, and MCP-1a, respectively. In this cytokine group, IL-17 showed a significant decrease with high-dose DA.

[0052] [Figure 44-50] Figures 44-50 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 summaries of high-dose (orange) and low-dose (blue) results, with asterisks indicating statistical significance.

[0054] [Figure 52] Figure 52 shows the change 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 and BID showed a significant effect on DSS-induced weight loss compared to the vehicle.

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

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

[0058] [Figure 56] Figure 56 shows the overall fecal score during the study period. DA administration showed a significant main effect on overall fecal status.

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

[0060] [Figure 59] Figure 59 shows the spleen weight on day 10. No significant effect was observed in mice, but high-dose DA administration tended to counteract the DSS-induced decrease in spleen weight.

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

[0062] [Figure 61] Figure 61 shows the significant difference between the treatment group and the control group at the departmental level.

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

[0064] [Figure 63] Figure 63 shows a significant enhancement of the unsaturated fatty acid biosynthesis pathway after 4 weeks of DA administration (upper panel: individual data, lower panel: group data).

[0065] [Figure 64] Figure 64 shows a significant enhancement of the arachidonic acid metabolic pathway after 4 weeks of DA administration (upper panel: individual data; lower panel: group data).

[0066] [Figure 65] Figure 65 shows significant enhancement of cofactor and vitamin metabolic pathways after 4 weeks of DA administration (upper panel: individual data; lower panel: group data).

[0067] [Figure 66] Figure 66 shows a significant enhancement of the lysine degradation pathway after 4 weeks of DA administration (upper panel: individual data; lower panel: group data).

[0068] [Figure 67] Figure 67 shows a significant enhancement of glycolysis and gluconeogenesis pathways after 4 weeks of DA administration (group data).

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

[0070] [Figure 69] Figure 69 shows a significant decrease in signaling for arginine and ornithine metabolism after 4 weeks of DA administration (upper panel: individual data; lower panel: group data).

[0071] [Figure 70A-C] Figures 70A-C show graphs comparing biomarkers across various studies by subject, demonstrating a decrease in the average percentage.

[0072] [Figure 71] In Figure 71, it should be noted that clusters of multiple biomarkers predict the effectiveness of each disease symptom, and are shown in separate groups in Figure 71.

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

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

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

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

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

[0078] [Figure 78] In Figure 78, although no statistically significant difference was observed, serum LDL levels on days 28 and 56 were significantly lower in the DA administration group compared to the vehicle control group.

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

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

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

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

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

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

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

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

[0087] (Detailed explanation) This 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 disorders (atherosclerosis, hypertension, and congestive heart failure); and (6) hyperphagia, e.g., Prader-Willi syndrome, and other monogenic symptomatic obesity disorders, including leptin pathway deficiency, each comprising orally administering a pharmaceutical composition comprising a denatonium salt. This disclosure is based on readouts from a series of studies tracking biomarker-level clusters to track mediators of inflammatory disorders and mediators of intestinal signaling hormones in response to orally administered denatonium salts. This disclosure further provides pharmaceutical compositions for treating and preventing various inflammatory conditions that can be tracked by pro-inflammatory biomarkers, comprising administering a pharmaceutical composition comprising a denatonium salt. Preferably, the pharmaceutical composition for daily oral administration comprises a denatonium salt delivered to a human adult in a total daily dose of about 20 mg to about 5000 mg by 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 (1) the discovery of a cluster of surprising results from an in vivo study of weight loss in a predictive ob / ob obese mouse model using denatonium salts and placebo controls. In several study data from various in vivo models, oral administration of denatonium salts with organic acid anions showed a therapeutic effect, demonstrating a significant anti-inflammatory effect by first measuring inflammatory cytokines in blood and other fluids (e.g., air sac exudate and pulmonary lavage fluid) as biomarkers, and then measuring intestinal signaling peptides. Oral administration (not intravenous administration) provided data demonstrating the effectiveness of a method for treating, preventing, and delaying disease progression in signs including metabolic syndrome (METS), obesity (inflammatory-mediated), 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 showing treatment and preventive effects mainly for signs including inflammatory bowel disease (IBD), including ulcerative colitis and Crohn's disease (Example 3); (3) a 4-week microbiome study in mice fed a high-fat diet showing treatment and preventive effects for atherosclerosis, hypertension, and congestive heart failure (Examples 4 onwards). Measurement of a cluster of pro-inflammatory cytokines achieved significant differences between drug-treated mice and control mice. Weight loss showed a strong trend in in vivo with DA administration, but similarly, the difference was not statistically significant.

[0089] The cytokine data provided herein demonstrate that the study drug DA showed therapeutic activity in three areas in an inflammatory bowel disease model (Example 3) and an air sac model of inflammatory disease: (1) treatment or prevention of METS; (2) treatment or prevention of common 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, based on microbiome data. Therefore, the data obtained in these studies tell a story, which is that denatonium salt pharmaceutical compositions demonstrate safety and efficacy for (1) treatment or prevention of METS; (2) treatment of obesity and weight loss effects; (3) treatment of autoimmune inflammatory conditions such as rheumatoid arthritis (RA), lupus and psoriasis; (4) treatment of Crohn's disease and inflammatory bowel disease (IBD); and (5) treatment or delay of disease progression in cardiovascular diseases such as 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 aforementioned conditions is administered orally to adults at a dose of about 25 mg to about 500 mg per day.

[0090] Furthermore, the test in Example 2 yielded a surprisingly statistically significant reduction in IL-5 production, demonstrating the efficacy of the pharmaceutical composition of the present invention, which includes DA and a denatonium salt, in the treatment of ARDS. TIFF0007857219000001.tif35151

[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 while stirring, and heat to 70-90°C. This solution needs to be heated and stirred at the values ​​given above for 24 hours, and then cooled to 30°C. Remove any unreacted reagents with 3 × 10 mL of toluene. Dissolve 65 g of sodium hydroxide in 65 mL of cold water while stirring, and add it to the aqueous solution while stirring over 3 hours. Filter the mixture, wash with water, and dry outdoors. Recrystallize in hot chloroform or hot ethanol. [ka] [ka]

[0092] Step 2: Preparation of denatonium acetate from denatonium hydroxide. 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 were added to a reflux apparatus. The mixture was stirred and heated at 35°C for 3 hours. Next, it was evaporated to dryness and recrystallized in hot acetone. [ka]

[0093] Formulation of DA tablets This provides a 50 mg granular formulation of denatonium acetate monohydrate (DA) as a free base, which is 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 stratification process was carried out in a fluid bed granulator (rotor-type granulator) equipped with a rotor insert. 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-type granulator. The final drug-loaded pellet was then dried in the rotor-type granulator for 10 minutes, discharged, and sieved through a #20 mesh.

[0096] 2. Seal coating process - seal coated pellets A seal coating dispersion was prepared by separately dissolving hypromellose E5 in a mixture of ethyl alcohol and purified water (1:1) until a clear solution was obtained. Next, the remaining ethyl alcohol was added to the solution, followed by talc. The dispersion was mixed for 20 minutes until the talc dispersion was homogenized. The seal coating dispersion was sprayed tangentially onto the drug-loaded pellets to achieve a 5% by weight increase. The seal coating 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 coating pellets were then sieved through a #20 mesh.

[0097] 3. Final Mixture - Denatonium Immediate Release (IR) Pellet Using a V-type mixer, talc sieved through a #60 mesh and seal-coated pellets were mixed for 10 minutes and then discharged. The mixed seal-coated beads and denatonium IR pellets were used for encapsulation.

[0098] 4. Encapsulation - Denatonium capsule, 50 mg Using an automated capsule filling machine, 50 mg of denatonium IR pellets were filled into size 1, white, opaque, rigid gelatin capsules. The capsules were then passed through a single-row capsule polishing machine and a metal detector. In-process control of capsule weight and appearance was performed during the encapsulation process. Quality assurance (QA) for the composite samples involved sampling inspections to meet acceptable quality levels (AQL) during the encapsulation process. Composite samples of the finished product were collected and analyzed according to the release test instructions.

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

[0100] The relationship between biomarkers and signs of disease Numerous examples provided herein demonstrate the effects of denatonium salts on various in vivo and in vitro models of various disease symptoms. Furthermore, blood samples were collected from test (and control) animals, and various biomarkers were measured and compared. Figures 70A–C are graphs comparing biomarkers across many studies. Table 2 groups the biomarkers by family, showing the average percentage reduction and indicating which disease symptoms are affected and predicted by each biomarker. It should be noted that clusters of multiple biomarkers predict the effectiveness of each disease symptom, and these clusters are shown in Figure 71.

[0101] [Table 2]

[0102] Microbiome In a mouse model fed a high-fat diet, microbiome changes were observed after 4 weeks in the group receiving oral DA compared to the group not receiving it. 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-administered group and the control group at 4 weeks.

[0103] At week 4, various organisms that had changed in the control and treatment groups were classified, and extensive changes were observed at the level of major or dominant bacterial phyla, as well as families and genera. For example, the phylum Firmicutes decreased dramatically in the treatment group, while Proteobacteria and Verrucomiclobia increased dramatically. Diversity at week 4 decreased in both the control and treatment groups over the study period due to the effects of diet. In the treatment group, overall diversity at week 4 was even more significantly decreased compared to the control group, indicating an increase in specialized populations.

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

[0105] Genetic possibility 1: Increased metabolism of unsaturated fatty acids. The biosynthetic pathway of unsaturated fatty acids was significantly enhanced. Accumulated evidence supports the benefits of dietary unsaturated fatty acids compared to saturated fatty acids, such as improved cardiovascular health (Front Pharmacol. 2018; 9:1082; Circulation. 2017; 136(3):e1-e23; Ann. Intern. Med. 2014; 160(6):398-406).

[0106] Genetic possibility 2: Increased arachidonic acid metabolism. Arachidonic acid metabolites are important factors in the initiation and resolution of inflammation and are associated with the pathophysiology of obesity, diabetes, non-alcoholic fatty liver disease (NAFLD) / non-alcoholic 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 changes in biological function associated with various human diseases (Nutrients. 2019;11(7):1578). Several 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).

[0108] Genetic possibility 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 intestinal barrier function (Nat. Microbiol. 2018; 3(12):1332-1333). Another end product, acetate, also has a similar anti-inflammatory 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 pirubate, acetyl coenzyme A (CoA), and finally to acetate, propionic acid, and butyrate (J. Lipid Res. 2016;57(6):943-54). This regulation is linked to the aforementioned pathway, which includes lysine degradation.

[0110] Genetic possibility 6: Increased phosphatidylinositol signaling. Upregulation of the phosphatidylinositol pathway was significant. The phosphatidylinositol pathway (e.g., PI3K / AKT, MAPK, and AMPK) has been documented as 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: Reduced arginine and ornithine metabolism. Significant reduction in the metabolic pathways of arginine and ornithine was observed. Randomized trials have suggested that high arginine levels are associated with a high risk of ischemic heart disease (Am. Heart J. 2016;182:54-61), and that ornithine accumulation is involved in the pathogenesis of several metabolic diseases (Biomed. Pharmacother. 2017; 86:185-194).

[0112] Figure 60 shows the phylum-level changes, indicating a >95% confidence level change in the microbiome at the phylum level at week 4: administration increased Proteobacteria*, Vercomiclovia*, and Cyanobacteria*. Administration decreased Bacteroidetes, Firmicutes*, Deferibacter, and Firmicutes*. **Significant difference from control or time 0**

[0113] Figure 61 shows significant differences between the treatment group and the control group at the family level. For each genus, there is a significant difference between the treatment group and baseline / control at week 4. Significant increase Parabacteroides Escherichia Erysipelatoclostridium Peptoclostridium - Sutterella Shigella Brenneria Significant decrease Lachnoclostridium Barnesiella Clostridium Oscillospira Dorea Candidatus soleaferrea Dehalobacterium Oscillibacter Flavonifractor

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

[0115] Figure 63 shows a significant enhancement of the unsaturated fatty acid biosynthesis pathway after 4 weeks of DA administration (upper panel: individual data, lower panel: group data).

[0116] Figure 64 shows a significant enhancement of the arachidonic acid metabolic pathway after 4 weeks of DA administration (upper panel: individual data; lower panel: group data).

[0117] Figure 65 shows significant enhancement of cofactor and vitamin metabolic pathways after 4 weeks of DA administration (upper panel: individual data; lower panel: group data).

[0118] Figure 66 shows a significant enhancement of the lysine degradation pathway after 4 weeks of DA administration (upper panel: individual data; lower panel: group data).

[0119] Figure 67 shows a significant enhancement of glycolysis and gluconeogenesis pathways after 4 weeks of DA administration (group data).

[0120] Figure 68 shows a significant enhancement of phosphatidylinositol signaling after 4 weeks of DA administration (group data).

[0121] Figure 69 shows a significant decrease in signaling for arginine and ornithine metabolism after 4 weeks of DA administration (upper panel: individual data; lower panel: group data). [Examples]

[0122] Example 1 This example describes an in vivo study of denatonium acetate on body weight in 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 force-fed distilled water via BID. The DA group (15 mice) was administered DA solution at a dose of 23.1 mg / kg via BID.

[0123] Body weight and weight change were measured on days 1, 3, 7, 10, 14, 21, 24, and 28. Feeding 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). Statistical analysis was performed using two-way repeated measures ANOVA followed by Tukey's multiple comparison post hoc test.

[0124] Table 3 and Figure 1 show the weight measurements from day 1 to day 28. [Table 3]

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

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

[0127] Drug administration did not show 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 between the two experimental groups on day 28.

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

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

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

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

[0133] Figure 8 shows the total blood cholesterol levels (LDL + HDL) on day 28. In animals administered 23.1 mg / kg of DA, the total blood cholesterol levels on day 28 showed an almost significant decrease compared to vehicles-administered animals.

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

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

[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 tended to increase in DA-treated animals compared to vehicle-treated controls.

[0137] Figure 12 shows the number and percentage of monocytes before administration and at day 28. Although there were no statistically significant differences, the changes in the number and percentage of monocytes tended to increase in DA-treated animals compared to vehicle-treated controls.

[0138] Figure 13 shows the changes in lymphocyte and white blood cell counts before administration and at day 28. Although there were no statistically significant differences, the changes in lymphocyte and white blood cell 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 between the two experimental groups over 28 days.

[0140] Figure 15 shows the analysis of various serum 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. KC / CXCL1 and M-CSF showed a significant decrease with DA administration.

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

[0142] Figure 17 shows the analysis of various serum 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. GM-CSF, IFNα, and IL-5 showed a significant decrease with DA administration.

[0143] There is a direct link between chronic inflammation and the progression 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 various immune cells, including macrophages, eosinophils, innate lymphoid cells (ILCs), T cells, and B cells. The accumulation of these immune cells induces chronic mild inflammation, which affects the metabolism of adipose tissue, promotes systemic inflammation, impairs insulin action, and causes harmful effects throughout the body (Wisse, J. Am. Soc. Nephrol. 2004: 15(11):2792-800). The overproduction of pro-inflammatory factors due to this accumulation of immune cells has been shown to play a role in this pathogenic context (Saltiel and Olefsky, J. Clin. Invest. 2017; 127(1):1-4). A broad range of pro-inflammatory factors, including cytokines and chemokines, show elevated circulating levels in individuals with metabolic syndrome, obesity, diabetes, or other metabolic disorders (Tchernof and Despres, Physiol. Rev. 2013; 93(1):359-404). Several 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 impaired fat storage (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 administered DA for 28 days showed a significant weight loss compared to vehicle-administered controls; there was no difference in individual average daily food intake between the two groups of animals. Nevertheless, in DA-administered mice, a panel of cytokines including GM-CSF, IFNγ, IL-5, IL-10, IL-12, KC, and M-CSF showed a significant decrease with DA administration. Therefore, the weight loss in the DA-administered group may be at least partially due to the fact that DA-induced agonism at TAS2R on immune cells inhibits the production of these cytokines, subsequently improving the inflammatory state in adipose tissue and correcting lipid metabolic dysfunction.

[0145] Example 2 This example presents results investigating the modulation of the immune response by DA in a mouse model of air sac inflammation. Eight C57BL / 6 mice were assigned to three groups: a control group (biotherapy with distilled water), a group receiving DA at a dose of 23.1 mg / kg (low-dose DA), and a group receiving DA at a dose of 96.4 mg / kg (high-dose DA). The number of infiltrating cells in air sac exudate and IL-6 levels in the air sac exudate were measured using ELISA assay (R&D Systems Cat.No.M6000B) and multiple multi-cytokine analyses (Mouse 32Plex Kit MilliporeSigma Cat.No.MCYTMAG70PMX32BK). Statistical analysis was performed using one-way ANOVA followed by Tukey's multiple comparison post hoc test for normally distributed data, and the Kruskal-Wallis test followed by Dunn's multiple comparison post hoc test for skewed distributed data. Outliers were identified using the ROUT method.

[0146] Duarte et al., Current Protocols in Pharmacology, 5.6.1-5.6.8 March 2012, states the following: "Subcutaneous air sacs are an in vivo model that can be used to study acute and chronic inflammation, resolution of inflammatory responses, and oxidative stress responses. Injecting an irritant into the air sac 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 widely used to identify potential anti-inflammatory drugs." This can be used to study local inflammation without affecting the entire body. However, in this case, the drug was administered via forced administration in a biopsy (BID). In a previous study using this model, R Romano et al. (1997) showed that forced administration of dexamethasone (a potent anti-inflammatory steroid with severe side effects) reduced TNF levels.

[0147] The test dose was administered at 5 ml / kg (body weight) via BID at 8-hour intervals. Air sacs were created in each test BL6 mouse by subcutaneous injection of 1.5 ml / mouse sterile air on day 0 and again on day 3. The compound (or control distilled water) was administered via 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 the study for all groups and from the exudate in the air sacs. Cell count analysis and IL-6 assays were performed in an animal facility, and plasma and exudate samples were sent for cytokine analysis. Each group consisted of 8 mice: distilled water control, 23.1 mg / kg DA, and 92.4 mg / kg DA.

[0148] Figure 18 shows the number of infiltrating cells in the air sac exudate, demonstrating that pre-administration of DA resulted in a dose-dependent decrease in the number of infiltrating cells in the air sac exudate after LPS induction. Among the results, animals pre-administered with 96.4 mg / kg of DA showed significantly lower infiltrating cell counts compared to animals pre-administered with a vehicle and animals pre-administered with a low dose of DA.

[0149] Figure 19 shows the IL-6 levels in the air sac exudate, demonstrating that pre-administration of DA resulted in a dose-dependent decrease in the number of infiltrating cells in the air sac exudate after LPS induction. Among the results, animals pre-administered with 96.4 mg / kg of DA showed significantly lower IL-6 levels compared to animals pre-administered with a vehicle and animals pre-administered with a low dose of 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. In 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. In this cytokine group, 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. In this cytokine group, 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 group of cytokines, TNF-1α showed a significant decrease with high-dose DA.

[0154] In summary, Figure 51 shows summaries of high-dose (orange) and low-dose (blue) regimens, with asterisks indicating significance. Furthermore, the pro-inflammatory biomarkers TNFα, IL-1β, IL-10, and IL-17 showed a significant dose-response decline 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 an unknown etiology. Numerous colitis mouse models 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 the 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 IBD-approved drugs have been studied in this model, allowing for the comparison of new potential drug compounds with existing drugs in approved IBD symptoms.

[0156] C5BL / 6 mice were divided into 5 groups of 3-10 mice each and fed a standard mouse chow diet (ad libitum) without dietary restrictions, with a maximum of 5 mice per cage. Dexamethasone 21-phosphate disodium salt (DMS; Alfa Aesar Catalog #J64083-1G, Lot R02F035) was used as a positive control. The 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) was also used. IBD-like symptoms were induced by supplementing the water of specific groups with 9011-18-1). Administration was started on day 3 before DSS delivery. All mice were pre-weighed on day 1 and given fresh 4-5% DSS in water daily for 5 days, then given water for the remainder of the study to induce disease. An additional control group was given water (no DSS) for the duration of the study (10 days). Body weight was measured daily, fecal occult blood status (hemocult) was measured three times a week, fecal viscosity was measured three times a week, and general health status was assessed daily. Mice were sacrificed on day 10, serum was collected, cytokine analysis was performed, and colon length and weight were measured. The two control groups were a water-only group and a DSS-only group. The two dose groups were 69.3 mg / kg (n=10) bid and 23.1 mg / kg bid (n=10).

[0157] Figure 52 shows the change 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 and BID showed a significant effect on DSS-induced weight loss compared to the vehicle.

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

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

[0161] Figure 56 shows the overall fecal score during the study period. DA administration showed a significant main effect on overall fecal status.

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

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

[0164] Example 4 Microbiome studies have shown that low levels of Parabacteroides (protective symbiotic bacteria) are associated with atherosclerosis, high levels of Escherichia are linked to coronary heart disease (CHD), Ruminococceae are often increased in patients with ACVD (atherosclerotic cardiovascular disease), and short-chain fatty acids (SCFAs) produced by microorganisms are linked to the reduction of atherosclerosis, inflammation, and moderate hypertension.

[0165] The effects of small molecular weight oral TAS2R agonists (DAs) on the microbial population in a non-alcoholic steatohepatitis (NASH) model mouse were investigated. Two groups of 4-week-old male C57BL / 6 mice (20 / group) were fed an amylin liver NASH (AMLN) diet and administered either a daily dose of ARD-101 (30 mg / mL in water) or a vehicle (water) via intragastric force-feeding. DNA was isolated from fecal samples collected at week 0 and week 4, and the microbial ecology was evaluated by 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 structures was analyzed using Qiime2. Differences were determined by repeated measures ANOVA and post hoc pairwise comparisons using Tukey's test. Taxonomic classification data was evaluated using a dual hierarchical dendrogram.

[0166] The AMLN diet induced changes in the microbial populations of both groups at week 4. Significant increases and decreases were observed at the phylum, family, and genus levels between the DA group and the vehicle group at week 4. For example, at the phylum level, there were significant increases in Proteobacteria, Verrucomiclovia, and Cyanobacteria, and significant decreases in Firmicutes, Deferibacter, and Firmicutes. Ecosystem and microbial community diversity was significantly lower at week 4 vs. week 0 in both administration groups, and at week 4 between DA and vehicle (p<0.05 for all comparisons). Genetic analysis showed that DA increased the 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 administration induced significant changes in physiological and metabolic pathways, and diet mitigated the decrease in fecal SCFAs. 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 exudate was obtained from Balb / c female mice by washing 4 days after intraperitoneal injection of 4 ml of sterile 4% thioglycolate broth. After washing with RPMI1640 medium, the cell suspension was centrifuged at 800 g, 4°C for 5 minutes. Red blood cells were removed with ACK buffer, the cells were washed, and resuspended in RPMI1640 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 (2x10) at 37°C in a 5% CO2 humidified atmosphere. 5 Cells were plated (cells / mL / well). Macrophages were pre-cultured for 24 hours in serum-free RPMI1640 medium to reduce mitogen effects. Macrophages were pre-treated with various concentrations of DA 1 hour prior to LPS administration and stimulated with LPS (100 ng / mL) for 24 hours. The treatment groups are shown in Table 4: [Table 5]

[0168] At 12h and 24h, approximately 200 μl of supernatant was taken, stored (-80°C), and cytokine analysis (13Plex) was performed. The cytokines analyzed were IL-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 in LPS did not induce the same significant difference as 12-hour incubation in LPS.

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

[0172] First, no biomarker (cytokine) effect was observed with intravenous administration of DA. It can be concluded that oral administration is necessary for DA to be effective. Furthermore, toxic side effects occurred with intravenous administration alone. Group #3 was the low-dose oral DA group, and group #4 was the high-dose oral DA group. In the low-dose DA group, significant decreases were observed in cytokines (compared to the control) of 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). In the high-dose DA group, significant decreases were observed in cytokines (compared to the control) of GM-CSF (p=0.03), IL-9 (p=0.003), KC (p=0.05), and VEGF (p=0.001). This study confirmed the effect of biomarkers in normal mice and confirmed that oral administration should be used rather than intravenous 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. The procedure involved administering three groups of CD-1 mice: (1) forced oral administration of physiological saline via BID, (2) oral administration of DA at a dose of 92.4 mg / kg BID, and (3) intravenous administration of DA as a 3 mg / kg IV bolus QD. Pulmonary lavage fluid was measured and cytokine analysis was performed. For statistical analysis, one-way ANOVA followed by Tukey's multiple comparison post-hoc test was used for normally distributed data, Kruskal-Wallis test followed by Dunn's multiple comparison post-hoc test was used for skewed data, and the ROUT method was used for outlier identification. After administering either the control or the drug for 3 days, 1 mg / ml of LPS was administered intratracheally using a 50L Penn Century needle. The mice were sacrificed 24 hours after LPS when the core temperature reached 39°C, and the protein concentration and serum cytokine levels of the lung lavage fluid were measured.

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

[0175] Example 8 This example provides the results of a second modified acute lung injury + hyperthermia test to evaluate the effect of denatonium acetate. The same procedure as in Example 7 was used. Three days prior to the induction of lung injury, each group of six CD-1 mice was prophylactically administered either vehicle or 92.4 mg / kg of denatonium acetate (DA) (administered twice daily (BID) by forced oral administration (PO)) or 3 mg / kg of DA (administered once daily (QD) by intraperitoneal injection (IP)). On day 0, lung injury was induced by intratracheal infusion 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 fluorescently labeled cell sorting (FACS)). Furthermore, lungs were harvested, fixed, stained with Masson's trichrome, and histologically evaluated. Female CD-1 mice were repeatedly administered 92.4 mg / kg DA as a PO (BID) or 3 mg / kg DA as an IP (QD) for 3 days, demonstrating good tolerability. Two mice (one administered via vehicle and one via DA (92.4 mg / kg)) died on day 1; however, the timing of these deaths (within 24 hours after LPS infusion) suggests that these deaths reflect the infusion process (not the test substance), high fever, or associated inflammation. This inference is consistent with the observation that deaths occurred with both vehicle and test substance administrations. No other adverse clinical observations were observed during the 3-day administration of the test substance. Oral administration of 92.4 mg / kg of DA significantly reduced the BALF concentrations of 7 of the 32 cytokines tested (including IL-2, IL-3, IL-10, MIP-1β, MCSF, and MIG) compared to the vehicle. Intravenous administration of 3 mg / kg of DA significantly reduced the BALF concentrations of 10 of the 32 cytokines tested (including G-CSF, eotaxin, IL2, IL-3, IL-4, IL-13, IP-10, MCP-1, M-CSF, and MIG) compared to the vehicle (see Figure 73).Oral and intravenous administration of DA at the indicated levels was associated with nominal (but not significant) changes in protein concentration in BALF; a nominal decrease in neutrophil count in BALF (by FACS assay); and a nominal decrease in the severity of pulmonary pathology (by histological scoring). Therefore, administration of 92.4 mg / kg of DA via BID PO or 3 mg / kg of DA via QD IP injection in these animals resulted in a significant reduction in the accumulation of multiple cytokines in the lungs of this mouse model of acute lung injury, along with nominal activity to counteract neutrophil infiltration and lung injury.

[0176] Example 9 This example presents study results on the effects of diet-induced (DIO) administration of DA + another compound (CQL) on body weight in mice. Adult C57BL / 6NTac mice were fed a high-fat diet (60%). The vehicle group (N=15) received distilled water in a forced-intake (BID), CQL (N=15) received a dose of 50 mg / kg in a forced-intake (BID), and DA (N=15) received a dose of 92.4 mg / kg in a forced-intake (BID). The study period was 56 days followed by a 2-3 day trial period. Body weight changes were measured three times a week, and diet and water intake 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 one hour after administration on day 1 and day 56, and two hours after administration on day 7 (suppression (from administration (up to blood collection), fasting >6 hours prior to administration); serum level of PPY on day 56).

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

[0178] Serum cytokine levels were measured on days 28 and 56 (28 / 56), revealing 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 samples were not available to demonstrate statistically significant differences.

[0179] Example 10 Leptin-deficient ob / ob mice exhibit hyperfeeding and obesity, as well as hyperglycemia and hypertriglyceridemia, which have also been observed in patients with hyperfeeding disorders, such as Prader-Willi syndrome, and monogenic symptomatic obesity disorder (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 predictable in vivo model of these signs. This example provides the results of a study on DA + another compound (CQL) against body weight in leptin-deficient (ob / ob) mice. The vehicle group (N=14) was administered distilled water in a forced BID, and the DA group (N=14) was administered a dose of 50 mg / kg in a forced BID. The study period was 56 days, followed by a 2-3 day trial period. 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 start and end of the study. Cytokine analysis was measured at the end of day 56.

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

[0181] In the DA group, the cytokines eotaxin (p=0.047) and MIG (p=0.026) were significantly reduced (compared to the control) on day 56. Although not statistically significant, the following cytokines showed decreased levels in the DA group compared to the vehicle group on day 56: RANTES (1.7% decrease), IL-1β (19.1% decrease), IL-6 (61.4% decrease), and MCP-1 (20.9% decrease). The present invention includes the following aspects and embodiments. [1] A method for treating, preventing and delaying the exacerbation of a group of signs of type 2 diabetes selected from the group consisting of metabolic syndrome (METS), obesity and hyperglycemia, comprising orally administering a pharmaceutical composition comprising a denatonium salt selected from the group consisting of denatonium acetate (DA), denatonium citrate, denatonium maleate, denatonium saccharide and denatonium tartrate. [2] The method according to [1], wherein the pharmaceutical composition further comprises about 0.5 g to about 5 g of acetic acid. [3] The method according to [1], wherein the daily dose of denatonium salt for adults is approximately 20 mg to approximately 5000 mg. [4] The method according to [3], wherein the daily dose of DA for adults is approximately 50 mg to approximately 1000 mg. [5] The method according to [4], wherein the daily dose of DA for adults is approximately 60 mg to approximately 500 mg, or a dose that achieves an intratube GI concentration of approximately 10 ppb to approximately 50 ppm. [6] The method according to [1], wherein the daily dose of denatonium salt is administered once, twice, or three times a day. [7] A method for treating, preventing and delaying the exacerbation of acute pulmonary inflammatory disorders, including ARDS, comprising orally administering a pharmaceutical composition comprising a denatonium salt selected from the group consisting of denatonium acetate (DA), denatonium citrate, denatonium maleate, denatonium saccharide and denatonium tartrate. [8] The method according to [7], wherein the pharmaceutical composition further comprises about 0.5 g to about 5 g of acetic acid. [9] The method according to [7], wherein the daily dose of denatonium salt for adults is approximately 20 mg to approximately 5000 mg.

[10] The method according to [9], wherein the daily dose of DA for adults is approximately 50 mg to approximately 1000 mg.

[11] The method according to

[10] , wherein the daily dose of DA for adults is approximately 60 mg to approximately 500 mg, or a dose that achieves an intratube concentration of approximately 10 ppb to approximately 50 ppm.

[12] The method according to [7], wherein the daily dose of denatonium salt is administered once, twice, or three times a day.

[13] A method for treating, preventing and delaying the exacerbation of a group of signs of chronic autoimmune inflammatory disorders selected from the group consisting of rheumatoid arthritis (RA), lupus and psoriasis, comprising orally administering a pharmaceutical composition comprising a denatonium salt selected from the group consisting of denatonium acetate (DA), denatonium citrate, denatonium maleate, denatonium saccharide and denatonium tartrate.

[14] The method according to

[13] , wherein the pharmaceutical composition further comprises about 0.5 g to about 5 g of acetic acid.

[15] The method according to

[13] , wherein the daily dose of denatonium salt for adults is approximately 20 mg to approximately 5000 mg.

[16] The method according to

[15] , wherein the daily dose of DA for adults is approximately 50 mg to approximately 1000 mg.

[17] The method according to

[13] , wherein the daily dose of DA for adults is approximately 60 mg to approximately 500 mg, or a dose that achieves an intratube GI concentration of approximately 10 ppb to approximately 50 ppm.

[18] The method according to

[17] , wherein the daily dose of denatonium salt is administered once, twice, or three times a day.

[19] A method for treating, preventing and delaying the exacerbation of a group of symptoms of chronic inflammatory bowel disease (IBD), selected from the group consisting of Crohn's disease and ulcerative colitis, comprising orally administering a pharmaceutical composition comprising a denatonium salt selected from the group consisting of denatonium acetate (DA), denatonium citrate, denatonium maleate, denatonium saccharide and denatonium tartrate.

[20] The method according to

[19] , wherein the pharmaceutical composition further comprises about 0.5 g to about 5 g of acetic acid.

[21] The method according to

[19] , wherein the daily dose of denatonium salt for adults is approximately 20 mg to approximately 5000 mg.

[22] The method according to

[21] , wherein the daily dose of DA for adults is approximately 50 mg to approximately 1000 mg.

[23] The method according to

[22] , wherein the daily dose of DA for adults is approximately 60 mg to approximately 500 mg, or a dose that achieves an intratube concentration of approximately 10 ppb to approximately 50 ppm.

[24] The method according to

[19] , wherein the daily dose of denatonium salt is administered once, twice, or three times a day.

[25] A method for treating, preventing and delaying the exacerbation of a group of metabolome-mediated symptoms selected from the group consisting of atherosclerosis, hypertension and congestive heart failure (CHF), comprising orally administering a pharmaceutical composition comprising a denatonium salt selected from the group consisting of denatonium acetate (DA), denatonium citrate, denatonium maleate, denatonium saccharide and denatonium tartrate.

[26] The method according to

[25] , wherein the pharmaceutical composition further comprises about 0.5 g to about 5 g of acetic acid.

[27] The method according to

[25] , wherein the daily dose of denatonium salt for adults is approximately 20 mg to approximately 5000 mg.

[28] The method according to

[27] , wherein the daily dose of DA for adults is approximately 50 mg to approximately 1000 mg.

[29] The method according to

[28] , wherein the daily dose of DA for adults is approximately 60 mg to approximately 500 mg, or a dose that achieves an intratube GI concentration of approximately 10 ppb to approximately 50 ppm.

[30] The method according to

[25] , wherein the daily dose of denatonium salt is administered once, twice, or three times a day.

[31] A method for treating or delaying the exacerbation of a group of hyperfeeding symptoms selected from the group consisting of Praderwilli and leptin pathway deficiency, comprising orally administering a pharmaceutical composition comprising a denatonium salt selected from the group consisting of denatonium acetate (DA), denatonium citrate, denatonium maleate, denatonium saccharide and denatonium tartrate.

[32] The method according to

[31] , wherein the pharmaceutical composition further comprises about 0.5 g to about 5 g of acetic acid.

[33] The method according to

[31] , wherein the daily dose of denatonium salt for adults is approximately 20 mg to approximately 5000 mg.

[34] The method according to

[33] , wherein the daily dose of DA for adults is approximately 50 mg to approximately 1000 mg.

[35] The method according to

[34] , wherein the daily dose of DA for adults is approximately 60 mg to approximately 500 mg, or a dose that achieves an intratube GI concentration of approximately 10 ppb to approximately 50 ppm.

[36] The method according to

[31] , wherein the daily dose of denatonium salt is administered once, twice, or three times a day.

Claims

1. A pharmaceutical composition comprising a denatonium salt selected from the group consisting of denatonium acetate (DA), denatonium citrate, denatonium maleate, denatonium saccharide, and denatonium tartrate, for the treatment, prevention, and / or delaying the worsening of metabolic syndrome (METS) or hyperglycemia, which is administered orally to humans.

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, or a dose that achieves an intra-gastrointestinal concentration of approximately 10 ppb to approximately 50 ppm.

14. The pharmaceutical composition according to any one of claims 1 to 13, wherein the daily dose of denatonium salt is administered once, twice, or three times a day.

15. A pharmaceutical composition according to any one of claims 1 to 14, wherein the treatment, prevention, and / or delay of exacerbation is for metabolic syndrome (METS).

16. A pharmaceutical composition according to any one of claims 1 to 14, wherein the treatment, prevention, and / or delay of exacerbation is of hyperglycemia.

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