GPR75 antagonists for use in the prevention or treatment of diet-induced obesity, cardiometabolic disease or cardiometabolic-related complications

Abstract

The present disclosure provides methods, compositions, and kits for treating or preventing diet-induced obesity, cardiometabolic diseases, and cardiometabolic-related complications, including liver diseases and their respective categories, including nonalcoholic fatty liver disease (NAFLD) (alternatively referred to herein as metabolic dysfunction-associated fatty / fibro fatty liver disease (MAFLD / MASLD)), nonalcoholic steatohepatitis (NASH), and cirrhosis, employing compounds that disrupt the activity of the 20-HETE receptor GPR75.

Description

Cross-reference to related applications

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 594,176, filed on October 30, 2023, which is incorporated herein by reference in its entirety. Technical Field

[0002] This disclosure provides methods, compositions, and kits for treating or preventing diet-induced obesity, cardiometabolic diseases, and cardiometabolic-related complications, including liver diseases (i.e., non-alcoholic fatty liver disease (NAFLD) (which may alternatively be referred to herein as metabolic dysfunction-associated steatosis / steatohepatitis (MAFLD / MASLD)), non-alcoholic steatohepatitis (NASH), and cirrhosis), using compounds that disrupt the activity of the 20-HETE receptor GPR75. Background Technology

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

[0004] To date, the role of GPR75 in fatty acid uptake has not been reported. Furthermore, there are currently no pharmacologically known compounds that can be used to prevent or treat NAFLD / MAFLD / MASLD or NASH. Therefore, such compounds are currently needed. Summary of the Invention

[0005] This disclosure relates to methods for inhibiting the activity of GPR75 in a subject, the method comprising administering to the subject an inhibitory amount of a GPR75 antagonist (which may alternatively be referred to herein as a "GPR75 antagonist / receptor blocker"), pharmaceutical 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] This disclosure provides a method for modulating a GPR75-mediated response by administering to a subject a compound that inhibits the activity of GPR75 (a GPR75 antagonist / receptor blocker). According to this disclosure, such inhibition can be achieved by any compound that inhibits the activity of GPR75. In a particular embodiment, the compound is disodium N-succinate-20-hydroxyeicosicosano-6(Z),15(Z)-dienecarboxamide (“AAA”), which is a disodium salt of N-(20-hydroxyeicosicosano-6(Z),15(Z)-dieneyl)-L-aspartic acid.

[0007] .

[0008] More specifically, the subject matter disclosed in this invention provides a method for treating diet-induced obesity, cardiometabolic diseases, and cardiometabolic-related complications, delaying their onset, alleviating their symptoms, and / or preventing their progression, including liver diseases (non-alcoholic fatty liver disease (NAFLD) / metabolic dysfunction-associated steatosis / steatohepatitis (MAFLD / MASLD), non-alcoholic steatohepatitis (NASH), and cirrhosis). The method comprises safely administering a therapeutically effective amount of a GPR75 antagonist to a subject in need. As demonstrated herein, the administration of such a GPR75 antagonist is designed to modulate GPR75 activity and inhibit fatty acid uptake, thereby preventing / reducing hepatic steatosis.

[0009] While aspects or implementations of the following disclosure may relate to the treatment of NAFLD / MAFLD / MASLD or NASH, this disclosure is equally applicable to the treatment of other diseases, delaying the onset of other diseases, alleviating the symptoms of other diseases, and / or preventing the progression of other diseases such as diet-induced obesity, cardiometabolic diseases (heart disease, stroke, type 2 diabetes, dyslipidemia), and cardiometabolic complications caused by GPR75 activation (coronary artery disease (CAD), atherosclerosis, diabetic neuropathy / retinopathy / nephropathy, chronic kidney disease, liver disease / cancer, pancreatitis, polycystic ovary syndrome (PCOS)).

[0010] In one embodiment, the subject matter of this invention provides a method for preventing, treating, or alleviating symptoms associated with GPR75 activation by administering a therapeutically effective amount of a GPR75 antagonist to a subject suspected of having liver disease or at risk of developing liver disease. Such subjects may be identified as those exhibiting symptoms of NAFLD / MAFLD / MASLD or NASH. Symptoms of NAFLD / MAFLD / MASLD may include fatigue, pain or discomfort in the right upper quadrant of the abdomen. Possible symptoms of NASH include advanced scarring (cirrhosis), abdominal swelling (ascites), enlarged blood vessels just below the skin surface, splenomegaly, red palms, and yellowing of the skin and eyes (jaundice). Additionally, patients may exhibit elevated levels of liver-related enzymes (serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST)). In one embodiment, the subject to be treated may be a subject with a condition that makes the subject more susceptible to developing metabolic disorders such as NAFLD / MAFLD / MASLD or NASH.

[0011] Pharmaceutical compositions and formulations are also disclosed, comprising a GPR75 antagonist alone or in combination with one or more other therapeutic agents, and physiologically compatible carriers, excipients, or stabilizers. The pharmaceutical compositions and formulations can be administered to subjects, such as human subjects, for therapeutic treatment, such as for the treatment or prevention of diet-induced obesity, cardiometabolic diseases, or cardiometabolic-related complications. For example, the pharmaceutical compositions and formulations can be used to treat or prevent liver disease. Depending on the severity of the subject and / or, for example, NAFLD / MAFLD / MASLD or NASH, the pharmaceutical compositions and formulations disclosed herein can be administered using a variety of methods known in the art. In one aspect, the GPR75 antagonist is administered, for example, orally (sublingually, orally), intravenously, intramuscularly, topically / transdermally, subcutaneously (abdomen, etc.), intranasally, by inhalation, or intravenously. Regardless of the chosen route of administration, the GPR75 antagonist pharmaceutical compositions are formulated into pharmaceutically acceptable dosage forms as described below, or formulated using other conventional methods known to those skilled in the art. In some embodiments, the GPR75 antagonist is AAA.

[0012] The subject matter of this invention also includes the use of GPR75 antagonists in the manufacture of medicaments for the treatment or prevention of diet-induced obesity, cardiometabolic diseases, or cardiometabolic complications (including liver disease). In some embodiments, the medicament is used to treat or prevent NAFLD / MAFLD / MASLD or NASH.

[0013] GPR75 antagonists or their pharmaceutical compositions or formulations can be assembled into kits or pharmaceutical systems for the treatment or prevention of diet-induced obesity, cardiometabolic diseases, or cardiometabolic-related complications, including liver disease. In some embodiments, the kits or pharmaceutical systems disclosed herein comprise a unit dosage form of the GPR75 antagonist. In other embodiments, the GPR75 antagonist may be present with pharmaceutically acceptable solvents, carriers, excipients, etc., as described herein. In some embodiments, the kits disclosed herein comprise one or more containers containing the GPR75 antagonist, including but not limited to vials, tubes, ampoules, bottles, etc. The kits or pharmaceutical systems disclosed herein may also include instructions for using the GPR75 antagonist or a composition containing the GPR75 antagonist to treat or prevent NAFLD / MAFLD / MASLD or NASH. Attached Figure Description

[0014] Figure 1A -B: In Cyp4a12-GPR75+ / + (WT) and Cyp4a12-GPR75- / - (KO) mice, in the presence and absence of doxycycline (DOX), in response to feeding mice with a control diet (CD) or a high-fat diet (HFD), Figure 1A Changes in systolic blood pressure (28 weeks) or Figure 1B Weight changes (28 weeks). Mean ± SEM (n=4–8). p<0.05, p<0.0001.

[0015] Figure 2 Representative microCT images from mice at baseline (week 0) and those fed CD or HFD for 28 weeks in the presence of DOX. (n=4–6). Visceral fat and subcutaneous fat, outlined.

[0016] Figure 3 Representative histological images (H&E staining) of liver sections from mice subjected to CD or HFD for 28 weeks in the presence and absence of DOX. (n=4–6). Cyp4a12-GPR75- / - (KO) mice showed a significant protective effect against the development of hepatic steatosis (NAFLD / MAFLD / MASLD or NASH) observed in Cyp4a12-GPR75+ / + mice (WT). In conclusion, these data suggest that GPR75 is a key regulator and contributor to the onset and progression of liver disease.

[0017] Figure 4A-C: Body weight change in GPR75+ / + (WT) or overall GPR75- / - (KO) mice after 6 weeks of administration of a methionine / choline-deficient (MCD) diet. Figure 4A ), liver histological changes ( Figure 4B ) and changes in metabolic cage activity ( Figure 4C (48 hours). n=7-9. p<0.05. GPR75 deficiency is sufficient to provide significant protection against MCD-induced liver injury, weight loss due to impaired lipid metabolism, and impaired activity due to muscle catabolism and metabolic imbalance.

[0018] Figure 5A -D: Figure 5A Dose response and IC50 calculation of the endothelial cell line AAA (N-succinic acid-20-hydroxyeicosicocarbon-6(Z),15(Z)-dienecarboxamide disodium salt) (EA.hy926). Fixed dose / concentration of 20-HETE (10 nM). n=6–8. p<0.05. Figure 5B GPR75 is expressed by hepatocytes and can be disrupted by gene silencing. Figure 5C Changes in intracellular calcium in the hepatocyte cell line (HepG2) in response to mediators (ethanol) and 20-HETE (10 nM). B) GPR75 expression in WT and GPR75 knockdown (KD) cells. Figure 5D Changes in intracellular calcium in WT and GPR75 knockdown (KD) cells treated with a medium (ethanol) and 20-HETE (10 nM) in the presence and absence of AAA (10 nM). Mean ± SEM (n=8). p < 0.0001. These data indicate that AAA effectively inhibits 20-HETE-dependent activation of GPR75 in both endothelial cells and hepatocytes, thereby disrupting downstream signaling events. AAA-mediated GPR75 blockade prevents alterations in intracellular calcium levels, which act as key signaling mediators and major indicators of GPR75 activity.

[0019] Figure 6A -C: Figure 6A Changes in fatty acid uptake in HEPG2 cells in the presence and absence of AAA (10 nM) in response to mediators (ethanol) and 20-HETE (10 nM). Figure 6BEffects of lipofermata (a specific FATP2 / SLC27A2 blocker) (10 μM) on 20-HETE-driven fatty acid uptake in WT cells. Figure 6C Changes in fatty acid uptake in GPR75 KD cells exposed to the mediator (ethanol) and 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). Cessation of excessive lipid transport can maintain hepatic lipid homeostasis, thus reducing the accumulation of lipotoxic substances that contribute to liver disease. By limiting dysregulated fatty acid uptake, GPR75 receptor blockade can alleviate cellular stress and prevent progression to liver fibrosis and cirrhosis, conditions often associated with excessive lipid deposition and inflammation.

[0020] Figure 7 Fatty acid uptake and lipid droplet formation (BODIPY) in HEPG2 cells pretreated with mediators (ethanol), 20-HETE (10 nM), AAA (10 nM), 20-HETE+AAA, calciphosphate protein C ((CC) PKC inhibitor (1 μM)), or 20-HETE+CC were detected before continuous oleic acid (1 mM) supplementation. Green (BODIPY), blue (Hoechst), and black (segmented BODIPY signal (lipid droplets)) (n=8). The ability of 20-HETE to drive increased fatty acid uptake (partially through activation of FATP2 / SCL27A2) exacerbated lipid droplet formation in hepatocytes. This process heavily depends on protein kinase C (PKC) activation. Notably, the use of PKC inhibitors prior to 20-HETE exposure effectively blocked lipid droplet accumulation. Furthermore, AAA antagonism of GPR75 completely eliminated the activation of the 20-HETE-GPR75-PKC signaling axis, thereby preventing lipid droplet formation. These findings highlight the ability of AAA to disrupt 20-HETE-GPR75-dependent signaling, thereby mitigating lipid-induced hepatotoxicity, particularly in the case of hepatic steatosis, where excessive lipid droplets are associated with cellular stress, inflammation, and progression to liver fibrosis.

[0021] Figure 8A -B: Figure 8ADetection of fatty acid uptake and lipid droplet formation (BODIPY) in GPR75 knockdown / deficient HEPG2 cells pretreated with a medium (ethanol) and 20-HETE (10 nM). Green (BODIPY), blue (Hoechst), red (segmented BODIPY signal (lipid droplets)) (n=8). Figure 8B A graph summarizing 20-HETE and GPR75-dependent changes in fatty acid uptake as a measure of body 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 quantitative data illustrate how GPR75 deficiency and antagonism disrupt lipid storage, prevent chronic lipid accumulation, and thus protect against hepatocellular damage, inflammation, fibrosis, cirrhosis, and liver failure.

[0022] Figure 9A -C: Figure 9A Daily body weight changes in WT mice fed intraperitoneally with a high-fat diet (HFD) + / - saline or AAA (50 mg / kg / day). Figure 9B Representative MicroCT images at week 4, and quantitative changes in total fat and lean body volume between saline and AAA. Figure 9C Representative histological images of white adipose tissue (WAT) and liver from WT mice fed HFD combined with daily saline (50 mg / kg / day) for 4 weeks. n=5. p < 0.01. Administration of AAA significantly absolved changes in body weight and fat content and reduced WAT adipocyte hypertrophy without affecting lean body volume. Furthermore, compared to their saline-treated counterparts, AAA-treated mice showed less hepatic steatosis and early lipid droplet formation (black arrows), key markers of fatty liver disease. These data highlight the ability of AAA to prevent adverse structural changes in both adipose tissue and liver induced by a high-fat diet, thereby inhibiting the early stages of hepatic steatosis by disrupting GPR75-dependent signaling mechanisms.

[0023] Figure 10A -D: Figure 10A Weekly body weight changes in WT mice fed HFD for 5 weeks followed by AAA (50 mg / kg / day). Bars indicate microCT imaging and GTT studies occurring during weeks 4–5, and arrows indicate the start of saline or AAA administration (week 5). Figure 10B Glucose tolerance test (GTT) in WT mice at week 8 of HFD, 3 weeks after treatment with AAA (50 mg / kg / day). Figure 10CRepresentative MicroCT images at week 8, and quantitative changes in total fat and lean body volume over 3 weeks between mice treated with saline and mice treated with AAA. Figure 10D Representative histological images of white adipose tissue (WAT) and liver from mice fed HFD + / - AAA. Quantitative steatosis of liver from AAA-treated mice. n=3–7. p < 0.01. A reversal study (in which mice were fed HFD for 5 weeks, followed by AAA (50 mg / kg / day) for 4 weeks while continuing the HFD regimen) demonstrated that AAA drove a rapid decrease in body weight. After 8 weeks of HFD, saline-treated mice exhibited impaired glucose handling, while AAA-treated mice cleared glucose challenge significantly faster. Further evaluation of AAA + HFD-treated mice revealed the efficacy of AAA in reversing changes in adipocyte size / hypertrophy and hepatic steatosis, changes clearly observed in saline-treated mice fed HFD. These data demonstrate a causal relationship between GPR75 and diet-driven metabolic consequences associated with GPR75 activation, highlighting the role of GPR75 as a key driver of metabolic dysfunction and liver disease progression. In summary, these data suggest that AAA can prevent and reverse liver disease progression and metabolically related changes contributing to liver injury, including excessive weight gain, insulin resistance, steatosis, and cellular inflammation / hypertrophy. Detailed Implementation

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

[0025] Unless the context clearly specifies otherwise, references to the singular form, such as “a / an” and “the”, include plural references.

[0026] As used herein, transitional terms such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” etc., should be understood as inclusive or open-ended (i.e., meaning including but not limited to), and they do not exclude elements, materials, or method steps not listed. Regarding the claims and exemplary embodiments herein, only the transitional phrases “consisting of…” and “substantially consisting of…” are closed or semi-closed transitional phrases, respectively. The transitional phrase “consisting of…” excludes any element, step, or component not specifically described. The transitional phrase “substantially consisting of…” limits the scope to the specified elements, materials, or steps and those elements, materials, or steps that do not materially affect the fundamental features of the invention disclosed and / or claimed herein.

[0027] As used herein, the phrase “and / or” should be understood to mean “any one or two” of the elements so combined, that is, elements that coexist in some cases and exist separately in others. Multiple elements listed with “and / or” should be interpreted in the same way, that is, “one or more” of the elements so combined. In addition to the elements specifically identified by the “and / or” clause, other elements may optionally exist, whether related to or unrelated to those specifically identified. Thus, as a non-limiting example, when used in conjunction with open-ended language such as “includes,” a reference to “A and / or B” may in one embodiment refer only to A (optionally including elements other than B); in another embodiment refer only to B (optionally including elements other than A); in yet another embodiment refer to both A and B (optionally including other elements); and so on.

[0028] Unless otherwise expressly stated herein, the expressions “one or more” and “at least one” (which may be used interchangeably) refer to the number of different entities, not the number of any particular entity.

[0029] Unless otherwise stated, as used herein, the term "alkyl" on its own or as part of another substituent means having any number of carbon atoms, and more particularly having a specified number of carbon atoms (e.g., C10, C20, C30, C40, C50, C60, C7 ... 1-6 Alkyl groups (meaning 1 to 6 carbon atoms) are straight-chain or branched, substituted or unsubstituted aliphatic groups. An exemplary "alkyl" group is a methyl group (-CH3; Me).

[0030] The term "cycloalkyl" refers to a compound derived from a carbon group having any number of carbon atoms, and more particularly having a specified number of carbon atoms (e.g., C10, C20, C30, C40, C50, C60, C7 ...60, C70, C60, C60, C 3-6 A monovalent group derived from cycloalkanes is a ring of 3 to 6 carbon atoms with hydrogen atoms removed.

[0031] The "lower alkyl" group is C 1-6 Alkyl or cycloalkyl groups.

[0032] As used herein, the terms “treat,” “treating,” and “treatment” are intended to reduce, inhibit, attenuate, diminish, stop, or reverse the etiology, development, or progression of diet-induced obesity, cardiometabolic disease, or cardiometabolic complications (including liver disease and / or related symptoms). As used herein, the terms “treat,” “treating,” and “treatment” may also refer to stabilizing the development or progression of NAFLD / MAFLD / MASLD, NASH, or cirrhosis. As used herein, the terms “treat,” “treating,” and “treatment” may refer to curative therapy, preventative therapy, and treatment-preventive therapy. Therefore, as used herein, “treatment” means slowing, stopping, or reversing the progression of metabolic disorders mediated by GPR75 activation and hepatic fatty acid uptake. For example, as used herein, “treatment” can mean slowing, stopping, or reversing the progression of NAFLD / MAFLD / MASLD or NASH, including reversing the progression to a point where the symptoms of NAFLD / MAFLD / MASLD or NASH are eliminated. It should be understood that treating a disease, condition, or symptom does not require the complete elimination of the disease, condition, symptom, or associated symptoms.

[0033] As used herein, the terms “prevent,” “preventing,” “prevention,” and “preventive treatment” refer to reducing or inhibiting the development of symptoms of diet-induced obesity, cardiometabolic disease, or cardiometabolic complications (including liver disease). For example, the terms “preventing,” “prevention,” and “preventive treatment” can refer to reducing the probability of developing symptoms of NAFLD / MAFLD / MASLD or NASH in a subject who does not have NAFLD / MAFLD / MASLD or NASH but is at risk or predisposed to developing NAFLD / MAFLD / MASLD or NASH. Therefore, in some implementations, GPR75 receptor antagonists may be administered prophylactically to prevent the onset of metabolic disorders or the recurrence of metabolic disorders (such as NAFLD / MAFLD / MASLD or NASH) in subjects.

[0034] As used herein, the “therapeutic effective amount” of a GPR75 antagonist generally refers to the amount of the agent necessary to elicit the desired biological response. For example, a therapeutic effective amount may be sufficient to prevent or treat NAFLD / MASLD / MAFLD or NASH and related symptoms. A “therapeutic effective amount” may also refer to, for example, an amount sufficient to reduce or improve the severity, duration, progression, or flare-up of symptoms associated with progression or stage of liver disease. The therapeutic effective amount of the agent can vary depending on factors such as the desired biological endpoint, the composition of the pharmaceutical composition, the target tissue or cells, and the health status of the subject being treated.

[0035] Methods for regulating the activity of GPR75 This disclosure provides methods for inhibiting the activity of GPR75 in a subject, the methods comprising administering an inhibitory amount of a GPR75 antagonist to the subject. These methods may be used to treat or prevent diet-induced obesity, cardiometabolic disorders, or cardiometabolic-related complications, including liver disease.

[0036] For example, in one embodiment, this disclosure relates to a method for treating or preventing liver disease (reflected herein as NAFLD / MAFLD / MASLD, NASH, or cirrhosis). This method is based on the understanding that activation of GPR75 can act as a stimulator of fatty acid uptake in the liver, which drives hepatic steatosis.

[0037] This disclosure also relates to methods, for example, for obtaining a less severe stage of disease in subjects with NAFLD / MAFLD / MASLD, NASH, or cirrhosis, and methods for delaying the progression of these liver disease stages by administering a GPR75 antagonist. In some embodiments, these methods are used to treat or prevent metabolic disorders such as NAFLD / MAFLD / MASLD or NASH. In one embodiment, the subject matter of this invention relates to a method for treating or preventing NAFLD / MAFLD / MASLD in subjects in need. The MASLD or NASH method involves administering a therapeutically effective amount of a GPR75 antagonist to the subject.

[0038] The “subject” in the method of the present invention can be a human or a non-human animal. Non-limiting examples of non-human animals that can be used in the method according to the present invention include companion animals, such as dogs or cats, as well as large animals, such as those used for livestock (e.g., sheep, cattle, pigs, llamas, buffalo, etc.) or wild animals, such as tigers, lions, elephants, etc.

[0039] "Subject" may include human subjects for medical purposes (such as for the treatment of NAFLD / MAFLD / MASLD or NASH or for preventive treatment to prevent the onset of NAFLD / MAFLD / MASLD or NASH), or animal subjects for medical, veterinary, or experimental purposes. Furthermore, "subject" may include patients who have or are suspected of having NAFLD / MAFLD / MASLD or NASH. Therefore, the terms "subject" and "patient" are used interchangeably herein.

[0040] As used in this article, “liver disease” means that the subject has symptoms associated with NAFLD / MAFLD / MASLD, NASH, or cirrhosis. Symptoms of NAFLD / MAFLD / MASLD may include fatigue, pain or discomfort in the right upper quadrant, and / or hepatomegaly. Clinical indicators include elevated liver enzyme levels (ALT, AST), increased liver fat visualized by CT or MRI, or confirmed steatosis / inflammation from a liver biopsy. Possible symptoms of NASH may include advanced scarring (cirrhosis), abdominal swelling (ascites), enlarged blood vessels just below the skin surface, splenomegaly, red palms, and yellowing of the skin and eyes (jaundice). In addition, 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 liver fat or liver fibrosis visualized by CT or MRI, or confirmed steatosis / inflammation / hepatocellular ballooning degeneration from a liver biopsy.

[0041] Treatment, administration, or therapy can be continuous or intermittent. Continuous treatment, administration, or therapy means treatment performed at least daily without interruption for one or more days. Intermittent treatment or administration, or treatment or administration in an intermittent manner, refers to treatment that is not continuous but rather periodic. Treatment according to the methods disclosed in this invention can lead to complete remission or cure of liver diseases (including NAFLD / MAFLD / MASLD, NASH, or cirrhosis), or partial improvement of one or more symptoms of these disease states / classes, which may be temporary or permanent.

[0042] In some embodiments, the subject matter disclosed herein also includes combination therapies. Additional therapeutic agents typically administered to treat or prevent, for example, NAFLD / MAFLD / MASLD, NASH, or cirrhosis, may be administered in combination with GPR75 antagonists as disclosed herein. For example, the GPR75 antagonist may optionally be administered in combination with other compounds (e.g., therapeutic agents) or treatments that can be used to treat liver disease and the development of NAFLD / MAFLD / MASLD, NASH, or cirrhosis. These additional agents may be administered separately from pharmaceutical compositions or formulations comprising GPR75 antagonists as part of a multi-dose regimen. Alternatively, these agents may be part of a single dosage form, mixed together with the GPR75 antagonist in a single composition.

[0043] "In combination with" means that a GPR75 antagonist is used in conjunction with, sequentially with, or in combination with one or more therapeutic agents. Therefore, a combination of a GPR75 antagonist and one or more therapeutic agents can be administered to a subject at the same time (i.e., simultaneously) or at different times (i.e., sequentially, in any order, on the same day or on different days), provided that the combined effect of the two agents is achieved in the subject. When a GPR75 antagonist and one or more therapeutic agents are administered concurrently, they can be administered to the subject as separate pharmaceutical compositions or formulations each containing a GPR75 antagonist or one or more therapeutic agents, or as a single pharmaceutical composition or formulation containing both agents.

[0044] When administered in combination, the effective concentration of each agent in the combination that elicits a specific biological response can be lower than the effective concentration of each agent when administered alone, thus allowing for a reduction in the dose of one or more agents relative to the dose required when the agent is administered as a single agent. The effects of multiple agents can, but do not necessarily, be additive or synergistic. Agents can 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 subsequent agents.

[0045] GPR75 antagonists can be administered using a variety of methods known in the art. More specifically, GPR75 antagonists can be administered via any suitable route of administration, including methods of administration such as local, parenteral, intramuscular, intravenous, nasal, oral, transdermal, mucosal, and subcutaneous, or other delivery methods known in the art.

[0046] The actual dose level of a GPR75 antagonist can be varied to obtain an amount of active ingredient that is effective in achieving the desired therapeutic response in a specific subject without toxicity to the subject. The chosen dose level will depend on a variety of factors, including route of administration, time of administration, excretion rate, duration of treatment, other medications used in combination with the GPR75 antagonist, the patient's age, sex, weight, condition, general health status, and prior medical history, as well as similar factors well known in the medical field.

[0047] Physicians with common skills in the field can easily identify and prescribe treatments (such as those for NAFLD / The effective amount of a given GPR75 antagonist or a pharmaceutical composition or formulation containing a GPR75 antagonist required for NAFLD / MASLD or NASH. For example, a physician may start with a dose of GPR75 antagonist lower than the dose required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved. Therefore, the range of doses administered will be adjusted by the physician as needed. It should be understood that the amount of GPR75 antagonist required to achieve the desired biological response (e.g., treatment or prevention of NAFLD / MAFLD / MASLD or NASH) may differ from the amount of GPR75 antagonist effective for another purpose.

[0048] Typically, the appropriate daily dose of a GPR75 antagonist will be the minimum dose required to produce an effective therapeutic effect. This effective dose usually depends on the factors mentioned above. The effective dose can often be determined based on the weight of the subject to be treated. If necessary, the effective daily dose of a GPR75 antagonist can be administered as one, two, three, four, five, six, or more sub-dose administered individually at appropriate intervals throughout the day, optionally in unit dosage form.

[0049] The application of GPR75 antagonists can produce several effects. For example, the application of GPR75 antagonists can: Protection: Weight loss caused by impaired lipid metabolism, muscle catabolism, or metabolic imbalance; Prevent changes in intracellular calcium levels; 20-HETE-GPR75-dependent activation inhibits fatty acid uptake; Reduce the accumulation of lipotoxic substances that lead to liver disease; Reduce cellular stress or prevent progression to liver fibrosis or cirrhosis; Disruption of 20-HETE-GPR75-dependent signaling reduces lipid-induced hepatotoxicity; Prevents liver cell damage, inflammation, fibrosis, cirrhosis, or liver failure; To eliminate changes in weight or body fat percentage, or to reduce WAT fat cell hypertrophy; To prevent adverse structural changes in the adipose tissue or liver of the subjects; Inhibiting the early stages of hepatic steatosis; Reversing changes in adipocyte size / hypertrophy or hepatic steatosis; and / or To prevent or reverse the progression of liver disease or metabolic-related changes that contribute to liver damage, such as excessive weight gain, insulin resistance, steatosis, and cellular inflammation / hypertrophy.

[0050] GPR75 antagonist The method disclosed herein employs a GPR75 antagonist. In some embodiments, the GPR75 antagonist is a small molecule or an antibody.

[0051] A variety of established methods well known to those skilled in the art can be used to identify GPR75 antagonists. For example, animal models can be used to evaluate the efficacy of potential antagonists, where fatty acid uptake is assessed using a range of advanced techniques. These include: histological staining for observing lipid accumulation; in situ hybridization for detecting mRNA expression of key lipid transporters; flow cytometry for quantifying cellular uptake of labeled fatty acids; and immunohistochemistry for locating and quantifying proteins involved in fatty acid metabolism. Additionally, bioassays including radiolabeled / fluorescent fatty acid uptake assays, lipidomics analysis, and colorimetric / fluorescent lipid quantification using human or animal samples can be used to evaluate changes in liver lipid accumulation / content.

[0052] In some instances, the amounts of fatty acid uptake and content in test animals exposed to or treated with the test compound (i.e., a possible GPR75 antagonist) are compared with those of a control. A “compound” or “test compound” is any substance or any combination of substances that can be used to achieve a target or result. Any compound with the potential to be modulated by inhibiting GPR75 can be tested using the methods of this disclosure.

[0053] 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. Suitable test compounds may be included in libraries, such as synthetic or natural compounds in combinatorial libraries. Many libraries are commercially available or readily generated; methods for the random and directed synthesis of a wide variety of organic compounds and biomolecules are also known. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are available or readily generated. Furthermore, naturally or synthetically generated libraries and compounds are readily modified using conventional chemical, physical, and biochemical methods and can be used to generate combinatorial libraries. Such libraries can be used to screen a large number of different compounds to identify GPR75 antagonists.

[0054] In screening methods, the amount of fatty acid uptake in the liver can be measured by analyzing test subjects or animals. Methods for detecting fatty acid uptake include, for example, QBT fatty acid uptake assays (Molecular Devices) or intraoperative fatty acid uptake using radiolabeled fatty acids (palmitates) in liver biopsies. A reduction in fatty acid uptake in the presence of one or more test compounds (e.g., a reduction of at least about 10%, about 20%, about 50%, about 80%, about 90%, or up to about 1 / 1.5, about 1 / 2, about 1 / 3, about 1 / 5, about 1 / 10, or less) compared to the absence of one or more test compounds indicates that the compound acts as a GPR75 antagonist to reduce fatty acid uptake.

[0055] GPR75 antagonists that can be used in this invention include, for example, compounds of formula (I) or (II): (I) (II), and physiologically acceptable salts, of which: R2 and R3 are each independently OH, C1-C3 alkyl, F or H; R4 is a C1-C3 alkyl group, 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 R1 is CO2H, NR8R9, C(O)R6, or tetrazolium, where: R6 represents OR7, NR8R9, D- / L- / D,L-α-amino acids (MW<250), and -NHS(O)2R. 10Polyethylene glycol (MW < 350) or its alkyl ethers, glycerol, monoglycerides or diglycerides (MW < 800) or isosteric or analogous carboxylic acid esters selected from the group consisting of: -P(O)(OH)2, -S(O)2OH, , in: The lower alkyl groups are C1-C6 alkyl groups or cycloalkyl groups; R7 is a C1-C6 alkyl or cycloalkyl, or benzyl; R8 and R9 are each independently H, C1-C6 alkyl or cycloalkyl, or benzyl, or R8 and R9 together with nitrogen form a 3-7 membered ring; and R 10 It is phenyl, C1-C5 alkyl or cycloalkyl, or CF3. The premise is: (i) At least one of R2, R3 and R4 is 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.

[0056] In some embodiments of compounds of formula (I) or (II), R1 is CO2H or C(O)R6, wherein R6 is a D- / L- / D,L-α-amino acid (MW < 250) or polyethylene glycol (MW < 350) or an alkyl ether thereof. In some embodiments, the D- / L- / D,L-α-amino acid (MW < 250) is glycine. or aspartic acid In some embodiments, polyethylene glycol (MW < 350) or its alkyl ether is... .

[0057] In some embodiments of the compound of formula (I) or (II), one of R2 and R3 is OH or F; one of R2 and R3 is H or -CH3; and R4 is H, -CH3 or CH2N3.

[0058] In some embodiments, the compound of formula (I) or (II) is a compound of the following formula:

[0059] , in It indicates a single bond or a double bond, provided there is at least one. It is a double bond.

[0060] In some embodiments, the compound of formula (I) or (II) is a compound of the following formula:

[0061] .

[0062] Non-limiting examples of GPR75 antagonists that can be used in this invention include: 20-hydroxyeicos ...

[0063] Other non-limiting examples of GPR75 antagonists that can be used as compounds of formula (I) or (II) or their physiologically acceptable salts include:

[0064]

[0065]

[0066]

[0067]

[0068]

[0069]

[0070]

[0071]

[0072]

[0073]

[0074] .

[0075] Compounds of formula (I) or (II) can be prepared by methods known to those skilled in the art. For example, methods for preparing selected compounds of formula (I) or (II) can be found in WO 2017 / 156164 A1, which is incorporated herein by reference in its entirety.

[0076] Some of the GPR75 antagonists disclosed herein have asymmetric carbon atoms (optical or chiral centers) and / or double bonds. Unless otherwise stated herein, the structures described herein are intended to include all stereochemical forms of the structure, such as the R and S configurations (D and L configurations of amino acids) for each asymmetric center. Optically active (R)- and (S)- or (D)- and (L)- isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. When the GPR75 antagonists of this disclosure contain olefinic bonds or other geometrically asymmetric centers, and unless otherwise specified, it is desirable for the compounds to contain both the E geometric isomer and the Z geometric isomer.

[0077] Some of the GPR75 antagonists disclosed herein can exist in tautomeric forms. Therefore, this disclosure also relates to the tautomeric forms of the GPR75 antagonists disclosed herein.

[0078] 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.

[0079] Physiologically acceptable acid addition salts of GPR75 antagonists can be prepared from inorganic and organic acids. Salts derived from inorganic acids include, but are not limited to, hydrochlorides, hydrobroms, sulfates, nitrates, and phosphates. Salts derived from organic acids include, but are not limited to, acetates, propionates, glycolates, pyruvates, oxalates, malates, malonates, succinates, glutarates, maleates, fumarates, tartrates, citrates, benzoates, cinnamates, mandelates, methanesulfonates, ethanesulfonates, p-toluenesulfonates, and salicylates.

[0080] In some embodiments of the GPR75 antagonists disclosed herein, the physiologically acceptable salt is a sodium, potassium, lithium, calcium, magnesium, or ammonium salt. In some embodiments of the GPR75 antagonists disclosed herein, the physiologically acceptable salt is a sodium salt.

[0081] Other examples of GPR75 antagonists include antibody molecules. As used herein, "antibody molecule" is intended to include intact antibodies, such as polyclonal antibodies or monoclonal antibodies (mAbs), and their proteolytic fragments, such as Fab or F(ab')2 fragments, chimeric antibodies, nanobodies, recombinant and engineered antibodies, single-chain antibodies and fragments thereof, and other molecules having at least one GPR75 antigen-binding site. In one embodiment, an antibody molecule that binds to GPR75 can be used to inhibit the activity of GPR75 in NAFLD / MAFLD / MASLD or NASH subjects.

[0082] Pharmaceutical compositions, formulations, dosage forms, drugs, and reagent kits The GPR75 antagonist that can be used in the disclosed methods may be included in the pharmaceutical composition or formulation.

[0083] The pharmaceutical compositions and formulations disclosed herein include pharmaceutical compositions comprising a GPR75 antagonist, alone or in combination with one or more other therapeutic agents, and a physiologically compatible carrier, excipient, or stabilizer. Such GPR75 antagonists may comprise small molecules that inhibit GPR75 activity. The pharmaceutical compositions and formulations may be administered to subjects, such as human subjects, for therapeutic or preventative treatment, such as for the treatment or prevention of diet-induced obesity, cardiometabolic disease, or cardiometabolic complications.

[0084] In some embodiments, pharmaceutical compositions and formulations comprising a GPR75 receptor antagonist that reduces fatty acid uptake are provided. As demonstrated herein, such inhibition of GPR75 can lead to a reduction in fatty acid uptake. Administration of such compositions and formulations is intended to inhibit the activity of GPR75 in a subject to be treated, thereby inhibiting fatty acid uptake.

[0085] In some embodiments, the pharmaceutical compositions and formulations are used to treat cardiometabolic liver diseases that cause NAFLD / MAFLD / MASLD, NASH, or cirrhosis phenotypes. In some embodiments, the pharmaceutical compositions and formulations are used to treat or prevent NAFLD / MAFLD / MASLD or NASH and can be administered to subjects, such as human subjects, for therapeutic or prophylactic treatment of NAFLD / MAFLD / MASLD or NASH. In a particular embodiment, the GPR75 antagonist is disodium N-succinate-20-hydroxyeicosicocarbon-6(Z),15(Z)-dienecarboxamide (AAA).

[0086] As used herein, "physiologically compatible carrier, excipient, or stabilizer" means an "acceptable" carrier, excipient, or stabilizer, i.e., compatible with the other components of the composition and harmless (e.g., non-toxic) to its recipient. Physiologically compatible carrier, excipient, or stabilizer may refer to physiologically acceptable diluents, including but not limited to water, phosphate-buffered saline, or brine, and in some embodiments, may include adjuvants.

[0087] Acceptable carriers, excipients, and stabilizers are non-toxic to recipients at the employed doses and concentrations and may include buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid, BHA, and BHT; low molecular weight (less than about 10 residues) peptides; 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 dextran; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as Tween, Pluronics, or PEG. Adjuvants suitable for the compositions disclosed in this invention include adjuvants known in the art, including but not limited to incomplete Freund's adjuvants, aluminum phosphate, aluminum hydroxide, and alum. The carriers, excipients, or stabilizers are known in the art and described in, for example, Remington: The Science and Practice of Pharmacy, 2000, Gennaro, AR ed., Eaton, Pa.: Mack Publishing Co. and Porter et al. ed., The Merck Manual, 19th edition, Merck and Co., Rahway, NJ, 2011, which are incorporated herein by reference in their entirety.

[0088] The pharmaceutical compositions and formulations disclosed in this invention can be administered using a variety of methods known in the art. More specifically, as described herein, GPR75 antagonists can be administered to subjects via any suitable route of administration to treat or prevent NAFLD / MAFLD / MASLD or NASH. These routes include oral, nasal, transmucosal, parenteral, intramuscular, subcutaneous, intramedullary injection, and intrathecal, direct intracardiac, intravenous, intra-articular, intrasternal, intrasynovial, intrahepatic, intralesional, intracranial, intraperitoneal, intranasal or intraocular injection, intracisional, local, such as by powder, ointment, including oral and sublingual, transdermal, by inhalation spray, or other delivery methods known in the art. Regardless of the chosen route of administration, GPR75 antagonists can be formulated into pharmaceutically acceptable dosage forms as described herein, or by other conventional methods known to those skilled in the art.

[0089] In some embodiments, the pharmaceutical compositions and formulations disclosed herein can be administered via rechargeable or biodegradable devices. For example, a variety of sustained-release polymer devices have been developed and controlled delivery of drugs has been tested in vivo. Suitable examples of sustained-release formulations include semi-permeable polymer matrices in the form of molded articles (e.g., membranes or microcapsules). Sustained-release matrices comprise polyesters, hydrogels, polylactide (US Patent No. 3,773,919; EP 58,481), copolymers of L-glutamic acid and γ-ethyl-L-glutamic acid esters (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., ibid.), or poly-D-(-)-3-hydroxybutyric acid (EP 133,988A). The sustained-release composition also comprises a liposome-embedded GPR75 antagonist, which can be prepared by methods known per se (Epstein et al., Proc. Natl. Acad. Sci. USA 82:3688, 1985; Hwang et al., Proc. Natl. Acad. Sci. USA 77:4030, 1980; U.S. Patents 4,485,045 and 4,544,545; and EP 102,324A). Typically, the liposomes are small (approximately 200–800 Å) monolayers containing a lipid content greater than approximately 30 mol% cholesterol, the selected proportions adjusted for optimal therapy. Such materials can be incorporated into implants, for example, for the sustained release of GPR75 antagonists.

[0090] The GPR75 antagonist that can be used in the disclosed method can be contained in nanoparticles.

[0091] In some embodiments, this disclosure provides nanoparticles comprising a GPR75 antagonist. The nanoparticles can be used to treat or prevent NAFLD / MAFLD / MASLD or NASH. These nanoparticles can be natural or synthetic. They can be produced from biomolecules or non-biomolecules. In some cases, a GPR75 antagonist is crosslinked with a polymer or lipid on the surface of the nanoparticle. In some embodiments, a GPR75 antagonist is adsorbed onto the surface of the nanoparticle. In some embodiments, a GPR75 antagonist is adsorbed onto the surface of the nanoparticle and then crosslinked to the surface of the nanoparticle. In some embodiments, a GPR75 antagonist is encapsulated within the nanoparticle.

[0092] In some embodiments, the nanoparticles are formed from biocompatible polymers. Examples of biocompatible polymers include polyethylene, polycarbonate, polyanhydride, polyhydroxy acid, polypropyl fumarate, polycaprolactone, polyamide, polyacetal, polyether, polyester, poly(orthoester), polycyanoacrylate, polyvinyl alcohol, polyurethane, polyphosphazene, polyacrylate, polymethacrylate, polycyanoacrylate, polyurea, polystyrene, or polyamine, or combinations thereof. In some embodiments, the nanoparticles are formed from polyethylene glycol (PEG), poly(lactide-co-glycolic acid) (PLGA), polyglycolic acid, poly-β-hydroxybutyrate, polyacrylate, or combinations thereof. In one embodiment, the nanoparticles are nanoliposomes. These nanoliposomes can be composed of phospholipids, such as 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-dispalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1,2-distearyl-sn-glycerol-3-phosphate-(1'-racemic-glycerol) (DSPG), 1,2-dispalmitoyl-sn-glycerol-3-phosphate-(1'-racemic-glycerol) (DPPG), and 1,2-dimyristoyl-sn-glycerol-3-phosphate-(1'-racemic-glycerol). (DMPG), 1,2-dioleoyl-sn-glycerol-3-phosphate-(1'-racemic-glycerol) (DOPG), dipalmitoylphosphatidylserine (DPPS), distearylphosphatidylserine (DSPS), dipalmitoylphosphatidylinositol (DPPI), distearylphosphatidylinositol (DSPI), dipalmitoylphosphatidic acid (DPPA), distearylphosphatidic acid (OSPA), 1,2-diacyl-3-trimethylammonium-propane (including but not limited to dioleoyl (DOTAP), 1,2-dipalmitoyl-sn-glycerol-3-phosphate ethanolamine-N-[methoxy(polyethylene glycol)-2000] (DPPE-PEG2000), 1,2-distearyl-sn-glycerol-3-phosphate ethanolamine-N-[methoxy(polyethylene glycol)-1000] (DSPE-PEG2000) and cholesterol.

[0093] In some embodiments, a crosslinking agent is used to coat the GPR75 antagonist onto the nanoparticles. In some embodiments, the GPR75 antagonist is adsorbed onto the surface of the nanoparticles. In some embodiments, the GPR75 antagonist is adsorbed onto the surface of the nanoparticles and then covalently crosslinked to the nanoparticle surface using a crosslinking agent.

[0094] Crosslinking agents suitable for crosslinking GPR75 antagonists to produce nanoparticles are known in the art and include those selected from the group consisting of: formaldehyde, formaldehyde derivatives, formalin, glutaraldehyde, glutaraldehyde derivatives, protein crosslinking agents, nucleic acid crosslinking agents, protein and nucleic acid crosslinking agents, primary amine reactive crosslinking agents, thiol reactive crosslinking agents, thiol addition or disulfide reduction, carbohydrate reactive crosslinking agents, carboxyl reactive crosslinking agents, photoreactive crosslinking agents, cleavable crosslinking agents, AEDP, APG, BASED, BM(PEO)3, BM(PEO)4, BMB, BMDB, BMH, BMOE, BS3, BSOCOES, DFDNB, DMA, DMP, DMS, DDPPB, DSG, DSP, DSS, DST, DTBP, DTME, DTSSP, EGS, HBVS, sulfonyl-BSOCOES, sulfonyl-DST, and sulfonyl-EGS.

[0095] In one embodiment, the nanoparticles are designed to target liver tissue. In this case, the nanoparticles may be coated with a liver-specific ligand that delivers the nanoparticles directly to the liver.

[0096] The subject matter of this invention also includes the use of GPR75 antagonists in the manufacture of medicaments for the treatment or prevention of diet-induced obesity, cardiometabolic diseases, or cardiometabolic complications (including liver disease). In some embodiments, the medicament is used to treat or prevent NAFLD / MAFLD / MASLD or NASH.

[0097] The GPR75 antagonist compositions disclosed in this invention can be assembled into kits or pharmaceutical systems for the treatment or prevention of diet-induced obesity, cardiometabolic diseases, or cardiometabolic-related complications, including liver disease. For example, the kits or pharmaceutical systems disclosed herein can be used to treat or prevent cardiometabolic-related liver diseases leading to NAFLD / MAFLD / MASLD, NASH, or the cirrhosis phenotype / liver dysfunction stage. In some embodiments, the kits or pharmaceutical systems disclosed herein comprise a unit dosage form of the GPR75 antagonist. In other embodiments, the GPR75 antagonist may be present with pharmaceutically acceptable solvents, carriers, excipients, etc., as described herein.

[0098] In some embodiments, the kits disclosed in this invention include one or more containers containing a GPR75 antagonist, including but not limited to vials, tubes, ampoules, bottles, etc. One or more containers may also be carried in a suitable carrier (such as a box, carton, tube, etc.). Such containers may be made of plastic, glass, laminated paper, metal foil, or other materials suitable for containing the drug.

[0099] The kits or pharmaceutical systems disclosed in this invention may also include instructions for using compositions containing a GPR75 antagonist to treat or prevent, for example, NAFLD / MAFLD / MASLD or NASH. In some embodiments, the instructions include one or more of the following: a description of the GPR75 antagonist; a dosage regimen; and instructions for administration for the treatment or prevention of NAFLD / MAFLD / MASLD or NASH; precautions; warnings; indications; contraindications; overdose information; adverse reactions; animal pharmacology; clinical studies; and references. In some embodiments, the instructions are for the treatment of diabetes. The instructions may be printed directly on the container (if present), as a label applied to the container, or as separate sheets of paper, brochures, cards, or folders provided in or with the container.

[0100] Embodiments of the present invention Specific embodiments of the present invention include, but are not limited to, the following: Implementation Scheme 1. A method for treating or preventing diet-induced obesity, cardiometabolic disease, or cardiometabolic-related complications in a subject, the method comprising administering a therapeutically effective amount of a GPR75 antagonist to the subject.

[0101] Implementation Scheme 2. The method of Implementation Scheme 1, wherein the administration of the GPR75 antagonist protects against weight loss due to impaired lipid metabolism, muscle catabolism, or metabolic imbalance.

[0102] Implementation Scheme 3. The method as described in Implementation Scheme 1, wherein the administration of the GPR75 antagonist prevents changes in intracellular calcium levels.

[0103] Implementation Scheme 4. The method of Implementation Scheme 1, wherein the application of the GPR75 antagonist inhibits 20-HETE-GPR75-dependent activation of fatty acid uptake.

[0104] Implementation Scheme 5. The method as described in Implementation Scheme 1, wherein the administration of the GPR75 antagonist reduces the accumulation of lipotoxic substances leading to liver disease.

[0105] Implementation Scheme 6. The method of Implementation Scheme 1, wherein the administration of the GPR75 antagonist reduces cellular stress or prevents progression to liver fibrosis or cirrhosis.

[0106] Implementation Scheme 7. The method of Implementation Scheme 1, wherein the administration of the GPR75 antagonist disrupts 20-HETE-GPR75-dependent signaling, thereby mitigating lipid-induced hepatotoxicity.

[0107] Implementation Scheme 8. The method of Implementation Scheme 1, wherein the administration of the GPR75 antagonist prevents hepatocellular damage, inflammation, fibrosis, cirrhosis, or liver failure.

[0108] Implementation Scheme 9. The method of Implementation Scheme 1, wherein the application of the GPR75 antagonist eliminates changes in body weight or fat content, or reduces WAT adipocyte hypertrophy.

[0109] Implementation Scheme 10. The method of Implementation Scheme 1, wherein the administration of the GPR75 antagonist prevents adverse structural changes in the subject's adipose tissue or liver.

[0110] Implementation Scheme 11. The method of Implementation Scheme 1, wherein the administration of the GPR75 antagonist inhibits the early stages of hepatic steatosis.

[0111] Implementation Scheme 12. The method of Implementation Scheme 1, wherein the administration of the GPR75 antagonist reverses changes in adipocyte size / hypertrophy or hepatic steatosis.

[0112] Implementation Scheme 13. The method of Implementation Scheme 1, wherein the administration of the GPR75 antagonist prevents or reverses metabolic-related changes that contribute to the progression of liver disease or liver injury.

[0113] Implementation Scheme 14. The method as described in Implementation Scheme 13, wherein the metabolic-related changes that contribute to liver injury include one or more of excessive weight gain, insulin resistance, steatosis, and cellular inflammation / hypertrophy.

[0114] Implementation Scheme 15. The method described in Implementation Scheme 1 is used for the treatment or prevention of non-alcoholic fatty liver disease (NAFLD), which may alternatively be referred to as metabolic dysfunction-associated steatosis / steatohepatitis (MAFLD / MASLD).

[0115] Implementation Scheme 16. The method described in Implementation Scheme 1 is used for the treatment or prevention of non-alcoholic steatohepatitis (NASH).

[0116] Implementation Scheme 17. The method of any one of Implementation Schemes 1 to 16, wherein the GPR75 antagonist is a small molecule.

[0117] Implementation Scheme 18. The method of any one of Implementation Schemes 1 to 17, wherein the GPR75 antagonist is a compound of formula (I) or (II): (I) (II), Or its physiologically acceptable salt, wherein: R2 and R3 are each independently OH, C1-C3 alkyl, F or H; R4 is a C1-C3 alkyl group, 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 R1 is CO2H, NR8R9, C(O)R6, or tetrazolium, where: R6 represents OR7, NR8R9, D- / L- / D,L-α-amino acids (MW<250), and -NHS(O)2R. 10 Polyethylene glycol (MW < 350) or its alkyl ethers, glycerol, monoglycerides or diglycerides (MW < 800) or isosteric or analogous carboxylic acid esters selected from the group consisting of: -P(O)(OH)2, -S(O)2OH, , in: The lower alkyl groups are C1-C6 alkyl groups or cycloalkyl groups; R7 is a C1-C6 alkyl or cycloalkyl, or benzyl; R8 and R9 are each independently H, C1-C6 alkyl or cycloalkyl, or benzyl, or R8 and R9 together with nitrogen form a 3-7 membered ring; and R 10 It is phenyl, C1-C5 alkyl or cycloalkyl, or -CF3. The premise is: (i) At least one of R2, R3 and R4 is 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.

[0118] Implementation Scheme 19. The method of Implementation Scheme 18, wherein the GPR75 antagonist is a compound of formula (I) or (II), wherein R1 is CO2H, C(O)R6, wherein R6 is a D- / L- / D,L-α-amino acid (MW<250) or polyethylene glycol (MW<350) or its alkyl ether, tetrazolium or , Or a physiologically acceptable salt.

[0119] Implementation Scheme 20. The method as described in Implementation Scheme 18 or 19, wherein the GPR75 antagonist is a compound of formula (I) or (II), wherein one of R2 and R3 is OH or F; one of R2 and R3 is H or -CH3; and R4 is H, -CH3 or CH2N3, or a physiologically acceptable salt thereof.

[0120] Implementation Scheme 21. The method of any one of Implementation Schemes 18 to 20, wherein the GPR75 antagonist is a compound having the following structure: (I) or (II)

[0121] , in It indicates a single bond or a double bond, provided there is at least one. It is a double bond, or a physiologically acceptable salt thereof.

[0122] Implementation Scheme 22. The method of any one of Implementation Schemes 18 to 21, wherein the GPR75 antagonist is a compound having the following structure: (I) or (II)

[0123] , Or a physiologically acceptable salt.

[0124] Implementation Scheme 23. The method of any one of Implementation Schemes 18 to 22, wherein the GPR75 antagonist is a compound of formula (I) or (II), wherein R1 is CO2H or C(O)R6, wherein R6 is glycine. Aspartic acid or or a physiologically acceptable salt thereof.

[0125] Implementation Scheme 24. The method of any one of Implementation Schemes 1 to 22, wherein the GPR75 antagonist is:

[0126]

[0127]

[0128]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136] .

[0137] Implementation Scheme 25. The method of any one of Implementation Schemes 1 to 22, wherein the GPR75 antagonist is 20-hydroxyeicos ...

[0138] Implementation Scheme 26. The method of any one of Implementation Schemes 1 to 22, wherein the GPR75 antagonist is disodium N-succinate-20-hydroxyeicosicocarbon-6(Z),15(Z)-dienecarboxamide (AAA).

[0139] Implementation Scheme 27. The method of any one of Implementation Schemes 1 to 16, wherein the GPR75 antagonist is an antibody.

[0140] Implementation Scheme 28. The method of any one of Implementation Schemes 1 to 27, further comprising administering to the subject a second therapeutic agent for treating liver disease categories including NAFLD / MAFLD / MASLD, NASH, or cirrhosis.

[0141] Implementation Scheme 29. A pharmaceutical composition for treating or preventing diet-induced obesity, cardiometabolic disease, or cardiometabolic-related complications, said pharmaceutical composition comprising a GPR75 antagonist and a physiologically compatible carrier, excipient, or stabilizer.

[0142] Implementation Scheme 30. The pharmaceutical composition of Implementation Scheme 29, wherein the GPR75 antagonist is defined as in any one of Implementation Schemes 17 to 27.

[0143] Implementation Scheme 31. The pharmaceutical composition as described in Implementation Scheme 29 or 30, further comprising one or more additional therapeutic agents.

[0144] Implementation Scheme 32. The pharmaceutical composition of any one of Implementation Schemes 29 to 31, for the treatment or prevention of cardiometabolic liver diseases resulting in NAFLD / MAFLD / MASLD, NASH, or cirrhosis phenotypes.

[0145] Implementation Scheme 33. The pharmaceutical composition as described in any one of Implementation Schemes 29 to 31, for the treatment or prevention of NAFLD / MAFLD / MASLD or NASH.

[0146] Implementation Plan 34. Use of GPR75 antagonists in the manufacture of medicines for the treatment or prevention of diet-induced obesity, cardiometabolic diseases, or cardiometabolic complications.

[0147] Implementation Scheme 35. Use as described in Implementation Scheme 34, wherein the drug is used to treat or prevent NAFLD / MAFLD / MASLD or NASH.

[0148] Implementation Scheme 36. Use as described in Implementation Scheme 34, wherein the drug is used to treat or prevent liver disease.

[0149] Implementation Scheme 37. Use as described in any one of Implementation Schemes 34 to 36, wherein the GPR75 antagonist is defined as in any one of Implementation Schemes 17 to 27.

[0150] Implementation Scheme 38. A kit comprising: (i) a GPR75 antagonist; and (ii) instructions for using said GPR75 antagonist to treat or prevent diet-induced obesity, cardiometabolic disease, or cardiometabolic-related complications.

[0151] Implementation Scheme 39. The kit as described in Implementation Scheme 38, wherein the description includes one or more of the following: (i) a description of the GPR75 antagonist; (ii) a dosage regimen; or (iii) instructions for administering the GPR75 antagonist to treat or prevent NAFLD / Explanation of MAFLD / MASLD or NASH.

[0152] Implementation Scheme 40. The kit as described in Implementation Scheme 38, wherein the instructions are for the treatment of diabetes.

[0153] Implementation Scheme 41. The kit as described in Implementation Scheme 38, wherein the description is for the treatment or prevention of cardiometabolic liver disease leading to NAFLD / MAFLD / MASLD, NASH, or the cirrhosis phenotype / liver dysfunction stage.

[0154] Implementation Scheme 42. The kit as described in any one of Implementation Schemes 38 to 41, wherein the GPR75 antagonist is as defined in any one of Implementation Schemes 17 to 27.

[0155] Although this disclosure and its advantages have been described in detail, it should be understood that various changes, substitutions and alternatives may be made herein without departing from the spirit and scope of the invention as defined in the claims.

[0156] This disclosure will be further illustrated in the following embodiments, which are given for illustrative purposes only and are not intended to limit the invention in any way. Example

[0157] A GPR75 defective transgenic mouse strain was developed (GPR75) - / - Whole knockout (KO) mice were used, and dominant 20-HETE synthase Cyp4a12 could be induced by administration of doxycycline (DOX) to drinking water to overproduce 20-HETE, thereby establishing Cyp4a12-GPR75. - / - Mouse model. This mouse model demonstrates that GPR75-deficient mice are protected from diet-induced obesity and liver injury. HTLA cells (HEK293 cell line stably expressing the tTA-dependent luciferase reporter gene and β-repressor 2-TEV fusion gene) [RRID: Addgene_66372] coupled with a GPR75-specific PRESTO-Tango overexpression construct, endothelial cell line EA.hy926 cells, and hepatocyte line Hep G2 cells served as in vitro tools for evaluating intracellular calcium measurements using the FLIPR Calcium 6 assay kit according to the manufacturer's protocol. Additionally, the human hepatocyte line Hep G2 was developed into a GPR75-deficient cell line via CRISPR-Cas9 with a knockdown efficiency of 90+%.

[0158] Figure 1A -B demonstrates the efficacy of Cyp4a12-GPR75 in the presence and absence of doxycycline (DOX). + / + Or Cyp4a12-GPR75 - / - Systolic blood pressure in mice (28 weeks) in response to control diet (CD) or high-fat diet (HFD) (28 weeks) Figure 1A ) or weight ( Figure 1B Variation of ) . Mean ± SEM (n=4-8). p<0.05, p<0.0001. Figure 2Representative microCT images of mice from baseline (week 0) and those fed CD or HFD for 28 weeks are presented, with or without DOX. (n=4–6). Visceral fat and subcutaneous fat are outlined. Figure 3 Representative histological images (H&E staining) of liver sections from mice subjected to CD or HFD for 28 weeks in the presence and absence of DOX are depicted (n=4–6). Overall, these data demonstrate the contributions of HFD and 20-HETE to the pathogenesis of hepatic steatosis and liver disease, and how GPR75 deficiency protects against various cardiometabolic parameters, including liver disease. Figure 4 illustrates how GPR75- / - mice are protected from weight loss, reduced activity, and liver injury associated with a methionine-choline-deficient diet, suggesting that pharmacological blockade of GPR75 can lead to equivalent levels of protection against HFD- or MCD-induced liver disease. Figure 5A The effect of N-succinate-20-hydroxyeicosicocarbon-6(Z),15(Z)-dienecarboxamide disodium (AAA) on 20-HETE-GPR75-mediated intracellular calcium changes was demonstrated, thus showing the calculated IC50 response of AAA as a competitive antagonist in the endothelial cell line EA.hy926. Although Figure 5A The free acid form of AAA was described, but N-succinicotinic acid-20-hydroxyeicosicocarbon-6(Z),15(Z)-dienecarbamate disodium (i.e., AAA) was used. Figure 5B -D highlights the ability of 20-HETE to drive intracellular calcium changes via GPR75 in the hepatocyte line HEPG2, a hallmark of GPR75-mediated Gq receptor activation. Furthermore, these data demonstrate that AAA can disrupt intracellular calcium changes observed via the 20-HETE-GPR75 axis. Figure 6A This study demonstrates the changes in fatty acid uptake in HEPG2 cells in response to mediators (ethanol) and 20-HETE (10 nM) in the presence and absence of AAA (10 nM) using the QBT fatty acid uptake kit. Figure 6B The effects of lipofermatta (a specific FATP2 blocker) (10 μM) on 20-HETE-driven fatty acid uptake in WT cells were demonstrated. Figure 6C The changes in fatty acid uptake in 20HR KD cells exposed to the mediator (ethanol) and 20-HETE (10 nM) are shown in the presence and absence of AAA (10 nM). Mean ± SEM (n=8). p < 0.0001. These data demonstrate that 20-HETE drives increased fatty acid uptake in hepatocytes, a process dependent on the presence of its receptor GPR75 and the fatty acid transporter FATP2 / SLC27A2. Furthermore, AAA can disrupt this signaling cascade, thereby blocking the changes in fatty acid uptake driven by the 20-HETE-GPR75 axis. Figure 7 The results show the detection of fatty acid uptake and lipid droplet formation (BODIPY) in HEPG2 cells pretreated with mediators (ethanol), 20-HETE (10 nM), AAA (10 nM), 20-HETE+AAA, calcium phosphate protein C ((CC) PKC inhibitor (1 μM)), or 20-HETE+CC before continuous oleic acid (1 mM) supplementation. Green (BODIPY), blue (Hoechst), and red (segmented BODIPY signal (lipid droplets)) (n=8). Figure 8A The detection of fatty acid uptake and lipid droplet formation (BODIPY) in GPR75 knockdown / deficient HEPG2 cells pretreated with a medium (ethanol) and 20-HETE (10 nM) was depicted. Green (BODIPY), blue (Hoechst), and red (segmented BODIPY signal (lipid droplets)) (n=8). Figure 8B This is a plot (n=8) summarizing the 20-HETE and GPR75-dependent changes in fatty acid uptake as a measure of body surface area. The effects of 20-HETE on fatty acid uptake via GPR75 and FATP2 drive significant changes in hepatocytes, leading to increased lipid droplet formation to accommodate oleic acid influx. These data support the role of GPR75-PKC as a key driver of fatty acid uptake and demonstrate the ability of AAA to inhibit molecular programming associated with the pairing of 20-HETE and GPR75. Figure 9A -C describes the effect of AAA in preventing diet-induced weight gain ( Figure 9A -B) and subsequent changes in adipocyte size and early hepatic steatosis ( Figure 9C Proficiency in ) aspect. Figure 10A -C demonstrates AAA's ability to reverse weight changes, glucose mismanagement, and adipose tissue volume associated with high-fat feeding programs. Figure 10D This highlights the ability of AAA to restore adipocyte size and hepatic steatosis. These mouse studies demonstrate that AAA can prevent and reverse diet-induced cardiometabolic changes that contribute to the development and pathogenesis of hepatic steatosis and liver disease.

Claims

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

2. The method of claim 1, used for the treatment or prevention of non-alcoholic fatty liver disease (NAFLD), alternatively referred to as metabolic dysfunction-associated steatosis / steatohepatitis (MAFLD / MASLD).

3. The method of claim 1, used for the treatment or prevention of non-alcoholic steatohepatitis (NASH).

4. The method of claim 1, wherein the GPR75 antagonist is applied: (i) Protection: Weight loss due to impaired lipid metabolism, muscle catabolism, or metabolic imbalance; (ii) Prevent changes in intracellular calcium levels; (iii) 20-HETE-GPR75-dependent activation that inhibits fatty acid uptake; (iv) Reduce the accumulation of lipotoxic substances that lead to liver disease; (v) Reduce cellular stress or prevent progression to liver fibrosis or cirrhosis; (vi) Disruption of 20-HETE-GPR75-dependent signaling, thereby reducing lipid-induced hepatotoxicity; (vii) Prevent liver cell damage, inflammation, fibrosis, cirrhosis, or liver failure; (viii) Eliminate changes in body weight or fat content, or reduce WAT fat cell hypertrophy; (ix) To prevent adverse structural changes in the adipose tissue or liver of the subjects; (x) inhibits the early stages of hepatic steatosis; (xi) Reversing changes in adipocyte size / hypertrophy or hepatic steatosis; and / or (xii) To prevent or reverse the progression of liver disease or metabolic-related changes that contribute to liver injury.

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 formula (I) or (II). (I) (II), Or its physiologically acceptable salt, wherein: R2 and R3 are each independently OH, C1-C3 alkyl, F or H; R4 is a C1-C3 alkyl group, 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 R1 is CO2H, NR8R9, C(O)R6, or tetrazolium, where: R6 represents OR7, NR8R9, D- / L- / D,L-α-amino acids (MW<250), and -NHS(O)2R. 10 Polyethylene glycol (MW < 350) or its alkyl ethers, glycerol, monoglycerides or diglycerides (MW < 800) or isosteric or analogous carboxylic acid esters selected from the group consisting of: -P(O)(OH)2, -S(O)2OH, ， in: The lower alkyl groups are C1-C6 alkyl groups or cycloalkyl groups; R7 is a C1-C6 alkyl or cycloalkyl, or benzyl; R8 and R9 are each independently H, C1-C6 alkyl or cycloalkyl, or benzyl, or R8 and R9 together with nitrogen form 3-7 membered rings; and R 10 It is phenyl, C1-C5 alkyl or cycloalkyl, or -CF3. The premise is: (i) At least one of R2, R3 and R4 is 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 formula (I) or (II) having the following structure: in: It indicates a single bond or a double bond, provided there is at least one. It is a double bond; R1 is CO2H or C(O)R6, where R6 is a D- / L- / D,L-α-amino acid (MW<250) or polyethylene glycol (MW<350) or its alkyl ether, tetrazolium or , One of R2 and R3 is OH or F; One of R2 and R3 is H or -CH3; and R4 can be H, -CH3, or CH2N3. Or a physiologically acceptable salt.

8. The method of any one of claims 1 to 7, wherein the GPR75 antagonist is: 。 9. The method according to any one of claims 1 to 7, wherein the GPR75 antagonist is 20-hydroxyeicos ...

10. The method according to any one of claims 1 to 7, wherein the GPR75 antagonist is disodium N-succinate-20-hydroxyeicosicocarbon-6(Z),15(Z)-dienecarboxamide (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 administering to the subject an antidote for treating NAFLD / MAFLD / Secondary treatment for liver diseases including MASLD, NASH, or cirrhosis.

13. A pharmaceutical composition for treating or preventing diet-induced obesity, cardiometabolic disease, or cardiometabolic-related complications, said 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 GPR75 antagonists in the manufacture of medicines for the treatment or prevention of diet-induced obesity, cardiometabolic diseases, or cardiometabolic complications.

16. The use as described in claim 16, wherein the drug is used to treat or prevent liver disease, preferably wherein the drug is used to treat or prevent NAFLD / MAFLD / MASLD or NASH.

17. A kit comprising: (i) a composition containing a GPR75 antagonist; and (ii) instructions for using the GPR75 antagonist to treat or prevent diet-induced obesity, cardiometabolic disease, or cardiometabolic-related complications.

18. The kit of claim 17, wherein the description is for the treatment of diabetes.

19. The kit of claim 17, wherein the description includes one or more of the following: (i) a description of the GPR75 antagonist; (ii) a dosage regimen; or (iii) instructions for administering the GPR75 antagonist to treat or prevent 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 defined in any one of claims 5 to 10.

Images