Use of mRNA expression to detect the induction of drug metabolising enzymes in rat and human hepatocytes.

It is important to investigate the induction of cytochrome P450 (CYP) enzymes by drugs. The most relevant end point is enzyme activity; however, this requires many cells and is low throughput. We have compared the CYP1A, CYP2B and CYP3A induction response to eight inducers in rat and human hepatocytes using enzyme activities (CYP1A2 (ethoxyresorufin), 2B (benzoxyresorufin for rat and bupropion for human) and CYP3A (testosterone)) and Taqman Low Density Array (TLDA) analysis. There was a good correlation between the induction of CYP1A2, CYP2B6 and CYP3A4 enzyme activities and mRNA expression in human hepatocytes. In contrast, BROD activities and mRNA expression in rat hepatocytes correlated poorly. However, bupropion hydroxylation correlated well with Cyp2b1 expression in rat hepatocytes. TLDA analysis of a panel of mRNAs encoding for CYPs, phase 2 enzymes, nuclear receptors and transporters revealed that the main genes induced by the 8 compounds tested were the CYPs. AhR ligands also induced UDP-glucuronosyltransferases and glutathione S-transferases in rat and human hepatocytes. The transporters, MDR1, MDR3 and OATPA were the only transporter genes significantly up-regulated in human hepatocytes. In rat hepatocytes Bsep, Mdr2, Mrp2, Mrp3 and Oatp2 were up-regulated. We could then show a good in vivo:in vitro correlation in the induction response of isolated rat hepatocytes and ex-vivo hepatic microsomes for the drug development candidate, EMD392949. In conclusion, application of TLDA methodology to investigate the potential of compounds to induce enzymes in rat and human hepatocytes increases the throughput and information gained from one assay, without reducing the predictive capacity.

Differential inhibition of rat and human hepatic cytochrome P450 by Andrographis paniculata extract and andrographolide.

The inhibitory effect of Andrographis paniculata extract (APE) and andrographolide (AND), the most medicinally active phytochemical in the extract, on hepatic cytochrome P450s (CYPs) activities was examined using rat and human liver microsomes. For this purpose, CYP1A2-dependent ethoxyresorufin-O-deethylation, CYP2B1-dependent benzyloxyresorufin-O-dealkylation, CYP2B6-dependent bupropion hydroxylation, CYP2C-dependent tolbutamide hydroxylation, CYP2E1-dependent p-nitrophenol hydroxylation and CYP3A-dependent testosterone 6 beta-hydroxylation activities, were determined in the presence and absence of APE or AND (0-200 microM). APE inhibited ethoxyresorufin-O-deethylation activity in rat and human liver microsomes, with apparent Ki values of 8.85 and 24.46 microM, respectively. In each case, the mode of inhibition was noncompetitive. APE also inhibited tolbutamide hydroxylation both in rat and human microsomes with apparent Ki values of 8.21 and 7.51 microM, respectively and the mode of inhibition was mixed type. In addition, APE showed a competitive inhibition only on CYP3A4 in human microsomes with Ki of 25.43 microM. AND was found to be a weak inhibitor of rat CYP2E1 with a Ki of 61.1 microM but did not affect human CYP2E1. In conclusion, it cannot be excluded from the present study that APE could cause drug-drug interactions in humans through CYP3A and 2C9 inhibition.