Study On Metabolism And Disposition Of TP In Vivo And In Vitro And Its Interaction With CYP Enzyme

Tripterygium wilfordii Hook.f.?TWHF?or thunder duke vine,sometimes called Huangteng,is a traditional Chinese medicine that has been marketed in China as Tripterygium wilfordii glycoside tablets since 1980s.TWHF is known to be a toxic herb drug and widely used in the treatment of various autoimmune disorders,including rheumatoid arthritis,systemic lupus erythematosus and transplant rejection.It also causes dose and time-dependent hepatotoxicity in clinic.Triptolide?TP?,a major active and hepatotoxic ingredient in TWHF,exerted many pharmacological effects,such as immunosuppressive activities,anti-inflammatory and anti-cancer.TP is a toxic component and is detoxified in liver mainly by CYP3A mediated metabolic elimination and P-gp mediated bile excretion.As part of TP metabolism and toxicology research project,the current study investigated metabolism and disposition of TP in vivo and in vitro,based on the previous findings.Inter-species differences in metabolic elimination were evaluated.The human PBPK model was established to predict human pharmacokinetics in health and liver injury populations.Effect of TP on CYP3A were explored after oral administration of different TP doses or different dosing duration.The study facilitates the better understanding of pharmacokinetic characterization of the drug and also the material basis of TP induced hepatotoxicity.The results of this study will be useful to guide further development and safe clinic use of the herb drug.A comparative study of metabolic clearance and enzymatic kinetics of triptolide in human and rat liver microsomes was conducted firstly.NADPH is essential to phase I metabolism of TP.Its metabolic rate in human liver microsomes was significantly lower than that in rat liver microsomes with the intrinsic clearance being 15.5-fold in human.Also,a panel of rCYP isoforms were used to identify the phenotyping of TP.It was showed that,several CYP isoforms contributed to TP metabolism,and CYP3A4was recognized as the major isoform who had predominant contribution assessed with TNR method to metabolism of TP,and next were CYP2C8 and CYP1A2 with contribution of 20.8%and 20.4%,respectively.The enzymatic kinetics in human recombinant CYP3A4 and rat recombinant 3A1,3A2 incubates were also determined.And the intrinsic clearance of TP in CYP3A1/2 was significantly higher than that in CYP3A4.The significant inter-species differences were observed in metabolic clearance and enzymatic kinetics of TP,resulted partially from the differences in affinity and reaction rate between human and rat CYP3A isoforms.Hence the need to take the inter-species difference into account while extrapolating results from experimental animals to human.The pharmacokinetic profile of TP in rat after an oral dose of 1.5 or 3.0 mg·kg^-1was investigated.The time to peak concentration was within 15 min,and the elimination half-life was 0.6 h,therefore the pharmacokinetic characteristics of TP was rapid absorption and elimination.Besides,TP was found to accumulate in liver at 15min post dose,which proved by a liver-to-plasma concentration ratio value of 3.4.After adjustment by the unbound fraction values of TP in plasma and liver,the ratio value was still higher than 1.7.Using GastroPlus^TM software,the PBPK models of TP in rat and human were successfully established and applied to predict the PK behaviors in healthy and liver injury populations,as well as effect of CYP3A inhibition on TP plasma exposure in human.It was showed that the exposure of TP was significantly elevated in the populations with severe liver trauma.The plasma exposure of TP in the concomitant groups with ketoconazole was significantly increased with the C_max and AUC being1.38-fold and 1.44-fold of the TP alone group,respectively.Therefore,it is necessary to monitor plasma exposure of the drug when it is used in patients of hepatic insufficiency,or in combination with the CYP inhibitors.After 3 days pretreatment with TP,the plasma exposure of CYP3A substrate midazolam was significantly escalated with the C_max being 1.5-fold of control group.And the formation rate of the metabolite?-hydroxymidazolam was lower than that in control group.What's more,the AUC value of experimental group was 3.1 times greater than the control one while extending the pretreatment time to 7 days,indicating an enhancement in CYP3A inhibition.The plasma exposure of TP in high-dose group was significantly increased after rats received oral TP doses for 28 days.Effect of TP on relative gene expression and activity of CYP3A1/2 in rat liver after 28 days oral administration were surveyed.It was showed that,relative gene expression of CYP3A1/2 were induced and the CYP3A1/2 activities of female rats were significantly activated in high-dose group.Dose and time dependent alterations in TP exposure and their effects on metabolic enzyme deserve further study.