title: Mechanism-based inhibition of cytochrome P450 (CYP)2A6 by chalepensin in recombinant systems, in human liver microsomes and in mice in vivo.
abstract: OBJECTIVE
  Chalepensin is a pharmacologically active furanocoumarin compound found in rue, a medicinal herb. Here we have investigated the inhibitory effects of chalepensin on cytochrome P450 (CYP) 2A6 in vitro and in vivo.
  
  
  METHODS
  Mechanism-based inhibition was studied in vitro using human liver microsomes and bacterial membranes expressing genetic variants of human CYP2A6. Effects in vivo were studied in C57BL/6J mice. CYP2A6 activity was assayed as coumarin 7-hydroxylation (CH) using HPLC and fluorescence measurements. Metabolism of chalepensin was assessed with liquid chromatography/mass spectrometry (LC/MS).
  
  
  RESULTS
  CYP2A6.1, without pre-incubation with NADPH, was competitively inhibited by chalepensin. After pre-incubation with NADPH, inhibition by chalepensin was increased (IC(50) value decreased by 98%). This time-dependent inactivation (k(inact) 0.044 min(-1) ; K(I) 2.64 µM) caused the loss of spectrally detectable P450 content and was diminished by known inhibitors of CYP2A6, pilocarpine or tranylcypromine, and by glutathione conjugation. LC/MS analysis of chalepensin metabolites suggested an unstable epoxide intermediate was formed, identified as the corresponding dihydrodiol, which was then conjugated with glutathione. Compared with the wild-type CYP2A6.1, the isoforms CYP2A6.7 and CYP2A6.10 were less inhibited. In mouse liver microsomes, pre-incubation enhanced inhibition of CH activity. Oral administration of chalepensin to mice reduced hepatic CH activity ex vivo.
  
  
  CONCLUSIONS
  Chalepensin was a substrate and a mechanism-based inhibitor of human CYP2A6. Formation of an epoxide could be a key step in this inactivation. 'Poor metabolizers' carrying CYP2A6*7 or *10 may be less susceptible to inhibition by chalepensin. Given in vivo, chalepensin decreased CYP2A activity in mice.
authors.affiliation: National Research Institute of Chinese Medicine, Taipei, Taiwan. ueng@nricm.edu.tw
authors.forename: Yune-Fang
authors.initials: YF
authors.lastname: Ueng
authors.forename: Chien-Chih
authors.initials: CC
authors.lastname: Chen
authors.forename: Yu-Ting
authors.initials: YT
authors.lastname: Chung
authors.forename: Tsung-Yun
authors.initials: TY
authors.lastname: Liu
authors.forename: Yu-Ping
authors.initials: YP
authors.lastname: Chang
authors.forename: Wei-Sheng
authors.initials: WS
authors.lastname: Lo
authors.forename: Norie
authors.initials: N
authors.lastname: Murayama
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Yamazaki
authors.forename: Pavel
authors.initials: P
authors.lastname: Souček
authors.forename: Gar-Yang
authors.initials: GY
authors.lastname: Chau
authors.forename: Chin-Wen
authors.initials: CW
authors.lastname: Chi
authors.forename: Ruei-Ming
authors.initials: RM
authors.lastname: Chen
authors.forename: Ding-Tzai
authors.initials: DT
authors.lastname: Li
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D065637:Cytochrome P-450 CYP2A6; D011564:Furocoumarins / Q000737:chemistry / Q000494:pharmacology*; D005978:Glutathione / Q000494:pharmacology; D006801:Humans; D008297:Male; D051379:Mice; D008862:Microsomes, Liver / Q000187:drug effects* / Q000378:metabolism; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D010862:Pilocarpine / Q000494:pharmacology; D017354:Point Mutation; D014191:Tranylcypromine / Q000494:pharmacology
chemical_list: D011564:Furocoumarins; D010862:Pilocarpine; C020740:3-(alpha,alpha-dimethylallyl)psoralen; D014191:Tranylcypromine; D001189:Aryl Hydrocarbon Hydroxylases; C585710:CYP2A6 protein, human; D065637:Cytochrome P-450 CYP2A6; D005978:Glutathione