Impact of Experimental Conditions on the Evaluation of Interactions between Multidrug and Toxin Extrusion Proteins and Candidate Drugs.
Multidrug and toxin extrusion transporters (MATEs) have a determining influence on the pharmacokinetic profiles of many drugs and are involved in several clinical drug-drug interactions (DDIs). Cellular uptake assays with recombinant cells expressing human MATE1 or MATE2-K are widely used to investigate MATE-mediated transport for DDI assessment; however, the experimental conditions and used test substrates vary among laboratories. We therefore initially examined the impact of three assay conditions that have been applied for MATE substrate and inhibitor profiling in the literature. One of the tested conditions resulted in significantly higher uptake rates of the three test substrates, [(14)C]metformin, [(3)H]thiamine, and [(3)H]1-methyl-4-phenylpyridinium (MPP(+)), but IC50 values of four tested MATE inhibitors varied only slightly among the three conditions (<2.5-fold difference). Subsequently, we investigated the uptake characteristics of the five MATE substrates: [(14)C]metformin, [(3)H]thiamine, [(3)H]MPP(+), [(3)H]estrone-3-sulfate (E3S), and rhodamine 123, as well as the impact of the used test substrate on the inhibition profiles of 10 MATE inhibitors at one selected assay condition. [(3)H]E3S showed atypical uptake characteristics compared with those observed with the other four substrates. IC50 values of the tested inhibitors were in a similar range (<4-fold difference) when [(14)C]metformin, [(3)H]thiamine, [(3)H]MPP(+), or [(3)H]E3S were used as substrates but were considerably higher with rhodamine 123 (9.8-fold and 4.1-fold differences compared with [(14)C]metformin with MATE1 and MATE2-K, respectively). This study demonstrated for the first time that the impact of assay conditions on IC50 determination is negligible, that kinetic characteristics differ among used test substrates, and that substrate-dependent inhibition exists for MATE1 and MATE2-K, giving valuable insight into the assessment of clinically relevant MATE-mediated DDIs in vitro.
Pharmacokinetics and Non-Clinical Safety Department, Nippon Boehringer Ingelheim Co., Ltd., Kobe, Japan (C.L., N.I., A.F., H.S., N.O., M.T., K.N., I.W., N.Y.); and Laboratory of Molecular Pharmacokinetics, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan (H.K.).
Christian
C
Lechner
Pharmacokinetics and Non-Clinical Safety Department, Nippon Boehringer Ingelheim Co., Ltd., Kobe, Japan (C.L., N.I., A.F., H.S., N.O., M.T., K.N., I.W., N.Y.); and Laboratory of Molecular Pharmacokinetics, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan (H.K.).
Naoki
N
Ishiguro
Pharmacokinetics and Non-Clinical Safety Department, Nippon Boehringer Ingelheim Co., Ltd., Kobe, Japan (C.L., N.I., A.F., H.S., N.O., M.T., K.N., I.W., N.Y.); and Laboratory of Molecular Pharmacokinetics, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan (H.K.).
Ayano
A
Fukuhara
Pharmacokinetics and Non-Clinical Safety Department, Nippon Boehringer Ingelheim Co., Ltd., Kobe, Japan (C.L., N.I., A.F., H.S., N.O., M.T., K.N., I.W., N.Y.); and Laboratory of Molecular Pharmacokinetics, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan (H.K.).
Hidetada
H
Shimizu
Pharmacokinetics and Non-Clinical Safety Department, Nippon Boehringer Ingelheim Co., Ltd., Kobe, Japan (C.L., N.I., A.F., H.S., N.O., M.T., K.N., I.W., N.Y.); and Laboratory of Molecular Pharmacokinetics, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan (H.K.).
Naoko
N
Ohtsu
Pharmacokinetics and Non-Clinical Safety Department, Nippon Boehringer Ingelheim Co., Ltd., Kobe, Japan (C.L., N.I., A.F., H.S., N.O., M.T., K.N., I.W., N.Y.); and Laboratory of Molecular Pharmacokinetics, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan (H.K.).
Masahito
M
Takatani
Pharmacokinetics and Non-Clinical Safety Department, Nippon Boehringer Ingelheim Co., Ltd., Kobe, Japan (C.L., N.I., A.F., H.S., N.O., M.T., K.N., I.W., N.Y.); and Laboratory of Molecular Pharmacokinetics, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan (H.K.).
Kotaro
K
Nishiyama
Pharmacokinetics and Non-Clinical Safety Department, Nippon Boehringer Ingelheim Co., Ltd., Kobe, Japan (C.L., N.I., A.F., H.S., N.O., M.T., K.N., I.W., N.Y.); and Laboratory of Molecular Pharmacokinetics, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan (H.K.).
Ikumi
I
Washio
Pharmacokinetics and Non-Clinical Safety Department, Nippon Boehringer Ingelheim Co., Ltd., Kobe, Japan (C.L., N.I., A.F., H.S., N.O., M.T., K.N., I.W., N.Y.); and Laboratory of Molecular Pharmacokinetics, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan (H.K.).
Norio
N
Yamamura
Pharmacokinetics and Non-Clinical Safety Department, Nippon Boehringer Ingelheim Co., Ltd., Kobe, Japan (C.L., N.I., A.F., H.S., N.O., M.T., K.N., I.W., N.Y.); and Laboratory of Molecular Pharmacokinetics, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan (H.K.) kusuhara@mol.f.u-tokyo.ac.jp.
Hiroyuki
H
Kusuhara
D015655:1-Methyl-4-phenylpyridinium / Q000378:metabolism*; D001692:Biological Transport; D002021:Buffers; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D004970:Estrone / Q000031:analogs & derivatives* / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D049990:Membrane Transport Modulators / Q000494:pharmacology; D008687:Metformin / Q000378:metabolism*; D027701:Organic Cation Transport Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D020112:Rhodamine 123 / Q000378:metabolism*; D018570:Risk Assessment; D013831:Thiamine / Q000378:metabolism*; D014162:Transfection
D002021:Buffers; D049990:Membrane Transport Modulators; D027701:Organic Cation Transport Proteins; C521869:SLC47A1 protein, human; C521870:SLC47A2 protein, human; D020112:Rhodamine 123; D004970:Estrone; D008687:Metformin; C017296:estrone sulfate; D015655:1-Methyl-4-phenylpyridinium; D013831:Thiamine