Effectiveness of The PPAR Agonist Saroglitazar in Nonalcoholic Steatohepatitis: Positive Data from Preclinical & Clinical Studies

Background & Aims: Saroglitazar, a novel peroxisome proliferator-activated receptor (PPAR) α/γ agonist, was evaluated in preclinical and clinical studies to evaluate its effectiveness in NASH. Methods: Preclinical studies included: (a) choline decient L-amino acid-dened high fat diet (CDAHFD) murine NASH model; (b) Long Evans and Wistar rats model for hepatotropic activity; (c) DIAMOND™ NASH mice model. Clinical studies included: Phase 2 and Phase 3 studies in patients with biopsy proven NASH in India. Results: In CDAHFD murine NASH model, Saroglitazar improved aspartate aminotransferase (AST), alanine aminotransferase (ALT) and prevented hepatocellular steatosis, hepatocyte ballooning and lobular inammation. In Long Evans and Wistar rats model, Saroglitazar was found to be hepatotropic. In the DIAMOND™ NASH mice model, Saroglitazar showed a systemic effect with resolution of steatohepatitis and improvement in all the key histological features of NASH, dyslipidemia and insulin sensitivity. In the phase 2 study, Saroglitazar 4 mg signicantly reduced ALT levels (U/L) from baseline (95.86±37.65) to week 12 (44.37±35.43). In the phase 3 study, there was a signicantly higher proportion of patients with decrease in NAS ≥ 2 spread across at least 2 of the NAS components without worsening of brosis at week 52 in Saroglitazar 4 mg group (52.3%) compared to placebo group (23.5%) (p value – 0.0427), achieving the primary ecacy endpoint. In the phase 3 study, there were no concerns with the safety prole of Saroglitazar. Conclusions: Positive results from the preclinical and clinical studies provide evidence for the effectiveness of Saroglitazar in the treatment of NASH. CDAHFD, choline decient L-amino acid-dened high fat diet; NASH, nonalcoholic steatohepatitis; AST, aspartate aminotransferase; ALT, alanine aminotransferase; TG, triglyceride; TC, total cholesterol; < 0.0001 p value was calculated for percent (%) changes from baseline to week-52 using paired t-test % change = (value at week-52 - baseline value) x 100/baseline unit/litre; Also, the effect of Saroglitazar 4 mg on various lipid parameters was studied in three individual clinical trials conducted in patients with NAFLD/NASH in the USA, Mexico, and India, respectively. Saroglitazar 4 mg improved triglycerides, total cholesterol, LDL-C, non-HDL-C, HDL-C from baseline in the USA, India and Mexico, respectively [abstract accepted at EAS 2020]. This signies that Saroglitazar can potentially reduce the CVD risk in different populations with NAFLD/NASH across a global population. preclinical globally.


Introduction
Nonalcoholic steatohepatitis (NASH) is the most severe form of nonalcoholic fatty liver disease (NAFLD) and is associated with a high risk of progression to NASH related cirrhosis and NASH related hepatocellular carcinoma. [1][2][3] NASH is considered as the hepatic component of the metabolic syndrome, which is a clinical syndrome characterized by obesity, dyslipidemia, type 2 diabetes mellitus (T2DM), and hypertension. [1][2][3] Patients with NASH are at risk not only for the liverrelated morbidity and mortality but also at a higher risk of morbidity and mortality due to cardiovascular diseases (CVDs). 1-Saroglitazar is a dual PPAR alpha/gamma (PPAR α/γ) agonist (predominant PPAR-α and moderate PPAR-γ). 5 Through its PPAR-α agonist action, Saroglitazar increases lipoprotein lipase activity and thereby reduces serum triglyceride levels and very low-density lipoprotein cholesterol (VLDL-C) levels, and increases high-density lipoprotein cholesterol (HDL-C) levels. 5 Saroglitazar, through its PPAR-γ agonist action, improves insulin sensitivity in peripheral tissues, increases glucose uptake and reduces blood glucose levels. 5 Overall, Saroglitazar improves lipid and glycemic pro les without signi cant increase in body weight and edema/ uid retention (commonly seen in PPAR-γ agonists such as thiazolidinediones). [5][6][7] Saroglitazar has the potential to provide therapeutic bene t all along the pathologic spectrum of insulin resistance, diabetic dyslipidemia, T2DM, NAFLD and NASH. [5][6][7][8] Clinical development has been completed in many of these indications and Saroglitazar has received marketing approval in a few countries. [5][6][7][8] Diabetic dyslipidemia was the rst indication for which clinical development was completed and in clinical studies completed in India, Saroglitazar improved dyslipidemia by reducing triglyceride, total cholesterol, low-density lipoprotein cholesterol (LDL-C), VLDL-C, non HDL-C and increasing HDL-C and improved glycemic indices by reducing fasting plasma glucose (FPG) and glycosylated hemoglobin (HbA1c). [5][6][7] Subsequent to the completion of the randomized controlled studies and marketing approval, numerous investigator initiated studies were performed across India leading to a considerable data of real world evidence. 8 In an integrated analysis of real world clinical studies, which included 18 such studies, involving about 5,800 patients, effects of Saroglitazar on lipid and glycemic parameters in patients with diabetic dyslipidemia were analysed. 8 Saroglitazar effectively improved lipid and glycemic parameters in patients with diabetic dyslipidemia from 12 weeks upto to 58 weeks of therapy in different IIT's. 8 Across these studies it reduced mean triglyceride levels by 45-62%, LDL-C levels by 11-27%, total cholesterol levels by 17-26%, non-HDL-C levels by 21-36%, HbA1c levels by 0.7-1.6%, and increased mean HDL-C levels (up to 9%) from baseline. 8 It also reduced mean alanine aminotransferase (ALT) levels by 28-67% in NAFLD patients with diabetic dyslipidemia. 8  This article aims to discuss the e cacy of Saroglitazar in NASH in the pre-clinical animal models and the phase 2 and 3 clinical trials. We believe that such a comprehensive discussion of the pre-clinical e cacy and the clinical e cacy in the same article, in which the effect on the typical pathological features of NASH can be correlated and understood, will provide the scienti c community a comprehensive and holistic understanding of the effect of Saroglitazar in NASH. 4,9,10 All the data depicted and discussed in this article are 'data on le'. The pre-clinical DIAMOND mice model study results have been published in Scienti c Reports (a nature research journal) 4 / presented at the Liver Meeting 2018, AASLD, the USA (Abstract # 1297, 1768, and 1795). The data from the phase 2 clinical trial though not published anywhere as on original investigation, but was presented at the 5th Annual Meeting of EASL NAFLD Study Group 2017. The data from the phase 3 clinical trial has not been published anywhere as on original investigation, but was accepted as an oral presentation at the 29th Annual Conference of Asian Paci c Association for the Study of the Liver (APASL) at Bali, Indonesia.

Saroglitazar E cacy In Nash Pre-clinical Animal Models
All the below mentioned pre-clinical animal model protocols were approved by Institutional Animal Ethics Committee of Zydus Research Centre, Cadila Healthcare Ltd. a) Choline de cient L-amino acid-de ned high fat diet (CDAHFD) murine NASH model In a murine NASH model, histological examination of liver tissue of mice fed with choline de cient L-amino acid-de ned high fat diet (CDAHFD) for 8 weeks revealed hepatocellular steatosis (grade 3), hepatocyte ballooning (grade 1-2), lobular in ammation (grade 1-2) and mild (stage 1) perisinusoidal or periportal brosis. 9 Serum aspartate aminotransferase (AST) and ALT levels were also increased signi cantly along with increased serum levels of MCP-1, an in ammatory marker. 9 Saroglitazar improved AST, ALT and other parameters [ Table 1]. 9 Saroglitazar 3 mg/kg/day completely prevented CDAHFD induced hepatocellular steatosis, hepatocyte ballooning and lobular in ammation. 9 The mild hepatic brosis observed in CDAHFD-fed mice was reduced by Saroglitazar treatment, but the effect was not signi cant. 9 Pioglitazone did not exhibit any improvement in hepatocellular steatosis, hepatocyte ballooning, lobular in ammation or brosis. 9 Fatty liver, steatohepatitis and brosis: Mice fed WDSW with vehicle control had developed grade 3 macro vesicular steatosis and some micro vesicular steatosis. 4 All WDSW mice had developed steatohepatitis. 4 Pioglitazone reduced mean SAF activity to 2 (± 0.6) and mean NAS to 4.9 (± 0.7). 4 Saroglitazar signi cantly reduced steatosis, lobular in ammation, and hepatocellular ballooning (p < 0.01). 4 In the Saroglitazar treated group, hepatocellular ballooning was absent in all mice, steatohepatitis was resolved in all the mice, 3 out of 12 mice had no histological evidence of NAFLD. 4 Saroglitazar reduced mean NAS to 1.45 (± 0.9), which was signi cantly lower than that in the WDSW vehicle group. 4 Saroglitazar reduced mean brosis stage to 0.54 and Pioglitazone reduced mean brosis stage to 0.6. 4 Overall, the effects of Saroglitazar were superior to Pioglitazone histologically. 4 Liver and lipid parameters: Saroglitazar had improved circulating cholesterol parameters and triglycerides compared to WDSW with or without vehicle control groups. 4 WDSW controls had increased levels of serum ALT and AST, which were reduced signi cantly by Saroglitazar and Pioglitazone at comparable levels. 4 Saroglitazar decreased TNF-α and increased circulating adiponectin compared to WDSW vehicle control. 4 Diet-induced obesity and insulin resistance: DIAMOND mice fed with WDSW gained weight rapidly along with development of insulin resistance. 4 Saroglitazar treatment for 12 weeks reduced the body weight, fasting insulin levels, and insulin resistance in DIAMOND mice. 4 The degree of improvement with Saroglitazar was at par with Pioglitazone. 4 Abbreviations: LSM = least square mean; SD = standard deviation; SE = standard error; N = number of subjects in the treatment group; ALT, alanine aminotransferase; Note: p-values < 0.05 indicates signi cant and from ANCOVA model; *= By applying Dixon Text, percent change of two patient were found to be an outlier and the percent change value were also > 3 SD. So data were presented including and excluding these patients.

Safety:
Overall, Saroglitazar 4 mg was safe and well tolerated. No deaths or serious adverse events (SAEs) were reported during in the study. There were no persistent changes from baseline in any laboratory parameters and no statistically signi cant change observed in body weight following treatment with Saroglitazar 4 mg. Five events of raised creatinine phosphokinase were reported during the study. These events were mild and none of these events were considered clinically signi cant by the investigator.

Phase 3 -NASH Study of 52 Weeks Duration in India
In India, a phase 3, double-blind, randomized trial of 52 weeks was conducted to determine e cacy and safety of Saroglitazar 4 mg compared to placebo in 102 adult patients with biopsy proven NASH without cirrhosis ( brosis stage 1, 2, or 3) with a NAFLD activity score (NAS) of ≥ 4 with a score of at least 1 in each component (steatosis, hepatocyte ballooning, lobular in ammation). 11  There was a signi cantly higher proportion of patients with decrease in NAS ≥ 2 spread across at least 2 of the NAS components without worsening of brosis at week 52 in Saroglitazar 4 mg group (52.3%) compared to placebo group (23.5%) (p value -0.0427), achieving the study primary e cacy endpoint [ Figure 1]. 11 Secondary E cacy Endpoints:

Summary
Lifestyle modi cations and weight loss are the only recommended modalities and no drug is yet approved for the treatment of patients with NASH by the USFDA or EMA. [1][2][3] The ideal drugs/therapies for the treatment of NASH should improve liver parameters and liver histology along with reducing the risk of CVDs. [1][2][3] Peroxisome proliferator-activated receptors, ligand-activated transcription factors, are involved in the transcriptional regulation of glucose homeostasis, lipid metabolism, atherosclerosis, in ammation, and energy balance. 2 Saroglitazar is a novel PPAR agonist with dual PPAR agonistic properties (predominant PPAR-α agonist with moderate PPAR-γ agonistic activity). 5 The PPAR agonistic properties of Saroglitazar signify its potential positive effects on liver histology, liver parameters, and lipid parameters in NASH. 2,5 In the preclinical DIAMOND™ NASH mice model, Saroglitazar exhibited a systemic effect with resolution of steatohepatitis and improvement in all of the key histological features of NASH, improving dyslipidemia (triglycerides and cholesterol) and insulin sensitivity, and reducing weight. 4 Preclinical effects of Saroglitazar look promising to reduce the CV risk along with improving liver histology and liver parameters. These provide a strong rationale for clinical trials of Saroglitazar in NASH. In the phase 2 clinical trial in 32 patients with NASH in India, Saroglitazar 4 mg signi cantly reduced ALT (U/L) from baseline (95.86 ± 37.65) to week-12 (44.37 ± 35.43), which is a clinically accepted endpoint for the NASH trials [Data on le]. In the phase 3 clinical trial in 102 patients with NASH in India, the primary study endpoint was achieved by demonstrating a signi cantly higher proportion of patients with decrease in NAS ≥ 2 spread across at least 2 of the NAS components without worsening of brosis at week 52 in Saroglitazar 4 mg (52.3%) compared to placebo group (23.5%) (p value: 0.0427). 11 Moreover, Saroglitazar 4 mg also signi cantly reduced the mean NAS score, steatosis, and hepatocyte ballooning at week 52. 11 Saroglitazar 4 mg also showed bene t by demonstrating a signi cant decrease in the liver enzymes, namely, ALT, AST, ALP and GGT. 11 Saroglitazar 4 mg signi cantly reduced lipid parameters such as triglyceride, LDL-C, sd-LDL, VLDL-C, total cholesterol, non-HDL-C and signi cantly increased HDL-C at week 52. 11 The drug was found to be safe in patients with NASH as there were no major cardiovascular events or mortality reported during the study period and subsequent 12 week follow-up. In this study, Saroglitazar 4 mg, given for a period of 52 weeks, resulted in improvement in liver histology [ Figure 4], liver biochemistry, and lipid parameters in adult patients with NASH. 11 These bene cial effects are likely to result in a reduction in the risk of CVDs.
Saroglitazar has also been studied in patients with NAFLD/NASH in the US. 12 In the US, a phase 2 study of 16 weeks duration was conducted to determine the e cacy and safety of Saroglitazar 1 mg, 2 mg, and 4 mg compared to placebo in 106 adult patients with NAFLD/NASH along with ALT ≥ 50 U/L (ClinicalTrials.gov Identi er: NCT03061721). 12 The change in mean ALT from baseline to week 16 (primary e cacy endpoint) was − 26.2 ± 33.4% with Saroglitazar 1 mg, 27.0 ± 26.5% with Saroglitazar 2 mg, and − 44.9 ± 26.2% with Saroglitazar 4 mg compared to 2.6 ± 32.1% with placebo (p < 0.001 for all). 12 Saroglitazar 4 mg, compared to placebo, signi cantly reduced mean liver fat content (LFC) [4.21 ± 6.23% versus − 0.28 ± 5.41%, p = 0.002] at week 16. 12 Saroglitazar 4 mg was associated with improvements in enhanced liver brosis score, atherogenic dyslipidemia, and glycemic parameters at week 16. 12 Also, the effect of Saroglitazar 4 mg on various lipid parameters was studied in three individual clinical trials conducted in patients with NAFLD/NASH in the USA, Mexico, and India, respectively. Saroglitazar 4 mg improved triglycerides, total cholesterol, LDL-C, non-HDL-C, HDL-C from baseline in the USA, India and Mexico, respectively [abstract accepted at EAS 2020]. This signi es that Saroglitazar can potentially reduce the CVD risk in different populations with NAFLD/NASH across a global population.
Overall, preclinical studies indicate that Saroglitazar reduces ALT, AST, and improves steatohepatitis, hepatocellular ballooning, and brosis. Clinical studies showed similar effects of Saroglitazar on liver parameters and components of NAS and brosis. Moreover, preclinical and clinical studies showed that Saroglitazar improved lipid parameters, which could reduce the CVD risk. Thus, Saroglitazar could be an ideal drug to treat NASH in different populations globally. Declarations