Effect Mechanism Of Panaxytriol On Midazolam1’-hydroxylation And4-hydroxylation Mediated By CYP3A4

Background:In our previous study, we ascertained panaxytriol had differential effect on themetabolic pathways of midazolam (MDZ), which could significantly enhance MDZ1′-hydroxylation but inhibit MDZ4-hydroxylation, through gradually separatingusing SPE (solid phase extraction) system, chromatographic analysis, spectrumanalysis and structure identification. We research the effect mechanism of Panaxytriolon midazolam1′-hydroxylation and4-hydroxylation mediated by CYP3A4, throughrat liver microsome, human liver microsome and rat primary hepatocyte metabolismmodel.Objectives:The aim of this test analyzed the effect of PXT on CYP3A enzyme kinetics fromthe perspective of enzyme multiple-sites, using liver microsome in vitro metabolism.We responsed the effect of PXT on CYP3A enzyme activity through determining thedifferent hydroxylation metabolites of CYP3A probe substrate midazolam in ratprimary hepatocyte. We finally determined the CYP3A1/2mRNA expressioncondition using RT-PCR experiment. We comprehensively clarified the effectmechanism of Panaxytriol on midazolam1′-hydroxylation and4-hydroxylationmediated by CYP3A4, providing scientific basis for clinical safe and rational use ofdrugs.Methods:1. Effect of PXT on midazolam1′-hydroxylation and4-hydroxylation mediated byCYP3A4in RLM and HLM.①To establish the incubation method of MDZ in rat and human liver microsomeand sample preparation method. To set up the HPLC detection method of4-OH-MDZand1′-OH-MDZ.②Analysis the effect of PXT with different concentrations (0,0.5,1.0,2.0μg/ml)on enzymatic kinetic of midazolam metabolism in liver microsome(Km, Vmaxand CLint).2. Effect of PXT on midazolam1′-hydroxylation and4-hydroxylation mediated byCYP3A4in rat primary hepatocytes.①Establishing the separation and purification methods of rat primaryhepatocytes. Determining the activity and production ratio of hepatocytes by trypanblue exclusion experiments. Observing the microstructure of hepatocytes usingoptical microscope. Acquainting the toxicity of MDZ, PXT on hepatocytes by MTTmethod. Setting up the LC-MS detection method of4-OH-MDZ and1′-OH-MDZ.Investigating the effect of culture time, hepatocytes inoculum density and substrateconcentration on the enzyme kinetics of4-hydroxylation and1′-hydroxylation inhepatocytes.②The influence of CYP3A inducer dexamethasone (DEX), inhibitor (KTZ) andpanaxytriol (PXT) to MDZ4-hydroxylation and1′-hydroxylation after culture45min,4h and6h with MDZ in rat primary hepatocytes, respectively.3. Effect of PXT on the expression of CYP3A1/2mRNA in rat primary hepatocytes.Investigating the effect of PXT on CYP3A1/2mRNA expression by RT-PCRtechnology after cluture45min and6h in hepatocytes, respectively.Results：1. Effect of PXT on midazolam1′-hydroxylation and4-hydroxylation mediated byCYP3A4in RLM and HLM.①Establishing efficient and sensitive HPLC detection method to meet testrequirements.②PXT presented to inhibit MDZ4-hydroxylation and activate1′-hydroxylationin liver microsomes. The enzyme kinetics parameter Vmaxdecreased from0.72to0.51nmol/min.mg pro, Kmincreased from5.12to7.26μM, and CLintdecreased from0.14to0.07ml/min.mg pro for4-hydroxylation in rat liver microsome, when theconcentration of PXT were0,0.5,1.0,2.0μg/ml respectively. According to two-sitemodel, the enzyme kinetics parameter Vmax1increased from0.38to0.84nmol/min.mg pro, Km1decreased from8.9to5.4μM, and Clintincreased from0.04to0.16ml/min.mg pro for1′-hydroxylation in rat liver microsome. The enzyme kineticsparameters of4-OH-MDZ and1′-OH-MDZ also presented the same trend in HLM. 2. Effect of PXT on midazolam1′-hydroxylation and4-hydroxylation mediated byCYP3A4in rat primary hepatocytes.①Setting up mature and steady rat primary hepatocytes Separation method. Thevigor of hepatocytes was above90%and the yield was approximately0.4-0.8ｘ108/liver. Establishing efficient LC-MS detection method of4-OH-MDZ and1′-OH-MDZ. Determining DEX, PXT had no hepatocytes toxicity in the range of1.0-40.0μM and0.5-20μg/ml by MTT test, respectively.4-OH-MDZ and1′-OH-MDZpresented linear increase when cultured for0-120min; inoculation density for(0.75-3.0)ｘ106/ml and MDZ concentration of1-10μM. The4-hydroxylation and1′-hydroxylation of MDZ approximately reached saturation when the concentrationabove10uM, meeting to the typical enzyme kinetics characteristics.②PXT (1.0,4.0,8.0μg/ml) had differential effect on the metabolic pathways ofMDZ after culture45min, which could enhance MDZ1′-hydroxylation but inhibitMDZ4-hydroxylation. However, PXT enhanced the inhibition effect on MDZ4-hydroxylation and also presented inhibition effect on MDZ1′-hydroxylation withthe extension of culture time. Compared with blank group, PXT (1.0,4.0,8.0μg/ml)inhibited MDZ1′-hydroxylation to82.3%,68.2%and59.0%after culture6h,expectively. It prompted PXT could inhibit CYP3A enzyme metabolic activity in ratprimary hepatocytes.3. Effect of PXT on the expression of CYP3A1/2mRNA in rat primary hepatocytes.PXT (2.0,4.0,8.0μg/ml) didn’t affect CYP3A1/2mRNA expression level afterculture45min; After culture6h, PXT (2.0μg/ml) didn’t affect CYP3A1/2mRNAexpression level, PXT (4.0,8.0μg/ml) decreased CYP3A1mRNA expression level to87%and80%, and decreased CYP3A2mRNA expression level to89%and85%,respectively.Conclusions:This test explained the effect of PXT on midazolam1′-hydroxylation and4-hydroxylation mediated by CYP3A4from the perspective of enzyme kineticsaccording to the enzyme multiple-sites in rat and human liver microsomes. Weproved that the inhibition effect of PXT on CYP3A enzyme showed well consistencyin enzyme metabolic activity level and CYP3A1/2mRNA expression level through rat primary hepatocytes and RT-PCR experiment. We comprehensively clarified theeffect mechanism of Panaxytriol on midazolam4-hydroxylation and1′-hydroxylationmediated by CYP3A through three aspects including enzyme kinetics, CYP3Aenzyme activity variation and CYP3A1/2mRNA expression level, providing scientificbasis for clinical safe and rational use of drugs.