title: Functional impact of ABCB1 variants on interactions between P-glycoprotein and methadone.
abstract: Methadone is a widely used substitution therapy for opioid addiction. Large inter-individual variability has been observed in methadone maintenance dosages and P-glycoprotein (P-gp) was considered to be one of the major contributors. To investigate the mechanism of P-gp's interaction with methadone, as well as the effect of genetic variants on the interaction, Flp-In™-293 cells stably transfected with various genotypes of human P-gp were established in the present study. The RNA and protein expression levels of human P-gp were confirmed by real-time quantitative RT-PCR and western blot, respectively. Utilizing rhodamine 123 efflux assay and calcein-AM uptake study, methadone was demonstrated to be an inhibitor of wild-type human P-gp via non-competitive kinetic (IC50 = 2.17±0.10 µM), while the variant-type human P-gp, P-gp with 1236T-2677T-3435T genotype and P-gp with 1236T-2677A-3435T genotype, showed less inhibition potency (IC50 = 2.97±0.09 µM and 4.43±1.10 µM, respectively) via uncompetitive kinetics. Methadone also stimulated P-gp ATPase and inhibited verapamil-stimulated P-gp ATPase activity under therapeutic concentrations. These results may provide a possible explanation for higher methadone dosage requirements in patients carrying variant-type of P-gp and revealed the possible drug-drug interactions in patients who receive concomitant drugs which are also P-gp substrates.
authors.affiliation: Department of Pharmacy, College of Pharmacy, China Medical University, Taichung, Taiwan. cc0206hung@gmail.com
authors.forename: Chin-Chuan
authors.initials: CC
authors.lastname: Hung
authors.forename: Mu-Han
authors.initials: MH
authors.lastname: Chiou
authors.forename: Yu-Ning
authors.initials: YN
authors.lastname: Teng
authors.forename: Yow-Wen
authors.initials: YW
authors.lastname: Hsieh
authors.forename: Chieh-Liang
authors.initials: CL
authors.lastname: Huang
authors.forename: Hsien-Yuan
authors.initials: HY
authors.lastname: Lane
mesh_terms: D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics / Q000378:metabolism*; D000704:Analysis of Variance; D015153:Blotting, Western; D005452:Fluoresceins / Q000378:metabolism; D014644:Genetic Variation; D057809:HEK293 Cells; D006801:Humans; D015151:Immunoblotting; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008691:Methadone / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D060888:Real-Time Polymerase Chain Reaction; D020112:Rhodamine 123 / Q000378:metabolism; D014700:Verapamil
chemical_list: C513055:ABCB1 protein, human; D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D005452:Fluoresceins; C085925:calcein AM; D020112:Rhodamine 123; D014700:Verapamil; D008691:Methadone