Simultaneous expression of plural forms of human cytochrome P450 at desired ratios in HepG2 cells: adenovirus-mediated tool for cytochrome P450 reconstitution.
Human hepatocyte culture is widely used to predict human drug metabolism for new drug development. The limited supply and lot-to-lot (donor-to-donor) variations in enzymatic activity, however, hamper its applicability. In the present study, we explore a new cell system with adenovirus-mediated expression of cytochrome P450s (P450s) as an alternative for hepatocytes. In this system, P450 apoprotein levels and catalytic activity increased depending on the amounts of adenoviruses infected for the individual expression of CYP3A4 or CYP2C19 in HepG2 cells. Similar results were observed in the system with co-expression of CYP3A4 and CYP2C19. When HepG2 cells were infected with adednovirus for CYP3A4 and that for CYP2C19 simultaneously at a ratio of 10:1, the ratio of their apoprotein levels was similar to that observed in human hepatocytes and the metabolic profile of diazepam in the system was almost identical to that observed in hepatocyets. These results indicate that this adenovirus-mediated system makes it possible to reproducibly prepare cells expressing multiple P450s at a desired ratio, suggesting a possible use of this system in preclinical metabolic tests for a drug candidate(s), particularly to assess the influence of inter-individual variation in P450 activity.
Aoyama
Kazunobu
K
Division of Drug Metabolism and Molecular Toxicology, Graduate School of Pharmaceutical Sciences, Tohoku University, Miyagi, Japan.
Yoshinari
Kouichi
K
Kim
Hye-Ji
HJ
Nagata
Kiyoshi
K
Yamazoe
Yasushi
Y
D000256:Adenoviridae / Q000235:genetics*; D001189:Aryl Hydrocarbon Hydroxylases / Q000378:metabolism; D006528:Carcinoma, Hepatocellular; D045744:Cell Line, Tumor; D065731:Cytochrome P-450 CYP2C19; D051544:Cytochrome P-450 CYP3A / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis*; D003975:Diazepam / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D008113:Liver Neoplasms; D014162:Transfection / Q000379:methods
D003577:Cytochrome P-450 Enzyme System; D001189:Aryl Hydrocarbon Hydroxylases; C045793:CYP2C19 protein, human; D065731:Cytochrome P-450 CYP2C19; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D003975:Diazepam