Mechanistic Investigation On The Metabolism Of Compound-3, A Novel Monophosphate Ester Prodrug Of Gemcitabine

Studies on the absorption,distribution,metabolism and excretion of compounds are important in selecting safe and effective drug candidates during the process of drug research and development.The research results of metabolic mechanisms of a compound play a critical role in determining whether a candidate posses drug-like properties.Compound-3 is a novel monophosphate ester prodrug of gemcitabine and previous data show that it is more effective than gemcitabine.As compared to the high systemic clearance of gemcitabine,compound-3 is metabolic stable.In the present study,we aimed to investigate the metabolic mechanism of compound-3,with focus on metabolites,the main metabolic enzyme and enzyme kinetic data,in various in vitro experiments.Liquid chromatography-tandem mass spectrometry?LC-MS/MS?methods were developed for compound-3 and its metabolites.During the incubations with various microsomes,compound-3 was found to be relatively stable in rat intestinal microsomes?RIM?,but unstable in human liver microsomes?HLM?and human intestinal microsomes?HIM?,suggesting a species difference in the stability of compound-3 in microsomes.Further experiments confrimed that the oxidative phase I reaction of compound-3 occurred in HLM and HIM.In order to identify key metabolites of compound-3,sample analysis was performed for plasma,cytosol,intestinal microsomes and liver microsomes by high resolution mass spectrometry?HRMS?.Data suggested that no metabolites generated significantly in plasma and cytosol samples,but three metabolites could be found in HLM and HIM.Based on HRMS analysis,M1 was the alcohol metabolite,M2 was the aldehyde metabolite and M3 was carboxylic acid metabolite of compound-3.To identify metabolic enzymes that mediated the oxidation of compound-3,recombinant human CYP enzymes and specific inhibitors of each CYP450 enzyme were used.Results showed that low level of M1 was found in CYP2D6,CYP3A4,CYP2B6 and CYP2C8.In CYP4F2,the generation of M1 and depletion of compound-3 were more obvious.Inhibition experiments indicated that HET-0016,a specific CYP4F2 inhibitor,could markedly inhibit the metabolism of compound-3 in HLM and CYP4F2.Study on enzyme kinetics is important to characterize enzyme-mediated reaction and define intrinsic clearance.Using HLM and recombinant human CYP4F2,data indicated that compound-3 was not only a substrate of CYP4F2,but also an inhibitor of CYP4F2.When pafuramidine was used as the substrate of CYP4F2,compound-3 had a strong inhibition against CYP4F2 activity(IC₅₀=0.12?M).On the other hand,pafuramidine also displayed a strong inhibition against CYP4F2 activity(IC₅₀=0.28?M).In conclusion,compound-3 can be metabolized by HLM.In the presence of CYP4F2,compound-3 was first oxidized into M1?alcohol?,and then to M2?aldehyde?and M3?carboxylic acid?.Compound-3 has a strong substrate inhibition effect on CYP4F2,so it is necessary to take into account the possibility of CYP4F2 mediated drug-drug interactions.