title: 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.
abstract: BACKGROUND
  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.
  
  
  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 × 10-8cm/s (decreased 1.24-fold) from 2.40 × 10-8and 3.54 × 10-8cm/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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: a School of Pharmacy, Ningxia Medical University , Yinchuan, Ningxia , PR China.
authors.forename: Liping
authors.initials: L
authors.lastname: Ma
authors.affiliation: b Institute of Clinical Pharmacology, General Hospital of Ningxia Medical University , Yinchuan, Ningxia , PR China.
authors.forename: Shijie
authors.initials: S
authors.lastname: Wei
authors.affiliation: a School of Pharmacy, Ningxia Medical University , Yinchuan, Ningxia , PR China.
authors.forename: Bei
authors.initials: B
authors.lastname: Yang
authors.affiliation: a School of Pharmacy, Ningxia Medical University , Yinchuan, Ningxia , PR China.
authors.forename: Wei
authors.initials: W
authors.lastname: Ma
authors.affiliation: a School of Pharmacy, Ningxia Medical University , Yinchuan, Ningxia , PR China.
authors.forename: Xiuli
authors.initials: X
authors.lastname: Wu
authors.affiliation: b Institute of Clinical Pharmacology, General Hospital of Ningxia Medical University , Yinchuan, Ningxia , PR China.
authors.forename: Hongyan
authors.initials: H
authors.lastname: Ji
authors.affiliation: a School of Pharmacy, Ningxia Medical University , Yinchuan, Ningxia , PR China.
authors.forename: Hong
authors.initials: H
authors.lastname: Sui
authors.affiliation: a School of Pharmacy, Ningxia Medical University , Yinchuan, Ningxia , PR China.
authors.forename: Jing
authors.initials: J
authors.lastname: Chen
mesh_terms: D018435:ATP Binding Cassette Transporter, Subfamily B / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D000818:Animals; D037621:Artemisinins / Q000378:metabolism*; D001692:Biological Transport; D015153:Blotting, Western; D018938:Caco-2 Cells; D002851:Chromatography, High Pressure Liquid; D003106:Colon / Q000187:drug effects* / Q000378:metabolism; D004347:Drug Interactions; D018432:Drug Resistance, Multiple / Q000187:drug effects; D005419:Flavonoids / Q000494:pharmacology*; D006801:Humans; D007421:Intestine, Small / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D008813:Mice, Inbred ICR; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D053719:Tandem Mass Spectrometry; D014162:Transfection; D015854:Up-Regulation; D000097202:ATP-Binding Cassette Sub-Family B Member 4
chemical_list: D018435:ATP Binding Cassette Transporter, Subfamily B; D037621:Artemisinins; D005419:Flavonoids; D012333:RNA, Messenger; D000097202:ATP-Binding Cassette Sub-Family B Member 4; C513055:ABCB1 protein, human; C570121:chrysosplenetin; C095122:multidrug resistance protein 3; C031327:artemisinin
keywords: ABC transporters; P-glycoprotein; Polymethoxylated flavonoids; antimalarial drugs