P115 Experimental conditions routinely used in human hepatocyte studies to improve the in vitro induction of CYP2D6 and CYP2C

Solenne Martin , EUROSAFE, SAINT GREGOIRE, France
Beatrice Lopez , EUROSAFE, SAINT GREGOIRE, France
Jenifer Carteret , EUROSAFE, SAINT GREGOIRE, France
Christophe Chesne , EUROSAFE, SAINT GREGOIRE, France
Human hepatocytes are routinely cultured in the presence of hydrocortisone or dexamethasone as a media supplement to promote cell survival and maintenance of normal morphology. JM. Pascussi previously demonstrated cross-talk between nuclear glucocorticoide-, CAR- and PXR‑receptor. M. Farooq has recently published that CYP2D6 is inducible in vitro but the routine presence of 100 nM dexamethasone in the culture medium masks this induction.

Based on these observations and the difficulty to evaluate mRNA induction of CYP2C8, 2C9, 2C19 and 2D6 on human hepatocytes in classical culture conditions, then we decided to modify the culture medium without any corticoid in order to investigate the induction of CYP2C and 2D6.

In order to select the best conditions on human hepatocytes, different concentrations of rifampicin, phenobarbital, dexamethasone and the mix dexamethasone/rifampicine were incubated during 24, 48 and 72 hours. At the end of each incubation time, CYPs mRNA was quantified by qRT-PCR. In parallel viability of hepatocytes was controlled. The evaluation of the induction potency was based on the EMA guideline considering that an in vitro induction is positive if incubations with a drug rise to a more than 100% increase in mRNA expression.

For CYP2C8, induction was observed with rifampicin at 25 µM, dexamethasone 0.1 to 10 µM, phenobarbital 0.5 and 1 mM and the mix (25µM rifampicine /0.1 and 1 µM dexamethasone) with a higher induction after 72h of incubation. The same results were observed for CYP2C9. For CYP2C19, induction was observed only with the mix (rifampicine /dexamethasone) and only after 72h of incubation. For CYP2D6, induction was observed with dexamethasone 0.1 to 10 µM and the mix (25µM rifampicine / 0.1 and 1 µM dexamethasone) only after 72h of incubation.

In conclusion the absence of corticoid in the culture medium allows to obtain significant induction with dexamethasone 0.1 to 10 µM for CYP2C8, 2C9 and 2D6 as well as with the mix (rifampicin/dexamethasone).