CYP1A1 MAP2K6
ATM p38
Table 2. Sequences of oligonucleotide primers used for PCR amplification
9. マイクロアレイ
マイクロアレイ解析に用いるサンプルは細胞からRNeasy Mini Kitを用いて メーカー推奨プロトコールに従って、RNA抽出および精製を行った。トータル RNA溶液の濃度および純度はNanoDrop 1000を用いてOD260およびA260/A280 比を測定することで算出した。得られた RNA サンプルをクラボウ社に送付し、
アフィメトリクス社 GeneChip® プローブアレイ受託解析サービスを利用した。
また、マイクロアレイの再現性確認のために同じくRNAサンプルをクラボウ社 に送付し、アジレントDNAマイクロアレイ解析サービスも利用した。
10. EmaxおよびEC50の計算
EmaxおよびEC50の算出はPhoenix® WinNonlin® (Certara) を用いて行った。
11. 統計解析
各群の有意差についてはスチューデントのt検定 (Student t-test)により解析を 行った。
謝辞
本研究にあたり、御懇篤なご指導並びにご鞭撻を賜りました東北医科薬科大 学薬学部機能病態分子学教室教授 井ノ口 仁一 先生ならびに終始ご指導、
ご校閲賜りました同環境衛生学教室教授 永田 清 先生、ご校閲やご助言を 賜りました同生化学教室教授 関 政幸 先生に衷心より感謝の意を申し上げ ます。
本研究を進めるにあたり、始終懇切なるご指導、ご助言を賜りました東北医 科薬科大学薬学部環境衛生学教室准教授 熊谷 健 先生、同環境衛生学教室 助教 佐々木 崇光 先生（現静岡県立大学薬学部衛生分子毒性学分野講師）、
同環境衛生学教室助教 進藤 佐和子 先生に厚く御礼申し上げます。
本研究にあたり、東北医科薬科大学薬学部環境衛生学教室 庄司 理恵 氏、
小田桐 玲生 氏、塩屋 安奈里 氏、渡辺 孝樹 氏をはじめとする東北薬 科大学環境衛生学教室の皆様には多くのご協力をいただきました。心より御礼 申し上げます。
本研究に対し、ご支援を賜りました東北医科薬科大学理事長・学長 高柳 元 明 先生に深く感謝の意を申し上げます。
また、大学での研究の機会を与えていただき、ご支援いただきました日本たば こ産業株式会社 医薬総合研究所 薬物動態研究所 小粥 基弘 所長、野村 幸弘 副所長、谷口 寿生 グループリーダーに厚く御礼申し上げます。
最後に私の研究に対して応援し、支えてくれた妻や子供たちにも深く感謝致し ます。
参考文献
1. Nebert, D. W., and Russell, D. W. (2002) Clinical importance of the cytochromes P450. Lancet. 360, 1155–1162
2. Gonzalez, F. J. (1992) Human cytochromes P450: problems and prospects.
Trends Pharmacol. Sci. 13, 346–352
3. Nelson, D. R., Koymans, L., Kamataki, T., Stegeman, J. J., Feyereisen, R., Waxman, D. J., Waterman, M. R., Gotoh, O., Coon, M. J., Estabrook, R. W., Gunsalus, I. C., and Nebert, D. W. (1996) P450 superfamily: update on new sequences, gene mapping, accession numbers and nomenclature.
Pharmacogenetics. 6, 1–42
4. Nelson, D. R., Kamataki, T., Waxman, D. J., Guengerich, F. P., Estabrook, R. W., Feyereisen, R., Gonzalez, F. J., Coon, M. J., Gunsalus, I. C., Gotoh, O., Okuda, K., and Nebert, D. W. (1993) The P450 Superfamily : Update on new sequences, gene-mapping, accession numbers, early trivial names of enzymes, and
nomenclature. DNA Cell Biol. 12, 1–51
5. Kolars, J. C., Lown, K. S., Schmiedlin-Ren, P., Ghosh, M., Fang, C., Wrighton, S.
A., Merion, R. M., and Watkins, P. B. (1994) CYP3A gene expression in human gut epithelium. Pharmacogenetics. 4, 247–59
6. Shimada, T., Yamazaki, H., Mimura, M., Inui, Y., and Guengerich, F. P. (1994) Interindividual Variations in Human Liver Cytochrome-P-450 Enzymes Involved in the Oxidation of Drugs, Carcinogens and Toxic-Chemicals - Studies With Liver-Microsomes of 30 Japanese and 30 Caucasians. J. Pharmacol. Exp. Ther.
270, 414–423
7. Wienkers, L. C., and Heath, T. G. (2005) Predicting in vivo drug interactions from in vitro drug discovery data. Nat. Rev. Drug Discov. 4, 825–833
8. Grub, S., Bryson, H., Goggin, T., Lüdin, E., and Jorga, K. (2001) The interaction of saquinavir (soft gelatin capsule) with ketoconazole, erythromycin and
rifampicin: Comparison of the effect in healthy volunteers and in HIV-infected patients. Eur. J. Clin. Pharmacol. 57, 115–121
9. Lin, J. H. (2006) CYP induction-mediated drug interactions: In vitro assessment and clinical implications. Pharm. Res. 23, 1089–1116
10. FDA (2017) In Vitro Metabolism and Transporter Mediated Drug-Drug Interaction Studies Guidance for Industry. FDA Guid.
ライン （ 案 ）
13. Sasaki, T., Sato, Y., Kumagai, T., Yoshinari, K., and Nagata, K. (2017) Effect of health foods on cytochrome P450-mediated drug metabolism. J. Pharm. Heal.
Care Sci. 3, 14–24
14. Moore, L. B., Goodwin, B., Jones, S. a, Wisely, G. B., Serabjit-Singh, C. J., Willson, T. M., Collins, J. L., and Kliewer, S. a (2000) St. John’s wort induces hepatic drug metabolism through activation of the pregnane X receptor. Proc Natl Acad Sci U S A. 97, 7500–7502
15. Kerns, E. H., and Di, L. (2003) Pharmaceutical profiling in drug discovery. Drug Discov. Today. 8, 316–323
16. 太田之弘 (2009) 創薬における薬物によるcyp誘導評価. 日薬理誌. 134, 330–333
17. Testino, S. A., and Patonay, G. (2003) High-throughput inhibition screening of major human cytochrome P450 enzymes using an in vitro cocktail and liquid chromatography-tandem mass spectrometry. J. Pharm. Biomed. Anal. 30, 1459–1467
18. Zhuang, X., and Lu, C. (2016) PBPK modeling and simulation in drug research and development. Acta Pharm. Sin. B. 6, 430–440
19. Fahmi, O. A., Hurst, S., Plowchalk, D., Cook, J., Guo, F., Youdim, K., Dickins, M., Phipps, A., Darekar, A., Hyland, R., and Obach, R. S. (2009) Comparison of different algorithms for predicting clinical drug-drug interactions, based on the use of CYP3A4 in vitro data: Predictions of compounds as precipitants of interaction. Drug Metab. Dispos. 37, 1658–1666
20. Kato, M., Shitara, Y., Sato, H., Yoshisue, K., Hirano, M., Ikeda, T., and Sugiyama, Y. (2008) The quantitative prediction of CYP-mediated drug
interaction by physiologically based pharmacokinetic modeling. Pharm. Res. 25, 1891–1901
21. Bertilsson, G., Heidrich, J., Svensson, K., Asman, M., Jendeberg, L.,
Sydow-Bäckman, M., Ohlsson, R., Postlind, H., Blomquist, P., and Berkenstam, A. (1998) Identification of a human nuclear receptor defines a new signaling pathway for CYP3A induction. Proc. Natl. Acad. Sci. U. S. A. 95, 12208–12213 22. Blumberg, B., Sabbagh, W., Juguilon, H., Bolado, J., Van Meter, C. M., Ong, E.
S., and Evans, R. M. (1998) SXR, a novel steroid and xenobiotic-sensing nuclear receptor. Genes Dev. 12, 3195–3205
23. Lehmann, J. M., McKee, D. D., Watson, M. A., Willson, T. M., Moore, J. T., and
compounds that regulate CYP3A4 gene expression and cause drug interactions. J.
Clin. Invest. 102, 1016–1023
24. Goodwin, B., Hodgson, E., Costa, D. J. D., and Robertson, G. R. (2002) Transcriptional Regulation of the Human CYP3A4 Gene by the Constitutive Androstane Receptor. Mol. Pharmacol. 62, 359–365
25. Drocourt, L., Ourlin, J. C., Pascussi, J. M., Maurel, P., and Vilarem, M. J. (2002) Expression of CYP3A4, CYP2B6, and CYP2C9 is regulated by the vitamin D receptor pathway in primary human hepatocytes. J. Biol. Chem. 277,
25125–25132
26. Li, A. P., Reith, M. K., Rasmussen, A., Gorski, J. C., Hall, S. D., Xu, L., Kaminski, D. L., and Cheng, L. K. (1997) Primary human hepatocytes as a tool for the evaluation of structure- activity relationship in cytochrome P450
induction potential of xenobiotics: Evaluation of rifampin, rifapentine and rifabutin. Chem. Biol. Interact. 107, 17–30
27. Turpeinen, M., Tolonen, A., Chesne, C., Guillouzo, A., Uusitalo, J., and Pelkonen, O. (2009) Functional expression, inhibition and induction of CYP enzymes in HepaRG cells. Toxicol. Vitr. 23, 748–753
28. Zimmermann, K., Wittman, M. D., Saulnier, M. G., Velaparthi, U., Sang, X., Frennesson, D. B., Struzynski, C., Seitz, S. P., He, L., Carboni, J. M., Li, A., Greer, A. F., Gottardis, M., Attar, R. M., Yang, Z., Balimane, P., Discenza, L. N., Lee, F. Y., Sinz, M., Kim, S., and Vyas, D. (2010) SAR of PXR transactivation in benzimidazole-based IGF-1R kinase inhibitors. Bioorganic Med. Chem. Lett.
20, 1744–1748
29. Gardner, D. S., Santella, J. B., Duncia, J. V., Carter, P. H., Dhar, T. G. M., Wu, H., Guo, W., Cavallaro, C., Van Kirk, K., Yarde, M., Briceno, S. W., Robert Grafstrom, R., Liu, R., Patel, S. R., Tebben, A. J., Camac, D., Khan, J., Watson, A., Yang, G., Rose, A., Foster, W. R., Cvijic, M. E., Davies, P., and Hynes, J.
(2013) The discovery of BMS-457, a potent and selective CCR1 antagonist.
Bioorganic Med. Chem. Lett. 23, 3833–3840
30. Schlegel, K. A. S., Yang, Z. Q., Reger, T. S., Shu, Y., Cube, R., Rittle, K. E., Bondiskey, P., Bock, M. G., Hartman, G. D., Tang, C., Ballard, J., Kuo, Y., Prueksaritanont, T., Nuss, C. E., Doran, S. M., Fox, S. V., Garson, S. L., Kraus, R. L., Li, Y., Uebele, V. N., Renger, J. J., and Barrow, J. C. (2010) Discovery and expanded SAR of 4,4-disubstituted quinazolin-2-ones as potent T-type calcium channel antagonists. Bioorganic Med. Chem. Lett. 20, 5147–5152
Paonessa, G., Marco, S. Di, Carfi, A., Giuliano, C., Padron, J., Bonelli, F., Migliaccio, G., Francesco, R. De, Laufer, R., Rowley, M., and Narjes, F. (2005) Potent Inhibitors of Subgenomic Hepatitis C Virus RNA Replication through Optimization of Indole- N -Acetamide Allosteric Inhibitors of the Viral NS5B Polymerase. J. Med. Chem. 48, 4547–4557
32. Synold, T. W., Dussault, I., and Forman, B. M. (2001) The orphan nuclear receptor SXR coordinately regulates drug metabolism and efflux. Nat. Med. 7, 584–590
33. Geick, A., Eichelbaum, M., and Burk, O. (2001) Nuclear Receptor Response Elements Mediate Induction of Intestinal MDR1 by Rifampin. J. Biol. Chem. 276, 14581–14587
34. Kamiguchi, K., Aoyama, E., Okuda, T., and Moriwaki, T. (2010) Short communication a 96-Well plate assay for CYP4503A induction using cryopreserved human hepatocytes. Drug Metab. Dispos. 38, 1912–1916 35. Wan, Y. Y., An, D., Cai, Y. A. N., Repa, J. J., Chen, T. I. M. H., Flores, M.,
Postic, C., Magnuson, M. A., Chen, J. U., Chien, K. R., French, S., Mangelsdorf, D. J., and Sucov, H. M. (2000) Hepatocyte-Specific Mutation Establishes
Retinoid X Receptor alpha as a Heterodimeric Integrator of Multiple Physiological Processes in the Liver. Mol. Cell. Biol. 20, 4436–4444
36. Luckert, C., Ehlers, A., Buhrke, T., Seidel, A., Lampen, A., and Hessel, S. (2013) Polycyclic aromatic hydrocarbons stimulate human CYP3A4 promoter activity via PXR. Toxicol. Lett. 222, 180–188
37. Kumagai, T., Suzuki, H., Sasaki, T., Sakaguchi, S., Miyairi, S., Yamazoe, Y., and Nagata, K. (2012) Polycyclic Aromatic Hydrocarbons Activate CYP3A4 Gene Transcription through Human Pregnane X Receptor. Drug Metab.
Pharmacokinet. 27, 200–206
38. Lin, W., Wu, J., Dong, H., Bouck, D., Zeng, F. Y., and Chen, T. (2008) Cyclin-dependent kinase 2 negatively regulates human pregnane X
receptor-mediated CYP3A4 gene expression in HepG2 liver carcinoma cells. J.
Biol. Chem. 283, 30650–30657
39. Sugatani, J., Osabe, M., Kurosawa, M., Kitamura, N., and Ikari, A. (2010) Induction of UGT1A1 and CYP2B6 by an Antimitogenic Factor in HepG2 Cells Is Mediated through Suppression of Cyclin- Dependent Kinase 2 Activity : Cell Cycle-Dependent Expression. DRUG Metab. Dispos. 38, 177–186
40. Goodwin, B., Hodgson, E., and Liddle, C. (1999) The orphan human pregnane X
through a distal enhancer module. Mol. Pharmacol. 56, 1329–1339
41. Toriyabe, T., Nagata, K., Takada, T., Aratsu, Y., Matsubara, T., Yoshinari, K., and Yamazoe, Y. (2009) Unveiling a New Essential Cis Element for the Transactivation of the CYP3A4 Gene by Xenobiotics. Mol. Pharmacol. 75, 677–684
42. Noracharttiyapot, W., Nagai, Y., Matsubara, T., Miyata, M., Shimada, M., Nagata, K., and Yamazoe, Y. (2006) Construction of several human derived stable cell lines displaying distinct profiles of CYP3A4 induction. Drug Metab.Pharmacokinet. 21, 99–108
43. Hellum, B. H., Hu, Z., and Nilsen, O. G. (2007) The induction of CYP1A2, CYP2D6 and CYP3A4 by six trade herbal products in cultured primary human hepatocytes. Basic Clin. Pharmacol. Toxicol. 100, 23–30
44. Adachi, J., Mori, Y., Matsui, S., Takigami, H., Fujino, J., Kitagawa, H., Miller, C.
A., Kato, T., Saeki, K., and Matsuda, T. (2001) Indirubin and Indigo are Potent Aryl Hydrocarbon Receptor Ligands Present in Human Urine. J. Biol. Chem. 276, 31475–31478
45. Parez, P., Pulgar, R., Olea-Serrano, F., Villalobos, M., Rivas, A., Metzler, M., Pedraza, V., and Olea, N. (1998) The estrogenicity of bisphenol A-related diphenylalkanes with various substituents at the central carbon and the hydroxy groups. Environ. Health Perspect. 106, 167–174
46. Vrzal, R., Zenata, O., Doricakova, A., and Dvorak, Z. (2015) Environmental pollutants parathion, paraquat and bisphenol A show distinct effects towards nuclear receptors-mediated induction of xenobiotics-metabolizing cytochromes P450 in human hepatocytes. Toxicol. Lett. 238, 43–53
47. Zenata, O., Dvorak, Z., and Vrzal, R. (2017) Profiling of bisphenol S towards nuclear receptors activities in human reporter cell lines. Toxicol. Lett. 281, 10–19 48. Kuzbari, O., Peterson, C. M., Franklin, M. R., Hathaway, L. B., Johnstone, E. B.,
Hammoud, A. O., and Lamb, J. G. (2013) Comparative analysis of human CYP3A4 and rat CYP3A1 induction and relevant gene expression by bisphenol A and diethylstilbestrol: Implications for toxicity testing paradigms. Reprod.
Toxicol. 37, 24–30
49. Nagai, Y. (1965) Selective Descriptions of Characteristics Compounds . of Polycyclic Aromatic. 有機合成化学. 23, 919–929
50. Hamburger, M. (2002) Isatis tinctoria - From the rediscovery of an ancient medicinal plant towards a novel anti-inflammatory phytopharmaceutical.
51. Ho, Y.-L., and Chang, Y.-S. (2002) Studies on the antinociceptive,
anti-inflammatory and anti pyretic effects of Isatis indigotica root. Phytomedicine Int. J. Phyther. Phytopharm. 9, 419–424
52. Zou, P., Hong, Y., and Koh, H. L. (2005) Chemical fingerprinting of Isatis indigotica root by RP-HPLC and hierarchical clustering analysis. J. Pharm.
Biomed. Anal. 38, 514–520
53. Liau, B. C., Jong, T. T., Lee, M. R., and Chen, S. S. (2007) LC-APCI-MS method for detection and analysis of tryptanthrin, indigo, and indirubin in Daqingye and Banlangen. J. Pharm. Biomed. Anal. 43, 346–351
54. Eisenbrand, G., Hippe, F., Jakobs, S., and Muehlbeyer, S. (2004) Molecular mechanisms of indirubin and its derivatives: Novel anticancer molecules with their origin in traditional Chinese phytomedicine. J. Cancer Res. Clin. Oncol.
130, 627–635
55. Sivertsson, L., Edebert, I., Palmertz, M. P., Ingelman-Sundberg, M., and Neve, E.
P. a (2013) Induced CYP3A4 expression in confluent Huh7 hepatoma cells as a result of decreased cell proliferation and subsequent pregnane X receptor activation. Mol. Pharmacol. 83, 659–670
56. Watson, A. J., and Hankinson, O. (1992) Dioxin- and Ah receptor-dependent protein binding to xenobiotic responsive elements and G-rich DNA studied by in vivo footprinting. J. Biol. Chem. 266, 6874–6878
57. Yamaori, S., Kinugasa, Y., Jiang, R., Takeda, S., Yamamoto, I., and Watanabe, K. (2015) Cannabidiol induces expression of human cytochrome P450 1A1 that is possibly mediated through aryl hydrocarbon receptor signaling in HepG2 cells.
Life Sci. 136, 87–93
58. Shizu, R., Osabe, M., Perera, L., Moore, R., Sueyoshi, T., and Negishi, M. (2017) Phosphorylated nuclear receptor CAR forms a homodimer to repress its
constitutive activity for ligand activation. Mol. Cell. Biol. 37, e00649-16 59. Alessi, D. R., Cuenda, A., Cohen, P., Dudley, D. T., and Saltiel, A. R. (1995)
Enzymology : PD 098059 Is a Specific Inhibitor of the Activation of
Mitogen-activated Protein Kinase Kinase in Vitro and in Vivo PD 098059 Is a Specific Inhibitor of the Activation of Mitogen-activated Protein Kinase Kinase in Vitro and in Vivo *. 270, 27489–27494
60. Bennett, B. L., Sasaki, D. T., Murray, B. W., O’Leary, E. C., Sakata, S. T., Xu, W., Leisten, J. C., Motiwala, A., Pierce, S., Satoh, Y., Bhagwat, S. S., Manning, A. M., and Anderson, D. W. (2001) SP600125, an anthrapyrazolone inhibitor of
61. Cuenda, A., Rouse, J., Doza, Y. N., Meier, R., Cohen, P., Gallagher, T. F., Young, P. R., and Lee, J. C. (1995) SB-203580 is a specific inhibitor of a MAP kinase homolog which is activated by cellular stresses and interleukin-1. FEBS Lett. 364, 229–233
62. Favata, M., Horiuchi, K., Manos, E., Daulerio, A., Stradley, D., Feeser, W., Van Dyk, D., Pitts, W., Earl, R., Hobbs, F., Copeland, R., Magolda, R., Scherle, P., and Trzaskos, J. (1998) Identification of a novel inhibitor of mitogen-activated protein kinase kinase. J Biol Chem. 273, 18623–18632
63. Takada, T., Ogino, M., Miyata, M., Shimada, M., Nagata, K., and Yamazoe, Y.
(2004) Differences in transactivation between rat CYP3A1 and human CYP3A4 genes by human pregnane X receptor. Drug Metab. Pharmacokinet. 19, 103–113 64. Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein-dye binding.
Anal. Biochem. 72, 248–254
65. Miyake, S., Makimura, M., Kanegae, Y., Harada, S., Sato, Y., Takamori, K., Tokuda, C., and Saito, and I. (1996) Efficient generation of recombinant adenoviruses using adenovirus DNA-terminal protein complex and a cosmid bearing the full-length virus genome. Proc. Natl. Acad. Sci. U. S. A. 93, 1320–1324