Determination of cytochrome P450 1A2 and cytochrome P450 3A4 induction in cryopreserved human hepatocytes.
Freshly prepared human hepatocytes are considered as the 'gold standard' for in vitro testing of drug candidates. However, several disadvantages are associated with the use of this model system. The availability of hepatocytes is often low and consequently the planning of the experiments rendered difficult. In addition, the quality of the available cells is in some cases poor. As an alternative, cryopreserved human hepatocytes were validated as a model to study cytochrome P450 1A2 (CYP1A2) and cytochrome P450 3A4 (CYP3A4) induction. In a single blinded experiment, hepatocytes from three separate lots were incubated with three concentrations of different compounds, and compared to non-treated cells and cells incubated with omeprazole or rifampicin. CYP1A2 and CYP3A4 induction was determined by measuring 7-ethoxyresorufin-O-deethylation activity and 6beta-hydroxytestosterone formation, respectively. CYP1A2 and CYP3A4 mRNA and protein expression were analyzed by TaqMan QRT-PCR and immunodetection. Cells responded well to the prototypical inducers with a mean 38.8- and 6.2-fold induction of CYP1A2 and CYP3A4 activity, respectively. Similar as with fresh human hepatocytes, high batch-to-batch variation of CYP1A2 and CYP3A4 induction was observed. Except for 1 and 10 microM rosiglitazone, the glitazones did not significantly affect CYP1A2. A similar result was observed for CYP3A4 activity although CYP3A4 mRNA and protein expression were dose-dependently upregulated. In conclusion, cryopreserved human hepatocytes may be a good alternative to fresh hepatocytes to study CYP1A and 3A induction.
Roymans
Dirk
D
Preclinical Pharmacokinetics, Johnson & Johnson Pharmaceutical Research & Development, Turnhoutseweg 30, B-2340 Beerse, Belgium. droymans@prdbe.jnj.com
Van Looveren
Cis
C
Leone
Angelique
A
Parker
J Brandon
JB
McMillian
Michael
M
Johnson
Mark D
MD
Koganti
Aruna
A
Gilissen
Ron
R
Silber
Paul
P
Mannens
Geert
G
Meuldermans
Willem
W
D002478:Cells, Cultured; D015925:Cryopreservation; D019388:Cytochrome P-450 CYP1A2 / Q000096:biosynthesis*; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis*; D004790:Enzyme Induction; D022781:Hepatocytes / Q000201:enzymology*; D006801:Humans
D003577:Cytochrome P-450 Enzyme System; C104464:CYP3A protein, human; D019388:Cytochrome P-450 CYP1A2; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human