Comparison of Inducibility of Sulfotransferase and UDP-Glucuronosyltransferase mRNAs by Prototypical Microsomal Enzyme Inducers in Primary Cultures of Human and Cynomolgus Monkey Hepatocytes

doi:10.2133/dmpk.23.45

Drug Metabolism and Pharmacokinetics

Volume 23, Issue 1, 2008, Pages 45-53

https://doi.org/10.2133/dmpk.23.45 Get rights and content

Summary:

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. Realtime 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.

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Comparison of mRNA expression profiles of drug-metabolizing enzymes and transporters in fresh and cryopreserved cynomolgus monkey hepatocytes
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Citation Excerpt :
In our laboratory, monkey hepatocytes of high quality have been isolated using the method for human hepatocyte isolation with some modification. These primary hepatocytes were used in induction studies of drug-metabolizing enzymes and to assess the mRNA expression fluctuation during short-term culture [14–16]. In general, primary hepatocytes are considered to lose liver function after isolation from the liver, and conventional monolayer cultures are limited to about 1 or 2 weeks of culture in which liver-specific markers (e.g., secretion of albumin), cell viability, and cell attachment efficiency are maintained [17–19].
In this study, freshly isolated and cryopreserved cynomolgus monkey hepatocytes were seeded on Cell-able^® plates with feeder cells to form spheroids and were cultured for 28 days. As a control, hepatocytes were also cultured with or without feeder cells on collagen-coated plates. We verified the mRNA expression levels of drug-metabolizing enzyme-related genes and the leakage of enzymes (AST, ALT, LDH, and γ-GTP) as indicators of cell survival. As a result, the patterns of target mRNA expression in fresh and cryopreserved hepatocytes were very similar during the culture period between culture methods. mRNA expression levels were highly maintained at day 28 using the 3D spheroid and co-culture methods, demonstrating that these methods are useful for maintenance of liver function. Leakage of AST and ALT was higher at day 3 but decreased at day 14. LDH was not detected, suggesting that the cell viability was also maintained during the culture period. Furthermore, the functional differences between fresh and cryopreserved hepatocytes were not clearly detected. The co-culture method was useful for long-term culture not requiring 3D structure, and the 3D spheroid culture method was effective as well. With these techniques, cynomolgus monkey hepatocytes are expected to exhibit smaller individual differences and high reproducibility.
Expression levels of drug-metabolizing enzyme, transporter, and nuclear receptor mRNAs in a novel three-dimensional culture system for human hepatocytes using micro-space plates
2011, Drug Metabolism and Pharmacokinetics
We evaluated a novel three-dimensional primary culture system using micro-space plates to determine the expression levels of 61 target (drug-metabolizing enzymes, transporters, and nuclear receptors) mRNAs in human hepatocytes. We measured mRNA expression levels of many target genes in four lots of cryopreserved human hepatocyte primary cells after 120 h of culture and compared differences in mRNA expression levels between cultures using traditional plates and those using micro-space plates. In this study, we show that the mRNA levels of many experimental targets in human hepatocytes before inoculation resemble the levels inside the human liver. Furthermore, we show that the rate of change of expression levels of many target mRNAs relative to the value before inoculation of the hepatocytes into micro-space plates was relatively smaller than the rate of change in hepatocytes inoculated into traditional plates. Pharmacokinetics-related examinations using this system are possible within a time frame of 120 h. We report that this novel three-dimensional culture system reproduces mRNA expression levels that are nearer to those in the liver in vivo and is an excellent platform for maintaining mRNA expression levels of drug-metabolizing enzymes and transporters when compared to common monolayer cultures.
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This study evaluates the induction potency of new drug candidates on mRNA levels of CYP1A2 and CYP3A4 in primary cultures of cryopreserved human hepatocytes. Analysis was performed by quantitative real-time RT-PCR using primers and TaqMan probes. Positive controls for CYP1A2 and CYP3A4 used β-naphthoflavone (β-NF) and rifampicin (Rif), respectively. In the first stage of the study, the lot showing the best induction of mRNA expression CYP1A2 and CYP3A4 from among eight lots of hepatocytes was selected. In the second stage, we evaluated the levels of CYP1A2 and CYP3A4 gene expression in hepatocytes after exposure to eight NO-1886 (ibrolipim) derivatives. A combination of real-time one-step RT-PCR and primary culture of cryopreserved human hepatocytes is suitable for evaluating of induction potency of a large number of new drug candidates.
Tissue-specific mRNA expression profiles of drug-metabolizing enzymes and transporters in the cynomolgus monkey
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Pairs of forward and reverse primers and TaqMan probes specific to each of 19 drug-metabolizing enzymes (cytochrome P450s, UDP-glucuronosyltransferases, glutathione S-transferases, and sulfotransferases) and 5 transporters (ABC and SLC transporters) in the cynomolgus monkey were prepared. The expression level of each target mRNA was analyzed in total RNA obtained from three specimens of various cynomolgus monkey tissues (adrenal gland, brain, heart, kidney, large intestine, liver, lung, pancreas, prostate, small intestine, spleen, testis, and thymus) by real-time reverse transcription PCR using an Applied Biosystems 7500 Fast Real-Time PCR System. The data obtained in the present study provide useful information on tissue-specific profiles of the expression of these target mRNAs in the cynomolgus monkey, and the results are expected to be valuable in establishing drug metabolism- and transporter-mediated screening systems using the cynomolgus monkey for the evaluation of new chemical entities in new drug development.
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Regular ArticleComparison of Inducibility of Sulfotransferase and UDP-Glucuronosyltransferase mRNAs by Prototypical Microsomal Enzyme Inducers in Primary Cultures of Human and Cynomolgus Monkey Hepatocytes

Summary:

Chem. Biol. Interact.

Gastroenterology

Drug Metab. Pharmacokinet.

Biochem. Pharmacol.

Food Chem. Toxicol.

Methods Cell Biol.

Drug Metab. Pharmacokinet.

Drug Metab. Pharmacokinet.

Biochem. Biophys. Res. Commun.

Drug Metab. Pharmacokinet.

Hepatology

J. Biol. Chem.

Rifampicin induction of lidocaine metabolism in cultured human hepatocytes

J. Pharmacol. Exp. Ther.

Regular Article
Comparison of Inducibility of Sulfotransferase and UDP-Glucuronosyltransferase mRNAs by Prototypical Microsomal Enzyme Inducers in Primary Cultures of Human and Cynomolgus Monkey Hepatocytes