Artemisinin-based Antimalarial Drug Metabolism And Pharmacokinetics Studies

Artemisinin derivatives are a kind of compounds with endoperoxide and applied well in antimalarial treatment due to that it has rapid effect,non-drugresistant and good toleration.But drug metabolism and pharmacokinetics was poorly understood due to the difficulties that have been encountered in establishing a sensitive and specific assay for Artemisinin and its derivatives.The metabolism mechanism and pharmacokinetics character of Artemisinin class compound are still not clear.The aim of this work is to evaluate the pharmacokinetics of artemisinin and major metabolite dihydroartemisinin in rats,a sensitive and specific liquid chromatography/tandem mass spectrometric(LC/MS/MS)method was developed and validated for the determination of artemisinin derivatives in rat plasma.The results show that artemisinin derivatives exhibite remarkable time-dependent pharmacokinetics after single or single dose per day for 5 days oral administrations.The work also attempt to recover auto-induction of drug metabolism characteristic for artemisinin derivatives,the in vitro metabolism study model of liver microsomes was established for artemisinin and dihydroartemisinin.1.The pharmacokinetics study of artemisinin in ratsTo evaluate the pharmacokinetics of artemisinin in rats,a sensitive and selective liquid chromatography-tandem mass spectrometric(LC/MS/MS)method for the determination of artemisinin in rat plasma was developed and validated.For detection,a Sciex API 4000 LC/MS/MS instrument with an electrospray ionization(ESI)TurboIonSpray inlet in the positive ion multiple reaction monitoring(MRM)mode was used to monitor precursor([M+NH₄]⁺)product ions of m/z 300.4-209.4 for artemisinin and m/z 316.4-163.4 for artemether,the internal standard(IS).The standard curve was linear(r＞0.99)over the artemisinin concentration range of 1.0～200.0 ng·ml^-1in plasma.The method had a lower limit of quantification of 1.0 ng·ml^-1for artemisinin in 100μL of plasma,which offered a satisfactory sensitivity for the determination of artemisinin.After single and single dose per day for 5 days oral administrations of 40 mg·kg^-1of artemisinin,mean peak plasma levels(C_max)of 84.24±21.25 ng·ml^-1and 18.03±10.21 ng·ml^-1and T_maxof 1.20±0.274 h were observed.The mean t_1/2value of 1.01±0.123 h and 1.08±0.124 h were obtained,AUC_0-t were calculated to be 125.68±36.92 h.ng.ml^-1and 39.62±20.12 h·ng·ml^-1.The method is sensitive and convenient,and is proved to be suitable for investigation of artemisinin pharmacokinetics.2.The pharmacokinetics study of dihydroartemisinin in ratsA sensitive and selective LC/MS/MS method for direct determination ofβ-dihydroartemisinin in rat plasma was developed and was used to study the pharmacokinetics of dihydroartemisinin.A Sciex API 4000 LC/MS/MS equipped with electrospray ionization(ESI)source was used as detector and was operated in the positive ion mode.Multiple reaction monitoring(MRM)using the precursor to product ion combinations of m/z 267.4-163.4 forβ-dihydroartemisinin and m/z 300.4 -209.4 for artemisinin,the internal standard(IS).The linear calibration curves were obtained in the concentration range of 0.2～100.0 ng·ml^-1in plasma.The method had a lower limit of quantification of 0.2 ng·ml^-1forβ-dihydroartemisinin.After single and single dose per day for 5 days oral administratios of 10 mg·kg^-1ofβ-dihydroartemisinin,the pharmacokinetic parameters ofβ-dihydroartemisinin were calculated by non-compartment model statistics,mean peak plasma levels(C_max)of 140.56±24.30 ng·ml^-1and 13.215±1.211 ng·ml^-1and T_maxof 0.75±0.25 h and 0.83±0.29 h were observed.The mean t_1/2value of 0.42±0.25 h and 0.52±0.33 h were obtained, AUC_0-twere calculated to be 146.83±30.86 h·ng·ml^-1and 10.80±3.664 h·ng·ml^-1.3.Investigated the effect on the activity of metabolism enzyme induced by artemisinin or dihydroartemisinin in rats liver microsomesThe liver microsomes of rats were prepared by applying ultracentrifugation approach.Ultraviolet spectrophotometry(UV)was used to determine the contents of protein and cytochrome P450(CYP450).The CYP450 contents for artemisinin and dihydroartemisnin increased from 0.81±0.07 nmol·ml^-1Protein of controlled group to 1.40±0.01 nmol·mg^-1Protein and 1.43±0.02 nmol·ml^-1Protein of the experimental group,respectively.This showed that the metabolism enzyme could be induced by artemisinin and dihydroartemisinin.The in vitro metabolism study model of liver microsomes was established for the investigation of artemisinin and dihydroartemisinin metabolism.The metabolism of artemisinin and dihydroartemisinin were investigated by incubation with control and induced rat contents at 37℃.The result suggested that artemisinin and dihydroartemisinin exhibited remarkable auto-induction of drug metabolism.