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WO2025106728A1 - Uses of lysophosphatidic acid receptor 1 antagonists - Google Patents

Uses of lysophosphatidic acid receptor 1 antagonists Download PDF

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WO2025106728A1
WO2025106728A1 PCT/US2024/055996 US2024055996W WO2025106728A1 WO 2025106728 A1 WO2025106728 A1 WO 2025106728A1 US 2024055996 W US2024055996 W US 2024055996W WO 2025106728 A1 WO2025106728 A1 WO 2025106728A1
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substituted
alkyl
group
epgn2154
liver
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Jashdeep Bhattacharjee
Rohit Kohli
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Saban Research Institute Children's Hospital Los Angeles
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Saban Research Institute Children's Hospital Los Angeles
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/42—Oxazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A61K31/366—Lactones having six-membered rings, e.g. delta-lactones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4192—1,2,3-Triazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/26—Glucagons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00

Definitions

  • BACKGROUND MASH metabolic dysfunction–associated steatohepatitis
  • NASH Nonalcoholic steatohepatitis
  • fatty liver disease a condition in which the liver builds up excessive fat deposits
  • This liver disease can worsen over time and bring potentially life-threatening consequences. With timely screening, detection, and management, it may be possible to stop or even reverse liver damage from MASH.
  • the additional active agent is one or more a glucagon-like peptide-1 receptor agonist(s), including, by not limited to, semaglutide, tirzepatide, dulaglutide, exenatide, liraglutide, lixisenatide, and/or albiglutide, and/or one or more lipid lowering agent.
  • the lipid lowering agent is a cholesterol absorption inhibitor, a statin, a PCSK9 inhibitor, an ACC inhibitor, an ApoC-III inhibitor, an ACL-inhibitor, fish oil, or a CETP inhibitor.
  • the statin is atorvastatin, rosuvastatin, simvastatin, pravastatin, lovastatin, fluvastatin, or pitavastatin.
  • the hepatic steatosis is non-alcoholic fatty liver disease (NAFLD) or metabolic dysfunction-associated steatohepatitis (MASH).
  • the additional active agent is one or more a glucagon-like peptide-1 receptor agonist(s), including, by not limited to, semaglutide, tirzepatide, dulaglutide, exenatide, liraglutide, lixisenatide, and/or albiglutide, and/or one or more lipid lowering agent.
  • the lipid lowering agent is a cholesterol absorption inhibitor, a statin, a PCSK9 inhibitor, an ACC inhibitor, an ApoC-III inhibitor, an ACL-inhibitor, fish oil, or a CETP inhibitor.
  • the statin is atorvastatin, rosuvastatin, simvastatin, pravastatin, lovastatin, fluvastatin, or pitavastatin.
  • the subject has liver disease.
  • the subject has hepatic steatosis, type IIb hyperlipidemia or familial combined hyperlipidemia.
  • the hepatic steatosis is non-alcoholic fatty liver disease (NAFLD) or metabolic dysfunction-associated steatohepatitis (MASH).
  • the subject's risk of developing liver disease is reduced.
  • the liver disease is metabolic dysfunction-associated steatohepatitis (MASH), or nonalcoholic fatty liver disease (NAFLD), alcoholic hepatic steatosis or primary biliary cirrhosis.
  • MASH metabolic dysfunction-associated steatohepatitis
  • NAFLD nonalcoholic fatty liver disease
  • alcoholic hepatic steatosis or primary biliary cirrhosis.
  • the additional active agent is one or more a glucagon- like peptide-1 receptor agonist(s), including, by not limited to, semaglutide, tirzepatide, dulaglutide, exenatide, liraglutide, lixisenatide, and/or albiglutide, and/or one or more lipid lowering agent.
  • the lipid lowering agent is a cholesterol absorption inhibitor, a statin, a PCSK9 inhibitor, an ACC inhibitor, an ApoC-III inhibitor, an ACL-inhibitor, fish oil, or a CETP inhibitor.
  • the statin is atorvastatin, rosuvastatin, simvastatin, pravastatin, lovastatin, fluvastatin, or pitavastatin.
  • the LPAR1 antagonist can be EPGN696 and/or EPGN2154.
  • the combination is administered daily or weekly. In some aspects, about 5 mg/kg to about 10 mg/kg body weight of the LPAR1 antagonist is administered. BRIEF DESCRIPTION OF THE FIGURES. 1A-1E. 1 EPGN2154 (WO 2019/246109 and US Pat No. 10,000459; incorporated herein by reference) and Sema combination therapy cause the maximum reduction in body weight.
  • EPGN2154 pharmacokinetics mean plasma concentration of EPGN2154 oral gavage (PO: 20 mg/kg) versus i.v. (1 mg/kg) over 24 hours.
  • B Body weight of WT mice at week 16 on HFHC or chow diet.
  • C Body weight change of WT mice from week 1 to week 16 when fed on HFHC or chow diet.
  • D Body weight of WT mice from week 16 to week 24 during drug dosing.
  • E Body weight change of WT mice from week 16 to week 24 during drug dosing. Mean ⁇ SEM. ****p ⁇ 0.0001, ***p ⁇ 0.001, **p ⁇ 0.01, *p ⁇ 0.05.
  • FIGS.2A-2F.2 EPGN2154 and Sema provide hepato-protection in the HFHC-fed WT mice.
  • A Body weight of mice at week 24.
  • B Fat mass percentage of mice at week 24.
  • C Lean mass percentage of mice at week 24.
  • D Liver weight of mice at week 24.
  • E Liver-to- body weight ratio of mice at week 24.
  • F Plasma ALT concentration of mice at week 24. Mean ⁇ SEM. ****p ⁇ 0.0001, ***p ⁇ 0.001.
  • FIGS. 3A-3C EPGN2154 and semaglutide improve hepatic injury and liver physiology.
  • A Representative image of hematoxylin and eosin–stained liver cross-section of experimental groups, “#” sign identifies the central hepatic veins.
  • B NAS of the liver cross- section.
  • C Oil Red O stain area percentage on the liver section of experimental groups at week 24. Mean ⁇ SEM. 0.0001, ***p ⁇ 0.001, **p ⁇ 0.01, *p ⁇ 0.05.
  • FIGS.4A-4D EPGN2154 imparts protection from the progression of hepatic fibrosis.
  • A Representative image of Sirius Red–stained liver cross section of experimental groups, “#” sign identifies the central hepatic veins.
  • B Percentage incidence of advanced-stage fibrosis (Fibrosis Score>2) in the liver cross-section of the experimental groups at week 24.
  • C Hepatic hydroxyproline concentration.
  • D Hepatic LPAR1 expression of the experimental groups at week 24.
  • EPGN2154 improves hepatic fibrosis in AMLN-fed ob/ob mice.
  • EPGN2154 improves hepatic fibrosis in AMLN-fed ob/ob mice by inhibiting macrophage migration and HSC proliferation.
  • A Representative image of Sirius Red staining of liver cross-sections.
  • B Hepatic fibrosis score of the liver cross-section of experimental groups; chi square has a significant p-value for all experimental groups compared to the AMLN+Veh group.
  • C Immunohistochemistry of the liver cross section for ⁇ SMA, Gal- 3, Col1, and Lam.
  • D Hepatic hydroxyproline concentration of the experimental groups.
  • E EPGN696 (WO 2019/246109 and US Pat No.
  • EPGN696 inhibits the LPA- stimulated proliferation of primary human HSCs in a dose-dependent manner, Mean ⁇ SEM, ****p ⁇ 0.0001 ***p ⁇ 0.001, **p ⁇ 0.01, *p ⁇ 0.05.
  • MASH-inducing diet increases the body weight of mice.6-8 weeks old male C57/Bl6J mice (WT) fed a MASH-inducing diet (MIO) for 16 weeks weigh more than chow fed mice. Mean ⁇ Sem, **** P ⁇ 0.0001.
  • FIG.9 MASH-inducing diet (MIO) increases the body weight of mice.6-8 weeks old male C57/Bl6J mice (WT) fed a MASH-inducing diet (MIO) for 16 weeks weigh more than chow fed mice. Mean ⁇ Sem, **** P ⁇ 0.0001.
  • MASH-inducing diet induce body weight gain in mice.6-8 weeks old male C57/Bl6J mice (WT) fed a MASH-inducing diet (MIO) for 16 weeks gain more body weigh chow fed mice. Mean ⁇ Sem, **** P ⁇ 0.0001.
  • FIG. 10. EPGN2154 lowers body weight. 6-8 weeks old male C57/Bl6J mice (WT) fed a MASH-inducing diet (MIO) for 16 weeks, received oral gavage of EPGN2154 doses for 8 weeks have lower body weight than MIO group. Mean ⁇ Sem, **** P ⁇ 0.0001, * P ⁇ 0.05.
  • FIG.11. EPGN2154 dose have negative correlation with body weight.
  • EPGN2154 6-8 weeks old male C57/Bl6J mice (WT) fed a MASH-inducing diet (MIO) for 16 weeks, received oral gavage of EPGN2154 doses for 8 weeks.
  • EPGN2154 dose have negative correlation with body weight.
  • FIG.12.10mg/kg EPGN2154 dose cause maximum body weight loss.
  • FIG. 13 EPGN2154 have dose dependent effect on body weight change.
  • FIG.14. EPGN2154 have no effect on adiposity. 6-8 weeks old male C57/Bl6J mice (WT) fed a MASH-inducing diet (MIO) for 16 weeks and received oral gavage of EPGN2154 for 8 weeks did not reduce adiposity. Mean ⁇ Sem, **** P ⁇ 0.0001.
  • FIG.15 EPGN2154 have no effect on lean mass.
  • EPGN2154 have no effect on food consumption. 6-8 weeks old male C57/Bl6J mice (WT) fed a MASH-inducing diet (MIO) for 16 weeks and received oral gavage of EPGN2154 for 8 weeks have no effect on food consumption. Mean ⁇ Sem. FIG. 17. EPGN2154 have no effect on water intake.
  • EPGN2154 treatment improved pathophysiology of the liver. 6-8 weeks old male WT mice fed a MIO diet for 16 weeks and received oral gavage of EPGN2154 for 8 weeks.
  • A Representative image of the liver cross-section of each experimental group
  • B EPGN2154 treatment reduced the steatosis and ballooning score and overall NAFLD Activity Score (NAS) than the MIO (no EPGN2154) group. Mean ⁇ SEM. **** P ⁇ 0.0001, *** P ⁇ 0.001, * P ⁇ 0.05.
  • the RNASeq of the liver of the mice revealed that EPGN2154 suppresses the expression of genes responsible for hepatic stellate cell activation and hepatic fibrosis signaling.
  • DESCRIPTION OF INVENTION Provided herein are methods to treat or prevent hepatic steatosis and/or fibrosis, comprising administering to a subject in need thereof an effective amount of a combination of a LPAR1 antagonist and an additional active agent.
  • the additional active agent is one or more a glucagon-like peptide-1 receptor agonist(s), including, by not limited to, semaglutide, tirzepatide, dulaglutide, exenatide, liraglutide, lixisenatide, and/or albiglutide, and/or one or more lipid lowering agent.
  • the lipid lowering agent is a cholesterol absorption inhibitor, a statin, a PCSK9 inhibitor, an ACC inhibitor, an ApoC-III inhibitor, an ACL-inhibitor, fish oil, or a CETP inhibitor.
  • the statin is atorvastatin, rosuvastatin, simvastatin, pravastatin, lovastatin, fluvastatin, or pitavastatin.
  • the LPAR1 antagonist comprises EPGN2154 and/or EPGN696.
  • the hepatic steatosis is non-alcoholic fatty liver disease (NAFLD) or metabolic dysfunction- associated steatohepatitis (MASH).
  • NAFLD non-alcoholic fatty liver disease
  • MASH metabolic dysfunction- associated steatohepatitis
  • the additional active agent is one or more a glucagon-like peptide-1 receptor agonist(s), including, by not limited to, semaglutide, tirzepatide, dulaglutide, exenatide, liraglutide, lixisenatide, and/or albiglutide, and/or one or more lipid lowering agent.
  • the lipid lowering agent is a cholesterol absorption inhibitor, a statin, a PCSK9 inhibitor, an ACC inhibitor, an ApoC-III inhibitor, an ACL-inhibitor, fish oil, or a CETP inhibitor.
  • the statin is atorvastatin, rosuvastatin, simvastatin, pravastatin, lovastatin, fluvastatin, or pitavastatin.
  • the additional active agent is one or more a glucagon- like peptide-1 receptor agonist(s), including, by not limited to, semaglutide, tirzepatide, dulaglutide, exenatide, liraglutide, lixisenatide, and/or albiglutide, and/or one or more lipid lowering agent.
  • the lipid lowering agent is a cholesterol absorption inhibitor, a statin, a PCSK9 inhibitor, an ACC inhibitor, an ApoC-III inhibitor, an ACL-inhibitor, fish oil, or a CETP inhibitor.
  • the statin is atorvastatin, rosuvastatin, simvastatin, pravastatin, lovastatin, fluvastatin, or pitavastatin.
  • Compounds/Active Agents comprise one or more lysophosphatidic acid receptor 1 antagonist, including, but not limited to, EPGN2154 and EPGN696.
  • Lysophosphatidic acid receptor 1 (LPA1) antagonists include, but are not limited to, inhibhitory RNAs (to inhibit expression or LPA1), BMS-986020, Ki6198, KI 16425, RO 6842262, and CHI, as well as compounds from WO 2010/51053, such as: .
  • Lysophosphatidic acid receptor 1 (LPA1) antagonists include, but are not limited to, compounds disclosed in US Pat No.10,000,459 and 10,570,103 (which are incorporated herein by reference), such as a compound Formula I having the structure or a pharmaceutically acceptable salt or prodrug thereof, wherein R A is —CO 2 H, —CO 2 R B , —CN, tetrazolyl, —C( ⁇ O)NH 2 , —C( ⁇ O)NHR B , — C( ⁇ O)NHSO2R B or —C( ⁇ O)NHCH2CH2SO3H or a carboxylic acid isostere; L 1 is absent or substituted or unsubstituted C1-C6 alkylene, substituted or unsubstituted C 1 -C 6 fluoroalkylene, substituted or unsubstituted C 3 -C 8 cycloalkylene, substituted or unsubstituted C 1 -C 6 heteroalkylene, or
  • R C and R D are —H, —CN, —F, —Cl, —Br, —I, —OC1-C4 alkyl, C1- C4 alkyl, C3-C6cycloalkyl, or C1-C4 fluoroalkyl, and the other R C or R D is —NR F C( ⁇ O)XCH(R G )—CY, —N(R F )C( ⁇ O)XC(R G ) 2 —CY, or —NR F C( ⁇ O)X—CY, —C( ⁇ O)—N(R F )—CH(R G )X—CY, or —C( ⁇ O)—N(R F )— C(R G )2X—CY, wherein X is absent, —O—, —NH— or —CH 2 -; R E is —H, —C1-C
  • Ring A has the structure of one of:
  • R C is —H, —CN, —F, —Cl, —Br, —I, —OC1-C4 alkyl, C1- C 4 alkyl, C 3 -C 6 cycloalkyl, or C 1 -C 4 fluoroalkyl and R D is —N(R F )—C( ⁇ O)XCH(R G )—CY, — N(R F )—C( ⁇ O)XC(R G ) 2 —CY or —N(R F )—C( ⁇ O)X—CY, wherein R F and each R G independently are —H or C1-C4 alkyl.
  • R A is —CO 2 H, —CO 2 R B , —CN, tetrazolyl, —C( ⁇ O)NH 2 , — C( ⁇ O)NHR B , C( ⁇ O)NHSO 2 R B or —C( ⁇ O)NHCH 2 CH 2 SO 3 H or a carboxylic acid isostere.
  • R A is —CO2H, —CO2R B , —CN, or —C( ⁇ O)NHSO2R B , wherein R B is substituted or unsubstituted C1-C4 alkyl or has the structure of one of:
  • L 1 is absent or substituted or unsubstituted C 1 -C 6 alkylene, C 1 - C6 fluoroalkylene, or substituted or unsubstituted C1-C6 heteroalkylene.
  • L 1 is absent or substituted or unsubstituted C1-C6 alkylene or — UV—Z—, wherein —UV— is defined by —OW—, —WO—, —N(R J )W—, —WN(R J )—, — N(R J )C( ⁇ O)—, —SW—, —S( ⁇ O) n W—, or —C( ⁇ O)N(R J )—, wherein W is substituted or unsubstituted C1-C3 alkylene, Z is substituted or unsubstituted C1-C6 alkylene or C1- C 6 fluoroalkylene; and n is 0, 1, or 2.
  • L 1 is —CH 2 —, dimethylmethane (i.e., —C(CH 3 ) 2 —), or —UV—Z— wherein —UV— is defined by —WO— , —WN(R J )—, or —C( ⁇ O)N(R J )—, wherein W is substituted or unsubstituted C 1 -C 3 alkylene; and Z is substituted or unsubstituted C1-C6 alkylene.
  • L 2 is absent, or substituted or unsubstituted C1-C6 alkylene, C1- C 6 fluoroalkylene, substituted or unsubstituted C 1 -C 6 heteroalkylene, —O—, —S—, — S( ⁇ O)—, S( ⁇ O)2—, —N(R B )—, or —C( ⁇ O)—.
  • L 2 is absent, —O—, —S—, —S( ⁇ O)—, S( ⁇ O)2—, —N(R J )—, or —C( ⁇ O)—.
  • Ring A is a 5 or 6 membered heteroarene having one of the structures of:
  • Formula I compounds have R C defined as —H, —CN, —F, — Cl, —Br, —I, —OC 1 -C 4 alkyl, C 1 -C 4 alkyl, C 3 -C 6 cycloalkyl, or C 1 -C 4 fluoroalkyl.
  • Formula I compounds have R C defined as —H, —F, —CN, — CH3, or —CF3.
  • Formula I compounds have R D defined as —N(R F )C( ⁇ O)— XCH(R G )—CY, —N(R F )C( ⁇ O)XC(R G ) 2 —CY, or —N(R F )C( ⁇ O)X—CY, wherein X is absent, —O—, —NH— or —CH2—, wherein R F is —H or C1-C4 alkyl and X, CY and R G are as previously defined.
  • Formula I compounds have R D defined as — N(R F )C( ⁇ O)OCH(R G )—CY, —N(R F )C( ⁇ O)NHC(R G )—CY, or —N(R F )C( ⁇ O)CH2—CY, wherein R F is —H or C 1 -C 4 alkyl and X, CY and R G are as previously defined.
  • Formula I compounds have R E defined as —H or C1-C4 alkyl, C1-C6 cycloalkyl or C1-C4 fluoroalkyl.
  • Formula I compounds have R E defined as —H, —CH 3 , cyclopropyl or —CF 3 .
  • Formula I compounds have R F defined as H, C1-C4 alkyl or C3- C 6 cycloalkyl.
  • Formula I compounds have R F defined as —H.
  • one R G is —C1-C4 alkyl and is taken together with CY and the carbon atom to which R G and CY is attached to define a substituted or unsubstituted carbocycle or a substituted or unsubstituted heterocycle and the other R G , if present is —H.
  • R G is independently —H or C1-C4 alkyl.
  • Ring B is substituted or unsubstituted C 3 -C 10 cycloalkylene, substituted or unsubstituted C 2 -C 10 heterocycloalkylene, a substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene, wherein if ring B is substituted then ring B is substituted with 1, 2, or 3 independently selected R H .
  • Ring C is substituted or unsubstituted C3-C10 cycloalkylene, substituted or unsubstituted C2-C10 heterocycloalkylene, a substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene, wherein if ring C is substituted then ring C is substituted with 1, 2, or 3 independently selected R H .
  • CY is substituted or unsubstituted C1- C6 alkyl, substituted or unsubstituted C3-C10 cycloalkyl, substituted or unsubstituted C2- C 10 heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, wherein if CY is substituted then CY is substituted with 1, 2, or 3 independently selected R H .
  • Ring A has the structure of one of:
  • Formula I compounds have Ring B and Ring C each independently defined as 1,4-substituted aryl or heteroaryl, R A is —CO 2 H, R C is —F or —CN, are as previously defined.
  • Formula I compounds have Ring B defined as 1,4-substituted aryl or heteroaryl, L 1 is —UV—Z— wherein —UV— is defined by —WO—, —WN(R J )—, or —C( ⁇ O)N(R J )—, wherein W is CH2, Z is substituted or unsubstituted C1-C6 alkylene, R A is —CO2H, R D is —N(R F )C( ⁇ O)OCH(R G )—CY, R E is —CH3, and R C , R F , R G , and CY are as previously defined.
  • U is substituted or unsubstituted C1-C6 alkylene, C1-C6 fluoroalkylene, or substituted or unsubstituted C 1 -C 6 heteroalkylene. In some embodiments U is .
  • Formula II compounds have R F defined as H, C 1 -C 4 alkyl or C 3 - C 6 cycloalkyl. In some embodiments, Formula II compounds have R F defined as -H. In some embodiments of Formula II compounds R G is independently -H or C 1 -C 4 alkyl. In some embodiments, Formula II compounds have R G defined as -CH 3 . In some embodiments of Formula II compounds CY is substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, wherein if CY is substituted then CY is substituted with 1, 2, or 3 independently selected R H .
  • R A is tetrazolyl
  • R c is -F or -Cl
  • R F , R G , and CY are as previously defined.
  • the LPA1R antagonist is as depicted in the below table:
  • lysophosphatidic acid receptor 1 (LPA1) antagonists can be administered alone or in combination with one or more additional active agents.
  • Additional active agents include, but are not limited to, lipid lower agents, such as statins (a class of drugs that inhibit the enzyme HMG-CoA reductase and are generally known to lower LDL cholesterol in patients).
  • statins include atorvastatin, rosuvastatin, simvastatin, pravastatin, lovastatin, fluvastatin, and pitavastatin.
  • Lipid lowering agents can also include a cholesterol absorption inhibitor, a PCSK9 inhibitor, an ACC inhibitor, an ApoC-III inhibitor, an ACL- inhibitor, fish oil, or a CETP inhibitor.
  • glucagon-like peptide-1 receptor agonist including, by not limited to, semaglutide, tirzepatide, dulaglutide, exenatide, liraglutide, lixisenatide, and/or albiglutide.
  • Chemical Definitions “Bond” or “single bond” as used herein means a chemical bond between two atoms, or two moieties when the atoms joined by the bond are considered to be part of larger substructure. As explicitly stated or implied by context, when a group described herein is a bond, the referenced group is absent thereby allowing a bond to be formed between the remaining identified groups.
  • “Membered ring” as used herein means any cyclic structure.
  • membered is meant to denote the number of skeletal atoms that constitute the ring.
  • those membered rings include cyclohexyl, pyridinyl, pyranyl and thiopyranyl, which are 6-membered rings and cyclopentyl, pyrrolyl, furanyl, and thienyl, which are 5-membered rings.
  • Moiety as used herein means a specific segment, fragment or functional group of a molecule or compound. Chemical moieties are sometimes indicated as chemical entities that are embedded in or appended (i.e., a substituent or variable group) to a molecule or compound.
  • Alkyl as used herein is a collection of carbon atoms that are covalently linked together in normal, secondary, tertiary or cyclic arrangements, i.e., in a linear, branched, cyclic arrangement or some combination thereof.
  • An alkyl substituent to a structure is that chain of carbon atoms that is covalently attached to the structure through a sp 3 carbon of the substituent.
  • the alkyl substituents contains one or more saturated moieties or groups and may additionally contain unsaturated alkyl moieties or groups, i.e., the substituent may comprise one, two, three or more independently selected double bonds or triple bonds of a combination thereof, typically one double or one triple bond if such unsaturated alkyl moieties or groups are present.
  • Unsaturated alkyl moieties or groups include moieties or groups as described below for alkenyl, alkynyl, cycloalkyl, and aryl moieties.
  • Saturated alkyl moieties contain saturated carbon atoms (sp 3 ) and no aromatic, sp 2 or sp carbon atoms.
  • the number of carbon atoms in an alkyl moiety or group can vary and typically is 1 to about 50, e.g., about 1-30 or about 1-20, unless otherwise specified, e.g., C1-8 alkyl or C1-C8 alkyl means an alkyl moiety containing 1, 2, 3, 4, 5, 6, 7 or 8 carbon atoms and C1-C6 alkyl or C1-C6 means an alkyl moiety containing 1, 2, 3, 4, 5 or 6 carbon atoms.
  • species may include methyl, ethyl, 1-propyl (n-propyl), 2-propyl (iso-propyl, —CH(CH3)2), 1-butyl (n-butyl), 2-methyl-1- propyl (iso-butyl, —CH 2 CH(CH 3 ) 2 ), 2-butyl (sec-butyl, —CH(CH 3 )CH 2 CH 3 ), 2-methyl-2- propyl (t-butyl, —C(CH 3 ) 3 ), amyl, isoamyl, sec-amyl and other linear, cyclic and branch chain alkyl moieties.
  • alkyl groups can contain species and groups described below for cycloalkyl, alkenyl, alkynyl groups, aryl groups, arylalkyl groups, alkylaryl groups and the like.
  • Cycloalkyl as used here is a monocyclic, bicyclic or tricyclic ring system composed of only carbon atoms.
  • the term “cycloalkyl” encompasses a monocyclic or polycyclic aliphatic, non-aromatic radical, wherein each of the atoms forming the ring (i.e. skeletal atoms) is a carbon atom.
  • the number of carbon atoms in an cycloalkyl substituent, moiety or group can vary and typically is 3 to about 50, e.g., about 1-30 or about 1-20, unless otherwise specified, e.g., C 3-8 alkyl or C 3 -C 8 alkyl means an cycloalkyl substituent, moiety or group containing 3, 4, 5, 6, 7 or 8 carbon atoms and C 3-6 alkyl or C 3 -C 6 means an cycloalkyl substituent, moiety or group containing 3, 4, 5 or 6 carbon atoms.
  • Cycloalkyl substituents, moieties or groups will typically have 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms and may contain exo or endo-cyclic double bonds or endo-cyclic triple bonds or a combination of both wherein the endo-cyclic double or triple bonds, or the combination of both, do not form a cyclic conjugated system of 4n+2 electrons; wherein the bicyclic ring system may share one (i.e., spiro ring system) or two carbon atoms and the tricyclic ring system may share a total of 2, 3 or 4 carbon atoms, typically 2 or 3.
  • a cycloalkyl substituent can be a monoradical or a diradical (i.e., a cycloalkylene, such as, but not limited to, cyclopropan-1,1-diyl, cyclobutan-1,1-diyl, cyclopentan-1,1-diyl, cyclohexan- 1,1-diyl, cyclohexan-1,4-diyl, cycloheptan-1,1-diyl, and the like).
  • a cycloalkylene such as, but not limited to, cyclopropan-1,1-diyl, cyclobutan-1,1-diyl, cyclopentan-1,1-diyl, cyclohexan- 1,1-diyl, cyclohexan-1,4-diyl, cycloheptan-1,1-diyl, and the like).
  • cycloalkyl When cycloalkyl is used as a Markush group (i.e., a substituent) the cycloalkyl is attached to a Markush formula with which it is associated through a carbon involved in a cyclic carbon ring system carbon of the cycloalkyl group that is not an aromatic carbon.
  • “Heteroalkylene” as used herein means an alkylene (i.e. alkanediyl) group, moiety or substituent in which one or more skeletal atoms of the alkyl are selected from an atom other than carbon, e.g., oxygen, nitrogen, sulfur, phosphorus or combinations thereof.
  • Heteroalkylene includes C1-C6 heteroalkylene or C1-C4 heteroalkylene.
  • Carboxylic acid bioisostere as used herein means a functional group, moiety or substituent that exhibits similar physical, biological and/or chemical properties as a carboxylic acid moiety.
  • carboxylic acid bioisosteres include, “Alkenyl” as used herein means a substituent, moiety or group that comprises one or more double bond moieties (e.g., —CH ⁇ CH—) or 1, 2, 3, 4, 5 or 6 or more, typically 1, 2 or 3 such moieties and can include an aryl moiety or group such as benzene, and additionally comprises linked normal, secondary, tertiary or cyclic carbon atoms, i.e., linear, branched, cyclic or any combination thereof unless the alkenyl moiety is a vinyl moiety (e.g., — CH ⁇ CH 2 ).
  • An alkenyl moiety, group or substituent with multiple double bonds may have the double bonds arranged contiguously (i.e. a 1,3 butadienyl moiety) or non-contiguously with one or more intervening saturated carbon atoms or a combination thereof, provided that a cyclic, contiguous arrangement of double bonds do not form a cyclically conjugated system of 4n+2 electrons (i.e., aromatic).
  • the number of carbon atoms in an alkenyl group or moiety can vary and typically is 2 to about 50, e.g., about 2-30 or about 2-20, unless otherwise specified, e.g., C2-8 alkenyl or C2-8 alkenyl means an alkenyl moiety containing 2, 3, 4, 5, 6, 7 or 8 carbon atoms and C 2-6 alkenyl or C2-6 alkenyl means an alkenyl moiety containing 2, 3, 4, 5 or 6 carbon atoms.
  • Alkenyl moieties or groups will typically have 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms.
  • species include, by way of example and not limitation, any of the alkyl or cycloalkyl, groups moieties or substituents described herein that has one or more double bonds, methylene ( ⁇ CH 2 ), methylmethylene ( ⁇ CH—CH3), ethylmethylene ( ⁇ CH—CH2—CH3), ⁇ CH—CH2—CH2—CH3, vinyl (— CH ⁇ CH2), allyl, 1-methylvinyl, butenyl, iso-butenyl, 3-methyl-2-butenyl, 1-pentenyl, cyclopentenyl, 1-methyl-cyclopentenyl, 1-hexenyl, 3-hexenyl, cyclohexenyl and other linear, cyclic and branched chained all carbon containing moieties containing at least one double bond.
  • alkenyl When alkenyl is used as a Markush group (i.e., a substituent) the alkenyl is attached to a Markush formula with which it is associated through an unsaturated carbon of a double bond of the alkenyl moiety or group unless specified otherwise.
  • Alkynyl as used herein means a substituent, moiety or group that comprises one or more triple bond moieties (i.e., —C ⁇ C—), e.g., 1, 2, 3, 4, 5, 6 or more, typically 1 or 2 triple bonds, optionally comprising 1, 2, 3, 4, 5, 6 or more double bonds, with the remaining bonds (if present) being single bonds and comprising linked normal, secondary, tertiary or cyclic carbon atoms, i.e., linear, branched, cyclic or any combination thereof, unless the alkynyl moiety is ethynyl.
  • triple bond moieties i.e., —C ⁇ C—
  • the number of carbon atoms in an alkenyl moiety or group can vary and typically is 2 to about 50, e.g., about 2-30 or about 2-20, unless otherwise specified, e.g., C 2- 8 alkynyl or C2-8 alkynyl means an alkynyl moiety containing 2, 3, 4, 5, 6, 7 or 8 carbon atoms.
  • Alkynyl groups will typically have 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms.
  • species include, by way of example and not limitation, any of the alkyl moieties, groups or substituents described herein that has one or more double bonds, ethynyl, propynyl, butynyl, iso-butynyl, 3-methyl-2-butynyl, 1- pentynyl, cyclopentynyl, 1-methyl-cyclopentynyl, 1-hexynyl, 3-hexynyl, cyclohexynyl and other linear, cyclic and branched chained all carbon containing moieties containing at least one triple bond.
  • alkynyl When an alkynyl is used as a Markush group (i.e., a substituent) the alkynyl is attached to a Markush formula with which it is associated through one of the unsaturated carbons of the alkynyl functional group.
  • “Aromatic” as used herein refers to a planar ring having a delocalized pi-electron system containing 4n+2 pi electrons, where n is a positive integer.
  • Aromatic rings can be formed from five, six, seven, eight, nine, ten, or more than ten atoms. Aromatics are optionally substituted.
  • aromatic includes both carboxcylic aryl (“aryl”, e.g., phenyl) and heterocyclic aryl (or “heteroaryl” or “heteroaromatic”) groups (e.g., pyridine).
  • aryl e.g., phenyl
  • heterocyclic aryl or “heteroaryl” or “heteroaromatic” groups (e.g., pyridine).
  • heteroaryl or “heteroaromatic” groups
  • Aryl as used here means an aromatic ring system or a fused ring system with no ring heteroatoms comprising 1, 2, 3 or 4 to 6 rings, typically 1 to 3 rings, wherein the rings are composed of only carbon atoms; and refers to a cyclically conjugated system of 4n+2 electrons (Huckel rule), typically 6, 10 or 14 electrons some of which may additionally participate in exocyclic conjugation (cross-conjugated (e.g., quinone).
  • Aryl substituents, moieties or groups are typically formed by five, six, seven, eight, nine, or more than nine, carbon atoms.
  • Aryl substituents, moieties or groups are optionally substituted.
  • Exemplary aryls include C 6 - C 10 aryls such as phenyl and naphthalenyl and phenanthryl.
  • an aryl group can be a monoradical or a diradical (i.e., an arylene group).
  • Exemplary arylenes include, but are not limited to, phenyl-1,2-ene, phenyl-1,3-ene, and phenyl-1,4-ene.
  • aryl is used as a Markush group (i.e., a substituent) the aryl is attached to a Markush formula with which it is associated through an aromatic carbon of the aryl group.
  • Arylalkyl as used herein means a substituent, moiety or group where an aryl moiety is bonded to an alkyl moiety, i.e., -alkyl-aryl, where alkyl and aryl groups are as described above, e.g., —CH 2 —C 6 H 5 or —CH 2 CH(CH 3 )—C 6 H 5 .
  • arylalkyl is used as a Markush group (i.e., a substituent) the alkyl moiety of the arylalkyl is attached to a Markush formula with which it is associated through a sp 3 carbon of the alkyl moiety.
  • Alkylaryl as used herein means a substituent, moiety or group where an alkyl moiety is bonded to an aryl moiety, i.e., -aryl-alkyl, where aryl and alkyl groups are as described above, e.g., —C 6 H 4 —CH 3 or —C 6 H 4 —CH 2 CH(CH 3 ).
  • alkylaryl is used as a Markush group (i.e., a substituent) the aryl moiety of the alkylaryl is attached to a Markush formula with which it is associated through a sp 2 carbon of the aryl moiety.
  • Substituted alkyl “substituted cycloalkyl”, “substituted alkenyl”, “substituted alkynyl”, substituted alkylaryl”, “substituted arylalkyl”, “substituted heterocycle”, “substituted aryl” and the like as used herein mean an alkyl, alkenyl, alkynyl, alkylaryl, arylalkyl heterocycle, aryl or other group or moiety as defined or disclosed herein that has a substituent(s) that replaces a hydrogen atom(s) or a substituent(s) that interrupts a carbon atom chain.
  • Alkenyl and alkynyl groups that comprise a substituent(s) are optionally substituted at a carbon that is one or more methylene moieties removed from the double bond.
  • “Optionally substituted alkyl”, “optionally substituted alkenyl”, “optionally substituted alkynyl”, “optionally substituted alkylaryl”, “optionally substituted arylalkyl”, “optionally substituted heterocycle”, “optionally substituted aryl”, “optionally substituted heteroaryl”, “optionally substituted alkylheteroaryl”, “optionally substituted heteroarylalkyl” and the like as used herein mean an alkyl, alkenyl, alkynyl, alkylaryl, arylalkyl heterocycle, aryl, heteroaryl, alkylheteroaryl, heteroarylalkyl, or other substituent, moiety or group as defined or disclosed herein that has a substituent(s) that optionally replaces a hydrogen atom(s) or
  • substituents are as described herein.
  • a phenyl moiety the arrangement of any two substituents present on the aromatic ring can be ortho (o), meta (m), or para (p).
  • An optionally substituted fluoroalkyl is an alkyl or cycloalkyl moiety, typically a linear alkyl, wherein one or more hydrogen atoms is replaced by fluorine and at least one other atom other than carbon and fluorine.
  • An optionally substituted or substituted substituent, moiety or group includes those having one or more additional group(s) that replace its hydrogen atom(s) individually and independently selected from alkyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy, alkylthio, arylthio, alkylsulfoxide, arylsulfoxide, alkylsulfone, arylsulfone, cyano, halo, nitro, haloalkyl, fluoroalkyl, fluoroalkoxy, and amino, including mono- and di- substituted amino groups, and the protected derivatives thereof.
  • an optional substituent(s) may be halide, —CN, —NO 2 , or LsRs, wherein each Ls is independently selected from a bond, —O—, —C( ⁇ O)—, —C( ⁇ O)O—, —S—, —S( ⁇ O)—, —S( ⁇ O)2—, —NH—, —NHC( ⁇ O)—, —C( ⁇ O)NH—, S( ⁇ O)2NH—, —NHS( ⁇ O)2, — OC( ⁇ O)NH—, —NHC( ⁇ O)O—, or —(C 1 -C 6 alkylene)-; and each Rs is selected from —H, alkyl, fluoroalkyl, heteroalkyl, cycloalkyl, aryl, heteroaryl, or heterocycloalkyl.
  • Optional substituents include those selected from the group consisting of halogen, —CN, —NH 2 , —OH, —N(CH 3 ) 2 , alkyl, fluoroalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxy, aryloxy, alkylthio, arylthio, alkylsulfoxide, arylsulfoxide, alkylsulfone, and arylsulfone, those selected from the group consisting of halogen, —CN, —NH 2 , —OH, NH(CH 3 ), —N(CH 3 ) 2 , —CO 2 H, —CO 2 alkyl, — C( ⁇ O)NH2, —C( ⁇ O)NHalkyl, —C( ⁇ O)N(
  • an optionally substituted, substituent, moiety or group is substituted with one or two of the preceding groups, or more typically with one of the preceding groups.
  • An optional substituent on an aliphatic carbon atom (acyclic or cyclic, saturated or unsaturated carbon atoms, excluding aromatic carbon atoms) further includes oxo ( ⁇ O).
  • Heterocycle or “heterocyclic” as used herein means a cycloalkyl or aromatic ring system wherein one or more, typically 1, 2 or 3, but not all of the carbon atoms comprising the ring system are replaced by a heteroatom which is an atom other than carbon, including, N, O, S, Se, B, Si, P, typically N, O or S wherein two or more heteroatoms may be adjacent to each other or separated by one or more carbon atoms, typically 1-17 carbon atoms, 1-7 atoms or 1- 3 atoms.
  • Heterocycles includes heteroaromatic rings (also known as heteroaryls) and heterocycloalkyl rings (also known as heteroalicyclic groups) containing one to four heteroatoms in the ring(s), where each heteroatom in the ring(s) is selected from O, S and N, wherein each heterocyclic group has from 4 to 10 atoms in its ring system, and with the proviso that the any ring does not contain two adjacent O or S atoms.
  • Non-aromatic heterocyclic, substituents, moieties or groups also known as heterocycloalkyls
  • aromatic heterocyclic groups have at least 5 atoms in their ring system and include benzo-fused ring systems.
  • Heterocyclics with 3, 4, 5, 6 and 10 atoms include aziridinyl azetidinyl, thiazolyl, pyridyl and quinolinyl, respectively.
  • Nonaromatic heterocyclic substituents, moieties or groups are pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, oxazolidinonyl, tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, piperidinyl, morpholinyl, thiomorpholinyl, thioxanyl, piperazinyl, aziridinyl, azetidinyl, oxetanyl, thietanyl, homopiperidinyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl, thiazepinyl
  • Aromatic heterocyclic includes, by way of example and not limitation, pyridinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, quinolinyl, isoquinolinyl, indolyl, benzimidazolyl, benzofuranyl, cinnolinyl, indazolyl, indolizinyl, phthalazinyl, pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl, thiadiazolyl, furazanyl, benzofurazanyl, benzo-thiophenyl, benzothiazolyl, benzoxazolyl, qui
  • Non-aromatic heterocycles may be substituted with one or two oxo ( ⁇ O) moieties and includes pyrrolidin-2-one.
  • heterocycle When heterocycle is used as a Markush group (i.e., a substituent) the heterocycle is attached to a Markush formula with which it is associated through a carbon or a heteroatom of the heterocycle, where such an attachment does not result in an unstable or disallowed formal oxidation state of that carbon or heteroatom.
  • a heterocycle that is C-linked is bonded to a molecule through a carbon atom include moieties such as —(CH2)n-heterocycle where n is 1, 2 or 3 or —C ⁇ heterocycle where C ⁇ represents a carbon atom in a heterocycle ring.
  • a heterocycle that is N-linked is a nitrogen containing heterocycle that is bonded a heterocycle ring nitrogen sometimes described as —N ⁇ heterocycle where N ⁇ represents a nitrogen atom in a heterocycle ring.
  • nitrogen-containing heterocycles may be C-linked or N-linked and include pyrrole substituents, which may be pyrrol-1-yl (N-linked) or pyrrol-3-yl (C-linked), imidazole substituents, which may be imidazol-1-yl or imidazol-3-yl (both N-linked) or imidazol-2-yl, imidazol-4-yl or imidazol-5-yl (all C-linked).
  • Heteroaryl as used herein means an aryl ring system wherein one or more, typically 1, 2 or 3, but not all of the carbon atoms comprising the aryl ring system are replaced by a heteroatom which is an atom other than carbon, including, N, O, S, Se, B, Si, P, typically, oxygen (—O—), nitrogen (—NX—) or sulfur (—S—) where X is —H, a protecting group or C 1-6 optionally substituted alkyl, wherein the heteroatom participates in the conjugated system either through pi-bonding with an adjacent atom in the ring system or through a lone pair of electrons on the heteroatom and may be optionally substituted on one or more carbons or heteroatoms, or a combination of both, in a manner which retains the cyclically conjugated system.
  • Heterocycles and heteroaryls include, by way of example and not limitation, heterocycles and heteroaryls described in Paquette, Leo A.; “Principles of Modern Heterocyclic Chemistry” (W. A. Benjamin, New York, 1968), particularly Chapters 1, 3, 4, 6, 7, and 9; “The Chemistry of Heterocyclic Compounds, A series of Monographs” (John Wiley & Sons, New York, 1950 to present), in particular Volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc. 1960, 82:5545-5473 particularly 5566-5573).
  • heteroaryls include by way of example and not limitation pyridyl, thiazolyl, pyrimidinyl, furanyl, thienyl, pyrrolyl, pyrazolyl, purinyl, imidazolyl, benzofuranyl, indolyl, isoindoyl, quinolinyl, isoquinolinyl, benzimidazolyl, pyridazinyl, pyrazinyl, benzothiopyran, benzotriazine, isoxazolyl, pyrazolopyrimidinyl, quinoxalinyl, thiadiazolyl, triazolyl and the like.
  • Heterocycles that are not heteroaryls include, by way of example and not limitation, tetrahydrothiophenyl, tetrahydrofuranyl, indolenyl, piperidinyl, pyrrolidinyl, 2-pyrrolidonyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, octahydroisoquinolinyl, 2H-pyrrolyl, 3H- indolyl, 4H-quinolizinyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, piperazinyl, quinuclidinyl, morpholinyl, oxazolidinyl and the like.
  • Other heteroaryls include, by way of example and not limitation, the following moieties:
  • Monocyclic heteroaryls include, by way of example and not limitation, pyridinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, pyridazinyl, triazinyl, oxadiazolyl, thiadiazolyl, and furazanyl.
  • Heteroaryls include those substituents, moieties or groups containing 0-3 N atoms, 1-3 N atoms or 0-3 N atoms, 0-1 O atoms and 0-1 S atoms.
  • a heteroaryl may be monocyclic or bicyclic.
  • the ring system of a heteroaryls ring typically contains 1-9 carbons (i.e., C1- C 9 heteroaryl).
  • Monocyclic heteroaryls include C 1 -C 5 heteroaryls.
  • Monocyclic heteroaryls include those having 5-membered or 6-membered ring systems.
  • Bicyclic heteroaryls include C6-C9 heteroaryls.
  • Heterocycloalkyls also referred to as non-aromatic heterocycles, include by way of example and not limitation: Heterocycloalkyl includes, by way of example and not limitation, oxazolidinonyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, tetrahydrothiopyranyl, piperidinyl, morpholinyl, thiomorpholinyl, piperazinyl, and indolinyl. Heteroalicyclics further includes all ring forms of carbohydrates, including but not limited to monosaccharides, disaccharides and oligosaccharides.
  • a heterocycloalkyl is a C 2 -C 10 heterocycloalkyl and includes C 4 -C 10 heterocycloalkyl.
  • a heterocycloalkyl may contain 0-2 N atoms, 0-2 O atoms or 0-1 S atoms.
  • “Heteroarylalkyl” as used herein means a substituent, moiety or group where a heteroaryl moiety is bonded to an alkyl moiety, i.e., -alkyl-heteroaryl, where alkyl and heteroaryl groups are as described above.
  • heteroarylalkyl When heteroarylalkyl is used as a Markush group (i.e., a substituent) the alkyl moiety of the heteroarylalkyl is attached to a Markush formula with which it is associated through a sp 3 carbon of the alkyl moiety.
  • Alkylheteroaryl as used herein means a substituent, moiety or group where a heteroaryl moiety is bonded to an alkyl moiety, i.e., -heteroaryl-alkyl, where heteroaryl and alkyl groups are as described above.
  • heteroarylalkyl When heteroarylalkyl is used as a Markush group (i.e., a substituent) the heteroaryl moiety of the heteroarylalkyl is attached to a Markush formula with which it is associated through a sp 2 carbon or heteroatom of the alkyl moiety.
  • “Halogen” or “halo” as used herein means fluorine, chlorine, bromine or iodine.
  • “Haloalkyl” as used herein means an alkyl substituent moiety or group in which one or more of its hydrogen atoms are replaced by one or more independently selected halide atoms.
  • Haloalkyl includes C1-C4 haloalkyl.
  • Example but non-limiting C1-C4 haloalkyls are —CH2Cl, CH2Br, —CH2I, —CHBrCl, —CHCl—CH2Cl and —CHCl—CH2I.
  • Haloalkylene as used herein means an alkylene substituent, moiety or group in which one or more hydrogen atoms are replaced by one or more halide atoms.
  • Haloalkylene includes C1-C6haloalkylenes or C1-C4 haloalkylenes.
  • “Fluoroalkyl” as used herein means an alkyl in which one or more hydrogen atoms are replaced by a fluorine atom.
  • Fluoroalkyl includes C 1 -C 6 and C 1 -C 4 fluoroalkyls.
  • Example but non-limiting fluoroalkyls include —CH3F, —CH2F2 and —CF3 and perfluroalkyls.
  • Fluoroalkylene as used herein means an alkylene in which one or more hydrogen atoms are replaced by a fluorine atom.
  • Fluoroalkylene includes C 1 -C 6 fluoroalkylenes or C 1 - C4 fluoroalkylenes.
  • heteroalkyl refers to an alkyl group in which one or more skeletal atoms of the alkyl are selected from an atom other than carbon, e.g., oxygen, nitrogen, sulfur, phosphorus or combinations thereof.
  • a heteroalkyl is a C1-C6 heteroalkyl.
  • Protecting group as used here means a moiety that prevents or reduces the ability of the atom or functional group to which it is linked from participating in unwanted reactions.
  • Non-limiting examples are for —OR PR , wherein R PR is a protecting group for the oxygen atom found in a hydroxyl, while for —C(O)—OR PR , R PR may be a carboxylic acid protecting group; for —SR PR , R PR may be a protecting group for sulfur in thiols and for —NHR PR or —N 2 — , at least one of R PR is a nitrogen atom protecting group for primary or secondary amines. Hydroxyl, amine, ketones and other reactive groups may require protection against reactions taking place elsewhere in the molecule.
  • the protecting groups for oxygen, sulfur or nitrogen atoms are usually used to prevent unwanted reactions with electrophilic compounds, such as acylating agents.
  • Protective groups for atoms or functional groups are given in Greene (1999), “Protective groups in organic synthesis, 3 rd ed.”, Wiley Interscience. “Ester” as used herein means a substituent, moiety or group that contains a —C(O)— O— structure (i.e., ester functional group) wherein the carbon atom of the structure is not directly connected to another heteroatom and is directly connected to —H or another carbon atom.
  • esters comprise or consist of an organic moiety containing 1-50 carbon atoms, 1-20 carbon atoms or 1-8 carbon atoms and 0 to 10 independently selected heteroatoms (e.g., O, S, N, P, Si), typically 0-2 where the organic moiety is bonded through the —C(O)—O— structure and include ester moieties such as organic moiety-C(O)—O—.
  • heteroatoms e.g., O, S, N, P, Si
  • the organic moiety usually comprises one or more of any of the organic groups described herein, e.g., C1-20 alkyl moieties, C 2-20 alkenyl moieties, C 2-20 alkynyl moieties, aryl moieties, C 3-8 heterocycles or substituted derivatives of any of these, e.g., comprising 1, 2, 3, 4 or more substituents, where each substituent is independently chosen.
  • Exemplary, non-limiting substitutions for hydrogen or carbon atoms in these organic groups are as described above for substituted alkyl and other substituted moieties and are independently chosen.
  • substitutions listed above are typically substituents that one can use to replace one or more carbon atoms, e.g., —O— or —C(O)—, or one or more hydrogen atom, e.g., halogen, —NH 2 or —OH.
  • exemplary esters include by way of example and not limitation, one or more independently selected acetate, propionate, isopropionate, isobutyrate, butyrate, valerate, isovalerate, caproate, isocaproate, hexanoate, heptanoate, octanoate, phenylacetate esters or benzoate esters.
  • ester When ester is used as a Markush group (i.e., a substituent) the single bonded oxygen of the ester functional group is attached to a Markush formula with which it is associated.
  • “Acetal”, “thioacetal”, “ketal”, “thioketal” and the like as used herein means a moiety, group or substituent comprising or consisting of a carbon to which is bonded two of the same or different heteroatoms wherein the heteroatoms are independently selected S and O.
  • the carbon For acetal the carbon has two bonded oxygen atoms, a hydrogen atom and an organic moiety.
  • the carbon has two bonded oxygen atoms and two independently selected organic moieties where the organic moiety is as described herein alkyl or optionally substituted alkyl group.
  • the organic moiety is as described herein alkyl or optionally substituted alkyl group.
  • thioacetals and thioketals one or both of the oxygen atoms in acetal or ketal, respectively, is replaced by sulfur.
  • the oxygen or sulfur atoms in ketals and thioketals are sometimes linked by an optionally substituted alkyl moiety.
  • the alkyl moiety is an optionally substituted C1-8 alkyl or branched alkyl structure such as —C(CH3)2—, — CH(CH3)—, —CH2—, —CH2—CH2—, —C[(C2-C4 alkyl)2]1, 2, 3- or [CH(C2-C4 alkyl)]1, 2, 3.
  • moieties can serve as protecting groups for an aldehyde or ketone include, by way of example and not limitation, acetals for aldehydes and ketals for ketones and contain — O—CH2—CH2—CH2—O— or —O—CH2—CH2—O— moieties that form a spiro ring with the carbonyl carbon, and can be removed by chemical synthesis methods or by metabolism in cells or biological fluids.
  • “Ether” as used herein means an organic moiety, group or substituent that comprises or consists of 1, 2, 3, 4 or more —O— moieties, usually 1 or 2, wherein no two —O— moieties are immediately adjacent (i.e., directly attached) to each other.
  • ethers comprise an organic moiety containing 1-50 carbon atoms, 1-20 carbon atoms or 1-8 carbon atoms and 0 to 10 independently selected heteroatoms (e.g., O, S, N, P, Si), typically 0-2.
  • An ether moiety, group or substituent includes organic moiety-O— wherein the organic moiety is as described herein for alkyl or optionally substituted alkyl group.
  • ether is used as a Markush group (i.e., a substituent) the oxygen of the ether functional group is attached to a Markush formula with which it is associated.
  • ether is a used as substituent in a Markush group it is sometimes designated as an “alkoxy” group.
  • Alkoxy includes C1-C4 ether substituents such as, by way of example and not limitation, methoxy, ethoxy, propoxy, iso-propoxy and butoxy. Ether further includes those substituents, moieties or groups that contain one (excluding ketal) or more —OCH 2 CH 2 O—, moieties in sequence (i.e., polyethylene or PEG moieties). “Carbonate” as used here means a substituent, moiety or group that contains a —O— C( ⁇ O)—O— structure (i.e., carbonate functional group).
  • carbonate groups as used here comprise or consist of an organic moiety containing 1-50 carbon atoms, 1-20 carbon atoms or 1-8 carbon atoms and 0 to 10 independently selected heteroatoms (e.g., O, S, N, P, Si), typically 0-2, bonded through the —O—C( ⁇ O)—O— structure, e.g., organic moiety-O— C( ⁇ O)—O—.
  • a Markush group i.e., a substituent
  • one of the singly bonded oxygen atoms of the carbonate functional group is attached to a Markush formula with which it is associated.
  • “Carbamate” or “urethane” as used here means a substituent, moiety or group that contains a —O—C( ⁇ O)N(R PR )—, —O—C( ⁇ O)N(R PR ) 2 , —O—C( ⁇ O)NH(optionally substituted alkyl) or —O—C( ⁇ O)N(optionally substituted alkyl) 2 -structure (i.e., carbamate functional group) where R PR and optionally substituted alkyl are independently selected and R PR are independently —H, a protecting group or an organic moiety as described for ester, alkyl or optionally substituted alkyl.
  • carbamate groups as used here comprise or consist of an organic moiety containing about 1-50 carbon atoms, 1-20 carbon atoms or 1-8 carbon atoms and 0 to 10 independently selected heteroatoms (e.g., O, S, N, P, Si), typically 0-2, bonded through the —O—C( ⁇ O)—NR PR - structure, e.g., organic moiety-O—C( ⁇ O)— NR PR — or —O—C( ⁇ O)—NR PR -organic moiety.
  • heteroatoms e.g., O, S, N, P, Si
  • carbamate When carbamate is used as a Markush group (i.e., a substituent) the singly bonded oxygen (O-linked) or nitrogen (N-linked) of the carbamate functional group is attached to a Markush formula with which it is associated.
  • the linkage of the carbamate substituent is either explicitly stated (N- or O-linked) or implicit in the context to which this substituent is referred.
  • the designated range means that any individual number of carbon atoms is described.
  • C1-C4 optionally substituted alkyl specifically means that a 1, 2, 3 or 4 carbon optionally substituted alkyl moiety as defined herein is present, or a 2, 3, 4, 5 or 6 carbon alkenyl, or a 3, 4, 5, 6, 7 or 8 carbon moiety comprising a heterocycle or optionally substituted alkenyl moiety as defined herein is present.
  • C1-C4 optionally substituted alkyl includes, e.g., 3 carbon alkyl, 4 carbon substituted alkyl and 4 carbon alkyl, including all positional isomers and the like are disclosed and can be expressly referred to or named.
  • esters, carbonates and carbamates defined by a given range of carbon atoms the designated range includes the carbonyl carbon of the respective functional group.
  • a C1 ester refers to a formate ester and a C2 ester refers to an acetate ester.
  • Disease/Disorders As used herein, “steatosis” is interchangeable with “fatty liver” which is an accumulation of fat in the liver. “Steatosis” and “hepatic steatosis” are used interchangeably herein.
  • lysophosphatidic acid receptor 1 LPA1 antagonist(s) alone or in combination with an additional active agent, such as a statin (e.g., atorvastatin, rosuvastatin, simvastatin, pravastatin, lovastatin, fluvastatin, or pitavastatin) or a glucagon-like peptide-1 receptor agonist.
  • a statin e.g., atorvastatin, rosuvastatin, simvastatin, pravastatin, lovastatin, fluvastatin, or pitavastatin
  • a glucagon-like peptide-1 receptor agonist e.g., a statin (e.g., atorvastatin, rosuvastatin, simvastatin, pravastatin, lovastatin, fluvastatin, or pitavastatin) or a glucagon-like peptide-1 receptor agonist.
  • the method comprises treating a subject to reduce
  • the liver disease is metabolic dysfunction-associated steatohepatitis (MASH). In some aspects the liver disease is alcoholic hepatic steatosis. In other aspects, the method comprises treating a subject to prevent or reduce the rate of progression of liver disease. In some aspects, the subject has type IIb hyperlipidemia. In some aspects, the subject has Familial combined hyperlipidemia (FCHL).
  • MASH metabolic dysfunction-associated steatohepatitis
  • FCHL Familial combined hyperlipidemia
  • the subject may have a risk factor for developing fatty liver (steatosis) wherein the risk factor is that the subject has metabolic syndrome, type-2 diabetes, impaired glucose tolerance, obesity, dyslipidemia, hepatitis B, hepatitis C, an HIV infection, or a metabolic disorder such as Wilson's disease, a glycogen storage disorder, or galactosemia.
  • the subject has diabetes.
  • the subject has an inflammatory condition.
  • the patient has an elevated body mass index above what is normal for gender, age and height.
  • type IIb hyperlipidemia or “type IIb” patient population means a patient population having a fasting LDL cholesterol blood plasma level ⁇ 130 mg/dl and a fasting triglyceride blood plasma level ⁇ 150 mg/dL. References to LDL-C, triglyceride or ApoB levels are fasting levels unless clearly indicated otherwise.
  • Type IIb hyperlipidemia is also known as Type IIb hyperlipoproteinemia. In some references type IIb hyperlipidemia is referred to as mixed dyslipidemia or is described as a subset of mixed dyslipidemia.
  • the methods for reducing the accumulation of liver fat reduce the subject's risk of developing a liver disease.
  • the fat is triglyceride.
  • the subject has liver disease.
  • the liver disease is nonalcoholic fatty liver disease (NAFLD).
  • the liver disease is metabolic dysfunction-associated steatohepatitis (MASH).
  • the liver disease is liver fibrosis.
  • the liver disease is inflammation of the liver.
  • LPA1 lysophosphatidic acid receptor 1
  • One aspect provides a method of reducing hepatic fibrosis in a subject in need thereof, comprising administering to the subject one or more lysophosphatidic acid receptor 1 (LPA1) antagonist(s) and optionally with one or more additional active agents.
  • Another aspect provides a method of reducing hepatic fibrosis in a subject in need thereof, comprising administering to the subject one or more lysophosphatidic acid receptor 1 (LPA1) antagonists, wherein the subject has MASH.
  • Treatment/Administration “API” is an abbreviation for active pharmaceutical ingredient. When referring to dosages and doses, the dosage or dose is calculated on the weight of the API. In some embodiments, the API may be administered as a pharmaceutically acceptable salt.
  • single dose formulation refers to a pharmaceutical composition in the form in which it is marketed for use, formulated with mixture of one or more APIs and one or more excipients, along with other optional non-reusable material that may not be considered either ingredient or packaging (e.g., a capsule shell).
  • single dose formulation and “fixed dose combination” are used interchangeably.
  • Common single dose formulations include pills, tablets, or capsules.
  • Formulations Compounds useful in the present invention can be formulated as pharmaceutical compositions and administered to a subject, such as a human subject, in a variety of forms adapted to the chosen route of administration, i.e., orally, transdermal, and parenterally.
  • a subject such as a human subject
  • Such compositions and methods for their preparation are well known and may be found, for example, in Remington's Pharmaceutical Sciences, 19th Edition (Mack Publishing Company, 1995).
  • LPA1R antagonist(s) are formulated alone, or in combination with another active agent, such as glucagon-like peptide-1 receptor agonists, and with common excipients and carriers such as starch, binders, diluents and the like, and molded into tablets, or encapsulated into gelatin capsules for convenient oral administration.
  • “Pharmaceutically acceptable formulation” as used herein means a composition comprising an active pharmaceutical ingredient, such as a compound having the formula of I or II in addition to one or more pharmaceutically acceptable excipients or refers to a composition prepared from an active pharmaceutical ingredient and one or more pharmaceutically acceptable excipients, wherein the composition is suitable for administration to a subject, such as a human or an animal, in need thereof.
  • the formulation For a pharmaceutically acceptable formulation to be suitable for administration to a human the formulation must have biological activity for treating or preventing a disease or condition disclosed herein or an expectation must exist that the formulation would have a desired activity towards an “intent to treat” disease or condition.
  • the “intent to treat” disease or condition is a lysophosphatidic acid receptor-mediated condition or disease. More typically the disease or condition to be treated or prevented is a lysophosphatidic acid lysophosphatidic acid type 1 receptor-mediated disease or condition.
  • a pharmaceutically acceptable formulation that is suitable for administration to an animal does not necessarily require a biological activity for treating or preventing a disease or condition and may be administered to the animal in order to evaluate a potential pharmacological or biological activity of a Formula I-II compound.
  • Those formulations must therefore be suitable for treating or preventing a disease or condition disclosed herein in an animal in need thereof or is suitable for evaluating a pharmacological or biological activity of a Formula I-II compound.
  • Solid formulation refers to a pharmaceutically acceptable formulation comprising the active agents and one or more pharmaceutically acceptable excipients in solid form(s) wherein the formulation is in a unit dosage form suitable for administration of a solid.
  • the dosage units include tablets, capsules, caplets, gelcaps, suspensions and other dosage units typically associated with parenteral or enteral (oral) administration of a solid.
  • “Liquid formulation” as used herein refers to a pharmaceutically acceptable formulation wherein at least one the active agent compound has been admixed or contacted with one or more pharmaceutically acceptable excipients, wherein at least one of the excipients is in liquid form in proportions required for a liquid formulation, i.e., such that a majority of the mass amount of the active agent(s) is dissolved into the non-solid excipient.
  • Dosage units containing a liquid formulation include syrups, gels, ointments and other dosage units typically associated with parenteral or enteral administration of a pharmaceutical formulation to a subject in need thereof in liquid form.
  • the active agent(s) such as, LPA1R antagonist(s) and/or other active agents, such as glucagon-like peptide-1 receptor agonists, are administered daily, weekly or biweekly or monthly.
  • 0.5 mg/kg to 15 mg/kg( body weight) of the active agent e.g., LPA1R antagonists
  • the active agent e.g., LPA1R antagonists
  • is administered to the subject including, 0.5 mg/kg, 1.0 mg/kg, 1.5 mg/kg, 2.0 mg/kg, 2.5 mg/kg, 3.0 mg/kg, 3.5 mg/kg, 4.0 mg/kg, 4.5 mg/kg mg/kg, 5.0 mg/kg, 5.5 mg/kg, 6.0 mg/kg, 6.5 mg/kg, 7.0 mg/kg, 7.5 mg/kg, 8.0 mg/kg, 8.5 mg/kg, 9.0 mg/kg, 9.5 mg/kg, 10.0 mg/kg, 10.5 mg/kg, 11.0 mg/kg, 11.5 mg/kg, 12.0 mg/kg, 12.5 mg/kg, 13.0 mg/kg, 13.5 mg/kg, 14.0 mg/kg, 14.5 mg/kg or 15.0 mg/kg.
  • kits comprising LPA1R antagonists and/or glucagon-like peptide-1 receptor agonist, and optionally with excipients and carriers; and instructions for the use thereof.
  • Example Overview MASH causes a tremendous health care burden in the United States. A glucagon-like peptide-1 agonist, semaglutide (Sema), treatment resulted in hepatic steatosis reduction in clinical trials of MASH. Lysophosphatidic acid receptor 1 antagonists are known to have antifibrotic effects in several organs.
  • Semaglutide Semaglutide
  • EPGN2154 a novel lysophosphatidic acid receptor 1 antagonist
  • Methods In the present study, (1) C57Bl6/J wild-type mice fed on a high-fat, high- carbohydrate (HFHC) diet for 16 weeks and (2) leptin-deficient mice (ob/ob) fed on an Amylin liver NASH diet for 16 weeks were used.
  • HFHC high-fat, high- carbohydrate
  • mice were randomly distributed in equal numbers in (1) no-drug, (2) EPGN2154, (3) Sema, and (4) EPGN2154+Sema treatment groups for 8 additional weeks at a dosage of 10 mg/kg body weight for EPGN2154 (oral gavage, 5 days a week) and 6.17 ⁇ g/kg body weight of Sema (subcutaneous injection every alternate day, 3 days a week).
  • Results In the wild-type-high-fat, high-carbohydrate model, the most body weight loss was seen in the EPGN2154+Sema combination group compared to the other treatment groups. All groups led to a significant reduction in alanine transaminase levels when compared to high- fat, high carbohydrate–fed wild type.
  • LPAR1 deficiency causes a reduction of infiltrating macrophages and expression of MCP-1 in response to lipopolysaccharide in a preclinical model of chronic lung diseases.
  • the infiltrating macrophages secrete proinflammatory cytokines in the liver, activating HSCs, thereby initiating the process of fibrosis.
  • HSCs constitute nearly 90% of the collagen-producing cells in a fibrotic liver.
  • LPA lysophosphatidic acid
  • Additional studies have shown that patients with type 2 diabetes have a higher prevalence of MASH.
  • Combination drug therapies for MASH have shown better outcomes in clinical trials.
  • EPGN2154 solution was prepared fresh in a vehicle formulation of 20% Solutol and 40% PEG400 in 40% water at a 5 mg/mL concentration. A total of 12 female CD1 mice were acclimatized to living conditions and then divided into 2 groups of 6 animals each. EPGN2154 was administered by oral gavage (dose 20 mg/kg). EPGN2154 was administered intravenously (dose 1 mg/kg) at a dose volume of 5 mL/kg based on mouse weight. Plasma samples were diluted with blank mouse plasma as needed. An aliquot of 20 ⁇ L of plasma sample was extracted with 100 ⁇ L of acetonitrile containing internal standard (terfenadine).
  • mice Six- to 8-week-old male C57Bl6/J WT and B6.Cg-Lep ob /J (ob/ob) mice (Jackson Laboratory, Bar Harbor, ME) were housed in a 12-hour light-dark cycle maintained in a (22 ⁇ 2°C) temperature-controlled room.
  • EPGN2154 was administered orally once daily for 5 days a week.
  • Sema BOC Sciences, Shirley, NY
  • mice After 8 weeks of treatment, the mice were euthanized, and tissues were harvested for assays and histological analysis.
  • EPGN2154 was administered orally once daily for 5 days a week. Sema was administered by subcutaneous injection every alternate day, that is, 3 days a week. After 8 weeks of treatment, the mice were euthanized, and tissues were harvested for assays and histological analysis. Body weights and food intake were recorded every week. Body composition Body composition analysis (fat mass and lean mass) was done using EchoMRI-100H Body Composition Analyzer (EchoMRI, TX). Plasma alanine transaminase assay Plasma isolated from the whole blood was used to estimate alanine transaminase (ALT) concentration using ALT Activity Assay kit (Sigma, St.Louis, MO) as per the manufacturer’s instruction.
  • ALT alanine transaminase
  • Oil Red O staining Oil Red O staining was performed on the 5 ⁇ m frozen liver tissue section using the Oil Red O Stain Kit (Lipid Stain) (ab150678) from Abcam (Boston, MA) according to the manufacturer’s instructions.
  • the Oil Red O staining area percentage in the liver sections was quantified by a single independent pathologist blinded to the experimental design and treatment groups.
  • Hydroxyproline assay Frozen liver tissue (100 mg) was used to quantify the hydroxyproline content using Hydroxyproline Assay Kit (Colorimetric) (ab222941) from Abcam (Boston, MA) according to the manufacturer’s instructions.
  • LPAR1 expression Total RNA was isolated from 10 mg of liver tissue by TRI Reagent Solution (Invitrogen, Waltham, MA).5 ⁇ g of total RNA was used for cDNA synthesis using SuperScript III Reverse Transcriptase (Invitrogen, Waltham, MA). cDNA was used to quantify the relative expression of LPAR1 using a Taqman probe for LPAR1 (Mm01346925_m1) (Invitrogen, Waltham, MA). The relative expression of LPAR1 was calculated by the ⁇ C t method using a Taqman probe for Rpl18 (Mm01197265_g1) (Invitrogen, Waltham, MA) as a housekeeping gene.
  • Liver histology analysis and immunohistochemistry Liver tissue was harvested from mice, fixed in 10% formalin, and sectioned in a microtome to generate 5- ⁇ m sections for histologic analyses. The sections were stained with eosin and hematoxylin and analyzed to determine the NAFLD Activity Score (NAS) by a single independent pathologist blinded to the experimental design and treatment groups.
  • NAS NAFLD Activity Score
  • liver histology is graded on steatosis (score 0–3), lobular inflammation (score 0–3), and ballooning (score 0–2). (32) Sirius Red staining evaluated hepatic fibrosis on a scale (0–4).
  • Immunohistochemistry was performed on the liver section of the ob/ob mice for alpha-smooth muscle actin, collagen I, laminin and galectin-3 using anti-alpha-smooth muscle actin antibody (Cat# ab5694, Abcam, Cambridge, UK), anti-Collagen I antibody (Cat# ab270993, Abcam, Cambridge, UK), anti-Laminin antibody (Cat# ab11575, Abcam, Cambridge, UK), and anti- galectin-3 antibody (Cat# ab76245, Abcam, Cambridge, UK) respectively.
  • anti-alpha-smooth muscle actin antibody Cat# ab5694, Abcam, Cambridge, UK
  • anti-Collagen I antibody Cat# ab270993, Abcam, Cambridge, UK
  • anti-Laminin antibody Cat# ab11575, Abcam, Cambridge, UK
  • anti- galectin-3 antibody Cat# ab76245, Abcam, Cambridge, UK
  • MCP-1 solution (10 -8 M, 26 ⁇ L) was added to the bottom chamber of the Boyden apparatus. A 5-micron polycarbonate membrane was applied, followed by the gasket and the donor chamber apparatus screwed into place. Once assembled, positive controls and test wells (compound plus cell incubations), all ⁇ 50 ⁇ L, were pipetted into the upper chamber. A set of background wells was established with a bottom chamber containing MEM only and the upper wells RAW264.7 cells (50 ⁇ L). Once complete, the setup is incubated at 35°C for 1 hour. After 1 hour, the apparatus was disassembled and inverted to allow for the removal of the upper chamber and gasket.
  • the filter was clamped, and the underside was rinsed and scraped in PBS before fixing it in methanol and drying on a glass slide.
  • the dried filter was hematoxylin and eosin stained, and cells were visualized by microscope. Cell count was determined for each well as the average of 5 fields under the microscope. Cell counts for the positive control (no antagonist) and test wells were corrected for background by subtraction of any cells counted on the membrane in the negative control (no MCP-1 in the receiving chamber).
  • Percent inhibition of migration was determined by (Cell count in test wells) ⁇ 100/(Cell count in positive control)
  • the effect of lysophosphatidic acid stimulated the proliferation of human stellate cells in response to the LPAR1 antagonist, EPGN696 HSCs were isolated as previously described.
  • DMEM, fetal calf serum, antimycotic/antibiotic, Cyquant cell proliferation kit, and Ca 2+ /Mg 2+ -free PBS were from Thermofisher (Waltham, MA).
  • Oleoyl-LPA-sodium salt was from Cayman Chemicals (Ann Arbor, MI). Thirty percent fatty acid-free bovine serum albumin was purchased from Sigma (St. Louis, MO).
  • LPA was dissolved in Ca 2+ /Mg 2+ -free PBS containing 0.1% fatty acid free bovine serum albumin to give a 1 mM stock solution. The solution was sonicated for 15 minutes (min) before use.
  • EPGN696 was diluted from a 10 mM solution in DMSO. HSCs were thawed and cultured in high glucose DMEM with 10% fetal calf serum and antibiotic/antimycotic at 37°C, 5% CO2. Cells were used between passages 4 and 7. Cells were plated at 1000 or 2000 cells/well of a 96-well plate and incubated overnight. The following day media was replaced with that containing AQ60.5% fetal bovine serum for 24 hours.
  • media was replaced with 100 ⁇ L media containing 0.5% fetal bovine serum and 0.1% DMSO or EPGN696 at 1 ⁇ M, 0.1 ⁇ M, or 0.01 ⁇ M for 15 minutes, followed by the addition of a further 100 ⁇ L media containing 0.5% fetal bovine serum, vehicle, 20 ⁇ M LPA (2x final concentration) and/or 1 ⁇ M, 0.1 ⁇ M, and 0.01 ⁇ M EPGN696.
  • Cells were incubated for 48 hours before removing the media and freezing the plate at ⁇ 80°C overnight. According to the manufacturer’s instructions, the relative cell number per well was assessed using the Cyquant cell proliferation kit.
  • the Cyquant assay determines cell density via a dye that fluoresces when bound to nucleic acids.
  • LPAR1 antagonists in vitro pharmacology and ADME-PK properties Abbreviations: LPAR1, lysophosphatidic acid receptor 1; MCP-1, monocyte chemoattractant protein-1, pharmacokinetics.
  • the HFHC+2154+Sem and the chow-fed group had no significant difference in body weight.
  • the HFHC+2154+Sem group had shown maximum weight loss among the drug treatment groups (Figure 1E).
  • the HFHC+2154+Sem group has shown the lowest body weight among the drug treatment groups ( Figure 2A).
  • the body mass composition revealed that the HFHC+Sem+2154 group has the lowest fat mass percentage among the drug treatment groups.
  • the HFHC+2154+Sem group has no significant difference in fat mass percentage from the chow group ( Figure 2B).
  • the HFHC+Sem+2154 group has the highest lean mass percentage among the drug treatment groups.
  • the HFHC+2154+Sem group has no significant difference in lean mass percentage from the chow group ( Figure 2C).
  • EPGN2154 and Sema cause a reduction in the liver weight and liver-to-body weight ratio
  • Both EPGN2154 and Sema cause a reduction in the liver weight compared to the no- drug treatment group.
  • the HFHC+Sem group has the lowest liver weight compared to the other treatment groups (HFHC+Sem vs. HFHC (no-drug) vs. HFHC+2154 vs. HFHC+2154+Sem: 1.623 ⁇ 0.093 g vs.
  • the HFHC+Sem group has a lower liver-to-body weight ratio of mice compared to the other treatment groups (HFHC+Sem vs. HFHC (no-drug) vs. HFHC+2154 vs. HFHC+2154+Sem: 0.0372 ⁇ 0.001 vs. 0.0694 ⁇ 0.003, p ⁇ 0.0001, 0.060 ⁇ 0.003, p ⁇ 0.0001, 0.053 ⁇ 0.001, p ⁇ 0.0001).
  • HFHC+Sem and the chow group
  • EPGN2154 and Sema improve hepatic injury and liver physiology Both EPGN2154 and Sema reduced the plasma ALT concentration compared to that of the HFHC group (HFHC+2154 vs. HFHC (no-drug) vs. HFHC+Sem vs. HFHC+2154+Sem: 62.109 ⁇ 10.746 vs. 116.205 ⁇ 11.950, p ⁇ 0.001, vs. 45.043 ⁇ 4.033, p>0.05, vs. 39.517 ⁇ 8.924, p>0.05). No significant difference was observed in plasma ALT levels between the drug treatment and chow groups (Figure 2F).
  • EPGN2154 imparts protection from the progression of hepatic fibrosis
  • Histological analysis of Sirius Red–stained liver cross section revealed that 50% of mice in the HFHC group have advanced-stage hepatic fibrosis (Fibrosis Grade >2).
  • HFHC vs. HFHC+2154 vs. HFHC+Sem 50% vs. 10%, ⁇ 2 p ⁇ 0.0001; 10%, ⁇ 2 p ⁇ 0.0001, Figure 4B).
  • No advanced-stage fibrosis was observed in the liver sections of mice from the HFHC+2154+Sem groups.
  • HFHC+2154 mice group has lower hydroxyproline concentration in the liver compared to the HFHC group (HFHC+2154 vs. HFHC: 0.22 ⁇ 0.038 ng/ ⁇ L per mg liver vs. 0.51 ⁇ 0.082 ng/ ⁇ L per mg liver, p ⁇ 0.001) ( Figure 4C).
  • the drug treatment mice groups did not show any significant differences in the hydroxyproline concentration in the liver.
  • HFHC+2154 mice group has lower lysophosphatidic acid receptor 1 (LPAR1) expression in the liver compared to the HFHC group (HFHC+2154 vs. HFHC: 0.66 ⁇ 0.062 A.U. vs.1.30 ⁇ 0.277 A.U.) ( Figure 4D).
  • LPAR1 lysophosphatidic acid receptor 1
  • mice gain similar body weight when fed either chow or AMLN diet
  • Genetically modified obese mice ob/ob
  • mice either fed a chow diet or an amylin diet AMLN
  • mice have similar body weight
  • no significant difference in the weight gain was observed between the chow- and AMLN diet-fed group (chow: 25.3842 ⁇ 0.401 vs. AMLN: 28.076 ⁇ 1.588, Figure 5B).
  • EPGN2154 reduces the body weight in B6.
  • Cg-Lepob/J (ob/ob) mice After 8 weeks of drug treatment, mice in AMLN+2154, AMLN+2154+Sem, and AMLN+Sem groups have shown lower body weight than the chow+Veh group (AMLN+2154: 64.188 ⁇ 0.594 g, p ⁇ 0.001; AMLN+2154+Sem: 60.255 ⁇ 0.850 g; AMLN+Sem: 63.493 ⁇ 0.841 g vs. chow+Veh: 70.422 ⁇ 1.034 g) (Figure 5C).
  • mice in the AMLN+2154 group gained the least body weight compared to the AMLN+2154+Sem and AMLN+Sem groups (AMLN+2154: 4.789 ⁇ 1.040 g, p ⁇ 0.05; AMLN+2154+Sem: 6.080 ⁇ 0.810 g; AMLN+Sem: 6.833 ⁇ 0.926 g vs. chow+Veh: 7.822 ⁇ 0.956 g) ( Figure 5D).
  • EPGN2154 improves NAS in AMLN-fed ob/ob mice After 8 weeks of treatment, the liver weight of AMLN+2154, AMLN+2154+Sem, and AMLN+Sem mice groups decreased compared to the AMLN+Veh group (AMLN+2154: 5.861 ⁇ 0.224 g; AMLN+2154+Sem: 4.812 ⁇ 0.203 g; AMLN+Sem: 5.562 ⁇ 0.240 g vs. AMLN+Veh: 6.419 ⁇ 0.206 g) (Figure 6A).
  • the AMLN+2154, AMLN+2154+Sem, and AMLN+Sem mice groups have lower liver-to-body weight ratio (arbitrary unit, AU) compared to the 16-week sentinel AMLN-fed mice group (AMLN 16 week) (AMLN+2154: 0.091 ⁇ 0.003 AU, p ⁇ 0.05; AMLN+2154+Sem: 0.080 ⁇ 0.003 AU; AMLN+Sem: 0.088 ⁇ 0.004 AU vs. AMLN 16 week: 0.104 ⁇ 0.002) (Figure 6B).
  • FIG. 6C the histological analysis of the liver section revealed that AMLN+2154 mice have a lower inflammation and ballooning score than AMLN+Sem mice (Inflammation score: 1.571 ⁇ 0.202 (AMLN+2154) versus 2.3 ⁇ 0.153 (AMLN+Sem); p ⁇ 0.05; Ballooning score: 0.286 ⁇ 0.184 (AMLN+2154) vs. 0.7 ⁇ 0.213 (AMLN+Sem)).
  • AMLN+2154 mice have a lower NAS than AMLN+Veh and AMLN+Sem groups (AMLN+2154: 4.571 ⁇ 0.430 vs.
  • AMLN+Veh 6.250 ⁇ 0.250, p ⁇ 0.0001; vs. AMLN+Sem: 6.000 ⁇ 0.298, p ⁇ 0.0001).
  • AMLN+2154 and AMLM+2154+Sem groups (4.125 ⁇ 0.295)
  • AMLN+2154 group has a lower Oil Red O stain area than AMLN+Sem group ( Figure 6E).
  • AMLN+2154 has no significant difference with AMLN+2154+Sem in the Oil Red O stain area.
  • EPGN2154 lowers the incidence of advanced-stage hepatic fibrosis in AMLN fed ob/ob mice
  • Mice in AMLN+2154 group (14%) have shown a lower incidence of advanced-stage hepatic fibrosis (Fibrosis Score ⁇ 2) than AMLN+Veh (100%, ⁇ 2 p ⁇ 0.0001), AMLN+Sem (100%, ⁇ 2 p ⁇ 0.0001), and AMLN 16-week sentinel (33%, ⁇ 2 p ⁇ 0.01).
  • a similar incidence of advanced-stage hepatic fibrosis was observed in AMLN+2154+Sem compared to the AMLN+2154 group ( Figure 7A, B).
  • the AMLN+2154 mice have a lower concentration of hepatic hydroxyproline compared to AMLN+Veh, AMLN+2154+Sem, and AMLN+Sem groups (AMLN+2154: 0.72 ⁇ 0.018 ng/ ⁇ L per mg liver vs. AMLN+Veh: 0.81 ⁇ 0.035 ng/ ⁇ L per mg liver, p ⁇ 0.05; vs. AMLN+2154+Sem: 0.97 ⁇ 0.075 ng/ ⁇ L per mg liver, p ⁇ 0.01; vs. AMLN+2154+Sem: 0.89 ⁇ 0.038 ng/ ⁇ L per mg liver, p ⁇ 0.01) (Figure 7D).
  • AMLN+2154 mice did not significantly differ in the hepatic hydroxyproline concentration compared to the AMLN-fed 16-week sentinel group (0.81 ⁇ 0.092, p> 0.05).
  • LPAR1 antagonists, EPGN2154 and EPGN696 inhibit the migration of macrophages and the proliferation of HSCs
  • EPGN2154 and EPGN696 inhibited MCP-1–mediated RAW264.7 cell migration across a concentration range of 0.0001–10 ⁇ M ( Figures 7E, F).
  • the calculated IC 50 for this inhibition was 1.76 nM for EPGN2154 and 0.95 nM for EPGN696.
  • BMS-986278 an Oral Lysophosphatidic Acid Receptor 1 (LPA1) Antagonist, for Patients With Idiopathic Pulmonary Fibrosis: Results From a Phase 2 Randomized Trial. www.abstractsonline.com/pp8/#!/10703/presentation/12283. 18. Baeck C, et al. Gut.2012;61:416–26. 19. Chen X, et al. Front Physiol.2017;8:155. 20. Marcher AB, et al. Scientific Rep.2019;9:1–13. 21. Mederacke I, et al.
  • mice MIO diet-fed mice were randomly distributed into four experimental groups where one group did not receive the EGN2154 dose, and the other three experimental groups received 10mg/kg, 5mg/kg, and 1 mg/kg body weight dose of EPGN2154 for eight weeks.
  • mice were euthanized to evaluate the effect of different doses on the remission of MASH-related pathogenesis.
  • the MIO-fed mice have gained significantly higher body weight than chow-fed mice (Body weight- MIO: 51.44 ⁇ 0.40g vs. chow: 32.87 ⁇ 2.12g, P>0.0001 (Figure 8); Body weight change- MIO:27.42 ⁇ 0.34 vs chow:10.12 ⁇ 1.07, P ⁇ 0.0001 ( Figure 9)).
  • the 10mg/kg EPGN2154 dose group has the lowest body weight compared to the other doses and no drug groups (10mg/kg: 50.57 ⁇ 0.91g vs 5mg/kg: 53.18 ⁇ 1.04g; 1mg/kg: 54.06 ⁇ 0.84g, P ⁇ 0.05); MIO: 54.55 ⁇ 0.95g (P ⁇ 0.05), Figure 10).
  • There was a negative correlation between the EPGN2154 dose and the body weight of the mice ( Figure 11). When compared with the body weight at the start of the drug dose (week 16), it was observed that mice that received 10mg/kg EPGN2154 dose gained the least body weight (Body weight change- 10mg/kg: -1.91 ⁇ 0.43g vs.
  • the term “comprising” or “comprises” is used in reference to compositions, methods, and respective component(s) thereof, that are useful to an embodiment, yet open to the inclusion of unspecified elements, whether useful or not. It will be understood by those within the art that, in general, terms used herein are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.).
  • a subject may be a mammal and the mammal may be, for example, a human, and human subjects include adult, adolescent and pediatric subjects. Mammals also include, but are not limited to, farm animals, sport animals and pets (companion animals).
  • the term “about,” as used herein, means approximately, in the region of, roughly, or around. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 10%. In one aspect, the term “about” means plus or minus 20% of the numerical value of the number with which it is being used.
  • biological sample refers to a sample obtained from an organism (e.g., a human patient) or from components (e.g., cells) of an organism.
  • the sample may be of any biological tissue or fluid.
  • the sample may be a "clinical sample” which is a sample derived from a patient.
  • Such samples include, but are not limited to, sputum, blood, blood cells (e.g., white cells), amniotic fluid, plasma, semen, bone marrow, circulating tumor cells, circulating DNA, circulating exosomes, and tissue or fine needle biopsy samples, urine, peritoneal fluid, aqueous humor, and pleural fluid, or cells therefrom.
  • Biological samples may also include sections of tissues such as frozen sections or formalin fixed paraffin embedded sections akin for histological purposes.
  • a biological sample may also be referred to as a "patient sample.”
  • “health care provider” includes either an individual or an institution that provides preventive, curative, promotional or rehabilitative health care services to a subject, such as a patient.
  • the data is provided to a health care provider so that they may use it in their diagnosis/treatment of the patient.
  • standard refers to something used for comparison, such as control or a healthy subject. All references cited herein are incorporated by reference in their entirety as though fully set forth. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology 3 rd ed., Revised, J. Wiley & Sons (New York, NY 2006); March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 7 th ed., J.

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Abstract

Provided herein are compositions and methods to treat obesity related liver disease (e.g., MASH/NAFLD) comprising administering a combination comprising one or more lysophosphatidic acid receptor 1 (LPAR1) antagonists and one or more additional agents, such as a glucagon-like peptide- 1 receptor agonist, to a subject in need thereof.

Description

USES OF LYSOPHOSPHATIDIC ACID RECEPTOR 1 ANTAGONISTS PRIORITY This application claims the benefit of the filing date of U.S. provisional application No.63/548,511, filed on November 14, 2023, the disclosures of which is incorporated by reference herein in its entirety. GOVERNMENT SUPPORT This invention was made with Government support under Grant No. DK129071 awarded by the National Institute of Health (NIH). The Government has certain rights in this invention. BACKGROUND MASH (metabolic dysfunction–associated steatohepatitis), formerly known as NASH (Nonalcoholic steatohepatitis), is an advanced form of fatty liver disease (a condition in which the liver builds up excessive fat deposits) that can go undetected, as there may be no symptoms with the disease and even when symptoms are present, they may not clearly indicate MASH. This liver disease can worsen over time and bring potentially life-threatening consequences. With timely screening, detection, and management, it may be possible to stop or even reverse liver damage from MASH. SUMMARY Provided herein are methods to treat or prevent hepatic steatosis and/or fibrosis, comprising administering to a subject in need thereof an effective amount of a combination of a lysophosphatidic acid receptor 1 (LPAR1) antagonists and an additional active agent. In some aspects, the additional active agent is one or more a glucagon-like peptide-1 receptor agonist(s), including, by not limited to, semaglutide, tirzepatide, dulaglutide, exenatide, liraglutide, lixisenatide, and/or albiglutide, and/or one or more lipid lowering agent. In some aspects, the lipid lowering agent is a cholesterol absorption inhibitor, a statin, a PCSK9 inhibitor, an ACC inhibitor, an ApoC-III inhibitor, an ACL-inhibitor, fish oil, or a CETP inhibitor. In some aspects, the statin is atorvastatin, rosuvastatin, simvastatin, pravastatin, lovastatin, fluvastatin, or pitavastatin. In some aspects, the hepatic steatosis is non-alcoholic fatty liver disease (NAFLD) or metabolic dysfunction-associated steatohepatitis (MASH). Provided herein are methods to reduce the amount of liver fat or accumulation of liver fat in a subject at risk for liver fat accumulation comprising administering to a subject in need thereof an effective amount of a combination of a LPAR1 antagonist and an additional active agent. In some aspects, the additional active agent is one or more a glucagon-like peptide-1 receptor agonist(s), including, by not limited to, semaglutide, tirzepatide, dulaglutide, exenatide, liraglutide, lixisenatide, and/or albiglutide, and/or one or more lipid lowering agent. In some aspects, the lipid lowering agent is a cholesterol absorption inhibitor, a statin, a PCSK9 inhibitor, an ACC inhibitor, an ApoC-III inhibitor, an ACL-inhibitor, fish oil, or a CETP inhibitor. In some aspects, the statin is atorvastatin, rosuvastatin, simvastatin, pravastatin, lovastatin, fluvastatin, or pitavastatin. In some aspects, the subject has liver disease. In some aspects, the subject has hepatic steatosis, type IIb hyperlipidemia or familial combined hyperlipidemia. In some aspects, the hepatic steatosis is non-alcoholic fatty liver disease (NAFLD) or metabolic dysfunction-associated steatohepatitis (MASH). In some aspects, the subject's risk of developing liver disease is reduced. In some aspects, the liver disease is metabolic dysfunction-associated steatohepatitis (MASH), or nonalcoholic fatty liver disease (NAFLD), alcoholic hepatic steatosis or primary biliary cirrhosis. Provided herein are methods to reduce hepatic fibrosis in a patient comprising to a subject in need thereof an effective amount of a combination of a LPAR1 antagonist and an additional active agent. In some aspects, the additional active agent is one or more a glucagon- like peptide-1 receptor agonist(s), including, by not limited to, semaglutide, tirzepatide, dulaglutide, exenatide, liraglutide, lixisenatide, and/or albiglutide, and/or one or more lipid lowering agent. In some aspects, the lipid lowering agent is a cholesterol absorption inhibitor, a statin, a PCSK9 inhibitor, an ACC inhibitor, an ApoC-III inhibitor, an ACL-inhibitor, fish oil, or a CETP inhibitor. In some aspects, the statin is atorvastatin, rosuvastatin, simvastatin, pravastatin, lovastatin, fluvastatin, or pitavastatin. In preferred aspects, the LPAR1 antagonist can be EPGN696 and/or EPGN2154. In some aspects, the combination is administered daily or weekly. In some aspects, about 5 mg/kg to about 10 mg/kg body weight of the LPAR1 antagonist is administered. BRIEF DESCRIPTION OF THE FIGURES FIGS. 1A-1E. 1 EPGN2154 (WO 2019/246109 and US Pat No. 10,000459; incorporated herein by reference) and Sema combination therapy cause the maximum reduction in body weight. (A) EPGN2154 pharmacokinetics mean plasma concentration of EPGN2154 oral gavage (PO: 20 mg/kg) versus i.v. (1 mg/kg) over 24 hours. (B) Body weight of WT mice at week 16 on HFHC or chow diet. (C) Body weight change of WT mice from week 1 to week 16 when fed on HFHC or chow diet. (D) Body weight of WT mice from week 16 to week 24 during drug dosing. (E) Body weight change of WT mice from week 16 to week 24 during drug dosing. Mean± SEM. ****p<0.0001, ***p<0.001, **p< 0.01, *p< 0.05. Abbreviations: HFHC, high-fat, high-carbohydrate; Sema, semaglutide. FIGS.2A-2F.2 EPGN2154 and Sema provide hepato-protection in the HFHC-fed WT mice. (A) Body weight of mice at week 24. (B) Fat mass percentage of mice at week 24. (C) Lean mass percentage of mice at week 24. (D) Liver weight of mice at week 24. (E) Liver-to- body weight ratio of mice at week 24. (F) Plasma ALT concentration of mice at week 24. Mean± SEM. ****p<0.0001, ***p<0.001. Abbreviations: ALT, alanine transaminase; AU, arbitrary unit; HFHC, high-fat, high-carbohydrate; Sema, semaglutide. FIGS. 3A-3C. EPGN2154 and semaglutide improve hepatic injury and liver physiology. (A) Representative image of hematoxylin and eosin–stained liver cross-section of experimental groups, “#” sign identifies the central hepatic veins. (B) NAS of the liver cross- section. (C) Oil Red O stain area percentage on the liver section of experimental groups at week 24. Mean± SEM.
Figure imgf000004_0001
0.0001, ***p<0.001, **p<0.01, *p<0.05. Abbreviations: HFHC, high-fat, high-carbohydrate; NAS, NAFLD Activity Score; Sema, semaglutide. FIGS.4A-4D. EPGN2154 imparts protection from the progression of hepatic fibrosis. (A) Representative image of Sirius Red–stained liver cross section of experimental groups, “#” sign identifies the central hepatic veins. (B) Percentage incidence of advanced-stage fibrosis (Fibrosis Score>2) in the liver cross-section of the experimental groups at week 24. (C) Hepatic hydroxyproline concentration. and (D) Hepatic LPAR1 expression of the experimental groups at week 24. Chi-square has a significant p-value for all experimental groups compared to the HFHC group. Mean±SEM, ***p<0.001, *p<0.05. Abbreviations: HFHC, high-fat, high- carbohydrate; LPAR1, lysophosphatidic acid receptor 1; Sema, semaglutide. FIGS. 5A-5D. EPGN2154 and semaglutide reduce body weight in B6.Cg-Lepob/J (ob/ob) (A) Body weight of 6- to 8-week-old male ob/ob mice fed AMLN and chow diet for 16 weeks. (B) Body weight change of ob/ob mice fed AMLN and chow from week 0 to week 16. (C) Body weight of ob/ob mice fed on AMLN diet from week 16 to week 24 with or without drug treatment. (D) Body weight change of ob/ob mice fed on AMLN diet from week 16 to week 24 with or without drug treatment. Mean±SEM. Abbreviations: AMLN, Amylin liver NASH; HFHC, high-fat, high-carbohydrate; Sema, semaglutide. FIGS. 6A-6E. EPGN2154 improves hepatic fibrosis in AMLN-fed ob/ob mice. (A) Liver weight of ob/ob mice at week 24. (B) Liver-to-body weight ratio of ob/ob mice at week 24. (C) Representative image of hematoxylin and eosin–stained liver cross-section of experimental groups. (D) NAS of liver cross-section of experimental groups at week 24 NAS of the liver cross-section. (E) Oil Red O stain area percentage on the liver section of experimental groups at week 24. Mean ± SEM. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05. Abbreviations: AMLN, Amylin liver NASH; A.U., arbitrary unit; NAS, NAFLD Activity Score; Sema, semaglutide; Veh, vehicle. FIGS. 7A-7G. EPGN2154 improves hepatic fibrosis in AMLN-fed ob/ob mice by inhibiting macrophage migration and HSC proliferation. (A) Representative image of Sirius Red staining of liver cross-sections. (B) Hepatic fibrosis score of the liver cross-section of experimental groups; chi square has a significant p-value for all experimental groups compared to the AMLN+Veh group. (C) Immunohistochemistry of the liver cross section for αSMA, Gal- 3, Col1, and Lam. (D) Hepatic hydroxyproline concentration of the experimental groups. (E) EPGN696 (WO 2019/246109 and US Pat No. 10,000459; incorporated herein by reference) (IC50 = 0.95 nM) inhibits migration of MCP-1-treated RAW264.7 cells (mouse macrophages) in a dose-dependent manner. (F) EPGN2154 (IC50 = 1.76 nM) inhibits migration of MCP-1- treated RAW264.7 cells in a dose-dependent manner. (G) EPGN696 inhibits the LPA- stimulated proliferation of primary human HSCs in a dose-dependent manner, Mean± SEM, ****p<0.0001 ***p<0.001, **p<0.01, *p<0.05. Abbreviations: AMLN, Amylin liver NASH; Col1a1, collagen1a1; Gal-3, galectin-3; Lam, laminin; LPA, lysophosphatidic acid; MCP-1, monocyte chemoattractant protein‐1; αSMA, α-smooth muscle actin; Veh, vehicle. FIG.8. MASH-inducing diet (MIO) increases the body weight of mice.6-8 weeks old male C57/Bl6J mice (WT) fed a MASH-inducing diet (MIO) for 16 weeks weigh more than chow fed mice. Mean±Sem, **** P< 0.0001. FIG.9. MASH-inducing diet (MIO) induce body weight gain in mice.6-8 weeks old male C57/Bl6J mice (WT) fed a MASH-inducing diet (MIO) for 16 weeks gain more body weigh chow fed mice. Mean±Sem, **** P< 0.0001. FIG. 10. EPGN2154 lowers body weight. 6-8 weeks old male C57/Bl6J mice (WT) fed a MASH-inducing diet (MIO) for 16 weeks, received oral gavage of EPGN2154 doses for 8 weeks have lower body weight than MIO group. Mean±Sem, **** P< 0.0001, * P<0.05. FIG.11. EPGN2154 dose have negative correlation with body weight. 6-8 weeks old male C57/Bl6J mice (WT) fed a MASH-inducing diet (MIO) for 16 weeks, received oral gavage of EPGN2154 doses for 8 weeks. EPGN2154 dose have negative correlation with body weight. FIG.12.10mg/kg EPGN2154 dose cause maximum body weight loss. 6-8 weeks old male C57/Bl6J mice (WT) fed a MASH-inducing diet (MIO) for 16 weeks, received oral gavage of EPGN2154 doses for 8 weeks.10mg/kg dose of EPGN2154 caused maximum body weight loss than other groups. Mean±Sem, **** P< 0.0001, **P<0.01. FIG. 13. EPGN2154 have dose dependent effect on body weight change. 6-8 weeks old male C57/Bl6J mice (WT) fed a MASH-inducing diet (MIO) for 16 weeks, received oral gavage of EPGN2154 doses for 8 weeks. EPGN2154 have a dose dependent effect on the body weight change (P-value=0.025, two tailed). FIG.14. EPGN2154 have no effect on adiposity. 6-8 weeks old male C57/Bl6J mice (WT) fed a MASH-inducing diet (MIO) for 16 weeks and received oral gavage of EPGN2154 for 8 weeks did not reduce adiposity. Mean±Sem, **** P< 0.0001. FIG.15. EPGN2154 have no effect on lean mass. 6-8 weeks old male C57/Bl6J mice (WT) fed a MASH-inducing diet (MIO) for 16 weeks and received oral gavage of EPGN2154 for 8 weeks have no difference in lean mass percentage than MIO group. Mean±Sem, **** P< 0.0001. FIG. 16. EPGN2154 have no effect on food consumption. 6-8 weeks old male C57/Bl6J mice (WT) fed a MASH-inducing diet (MIO) for 16 weeks and received oral gavage of EPGN2154 for 8 weeks have no effect on food consumption. Mean±Sem. FIG. 17. EPGN2154 have no effect on water intake. 6-8 weeks old male C57/Bl6J mice (WT) fed a MASH-inducing diet (MIO) for 16 weeks and received oral gavage of EPGN2154 for 8 weeks have no effect on water intake. Mean±Sem. FIG.18. EPGN2154 improve liver injury. 6-8 weeks old male C57/Bl6J mice (WT) fed a MASH-inducing diet (MIO) for 16 weeks and received oral gavage of EPGN2154 at 10mg/kg and 5 mg/kg dose for 8 weeks have lower levels of plasma alanine transaminase (ALT) than MIO (no EPGN2154) group. Mean±Sem. *P <0.05. FIGS. 19A-19B. EPGN2154 treatment improved pathophysiology of the liver. 6-8 weeks old male WT mice fed a MIO diet for 16 weeks and received oral gavage of EPGN2154 for 8 weeks. (A) Representative image of the liver cross-section of each experimental group, (B) EPGN2154 treatment reduced the steatosis and ballooning score and overall NAFLD Activity Score (NAS) than the MIO (no EPGN2154) group. Mean±SEM. **** P< 0.0001, *** P< 0.001, * P < 0.05. FIGS.20A-20B. EPGN2154 inhibit expression of hepatic profibrotic genes. 6-8 weeks old male WT mice fed a MIO diet for 16 weeks and received oral gavage of EPGN2154 for 8 weeks. The qPCR of the liver of the mice revealed that EPGN2154 suppresses the expression of transforming growth factor β (TGFβ) target genes such as (A) collagen 1A2 (Col1A2) and (B) α-smooth muscle actin (αSMA) responsible for hepatic fibrosis. Mean±SEM. *** P< 0.001, ** P< 0.01, *P<0.05. FIG.21. EPGN2154 provide hepato-protection during MASH progression.6-8 weeks old male WT mice fed a MIO diet for 16 weeks and received oral gavage of EPGN2154 for 8 weeks. The RNASeq of the liver of the mice revealed that EPGN2154 suppresses the expression of genes responsible for hepatic stellate cell activation and hepatic fibrosis signaling. DESCRIPTION OF INVENTION Provided herein are methods to treat or prevent hepatic steatosis and/or fibrosis, comprising administering to a subject in need thereof an effective amount of a combination of a LPAR1 antagonist and an additional active agent. In some aspects, the additional active agent is one or more a glucagon-like peptide-1 receptor agonist(s), including, by not limited to, semaglutide, tirzepatide, dulaglutide, exenatide, liraglutide, lixisenatide, and/or albiglutide, and/or one or more lipid lowering agent. In some aspects, the lipid lowering agent is a cholesterol absorption inhibitor, a statin, a PCSK9 inhibitor, an ACC inhibitor, an ApoC-III inhibitor, an ACL-inhibitor, fish oil, or a CETP inhibitor. In some aspects, the statin is atorvastatin, rosuvastatin, simvastatin, pravastatin, lovastatin, fluvastatin, or pitavastatin. In some aspects, the LPAR1 antagonist comprises EPGN2154 and/or EPGN696. In some aspects, the hepatic steatosis is non-alcoholic fatty liver disease (NAFLD) or metabolic dysfunction- associated steatohepatitis (MASH). Provided herein are methods to reduce the amount of liver fat or accumulation of liver fat in a subject at risk for liver fat accumulation comprising administering to a subject in need thereof an effective amount of a combination of a LPAR1 antagonist and an additional active agent. In some aspects, the additional active agent is one or more a glucagon-like peptide-1 receptor agonist(s), including, by not limited to, semaglutide, tirzepatide, dulaglutide, exenatide, liraglutide, lixisenatide, and/or albiglutide, and/or one or more lipid lowering agent. In some aspects, the lipid lowering agent is a cholesterol absorption inhibitor, a statin, a PCSK9 inhibitor, an ACC inhibitor, an ApoC-III inhibitor, an ACL-inhibitor, fish oil, or a CETP inhibitor. In some aspects, the statin is atorvastatin, rosuvastatin, simvastatin, pravastatin, lovastatin, fluvastatin, or pitavastatin. Provided herein are methods to reduce hepatic fibrosis in a patient comprising to a subject in need thereof an effective amount of a combination of a LPAR1 antagonist and an additional active agent. In some aspects, the additional active agent is one or more a glucagon- like peptide-1 receptor agonist(s), including, by not limited to, semaglutide, tirzepatide, dulaglutide, exenatide, liraglutide, lixisenatide, and/or albiglutide, and/or one or more lipid lowering agent. In some aspects, the lipid lowering agent is a cholesterol absorption inhibitor, a statin, a PCSK9 inhibitor, an ACC inhibitor, an ApoC-III inhibitor, an ACL-inhibitor, fish oil, or a CETP inhibitor. In some aspects, the statin is atorvastatin, rosuvastatin, simvastatin, pravastatin, lovastatin, fluvastatin, or pitavastatin. Compounds/Active Agents The methods and compositions described herein comprise one or more lysophosphatidic acid receptor 1 antagonist, including, but not limited to, EPGN2154 and EPGN696. Lysophosphatidic acid receptor 1 (LPA1) antagonists include, but are not limited to, inhibhitory RNAs (to inhibit expression or LPA1), BMS-986020, Ki6198, KI 16425, RO 6842262, and CHI, as well as compounds from WO 2010/51053, such as:
Figure imgf000008_0001
. Lysophosphatidic acid receptor 1 (LPA1) antagonists include, but are not limited to, compounds disclosed in US Pat No.10,000,459 and 10,570,103 (which are incorporated herein by reference), such as a compound Formula I having the structure
Figure imgf000009_0001
or a pharmaceutically acceptable salt or prodrug thereof, wherein RA is —CO2H, —CO2RB, —CN, tetrazolyl, —C(═O)NH2, —C(═O)NHRB, — C(═O)NHSO2RB or —C(═O)NHCH2CH2SO3H or a carboxylic acid isostere; L1 is absent or substituted or unsubstituted C1-C6 alkylene, substituted or unsubstituted C1-C6 fluoroalkylene, substituted or unsubstituted C3-C8cycloalkylene, substituted or unsubstituted C1-C6 heteroalkylene, or —UV—Z—, wherein —UV— is defined by —OW—, —WO—, —N(RJ)W—, —WN(RJ)—, —N(RJ)C(═O)—, —SW—, —S(═O)nW—, or — C(═O)N(RJ)—, wherein W is substituted or unsubstituted C1-C3 alkylene, or W is —C(RL)2— ; Z is substituted or unsubstituted C1-C6 alkylene, substituted or unsubstituted C3- C8 cycloalkylene, or C1-C6 fluoroalkylene or Z is —C(RL)2—; and n is 0, 1, or 2; L2 is absent, or substituted or unsubstituted C1-C6 alkylene, substituted or unsubstituted C3-C8 cycloalkylene, C1-C6 fluoroalkylene, substituted or unsubstituted C3-C8 cycloalkylene, substituted or unsubstituted C1-C6 heteroalkylene, —O—, —S—, —SO—, —SO2—, —NRJ— , —C(═O)—, or —C(═O)N(RJ)—; wherein RB is substituted or unsubstituted C1-C4 alkyl, or has the structure of one of:
Figure imgf000009_0002
Ring A is a 5 or 6 membered heteroarene having the structure of one of:
wherein the dashed line indicates the point of attachment of Ring A to Ring B; wherein one of RC and RD is —H, —CN, —F, —Cl, —Br, —I, —OC1-C4 alkyl, C1- C4 alkyl, C3-C6cycloalkyl, or C1-C4 fluoroalkyl, and the other RC or RD is —NRFC(═O)XCH(RG)—CY, —N(RF)C(═O)XC(RG)2—CY, or —NRFC(═O)X—CY, —C(═O)—N(RF)—CH(RG)X—CY, or —C(═O)—N(RF)— C(RG)2X—CY, wherein X is absent, —O—, —NH— or —CH2-; RE is —H, —C1-C4 alkyl or —C1-C4 fluoroalkyl, RF is —H or C1-C4 alkyl, and RG is independently selected RE or one RG is C1-C4 alkyl and is taken together with CY and the carbon atom to which RG and CY is attached to define a substituted or unsubstituted carbocycle or substituted or unsubstituted heterocycle and the other RG, if present, is as defined for RE; wherein CY is substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3- C10 cycloalkyl, substituted or unsubstituted C2-C10 heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, wherein if CY is substituted then CY is substituted with 1, 2, or 3 independently selected RH, RH is independently —H, halogen, —CN, —NO2, —OH, —ORJ, —SRJ, —S(═O)RJ, —S(═O)2RJ, —N(RJ)S(═O)2RJ, —S(═O)2N(RL)2, —C(═O)RJ, OC(═O)RJ, —CO2RJ, — OCO2RJ, —N(RL)2, —C(═O)N(RL)2, —OC(═O)N(RL)2, N(RJ)C(═O)N(RL)2, — N(RJ)C(═O)RJ, —N(RJ)C(═O)ORJ, C1-C4 alkyl, C1-C4 fluoroalkyl, C1-C4 fluoroalkoxy, C1- C4 alkoxy, or C1-C4 heteroalkyl, wherein each RJ is independently substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 heteroalkyl, substituted or unsubstituted C1-C6 fluoroalkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, —C1-C4 alkylene-(substituted or unsubstituted C3-C6 cycloalkyl), —C1-C4 alkylene-(substituted or unsubstituted heterocycloalkyl), —C1-C4 alkylene-(substituted or unsubstituted aryl), and —C1-C4 alkylene- (substituted or unsubstituted heteroaryl), and wherein each RL is independently —H, C1-C6 alkyl, C1-C6 heteroalkyl, C1- C6 fluoroalkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, — C1-C4 alkylene-(substituted or unsubstituted C3-C6 cycloalkyl), —C1-C4 alkylene-(substituted or unsubstituted heterocycloalkyl), —C1-C4 alkylene(substituted or unsubstituted aryl), or — C1-C4 alkylene-(substituted or unsubstituted heteroaryl), or when RH is —S(═O)2N(RL)2, —N(RL)2, —C(═O)N(RL)2, —OC(═O)N(RL)2 or — N(RJ)C(═O)N(RL)2, each RL is independently —H or C1-C6 alkyl, or the RL groups independently are C1-C6 alkyl which are taken together with the N atom to which they are attached to define a substituted or unsubstituted heterocycle, or when W or Z is —C(RL)2— each RL is independently —H, C1-C6 alkyl, or the RL groups independently are C1-C6 alkyl which are taken together with the carbon atom to which they are attached to define a carbocycle; Ring B is substituted or unsubstituted C3-C10 cycloalkylene, substituted or unsubstituted C2-C10 heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene, where if ring B is substituted then ring B is substituted with 1, 2, or 3 independently selected RH, wherein RH is as previously defined; and Ring C is absent or substituted or unsubstituted C3-C10 cycloalkylene, substituted or unsubstituted C2-C10 heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene, wherein if ring C is substituted then ring C is substituted with 1, 2, or 3 independently selected RH, wherein RH is as previously defined, wherein when Ring B is substituted or unsubstituted arylene, Ring C is absent, L2 is absent, L1 is —UV—Z, wherein —N(RJ)C(═O)—, wherein RF is —H, RD is — N(RF)C(═O)XCH(RG)—CY, wherein X is —O—, RG is —CH3 and RF is —H, and RC is —H, —CH3 or —CF3, or when Ring B is substituted or unsubstituted arylene and Ring C is substituted or unsubstituted arylene or is substituted or unsubstituted C3-C10 cycloalkylene, or Ring B is substituted or unsubstituted C3-C10 cycloalkylene and Ring C is substituted or unsubstituted arylene, L2 is absent, L1 is C1-C6 alkylene, and RC is —H or —CH3 and RA is —CO2H or —CO2RB, then Ring A has the structure of one of:
and when Ring B is C2-C10 heterocycloalkylene, Ring C is substituted or unsubstituted arylene, L2 is absent, L1 is C1-C6 alkylene, RC is —CH3 and RA is —CO2H or —CO2RB, then Ring A has the structure of one of:
In some embodiments RC is —H, —CN, —F, —Cl, —Br, —I, —OC1-C4 alkyl, C1- C4 alkyl, C3-C6cycloalkyl, or C1-C4 fluoroalkyl and RD is —N(RF)—C(═O)XCH(RG)—CY, — N(RF)—C(═O)XC(RG)2—CY or —N(RF)—C(═O)X—CY, wherein RF and each RG independently are —H or C1-C4 alkyl. In some embodiments RA is —CO2H, —CO2RB, —CN, tetrazolyl, —C(═O)NH2, — C(═O)NHRB, C(═O)NHSO2RB or —C(═O)NHCH2CH2SO3H or a carboxylic acid isostere. In preferred embodiments RA is —CO2H, —CO2RB, —CN, or —C(═O)NHSO2RB, wherein RB is substituted or unsubstituted C1-C4 alkyl or has the structure of one of:
In some embodiments L1 is absent or substituted or unsubstituted C1-C6 alkylene, C1- C6 fluoroalkylene, or substituted or unsubstituted C1-C6 heteroalkylene. In some embodiments L1 is absent or substituted or unsubstituted C1-C6 alkylene or — UV—Z—, wherein —UV— is defined by —OW—, —WO—, —N(RJ)W—, —WN(RJ)—, — N(RJ)C(═O)—, —SW—, —S(═O)nW—, or —C(═O)N(RJ)—, wherein W is substituted or unsubstituted C1-C3 alkylene, Z is substituted or unsubstituted C1-C6 alkylene or C1- C6 fluoroalkylene; and n is 0, 1, or 2. In some embodiments L1 is —CH2—,
Figure imgf000015_0001
dimethylmethane (i.e., —C(CH3)2—), or —UV—Z— wherein —UV— is defined by —WO— , —WN(RJ)—, or —C(═O)N(RJ)—, wherein W is substituted or unsubstituted C1-C3 alkylene; and Z is substituted or unsubstituted C1-C6 alkylene. In some embodiments L2 is absent, or substituted or unsubstituted C1-C6 alkylene, C1- C6 fluoroalkylene, substituted or unsubstituted C1-C6 heteroalkylene, —O—, —S—, — S(═O)—, S(═O)2—, —N(RB)—, or —C(═O)—. In some embodiments L2 is absent, —O—, —S—, —S(═O)—, S(═O)2—, —N(RJ)—, or —C(═O)—. In some embodiments Ring A is a 5 or 6 membered heteroarene having one of the structures of:
In some embodiments, Formula I compounds have RC defined as —H, —CN, —F, — Cl, —Br, —I, —OC1-C4 alkyl, C1-C4 alkyl, C3-C6 cycloalkyl, or C1-C4 fluoroalkyl. In some embodiments, Formula I compounds have RC defined as —H, —F, —CN, — CH3, or —CF3. In some embodiments, Formula I compounds have RD defined as —N(RF)C(═O)— XCH(RG)—CY, —N(RF)C(═O)XC(RG)2—CY, or —N(RF)C(═O)X—CY, wherein X is absent, —O—, —NH— or —CH2—, wherein RF is —H or C1-C4 alkyl and X, CY and RG are as previously defined. In some embodiments, Formula I compounds have RD defined as — N(RF)C(═O)OCH(RG)—CY, —N(RF)C(═O)NHC(RG)—CY, or —N(RF)C(═O)CH2—CY, wherein RF is —H or C1-C4 alkyl and X, CY and RG are as previously defined. In some embodiments, Formula I compounds have RE defined as —H or C1-C4 alkyl, C1-C6 cycloalkyl or C1-C4 fluoroalkyl. In some embodiments, Formula I compounds have RE defined as —H, —CH3, cyclopropyl or —CF3. In some embodiments, Formula I compounds have RF defined as H, C1-C4 alkyl or C3- C6 cycloalkyl. In some embodiments, Formula I compounds have RF defined as —H. In some embodiments of Formula I compounds one RG is —C1-C4 alkyl and is taken together with CY and the carbon atom to which RG and CY is attached to define a substituted or unsubstituted carbocycle or a substituted or unsubstituted heterocycle and the other RG, if present is —H. In other embodiments of Formula I compounds RG is independently —H or C1-C4 alkyl. In some embodiments of Formula I compounds Ring B is substituted or unsubstituted C3-C10 cycloalkylene, substituted or unsubstituted C2-C10 heterocycloalkylene, a substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene, wherein if ring B is substituted then ring B is substituted with 1, 2, or 3 independently selected RH. In some embodiments of Formula I compounds Ring C is substituted or unsubstituted C3-C10 cycloalkylene, substituted or unsubstituted C2-C10 heterocycloalkylene, a substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene, wherein if ring C is substituted then ring C is substituted with 1, 2, or 3 independently selected RH. In some embodiments of Formula I compounds CY is substituted or unsubstituted C1- C6 alkyl, substituted or unsubstituted C3-C10 cycloalkyl, substituted or unsubstituted C2- C10 heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, wherein if CY is substituted then CY is substituted with 1, 2, or 3 independently selected RH. In some embodiments Ring A has the structure of one of:
In some embodiments, Formula I compounds have Ring B and Ring C each independently defined as 1,4-substituted aryl or heteroaryl, RA is —CO2H, RC is —F or —CN,
Figure imgf000018_0001
are as previously defined. In some embodiments, Formula I compounds have Ring B defined as 1,4-substituted aryl or heteroaryl, L1 is —UV—Z— wherein —UV— is defined by —WO—, —WN(RJ)—, or —C(═O)N(RJ)—, wherein W is CH2, Z is substituted or unsubstituted C1-C6 alkylene, RA is —CO2H, RD is —N(RF)C(═O)OCH(RG)—CY, RE is —CH3, and RC, RF, RG, and CY are as previously defined. Lysophosphatidic acid receptor 1 (LPA1) antagonists include, but are not limited to, compounds disclosed in WO 2019/246109 (which is incorporated herein by reference), such as: a compound of Formula II having the structure:
Figure imgf000018_0002
Formula II or a pharmaceutically acceptable salt or prodrug thereof, wherein RA is -CO2H, -CO2RB, -CN, tetrazolyl, -C(=O)NH2, -C(=O)NHRB, C(=O)NHSO2RB or -C(=O)NHCH2CH2SO3H or has the structure; RB is optionally substituted C1-C4 alkyl or has the structure of one of
Figure imgf000019_0001
wherein RB is substituted or unsubstituted C1-C4 alkyl; L1 is substituted or unsubstituted C1-C6 alkylene, substituted or unsubstituted C1- C6 fluoroalkylene, substituted or unsubstituted C3-C8 cycloalkylene, substituted or unsubstituted C1-C6 heteroalkylene; wherein A1 is -N= or -CH; wherein Ring A has the structure of one of
Figure imgf000019_0002
wherein Rc is -CN, -F, -Cl, -Br, -I, -OC1-C4 alkyl, C3-C6 cycloalkyl, or C1- C4 fluoroalkyl; and RD is -N(RF)-C(=O)XCH(RG)-CY, wherein X is O and CY is phenyl substituted with one RH:
Figure imgf000019_0003
RE, RF and RG independently are -H or C1-C4 alkyl or C3-C6 cycloalkyl or RE and RF independently are -H or C1-C4 alkyl or C1-C6 cycloalkyl and one RG is -C1-C4 alkyl and is taken together with the RH phenyl moiety of the Ring A RD substituent and the carbon atom to which RG and said phenyl moiety is attached to define a substituted or unsubstituted carbocycle or a substituted or unsubstituted heterocycle; RH is independently -H, halogen, -CN, -NO2, -OH, C1-C4 alkyl, C1-C4 fluoroalkyl, C1- C4 fluoroalkoxy, or C1-C4 alkoxy, In some embodiments Rc is -CN, -F, -Cl, -Br, -I, -OC1-C4 alkyl, C1-C4 alkyl, C3- C6 cycloalkyl, or C1-C4 fluoroalkyl and RD is -N(RF)-C(=O)OCH(RG)-CY, wherein RF and each RG independently are -H or C1-C4 alkyl. In some embodiments RA is -CO2H, -CONHCN, tetrazolyl, or -C(=O)NHSO2Rb, wherein RB is substituted or unsubstituted C1-C4 alkyl. In some embodiments RA is -CO2H, -CONHCN, tetrazolyl, or -C(=O)NHSO2Rb, wherein RB is -CH3. In some embodiments U is substituted or unsubstituted C1-C6 alkylene, C1-C6 fluoroalkylene, or substituted or unsubstituted C1-C6 heteroalkylene. In some embodiments U is
Figure imgf000020_0001
. In some embodiments, Formula II compounds have Rc defined as -CN, -F, -Cl, or C1- C4 fluoroalkyl. In some embodiments, Formula II compounds have Rc defined as -F, or -Cl. In some embodiments, Formula II compounds have RD defined as -N(RF)C(=O)- OCH(RG)-CY, wherein RF is -H or C1-C4 alkyl and X, CY and RG are as previously defined. In some embodiments, Formula II compounds have RD defined as - N(RF)C(=O)OCH(RG)-CY, wherein RF is -H and CY and RG are as previously defined. In some embodiments, Formula II compounds have RF defined as H, C1-C4 alkyl or C3- C6 cycloalkyl. In some embodiments, Formula II compounds have RF defined as -H. In some embodiments of Formula II compounds RG is independently -H or C1-C4 alkyl. In some embodiments, Formula II compounds have RG defined as -CH3. In some embodiments of Formula II compounds CY is substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, wherein if CY is substituted then CY is substituted with 1, 2, or 3 independently selected RH. In some embodiments Formula II compounds have RA is -CO2H, Rc is -F or -Cl, RD is -NRFC(=O)OCH(RG)-CY, and RF, RG, and CY are as previously defined. In some embodiments Formula II compounds RA is tetrazolyl, Rc is -F or -Cl, RD is - NRFC(=O)OCH(RG)-CY, and RF, RG, and CY are as previously defined. In some embodiments Formula II compounds RA is -C(=O)NHSCH2RB, Rc is -F or -Cl, RD is -NRFC(=O)OCH(RG)-CY, and RB, RF, RG, and CY are as previously defined. In some embodiments Formula II compounds RA is -CONHCN, Rc is -F or -Cl, RD is - NRFC(=O)OCH(RG)-CY, and RF, RG, and CY are as previously defined. In some embodiments, the LPA1R antagonist is as depicted in the below table:
Figure imgf000022_0001
Figure imgf000023_0001
Additional compounds One or more lysophosphatidic acid receptor 1 (LPA1) antagonists can be administered alone or in combination with one or more additional active agents. Additional active agents, include, but are not limited to, lipid lower agents, such as statins (a class of drugs that inhibit the enzyme HMG-CoA reductase and are generally known to lower LDL cholesterol in patients). Examples of statins include atorvastatin, rosuvastatin, simvastatin, pravastatin, lovastatin, fluvastatin, and pitavastatin. Lipid lowering agents can also include a cholesterol absorption inhibitor, a PCSK9 inhibitor, an ACC inhibitor, an ApoC-III inhibitor, an ACL- inhibitor, fish oil, or a CETP inhibitor. Other additional active agents include, but are not limited to, glucagon-like peptide-1 receptor agonist(s), including, by not limited to, semaglutide, tirzepatide, dulaglutide, exenatide, liraglutide, lixisenatide, and/or albiglutide. Chemical Definitions “Bond” or “single bond” as used herein means a chemical bond between two atoms, or two moieties when the atoms joined by the bond are considered to be part of larger substructure. As explicitly stated or implied by context, when a group described herein is a bond, the referenced group is absent thereby allowing a bond to be formed between the remaining identified groups. “Membered ring” as used herein means any cyclic structure. The term “membered” is meant to denote the number of skeletal atoms that constitute the ring. Thus, by way of example and not limitation, those membered rings include cyclohexyl, pyridinyl, pyranyl and thiopyranyl, which are 6-membered rings and cyclopentyl, pyrrolyl, furanyl, and thienyl, which are 5-membered rings. “Moiety” as used herein means a specific segment, fragment or functional group of a molecule or compound. Chemical moieties are sometimes indicated as chemical entities that are embedded in or appended (i.e., a substituent or variable group) to a molecule or compound. “Alkyl” as used herein is a collection of carbon atoms that are covalently linked together in normal, secondary, tertiary or cyclic arrangements, i.e., in a linear, branched, cyclic arrangement or some combination thereof. An alkyl substituent to a structure is that chain of carbon atoms that is covalently attached to the structure through a sp3 carbon of the substituent. The alkyl substituents, as used herein, contains one or more saturated moieties or groups and may additionally contain unsaturated alkyl moieties or groups, i.e., the substituent may comprise one, two, three or more independently selected double bonds or triple bonds of a combination thereof, typically one double or one triple bond if such unsaturated alkyl moieties or groups are present. Unsaturated alkyl moieties or groups include moieties or groups as described below for alkenyl, alkynyl, cycloalkyl, and aryl moieties. Saturated alkyl moieties contain saturated carbon atoms (sp3) and no aromatic, sp2 or sp carbon atoms. The number of carbon atoms in an alkyl moiety or group can vary and typically is 1 to about 50, e.g., about 1-30 or about 1-20, unless otherwise specified, e.g., C1-8 alkyl or C1-C8 alkyl means an alkyl moiety containing 1, 2, 3, 4, 5, 6, 7 or 8 carbon atoms and C1-C6 alkyl or C1-C6 means an alkyl moiety containing 1, 2, 3, 4, 5 or 6 carbon atoms. When an alkyl substituent, moiety or group is specified, species may include methyl, ethyl, 1-propyl (n-propyl), 2-propyl (iso-propyl, —CH(CH3)2), 1-butyl (n-butyl), 2-methyl-1- propyl (iso-butyl, —CH2CH(CH3)2), 2-butyl (sec-butyl, —CH(CH3)CH2CH3), 2-methyl-2- propyl (t-butyl, —C(CH3)3), amyl, isoamyl, sec-amyl and other linear, cyclic and branch chain alkyl moieties. Unless otherwise specified, alkyl groups can contain species and groups described below for cycloalkyl, alkenyl, alkynyl groups, aryl groups, arylalkyl groups, alkylaryl groups and the like. Cycloalkyl as used here is a monocyclic, bicyclic or tricyclic ring system composed of only carbon atoms. The term “cycloalkyl” encompasses a monocyclic or polycyclic aliphatic, non-aromatic radical, wherein each of the atoms forming the ring (i.e. skeletal atoms) is a carbon atom. The number of carbon atoms in an cycloalkyl substituent, moiety or group can vary and typically is 3 to about 50, e.g., about 1-30 or about 1-20, unless otherwise specified, e.g., C3-8 alkyl or C3-C8 alkyl means an cycloalkyl substituent, moiety or group containing 3, 4, 5, 6, 7 or 8 carbon atoms and C3-6 alkyl or C3-C6 means an cycloalkyl substituent, moiety or group containing 3, 4, 5 or 6 carbon atoms. Cycloalkyl substituents, moieties or groups will typically have 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms and may contain exo or endo-cyclic double bonds or endo-cyclic triple bonds or a combination of both wherein the endo-cyclic double or triple bonds, or the combination of both, do not form a cyclic conjugated system of 4n+2 electrons; wherein the bicyclic ring system may share one (i.e., spiro ring system) or two carbon atoms and the tricyclic ring system may share a total of 2, 3 or 4 carbon atoms, typically 2 or 3. Unless otherwise specified, cycloalkyl substituents, moieties or groups can contain moieties and groups described for alkenyl, alkynyl, aryl, arylalkyl, alkylaryl and the like and can contain one or more other cycloalkyl moieties. Thus, cycloalkyls may be saturated, or partially unsaturated. Cycloalkyls may be fused with an aromatic ring, and the points of attachment to the aromatic ring are at a carbon or carbons of the cycloalkyl substituent, moiety or group that is not an aromatic ring carbon atom. Cycloalkyl groups include groups having from 3 to 10 ring atoms. Cycloalkyl substituents, moieties or groups include cyclopropyl, cyclopentyl, cyclohexyl, adamantly or other cyclic all carbon containing moieties. Cycloalkyls further include cyclobutyl, cyclopentenyl, cyclohexenyl, cycloheptyl and cyclooctyl. Cycloalkyl groups may be substituted or unsubstituted. Depending on the substituent structure, a cycloalkyl substituent can be a monoradical or a diradical (i.e., a cycloalkylene, such as, but not limited to, cyclopropan-1,1-diyl, cyclobutan-1,1-diyl, cyclopentan-1,1-diyl, cyclohexan- 1,1-diyl, cyclohexan-1,4-diyl, cycloheptan-1,1-diyl, and the like). When cycloalkyl is used as a Markush group (i.e., a substituent) the cycloalkyl is attached to a Markush formula with which it is associated through a carbon involved in a cyclic carbon ring system carbon of the cycloalkyl group that is not an aromatic carbon. “Alkylamine” as used herein means an —N(alkyl)xHy group, moiety or substituent where x and y are independently selected from the group x=1, y=1 and x=2, y=O. Alkylamine includes those —N(alkyl)xHy groups wherein x=2 and y=0 and the alkyl groups taken together with the nitrogen atom to which they are attached form a cyclic ring system. “Heteroalkylene” as used herein means an alkylene (i.e. alkanediyl) group, moiety or substituent in which one or more skeletal atoms of the alkyl are selected from an atom other than carbon, e.g., oxygen, nitrogen, sulfur, phosphorus or combinations thereof. Heteroalkylene includes C1-C6 heteroalkylene or C1-C4 heteroalkylene. Exemplary heteroalkylenes include, but are not limited to, —OCH2—, —OCH(CH3)—, —OC(CH3)2—, —OCH2CH2—, —CH2O—, —CH(CH3)O—, C(CH3)2O—, —CH2CH2O—, —CH2OCH2—, —CH2OCH2CH2—, —CH2CH2OCH2—, —SCH2—, —SCH(CH3)—, —SC(CH3)2—, — SCH2CH2—, —CH2S—, —CH(CH3)S—, —C(CH3)2S—, —CH2CH2S—, —CH2SCH2—, — CH2SCH2CH2—, —CH2CH2SCH2—, —S(═O)2CH2—, —S(═O)2CH(CH3)—, — S(═O)2C(CH3)2—, —S(═O)2CH2CH2—, —CH2S(═O)2—, —CH(CH3)S(═O)2—, — C(CH3)2S(═O)2—, —CH2CH2S(═O)2—, —CH2S(═O)2CH2—, —CH2S(═O)2CH2CH2—, CH2CH2S(═O)2CH2—, —NHCH2—, —NHCH(CH3)—, —NHC(CH3)2—, —NHCH2CH2—, —CH2NH—, —CH(CH3)NH—, —C(CH3)2NH—, —CH2CH2NH—, —CH2NHCH2—, — CH2NHCH2CH2—, —CH2CH2NHCH2—, and the like. “Carboxylic acid bioisostere” as used herein means a functional group, moiety or substituent that exhibits similar physical, biological and/or chemical properties as a carboxylic acid moiety. By way of example and not limitation, carboxylic acid bioisosteres include,
Figure imgf000026_0001
“Alkenyl” as used herein means a substituent, moiety or group that comprises one or more double bond moieties (e.g., —CH═CH—) or 1, 2, 3, 4, 5 or 6 or more, typically 1, 2 or 3 such moieties and can include an aryl moiety or group such as benzene, and additionally comprises linked normal, secondary, tertiary or cyclic carbon atoms, i.e., linear, branched, cyclic or any combination thereof unless the alkenyl moiety is a vinyl moiety (e.g., — CH═CH2). An alkenyl moiety, group or substituent with multiple double bonds may have the double bonds arranged contiguously (i.e. a 1,3 butadienyl moiety) or non-contiguously with one or more intervening saturated carbon atoms or a combination thereof, provided that a cyclic, contiguous arrangement of double bonds do not form a cyclically conjugated system of 4n+2 electrons (i.e., aromatic). The number of carbon atoms in an alkenyl group or moiety can vary and typically is 2 to about 50, e.g., about 2-30 or about 2-20, unless otherwise specified, e.g., C2-8 alkenyl or C2-8 alkenyl means an alkenyl moiety containing 2, 3, 4, 5, 6, 7 or 8 carbon atoms and C2-6 alkenyl or C2-6 alkenyl means an alkenyl moiety containing 2, 3, 4, 5 or 6 carbon atoms. Alkenyl moieties or groups will typically have 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms. When an alkenyl moiety, group or substituent is specified, species include, by way of example and not limitation, any of the alkyl or cycloalkyl, groups moieties or substituents described herein that has one or more double bonds, methylene (═CH2), methylmethylene (═CH—CH3), ethylmethylene (═CH—CH2—CH3), ═CH—CH2—CH2—CH3, vinyl (— CH═CH2), allyl, 1-methylvinyl, butenyl, iso-butenyl, 3-methyl-2-butenyl, 1-pentenyl, cyclopentenyl, 1-methyl-cyclopentenyl, 1-hexenyl, 3-hexenyl, cyclohexenyl and other linear, cyclic and branched chained all carbon containing moieties containing at least one double bond. When alkenyl is used as a Markush group (i.e., a substituent) the alkenyl is attached to a Markush formula with which it is associated through an unsaturated carbon of a double bond of the alkenyl moiety or group unless specified otherwise. “Alkynyl” as used herein means a substituent, moiety or group that comprises one or more triple bond moieties (i.e., —C≡C—), e.g., 1, 2, 3, 4, 5, 6 or more, typically 1 or 2 triple bonds, optionally comprising 1, 2, 3, 4, 5, 6 or more double bonds, with the remaining bonds (if present) being single bonds and comprising linked normal, secondary, tertiary or cyclic carbon atoms, i.e., linear, branched, cyclic or any combination thereof, unless the alkynyl moiety is ethynyl. The number of carbon atoms in an alkenyl moiety or group can vary and typically is 2 to about 50, e.g., about 2-30 or about 2-20, unless otherwise specified, e.g., C2- 8 alkynyl or C2-8 alkynyl means an alkynyl moiety containing 2, 3, 4, 5, 6, 7 or 8 carbon atoms. Alkynyl groups will typically have 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms. When an alkynyl moiety or group is specified, species include, by way of example and not limitation, any of the alkyl moieties, groups or substituents described herein that has one or more double bonds, ethynyl, propynyl, butynyl, iso-butynyl, 3-methyl-2-butynyl, 1- pentynyl, cyclopentynyl, 1-methyl-cyclopentynyl, 1-hexynyl, 3-hexynyl, cyclohexynyl and other linear, cyclic and branched chained all carbon containing moieties containing at least one triple bond. When an alkynyl is used as a Markush group (i.e., a substituent) the alkynyl is attached to a Markush formula with which it is associated through one of the unsaturated carbons of the alkynyl functional group. “Aromatic” as used herein refers to a planar ring having a delocalized pi-electron system containing 4n+2 pi electrons, where n is a positive integer. Aromatic rings can be formed from five, six, seven, eight, nine, ten, or more than ten atoms. Aromatics are optionally substituted. The term “aromatic” includes both carboxcylic aryl (“aryl”, e.g., phenyl) and heterocyclic aryl (or “heteroaryl” or “heteroaromatic”) groups (e.g., pyridine). The term includes monocyclic or fused-ring polycyclic (i.e., rings which share adjacent pairs of carbon atoms) groups. “Aryl” as used here means an aromatic ring system or a fused ring system with no ring heteroatoms comprising 1, 2, 3 or 4 to 6 rings, typically 1 to 3 rings, wherein the rings are composed of only carbon atoms; and refers to a cyclically conjugated system of 4n+2 electrons (Huckel rule), typically 6, 10 or 14 electrons some of which may additionally participate in exocyclic conjugation (cross-conjugated (e.g., quinone). Aryl substituents, moieties or groups are typically formed by five, six, seven, eight, nine, or more than nine, carbon atoms. Aryl substituents, moieties or groups are optionally substituted. Exemplary aryls include C6- C10 aryls such as phenyl and naphthalenyl and phenanthryl. Depending on the structure, an aryl group can be a monoradical or a diradical (i.e., an arylene group). Exemplary arylenes include, but are not limited to, phenyl-1,2-ene, phenyl-1,3-ene, and phenyl-1,4-ene. When aryl is used as a Markush group (i.e., a substituent) the aryl is attached to a Markush formula with which it is associated through an aromatic carbon of the aryl group. “Arylalkyl” as used herein means a substituent, moiety or group where an aryl moiety is bonded to an alkyl moiety, i.e., -alkyl-aryl, where alkyl and aryl groups are as described above, e.g., —CH2—C6H5 or —CH2CH(CH3)—C6H5. When arylalkyl is used as a Markush group (i.e., a substituent) the alkyl moiety of the arylalkyl is attached to a Markush formula with which it is associated through a sp3 carbon of the alkyl moiety. “Alkylaryl” as used herein means a substituent, moiety or group where an alkyl moiety is bonded to an aryl moiety, i.e., -aryl-alkyl, where aryl and alkyl groups are as described above, e.g., —C6H4—CH3 or —C6H4—CH2CH(CH3). When alkylaryl is used as a Markush group (i.e., a substituent) the aryl moiety of the alkylaryl is attached to a Markush formula with which it is associated through a sp2 carbon of the aryl moiety. “Substituted alkyl”, “substituted cycloalkyl”, “substituted alkenyl”, “substituted alkynyl”, substituted alkylaryl”, “substituted arylalkyl”, “substituted heterocycle”, “substituted aryl” and the like as used herein mean an alkyl, alkenyl, alkynyl, alkylaryl, arylalkyl heterocycle, aryl or other group or moiety as defined or disclosed herein that has a substituent(s) that replaces a hydrogen atom(s) or a substituent(s) that interrupts a carbon atom chain. Alkenyl and alkynyl groups that comprise a substituent(s) are optionally substituted at a carbon that is one or more methylene moieties removed from the double bond. “Optionally substituted alkyl”, “optionally substituted alkenyl”, “optionally substituted alkynyl”, “optionally substituted alkylaryl”, “optionally substituted arylalkyl”, “optionally substituted heterocycle”, “optionally substituted aryl”, “optionally substituted heteroaryl”, “optionally substituted alkylheteroaryl”, “optionally substituted heteroarylalkyl” and the like as used herein mean an alkyl, alkenyl, alkynyl, alkylaryl, arylalkyl heterocycle, aryl, heteroaryl, alkylheteroaryl, heteroarylalkyl, or other substituent, moiety or group as defined or disclosed herein that has a substituent(s) that optionally replaces a hydrogen atom(s) or a substituent(s) that interrupts a carbon atom chain. Such substituents are as described herein. For a phenyl moiety, the arrangement of any two substituents present on the aromatic ring can be ortho (o), meta (m), or para (p). An optionally substituted fluoroalkyl is an alkyl or cycloalkyl moiety, typically a linear alkyl, wherein one or more hydrogen atoms is replaced by fluorine and at least one other atom other than carbon and fluorine. An optionally substituted or substituted substituent, moiety or group includes those having one or more additional group(s) that replace its hydrogen atom(s) individually and independently selected from alkyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy, alkylthio, arylthio, alkylsulfoxide, arylsulfoxide, alkylsulfone, arylsulfone, cyano, halo, nitro, haloalkyl, fluoroalkyl, fluoroalkoxy, and amino, including mono- and di- substituted amino groups, and the protected derivatives thereof. By way of example and not limitation an optional substituent(s) may be halide, —CN, —NO2, or LsRs, wherein each Ls is independently selected from a bond, —O—, —C(═O)—, —C(═O)O—, —S—, —S(═O)—, —S(═O)2—, —NH—, —NHC(═O)—, —C(═O)NH—, S(═O)2NH—, —NHS(═O)2, — OC(═O)NH—, —NHC(═O)O—, or —(C1-C6 alkylene)-; and each Rs is selected from —H, alkyl, fluoroalkyl, heteroalkyl, cycloalkyl, aryl, heteroaryl, or heterocycloalkyl. The protecting groups that may form the protective derivatives of the above substituents may be found in sources such as Greene and Wuts, above. Optional substituents include those selected from the group consisting of halogen, —CN, —NH2, —OH, —N(CH3)2, alkyl, fluoroalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxy, aryloxy, alkylthio, arylthio, alkylsulfoxide, arylsulfoxide, alkylsulfone, and arylsulfone, those selected from the group consisting of halogen, —CN, —NH2, —OH, NH(CH3), —N(CH3)2, —CO2H, —CO2alkyl, — C(═O)NH2, —C(═O)NHalkyl, —C(═O)N(alkyl)2, —S(═O)2NH2, —S(═O)2NH(alkyl), — S(═O)2N(alkyl)2, alkyl, cycloalkyl, fluoroalkyl, heteroalkyl, alkoxy, fluoroalkoxy, —S-alkyl and —S(═O)2alkyl or those selected from the group consisting of halogen, —CN, —NH2, — OH, —NH(CH3), —N(CH3)2, —CH3, —CH2CH3, —CF3, —OCH3, and —OCF3. Typically, an optionally substituted, substituent, moiety or group is substituted with one or two of the preceding groups, or more typically with one of the preceding groups. An optional substituent on an aliphatic carbon atom (acyclic or cyclic, saturated or unsaturated carbon atoms, excluding aromatic carbon atoms) further includes oxo (═O). “Heterocycle” or “heterocyclic” as used herein means a cycloalkyl or aromatic ring system wherein one or more, typically 1, 2 or 3, but not all of the carbon atoms comprising the ring system are replaced by a heteroatom which is an atom other than carbon, including, N, O, S, Se, B, Si, P, typically N, O or S wherein two or more heteroatoms may be adjacent to each other or separated by one or more carbon atoms, typically 1-17 carbon atoms, 1-7 atoms or 1- 3 atoms. Heterocycles includes heteroaromatic rings (also known as heteroaryls) and heterocycloalkyl rings (also known as heteroalicyclic groups) containing one to four heteroatoms in the ring(s), where each heteroatom in the ring(s) is selected from O, S and N, wherein each heterocyclic group has from 4 to 10 atoms in its ring system, and with the proviso that the any ring does not contain two adjacent O or S atoms. Non-aromatic heterocyclic, substituents, moieties or groups (also known as heterocycloalkyls) have at least 3 atoms in their ring system, and aromatic heterocyclic groups have at least 5 atoms in their ring system and include benzo-fused ring systems. Heterocyclics with 3, 4, 5, 6 and 10 atoms include aziridinyl azetidinyl, thiazolyl, pyridyl and quinolinyl, respectively. Nonaromatic heterocyclic substituents, moieties or groups are pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, oxazolidinonyl, tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, piperidinyl, morpholinyl, thiomorpholinyl, thioxanyl, piperazinyl, aziridinyl, azetidinyl, oxetanyl, thietanyl, homopiperidinyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl, thiazepinyl, 1,2,3,6-tetrahydropyridinyl, pyrrolin-2-yl, pyrrolin-3-yl, indolinyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolinyl, dithianyl, dithiolanyl, dihydropyranyl, dihydrothienyl, dihydrofuranyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, 3-azabicyclo[3.1.0)hexanyl, 3azabicyclo[4.1.0)heptanyl, 3H-indolyl and quinolizinyl. Aromatic heterocyclic includes, by way of example and not limitation, pyridinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, quinolinyl, isoquinolinyl, indolyl, benzimidazolyl, benzofuranyl, cinnolinyl, indazolyl, indolizinyl, phthalazinyl, pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl, thiadiazolyl, furazanyl, benzofurazanyl, benzo-thiophenyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl, and furopyridinyl. Non-aromatic heterocycles may be substituted with one or two oxo (═O) moieties and includes pyrrolidin-2-one. When heterocycle is used as a Markush group (i.e., a substituent) the heterocycle is attached to a Markush formula with which it is associated through a carbon or a heteroatom of the heterocycle, where such an attachment does not result in an unstable or disallowed formal oxidation state of that carbon or heteroatom. A heterocycle that is C-linked is bonded to a molecule through a carbon atom include moieties such as —(CH2)n-heterocycle where n is 1, 2 or 3 or —C<heterocycle where C< represents a carbon atom in a heterocycle ring. A heterocycle that is N-linked is a nitrogen containing heterocycle that is bonded a heterocycle ring nitrogen sometimes described as —N<heterocycle where N< represents a nitrogen atom in a heterocycle ring. Thus, nitrogen-containing heterocycles may be C-linked or N-linked and include pyrrole substituents, which may be pyrrol-1-yl (N-linked) or pyrrol-3-yl (C-linked), imidazole substituents, which may be imidazol-1-yl or imidazol-3-yl (both N-linked) or imidazol-2-yl, imidazol-4-yl or imidazol-5-yl (all C-linked). “Heteroaryl” as used herein means an aryl ring system wherein one or more, typically 1, 2 or 3, but not all of the carbon atoms comprising the aryl ring system are replaced by a heteroatom which is an atom other than carbon, including, N, O, S, Se, B, Si, P, typically, oxygen (—O—), nitrogen (—NX—) or sulfur (—S—) where X is —H, a protecting group or C1-6 optionally substituted alkyl, wherein the heteroatom participates in the conjugated system either through pi-bonding with an adjacent atom in the ring system or through a lone pair of electrons on the heteroatom and may be optionally substituted on one or more carbons or heteroatoms, or a combination of both, in a manner which retains the cyclically conjugated system. Heterocycles and heteroaryls, include, by way of example and not limitation, heterocycles and heteroaryls described in Paquette, Leo A.; “Principles of Modern Heterocyclic Chemistry” (W. A. Benjamin, New York, 1968), particularly Chapters 1, 3, 4, 6, 7, and 9; “The Chemistry of Heterocyclic Compounds, A series of Monographs” (John Wiley & Sons, New York, 1950 to present), in particular Volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc. 1960, 82:5545-5473 particularly 5566-5573). Examples of heteroaryls include by way of example and not limitation pyridyl, thiazolyl, pyrimidinyl, furanyl, thienyl, pyrrolyl, pyrazolyl, purinyl, imidazolyl, benzofuranyl, indolyl, isoindoyl, quinolinyl, isoquinolinyl, benzimidazolyl, pyridazinyl, pyrazinyl, benzothiopyran, benzotriazine, isoxazolyl, pyrazolopyrimidinyl, quinoxalinyl, thiadiazolyl, triazolyl and the like. Heterocycles that are not heteroaryls include, by way of example and not limitation, tetrahydrothiophenyl, tetrahydrofuranyl, indolenyl, piperidinyl, pyrrolidinyl, 2-pyrrolidonyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, octahydroisoquinolinyl, 2H-pyrrolyl, 3H- indolyl, 4H-quinolizinyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, piperazinyl, quinuclidinyl, morpholinyl, oxazolidinyl and the like. Other heteroaryls include, by way of example and not limitation, the following moieties:
Monocyclic heteroaryls include, by way of example and not limitation, pyridinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, pyridazinyl, triazinyl, oxadiazolyl, thiadiazolyl, and furazanyl. Heteroaryls include those substituents, moieties or groups containing 0-3 N atoms, 1-3 N atoms or 0-3 N atoms, 0-1 O atoms and 0-1 S atoms. A heteroaryl may be monocyclic or bicyclic. The ring system of a heteroaryls ring typically contains 1-9 carbons (i.e., C1- C9 heteroaryl). Monocyclic heteroaryls include C1-C5 heteroaryls. Monocyclic heteroaryls include those having 5-membered or 6-membered ring systems. Bicyclic heteroaryls include C6-C9 heteroaryls. Depending on the structure, a heteroaryl group can be a monoradical or a diradical (i.e., a heteroarylene group). “Heterocycloalkyl” or “heteroalicyclic” as used herein means a cycloalkyl group, moiety or substituent wherein at least on carbon of the cycloalkyl chain is replaces with a heteroatom selected from the group consisting of nitrogen, oxygen and sulfur. The heterocycloalkyl may be fused with an aryl or heteroaryl. Heterocycloalkyls, also referred to as non-aromatic heterocycles, include by way of example and not limitation:
Figure imgf000033_0001
Heterocycloalkyl includes, by way of example and not limitation, oxazolidinonyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, tetrahydrothiopyranyl, piperidinyl, morpholinyl, thiomorpholinyl, piperazinyl, and indolinyl. Heteroalicyclics further includes all ring forms of carbohydrates, including but not limited to monosaccharides, disaccharides and oligosaccharides. Typically, a heterocycloalkyl is a C2-C10 heterocycloalkyl and includes C4-C10 heterocycloalkyl. A heterocycloalkyl may contain 0-2 N atoms, 0-2 O atoms or 0-1 S atoms. “Heteroarylalkyl” as used herein means a substituent, moiety or group where a heteroaryl moiety is bonded to an alkyl moiety, i.e., -alkyl-heteroaryl, where alkyl and heteroaryl groups are as described above. When heteroarylalkyl is used as a Markush group (i.e., a substituent) the alkyl moiety of the heteroarylalkyl is attached to a Markush formula with which it is associated through a sp3 carbon of the alkyl moiety. “Alkylheteroaryl” as used herein means a substituent, moiety or group where a heteroaryl moiety is bonded to an alkyl moiety, i.e., -heteroaryl-alkyl, where heteroaryl and alkyl groups are as described above. When heteroarylalkyl is used as a Markush group (i.e., a substituent) the heteroaryl moiety of the heteroarylalkyl is attached to a Markush formula with which it is associated through a sp2 carbon or heteroatom of the alkyl moiety. “Halogen” or “halo” as used herein means fluorine, chlorine, bromine or iodine. “Haloalkyl” as used herein means an alkyl substituent moiety or group in which one or more of its hydrogen atoms are replaced by one or more independently selected halide atoms. Haloalkyl includes C1-C4 haloalkyl. Example but non-limiting C1-C4 haloalkyls are —CH2Cl, CH2Br, —CH2I, —CHBrCl, —CHCl—CH2Cl and —CHCl—CH2I. “Haloalkylene” as used herein means an alkylene substituent, moiety or group in which one or more hydrogen atoms are replaced by one or more halide atoms. Haloalkylene includes C1-C6haloalkylenes or C1-C4 haloalkylenes. “Fluoroalkyl” as used herein means an alkyl in which one or more hydrogen atoms are replaced by a fluorine atom. Fluoroalkyl includes C1-C6 and C1-C4 fluoroalkyls. Example but non-limiting fluoroalkyls include —CH3F, —CH2F2 and —CF3 and perfluroalkyls. “Fluoroalkylene” as used herein means an alkylene in which one or more hydrogen atoms are replaced by a fluorine atom. Fluoroalkylene includes C1-C6 fluoroalkylenes or C1- C4 fluoroalkylenes. The term “heteroalkyl” refers to an alkyl group in which one or more skeletal atoms of the alkyl are selected from an atom other than carbon, e.g., oxygen, nitrogen, sulfur, phosphorus or combinations thereof. In one aspect, a heteroalkyl is a C1-C6 heteroalkyl. “Protecting group” as used here means a moiety that prevents or reduces the ability of the atom or functional group to which it is linked from participating in unwanted reactions. Non-limiting examples are for —ORPR, wherein RPR is a protecting group for the oxygen atom found in a hydroxyl, while for —C(O)—ORPR, RPR may be a carboxylic acid protecting group; for —SRPR, RPR may be a protecting group for sulfur in thiols and for —NHRPR or —N
Figure imgf000034_0001
2— , at least one of RPR is a nitrogen atom protecting group for primary or secondary amines. Hydroxyl, amine, ketones and other reactive groups may require protection against reactions taking place elsewhere in the molecule. The protecting groups for oxygen, sulfur or nitrogen atoms are usually used to prevent unwanted reactions with electrophilic compounds, such as acylating agents. Typical protecting groups for atoms or functional groups are given in Greene (1999), “Protective groups in organic synthesis, 3rd ed.”, Wiley Interscience. “Ester” as used herein means a substituent, moiety or group that contains a —C(O)— O— structure (i.e., ester functional group) wherein the carbon atom of the structure is not directly connected to another heteroatom and is directly connected to —H or another carbon atom. Typically, esters comprise or consist of an organic moiety containing 1-50 carbon atoms, 1-20 carbon atoms or 1-8 carbon atoms and 0 to 10 independently selected heteroatoms (e.g., O, S, N, P, Si), typically 0-2 where the organic moiety is bonded through the —C(O)—O— structure and include ester moieties such as organic moiety-C(O)—O—. The organic moiety usually comprises one or more of any of the organic groups described herein, e.g., C1-20 alkyl moieties, C2-20 alkenyl moieties, C2-20 alkynyl moieties, aryl moieties, C3-8 heterocycles or substituted derivatives of any of these, e.g., comprising 1, 2, 3, 4 or more substituents, where each substituent is independently chosen. Exemplary, non-limiting substitutions for hydrogen or carbon atoms in these organic groups are as described above for substituted alkyl and other substituted moieties and are independently chosen. The substitutions listed above are typically substituents that one can use to replace one or more carbon atoms, e.g., —O— or —C(O)—, or one or more hydrogen atom, e.g., halogen, —NH2 or —OH. Exemplary esters include by way of example and not limitation, one or more independently selected acetate, propionate, isopropionate, isobutyrate, butyrate, valerate, isovalerate, caproate, isocaproate, hexanoate, heptanoate, octanoate, phenylacetate esters or benzoate esters. When ester is used as a Markush group (i.e., a substituent) the single bonded oxygen of the ester functional group is attached to a Markush formula with which it is associated. “Acetal”, “thioacetal”, “ketal”, “thioketal” and the like as used herein means a moiety, group or substituent comprising or consisting of a carbon to which is bonded two of the same or different heteroatoms wherein the heteroatoms are independently selected S and O. For acetal the carbon has two bonded oxygen atoms, a hydrogen atom and an organic moiety. For ketal, the carbon has two bonded oxygen atoms and two independently selected organic moieties where the organic moiety is as described herein alkyl or optionally substituted alkyl group. For thioacetals and thioketals one or both of the oxygen atoms in acetal or ketal, respectively, is replaced by sulfur. The oxygen or sulfur atoms in ketals and thioketals are sometimes linked by an optionally substituted alkyl moiety. Typically, the alkyl moiety is an optionally substituted C1-8 alkyl or branched alkyl structure such as —C(CH3)2—, — CH(CH3)—, —CH2—, —CH2—CH2—, —C[(C2-C4 alkyl)2]1, 2, 3- or [CH(C2-C4 alkyl)]1, 2, 3. Some of these moieties can serve as protecting groups for an aldehyde or ketone include, by way of example and not limitation, acetals for aldehydes and ketals for ketones and contain — O—CH2—CH2—CH2—O— or —O—CH2—CH2—O— moieties that form a spiro ring with the carbonyl carbon, and can be removed by chemical synthesis methods or by metabolism in cells or biological fluids. “Ether” as used herein means an organic moiety, group or substituent that comprises or consists of 1, 2, 3, 4 or more —O— moieties, usually 1 or 2, wherein no two —O— moieties are immediately adjacent (i.e., directly attached) to each other. Typically, ethers comprise an organic moiety containing 1-50 carbon atoms, 1-20 carbon atoms or 1-8 carbon atoms and 0 to 10 independently selected heteroatoms (e.g., O, S, N, P, Si), typically 0-2. An ether moiety, group or substituent includes organic moiety-O— wherein the organic moiety is as described herein for alkyl or optionally substituted alkyl group. When ether is used as a Markush group (i.e., a substituent) the oxygen of the ether functional group is attached to a Markush formula with which it is associated. When ether is a used as substituent in a Markush group it is sometimes designated as an “alkoxy” group. Alkoxy includes C1-C4 ether substituents such as, by way of example and not limitation, methoxy, ethoxy, propoxy, iso-propoxy and butoxy. Ether further includes those substituents, moieties or groups that contain one (excluding ketal) or more —OCH2CH2O—, moieties in sequence (i.e., polyethylene or PEG moieties). “Carbonate” as used here means a substituent, moiety or group that contains a —O— C(═O)—O— structure (i.e., carbonate functional group). Typically, carbonate groups as used here comprise or consist of an organic moiety containing 1-50 carbon atoms, 1-20 carbon atoms or 1-8 carbon atoms and 0 to 10 independently selected heteroatoms (e.g., O, S, N, P, Si), typically 0-2, bonded through the —O—C(═O)—O— structure, e.g., organic moiety-O— C(═O)—O—. When carbonate is used as a Markush group (i.e., a substituent) one of the singly bonded oxygen atoms of the carbonate functional group is attached to a Markush formula with which it is associated. “Carbamate” or “urethane” as used here means a substituent, moiety or group that contains a —O—C(═O)N(RPR)—, —O—C(═O)N(RPR)2, —O—C(═O)NH(optionally substituted alkyl) or —O—C(═O)N(optionally substituted alkyl)2-structure (i.e., carbamate functional group) where RPR and optionally substituted alkyl are independently selected and RPR are independently —H, a protecting group or an organic moiety as described for ester, alkyl or optionally substituted alkyl. Typically, carbamate groups as used here comprise or consist of an organic moiety containing about 1-50 carbon atoms, 1-20 carbon atoms or 1-8 carbon atoms and 0 to 10 independently selected heteroatoms (e.g., O, S, N, P, Si), typically 0-2, bonded through the —O—C(═O)—NRPR- structure, e.g., organic moiety-O—C(═O)— NRPR— or —O—C(═O)—NRPR-organic moiety. When carbamate is used as a Markush group (i.e., a substituent) the singly bonded oxygen (O-linked) or nitrogen (N-linked) of the carbamate functional group is attached to a Markush formula with which it is associated. The linkage of the carbamate substituent is either explicitly stated (N- or O-linked) or implicit in the context to which this substituent is referred. For any substituent group or moiety described by a given range of carbon atoms, the designated range means that any individual number of carbon atoms is described. Thus, reference to, e.g., “C1-C4 optionally substituted alkyl”, “C2-C6 alkenyl optionally substituted alkenyl”, “C3-C8 optionally substituted heterocycle” specifically means that a 1, 2, 3 or 4 carbon optionally substituted alkyl moiety as defined herein is present, or a 2, 3, 4, 5 or 6 carbon alkenyl, or a 3, 4, 5, 6, 7 or 8 carbon moiety comprising a heterocycle or optionally substituted alkenyl moiety as defined herein is present. All such designations are expressly intended to disclose all of the individual carbon atom groups and thus “C1-C4 optionally substituted alkyl” includes, e.g., 3 carbon alkyl, 4 carbon substituted alkyl and 4 carbon alkyl, including all positional isomers and the like are disclosed and can be expressly referred to or named. For esters, carbonates and carbamates defined by a given range of carbon atoms, the designated range includes the carbonyl carbon of the respective functional group. Thus, a C1 ester refers to a formate ester and a C2 ester refers to an acetate ester. The organic substitutents, moieties and groups described herein, and for other any other moieties described herein, usually will exclude unstable moieties except where such unstable moieties are transient species that one can use to make a compound with sufficient chemical stability for the one or more of the uses described herein. Substituents, moieties or groups by operation of the definitions herein that results in those having a pentavalent carbon are specifically excluded. Disease/Disorders As used herein, “steatosis” is interchangeable with “fatty liver” which is an accumulation of fat in the liver. “Steatosis” and “hepatic steatosis” are used interchangeably herein. Provided herein are methods for reducing the amount of liver fat or the accumulation of liver fat in a subject comprising administering one or more lysophosphatidic acid receptor 1 (LPA1) antagonist(s) alone or in combination with an additional active agent, such as a statin (e.g., atorvastatin, rosuvastatin, simvastatin, pravastatin, lovastatin, fluvastatin, or pitavastatin) or a glucagon-like peptide-1 receptor agonist. In some aspects, the method comprises treating a subject to reduce or prevent steatosis. In some aspects, the method of comprises treating fatty liver disease in the subject. In some aspects, the liver disease is non-alcoholic fatty liver disease (NAFLD). In some aspects, the liver disease is metabolic dysfunction-associated steatohepatitis (MASH). In some aspects the liver disease is alcoholic hepatic steatosis. In other aspects, the method comprises treating a subject to prevent or reduce the rate of progression of liver disease. In some aspects, the subject has type IIb hyperlipidemia. In some aspects, the subject has Familial combined hyperlipidemia (FCHL). In any of aspects, the subject may have a risk factor for developing fatty liver (steatosis) wherein the risk factor is that the subject has metabolic syndrome, type-2 diabetes, impaired glucose tolerance, obesity, dyslipidemia, hepatitis B, hepatitis C, an HIV infection, or a metabolic disorder such as Wilson's disease, a glycogen storage disorder, or galactosemia. In some aspects, the subject has diabetes. In some aspects, the subject has an inflammatory condition. In some aspects, the patient has an elevated body mass index above what is normal for gender, age and height. As used herein “type IIb hyperlipidemia” or “type IIb” patient population means a patient population having a fasting LDL cholesterol blood plasma level ≥130 mg/dl and a fasting triglyceride blood plasma level ≥150 mg/dL. References to LDL-C, triglyceride or ApoB levels are fasting levels unless clearly indicated otherwise. Type IIb hyperlipidemia is also known as Type IIb hyperlipoproteinemia. In some references type IIb hyperlipidemia is referred to as mixed dyslipidemia or is described as a subset of mixed dyslipidemia. In some aspects, the methods for reducing the accumulation of liver fat reduce the subject's risk of developing a liver disease. In some aspects, the fat is triglyceride. In some aspects, the subject has liver disease. In some aspects, the liver disease is nonalcoholic fatty liver disease (NAFLD). In some aspects, the liver disease is metabolic dysfunction-associated steatohepatitis (MASH). In some aspects, the liver disease is liver fibrosis. In some aspects, the liver disease is inflammation of the liver. In some aspects, the liver disease is cirrhosis of the liver. Aspects provide methods for reducing fibrosis in a patient comprising administering one or more lysophosphatidic acid receptor 1 (LPA1) antagonist(s) alone or in combination with an additional active agent as disclosed herein. One aspect provides a method of reducing hepatic fibrosis in a subject in need thereof, comprising administering to the subject one or more lysophosphatidic acid receptor 1 (LPA1) antagonist(s) and optionally with one or more additional active agents. Another aspect provides a method of reducing hepatic fibrosis in a subject in need thereof, comprising administering to the subject one or more lysophosphatidic acid receptor 1 (LPA1) antagonists, wherein the subject has MASH. Treatment/Administration “API” is an abbreviation for active pharmaceutical ingredient. When referring to dosages and doses, the dosage or dose is calculated on the weight of the API. In some embodiments, the API may be administered as a pharmaceutically acceptable salt. Where the API is administered as a salt, the dose is still calculated on the basis of the API. As used herein, the term “single dose formulation” refers to a pharmaceutical composition in the form in which it is marketed for use, formulated with mixture of one or more APIs and one or more excipients, along with other optional non-reusable material that may not be considered either ingredient or packaging (e.g., a capsule shell). As used herein, the terms “single dose formulation” and “fixed dose combination” are used interchangeably. Common single dose formulations include pills, tablets, or capsules. Formulations Compounds useful in the present invention can be formulated as pharmaceutical compositions and administered to a subject, such as a human subject, in a variety of forms adapted to the chosen route of administration, i.e., orally, transdermal, and parenterally. Such compositions and methods for their preparation are well known and may be found, for example, in Remington's Pharmaceutical Sciences, 19th Edition (Mack Publishing Company, 1995). In one embodiment, LPA1R antagonist(s) are formulated alone, or in combination with another active agent, such as glucagon-like peptide-1 receptor agonists, and with common excipients and carriers such as starch, binders, diluents and the like, and molded into tablets, or encapsulated into gelatin capsules for convenient oral administration. “Pharmaceutically acceptable formulation” as used herein means a composition comprising an active pharmaceutical ingredient, such as a compound having the formula of I or II in addition to one or more pharmaceutically acceptable excipients or refers to a composition prepared from an active pharmaceutical ingredient and one or more pharmaceutically acceptable excipients, wherein the composition is suitable for administration to a subject, such as a human or an animal, in need thereof. For a pharmaceutically acceptable formulation to be suitable for administration to a human the formulation must have biological activity for treating or preventing a disease or condition disclosed herein or an expectation must exist that the formulation would have a desired activity towards an “intent to treat” disease or condition. Typically, the “intent to treat” disease or condition is a lysophosphatidic acid receptor-mediated condition or disease. More typically the disease or condition to be treated or prevented is a lysophosphatidic acid lysophosphatidic acid type 1 receptor-mediated disease or condition. A pharmaceutically acceptable formulation that is suitable for administration to an animal does not necessarily require a biological activity for treating or preventing a disease or condition and may be administered to the animal in order to evaluate a potential pharmacological or biological activity of a Formula I-II compound. Those formulations must therefore be suitable for treating or preventing a disease or condition disclosed herein in an animal in need thereof or is suitable for evaluating a pharmacological or biological activity of a Formula I-II compound. “Solid formulation” as used herein refers to a pharmaceutically acceptable formulation comprising the active agents and one or more pharmaceutically acceptable excipients in solid form(s) wherein the formulation is in a unit dosage form suitable for administration of a solid. The dosage units include tablets, capsules, caplets, gelcaps, suspensions and other dosage units typically associated with parenteral or enteral (oral) administration of a solid. “Liquid formulation” as used herein refers to a pharmaceutically acceptable formulation wherein at least one the active agent compound has been admixed or contacted with one or more pharmaceutically acceptable excipients, wherein at least one of the excipients is in liquid form in proportions required for a liquid formulation, i.e., such that a majority of the mass amount of the active agent(s) is dissolved into the non-solid excipient. Dosage units containing a liquid formulation include syrups, gels, ointments and other dosage units typically associated with parenteral or enteral administration of a pharmaceutical formulation to a subject in need thereof in liquid form. In some aspects, the active agent(s), such as, LPA1R antagonist(s) and/or other active agents, such as glucagon-like peptide-1 receptor agonists, are administered daily, weekly or biweekly or monthly. In aspects, 0.5 mg/kg to 15 mg/kg( body weight) of the active agent, e.g., LPA1R antagonists , is administered to the subject, including, 0.5 mg/kg, 1.0 mg/kg, 1.5 mg/kg, 2.0 mg/kg, 2.5 mg/kg, 3.0 mg/kg, 3.5 mg/kg, 4.0 mg/kg, 4.5 mg/kg mg/kg, 5.0 mg/kg, 5.5 mg/kg, 6.0 mg/kg, 6.5 mg/kg, 7.0 mg/kg, 7.5 mg/kg, 8.0 mg/kg, 8.5 mg/kg, 9.0 mg/kg, 9.5 mg/kg, 10.0 mg/kg, 10.5 mg/kg, 11.0 mg/kg, 11.5 mg/kg, 12.0 mg/kg, 12.5 mg/kg, 13.0 mg/kg, 13.5 mg/kg, 14.0 mg/kg, 14.5 mg/kg or 15.0 mg/kg. Also provided herein is a kit comprising LPA1R antagonists and/or glucagon-like peptide-1 receptor agonist, and optionally with excipients and carriers; and instructions for the use thereof. Example Overview MASH causes a tremendous health care burden in the United States. A glucagon-like peptide-1 agonist, semaglutide (Sema), treatment resulted in hepatic steatosis reduction in clinical trials of MASH. Lysophosphatidic acid receptor 1 antagonists are known to have antifibrotic effects in several organs. Semaglutide (Sema) and a novel lysophosphatidic acid receptor 1 antagonist, EPGN2154, were tested individually and in combination to evaluate their efficacy for MASH remission in preclinical models. Methods: In the present study, (1) C57Bl6/J wild-type mice fed on a high-fat, high- carbohydrate (HFHC) diet for 16 weeks and (2) leptin-deficient mice (ob/ob) fed on an Amylin liver NASH diet for 16 weeks were used. After 16 weeks, the mice were randomly distributed in equal numbers in (1) no-drug, (2) EPGN2154, (3) Sema, and (4) EPGN2154+Sema treatment groups for 8 additional weeks at a dosage of 10 mg/kg body weight for EPGN2154 (oral gavage, 5 days a week) and 6.17 μg/kg body weight of Sema (subcutaneous injection every alternate day, 3 days a week). Results: In the wild-type-high-fat, high-carbohydrate model, the most body weight loss was seen in the EPGN2154+Sema combination group compared to the other treatment groups. All groups led to a significant reduction in alanine transaminase levels when compared to high- fat, high carbohydrate–fed wild type. However, no significant difference in alanine transaminase levels was observed among the treatment groups. In the ob/ob mice study, Sema did not cause body weight loss. Moreover, the EPGN2154 and the combination groups had a lower NAFLD Activity Score and incidence of advanced-stage hepatic fibrosis than the Sema group. Conclusions: EPGN2154 demonstrated a hepato-protective effect independent of body weight loss in preclinical MASH models. Introduction NASH, now called MASH, has imposed a sizable health care burden globally. (1) Studies have found many causal factors for MASH, including genetic polymorphisms, type 2 diabetes, dyslipidemia, environment, and lifestyle of patients. (2) As a result of its multifaceted nature, researchers have tried MASH remission approaches targeting various receptors of molecular signaling pathways, including bile acid receptors, (3) peroxisome proliferator– activated receptors, (4) liver-specific thyroid hormone receptors, (5) chemokine receptors, (6) and hepatic lipid metabolism. (7) Despite promising preclinical and clinical data, the Food and Drug Administration (FDA) of the United States has yet to approve any medication specifically for MASH. (8) A glucagon-like peptide-1 receptor agonist, semaglutide (Sema), has shown the ability to induce weight loss in humans. (9) In a placebo-controlled phase 2 clinical trial in patients with MASH, Sema treatment resulted in a significantly higher percentage of patients with MASH resolution than placebo. However, the trial did not show a significant between-group difference in the percentage of patients with improved fibrosis. (10) Small molecule lysophosphatidic acid receptor 1 (LPAR1) antagonists have shown therapeutic efficacy in preclinical models of lung and kidney fibrosis as well as clinical efficacy in idiopathic pulmonary fibrosis. (11–17) Further, pharmacological inhibition of a pro-fibrogenic protein like monocyte chemoattractant protein‐1 (MCP-1) has shown the reduction of macrophage infiltration in the liver of the preclinical MASH models. (18) Interestingly, LPAR1 deficiency causes a reduction of infiltrating macrophages and expression of MCP-1 in response to lipopolysaccharide in a preclinical model of chronic lung diseases.[19] During NASH progression, the infiltrating macrophages secrete proinflammatory cytokines in the liver, activating HSCs, thereby initiating the process of fibrosis. (20) HSCs constitute nearly 90% of the collagen-producing cells in a fibrotic liver. (21) Studies have shown that lysophosphatidic acid (LPA) induces the proliferation of HSC. (22) Additional studies have shown that patients with type 2 diabetes have a higher prevalence of MASH. (23–25) Combination drug therapies for MASH have shown better outcomes in clinical trials. (26) Provided herein is the efficacy of a novel small molecule LPAR1 antagonist, namely EPGN2154, and the glucagon-like peptide-1 receptor agonist Sema, either individually or in combination, in preclinical MASH models. Previous studies reported that wild-type (WT), C57Bl6/J, mice fed a high-fat and high- carbohydrate diet (HFHC) develop hepatic fibrosis. (27–29) Leptin deficient mice (ob/ob) develop a type 2 diabetes phenotype with mild hepatic fibrosis. (30) Ob/ob mice, when fed the amylin liver MASH (AMLN) diet, develop a severe MASH phenotype, including hepatic fibrosis. (31) Both WT and ob/ob mice MASH models allow for the evaluation of the efficacy of EPGN2154 and Sema, to determine (1) the effect of LPAR1 antagonist, EPGN2154, on body weight and regression of hepatic fibrosis; (2) the effect of Sema in hepatic fibrosis regression in the absence/presence of body weight loss; and (3) the efficacy of EPGN2154 compared to Sema in hepatic fibrosis regression. Materials and Methods Determination of mouse pharmacokinetics and oral bioavailability of EPGN2154 EPGN2154 solution was prepared fresh in a vehicle formulation of 20% Solutol and 40% PEG400 in 40% water at a 5 mg/mL concentration. A total of 12 female CD1 mice were acclimatized to living conditions and then divided into 2 groups of 6 animals each. EPGN2154 was administered by oral gavage (dose 20 mg/kg). EPGN2154 was administered intravenously (dose 1 mg/kg) at a dose volume of 5 mL/kg based on mouse weight. Plasma samples were diluted with blank mouse plasma as needed. An aliquot of 20 μL of plasma sample was extracted with 100 μL of acetonitrile containing internal standard (terfenadine). The mixture was vortexed on a shaker for 15 minutes and subsequently centrifuged at 4000 rpm for 15 minutes. An aliquot of 70 μL of the supernatant was mixed with 70 μL of water for the injection into the LC/MS/MS. Calibration standards and quality control samples were prepared by spiking the test compound into blank mouse plasma and then processed with the unknown samples. Mice experiments Six- to 8-week-old male C57Bl6/J WT and B6.Cg-Lepob/J (ob/ob) mice (Jackson Laboratory, Bar Harbor, ME) were housed in a 12-hour light-dark cycle maintained in a (22± 2°C) temperature-controlled room. WT mice were randomized to chow (n = 4) (Research Diets Inc., New Brunswick, NJ) or high-fat, high-carbohydrate (n = 40) (HFHC) diet (D12331i, Research Diets Inc., 58 kcal % fat; Research Diets, New Brunswick, NJ) and drinking water with high fructose (55% fructose by weight; Acros Organics, Morris Plains, NJ) and sucrose (45% sucrose by weight; Acros Organics, Morris Plains, NJ) mixture at a concentration of 42 g/L. (5)] Animals were provided ad-lib access to diets for 24 weeks. Ob/ob mice were randomized to chow (n = 5) (Research Diets Inc., New Brunswick, NJ) or AMLN diet (n = 50) (D09100310i, 40% fat, 22% fructose, and 2% cholesterol, Research Diets, New Brunswick, NJ). After 16 weeks on the AMLN diet, a portion of the ob/ob mice cohort (n = 10) was euthanized to serve as a 16-week sentinel group (AMLN 16 wk). WT and ob/ob mice were provided ad-lib diet access for 24 weeks. In the experiments with WT mice, after 16 weeks on the HFHC diet, mice were randomly distributed in each experimental group (n = 10): (1) HFHC (no-drug treatment), (2) HFHC+2154 (EPGN2154 treatment at 10 mg/kg body weight), (3) HFHC+Sema (Sema treatment at 6.17 μg/kg body weight), and (4) HFHC +2154+Sema (combination therapy of EPGN2154 at 10 mg/kg body weight and Sema treatment at 6.17 μg/kg body weight). EPGN2154 was administered orally once daily for 5 days a week. Sema (BOC Sciences, Shirley, NY) was administered by subcutaneous injection every alternate day, that is, 3 days a week. After 8 weeks of treatment, the mice were euthanized, and tissues were harvested for assays and histological analysis. In the experiments with ob/ob mice, after 16 weeks on the AMLN diet, the mice were randomly distributed in each experimental group (n = 10): (1) AMLN +Vehicle (Vehicle treatment group), (2) AMLN+2154 (EPGN2154 treatment at 10 mg/kg body weight), (3) AMLN+Sema (Sema treatment at 6.17 μg/kg body weight), and (4) AMLN+2154+Sema (combination therapy of EPGN2154 at 10 mg/kg body weight and Sema treatment at 6.17 μg/ kg body weight). EPGN2154 was administered orally once daily for 5 days a week. Sema was administered by subcutaneous injection every alternate day, that is, 3 days a week. After 8 weeks of treatment, the mice were euthanized, and tissues were harvested for assays and histological analysis. Body weights and food intake were recorded every week. Body composition Body composition analysis (fat mass and lean mass) was done using EchoMRI-100H Body Composition Analyzer (EchoMRI, TX). Plasma alanine transaminase assay Plasma isolated from the whole blood was used to estimate alanine transaminase (ALT) concentration using ALT Activity Assay kit (Sigma, St.Louis, MO) as per the manufacturer’s instruction. Oil Red O staining Oil Red O staining was performed on the 5 μm frozen liver tissue section using the Oil Red O Stain Kit (Lipid Stain) (ab150678) from Abcam (Boston, MA) according to the manufacturer’s instructions. The Oil Red O staining area percentage in the liver sections was quantified by a single independent pathologist blinded to the experimental design and treatment groups. Hydroxyproline assay Frozen liver tissue (100 mg) was used to quantify the hydroxyproline content using Hydroxyproline Assay Kit (Colorimetric) (ab222941) from Abcam (Boston, MA) according to the manufacturer’s instructions. LPAR1 expression Total RNA was isolated from 10 mg of liver tissue by TRI Reagent Solution (Invitrogen, Waltham, MA).5 μg of total RNA was used for cDNA synthesis using SuperScript III Reverse Transcriptase (Invitrogen, Waltham, MA). cDNA was used to quantify the relative expression of LPAR1 using a Taqman probe for LPAR1 (Mm01346925_m1) (Invitrogen, Waltham, MA). The relative expression of LPAR1 was calculated by the ΔΔCt method using a Taqman probe for Rpl18 (Mm01197265_g1) (Invitrogen, Waltham, MA) as a housekeeping gene. Liver histology analysis and immunohistochemistry Liver tissue was harvested from mice, fixed in 10% formalin, and sectioned in a microtome to generate 5-μm sections for histologic analyses. The sections were stained with eosin and hematoxylin and analyzed to determine the NAFLD Activity Score (NAS) by a single independent pathologist blinded to the experimental design and treatment groups. In NAS, liver histology is graded on steatosis (score 0–3), lobular inflammation (score 0–3), and ballooning (score 0–2). (32) Sirius Red staining evaluated hepatic fibrosis on a scale (0–4). Immunohistochemistry was performed on the liver section of the ob/ob mice for alpha-smooth muscle actin, collagen I, laminin and galectin-3 using anti-alpha-smooth muscle actin antibody (Cat# ab5694, Abcam, Cambridge, UK), anti-Collagen I antibody (Cat# ab270993, Abcam, Cambridge, UK), anti-Laminin antibody (Cat# ab11575, Abcam, Cambridge, UK), and anti- galectin-3 antibody (Cat# ab76245, Abcam, Cambridge, UK) respectively. The biotinylated Goat Anti-Rabbit IgG (H+L) (Cat# ab64256, Abcam, Cambridge, UK) and streptavidin HRP (Cat# ab64269, Abcam, Cambridge, UK) were used for detection. The effect on the migration of MCP-1 stimulated RAW264.7 in response to LPAR1 antagonists EPGN696 and EPGN2154 EPGN2154 and EPGN696 were prepared as a stock solution in DMSO at 10 mM concentration. The stock solution was diluted 50-fold with PBS to the final concentration to be used in the migration assay. For the migration assay, briefly, each test solution (1 μL) was diluted 50-fold in RAW264.7 cell suspension (49 μL). Final concentrations in a dose range of 10 fM to 10 μM were tested. MCP-1 solution (10-8M, 26 μL) was added to the bottom chamber of the Boyden apparatus. A 5-micron polycarbonate membrane was applied, followed by the gasket and the donor chamber apparatus screwed into place. Once assembled, positive controls and test wells (compound plus cell incubations), all ~50 μL, were pipetted into the upper chamber. A set of background wells was established with a bottom chamber containing MEM only and the upper wells RAW264.7 cells (50 μL). Once complete, the setup is incubated at 35°C for 1 hour. After 1 hour, the apparatus was disassembled and inverted to allow for the removal of the upper chamber and gasket. The filter was clamped, and the underside was rinsed and scraped in PBS before fixing it in methanol and drying on a glass slide. The dried filter was hematoxylin and eosin stained, and cells were visualized by microscope. Cell count was determined for each well as the average of 5 fields under the microscope. Cell counts for the positive control (no antagonist) and test wells were corrected for background by subtraction of any cells counted on the membrane in the negative control (no MCP-1 in the receiving chamber). Percent inhibition of migration was determined by (Cell count in test wells) × 100/(Cell count in positive control) The effect of lysophosphatidic acid stimulated the proliferation of human stellate cells in response to the LPAR1 antagonist, EPGN696 HSCs were isolated as previously described. (33,34) DMEM, fetal calf serum, antimycotic/antibiotic, Cyquant cell proliferation kit, and Ca2+/Mg2+-free PBS were from Thermofisher (Waltham, MA). Oleoyl-LPA-sodium salt was from Cayman Chemicals (Ann Arbor, MI). Thirty percent fatty acid-free bovine serum albumin was purchased from Sigma (St. Louis, MO). LPA was dissolved in Ca2+/Mg2+-free PBS containing 0.1% fatty acid free bovine serum albumin to give a 1 mM stock solution. The solution was sonicated for 15 minutes (min) before use. EPGN696 was diluted from a 10 mM solution in DMSO. HSCs were thawed and cultured in high glucose DMEM with 10% fetal calf serum and antibiotic/antimycotic at 37°C, 5% CO2. Cells were used between passages 4 and 7. Cells were plated at 1000 or 2000 cells/well of a 96-well plate and incubated overnight. The following day media was replaced with that containing AQ60.5% fetal bovine serum for 24 hours. The following day media was replaced with 100 μL media containing 0.5% fetal bovine serum and 0.1% DMSO or EPGN696 at 1 μM, 0.1 μM, or 0.01 μM for 15 minutes, followed by the addition of a further 100 μL media containing 0.5% fetal bovine serum, vehicle, 20 μM LPA (2x final concentration) and/or 1 μM, 0.1 μM, and 0.01 μM EPGN696. Cells were incubated for 48 hours before removing the media and freezing the plate at −80°C overnight. According to the manufacturer’s instructions, the relative cell number per well was assessed using the Cyquant cell proliferation kit. The Cyquant assay determines cell density via a dye that fluoresces when bound to nucleic acids. Fluorescence was determined using a Clariostar plate reader. Statistical analysis Statistical comparison between the experimental groups was performed using one-way or two-way ANOVA and post hoc Bonferroni test. Student t test was used to determine a statistical comparison between the 2 experimental groups. The p-value of <0.05 was considered statistically significant. Results were presented as mean ± SEM. Results EPGN696 and EPGN2154 EPGN2154 has demonstrated superior pharmacokinetics and distribution profiles that afford efficacy with much lower doses, leading to an improved risk-benefit profile compared to EPGN696 (Table 1). Furthermore, in preclinical models of diabetic kidney disease, EPGN2154 has demonstrated efficacy in the remission of kidney fibrosis and diabetic nephropathy. Data from ob/ob mice show that EPGN2154 reaches a steady state, and the half- life is appropriate for the once daily dosing regimen used in this study (data not shown). Oral gavage (PO) administration of EPGN2154 has shown absolute oral bioavailability of 40% and half-life of ~3 hours (Figure 1A). Table 1. LPAR1 antagonists—in vitro pharmacology and ADME-PK properties
Figure imgf000046_0001
Figure imgf000047_0001
Abbreviations: LPAR1, lysophosphatidic acid receptor 1; MCP-1, monocyte chemoattractant protein-1, pharmacokinetics. EPGN2154 and Sema cause a reduction in body weight WT mice fed the HFHC diet for 16 weeks had body weight higher than that of chow- fed mice (HFHC vs. Chow: 47.383 ± 0.843 g vs.33.650± 0.833 g; p< 0.0001) (Figure 1B). The HFHC-fed mice showed a body weight change of 24.021 ±0.734 g, whereas the chow-fed mice gained 10.15± 1.569 g in 16 weeks (Figure 1C). After 8 weeks of drug administration, the HFHC+2154+Sem group had the lowest body weight compared to the HFHC (no drug), HFHC+2154, and HFHC+Sem groups (Figure 1D). The HFHC+2154+Sem and the chow-fed group had no significant difference in body weight. The HFHC+2154+Sem group had shown maximum weight loss among the drug treatment groups (Figure 1E). At 24 weeks of the study, the HFHC+2154+Sem group has shown the lowest body weight among the drug treatment groups (Figure 2A). The body mass composition revealed that the HFHC+Sem+2154 group has the lowest fat mass percentage among the drug treatment groups. The HFHC+2154+Sem group has no significant difference in fat mass percentage from the chow group (Figure 2B). The HFHC+Sem+2154 group has the highest lean mass percentage among the drug treatment groups. The HFHC+2154+Sem group has no significant difference in lean mass percentage from the chow group (Figure 2C). EPGN2154 and Sema cause a reduction in the liver weight and liver-to-body weight ratio Both EPGN2154 and Sema cause a reduction in the liver weight compared to the no- drug treatment group. However, the HFHC+Sem group has the lowest liver weight compared to the other treatment groups (HFHC+Sem vs. HFHC (no-drug) vs. HFHC+2154 vs. HFHC+2154+Sem: 1.623±0.093 g vs. 3.775±0.190 g, p<0.0001, 2.849±0.252 g, p<0.0001, 1.943±0.077 g, p>0.05) (Figure 2D). Further, the HFHC+Sem group has a lower liver-to-body weight ratio of mice compared to the other treatment groups (HFHC+Sem vs. HFHC (no-drug) vs. HFHC+2154 vs. HFHC+2154+Sem: 0.0372±0.001 vs. 0.0694±0.003, p<0.0001, 0.060±0.003, p<0.0001, 0.053±0.001, p<0.0001). There was no observed significant difference between HFHC+Sem and the chow group (Figure 2E). EPGN2154 and Sema improve hepatic injury and liver physiology Both EPGN2154 and Sema reduced the plasma ALT concentration compared to that of the HFHC group (HFHC+2154 vs. HFHC (no-drug) vs. HFHC+Sem vs. HFHC+2154+Sem: 62.109±10.746 vs. 116.205±11.950, p<0.001, vs. 45.043±4.033, p>0.05, vs. 39.517± 8.924, p>0.05). No significant difference was observed in plasma ALT levels between the drug treatment and chow groups (Figure 2F). The analysis of liver histology (Figure 3A) by NAS revealed that HFHC+2154 and HFHC+Sem groups have no significant difference in the steatosis score (2.100±0.314 vs.1.400±0.221, p>0.05) and ballooning score (0.800±0.200 vs. 0.300±0.153, p>0.05) (Figure 3B). The liver sections of HFHC+2154 and HFHC+Sem groups have shown no difference in the Oil Red O stain area (Figure 3C). However, both HFHC+2154 and HFHC+Sem groups have lower Oil Red O stain area than HFHC. EPGN2154 imparts protection from the progression of hepatic fibrosis Histological analysis of Sirius Red–stained liver cross section (Figure 4A) revealed that 50% of mice in the HFHC group have advanced-stage hepatic fibrosis (Fibrosis Grade >2). However, only 10% of mice in the HFHC+2154 and HFHC+Sem groups have advanced-stage hepatic fibrosis (HFHC vs. HFHC+2154 vs. HFHC+Sem: 50% vs. 10%, χ2 p<0.0001; 10%, χ2 p<0.0001, Figure 4B). No advanced-stage fibrosis was observed in the liver sections of mice from the HFHC+2154+Sem groups. HFHC+2154 mice group has lower hydroxyproline concentration in the liver compared to the HFHC group (HFHC+2154 vs. HFHC: 0.22±0.038 ng/μL per mg liver vs. 0.51±0.082 ng/μL per mg liver, p<0.001) (Figure 4C). The drug treatment mice groups did not show any significant differences in the hydroxyproline concentration in the liver. HFHC+2154 mice group has lower lysophosphatidic acid receptor 1 (LPAR1) expression in the liver compared to the HFHC group (HFHC+2154 vs. HFHC: 0.66±0.062 A.U. vs.1.30±0.277 A.U.) (Figure 4D). The drug treatment and chow-fed groups have no significant difference in the hepatic LPAR1 expression. B6.Cg-Lepob/J (ob/ob) mice gain similar body weight when fed either chow or AMLN diet Genetically modified obese mice (ob/ob), mice either fed a chow diet or an amylin diet (AMLN), have similar body weight (chow: 61.802 ±0.936 vs. AMLN: 60.680± 0.380, Figure 5A). Likewise, no significant difference in the weight gain was observed between the chow- and AMLN diet-fed group (chow: 25.3842± 0.401 vs. AMLN: 28.076 ±1.588, Figure 5B). EPGN2154 reduces the body weight in B6. Cg-Lepob/J (ob/ob) mice After 8 weeks of drug treatment, mice in AMLN+2154, AMLN+2154+Sem, and AMLN+Sem groups have shown lower body weight than the chow+Veh group (AMLN+2154: 64.188±0.594 g, p<0.001; AMLN+2154+Sem: 60.255±0.850 g; AMLN+Sem: 63.493±0.841 g vs. chow+Veh: 70.422±1.034 g) (Figure 5C). Mice in the AMLN+2154 group gained the least body weight compared to the AMLN+2154+Sem and AMLN+Sem groups (AMLN+2154: 4.789±1.040 g, p<0.05; AMLN+2154+Sem: 6.080±0.810 g; AMLN+Sem: 6.833±0.926 g vs. chow+Veh: 7.822±0.956 g) (Figure 5D). EPGN2154 improves NAS in AMLN-fed ob/ob mice After 8 weeks of treatment, the liver weight of AMLN+2154, AMLN+2154+Sem, and AMLN+Sem mice groups decreased compared to the AMLN+Veh group (AMLN+2154: 5.861±0.224 g; AMLN+2154+Sem: 4.812±0.203 g; AMLN+Sem: 5.562±0.240 g vs. AMLN+Veh: 6.419±0.206 g) (Figure 6A). The AMLN+2154, AMLN+2154+Sem, and AMLN+Sem mice groups have lower liver-to-body weight ratio (arbitrary unit, AU) compared to the 16-week sentinel AMLN-fed mice group (AMLN 16 week) (AMLN+2154: 0.091±0.003 AU, p<0.05; AMLN+2154+Sem: 0.080±0.003 AU; AMLN+Sem: 0.088±0.004 AU vs. AMLN 16 week: 0.104±0.002) (Figure 6B). Further, the histological analysis of the liver section (Figure 6C) revealed that AMLN+2154 mice have a lower inflammation and ballooning score than AMLN+Sem mice (Inflammation score: 1.571±0.202 (AMLN+2154) versus 2.3±0.153 (AMLN+Sem); p<0.05; Ballooning score: 0.286±0.184 (AMLN+2154) vs. 0.7±0.213 (AMLN+Sem)). AMLN+2154 mice have a lower NAS than AMLN+Veh and AMLN+Sem groups (AMLN+2154: 4.571±0.430 vs. AMLN+Veh: 6.250±0.250, p<0.0001; vs. AMLN+Sem: 6.000±0.298, p<0.0001). There was no observed significant difference between the AMLN+2154 and AMLM+2154+Sem groups (4.125±0.295) (Figure 6D). AMLN+2154 group has a lower Oil Red O stain area than AMLN+Sem group (Figure 6E). AMLN+2154 has no significant difference with AMLN+2154+Sem in the Oil Red O stain area. EPGN2154 lowers the incidence of advanced-stage hepatic fibrosis in AMLN fed ob/ob mice Mice in AMLN+2154 group (14%) have shown a lower incidence of advanced-stage hepatic fibrosis (Fibrosis Score <2) than AMLN+Veh (100%, χ2 p <0.0001), AMLN+Sem (100%, χ2 p < 0.0001), and AMLN 16-week sentinel (33%, χ2 p< 0.01). In addition, a similar incidence of advanced-stage hepatic fibrosis was observed in AMLN+2154+Sem compared to the AMLN+2154 group (Figure 7A, B). Immunophenotyping of the liver cross section revealed that the AMLN+2154 group has a lower abundance of hepatic fibrosis markers like α-smooth muscle actin, galectin-3, collagen1a1, and laminin than that of the AMLN+Sem and AMLN+Veh groups (Figure 7C). The differences between the AMLN +2154 and AMLN+2154+Sem groups in α-smooth muscle actin, galectin-3, collagen1a1, and laminin abundance were insignificant. The AMLN+2154 mice have a lower concentration of hepatic hydroxyproline compared to AMLN+Veh, AMLN+2154+Sem, and AMLN+Sem groups (AMLN+2154: 0.72± 0.018 ng/μL per mg liver vs. AMLN+Veh: 0.81 ±0.035 ng/μL per mg liver, p< 0.05; vs. AMLN+2154+Sem: 0.97 ±0.075 ng/ μL per mg liver, p< 0.01; vs. AMLN+2154+Sem: 0.89± 0.038 ng/μL per mg liver, p <0.01) (Figure 7D). AMLN+2154 mice did not significantly differ in the hepatic hydroxyproline concentration compared to the AMLN-fed 16-week sentinel group (0.81 ± 0.092, p> 0.05). LPAR1 antagonists, EPGN2154 and EPGN696, inhibit the migration of macrophages and the proliferation of HSCs EPGN2154 and EPGN696 inhibited MCP-1–mediated RAW264.7 cell migration across a concentration range of 0.0001–10 μM (Figures 7E, F). The calculated IC50 for this inhibition was 1.76 nM for EPGN2154 and 0.95 nM for EPGN696. The data obtained successfully demonstrated the inhibition of hepatic fibrosis by the LPAR1 antagonist, EPGN2154. Treatment of human HSCs with a prototypical LPAR1 antagonist, EPGN696, at 1 μM significantly reduced the proliferation of HSCs stimulated by 10μM LPA after 48 hours. No effect was observed with 0.010 μM EPGN696 ex vivo. These data demonstrate a dose- dependent response of EPGN696 on HSC proliferation (Figure 7G). Discussion The FDA has approved Sema, a GLP-1 analog, for the treatment of obesity. (35,36) However, there continues to be a lack of FDA-approved therapeutics for obesity related liver disease (MASH/NAFLD). Lysophosphatidic acid receptor (LPAR1) antagonists have shown antifibrotic effects in patients with idiopathic pulmonary fibrosis. (14,17) This study demonstrated that a novel LPAR1 antagonist, EPGN2154, reverses MASH-related liver fibrosis. Both diet-induced WT and ob/ob mice, when treated with EPGN2154, have reduced liver inflammation like ALT, lower NAS, and lower incidence of advanced-stage fibrosis compared to the nontreatment group and, more interestingly, both in ob/ob mice and HFHC- fed WT mice, the EPGN2154 treatment groups had lower NAS and a lower incidence of advanced-stage fibrosis. Bibliography 1. Li Z, et al. Diabetol Metab Syndr.2023;15:6. 2. Younossi ZM, et al. Hepatology.2016;64:73–84. 3. Wang XX, et al. J Biol Chem.2022;298:102530. 4. Staels B, et al. Hepatology [Internet].2013;58:1941–52. 5. Cable EE, et al. Hepatology.2009;49:407–17. 6. Lefebvre E, et al. PLoS One.2016;11:e0158156. 7. Safadi R, et al. Clin Gastroenterol Hepatol.2014;12:2085–91.e1. 8. Chalasani N, et al. Hepatology.2018;67:328–57. 9. Mahapatra MK, Karuppasamy M, Sahoo BM. Rev Endocr Metab Disord.2022;23:521–39. 10. Newsome PN, et al. N Engl J Med.2021;384: 1113–24. 11. Allanore Y, et al. Arthritis Rheumatol.2018;70:1634–43. 12. Palmer SM, et al. Chest.2018;154: 1061–9. 13. Swaney JS, et al. J Pharmacol Exp Ther.2011;336:693–700. 14. Decato BE, et al. Respir Res.2022;23:1–12. 15. Swaney JS, et al. Br J Pharmacol.2010;160:1699–713. 16. Naruse T, Otake H, Takahashi T. J Pharmacol Sci.2022;149:179–88. 17. BMS-986278, an Oral Lysophosphatidic Acid Receptor 1 (LPA1) Antagonist, for Patients With Idiopathic Pulmonary Fibrosis: Results From a Phase 2 Randomized Trial. www.abstractsonline.com/pp8/#!/10703/presentation/12283. 18. Baeck C, et al. Gut.2012;61:416–26. 19. Chen X, et al. Front Physiol.2017;8:155. 20. Marcher AB, et al. Scientific Rep.2019;9:1–13. 21. Mederacke I, et al. Nat Commun.2013;4:2823. 22. Ikeda H, et al. Biochem Biophys Res Commun.1998;248:436–0. 23. Williamson RM, QJM.2012;105: 425–32. 24. Portillo-Sanchez P, et al. J Clin Endocrinol Metab.2015; 100:2231–8. 25. Leite NC, et al. Liver International.2011; 31:700–6. 26. Pockros PJ, et al. Liver Int.2019;39:2082–93. 27. Bhattacharjee J, et al. Hepatol Commun.2017;1:299–310. 28. Xi D, et al. Sci Rep.2020;10:6689. 29. Kohli R, et al. Hepatology.2010;52:934–44. 30. Trak-Smayra V, et al. Int J Exp Pathol.2011;92:413–21. 31. Kristiansen MNB, et al. World J Hepatol.2016;8:673. 32. Kleiner DE, et al. Hepatology.2005;41:1313–21. 33. Yoshida S, et al. Gastroenterology.2014;147: 1378–92. 34. Schwabe RF, Bataller R, Brenner DA. Am J Physiol Gastrointest Liver Physiol [Internet]. 2003;285:G949-58. 35. Wilding JPH, et al. N Engl J Med.2021;384:989–1002. 36. Garvey WT, et al. Nature Med.2022;28:2083–91. 37. Dobie R, et al. Cell Rep.2019;29:1832–47.e8. 38. Watanabe N, et al. J Clin Gastroenterol.2007;41:616–23. Example II Example I demonstrated MASH-related hepatic fibrosis remission of an LPAR1 antagonist, EPGN2154, in a diet-induced preclinical MASH model. The studies used a 10mg/kg body weight dose of EPGN2154 administered to mice through the oral route five days a week. To determine an optimum dose for EPGN2154 for the combination therapy with tirzepatide, 6-8 weeks old male C57Bl6/J (wild type, WT) were fed a MASH-inducing obesogenic (MIO) diet for 16 weeks. After 16 weeks, mice MIO diet-fed mice were randomly distributed into four experimental groups where one group did not receive the EGN2154 dose, and the other three experimental groups received 10mg/kg, 5mg/kg, and 1 mg/kg body weight dose of EPGN2154 for eight weeks. After eight weeks of EPGN2154 dose, mice were euthanized to evaluate the effect of different doses on the remission of MASH-related pathogenesis. The MIO-fed mice have gained significantly higher body weight than chow-fed mice (Body weight- MIO: 51.44±0.40g vs. chow: 32.87±2.12g, P>0.0001 (Figure 8); Body weight change- MIO:27.42±0.34 vs chow:10.12±1.07, P<0.0001 (Figure 9)). After eight weeks of EPGN2154 dose, the 10mg/kg EPGN2154 dose group has the lowest body weight compared to the other doses and no drug groups (10mg/kg: 50.57±0.91g vs 5mg/kg: 53.18±1.04g; 1mg/kg: 54.06±0.84g, P<0.05); MIO: 54.55±0.95g (P<0.05), Figure 10). There was a negative correlation between the EPGN2154 dose and the body weight of the mice (Figure 11). When compared with the body weight at the start of the drug dose (week 16), it was observed that mice that received 10mg/kg EPGN2154 dose gained the least body weight (Body weight change- 10mg/kg: -1.91±0.43g vs. 5mg/kg: 0.52±0.52g, P<0.0001; 1mg/kg: 2.75±0.54g, P<0.0001; MIO: 3.00±0.47g, P<0.0001 (Figure 12)). Like the body weight, a negative correlation was observed between the EPGN2154 dose and the body weight change of the mice (r =-0.9992, P<0.05, (Figure 13)). Hence, EPGN2154 has a dose-dependent effect on body weight loss. No difference was observed in the adiposity (Figure 14) and lean mass percentage (Figure 15) among the EPGN2154-administered mice and MIO groups. Furthermore, during the 8th week of the EPGN2154 dose, the food and water intake of the experimental groups was determined. There was no difference in the food (Figure 16) and water (Figure 17) intake among the EPGN2154 administered and MIO groups. However, a lower plasma alanine transaminase (ALT) concentration was observed in the 10mg/kg and 5mg/kg treatment groups compared to the MIO group (Figure 18). There was no significant difference in ALT concentration between the 1mg/kg and no-drug treatment groups. In the histological analysis of the liver sections of the experimental groups (Figure 19A), the EPGN2154 treatment groups have lower NAFLD Activity Score (NAS) than the MIO group (Figure 19B). However, among the EPGN2154 treatment groups, there was no statistically significant difference between the 10mg/kg and 5mg/kg groups. In addition, a lower expression of profibrotic genes such as alpha-smooth muscle actin (αSMA) (Figure 20A) and collagen 1A2 (Col1A2) (Figure 20B) was observed in the liver of mice of EPGN2154 treatment groups compared to the MIO group. The RNASeq of the whole liver revealed differentially expressed genes that (i) downregulated hepatic stellate cell activation and hepatic fibrosis signaling pathway and (ii) upregulated fatty acid β-oxidation and cholesterol biosynthesis in the liver of 10mg/kg treatment group compared to the MIO group (Figure 21). Thus, EPGN2154 treatment provides hepato-protection from MASH. Although the 10mg/kg dose resulted in the maximum weight loss among the EPGN2154 treatment groups, the 5mg/kg dose of EPGN2154 resulted in lower ALT, lower NAS, and lower expression of hepatic profibrotic genes similar to the 10mg/kg dose. Thus, orally administered EPGN2154 at a dose of 7.5mg/kg body weight was sufficient. Various embodiments of the invention are described above in the Detailed Description. While these descriptions directly describe the above embodiments, it is understood that those skilled in the art may conceive modifications and/or variations to the specific embodiments shown and described herein. Any such modifications or variations that fall within the purview of this description are intended to be included therein as well. Unless specifically noted, it is the intention of the inventors that the words and phrases in the specification and claims be given the ordinary and accustomed meanings to those of ordinary skill in the applicable art(s). The foregoing description of various embodiments of the invention known to the applicant at this time of filing the application has been presented and is intended for the purposes of illustration and description. The present description is not intended to be exhaustive nor limit the invention to the precise form disclosed and many modifications and variations are possible in the light of the above teachings. The embodiments described serve to explain the principles of the invention and its practical application and to enable others skilled in the art to utilize the invention in various embodiments and with various modifications as are suited to the particular use contemplated. Therefore, it is intended that the invention not be limited to the particular embodiments disclosed for carrying out the invention. While particular embodiments of the present invention have been shown and described, it will be obvious to those skilled in the art that, based upon the teachings herein, changes and modifications may be made without departing from this invention and its broader aspects and, therefore, the appended claims are to encompass within their scope all such changes and modifications as are within the true spirit and scope of this invention. It will be understood by those within the art that, in general, terms used herein are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). As used herein the term “comprising” or “comprises” is used in reference to compositions, methods, and respective component(s) thereof, that are useful to an embodiment, yet open to the inclusion of unspecified elements, whether useful or not. It will be understood by those within the art that, in general, terms used herein are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). Although the open-ended term “comprising,” as a synonym of terms such as including, containing, or having, is used herein to describe and claim the invention, the present invention, or embodiments thereof, may alternatively be described using alternative terms such as “consisting of” or “consisting essentially of.” The articles “a” and “an” are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. “Plurality” means at least two. As used herein “subject” and “patient” are used interchangeably. A subject may be a mammal and the mammal may be, for example, a human, and human subjects include adult, adolescent and pediatric subjects. Mammals also include, but are not limited to, farm animals, sport animals and pets (companion animals). The term "about,” as used herein, means approximately, in the region of, roughly, or around. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 10%. In one aspect, the term “about” means plus or minus 20% of the numerical value of the number with which it is being used. Therefore, about 50% means in the range of 45%-55%. Numerical ranges recited herein by endpoints include all numbers and fractions subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). It is also to be understood that all numbers and fractions thereof are presumed to be modified by the term “about.” The term "biological sample" refers to a sample obtained from an organism (e.g., a human patient) or from components (e.g., cells) of an organism. The sample may be of any biological tissue or fluid. The sample may be a "clinical sample" which is a sample derived from a patient. Such samples include, but are not limited to, sputum, blood, blood cells (e.g., white cells), amniotic fluid, plasma, semen, bone marrow, circulating tumor cells, circulating DNA, circulating exosomes, and tissue or fine needle biopsy samples, urine, peritoneal fluid, aqueous humor, and pleural fluid, or cells therefrom. Biological samples may also include sections of tissues such as frozen sections or formalin fixed paraffin embedded sections akin for histological purposes. A biological sample may also be referred to as a "patient sample." As used herein, “health care provider” includes either an individual or an institution that provides preventive, curative, promotional or rehabilitative health care services to a subject, such as a patient. In one embodiment, the data is provided to a health care provider so that they may use it in their diagnosis/treatment of the patient. The term “standard,” as used herein, refers to something used for comparison, such as control or a healthy subject. All references cited herein are incorporated by reference in their entirety as though fully set forth. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology 3rd ed., Revised, J. Wiley & Sons (New York, NY 2006); March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 7th ed., J. Wiley & Sons (New York, NY 2013); and Sambrook and Russel, Molecular Cloning: A Laboratory Manual 4th ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2012), provide one skilled in the art with a general guide to many of the terms used in the present application. All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

Claims

WHAT IS CLAIMED IS: 1. A method to treat or prevent hepatic steatosis comprising administering to a subject in need thereof an effective amount of a combination of one or more lysophosphatidic acid receptor 1 (LPAR1) antagonists and an additional active agent.
2. The method to treat or prevent hepatic steatosis of claim 1, wherein the hepatic steatosis is non-alcoholic fatty liver disease (NAFLD) or metabolic dysfunction-associated steatohepatitis (MASH).
3. A method to reduce the amount of liver fat or accumulation of liver fat in a subject at risk for liver fat accumulation comprising administering to a subject in need thereof an effective amount of a combination of one or more lysophosphatidic acid receptor 1 (LPAR1) antagonists and an additional active agent.
4. The method according to claim 3, wherein the subject has liver disease.
5. The method of claim 3 or 4, wherein the subject has hepatic steatosis, type IIb hyperlipidemia or familial combined hyperlipidemia.
6. The method of claim 5, wherein the hepatic steatosis is non-alcoholic fatty liver disease (NAFLD) or metabolic dysfunction-associated steatohepatitis (MASH).
7. The method of claim 3, wherein the subject's risk of developing liver disease is reduced.
8. The method of claim 7, wherein the liver disease is metabolic dysfunction-associated steatohepatitis (MASH), or nonalcoholic fatty liver disease (NAFLD), alcoholic hepatic steatosis or primary biliary cirrhosis.
9. A method to reduce hepatic fibrosis in a patient comprising administering to a subject in need thereof an effective amount of a combination of one or more lysophosphatidic acid receptor 1 (LPAR1) antagonists and an additional active agent.
10. The method of any one of claims 1-9, wherein the combination is administered daily or weekly.
11. The method of claim 10, wherein about 5 mg/kg to about 10 mg/kg body weight of the LPAR1 antagonist and/or additional active agent is administered.
12. The method of any one of claims claim 1 to 11, wherein the additional active agent comprises a glucagon-like peptide-1 receptor agonist, a lipid lowering agent or a combination thereof.
13. The method of claim 12, wherein the glucagon-like peptide-1 receptor agonist comprises semaglutide, tirzepatide, dulaglutide, exenatide, liraglutide, lixisenatide, and/or albiglutide 14. The method of claim 12 or 13, wherein the lipid lowering agent comprises a cholesterol absorption inhibitor, a statin, a PCSK9 inhibitor, an ACC inhibitor, an ApoC-III inhibitor, an ACL-inhibitor, fish oil, and/or a CETP inhibitor. 15. The method of claim 14, wherein the statin comprises atorvastatin, rosuvastatin, simvastatin, pravastatin, lovastatin, fluvastatin, and/or pitavastatin. 16. The method of any one of claims 1 to 15, wherein the LPAR1 antagonist is a compound of Formula I, Formula II or a combination thereof, as disclosed herein.
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WO2019111048A1 (en) * 2017-12-06 2019-06-13 Basf As Fatty acid derivatives for treating non-alcoholic steatohepatitis
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