小括

カニクイザル新鮮及び凍結肝細胞を用い，3種類の培養条件で典型的な酵素誘 導剤を曝露した時の主なCYP mRNA発現レベルを比較した．フィーダー細胞が 共存する共培養及び3Dスフェロイド培養では，長期培養期間中においてmRNA 発現レベルが維持されていることが確認されている．実際，共培養と3Dスフェ ロイド培養は似通った結果を示した．単層培養においてわずかに誘導率が高く なる傾向を示したが，これは無処理の細胞においてmRNA発現レベルが減少す

60

ることで，相対的にmRNA発現レベルが高くなったためと考えられた．以上か ら，カニクイザルの新鮮及び凍結肝細胞のいずれも，新規化合物の誘導能を評 価するのに有用であることが示唆された．酵素誘導剤により核内レセプターの mRNA 発現レベルが減少する理由についてはさらなる検証で明らかにする必要 がある．

61

第四章 総括

本研究において，カニクイザルの新鮮肝細胞及び凍結肝細胞で，mRNA発現 レベルを指標とした細胞の機能に明確な違いはみられなかった．長期培養にお けるmRNA発現レベルの維持には，フィーダー細胞の存在が重要であることが 示唆され，また共培養法は細胞数や培養基材の面から，費用対効果の高い方法 であると考えられた．カニクイザル肝細胞は，新規化合物の酵素誘導能を評価 するのに有用なツールとなる可能性が示唆された．

62

謝辞

本研究を遂行するにあたり，終始ご懇篤な御指導，御鞭撻を賜り，また本論 文の御校閲を賜りました名古屋市立大学大学院 薬学研究科 臨床薬学分野 松永 民秀 教授に深甚なる謝意を表します．

本論文の作成にあたり，御校閲，御指導を賜りました名古屋市立大学大学院 薬学研究科 頭金 正博 教授，湯浅 博昭 教授，井上 靖道 准教授に深く感謝致 します．

本研究の遂行に際し，親身なる御指導と御鞭撻を賜りました名古屋市立大学 大学院 薬学研究科 臨床薬学分野 岩尾 岳洋 准教授に厚く御礼申し上げます．

本研究を遂行に際し，御指導，御助言を賜りました琉球大学医学部付属病院 薬剤部 教授・薬剤部長 中村 克徳 教授に厚く御礼申し上げます．

本研究の遂行に際し，終始御協力と御激励を賜りました株式会社イナリサー チの皆様に深く感謝致します．

63

引用文献

1) Shiina T, Yamada Y, Aarnink A, Suzuki S, Masuya A, Ito S, et al. Discovery of novel MHC-class I alleles and haplotypes in Filipino cynomolgus macaques (Macaca fascicularis) by pyrosequencing and Sanger sequencing: Mafa-class I polymorphism. Immunogenetics. 2015;67:563–578.

2) Shiina T, Tanaka K, Katsuyama Y, Otabe K, Sakamoto K, Kurata M, et al.

Mitochondrial DNA diversity among three subpopulations of cynomolgus macaques (Macaca fascicularis) originating from the Indochinese region. Exp Anim. 2010;59:567-78.

3) Shiba Y, Gomibuchi T, Seto T, Wada Y, Ichimura H, Tanaka Y, et al. Allogeneic transplantation of iPS cell-derived cardiomyocytes regenerates primate hearts.

Nature. 2016;538:388-91.

4) Seglen PO. Preparation of rat liver cells. I. Effect of Ca 2+ on enzymatic dispersion of isolated, perfused liver. Exp Cell Res. 1972;74:450–454.

5) Godoy P, Hewitt NJ, Albrecht U, Andersen ME, Ansari N, Bhattacharya S, et al.

Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME. Arch Toxicol. 2013;87:1315–1530.

6) Pfeiffer E, Kegel V, Zeilinger K, Hengstler JG, Nüssler AK, Seehofer D, et al.

Featured Article: Isolation, characterization, and cultivation of human hepatocytes and non-parenchymal liver cells. Exp Biol Med (Maywood).

2015;240:645–656.

64

7) In Vitro Metabolism- and Transporter- Mediated Drug-Drug Interaction Studies.

Office of Communications, Division of Drug Information, Center for Drug Evaluation and Research, Food and Drug Administration. October 2017.

https://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformat ion/Guidances/UCM581965.pdf

8) Aninat C, Piton A, Glaise D, Le Charpentier T, Langouët S, Morel F, et al.

Expression of cytochromes P450, conjugating enzymes and nuclear receptors in human hepatoma HepaRG cells. Drug Metab Dispos. 2006;34:75–83.

9) Jossé R, Aninat C, Glaise D, Dumont J, Fessard V, Morel F, et al. Long-term functional stability of human HepaRG hepatocytes and use for chronic toxicity and genotoxicity studies. Drug Metab Dispos. 2008;36:1111–1118.

10) Kanebratt KP, Andersson TB. Evaluation of HepaRG cells as an in vitro model for human drug metabolism studies. Drug Metab Dispos. 2008;36:1444–1452.

11) Ikeda Y, Jomura T, Horiuchi U, Saeki J, Yoshimoto K, Ikeya T, et al. Long-term survival and functional maintenance of hepatocytes by using a microfabricated cell array. Colloids Surf B Biointerfaces. 2012;97:97–100.

12) Otsuka H, Sasaki K, Okimura S, Nagamura M, Nakasone Y. Micropatterned co-culture of hepatocyte spheroids layered on non-parenchymal cells to understand heterotypic cellular interactions. Sci Technol Adv Mater.

2013;14:065003.

13) Bissell DM, Arenson DM, Maher JJ, Roll FJ. Support of cultured hepatocytes by a laminin-rich gel. Evidence for a functionally significant subendothelial matrix in normal rat liver. J Clin Invest. 1987;79:801–812.

14) Bachmann A, Moll M, Gottwald E, Nies C, Zantl R, Wagner H, et al. 3D

65

cultivation techniques for primary human hepatocytes. Microarrays (Basel).

2015;4:64–83.

15) OECD Guidelines for the Testing of Chemicals, Section 4, Test No. 431: In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method. Adopted:

29 July 2016.

http://www.oecd-ilibrary.org/environment/test-no-431-in-vitro-skin-corrosion-re constructed-human-epidermis-rhe-test-method_9789264264618-en

16) OECD Guidelines for the Testing of Chemicals, Section 4, Test No. 492:

Reconstructed human Cornea-like Epithelium (RhCE) test method for identifying chemicals not requiring classification and labelling for eye irritation or serious eye damage. Adapted: 9 October 2017.

http://www.oecd-ilibrary.org/environment/test-no-492-reconstructed-human-cor nea-like-epithelium-rhce-test-method-for-identifying-chemicals-not-requiring-cl assification-and-labelling-for-eye-irritation-or-serious-eye-damage_978926424 2548-en

17) OECD Guidelines for the Testing of Chemicals, Section 4, Test No. 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method. Adopted: 28 July 2015.

https://www.oecd-ilibrary.org/environment/test-no-439-in-vitro-skin-irritation-re constructed-human-epidermis-test-method_9789264242845-en

18) Swales NJ, Utesch D. Metabolic activity of fresh and cryopreserved dog hepatocyte suspensions. Xenobiotica. 1998;28:937–948.

19) Li AP, Lu C, Brent JA, Pham C, Fackett A, Ruegg CE, et al. Cryopreserved human hepatocytes: characterization of drug-metabolizing enzyme activities and

66

applications in higher throughput screening assays for hepatotoxicity, metabolic stability, and drug-drug interaction potential. Chem Biol Interact. 1999;121:17–

35.

20) Li AP, Roque DJ, Beck DJ, Kaminski DL. Isolation and culturing of hepatocytes from human livers. J Tiss Cult Meth 1992; 14:139–146

21) Hewitt NJ, Utesch D. Cryopreserved rat, dog and monkey hepatocytes:

measurement of drug metabolizing enzymes in suspensions and cultures. Hum Exp Toxicol. 2004;23:307–316.

22) Hewitt NJ, Fischer T, Zuehlke U, Oesch F, Utesch D. Metabolic activity of fresh and cryopreserved cynomolgus monkey (Macaca fascicularis) hepatocytes.

Xenobiotica. 2000;30:665–681.

23) Nishimura M, Koeda A, Suganuma Y, Suzuki E, Shimizu T, Nakayama M, et al.

Comparison of inducibility of CYP1A and CYP3A mRNAs by prototypical inducers in primary cultures of human, cynomolgus monkey, and rat hepatocytes.

Drug Metab Pharmacokinet. 2007;22:178–186.

24) Nishimura M, Koeda A, Morikawa H, Satoh T, Narimatsu S, Naito S.

Comparison of inducibility of multidrug resistance (MDR)1, multidrug resistance-associated protein (MRP)1, and MRP2 mRNAs by prototypical microsomal enzyme inducers in primary cultures of human and cynomolgus monkey hepatocytes. Biol Pharm Bull. 2008;31:2068–2072.

25) Nishimura M, Koeda A, Shimizu T, Nakayama M, Satoh T, Narimatsu S, et al.

Comparison of inducibility of sulfotransferase and UDP-glucuronosyltransferase mRNAs by prototypical microsomal enzyme inducers in primary cultures of human and cynomolgus monkey hepatocytes. Drug Metab Pharmacokinet.

67

2008;23:45–53.

26) Shitara Y, Li AP, Kato Y, Lu C, Ito K, Itoh T, et al. Function of uptake transporters for taurocholate and estradiol 17beta-D-glucuronide in cryopreserved human hepatocytes. Drug Metab Pharmacokinet. 2003;18:33–41.

27) Fischer T, Hoepner U, Roos-Poppe U, Ikeda T, Mueller J. Use of human liver tissue in predicting drug metabolism and enzyme induction. Annu Rep Sankyo Res Lab. 2002;54:105–117

28) Nishimura M, Koeda A, Morikawa H, Satoh T, Narimatsu S, Naito S.

Tissue-specific mRNA expression profiles of drug-metabolizing enzymes and transporters in the cynomolgus monkey. Drug Metab Pharmacokinet.

2009;24:139–144.

29) Bullock P, Pearce R, Draper A, Podval J, Bracken W, Veltman J, et al. Induction of liver microsomal cytochrome P450 in cynomolgus monkeys. Drug Metab Dispos. 1995;23:736–748.

30) Ise R, Nakanishi Y, Kohara S, Yamashita H, Yoshikawa T, Iwasaki K, et al.

Expression profile of hepatic genes in cynomolgus macaques bred in Cambodia, China, and Indonesia: implications for cytochrome P450 genes. Drug Metab Pharmacokinet. 2012;27:307–316.

31) Niwa T, Kaneko H, Naritomi Y, Togawa A, Shiraga T, Iwasaki K, et al. Species and sex differences of testosterone and nifedipine oxidation in liver microsomes of rat, dog and monkey. Xenobiotica. 1995;25:1041–1049.

32) Uno Y, Ohuchi T, Uehara S, Kito G, Kamataki T, Nagata R. Sex-related differences in the expression of mfGSTA2, a novel GST identified in cynomolgus monkey (Macaca fascicularis). Drug Metab Dispos. 2009;37:453–

68

456.

33) Senoo H, Tsukada Y, Sato T, Hata R. Co-culture of fibroblasts and hepatic parenchymal cells induces metabolic changes and formation of a three-dimensional structure. Cell Biol Int Rep. 1989;13:197–206.

34) Sato H, Funahashi M, Kristensen DB, Tateno C, Yoshizato K. Pleiotrophin as a Swiss 3T3 cell-derived potent mitogen for adult rat hepatocytes. Exp Cell Res.

1999;246:152–164.

35) Ooe H, Kon J, Miyamoto S, Ozone Y, Ninomiya S, Mitaka T. Cytochrome p450 expression of cultured rat small hepatocytes after long-term cryopreservation.

Drug Metab Dispos. 2006;34:1667–1671.

36) Ishii M, Kino J, Ichinohe N, Tanimizu N, Ninomiya T, Suzuki H, et al.

Hepatocytic parental progenitor cells of rat small hepatocytes maintain self-renewal capability after long-term culture. Sci Rep. 2017;7:46177.

37) Rodríguez-Antona C, Donato MT, Pareja E, Gómez-Lechón MJ, Castell JV.

Cytochrome P-450 mRNA expression in human liver and its relationship with enzyme activity. Arch Biochem Biophys. 2001;393:308–315.

38) Donato MT, Castell JV. Strategies and molecular probes to investigate the role of cytochrome P450 in drug metabolism: focus on in vitro studies. Clin Pharmacokinet. 2003;42:153–178.

39) Iwasaki K, Uno Y. Cynomolgus monkey CYPs: a comparison with human CYPs.

Xenobiotica. 2009;39:578-581.

40) Uno Y, Uehara S, Yamazaki H. Utility of non-human primates in drug development: Comparison of non-human primate and human drug-metabolizing cytochrome P450 enzymes. Biochem Pharmacol. 2016;121:1-7.

69

41) Emoto C, Yoda N, Uno Y, Iwasaki K, Umehara K, Kashiyama E, Yamazaki H.

Comparison of P450 enzymes between cynomolgus monkeys and humans: P450 identities, protein contents, kinetic parameters, and potential for inhibitory profiles. Curr Drug Metab. 2013;14:239-252.

42) Drug Development and Drug Interactions: Table of Substrates, Inhibitors and Inducers

https://www.fda.gov/drugs/drug-interactions-labeling/drug-development-and-dr ug-interactions-table-substrates-inhibitors-and-inducers.

43) Denison MS, Nagy SR. Activation of the aryl hydrocarbon receptor by structurally diverse exogenous and endogenous chemicals. Annu Rev Pharmacol Toxicol. 2003;43:309-334.

44) Stejskalova L, Dvorak Z, Pavek P. Endogenous and exogenous ligands of aryl hydrocarbon receptor: current state of art. Curr Drug Metab. 2011;12:198-212.

45) Kim S, Dinchuk JE, Anthony MN, Orcutt T, Zoeckler ME, Sauer MB, Mosure KW, Vuppugalla R, Grace JE jr, Simmermacher J, Dulac HA, Pizzano, Sinz M.

Evaluation of cynomolgus monkey pregnane X receptor, primary hepatocyte, and in vivo pharmacokinetic changes in predicting human CYP3A4 induction.

Drug Metab Dispos. 2010;38:16-24.

46) Faucette SR, Zhang TC, Moore R, Sueyoshi T, Omiecinski CJ, LeCluyse EL, Negishi M, Wang H. Relative activation of human pregnane X receptor versus constitutive androstane receptor defines distinct classes of CYP2B6 and CYP3A4 inducers. J Pharmacol Exp Ther. 2007;320:72-80.

47) LeCluyse EL. Pregnane X receptor: molecular basis for species differences in CYP3A induction by xenobiotics. Chem Biol Interact. 2001;134:283-289.

70

48) Moore JT, Moore LB, Maglich JM, Kliewer SA. Functional and structural comparison of PXR and CAR. Biochim Biophys Acta. 2003;1619:235-238.

49) Koeda A, Iwao T, Nakanishi A, Mizuno S, Yamashita M, Sakai Y, Nakamura K, Matsunaga T. Comparison of mRNA expression profiles of drug-metabolizing enzymes and transporters in fresh and cryopreserved cynomolgus monkey hepatocytes. Drug Metab Pharmacokinet. 2019;34:253-263.

50) Moscovitz JE, Kalgutkar AS, Nulick K, Johnson N, Lin Z, Goosen TC, Weng Y.

Establishing Transcriptional Signatures to Differentiate PXR-, CAR-, and AhR-Mediated Regulation of Drug Metabolism and Transport Genes in Cryopreserved Human Hepatocytes. J Pharmacol Exp Ther. 2018;365:262-271.

51) Sueyoshi T, Kawamoto T, Zelko I, Honkakoski P, Negishi M. The repressed nuclear receptor CAR responds to phenobarbital in activating the human CYP2B6 gene. J Biol Chem. 1999;274:6043-6046.

52) Yajima K, Uno Y, Murayama N, Uehara S, Shimizu M, Nakamura C, et al.

Evaluation of 23 lots of commercially available cryopreserved hepatocytes for induction assays of human cytochromes P450. Drug Metab Dispos.

2014;42:867-871.

53) Feidt DM, Klein K, Hofmann U, Riedmaier S, Knobeloch D, Thasler WE, Weiss TS, Schwab M, Zanger UM. Profiling induction of cytochrome P450 enzyme activity by statins using a new liquid chromatography-tandem mass spectrometry cocktail assay in human hepatocytes. Drug Metab Dispos.

2010;38:1589-1597.

54) Uehara S, Uno Y, Suzuki T, Inoue T, Utoh M, Sasaki E, Yamazaki H. Strong induction of cytochrome P450 1A/3A, but not P450 2B, in cultured hepatocytes

71

from common marmosets and cynomolgus monkeys by typical human P450 inducing agents. Drug Metab Lett. 2017;10:244-253.

55) Uno Y, Iwasaki K, Yamazaki H, Nelson DR. Macaque cytochromes P450:

nomenclature, transcript, gene, genomic structure, and function. Drug Metab Rev. 2011;43:346-361.

56) Kostrubsky VE, Ramachandran V, Venkataramanan R, Dorko K, Esplen JE, Zhang S, Sinclair JF. The use of human hepatocyte cultures to study the induction of cytochrome P-450. Drug Metab Dispos. 1999;27:887-894.

57) Bullock P, Pearce R, Draper A, Podval J, Bracken W, Veltman J, Thomas P, Parkinson A. Induction of liver microsomal cytochrome P450 in cynomolgus monkeys. Drug Metab Dispos. 1995;23:736-748.

58) Jones CR, Guengerich FP, Rice JM, Lubet RA. Induction of various cytochromes CYP2B, CYP2C and CYP3A by phenobarbitone in non-human primates. Pharmacogenetics. 1992;2(4):160-172.

59) Martignoni M, Groothuis GM, de Kanter R. Species differences between mouse, rat, dog, monkey and human CYP-mediated drug metabolism, inhibition and induction. Expert Opin Drug Metab Toxicol. 2006;2:875-894.

60) Uno Y, Yamazaki H. Expression of cytochrome P450 regulators in cynomolgus macaque. Xenobiotica. 2018;48:695-703.

61) Sinz M, Wallace G, Sahi J. Current industrial practices in assessing CYP450 enzyme induction: preclinical and clinical. AAPS J. 2008;10:391-400.

62) Pascussi JM, Drocourt L, Fabre JM, Maurel P, Vilarem MJ. Dexamethasone induces pregnane X receptor and retinoid X receptor-alpha expression in human hepatocytes: synergistic increase of CYP3A4 induction by pregnane X receptor