Gpr75 antagonists for the prevention or treatment of diet-induced obesity, cardiometabolic disease or cardiometabolic-associated complications

Abstract

The present disclosure provides methods, compositions and kits for the treatment or prevention of diet-induced obesity, cardiometabolic disease and cardiometabolic-associated complications including liver disease and its respective categories including nonalcoholic fatty liver disease (NAFLD) (alternatively termed herein Metabolic Dysfunction-associated Fatty / Steatotic Liver Disease (MAFLD / MASLD)), nonalcoholic steatohepatitis (NASH) and cirrhosis that employ a compound that disrupts the activity of the 20-HETE receptor, GPR75.

Description

DESCRIPTION GPR75 ANTAGONISTS FOR THE PREVENTION OR TREATMENT OF DIET- INDUCED OBESITY, CARDIOMETABOLIC DISEASE OR CARDIOMETABOLIC- ASSOCIATED COMPLICATIONS CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to US Provisional Patent Application No.63 / 594,176, filed October 30, 2023, which is incorporated by reference in its entirety. FIELD OF THE INVENTION

[0002] The present disclosure provides methods, compositions and kits for the treatment or prevention of diet-induced obesity, cardiometabolic disease and cardiometabolic-associated complications including liver disease (i.e., nonalcoholic fatty liver disease (NAFLD) (alternatively termed herein Metabolic Dysfunction-associated Fatty / Steatotic Liver Disease (MAFLD / MASLD)), nonalcoholic steatohepatitis (NASH) and cirrhosis) that employ a compound that disrupts the activity of the 20-HETE receptor, GPR75. BACKGROUND OF THE INVENTION

[0003] GPR75 is a G protein-coupled receptor and member of the Gq-coupled Rhodopsin Class A family of GPCRs. The vasoactive eicosanoid 20-hydroxyeicosatetraenoic acid (20- HETE) is a high-affinity ligand for GPR75. This ligand-receptor pairing has been shown to be a prominent pro-inflammatory driver of endothelial function, vascular tone, vascular remodeling, blood pressure, cancer, renal disease and heart failure.

[0004] To date a role for GPR75 in fatty acid uptake has not been reported. Moreover, presently there are no pharmacological compounds available for the prevention or treatment of NAFLD / MAFLD / MASLD or NASH. Accordingly, there is a present need for such compounds. SUMMARY OF THE INVENTION

[0005] The present disclosure relates to methods for inhibiting the activity of GPR75 in a subject, which comprise administering to said subject an inhibiting amount of a GPR75 antagonist (alternatively termed herein "a GPR75 antagonist / receptor blocker"), as well as topharmaceutical compositions or formulations and kits for use in such methods, and the use of a GPR75 antagonist in the manufacture of a medicament for use in such methods.

[0006] The present disclosure provides methods for regulating GPR75 mediated responses by administering to a subject a compound that inhibits the activity of GPR75 (a GPR75 antagonist / receptor blocker). According to the present disclosure, this inhibition may be achieved by any compound that inhibits the activity of GPR75. In a specific embodiment, the compound is disodium N-succinate-20-hydroxyeicosa-6(Z),15(Z)-diencarboxamide ("AAA"), which is the disodium salt of N-(20-hydroxyeicosa-6(Z),15(Z)-dienoyl)-L-aspartic acid:

[0007] More specifically, the presently disclosed subject matter provides a method of treating, delaying the onset, alleviating a symptom, and / or preventing the progression of diet-induced obesity, cardiometabolic disease and cardiometabolic-associated complications including liver disease (nonalcoholic fatty liver disease (NAFLD) / Metabolic Dysfunction-associated Fatty / Steatotic Liver Disease (MAFLD / MASLD), nonalcoholic steatohepatitis (NASH) and cirrhosis). The method includes administering safely to a subject in need thereof a therapeutically effective amount of a GPR75 antagonist. The administration of such GPR75 antagonists is intended to, as demonstrated herein, modulate the activity of GPR75 and inhibit fatty acid uptake, preventing / reducing liver steatosis.

[0008] While aspects or embodiments of the disclosure below may be directed to treatment of NAFLD / MAFLD / MASLD or NASH, the disclosure applies equally as well to treating, delaying the onset, alleviating a symptom, and / or deterring the progression of other diseases such as diet-induced obesity, cardiometabolic disease (heart disease, stroke, type-2 diabetes, dyslipidemia) and cardiometabolic-associated complications (coronary artery disease (CAD), atherosclerosis, diabetic neuropathy / retinopathy / nephropathy, chronic kidney disease, liver disease / cancer, pancreatitis, polycystic ovary syndrome (PCOS)) that result from activation of GPR75.

[0009] In one embodiment, the presently disclosed subject matter provides a method for preventing, treating or alleviating, the symptoms associated with activation of GPR75 throughadministration of a GPR75 antagonist in a therapeutically effective amount to a subject suspected of suffering from, or at risk of developing liver disease. Such subjects may be identified as those having the symptoms of NAFLD / MAFLD / MASLD or NASH. Symptoms of NAFLD / MAFLD / MASLD may include fatigue, pain or discomfort in the upper right abdomen. Possible symptoms of NASH include advanced scarring (cirrhosis), abdominal swelling (ascites), enlarged blood vessels just beneath the skin's surface, enlarged spleen, red palms and yellowing of the skin and eyes (jaundice). Additionally, patients may exhibit elevated liver-related enzymes (serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST). In an embodiment, the subject to be treated may be one suffering from disorders that render a subject more prone for development of metabolic disorders such as NAFLD / MAFLD / MASLD or NASH.

[0010] Also disclosed are pharmaceutical compositions and formulations comprising a GPR75 antagonist, alone or in combination with one or more additional therapeutic agents, and a physiologically compatible carrier, excipient or stabilizer. The pharmaceutical compositions and formulations can be administered to a subject, for example, a human subject, for therapeutic treatment, such as for treating or preventing diet-induced obesity, cardiometabolic disease or cardiometabolic-associated complications. For example, the pharmaceutical compositions and formulations may be for use in treating or preventing liver disease. The presently disclosed pharmaceutical compositions and formulations can be administered using a variety of methods known in the art depending on the subject and / or the severity of the, for example, NAFLD / MAFLD / MASLD or NASH. In one aspect, the GPR75 antagonist is administered, for example, orally (sublingual, buccal), intravenously, intramuscularly, topical / transdermal, subcutaneously (abdomen etc.), intranasally, by inhalation or intravenously. Regardless of the route of administration selected, the GPR75 antagonist pharmaceutical compositions are formulated into pharmaceutically acceptable dosage forms such as described below or by other conventional methods known to those of skill in the art. In some embodiments, the GPR75 antagonist is AAA.

[0011] The presently disclosed subject matter also includes the use of a GPR75 antagonist in the manufacture of a medicament for treating or preventing diet-induced obesity, cardiometabolic disease or cardiometabolic-associated complications including liver disease. In some embodiments, the medicament is for treating or preventing NAFLD / MAFLD / MASLD or NASH.

[0012] The GPR75 antagonists or pharmaceutical compositions or formulations thereof can be assembled into kits or pharmaceutical systems for use for treatment or prevention of diet- induced obesity, cardiometabolic disease or cardiometabolic-associated complications including liver disease. In some embodiments, the presently disclosed kits or pharmaceutical systems include a GPR75 antagonist in unit dosage form. In further embodiments, the GPR75 antagonist can be present together with a pharmaceutically acceptable solvent, carrier, excipient, or the like, as described herein. In some embodiments, the presently disclosed kits include one or more containers, including, but not limited to a vial, tube, ampule, bottle, and the like, containing the GPR75 antagonist. The presently disclosed kits or pharmaceutical systems also can include associated instructions for using the GPR75 antagonist or GPR75 antagonist containing compositions for treating or preventing NAFLD / MAFLD / MASLD or NASH. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG.1A-B: FIG.1A Changes in systolic blood pressure (28 weeks) or FIG.1B body weight in response to control diet (CD) or high fat diet (HFD) fed mice (28 weeks) in the presence and absence of doxycycline (DOX) in Cyp4a12-GPR75+ / + (WT) and Cyp4a12- GPR75- / - (KO) mice. Mean ± SEM (n=4-8). * p<0.05 , **** p<0.0001.

[0014] FIG.2. Representative MicroCT images from mice at baseline (Week 0) and 28 Weeks of CD or HFD feeding in the presence of DOX. (n=4-6). Visceral Fat, Subcutaneous Fat, outlined.

[0015] FIG.3. Representative histological images (H&E staining) of liver sections from mice under CD or HFD in the presence and absence of DOX for 28 weeks. (n=4-6). Cyp4a12-GPR75- / - (KO) mice exhibit a clear protection from the development of liver steatosis (NAFLD / MAFLD / MASLD or NASH) observed in Cyp4a12-GPR75+ / + mice (WT). Taken together, these data suggest GPR75 as a key regulator and contributor to the onset and progression of liver disease.

[0016] FIG.4A-C: Changes to body weight FIG.4A, liver histology FIG.4B, and metabolic cage activity FIG. 4C (48 hours) of GPR75+ / + (WT) or global GPR75- / - (KO) mice on methionine / choline deficient (MCD) diet for 6 weeks. n=7-9. * p<0.05. A GPR75 deficiency is sufficient to provide significant protection again MCD-induced liver damage, body-weight loss due to impaired lipid metabolism and impairments to activity due to muscle catabolism and metabolic imbalances.

[0017] FIG. 5A-D: FIG. 5A. Endothelial cell line (EA.hy926) dose responses and IC50 calculation for AAA (disodium N-succinate-20-hydroxyeicosa-6(Z),15(Z)-diencarboxamide). Fixed dose / concentration of 20-HETE (10nM). n=6-8. * p<0.05. FIG.5B. GPR75 is expressed by hepatocytes and can be disrupted by gene silencing. FIG. 5C. Changes in intracellular calcium across the hepatocyte cell line (HepG2) in response to Vehicle (ethanol), 20-HETE (10 nM). B) GPR75 expression in WT vs GPR75 knockdown (KD) cells. FIG.5D. Changes in intracellular calcium across WT and GPR75 knockdown (KD) cells treated with Vehicle (ethanol), 20-HETE (10 nM) in the presence and absence of AAA (10nM). Mean ± SEM (n=8). **** p<0.0001. These data illustrate AAA’s ability to effectively inhibit the 20-HETE- dependent activation of GPR75 in both endothelial cells and hepatocytes, thereby disrupting downstream signaling events. The AAA-mediated blockade of GPR75 prevents alterations in intracellular calcium levels, which serve as a pivotal signaling mediator and a primary indicator of GPR75 activity.

[0018] FIG.6A-C: FIG.6A Changes in fatty acid uptake across HEPG2 cells in response to vehicle (ethanol), 20-HETE (10 nM) in the presence and absence of AAA (10 nM). FIG.6B Effects of lipofermata (a specific FATP2 / SLC27A2 blocker) (10 μM) on 20-HETE-driven fatty acid uptake in WT cells. FIG.6C Changes in fatty acid uptake in GPR75 KD cells exposed to Vehicle (ethanol), 20-HETE (10 nM) in the presence and absence of AAA (10 nM). Mean ± SEM (n=8). **** p<0.0001. AAA-mediated GPR75 receptor blockade inhibits the 20-HETE- GPR75-dependent activation of fatty acid uptake in hepatocytes via FATP2 (SLC27A2). Halting excess lipid transport can maintain hepatic lipid homeostasis, therefore attenuating the accumulation of lipotoxic species that contribute to liver diseases. By limiting the dysregulated uptake of fatty acids, GPR75 receptor blockade can mitigate cellular stress and prevent the progression to liver fibrosis and cirrhosis, conditions often linked to excessive lipid deposition and inflammation.

[0019] FIG.7. Detection of fatty acid uptake and lipid droplet formation (BODIPY) in HEPG2 pre-treated with either vehicle (ethanol), 20-HETE (10 nM), AAA (10 nM), 20-HETE+AAA, Calphostin C ((CC) PKC inhibitor (1 μM)) or 20-HETE+CC prior to oleic acid supplementation (1 mM) for 4 h. Green (BODIPY), Blue (Hoechst), Black (Segmented BODIPY Signal (Lipid Droplets)) (n=8). 20-HETE’s ability to drive increases in fatty acid uptake, in part through the activation of FATP2 / SCL27A2, exacerbates the formations of lipid droplets in hepatocytes. This process is critically dependent on the activation of protein kinase C (PKC). Notably, the use of a PKC inhibitor prior to 20-HETE exposure can effectively block lipid droplet accumulation. Furthermore, antagonism of GPR75 via AAA fully abrogates theactivation of the 20-HETE-GPR75-PKC signaling axis, thereby preventing lipid droplet formation. These findings highlight AAA’s ability to disrupt 20-HETE-GPR75-dependent signaling, mitigating lipid-induced hepatoxicity, particularly in the context of hepatic steatosis, where excessive lipid droplets are linked to cellular stress, inflammation, and progression to liver fibrosis.

[0020] FIG. 8A-B: FIG. 8A Detection of fatty acid uptake and lipid droplet formation (BODIPY) in GPR75 knockdown / deficient HEPG2 cells pre-treated with either vehicle (ethanol), 20-HETE (10 nM). Green (BODIPY), Blue (Hoechst), Red (Segmented BODIPY Signal (Lipid Droplets)) (n=8). FIG.8B Graph summarizing 20-HETE- and GPR75-dependent changes in fatty acid uptake as a measure of BODIPY surface area (n=8). In metabolic disorders such as NAFLD / MAFLD / MASLD, NASH and cirrhosis, unregulated fatty acid uptake and lipid droplet formation are major contributors to disease progression. These images and quantified data illustrate how GPR75 deficiency and antagonism can interrupt lipid storage, arresting chronic lipid accumulation, thus preventing hepatocyte injury, inflammation, fibrosis, cirrhosis and liver failure.

[0021] FIG.9A-C: FIG.9A Daily body weight change of WT mice fed a high-fat diet (HFD) + / - Saline or AAA (50 mg / kg / day) intraperitoneally. FIG.9B Representative MicroCT images at week 4 alongside quantified total adipose and lean body volume changes between Saline and AAA. FIG.9C Representative histological images of white adipose tissue (WAT) and livers from WT mice fed a HFD for 4 weeks in conjunction with either daily Saline of AAA (50mg / kg / day). n=5. ** p<0.01. The administration of AAA significantly abrogated changes to body weight, adipose content and reduced WAT adipocyte hypertrophy while unaffecting lean body mass volume. Moreover, AAA-treated mice displayed less hepatic steatosis and early lipid droplet formation (black arrows), a key marker of fatty liver disease, compared to their saline-treated counterparts. These data highlight AAA’s ability to prevent the adverse structural changes in both adipose tissue and the liver induced by high-fat feeding, thus impeding the early stages of liver steatosis via disruptions in GPR75-dependent signaling mechanisms.

[0022] FIG. 10A-D: FIG. 10A Weekly body weight change of WT mice fed a HFD for 5 weeks followed by AAA (50 mg / kg / day). Bar denotes microCT imaging and GTT studies occurring during week 4-5, arrow denotes start of Saline or AAA administrations (Week 5). FIG.10B Glucose tolerance test (GTT) of WT mice at week 8 of HFD week 8 after 3 weeks of AAA (50 mg / kg / day) treatment. FIG. 10C Representative MicroCT images at week 8 alongside quantified total adipose and lean body volume changes between Saline- and AAA-treated mice for 3 weeks. FIG.10D Representative histological images of white adipose tissue (WAT) and livers of mice fed a HFD + / - AAA. Quantified steatosis of livers from AAA-treated mice. n=3-7, ** p<0.01. Reversal studies in which mice fed HFD for 5 weeks and were then given AAA (50 mg / kg / day) for 4 weeks while continuing to be fed a HFD regiment showcases AAA’s ability to drive a rapid decrease in body weight. After 8 weeks of HFD, saline-treated mice exhibit impaired glucose handling while AAA-treated mice were able to clear the glucose challenge significantly faster. Further evaluation of AAA + HFD-treated mice revealed AAA’s efficacy in reversing changes in adipocyte size / hypertrophy and liver steatosis clearly observed in saline-treated mice on HFD. These data suggest a causal relationship between GPR75, and diet-driven metabolic consequences associated with the activation of GPR75, highlighting GPR75’s role as a key driver of metabolic dysfunction and liver disease progression. Taken together, these data showcase AAA’s ability to prevent and reverse liver disease progression alongside the metabolic-associated changes that precipitate liver damage including excess body weight gain, insulin resistance, steatosis and cellular inflammation / hypertrophy. DETAILED DESCRIPTION OF THE INVENTION

[0023] Definitions

[0024] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure pertains.

[0025] Reference to singular forms, for example, "a", "an" and "the", include plural reference unless the context clearly dictates otherwise.

[0026] The transitional terms "comprising", "including", "carrying", "having", "containing", "involving", and the like, as used herein, are to be understood as being inclusive or open-ended (i.e., to mean including but not limited to), and they do not exclude unrecited elements, materials or method steps. Only the transitional phrases "consisting of" and "consisting essentially of", respectively, are closed or semi-closed transitional phrases with respect to claims and exemplary embodiments herein. The transitional phrase "consisting of" excludes any element, step, or ingredient which is not specifically recited. The transitional phrase "consisting essentially of" limits the scope to the specified elements, materials or steps and to those that do not materially affect the basic characteristic(s) of the invention disclosed and / or claimed herein.

[0027] The phrase "and / or", as used herein, should be understood to mean "either or both" ofthe elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with "and / or" should be construed in the same fashion, i.e., "one or more" of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the "and / or" clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to "A and / or B", when used in conjunction with open-ended language such as "comprising" can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0028] The expressions "one or more" and "at least one" (which may be used interchangeably), unless explicitly stated otherwise herein, refer to the number of different entities, and not to the quantity of any particular entity.

[0029] The term "alkyl", as used herein, by itself or as part of another substituent refers to, unless otherwise stated, a straight or branched chain, substituted or unsubstituted, aliphatic group having any number of carbons, and more particularly having the number of carbon atoms as designated (e.g., C1-6meaning 1 to 6 carbon atoms). An exemplary "alkyl" group is a methyl group (-CH3; Me).

[0030] The term "cycloalkyl" refers to a univalent group derived from a cycloalkane by removal of a hydrogen atom from a ring carbon atom having any number of carbons, and more particularly having the number of carbon atoms as designated (e.g., C3-6meaning 3 to 6 carbon atoms).

[0031] A "lower alkyl" group is a C1-6alkyl or cycloalkyl group.

[0032] The terms "treat", "treating", "treatment" and the like, as used herein, are meant to denote a decrease, suppression, attenuation, diminishing, arresting, reversal, of the cause, development or progression of diet-induced obesity, cardiometabolic disease or cardiometabolic-associated complications including liver disease and / or symptoms associated therewith. The terms "treat", "treating", "treatment" and the like, as used herein , can mean to stabilize the development or progression of NAFLD / MAFLD / MASLD, NASH or liver cirrhosis. The terms "treat", "treating", "treatment" and the like, as used herein can refer to curative therapy, prophylactic therapy, and preventative therapy. Accordingly, as used herein, "treating" means either slowing, stopping or reversing the progression of the metabolic disorders mediated by GPR75 activation and uptake of fatty acids by the liver. For example, as used herein, "treating" can mean either slowing, stopping or reversing the progression of NAFLD / MAFLD / MASLD or NASH, including reversing the progression to the point ofeliminating the symptoms of NAFLD / MAFLD / MASLD or NASH. It should be appreciated that treating a disease, disorder or condition does not require that the disease, disorder, condition, or symptoms associated therewith be completely eliminated.

[0033] As used herein, the terms "prevent", "preventing", "prevention", "prophylactic treatment" and the like refer to reducing or inhibiting the probability of developing symptoms of the diet-induced obesity, cardiometabolic disease or cardiometabolic-associated complications including liver disease. For example, the terms "prevent," "preventing," "prevention," "prophylactic treatment" and the like may refer to reducing the probability of developing symptoms of NAFLD / MAFLD / MASLD or NASH in a subject, who does not have, but is at risk of or susceptible to developing NAFLD / MAFLD / MASLD or NASH. Thus, in some embodiments, a GPR75 receptor antagonist can be administered prophylactically to prevent the onset of metabolic disorders or to prevent the recurrence of metabolic disorders, such as NAFLD / MAFLD / MASLD or NASH, in a subject.

[0034] As used herein, in general, a "therapeutically effective amount" of a GPR75 antagonist refers to the amount of the agent necessary to elicit the desired biological response. For example, a therapeutically effective amount may be an amount sufficient for prevention or treatment of NAFLD / MASLD / MAFLD or NASH and associated symptoms. A "therapeutically effective amount" may also refer to, for example, an amount sufficient to reduce or ameliorate the severity, duration, progression, or onset of symptoms associated with liver disease progression or stage. The therapeutically effective amount of an agent may vary depending on such factors as the desired biological endpoint, the composition of the pharmaceutical composition, the target tissue or cell, the health of the subject to be treated and the like.

[0035] Methods for Modulating the Activity of GPR75

[0036] The present disclosure provides methods for inhibiting the activity of GPR75 in a subject, which comprise administering to said subject an inhibiting amount of a GPR75 antagonist. The methods may be for treating or preventing diet-induced obesity, cardiometabolic disease or cardiometabolic-associated complications including liver disease.

[0037] For example, in one embodiment, the present disclosure relates a method for treating or preventing liver disease (herein reflecting NAFLD / MAFLD / MASLD, NASH or liver cirrhosis). The method is based on the recognition that activation of GPR75 can act as a stimulator of fatty acid uptake in the liver driving hepatic steatosis.

[0038] The disclosure also relates to, for example, methods for obtaining a less severe disease stage in a subject suffering from NAFLD / MAFLD / MASLD, NASH or liver cirrhosis, as wellas methods of delaying the progression of these liver disease stages through administration of a GPR75 antagonist. In some embodiments, the methods are for the treatment or prevention of metabolic disorders such as NAFLD / MAFLD / MASLD or NASH. In one embodiment, the presently disclosed subject matter relates to a method of treating or preventing NAFLD / MAFLD / MASLD or NASH in a subject in need thereof, the method including administering to the subject a therapeutically effective amount of a GPR75 antagonist.

[0039] The "subject" of the inventive methods may be a human or a non-human animal. Non- limiting examples of non-human animals that may be used in the methods according to the invention include companion animals, such as dogs or cats, and large animals such as those used in animal husbandry (for example, sheep, cattle, pigs, llamas, buffalo etc.) or wild animals such as tigers, lions, elephants, etc.

[0040] A "subject" can include a human subject for medical purposes, such as for the treatment of NAFLD / MAFLD / MASLD or NASH or the prophylactic treatment for preventing the onset of NAFLD / MAFLD / MASLD or NASH, or an animal subject for medical, veterinary purposes, or experimental purposes. Further, a "subject" can include a patient afflicted with or suspected of being afflicted with NAFLD / MAFLD / MASLD or NASH. Thus, the terms "subject" and "patient" are used interchangeably herein.

[0041] As used herein, "liver disease" means that the subject has symptoms associated with NAFLD / MAFLD / MASLD, NASH or liver cirrhosis. Symptoms of NAFLD / MAFLD / MASLD may include fatigue, pain or discomfort in the upper right abdomen and / or hepatomegaly. Clinical indicators include elevated liver enzyme levels (ALT, AST), increased hepatic fat visualized via CT or MRI, or confirmed steatosis / inflammation from liver biopsy. Possible symptoms of NASH can include advanced scarring (cirrhosis), abdominal swelling (ascites), enlarged blood vessels just beneath the skin's surface, enlarged spleen, red palms and yellowing of the skin and eyes (jaundice). Additionally, clinical indicators of NASH and cirrhosis include elevated liver enzyme levels (ALT, AST), elevated alkaline phosphatase (ALP) and gamma-glutamyl transferase (GGT), altered bilirubin / albumin levels, increased hepatic fat or liver fibrosis visualized via CT or MRI, or confirmed steatosis / inflammation / hepatocyte ballooning from liver biopsy.

[0042] The treatment, administration, or therapy can be continuous or intermittent. Continuous treatment, administration, or therapy refers to treatment on at least a daily basis without interruption in treatment by one or more days. Intermittent treatment or administration, or treatment or administration in an intermittent fashion, refers to treatmentthat is not continuous, but rather cyclic in nature. Treatment according to the presently disclosed methods can result in complete relief or cure from liver disease including NAFLD / MAFLD / MASLD, NASH or cirrhosis or partial amelioration of one or more symptoms of these disease states / categories can be temporary or permanent.

[0043] In certain embodiments, the presently disclosed subject matter also includes combination therapies. Additional therapeutic agents, which are normally administered to treat or prevent, for example, NAFLD / MAFLD / MASLD, NASH or cirrhosis may be administered in combination with a GPR75 antagonist as disclosed herein. For example, the GPR75 antagonist may optionally be administered in conjunction with other compounds (e.g., therapeutic agents) or treatments useful in treating liver disease and the development of NAFLD / MAFLD / MASLD, NASH or cirrhosis. These additional agents may be administered separately, as part of a multiple dosage regimen, from a pharmaceutical composition or formulation comprising a GPR75 antagonist as disclosed herein. Alternatively, these agents may be part of a single dosage form, mixed together with a GPR75 antagonist, in a single composition.

[0044] By "in combination with" it is meant that the GPR75 antagonist is for administration with one or more therapeutic agents, either simultaneously, sequentially, or a combination thereof. Therefore, a subject can be administered a combination of a GPR75 antagonist and one or more therapeutic agents at the same time (i.e., simultaneously) or at different times (i.e., sequentially, in either order, on the same day or on different days), so long as the effect of the combination of both agents is achieved in the subject. Where the GPR75 antagonist and one or more therapeutic agents are administered simultaneously, they can be administered to the subject as separate pharmaceutical compositions or formulations, each containing either a GPR75 antagonist or one or more therapeutic agents or be administered to a subject as a single pharmaceutical composition or formulation comprising both agents.

[0045] When administered in combination, the effective concentration of each of the agents to elicit a particular biological response may be less than the effective concentration of each agent when administered alone, thereby allowing a reduction in the dose of one or more of the agents relative to the dose that would be needed if the agent was administered as a single agent. The effects of multiple agents may, but need not be, additive or synergistic. The agents may be administered multiple times. In such combination therapies, the therapeutic effect of the first administered agent is not diminished by the sequential, simultaneous or separate administration of the subsequent agent(s).

[0046] The GPR75 antagonist can be administered using a variety of methods known in the art. More particularly, the GPR75 antagonist can be administered by any suitable route of administration, including administration methods such as topical, parenteral, intramuscular, intravenous, nasal, oral, transdermal, mucosal, and subcutaneous or other modes of delivery known in the art.

[0047] Actual dosage levels of the GPR75 antagonist can be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular subject without being toxic to the subject. The selected dosage level will depend on a variety of factors including the route of administration, the time of administration, the rate of excretion, the duration of the treatment, other drugs used in combination with the GPR75 antagonist, the age, sex, weight, condition, general health, and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0048] A physician having ordinary skill in the art can readily determine and prescribe the effective amount of a given GPR75 antagonist or GPR75 antagonist-containing pharmaceutical composition or formulation required for treatment, such as for the treatment of NAFLD / MAFLD / MASLD or NASH. For example, the physician could start doses of the GPR75 antagonist lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. Accordingly, the dosage range for administration will be adjusted by the physician, as necessary. It will be appreciated that the amount of a GPR75 antagonist required for achieving the desired biological response, e.g., treatment or prevention of NAFLD / MAFLD / MASLD or NASH, may be different from the amount of a GPR75 antagonist effective for another purpose.

[0049] In general, a suitable daily dose of a GPR75 antagonist will be the amount that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above. Effective dosages may be determined based generally on the weight of the subject to be treated. If desired, the effective daily dose of the GPR75 antagonist can be administered as one, two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.

[0050] Administration of the GPR75 antagonist can bring about several effects. For example, administration of the GPR75 antagonist may:• Protect against: body-weight loss due to impaired lipid metabolism, muscle catabolism or metabolic imbalances; • Prevent alteration in intracellular calcium levels; • Inhibit the 20-HETE-GPR75-dependent activation of fatty acid uptake; • Attenuate the accumulation of lipotoxic species that contribute to liver diseases; • Mitigate cellular stress or prevent progression to liver fibrosis or cirrhosis; • Disrupt 20-HETE-GPR75-dependent signaling, mitigating lipid-induced hepatoxicity; • Prevent hepatocyte injury, inflammation, fibrosis, cirrhosis or liver failure; • Abrogate changes to body weight or adipose content, or reduce WAT adipocyte hypertrophy; • Prevent adverse structural changes in adipose tissue or the liver of a subject; • Impede the early stages of liver steatosis; • Reverse changes in adipocyte size / hypertrophy or liver steatosis; and / or • Prevent or reverse liver disease progression or the metabolic-associated changes that precipitate liver damage such as excess body weight gain, insulin resistance, steatosis and cellular inflammation / hypertrophy.

[0051] GPR75 Antagonists

[0052] The methods of the disclosure employ a GPR75 antagonist. In some embodiments, the GPR75 antagonist is a small molecule or an antibody.

[0053] Various established methods, well known to those skilled in the art, can be employed to identify antagonists of GPR75. For example, the efficacy of potential antagonists can be evaluated using animal models, wherein fatty acid uptake is assessed through a range of advanced techniques. These include histological staining to visualize lipid accumulation, in situ hybridization to detect mRNA expression of key lipid transporters, flow cytometry for quantifying cellular uptake of labeled fatty acids, and immunohistochemistry to localize and quantify proteins involved in fatty acid metabolism. Additionally, bioassays including radiolabeled / fluorescent fatty acid uptake assays, lipidomic profiling and colorimetric / fluorometric lipid quantification assays using human or animal samples can be used to evaluate changes to hepatic lipid accumulation / content.

[0054] In some examples, the amount of fatty acid uptake and content in a test animal contacted or treated with a test compound, i.e., a possible GPR75 antagonist, is compared to a control. A "compound" or "test compound" is any substance or any combination ofsubstances that is useful for achieving an end or result. Any compound that has the potential to modulate through inhibition of GPR75 can be tested using the methods of this disclosure.

[0055] Exemplary test compounds include, but are not limited to, peptides, such as soluble peptides, including but not limited to members of random peptide libraries, antibodies and antibody fragments and small organic or inorganic molecules. Appropriate test compounds can be contained in libraries, for example, synthetic or natural compounds in a combinatorial library. Numerous libraries are commercially available or can be readily produced; means for random and directed synthesis of a wide variety of organic compounds and biomolecules also are known. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or can be readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Such libraries are useful for the screening of a large number of different compounds for the identification of GPR75 antagonists.

[0056] In screening methods, the amount of fatty acid uptake in the liver can be measured through analysis of a test subject or animal. Methods detecting fatty acid uptake include, for example, the QBT Fatty Acid Uptake Assay (Molecular Devices) or intraoperative fatty acid uptake using radiolabeled fatty acids (palmitate) across liver biopsies. A decrease in fatty acid uptake (such as a decrease of at least about 10%, about 20%, about 50%, about 80%, about 90%, about 1.5-fold, about 2-fold, about 3-fold, about 5-fold, about 10-fold or more) in the presence of one or more test compounds as compared to in the absence of the one or more test compounds indicates that the compound acts as an GPR75 antagonist to reduce fatty acid uptake.

[0057] GPR75 antagonists that can be used in the present invention, include, for example, compounds of the formula (I) or (II):and physiologically acceptable salts thereof, wherein: R2and R3are each, independently, OH, C1-C3alkyl, F, or H; R4is C1-C3alkyl, H, F, or -CH2N3(azide); X is C or O; m and p are 0 or 1; n and q are 1 to 3; and R1is CO2H, NR8R9, C(O)R6or a tetrazole, wherein: R6is OR7, NR8R9, a D- / L- / D,L-α-amino acid (MW<250), -NHS(O)2R10, a polyethylene glycol (MW<350) or alkyl ether thereof, glycerol, glyceride mono- or diester (MW<800), or a carboxylate isostere or mimetic selected from the group consisting of: -P(O)(OH)2, -S(O)2OH, wherein: lower alkyl is a C1-C6alkyl or cycloalkyl; R7is C1-C6alkyl or cycloalkyl, or benzyl; R8and R9are each, independently, H, C1-C6alkyl or cycloalkyl, or benzyl, or R8and R9together constitute a 3-7 membered ring with the nitrogen; and R10is phenyl, C1-C5alkyl or cycloalkyl, or CF3, provided that: (i) at least one of R2, R3and R4is OH or F; (ii) the sum of the number of carbon atoms provided by n and m is 3 or 4; and (iii) the sum of the number of carbon atoms provided by p and q is 3 or 4.

[0058] In some embodiments of the compounds of the formula (I) or (II), R1is CO2H or C(O)R6, wherein R6is a D- / L- / D,L-α-amino acid (MW<250) or a polyethylene glycol (MW<350) or alkyl ether thereof. In some embodiments, the D- / L- / D,L-α-amino acid (MW<250) is glycor aspartic acid. In some embodiments, the polyethylene glycol (MW<350) or alkyl ether thereof is.

[0059] In some embodiments of the compounds of the formula (I) or (II), one of R2and R3is OH or F; one of R2and R3is H or -CH3; and R4is H, -CH3or CH2N3.

[0060] In some embodiments, the compounds of the formula (I) or (II) are compounds of the formula:, wherein represents a single or a double bond, provided that at least one is a double bond.

[0061] In some embodiments, the compounds of the formula (I) or (II) are compounds of the formula:.

[0062] Non-limiting examples of the GPR75 antagonists that can be used in the present invention include: 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (20-6,15-HEDE, 20-HEDE), N-[20-hydroxyeicosa-6(Z),15(Z)- dienoyl]glycine (20-HEDGE), [2,5,8,11,14,17- hexaoxanonadecan-19-yl 20-hydroxyicosa-6(Z),15(Z)-dienoate] (20-SOLA), 19-HETE analogues and disodium N-succinate-20-hydroxyeicosa-6(Z),15(Z)-diencarboxamide (AAA). Chemical structures of such GPR75 receptor antagonists that are compounds of the formula (I) or (II), or a physiologically acceptable salt thereof, include:

[0063] Other non-limiting examples of GPR75 antagonists that are compounds of the formula (I) or (II), or a physiologically acceptable salt thereof, that can be used in the present invention include:

[0064] Compounds of the formula (I) or (II) can be made by methods known to those of skill in the art. For example, methods of preparing select compounds of the formula (I) or (II) can be found in WO 2017 / 156164 A1, which is incorporated by reference in its entirety.

[0065] Certain GPR75 antagonists disclosed herein possess asymmetric carbon atoms (optical or chiral centers) and / or double bonds. Unless otherwise indicated herein, structures depicted herein are meant to include all stereochemical forms of the structure, for example the R and S configurations for each asymmetric center (D and L configurations for amino acids). Optically active (R)- and (S)- or (D)- and (L)-isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. When GPR75 antagonists of the disclosure contain olefinic bonds or other centers of geometric asymmetry, and unless indicated otherwise, it is intended that the compounds include both E and Z geometric isomers.

[0066] Certain GPR75 antagonists disclosed herein may exist in tautomeric forms. Accordingly, the present disclosure also relates to tautomers of the GPR75 antagonists of the disclosure.

[0067] Physiologically acceptable base addition salts of GPR75 antagonists can be prepared from inorganic and organic bases. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, calcium and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary and tertiary amines.

[0068] Physiologically acceptable acid addition salts of GPR75 antagonists may be prepared from inorganic and organic acids. Salts derived from inorganic acids include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid and phosphoric acid salts, and the like. Salts derived from organic acids include, but are not limited to, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, glutaric acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluene sulfonic acid and salicylic acid salts, and the like.

[0069] In some embodiments of the GPR75 antagonists disclosed herein, the physiologically acceptable salt is a sodium salt, a potassium salt, a lithium salt, a calcium salt, a magnesium salt or an ammonium salt. In certain embodiments of the GPR75 antagonists disclosed herein, the physiologically acceptable salt is a sodium salt.

[0070] Other examples of GPR75 antagonists include antibody molecules. "Antibody molecule", as used herein, is intended to include intact antibodies, such as polyclonal antibodies or monoclonal antibodies (mAbs), as well as proteolytic fragments thereof such as the Fab orF(ab')2fragments, chimeric antibodies, nanobodies, recombinant and engineered antibodies, single-chain antibodies and fragments thereof, as well as other molecules having at least one GPR75 antigen-binding site. In an embodiment, antibody molecules that bind to GPR75 may be used to inhibit the activity of GPR75 in NAFLD / MAFLD / MASLD or NASH subjects.

[0071] Pharmaceutical Compositions, Formulations, Dosage Forms, Medicaments and Kits

[0072] The GPR75 antagonists useful in the disclosed methods may be included in a pharmaceutical composition or formulation.

[0073] Pharmaceutical compositions and formulations of the disclosure include pharmaceutical compositions comprising a GPR75 antagonist, alone or in combination with one or more additional therapeutic agents, and a physiologically compatible carrier, excipient, or stabilizer. Such GPR75 antagonists may include small molecules that function to inhibit the activity of GPR75. The pharmaceutical compositions and formulations can be administered to a subject, for example, a human subject, for therapeutic or prophylactic treatment, such as for treating or preventing diet-induced obesity, cardiometabolic disease or cardiometabolic-associated complications.

[0074] In some embodiments, pharmaceutical compositions and formulations are provided comprising a GPR75 receptor antagonist that reduces fatty acid uptake. Such inhibition of GPR75, as demonstrated herein, can result in a decrease in fatty acid uptake. The administration of such compositions and formulations is intended to inhibit the activity of GPR75 in the subject to be treated thereby inhibiting fatty acid uptake.

[0075] In some embodiments, the pharmaceutical compositions and formulations are for use in treatment of cardiometabolic associated liver disease resulting in NAFLD / MAFLD / MASLD, NASH or cirrhosis phenotypes. In some embodiments, the pharmaceutical compositions and formulations are for use in treating or preventing NAFLD / MAFLD / MASLD or NASH, and can be administered to a subject, for example, a human subject, for therapeutic or prophylactic treatment of NAFLD / MAFLD / MASLD or NASH. In a specific embodiment, the GPR75 antagonist is disodium N-succinate-20- hydroxyeicosa-6(Z),15(Z)-diencarboxamide (AAA).

[0076] As used herein, "physiologically compatible carrier, excipient or stabilizer" refers to carriers, excipients and stabilizers that are ‘acceptable’ in the sense of being compatible with the other ingredients of a composition and not deleterious (e.g., toxic) to the recipient thereof. Physiologically compatible carrier, excipient or stabilizer can refer to a physiologicallyacceptable diluent including, but not limited to water, phosphate buffered saline, or saline, and, in some embodiments, can include an adjuvant.

[0077] Acceptable carriers, excipients, and stabilizers are nontoxic to recipients at the dosages and concentrations employed, and can include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid, BHA, and BHT; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrans; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counter-ions such as sodium; and / or nonionic surfactants such as Tween, Pluronics, or PEG. Adjuvants suitable for use with the presently disclosed compositions include adjuvants known in the art including, but not limited to, incomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide, and alum. Carriers, excipients or stabilizers are known in the art and are described in, for example, Remington: The Science and Practice of Pharmacy, 2000, Gennaro, A R ed., Eaton, Pa.: Mack Publishing Co., and Porter et al., eds., The Merck Manual, 19th edition, Merck and Co., Rahway, N.J., 2011, which are incorporated by reference in their entirety.

[0078] The presently disclosed pharmaceutical compositions and formulations can be administered using a variety of methods known in the art. More particularly, as described herein, the GPR75 antagonist can be administered to a subject for treating or preventing NAFLD / MAFLD / MASLD or NASH by any suitable route of administration, including orally, nasally, transmucosally, parenterally, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intra- articular, intra-sternal, intra-synovial, intra-hepatic, intralesional, intracranial, intraperitoneal, intranasal, or intraocular injections, intracisternally, topically, as by powders, ointments, including buccally and sublingually, transdermally, through an inhalation spray, or other modes of delivery known in the art. Regardless of the route of administration selected, the GPR75 antagonist can be formulated into pharmaceutically acceptable dosage forms such as described herein or by other conventional methods known to those of skill in the art.

[0079] In some embodiments, the presently disclosed pharmaceutical compositions and formulations can be administered by rechargeable or biodegradable devices. For example, a variety of slow-release polymeric devices have been developed and tested in vivo for the controlled delivery of drugs. Suitable examples of sustained release preparations include semipermeable polymer matrices in the form of shaped articles, e.g., films or microcapsules.Sustained release matrices include polyesters, hydrogels, polylactides (U.S. Pat. No. 3,773,919; EP 58,481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., Biopolymers 22:547, 1983), poly (2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed. Mater. Res.15:167, 1981), ethylene vinyl acetate (Langer et al., Id), or poly- D-(-)-3-hydroxybutyric acid (EP 133,988A). Sustained release compositions also include liposomally entrapped a GPR75 antagonist, which can be prepared by methods known per se (Epstein et al., Proc. Natl. Acad. Sci. U.S.A.82:3688, 1985; Hwang et al., Proc. Natl. Acad. Sci. U.S.A.77:4030, 1980; U.S. Pat. Nos.4,485,045 and 4,544,545; and EP 102,324A). Ordinarily, the liposomes are of the small (about 200-800 Angstroms) unilamelar type in which the lipid content is greater than about 30 mol % cholesterol, the selected proportion being adjusted for the optimal therapy. Such materials can include an implant, for example, for sustained release of the GPR75 antagonist.

[0080] The GPR75 antagonists useful in the disclosed methods may be comprised in a nanoparticle.

[0081] In some embodiments, the present disclosure provides a nanoparticle comprising a GPR75 antagonist. The nanoparticle may be for use in treating or preventing NAFLD / MAFLD / MASLD or NASH. Such nanoparticles can be natural or synthetic. They can be created from biological molecules or from non-biological molecules. In some cases, the GPR75 antagonist is crosslinked to a polymer or lipid on nanoparticle surface. In embodiments, the GPR75 antagonist is adsorbed onto the nanoparticle surface. In some embodiments, the GPR75 antagonist is adsorbed onto the nanoparticle surface and then crosslinked to the nanoparticle surface. In some embodiments, the GPR75 antagonist is encapsulated into the nanoparticle.

[0082] In certain embodiments, the nanoparticle is formed from a biocompatible polymer. Examples of biocompatible polymers include polyethylenes, polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumerates, polycaprolactones, polyamides, polyacetals, polyethers, polyesters, poly(orthoesters), polycyanoacrylates, polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates, polycyanoacrylates, polyureas, polystyrenes, or polyamines, or combinations thereof. In some embodiments, the nanoparticle is formed from a polyethylene glycol (PEG), poly(lactide-co-glycolide) (PLGA), polyglycolic acid, poly-beta-hydroxybutyrate, polyacrylic acid ester, or a combination thereof. In one embodiment, the nanoparticle is a nanoliposome. Such nanoliposomes may be composed of phospholipids such as 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-distearoyl- sn-glycero-3-phospho-(1'-rac-glycerol) (DSPG), 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'- rac-glycerol) (DPPG), 1,2-dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DMPG), 1,2- dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG), dipalmitoyl phosphatidylserine (DPPS), distearoyl phosphatidylserine (DSPS), dipalmitoyl phosphatidylinositol (DPPI), distearoyl phosphatidylinositol (DSPI), dipalmitoyl phosphatidic acid (DPPA), distearoyl phosphatidic acid (OSPA), 1,2-diacyl-3-trimethylammonium-propanes, (including but not limited to, dioleoyl (DOTAP),1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N [methoxy(polyethylene glycol)-2000] (DPPE-PEG2000), 1,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000] (DSPE-PEG2000), and cholesterol.

[0083] In some embodiments, the GPR75 antagonist is coated on the nanoparticle using a crosslinking agent. In some embodiments, the GPR75 antagonist is adsorbed onto the nanoparticle surface. In some embodiments, the GPR75 antagonist is adsorbed onto the nanoparticle surface followed by covalent crosslinking of the GPR75 antagonist to the nanoparticle surface using a crosslinking agent.

[0084] Crosslinking agents suitable for crosslinking the GPR75 antagonist to produce the nanoparticle, are known in the art, and include those selected from the group consisting of formaldehyde, formaldehyde derivatives, formalin, glutaraldehyde, glutaraldehyde derivatives, a protein cross-linker, a nucleic acid cross-linker, a protein and nucleic acid cross-linker, primary amine reactive crosslinkers, sulfhydryl reactive crosslinkers, sulfydryl addition or disulfide reduction, carbohydrate reactive crosslinkers, carboxyl reactive crosslinkers, photoreactive crosslinkers, cleavable crosslinkers, AEDP, APG, BASED, BM(PEO)3, BM(PEO)4, BMB, BMDB, BMH, BMOE, BS3, BSOCOES, DFDNB, DMA, DMP, DMS, DPDPB, DSG, DSP, DSS, DST, DTBP, DTME, DTSSP, EGS, HBVS, sulfo- BSOCOES, Sulfo-DST, and Sulfo-EGS.

[0085] In an embodiment, the nanoparticle is designed for targeting liver tissue. In such an instance the nanoparticle may be coated with a liver specific ligand that delivers the nanoparticle directly to the liver.

[0086] The presently disclosed subject matter also includes the use of a GPR75 antagonist, in the manufacture of a medicament for treating or preventing diet-induced obesity, cardiometabolic disease or cardiometabolic-associated complications including liver disease. In some embodiments, the medicament is for treating or preventing NAFLD / MAFLD / MASLD or NASH.

[0087] The presently disclosed compositions of a GPR75 antagonist can be assembled into kits or pharmaceutical systems for use in treating or preventing diet-induced obesity, cardiometabolic disease or cardiometabolic-associated complications including liver disease. For example, kits or pharmaceutical systems of the disclosure may be for use in treating or preventing cardiometabolic associated liver disease resulting in NAFLD / MAFLD / MASLD, NASH or cirrhosis phenotypes / stages of liver dysfunction. In some embodiments, the presently disclosed kits or pharmaceutical systems include a GPR75 antagonist in unit dosage form. In further embodiments, the GPR75 antagonist can be present together with a pharmaceutically acceptable solvent, carrier, excipient, or the like, as described herein.

[0088] In some embodiments, the presently disclosed kits include one or more containers, including, but not limited to a vial, tube, ampule, bottle, and the like, for containing the GPR75 antagonist. The one or more containers also can be carried within a suitable carrier, such as a box, carton, tube, or the like. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.

[0089] The presently disclosed kits or pharmaceutical systems also can include associated instructions for using the GPR75 antagonist containing compositions for treating or preventing, for example, NAFLD / MAFLD / MASLD or NASH. In some embodiments, the instructions include one or more of the following: a description of a GPR75 antagonist; a dosage schedule; and instructions for administration for treating or preventing NAFLD / MAFLD / MASLD or NASH; precautions; warnings; indications; counter- indications; overdosage information; adverse reactions; animal pharmacology; clinical studies; and references. In some embodiments, the instructions are for treating diabetes. The instructions can be printed directly on a container (when present), as a label applied to the container, as a separate sheet, pamphlet, card, or folder supplied in or with the container.

[0090] Embodiments of the Invention

[0091] Particular embodiments of the invention include, without limitation, the following:

[0092] Embodiment 1. A method of treating or preventing diet-induced obesity, cardiometabolic disease or cardiometabolic-associated complications in a subject, the method comprising administration of a therapeutically effective amount of a GPR75 antagonist to the subject.

[0093] Embodiment 2. The method of embodiment 1, wherein administration of the GPR75 antagonist protects against: body-weight loss due to impaired lipid metabolism, muscle catabolism or metabolic imbalances.

[0094] Embodiment 3. The method of embodiment 1, wherein administration of the GPR75 antagonist prevents alterations in intracellular calcium levels.

[0095] Embodiment 4. The method of embodiment 1, wherein administration of the GPR75 antagonist inhibits the 20-HETE-GPR75-dependent activation of fatty acid uptake.

[0096] Embodiment 5. The method of embodiment 1, wherein administration of the GPR75 antagonist attenuates the accumulation of lipotoxic species that contribute to liver diseases.

[0097] Embodiment 6. The method of embodiment 1, wherein administration of the GPR75 antagonist mitigates cellular stress or prevents progression to liver fibrosis and cirrhosis.

[0098] Embodiment 7. The method of embodiment 1, wherein administration of the GPR75 antagonist disrupts 20-HETE-GPR75-dependent signaling, mitigating lipid-induced hepatoxicity.

[0099] Embodiment 8. The method of embodiment 1, wherein administration of the GPR75 antagonist prevents hepatocyte injury, inflammation, fibrosis, cirrhosis or liver failure.

[0100] Embodiment 9. The method of embodiment 1, wherein administration of the GPR75 antagonist abrogates changes to body weight or adipose content, or reduces WAT adipocyte hypertrophy.

[0101] Embodiment 10. The method of embodiment 1, wherein administration of the GPR75 antagonist prevents adverse structural changes in adipose tissue or the liver of the subject.

[0102] Embodiment 11. The method of embodiment 1, wherein administration of the GPR75 antagonist impedes the early stages of liver steatosis.

[0103] Embodiment 12. The method of embodiment 1, wherein administration of the GPR75 antagonist reverses changes in adipocyte size / hypertrophy or liver steatosis.

[0104] Embodiment 13. The method of embodiment 1, wherein administration of the GPR75 antagonist prevents or reverses liver disease progression or the metabolic-associated changes that precipitate liver damage.

[0105] Embodiment 14. The method of embodiment 13, wherein the metabolic-associated changes that precipitate liver damage comprise one or more of excess body weight gain, insulin resistance, steatosis and cellular inflammation / hypertrophy.

[0106] Embodiment 15. The method of embodiment 1, which is for treating or preventing nonalcoholic fatty liver disease (NAFLD), alternatively termed Metabolic Dysfunction- associated Fatty / Steatotic Liver Disease (MAFLD / MASLD).

[0107] Embodiment 16. The method of embodiment 1, which is for treating or preventing nonalcoholic steatohepatitis (NASH).

[0108] Embodiment 17. The method of any one of embodiments 1 to 16, wherein the GPR75 antagonist is a small molecule.

[0109] Embodiment 18. The method of any one of embodiments 1 to 17, wherein the GPR75 antagonist is a compound of the formula (I) or (II):or a physiologically acceptable salt thereof, wherein: R2and R3are each, independently, OH, C1-C3alkyl, F, or H; R4is C1-C3alkyl, H, F, or -CH2N3(azide); X is C or O; m and p are 0 or 1; n and q are 1 to 3; and R1is CO2H, NR8R9, C(O)R6or a tetrazole, wherein: R6is OR7, NR8R9, a D- / L- / D,L-α-amino acid (MW<250), -NHS(O)2R10, a polyethylene glycol (MW<350) or alkyl ether thereof, glycerol, glyceride mono- or diester (MW<800), or a carboxylate isostere or mimetic selected from the groupconsisting of: -P(O)(OH)2, -S(O)2OH,,wherein: lower alkyl is a C1-C6alkyl or cycloalkyl;R7is C1-C6alkyl or cycloalkyl, or benzyl; R8and R9are each, independently, H, C1-C6alkyl or cycloalkyl, or benzyl, or R8and R9together constitute a 3-7 membered ring with the nitrogen; and R10is phenyl, C1-C5alkyl or cycloalkyl, or -CF3, provided that: (i) at least one of R2, R3and R4is OH or F; (ii) the sum of the number of carbon atoms provided by n and m is 3 or 4; and (iii) the sum of the number of carbon atoms provided by p and q is 3 or 4.

[0110] Embodiment 19. The method of embodiment 18, wherein the GPR75 antagonist is a compound of the formula (I) or (II) in which R1is CO2H, C(O)R6, wherein R6a D- / L- / D,L- α-amino acid (MW<250) or a polyethylene glycol (MW<350) or alkyl ether thereof, a tetrazoleor , or a physiologically acceptable salt thereof.

[0111] Embodiment 20. The method of embodiment 18 or 19, wherein the GPR75 antagonist is a compound of the formula (I) or (II) in which one of R2and R3is OH or F; one of R2and R3is H or -CH3; and R4is H, -CH3or CH2N3, or a physiologically acceptable salt thereof.

[0112] Embodiment 21. The method of any one of embodiments 18 to 20, wherein the GPR75 antagonist is a compound of the formula (I) or (II) having a structure of:, wherein represents a single or a double bond, provided that at least one is a double bond, or a physiologically acceptable salt thereof.

[0113] Embodiment 22. The method of any one of embodiments 18 to 21, wherein the GPR75 antagonist is a compound of the formula (I) or (II) having a structure of:, or a physiologically acceptable salt thereof.

[0114] Embodiment 23. The method of any one of embodiments 18 to 22, wherein the GPR75 antagonist is a compound of the formula (I) or (II) in which R1is CO2H or C(O)R6, wherein R6is glycine , aspartic acid, or , or a physiologically acceptable salt thereof.

[0115] Embodiment 24. The method of any one of embodiments 1 to 22, wherein the GPR75 antagonist is:

[0116] Embodiment 25. The method of any one of embodiments 1 to 22, wherein the GPR75 antagonist is 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (20-6,15-HEDE, 20-HEDE), N-[20- hydroxyeicosa-6(Z),15(Z)- dienoyl]glycine (20-HEDGE), [2,5,8,11,14,17- hexaoxanonadecan-19-yl 20-hydroxyicosa-6(Z),15(Z)-dienoate] (20-SOLA), a 19-HETE analogue, or disodium N-succinate-20-hydroxyeicosa-6(Z),15(Z)-diencarboxamide (AAA).

[0117] Embodiment 26. The method of any one of embodiments 1 to 22, wherein the GPR75 antagonist is disodium N-succinate-20-hydroxyeicosa-6(Z),15(Z)-diencarboxamide (AAA).

[0118] Embodiment 27. The method of any one of embodiments 1 to 16, wherein the GPR75 antagonist is an antibody.

[0119] Embodiment 28. The method of any one of embodiments 1 to 27, further comprising administration of a second therapeutic agent for treatment of liver disease categories including NAFLD / MAFLD / MASLD, NASH or cirrhosis to the subject.

[0120] Embodiment 29. A pharmaceutical composition for use for treating or preventing diet-induced obesity, cardiometabolic disease or cardiometabolic-associated complications, the pharmaceutical composition comprising a GPR75 antagonist and a physiologically compatible carrier, excipient, or stabilizer.

[0121] Embodiment 30. The pharmaceutical composition of embodiment 29, wherein the GPR75 antagonist is as defined in any one of embodiments 17 to 27.

[0122] Embodiment 31. The pharmaceutical composition of embodiment 29 or 30, further comprising one or more additional therapeutic agents.

[0123] Embodiment 32. The pharmaceutical composition of any one of embodiments 29 to 31, which is for treating or preventing a cardiometabolic associated liver disease resulting in NAFLD / MAFLD / MASLD, NASH or cirrhosis phenotypes.

[0124] Embodiment 33. The pharmaceutical composition of any one of embodiments 29 to 31, which is for treating or preventing NAFLD / MAFLD / MASLD or NASH.

[0125] Embodiment 34. Use of a GPR75 antagonist in the manufacture of a medicament for treating or preventing diet-induced obesity, cardiometabolic disease or cardiometabolic- associated complications.

[0126] Embodiment 35. The use of embodiment 34, wherein the medicament is for treating or preventing NAFLD / MAFLD / MASLD or NASH.

[0127] Embodiment 36. The use of embodiment 34, wherein the medicament is for treating or preventing liver disease.

[0128] Embodiment 37. The use of any one of embodiments 34 to 36, wherein the GPR75 antagonist is as defined in any one of embodiments 17 to 27.

[0129] Embodiment 38. A kit comprising: (i) a GPR75 antagonist; and (ii) instructions for using the GPR75 antagonist for treating or preventing diet-induced obesity, cardiometabolic disease or cardiometabolic-associated complications.

[0130] Embodiment 39. The kit of embodiment 38, wherein the instructions include one or more of the following: (i) a description of the GPR75 antagonist; (ii) a dosage schedule; or (iii) instructions for administration of the GPR75 antagonist for treating or preventing NAFLD / MAFLD / MASLD or NASH.

[0131] Embodiment 40. The kit of embodiment 38, wherein the instructions are for treating diabetes.

[0132] Embodiment 41. The kit of embodiment 38, wherein the instructions are treating or preventing cardiometabolic associated liver disease resulting in NAFLD / MAFLD / MASLD, NASH or cirrhosis phenotypes / stages of liver dysfunction.

[0133] Embodiment 42. The kit of any one of embodiments 38 to 41, wherein the GPR75 antagonist is as defined in any one of embodiments 17 to 27.

[0134] Although the present disclosure and its advantages have been described in detail, it should be understood that various changes, substitutions, and alterations can be made herein without departing from the spirit and scope of the invention as defined in the claims.

[0135] The present disclosure will be further illustrated in the following Example, which is given for illustration purposes only and is not intended to limit the invention in any way. EXAMPLE

[0136] A transgenic mouse line was developed that is deficient in GPR75 (GPR75- / -global knockout (KO) mice) and in which the dominant 20-HETE synthase, Cyp4a12, can be induced to overproduce 20-HETE globally via doxycycline (DOX) administered in the drinking water, thus establishing the Cyp4a12-GPR75- / -mouse model. This mouse model shows that mice deficient in GPR75 are protected from diet-induced obesity and liver damage. HTLA cells (an HEK293 cell line stably expressing a tTA-dependent luciferase reporter and a β-arrestin2-TEV fusion gene) coupled to a GPR75-specific PRESTO-Tango overexpression construct [RRID:Addgene_66372], the endothelial cell line, EA.hy926 cells, and hepatocyte cell line, Hep G2 cells serve as an in vitro tools for the evaluation of intracellular calcium measurements using the FLIPR Calcium 6 Assay Kit according to the manufacturer's protocol. Additionally, the human hepatocyte cell line, Hep G2 cells, were developed to be deficient in GPR75 via CRISPR-Cas9 with a 90+% knockdown efficiency.

[0137] FIG.1A-B demonstrates changes in systolic blood pressure (28 weeks) (FIG.1A) or body weight (FIG.1B) in response to control diet (CD) or high fat diet (HFD) in Cyp4a12- GPR75+ / +or Cyp4a12-GPR75- / -mice (28 weeks) in the presence and absence of doxycycline (DOX). Mean ± SEM (n=4-8). * p<0.05 , **** p<0.0001. FIG.2 demonstrates representative MicroCT images from mice at baseline (Week 0) and 28 Weeks of CD or HFD feeding in the presence or absence of DOX. (n=4-6). Visceral Fat, Subcutaneous Fat, outlined. FIG.3 depicts representative histological images (H&E staining) of liver sections from mice under CD or HFD in the presence and absence of DOX for 28 weeks. (n=4-6). Collectively, these datashowcase HFD’s and 20-HETE’s contribution to the pathogenesis of hepatic steatosis and liver disease and how a deficiency GPR75 protects against various cardiometabolic parameters including liver disease. FIG. 4 illustrates how GPR75- / - mice are protected from the body weight loss, reductions in activity and liver damage associated with methionine-choline- deficient diet, suggesting that pharmacological blockade of GPR75 may lead to an equivalent level of protection from HFD- or MCD-induced liver disease. FIG.5A. demonstrates disodium N-succinate-20-hydroxyeicosa-6(Z),15(Z)-diencarboxamide’s (AAA’s) influence on 20- HETE-GPR75-mediated changes in intracellular calcium, showcasing AAA’s calculated IC50 response as a competitive antagonist in the endothelial cell line, EA.hy926. While FIG. 5A depicts the free acid form of AAA, it is disodium N-succinate-20-hydroxyeicosa-6(Z),15(Z)- diencarboxamide (i.e., AAA) that was used. FIG.5B-D highlight 20-HETE’s ability to drive changes in intracellular calcium via GPR75 in the hepatocyte cells line HEPG2, a hallmark of GPR75-mediated Gq-receptor activation. Moreover, these data demonstrate AAA’s ability to disrupt changes to intracellular calcium observed via the 20-HETE-GPR75 axis. FIG. 6A demonstrates changes in fatty acid uptake across HEPG2 cells in response to Vehicle (ethanol), 20-HETE (10 nM) in the presence and absence of AAA (10nM) using the QBT Fatty Acid Uptake Kit. FIG. 6B demonstrates the effects of lipofermata (a specific FATP2 blocker) (10μM) on 20-HETE-driven fatty acid uptake in WT cells. FIG.6C demonstrates changes in fatty acid uptake in 20HR KD cells exposed to Vehicle (ethanol), 20-HETE (10 nM) in the presence and absence of AAA (10nM). Mean ± SEM (n=8). **** p<0.0001. These data demonstrate 20-HETE’s ability to drive increases in fatty acid uptake in hepatocytes, a process that is dependent on the presence of its receptor, GPR75, and the fatty acid transport protein, FATP2 / SLC27A2. Moreover, AAA is able to abrogate this signaling cascade, thus blocking the 20-HETE-GPR75-axis from driving changes to fatty acid uptake. FIG.7 shows detection of fatty acid uptake and lipid droplet formation (BODIPY) in HEPG2 pre-treated with either Vehicle (ethanol), 20-HETE (10nM), AAA (10nM), 20-HETE+AAA, Calphostin C ((CC) PKC inhibitor (1μM)) or 20-HETE+CC prior to oleic acid supplementation (1mM) for 4 h. Green (BODIPY), Blue (Hoechst), Red (Segmented BODIPY Signal (Lipid Droplets)) (n=8). FIG.8A depicts detection of fatty acid uptake and lipid droplet formation (BODIPY) in GPR75 knockdown / deficient HEPG2 cells pre-treated with either vehicle (ethanol), 20-HETE (10nM). Green (BODIPY), Blue (Hoechst), Red (Segmented BODIPY Signal (Lipid Droplets)) (n=8). FIG.8B is a graph summarizing 20-HETE- and GPR75-dependent changes in fatty acid uptake as a measure of BODIPY surface area (n=8). 20-HETE’s influence on fatty acid uptake via GPR75 and FATP2 drives dramatic changes to hepatocytes resulting increases in lipid dropletformations in order to accommodate the influx of oleic acid. These data support the role of GPR75-PKC as a key driver in fatty acid uptake and illustrate AAA’s ability to prevent the molecular programing associated with the pairing of 20-HETE and GPR75. FIG.9A-C depict AAA’s proficiency in preventing diet induced weight gain (FIG. 9A-B) and subsequent changes to adipocyte size and early liver steatosis (FIG.9C). FIG.10A-C demonstrate AAA’s capacity to reverse changes to body weight, glucose mismanagement, and adipose tissue volume associated with a high-fat feeding protocol. FIG. 10D highlights AAA’s ability to restore adipocyte size, and liver steatosis. These mouse studies showcase AAA capacity to both prevent and reverse the diet-induced cardiometabolic associated changes that contribute to the development and pathogenesis of liver steatosis and liver disease.

Claims

WHAT IS CLAIMED IS:

1. A method of treating or preventing diet-induced obesity, cardiometabolic disease or cardiometabolic-associated complications in a subject, the method comprising administration of a therapeutically effective amount of a GPR75 antagonist to the subject.

2. The method of claim 1, which is for treating or preventing nonalcoholic fatty liver disease (NAFLD), alternatively termed Metabolic Dysfunction-associated Fatty / Steatotic Liver Disease (MAFLD / MASLD).

3. The method of claim 1, which is for treating or preventing nonalcoholic steatohepatitis (NASH).

4. The method of claim 1, wherein administration of the GPR75 antagonist: (i) protects against: body-weight loss due to impaired lipid metabolism, muscle catabolism or metabolic imbalances; (ii) prevents alteration in intracellular calcium levels; (iii) inhibits the 20-HETE-GPR75-dependent activation of fatty acid uptake; (iv) attenuates the accumulation of lipotoxic species that contribute to liver diseases; (v) mitigates cellular stress or prevents progression to liver fibrosis or cirrhosis; (vi) disrupts 20-HETE-GPR75-dependent signaling, mitigating lipid-induced hepatoxicity; (vii) prevents hepatocyte injury, inflammation, fibrosis, cirrhosis or liver failure; (viii) abrogates changes to body weight or adipose content, or reduces WAT adipocyte hypertrophy; (ix) prevents adverse structural changes in adipose tissue or the liver of the subject; (x) impedes the early stages of liver steatosis; (xi) reverses changes in adipocyte size / hypertrophy or liver steatosis; and / or (xii) prevents or reverses liver disease progression or the metabolic-associated changes that precipitate liver damage.

5. The method of any one of claims 1 to 4, wherein the GPR75 antagonist is a small molecule.

6. The method of any one of claims 1 to 5, wherein the GPR75 antagonist is a compound of the formula (I) or (II)or a physiologically acceptable salt thereof, wherein: R2and R3are each, independently, OH, C1-C3alkyl, F, or H; R4is C1-C3alkyl, H, F, or -CH2N3(azide); X is C or O; m and p are 0 or 1; n and q are 1 to 3; and R1is CO2H, NR8R9, C(O)R6or a tetrazole, wherein: R6is OR7, NR8R9, a D- / L- / D,L-α-amino acid (MW<250), -NHS(O)2R10, a polyethylene glycol (MW<350) or alkyl ether thereof, glycerol, glyceride mono- or diester (MW<800), or a carboxylate isostere or mimetic selected from the group consisting of: -P(O)(OH)2, -S(O)2OH,, wherein: lower alkyl is a C1-C6alkyl or cycloalkyl; R7is C1-C6alkyl or cycloalkyl, or benzyl; R8and R9are each, independently, H, C1-C6alkyl or cycloalkyl, or benzyl, or R8and R9together constitute a 3-7 membered ring with the nitrogen; and R10is phenyl, C1-C5alkyl or cycloalkyl, or -CF3, provided that: (i) at least one of R2, R3and R4is OH or F; (ii) the sum of the number of carbon atoms provided by n and m is 3 or 4; and (iii) the sum of the number of carbon atoms provided by p and q is 3 or 4.

7. The method of claim 6, wherein the GPR75 antagonist is a compound of the formula (I) or (II) having a structure of:wherein: represents a single bond or a double bond, provided that at least one is a double bond;R1is CO2H, C(O)R6, wherein R6is a D- / L- / D,L-α-amino acid (MW<250) or a polyethylene glycol (MW<350) or alkyl ether thereof, a tetrazole or, one of R2and R3is OH or F; one of R2and R3is H or -CH3; and R4is H, -CH3or CH2N3, or a physiologically acceptable salt thereof.

8. The method of any one of claims 1 to 7, wherein the GPR75 antagonist is:

9. The method of any one of claims 1 of 7, wherein the GPR75 antagonist is 20- hydroxyeicosa-6(Z),15(Z)-dienoic acid (20-6,15-HEDE, 20-HEDE), N-[20-hydroxyeicosa- 6(Z),15(Z)- dienoyl]glycine (20-HEDGE), [2,5,8,11,14,17-hexaoxanonadecan-19-yl 20- hydroxyicosa-6(Z),15(Z)-dienoate] (20-SOLA), a 19-HETE analogue, or disodium N- succinate-20-hydroxyeicosa-6(Z),15(Z)-diencarboxamide (AAA).

10. The method of any one of claims 1 to 7, wherein the GPR75 antagonist is disodium N-succinate-20-hydroxyeicosa-6(Z),15(Z)-diencarboxamide (AAA).

11. The method of any one of claims 1 to 4, wherein the GPR75 antagonist is an antibody.

12. The method of any one of claims 1 to 11, further comprising administration of a second therapeutic agent for treatment of liver disease categories includingNAFLD / MAFLD / MASLD, NASH or cirrhosis to the subject.

13. A pharmaceutical composition for use for treating or preventing diet-induced obesity, cardiometabolic disease or cardiometabolic-associated complications, the pharmaceutical composition comprising a GPR75 antagonist and a physiologically compatible carrier, excipient, or stabilizer.

14. The pharmaceutical composition of claim 13, further comprising one or more additional therapeutic agents.

15. Use of a GPR75 antagonist in the manufacture of a medicament for treating or preventing diet-induced obesity, cardiometabolic disease or cardiometabolic-associated complications.

16. The use of claim 16, wherein the medicament is for treating or preventing liver disease, preferably wherein the medicament is for treating or preventing NAFLD / MAFLD / MASLD or NASH.

17. A kit comprising: (i) a GPR75 antagonist containing compositions; and (ii) instructions for using the GPR75 antagonist for treating or preventing diet-induced obesity, cardiometabolic disease or cardiometabolic-associated complications.

18. The kit of claim 17, wherein the instructions are for treating diabetes.

19. The kit of claim 17, wherein the instructions include one or more of the following: (i) a description of the GPR75 antagonist; (ii) a dosage schedule; or (iii) instructions for administration of the GPR75 antagonist for treating or preventing NAFLD / MAFLD / MASLD or NASH.

20. The pharmaceutical composition of claim 13 or 14, the use of claim 15 or 16, or the kit of any one of claims 17 to 19, wherein the GPR75 antagonist is as defined in any one of claims 5 to 10.

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