Preclinical Efficacy Study Of Acetyl-CoA Carboxylase Inhibitor For The Treatment Of Nonalcoholic Fatty Liver Disease

Background:Nonalcoholic fatty liver disease?NAFLD?is characterized by excessive accumulation of fat in hepatocytes.The prevalence of NAFLD in adults was 25%worldwide and 29.6%in China in the past two decades.It is also the most common disease among children and adolescents.NAFLD can be divided into nonalcoholic fatty liver and nonalcoholic steatohepatitis?NASH?.Some of these patients will develop into progressive fibrosis,which may lead to cirrhosis and Hepatocellular Carcinoma?HCC?.HCC and cardiovascular complications are life-threatening complications of NAFLD.It is predicted that NASH will replace hepatitis C as the main cause of liver transplantation in 2025.But there is no drug approved by FDA for NAFLD treatment,it can be improved by changing lifestyle,restricting diet and,possibly,bariatric surgery in clinic.There is still a large number of unmet clinical demands,so it is very urgent to develop new drugs.Lipotoxic lipids,which are derived from the excessive accumulation of fatty acids in hepatocytes,mediate cellular injury and inflammatory and fibrotic response.And these deteriorating changes constitute the histological phenotype of NASH.Acetyl coenzyme A Carboxylase?ACC?is a biotin-dependent carboxylase that catalyzes the conversion of acetyl CoA to malonyl CoA.In humans and other mammals,there are two isoforms of the ACC enzyme,ACC1 and ACC2,which are involved in the de novo synthesis of fatty acids and the regulation of fatty acid?oxidation respectively.The inhibition of ACC activity can improve hepatic steatosis and reduce the lipotoxicity,so it is a potential target for the treatment of NAFLD.At present,the development of ACC inhibitors GS0976 and PF-05221304 for NAFLD treatment has progressed to clinical phase 2,both of which show significant anti-steatosis effects in preclinical and clinical trials.Based on the general structure of GS0976,we modified a series of compounds for the following research.Objective:To evaluate the preclinical efficacy of ACC inhibitors through reliable screening platform in vitro and animal model in vivo,compared with reference compound GS0976.1.In vitro activity screening of candidate compounds:Methods:1)Screened a series of compounds by ACC1 and ACC2 enzyme activity experiments;2)Measured the effect of compounds on DNL ability of Hep G2 cells.Results:1)We screened 22compounds in ACC enzyme activity test,and 11 of them showed a strong effect in inhibiting both ACC1 and ACC2 activity,namely C1002,C1006,C1008,C1010,C1013,C1014,C1016,C1018,C1019,C1020 and C1021.The IC₅₀ of these compounds was equivalent to the reference compound GS0976;2)Cellular DNL test was performed on 9compounds with great ACC enzyme activity,of which 8 compounds?C1002,C1006,C1013,C1016,C1018,C1019,C1020,C1021?had better inhibitory effect of DNL than that of GS0976,and their IC₅₀ were 54.3 10.9,7.1,64.4,30.5,4.6,3.8 and 5.6 n M,respectively.2.In vivo activity screening of candidate compounds:Methods:According to the results of in vitro screening,three compounds C1016,C1018 and C1019 were selected for the following experiments:C57BL/6 mice fatty liver model was established by high-fat diet and employed to estimate the effects of 3 compounds on steatosis.Results:In the fatty liver model,GS0976,C1016,C1018 and C1019 could significantly reduce the liver TG content at a dose of 30 mg/kg,and the reduction rates of them were 58.54%,61.17%,51.40%and 32.67%,respectively.The effects of C1016 are similar to GS0976.3.Pharmacodynamics of NASH model in vivo:Methods:According to the above experiments,the best candidate compound C1016 was selected and investigated by the following experiments:Determinated the effcet of C1016 in the following models:1).the NASH model of ob/ob mice induced by Western diet,2)the NASH model of C57BL/6mice induced by CDAHF diet and 3)the liver fibrosis model of C57BL/6 mice induced by CCL₄;Results:1)In the NASH model of ob/ob mice induced by Western diet,C1016 10mg/kg and 30 mg/kg had no effect on the score of steatosis,inflammation and ballooning in hepatic histopathology,but could reduce the level of liver TG by 33.79%and 42.13%?149.65±24.27 and 130.80±11.97 vs model 226.02±27.26 mg/g,P<0.001?,and decrease liver CHO contend by 38.65%and 37.93%?24.09±7.89 and 24.37±4.40 vs model 39.26±4.35 mg/g,P<0.001?,respectively.In addition,C1016 showed a dose-dependent reduction in ALT?P<0.05?and AST?P<0.05?level.2)In the NASH model of C57BL/6 mice induced by CDAHF diet,C1016 10 mg/kg could significantly reduce the liver TG content by 35%?136.94±32.07 vs model210.51±28.20 mg/g,P<0.001?and Liver CHO content by 19.03%?3.02±0.64 vs model3.73±0.67 mg/g,P<0.05?,and at the same time it could also reduce serum ALT?P<0.05?and AST?P<0.05?levels.The effect of C1016 on liver lipid content reduction was stronger than GS0976,while the improvement of liver function was weaker.Both doses had no effect on the score of steatosis and inflammation,as well as fibrosis area in histopathology;3)In CCL₄ induced liver fibrosis model,C1016 30 mg/kg could significantly reduce the levels of serum ALT?P<0.001?,AST?P<0.001?and TBIL?P<0.001?.It also significantly reduced the liver fibrosis area by 46.73%?1.24±0.31 vs model 1.74±0.10%,P<0.001?and the score of cellular necrosis?0.25±0.46,P<0.001?.The overall effect was better than the reference compound GS0976 in this model.Conclusion:Candidate compound C1016 is a dual ACC1 and ACC2 inhibitor,it showed great inhibition activity of DNL and reduction of live lipids levels both in in vitro and in vivo assay.In addition,C1016 had strong anti-fibrosis effect on chemical-induced liver fibrosis model.