title: Absolute quantification and differential expression of drug transporters, cytochrome P450 enzymes, and UDP-glucuronosyltransferases in cultured primary human hepatocytes.
abstract: The levels of metabolizing enzymes and transporters expressed in hepatocytes are decisive factors for hepatobiliary disposition of most drugs. Induction via nuclear receptor activation can significantly alter those levels, with the coregulation of multiple enzymes and transporters occurring to different extents. Here, we report the use of a targeted liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) method for concurrent quantification of multiple cytochrome P450 (P450), UDP-glucuronosyltransferase (UGT), and transporter proteins in cultured primary human hepatocytes. The effects of culture format (i.e., sandwich culture versus conventional culture) and of dexamethasone (DEX) media concentrations on mRNA, protein, and activity levels were determined for three donors, and protein expression was compared with that in liver. In general, P450 and UGT expression was lower in hepatocyte cultures than that in liver, and CYP2C9 was found to be the most abundant P450 isoform expressed in cultured hepatocytes. The sandwich culture format and 0.1 μM DEX in media retained the protein expression in the hepatocytes closest to the levels found in liver. However, higher in vitro expression was observed for drug transporters, especially for multidrug resistance protein 1 and breast cancer resistance protein. Direct protein quantification was applied successfully to study in vitro induction in sandwich cultured primary hepatocytes in a 24-well format using the prototypical inducers rifampicin, omeprazole, and phenobarbital. We conclude that targeted absolute LC-MS/MS quantification of drug-metabolizing enzymes and transporters can broaden the scope and significantly increase the impact of in vitro drug metabolism studies, such as induction, as an important supplement or future alternative to mRNA and activity data.
authors: Department of Pharmacokinetics and Nonclinical Safety, Kobe Pharma Research Institute, Nippon Boehringer Ingelheim Co., Ltd., 6-5-7 Minatojima-Minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan. olaf.schaefer@boehringer-ingelheim.com
authors: Olaf
authors: O
authors: Schaefer
authors: Sumio
authors: S
authors: Ohtsuki
authors: Hirotaka
authors: H
authors: Kawakami
authors: Tae
authors: T
authors: Inoue
authors: Stephanie
authors: S
authors: Liehner
authors: Asami
authors: A
authors: Saito
authors: Atsushi
authors: A
authors: Sakamoto
authors: Naoki
authors: N
authors: Ishiguro
authors: Takehisa
authors: T
authors: Matsumaru
authors: Tetsuya
authors: T
authors: Terasaki
authors: Thomas
authors: T
authors: Ebner
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D003907:Dexamethasone / Q000737:chemistry / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D014453:Glucuronosyltransferase / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D022781:Hepatocytes / Q000737:chemistry* / Q000201:enzymology / Q000378:metabolism; D006801:Humans; D026901:Membrane Transport Proteins / Q000096:biosynthesis / Q000737:chemistry*; D051381:Rats; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D026901:Membrane Transport Proteins; D003907:Dexamethasone; D003577:Cytochrome P-450 Enzyme System; D014453:Glucuronosyltransferase