Cryopreserved human hepatocytes as alternative in vitro model for cytochrome p450 induction studies.
Induction of cytochrome P450 (CYP) by drugs is one of major concerns for drug-drug interactions. Thus, the assessment of CYP induction by novel compounds is a vital component in the drug discovery and development processes. Primary human hepatocytes are the preferred in vitro model for predicting CYP induction in vivo. However, their use is hampered by the erratic supply of human tissue and donor-to-donor variability. Although cryopreserved hepatocytes have been recommended for short-term applications in suspension, their use in studies on induction of enzyme activity has been limited because of poor attachment and response to enzyme inducers. In this study, we report culture conditions that allowed the attachment of cryopreserved human hepatocytes and responsiveness to CYP inducers. We evaluated the inducibility of CYP1A1/2 and CYP3A4 enzymes in cryopreserved hepatocytes from three human donors. Cryopreserved human hepatocytes were cultured in serum-free medium for 4 d. They exhibited normal morphology and measurable viability as evaluated by the reduction of tetrazolium salts (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) by cellular dehydrogenases. Treatment with beta-naphthoflavone (10 microM) for 3 d increased ethoxyresorufin-O-deethylase activity (CYP1A1/2) by 6- to 11-fold over untreated cultures and increased CYP1A2 messenger ribonucleic acid (mRNA) expression by three- to eightfold. Similarly, treatment of cryopreserved human hepatocytes with rifampicin (25 microM) for 3 d increased testosterone 6 beta-hydroxylase activity (CYP3A4) by five- to eightfold over untreated cultures and increased CYP3A4 mRNA expression by four- to eightfold. The results suggest that cryopreserved human hepatocytes can be a suitable in vitro model for evaluating xenobiotics as inducers of CYP1A1/2 and CYP3A4 enzymes.
Absorption Systems, LP, Oaklands Corporate Center, 440 Creamery Way, Suite 300, Exton, Pennsylvania 19341, USA. garcia@absorption.com
Martha
M
Garcia
Joseph
J
Rager
Qing
Q
Wang
Robert
R
Strab
Ismael J
IJ
Hidalgo
Albert
A
Owen
Jibin
J
Li
D000328:Adult; D000368:Aged; D000818:Animals; D048429:Cell Size; D002470:Cell Survival; D002478:Cells, Cultured; D015925:Cryopreservation; D016895:Culture Media, Serum-Free; D019363:Cytochrome P-450 CYP1A1 / Q000235:genetics / Q000378:metabolism*; D019388:Cytochrome P-450 CYP1A2 / Q000235:genetics / Q000378:metabolism*; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D005786:Gene Expression Regulation; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D006915:Hydroxytestosterones / Q000737:chemistry / Q000378:metabolism; D066298:In Vitro Techniques; D008297:Male; D008875:Middle Aged; D008954:Models, Biological; D010078:Oxazines / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D012293:Rifampin / Q000494:pharmacology
D016895:Culture Media, Serum-Free; D004791:Enzyme Inhibitors; D006915:Hydroxytestosterones; D010078:Oxazines; D012333:RNA, Messenger; C014180:resorufin; D003577:Cytochrome P-450 Enzyme System; C104464:CYP3A protein, human; D019363:Cytochrome P-450 CYP1A1; D019388:Cytochrome P-450 CYP1A2; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D012293:Rifampin