Chrysosplenetin inhibits artemisinin efflux in P-gp-over-expressing Caco-2 cells and reverses P-gp/MDR1 mRNA up-regulated expression induced by artemisinin in mouse small intestine

Context: CYP3A4 and P-gp together form a highly efficient barrier for orally absorbed drugs and always share the same substrates. Our previous work revealed that chrysosplenetin (CHR) significantly augmented the rat plasma level and anti-malarial efficacy of artemisinin (ART), partially due to the uncompetitive inhibition effect of CHR on rat CYP3A. But the impact of CHR on P-gp is still unknown. Objective: The present study investigates whether CHR interferes with P-gp-mediated efflux of ART and elucidates the underlying mechanism. Materials and methods: P-gp-over-expressing Caco-2 cells were treated with ART (10 mu M) or ART-CHR (1:2, 10:20 mu M) in ART bidirectional transport experiment. ART concentration was determined by UHPLC-MS/MS method. Healthy male ICR mice were randomly divided into nine groups (n = 6) including negative control (0.5% CMC-Na solution, 13 mL/kg), ART alone (40mg/kg), verapamil (positive control, 40 mg/kg), ART-verapamil (1:1, 40:40 mg/kg), CHR alone (80 mg/kg), ART-CHR (1:0.1, 40:4 mg/kg), ART-CHR (1:1, 40:40 mg/kg), ART-CHR (1: 2, 40:80 mg/kg) and ART-CHR (1:4, 40:160 mg/kg). The drugs were administrated intragastrically for seven consecutive days. MDR1 and P-gp expression levels in mice small intestine were examined by performing RT-PCR and western blot analysis. ABC coupling ATPase activity was also determined. Results: After combined with CHR (1: 2), P-app (AP-BL) and P-app (BL-AP) of ART changed to 4.29 x 10(-8) (increased 1.79-fold) and 2.85 x 10(-8) cm/s (decreased 1.24-fold) from 2.40 x 10(-8) and 3.54 x 10(-8) cm/s, respectively. Efflux ratio (P-BA/P-AB) declined 2.21-fold (p < 0.01) versus to ART alone. ART significantly up-regulated both MDR1 mRNA and P-gp levels compared with vehicle, while CHR in combination ratio of 0:1, 0.1:1, 1:1, 2:1 and 4:1 with ART, reversed them to normal levels as well as negative control (p < 0.05). The ATPase activities in ART-CHR 1:4 and CHR alone groups achieved a slight increase (p < 0.05) when compared with ART alone. Discussion and conclusion: These results confirm that CHR inhibited P-gp activity and reverse the upregulated P-gp and MDR1 levels induced by ART. It suggested that CHR potentially can be used as a P-gp reversal agent to obstruct ART multidrug resistance.