This study was conducted to investigate the effects of treatment with the prototypical inducers rifampicin (Rif), dexamethasone (Dex), and omeprazole (Ome) on the mRNA levels of drug-metabolizing enzymes in primary cultures of cryopreserved human, cynomolgus monkey, and rat hepatocytes. Analysis was performed by quantitative real-time RT-PCR using primers and TaqMan probes. Treatment with Ome substantially increased the mRNA levels of both CYP1A1 and CYP1A2 in human hepatocytes, but increased only the mRNA level of CYP1A1 in monkey hepatocytes, whereas it had no marked effect on the mRNA levels of CYP1A1 or CYP1A2 in rat hepatocytes. Treatment with Rif or Dex did not markedly affect the mRNA level of CYP1A in any of the hepatocyte cultures under the conditions used. All three inducers increased the mRNA level of CYP3A8 in monkey hepatocytes (in the order Rif>Dex>or=Ome), and a similar profile was observed for the mRNA level of CYP3A4 in human hepatocytes, but the potency of induction was markedly attenuated. In contrast, only Dex substantially increased the mRNA level of CYP3A1 in rat hepatocytes, with Rif and Ome showing no effects. These results indicate that the molecular mechanisms responsible for the regulation of CYP1A2 genes differ between humans and cynomolgus monkeys, although the regulatory mechanisms for CYP1A1 and CYP3A genes are similar.

We investigated the utility of three-dimensional (3D) spheroid cultures of human hepatocytes in discovering drug metabolites. Metabolites of acetaminophen, diclofenac, lamotrigine, midazolam, propranolol and salbutamol were analyzed by liquid chromatography-tandem mass spectrometry (LC/MS/MS) to measure enzyme activities in this system cultured for 2 and 7 days. Sequential metabolic reactions by Phase I and then Phase II enzymes were found in diclofenac [CYP2C9 and UDP-glucuronyltransferases (UGTs)], midazolam (CYP3A4 and UGTs) and propranolol (CYP1A2/2D6 and UGTs). Moreover, lamotrigine and salbutamol were metabolized to lamotrigine-N-glucuronide and salbutamol 4-O-sulfate, respectively. These metabolites, which are human specific, could be observed in clinical studies, but not in conventional hepatic culture systems as in previous reports. Acetaminophen was metabolized to glucuronide and sulfate conjugates, and N-acetyl-p-benzo-quinoneimine (NAPQI) and its metabolites were not observed. In addition, mRNA of drug-metabolism enzymes [CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, UGT1A1, UGT2B7, sulfotransferase 1A1 (SULT1A1) and glutathione S-transferase pi 1 (GSTP1)], which were measured by qRT-PCR, were expressed in the human hepatocyte spheroids. In conclusion, these results suggest that human hepatocyte spheroids are useful in discovering drug metabolites.

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The mRNA induction of various transporters by rifampicin (Rif), dexamethasone (Dex) and omeprazole (Ome) was investigated in primary cultures of cryopreserved human and rat hepatocytes. Analysis was performed by quantitative real-time RT-PCR using primers and TaqMan probes. In primary cultures of human hepatocytes, mRNA levels of MDR and MRP1 were increased by about 1.5 fold and 1.3 fold, respectively, by exposure to Rif at 2 to 50 microM as compared with 0.1% DMSO-treated controls. MRP2 mRNA levels in the same human hepatocytes were significantly increased by 1.2 to 1.8 fold by exposure to Rif at 50 microM as compared with controls. In primary cultures of rat hepatocytes, Mdr1a and Mdr1b mRNA levels were not increased or only slightly increased at 24 hr by exposure to any of the inducers at 2, 10 or 50 microM. Mrp2 mRNA levels in the same rat hepatocytes were significantly increased by 7 to 45 fold by exposure to Dex at 2 microM as compared with controls. Based on the species differences observed in the present study, primary cultures of cryopreserved hepatocytes from both the human and rat should be useful in preclinical drug development for evaluating candidate drugs for transporter induction.

We investigated the change of the mRNA levels of sulfotransferase and UDP-glucuronosyltransferase isoforms by the prototypical microsomal enzyme inducers rifampicin (Rif), dexamethasone (Dex), and omeprazole (Ome) in primary cultures of cryopreserved human and cynomolgus monkey hepatocytes. Real-time RT-PCR analysis was performed using primers and TaqMan probes. Rif, Dex, and Ome increased SULT2A1 mRNA level in both human and cynomolgus monkey hepatocytes in dose-dependent manner, but not SULT1A1 mRNA level. Rif, Dex, and Ome increased the mRNA level of UGT1A1 in both human and cynomolgus monkey hepatocytes, Ome more potently in humans and Rif and Ome more potently in monkeys. They also increased the mRNA levels of UGT1A6 and UGT1A9 in cynomolgus monkey hepatocytes, though the extent of elevation of UGT1A6 and UGT1A9 mRNA levels was smaller than that of UGT1A1 mRNA level. Furthermore, these inducers scarcely affected UGT1A6 and UGT1A9 in human hepatocytes. Rif, Dex, and Ome also showed no remarkable effect on the mRNA levels of UGT2Bs in human or cynomolgus monkey hepatocytes. We also studied in detail the time course of mRNA expression of these enzymes in primary cultures of hepatocytes. In conclusion, the results of the present study show that primary cultures of hepatocytes isolated from the cynomolgus monkey liver are as useful as human hepatocytes for evaluating the induction of drug-metabolizing enzymes in preclinical studies.