Regular Article Comparison of Inducibility of CYP1A and CYP3A Mrnas by Prototypical Inducers in Primary Cultures of Human, Cynomolgus Monkey, and Rat Hepatocytes

Drug Metab. Pharmacokinet. 22 (3): 178–186 (2007). Regular Article Comparison of Inducibility of CYP1A and CYP3A mRNAs by Prototypical Inducers in Primary Cultures of Human, Cynomolgus Monkey, and Rat Hepatocytes Masuhiro NISHIMURA1, Akiko KOEDA2, Yasuyuki SUGANUMA2,EmakoSUZUKI2, Takefumi SHIMIZU2,MitsuoNAKAYAMA1,TetsuoSATOH2,3, Shizuo NARIMATSU4 and Shinsaku NAITO1,* 1Department of Drug Metabolism, Division of Pharmacology, Drug Safety and Metabolism, Otsuka Pharmaceutical Factory, Inc., Tokushima, Japan 2Ina Research Inc., Nagano, Japan 3Non-Proˆt Organization Human & Animal Bridging Research Organization, Chiba, Japan 4Laboratory of Health Chemistry, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Japan Full text of this paper is available at http://www.jstage.jst.go.jp/browse/dmpk Summary: This study was conducted to investigate the eŠects 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 eŠect on the mRNA levels of CYP1A1 or CYP1A2 in rat hepatocytes. Treatment with Rif or Dex did not markedly aŠect 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 RifÀDexÆOme), and a similar proˆle 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 eŠects. These results indicate that the molecular mechanisms responsible for the regulation of CYP1A2 genes diŠer between humans and cynomolgus monkeys, although the regulatory mechanisms for CYP1A1 and CYP3A genes are similar. Key words: induction, cytochrome P450, cynomolgus monkey, human, rat, hepatocytes an experimental animal species because the monkey Introduction belongs to the order Primates, which also includes man. Drug discovery and development depend greatly on Various monkey species such as cynomolgus monkeys, the successful extrapolation of drug metabolism and rhesus monkeys, marmoset monkeys, and so on have toxicity data obtained from experimental animals to been used in a wide variety of experiments. Among humans. It is therefore important to understand the these species, cynomolgus monkeys have often been similarities and diŠerences between experimental employed in studies of drug metabolism and toxicity. animals and humans in terms of the ‰uctuation proˆles Recently, we have been studying the changes in the of the cytochrome P450 (CYP) subfamilies following mRNA levels of various drug-metabolizing enzymes in exposure to xenobiotics. From the viewpoint of data primary cultures of human and rat hepatocytes follow- extrapolation, the monkey is a promising candidate for ing treatment with prototypical inducers such as rifam- Received; January 10, 2007, Accepted; May 2, 2007 *To whom correspondence should be addressed: Shinsaku NAITO,Ph.D.,Division of Pharmacology, Drug Safety and Metabolism, Otsuka Pharmaceutical Factory, Inc., Naruto, Tokushima 772-8601, Japan. Tel. ＋81-88-685-1151, Fax. ＋81-88-686-8176, E-mail: naitousn＠otsukakj. co.jp 178 CYP1A and CYP3A Induction in Primary Cultures of Hepatocytes 179 Table 1. Characteristics of hepatocytes and preparations. reagent grade. Animals: Adult male and female cynomolgus Age Sex RaceWStrain Viability (z) monkeys were obtained from Ina Research Philippines, Human Inc. (Batangas, Philippines) and adult female rats were Donor #1 23 years female Caucasian 93.8 obtained from Charles River Japan, Inc. (Kanagawa, Donor #2 74 years female Caucasian 91.8 Donor #3 2 years female Caucasian 91.2 Japan) (Table 1). These animals were allowed free Monkey access to food and water. The study was approved by Monkey #1 3 years male Cynomolgus 80.3 the Committee on the Care and Use of Laboratory Monkey #2 2–3 years male Cynomolgus 99.3 Animals of Ina Research Inc. and the Committee on the Monkey #3 3 years female Cynomolgus 88.3 Care and Use of Laboratory Animals of Otsuka Phar- Rat Rat #1 7 weeks female Sprague-Dawley 91.8 maceutical Factory, Inc. Rat #2 7 weeks female Sprague-Dawley 89.3 Isolation of cynomolgus monkey hepatocytes: Per- Rat #3 7 weeks female Sprague-Dawley 88.1 fusion of the cynomolgus monkey liver was performed according to the Seglen method with some modiˆca- Viability was determined by the trypan blue exclusion test. tions.8) Brie‰y, the liver was perfused with Ca2＋-free Hanks solution containing 0.6 mM EGTA at 379Cfor picin (Rif), dexamethasone (Dex), and omeprazole 4 min and further perfused with Hanks solution con- (Ome). Rif induces CYP3A4 in primary cultures of taining 0.025z collagenase at 379C for 6 min. Hepato- human hepatocytes,1–3) while this drug is not an inducer cytes were dispersed from the perfused liver in ice-cold of CYP3A in rat hepatocytes,2–4) with the rat being the HEPES solution. After ˆltration through gauze, the most commonly used laboratory animal species for hepatocytes were separated from nonparenchymal cells toxicological studies. Dex is a potent inducer of CYP3A by centrifugation at 50×g for 2 min at 49C. The cells in rat hepatocytes, whereas it is a weak inducer of were washed and resuspended in HEPES solution three CYP3A in human hepatocytes.3) Ome is a potent in- times, then washed with Williams' medium E containing ducer of CYP1A in primary cultures of human hepato- 20z fetal bovine serum, 2.5z polyvinylpyrrolidone, cytes.5) and 2z bovine serum albumin7) (with the pH adjusted Although various studies on the catalytic properties to approximately 7.4 with NaHCO3). Finally, the cells of CYP enzymes in cynomolgus monkey hepatocytes were resuspended in the same medium and cell viability have been reported,6,7) there is little information availa- was determined by the trypan blue exclusion test. After ble on the induction of CYP enzymes in cynomolgus the addition of dimethylsulfoxide (DMSO, ˆnal concen- monkey hepatocytes. In the present study, we conduct- tration of 10z) to the cell suspension, the cell density ed comparative experiments on the inducibility of was approximately 1.5 to 2.5×107 viable cellsWmL. The CYP1A and CYP3A mRNAs by the prototypical cynomolgus monkey hepatocytes were cryopreserved at inducers Rif, Dex, and Ome in primary cultures of －309C for 40 min, at －809C for 15 min, and then in cryopreserved human, cynomolgus monkey, and rat liquid nitrogen until use. hepatocytes. Isolation of rat hepatocytes: Perfusion of the rat liver was performed according to the Seglen method8) Materials and Methods with some modiˆcations.9,10) The rat hepatocytes were Materials: Rif, Dex and Ome were purchased from cryopreserved at －309C for 40 min, at －809Cfor15 Wako Pure Chemical Industries, Ltd. (Osaka, Japan). min, and then in liquid nitrogen until use. Collagenase S-1 was purchased from Nitta Gelatin, Inc. Monolayer culture of human and rat hepatocytes: (Osaka, Japan); bovine serum albumin (fraction V) Monolayer cultures of the cryopreserved human and rat from Sigma-Aldrich (St. Louis, MO); and trypan blue hepatocytes were obtained according to the method of from Merck (Darmstadt, Germany) and Flow Labora- Nishimura et al.9,10) Human cell suspensions with tories, Ltd. (Irvine, UK). Cryopreserved human hepato- viability rates of 91z to 94z and rat cell suspensions cytes (donor #1 [Lot 082], donor #2 [Lot 100], and with viability rates of 88z to 92z, as assessed by the donor #3[Lot130])(Table 1) were purchased from trypan blue exclusion test, were used for the experi- In Vitro Technologies, Inc. (Baltimore, MD). Hepato- ments (Table 1). The cells were used for experiments at cyte Culture Medium (CC-3198) was purchased from 48 hr after inoculation. BioWhittaker, Inc. (Walkersville, MD); the RNeasy} Monolayer culture of cynomolgus monkey hepato- Mini Kit and QIAshredderTM from QIAGEN (Hilden, cytes: Monolayer cultures of the cryopreserved Germany); yeast tRNA from Life Technologies, Inc. cynomolgus monkey hepatocytes were obtained accord- (Rockville, MD); and TaqMan One-Step RT-PCR ing to the method of Nishimura et al.11) with some Master Mix Reagents from Applied Biosystems (Foster modiˆcations. The cryopreserved hepatocytes were City, CA). All other chemicals used in this study were of suspended in Hepatocyte Culture Medium. The hepato- 180 Masuhiro NISHIMURA, et al. cytes were then centrifuged at 45×g for 3 min at 49C been reported previously. The primers and TaqMan and resuspended in the same medium. The number of probes were synthesized by Sigma-Aldrich Japan K.K. cells was counted using a Coulter Counter (Beckman Genosys Division (Ishikari, Japan). The TaqMan Coulter, Inc., Fullerton, CA). Cell suspensions with probes contained 6-carboxy‰uorescein (FAM) at the 5? viability rates of 80z to 99z were used for the end and 6-carboxytetramethylrhodamine (TAMRA) at experiments (Table 1). The cell suspensions were diluted the 3? end and were designed to hybridize to a sequence to a ˆnal concentration of 2.5×105 viable cellsWmL with located between the PCR primers. Hepatocyte Culture Medium, and inocula of 1×105 TaqMan RT-PCR conditions: Total RNA was viable cellsW0.4 mLWwell were introduced into 24-well diluted to about 4 mgWmL with 50 mgWmL yeast tRNA. platesthathadbeencoatedwithtypeIcollagen.The The RT-PCR assay was performed in 50 mLofTaqMan cells were cultured for 3 hr after inoculation under 5z One-Step RT-PCR Master Mix reagents containing 300 CO2 and 95z air at 379C. The medium was then nM forward primer, 900 nM reverse primer, 200 nM replaced with fresh medium, and the cells were cultured TaqMan probe, and about 20 ng of total RNA accord- 14,15) for 21 hr under 5z CO2 and 95z air at 379C.

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