Lysophosphatidic acid receptor 1 antagonist (EPGN2154) causes regression of NASH in preclinical NASH models

Abstract

Background: NASH 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 NASH. Lysophosphatidic acid receptor 1 antagonists are known to have antifibrotic effects in several organs. We tested Sema and a novel lysophosphatidic acid receptor 1 antagonist, EPGN2154, individually and in combination to evaluate their efficacy for NASH remission in preclinical models.

Methods: In the present study, we used (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. 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, we observed the most body weight loss 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 NASH models.

Graphical abstract

FIGURE 1

EPGN2154 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; PO, per-os; Sema, semaglutide.

FIGURE 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.

FIGURE 3

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. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05. Abbreviations: HFHC, high-fat, high-carbohydrate; NAS, NAFLD Activity Score; Sema, semaglutide.

FIGURE 4

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.

FIGURE 5

EPGN2154 and semaglutide reduce body weight in B6.Cg-Lep ^ob /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.

FIGURE 6

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.

FIGURE 7

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 (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: αSMA, α-smooth muscle actin; AMLN, Amylin liver NASH; Col1a1, collagen1a1; Gal-3, galectin-3; Lam, laminin; LPA, lysophosphatidic acid; MCP-1, monocyte chemoattractant protein‐1; mMCP-1, murine monocyte chemoattractant protein-1; Veh, vehicle.