Preparation Of Human And Rat Liver Microsomes And Determination Of Metabolite Profile (Species Comparison) In Liver Microsomes

Human liver with responsibility for metabolizing on endogenous and exogenous compounds,was the important lieu for biotransformation.The liver microsomes were subcellular fractions(mainly endoplasmatic reticulam)containing many drug -metabolizing enzymes,the most prominent group of drug metabolizing enzymes was phase I drug metabolizing enzymes,e.g.cytochromo P450(CYP)and flavin-containing monoozygenase(FMO).Therefore they were widely used to investigate drug metabolism in vitro,includingâ…°).CYP reaction phenotyping study;â…±). CYP-based drug-drug interaction and drug metabolic stability study.Drug metabolism in vitro and in vivo data have some relevance,the research of in vitro liver models was helpful to explain clinical data to evaluate in vivo drug metabolism. Using in vitro metabolizing model to find the way and products of drug metabolism, which can provide a clear direction for pre-ctinical studies.Aim of this study was to preparation of human and rat liver microsomes and determination of metabolite profile in liver.SD rat liver microsome(RLM)and pooled human liver microsome(HLM)were obtained by modefied Omura method,the P450 contents were assayed by reduced CO difference spectrum firstly,then the microsomes were used to measure enzyme activity in real-time assays using fluorogenic substrates CEC,DBF,AMMC and EFC, preliminary judgement the P450 enzyme activity.The result of reduced CO difference spectrum show that there was a 450-nm peak observed in rat and human liver microsome,and their P450 enzyme content were 593±27pmol/mg and 964.67± 32pmol/mg respectively,after several times repeating,in addition to substrate EFC no obvious catalytic activity and weak catalytic activity AMMC of human liver microsomes,the human liver and rat liver microsomes show good catalytic activity to CEC and DBF,the fluorescence signal increases with the higher enzyme concentration and longer time.Followed by high-performance liquid chromatography technology for furthe evaluation of liver microsomes to the reliability of the model,liver microsomes and specificity probe substrate bufuralol,coumarin,s-mephenytoin and nifedipine reaction,calculated Km and Vmax values,according to CLint = Vmax / Km calculated intrinsic clearance rate.then we choose 2D6,2A6,2C19 and 3A4 special inhibitor drug quininedine,tranylcypromine and ketoconazole to do inhibition assay,and inspected inhibitory effect strong or weak through IC₅₀value.The results showed that rat liver microsomal probe with the substrate nifedipine bufuralol have significant catalytic activity,but for coumarin and s-mephenytoin,however,the four probe substrate have specificity and stability activity in the human liver microsomes,the catalytic activity sought by the Km and Vmax and CLint values consistent with the reported,specific inhibitors of rat liver microsomal no inhibitory effect,but the IC₅₀values is small on the human liver microsomes, which is showing a strong inhibitory effect.Through a multidimensional compared to prove the feasibility and reliability of in vitro drug metabolism platform of human and rat liver microsomes.High performance liquid chromatography and fluorescence detection results show good consistency, activity and inhibition assay results showed that the rat and human liver microsomal enzyme can be used for CYP450 and related in vitro drug metabolism model,during the comparison of human liver microsomes and recombinant human cytochrome P450 enzymes to prove the combination of enzyme activity and its selective inhibition assay are reasonable models.In conclusion of this study,we successfully established and primary applied in vitro metabolizing model of human and rat liver microsomal.