title: Human HepaRG Cells can be Cultured in Hanging-drop Plates for Cytochrome P450 Induction and Function Assays.
abstract: Recent guidance/guidelines for industry recommend that cytochrome P450 induction can be assessed using human hepatocyte enzyme activity and/or mRNA levels to evaluate potential drug- drug interactions. To evaluate time-dependent cytochrome P450 induction precisely, induction of CYP1A2, CYP2B6, and CYP3A4 mRNA was confirmed (>2-fold) by the treatment with omeprazole, phenobarbital, and rifampicin, respectively, for 24 or 48 h on day 3 from the start of culture. After 24 h, the fold induction of CYP1A2 with 3.6 and 1.8x10(4) HepaRG cells per well was lower than that for 7.2x10(4) cells. CYP1A2 induction levels at 24 h were higher than those after 48 h. In contrast, higher CYP2B6 inductions were confirmed after 48 h exposure than after 24 h, independent of the number of cells per well. To help reduce the use of human cryopreserved hepatocytes, typical P450-dependent enzyme activities were investigated in human HepaRG cells cultured in commercial hanging-drop plates. Newly designed 96-well hanging-drop plates were capable of maintaining human CYP3A-dependent midazolam hydroxylation activities for up to 4 days using only 10% of the recommended initial 7.2x10(4) cells per well. Favorable HepaRG function using hanging-drop plates was confirmed by detecting 1'- hydroxymidazolam O-glucuronide on day 3, suggesting an improvement over traditional control plates in which this metabolite can be detected for 24-well plates. These results suggest that the catalytic function and/or induction of CYP1A2, CYP2B6, and CYP3A4 can be readily assessed with reduced numbers of starting HepaRG cells cultured in three-dimensional cultures in drops prepared with hanging-drop plates.
authors.forename: Norie
authors.initials: N
authors.lastname: Murayama
authors.forename: Takashi
authors.initials: T
authors.lastname: Usui
authors.forename: Nicky
authors.initials: N
authors.lastname: Slawny
authors.forename: Christophe
authors.initials: C
authors.lastname: Chesné
authors.affiliation: Laboratory of Drug Metabolism and Pharmacokinetics, Showa Pharmaceutical University, 3-3165 Higashi-tamagawa Gakuen, Machida, Tokyo 194-8543, Japan. hyamazak@ac.shoyaku.ac.jp.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Yamazaki
mesh_terms: D018929:Cell Culture Techniques; D019388:Cytochrome P-450 CYP1A2 / Q000096:biosynthesis; D065702:Cytochrome P-450 CYP2B6 / Q000096:biosynthesis; D051544:Cytochrome P-450 CYP3A / Q000096:biosynthesis; D065693:Cytochrome P-450 Enzyme Inducers / Q000494:pharmacology*; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000235:genetics; D004347:Drug Interactions; D004790:Enzyme Induction; D056945:Hep G2 Cells; D022781:Hepatocytes / Q000187:drug effects* / Q000201:enzymology; D006801:Humans; D012333:RNA, Messenger / Q000096:biosynthesis; D018570:Risk Assessment; D012307:Risk Factors; D013379:Substrate Specificity; D013997:Time Factors
chemical_list: D065693:Cytochrome P-450 Enzyme Inducers; D012333:RNA, Messenger; D003577:Cytochrome P-450 Enzyme System; C504573:CYP1A2 protein, human; C585599:CYP2B6 protein, human; D019388:Cytochrome P-450 CYP1A2; D065702:Cytochrome P-450 CYP2B6; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human