Abstract
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 μM) or ART-CHR (1:2, 10:20 μM) 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 (40 mg/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), Papp (AP-BL) and Papp (BL-AP) of ART changed to 4.29 × 10 − 8 (increased 1.79-fold) and 2.85 × 10 − 8 cm/s (decreased 1.24-fold) from 2.40 × 10 − 8 and 3.54 × 10 − 8 cm/s, respectively. Efflux ratio (PBA/PAB) 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 up-regulated 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.
Highlights
To date, artemisinin (ART, Figure 1(A)) antimalarial drugs are still of the utmost importance in the worldwide combination therapy of resistant Plasmodium falciparum (Tripathi et al 2013)
CHR belongs to the polymethoxylated flavonoids together with ART in the leaves and flowers of A. annua
Accumulating evidence suggested that many flavonoids have the ability to inhibit CYP3A4 and Pgp activity (Sandor et al 1998; Middleton et al 2000; Daddam et al 2014)
Summary
Artemisinin (ART, Figure 1(A)) antimalarial drugs are still of the utmost importance in the worldwide combination therapy of resistant Plasmodium falciparum (Tripathi et al 2013). P-gp usually shares the identical substrates with human CYP3A4/rat CYP3A (Pal et al 2011; Nabekura et al 2015; Wang et al 2015). This causes the low bioavailability and blood concentration for terminal drugs (Meng et al 2014). Burk et al (2005) suggested that ART induced the expression of CYP2B6, CYP3A4 and MDR1 through activating human PXR as well as human and mouse CAR as a ligand of both two nuclear receptors
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