Study On Metabolic Interaction Among Artemisinin Derivatives, Piperaquine Phosphate And Benflumetol In Vitro

Artemisinin derivatives are a kind of antimalarial compounds which was applied well in treatment due to their rapid effect, non-drug resistant. In order to reduce recurrence rate and prevent drug resistant, WHO recommend combinations of artemisinin derivatives for clinical treatment. But the metabolism mechanism and pharmacokinetics were poorly understood due to the difficulties that have been encountered in establishing sensitive and specific ananlysis methods for the determination of artemisinin and its derivatives. Artemisinin derivatives exhibited time-dependency pharmacokinetics with unknown mechanism and auto-induction metabolism which are easy to bring about drug interaction. It is necessary to study the interaction mechanism of drug combination because artemisinin-based combinations have been widely used in malarial therapy. The aim of this work is to develop a sensitive and specific high-performance liquid chromatographic method with post-column alkali decomposition and UV detection. The method was suitable to determine preparation content of artemisinin and its major derivatives dihydroartemisinin and artesunate and piperaquine phosphate. At the same time, a sensitive and specific high-performance liquid chromatographic method was developed to determine preparation content of benflumetol. The work also investigated the in vitro metabolism study model of human liver microsomes for artemisinin derivatives. In addition, the work also investigated the in vitro metabolism study model of human liver microsomes for artemisinin derivatives and other antimalarial drugs to investigate interactions between artemisinin derivatives and other antimalarial drugs.1. To establish analysis methods for the determination the content of artemisinin derivatives and other antimalarial drugsTo quantificate artemisinin derivatives, high- performance liquid chromatographic (HPLC) methods with post-column alkali derivation and UV detection were estabolished. The first one is a method to determine dihydroartemisinin and piperaquine phosphate simultaneously. For dihydroartemisinin, the linearity was 0.9999 over the concentration range of 3.0-300μmol/L , the lower limit of quantification was 3.0μmol/L and detection limit was 0.6μmol/L. For piperaquine phosphate, the linearity was 0.9996 over the concentration range of 6.8-680μmol/L. The second method is to simultaneously determine artemisinin and piperaquine phosphate. For artemisinin, the linearity was 0.9998 over the concentration range of 3-300μmol /L, the lower limit of quantification was 3μmol/L and the detection limit was 0.3μmol/L. For piperaquine phosphate, the linearity was 0.9997 over the concentration range of 6.8-680μmol /L. The third method is to simultaneously determine artesunate and its metabolite dihydroartemisinin. For artesunate, the linearity was 0.9997 over the concentration range of 5-500μmol /L,the lower limit of quantification was 5μmol/L and the detection limit was 1.5μmol/L; For dihydroartemisinin, the linearity was 0.9999 over the concentration range of 5-500μmol /L. In addition, a sensitive and specific high-performance liquid chromatographic method was developed to determine preparation content of benflumetol. the linearity was 0.9998 over the concentration range of 3.78-378μmol /L,the lower limit of quantification was 3.78μmol/L. All methods are stable, cheap and convenient, and were proved to be suitable for the investigation of artemisinin derivatives and other antimalarial drugs quality control.2. Investigation of the in vitro metabolism kinetics of artemisinin derivatives in human liver microsomesTo evaluate the metabolism of artemisinin derivatives in human liver microsomes, HPLC method with post-column derivatization and UV detection was developed. The in vitro metabolism study model of liver microsomes was established for the investigation of artemisinin, dihydroartemisinin and artesunate. After incubation of artemisinin for 60 min in microsomes, the parameters Km, Vmax and Clint of artemisinine were as follows: 99.99±39.29 (μmol·L-1), 2.73±0.56 (μmol·L-1·min-1·mg pro-1) and (2.70±0.54)×10-2 (ml·min-1·mg pro-1), respectively. At the same time, the parameters Km, Vmax and Clint of dihydroartemisinine were as follows: 99.73±50.94 (μmol·L-1), 1.12±0.30 (μmol·L-1·min-1·mg pro-1) and (1.10±0.20)×10-2 (ml·min-1·mg pro-1), respectively. The parameters Km, Vmax and Clint of artesunate were as follows: 49.47±18.75 (μmol·L-1), 7.16±0.56 (μmol·L-1·min-1·mg pro-1) and (0.14±0.04)×10-2 (μmol·L-1·min-1·mg pro-1), respectively. The results showed that biological metabolism of artemisinin derivatives were happened, in stead of chemical degradation.3. Investigation of the in vitro metabolism of artemisinin derivatives and other antimalarial drugs in human liver microsomes The metabolism differences of combination containing artemisinin derivatives and other antimalarial drugs were investigated in human liver microsomes. Through comparing variations of metabolism parameters between drugs breeding solely and breeding together, we conclueded that: (1) piperaquine phosphate and benflumetol maked the metabolism of artemisinin slower in human liver microsomes. (2) piperaquine phosphate maked the metabolism of dihydroartemisinin faster in human liver microsomes. (3) benflumetol had no significant influence on the metabolism of dihydroartemisinin in human liver microsomes. (4) artemisinin and dihydroartemisinin had no significant influence on the metabolism of piperaquine phosphate in human liver microsomes. (5) artemisinin and dihydroartemisinin could increase the metabolism of benflumetol in human liver microsomes.