Abstract
BackgroundArtemisinin (ART) is an efficacious and safe anti-malarial drugs but has low oral bioavailability and auto-induction profiles during multiple dosing. The pharmacokinetic disadvantages have been found to partially depend on the induction of cytochrome P-450 enzymes by ART and resulted in the therapeutic failure due to insufficient drug levels. The present study, therefore, investigated the impacts of chrysosplenetin (CHR), a polymethoxylated flavonoid from Artemisia annua, on the pharmacokinetics and the anti-malarial efficacy of ART against Plasmodium berghei. The inhibition of CHR on enzymatic activity of CYP1A2, CYP2A, CYP2C19, CYP2D6, CYP2E1, and CYP3A in rat liver microsome was also investigated. IC50, Km, Ki, and inhibitory type of CHR were respectively calculated.MethodsTwenty rats were randomly divided into four groups and received three-day oral doses of ART in absence or presence of CHR (in ratio of 1:0, 1:1, 1:2, and 1:4, respectively). Plasma samples were separately harvested for ART pharmacokinetics analysis using a valid liquid chromatography tandem mass spectrometric (LC–MS/MS) method. Female Kunming mice were inoculated by P. berghei K173 strain and pre-exposed to three-day oral administration of ART with or without CHR as pharmacokinetics protocol. Giemsa staining method was applied to calculate percent parasitaemia (%) and inhibition (%). In vitro rat liver microsomal model was employed to elucidate the inhibitory effect of CHR on CYP1A2, CYP2A, CYP2C19, CYP2D6, CYP2E1, and CYP3A.ResultsThe AUC0–t, Cmax, and t1/2 of ART increased significantly (P < 0.05 or P < 0.01) as well as declined CLz (P < 0.05 or P < 0.01) after three-day oral doses of ART in presence of CHR (1:2) when compared with ART alone. Also, parasitaemia (%) remarkably attenuated 1.59 folds with 1.63-fold augmented inhibition (%) when the ratio between ART and CHR reached 1:2. CHR itself had no anti-malarial efficacy (P > 0.05). CHR inhibited in vitro activity of CYP1A2 and CYP2C19 (P < 0.01, IC50 = 4.61 and 6.23 μM) in a concentration–response manner. The inhibition did not emerge on CYP2E1 and CYP3A until the CHR concentration exceeded 4.0 μM (P < 0.01, IC50 = 28.17 and 3.38 µM). CHR has no impact on CYP 2A and CYP2D6 (P > 0.05). The inhibition types of CHR on CYP1A2 and CYP3A belonged to noncompetitive and uncompetitive, respectively.ConclusionsCo-administration of ART with CHR in ratio of 1:2 achieved a synergic anti-malarial effect partly because of the noncompetitive or uncompetitive inhibition of CHR of drug-metabolism enzymes, especially CYP3A which is closely related to the auto-induction of ART.
Highlights
Artemisinin (ART) is an efficacious and safe anti-malarial drugs but has low oral bioavailability and auto-induction profiles during multiple dosing
Co-administration of ART with CHR in ratio of 1:2 achieved a synergic anti-malarial effect partly because of the noncompetitive or uncompetitive inhibition of CHR of drug-metabolism enzymes, especially CYP3A which is closely related to the auto-induction of ART
The auto-induction of both phase I and phase II metabolism of ART was demonstrated to be present in healthy Chinese subjects after a recommended two-day oral dose of ART-piperaquine probably due to the induction of CYP2B6 and CYP3A4 enzyme activity [7]
Summary
Chemicals and reagents Artemisinin and internal standard artemether (ARM, Fig. 1b) were purchased from Chongqing Huali Konggu Co., Ltd. (purity >99.0 %, Chongqing, China). Stock solutions of chemicals ART and CHR were separately suspended in 0.5 % carboxy methyl cellulose (CMC-Na) by sufficient emulsification to get the stock solution of 2 mg/mL strength and diluted to get the desired concentrations for each drug before it was administrated by the intramuscular injection or gavage perfusion. Master stock solutions for assay of blood concentration were individually prepared by dissolving ARN and ARM standards in acetonitrile at equivalent concentrations of 1000 μg/mL and were gradually diluted to 2 μg/mL by mobile phase for the preparation of calibration curve (0.2–200 ng/mL) and quality control (QC) samples (0.5, 10 and 160 ng/mL for ART), respectively. The t1/2 in ART-CHR-M (1:2) group enhanced 1.68 folds compared with ART alone (P < 0.05) It showed that oral co-administration of CHR in combinatory ratio of 1:2 prior to intramuscular injection of ART achieved a longer t1/2, a higher AUC and Cmax, and lower CLz than ART alone
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