| Co-Investigator(Kenkyū-buntansha) |
EMI Yoshikazu Himeji Institute of Technology, Faculty of Science, Assistant Professor, 理学部, 助手 (60232980)
IKUSHIRO Shin-ichi Himeji Institute of Technology, Faculty of Science, Assistant Professor, 理学部, 助手 (50244679)
KIMURA Shigenobu Himeji Institute of Technology, Faculty of Science, Associate Professor, 理学部, 助教授 (90291608)
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| Research Abstract |
The drugs are transformed by a variety of pathways in two distinct stages. Phase I reactions serve to introduce a suitable functional group into the drug molecule, and the product may then act as the substrate for phase II metabolism, resulting in conjugation with endogenous substrates, increased water solubility and polarity, and drug elimination or excretion from the body. Phase I metabolism includes oxidation, reduction and hydroysis reactions. Phase II metabolism includes glucuronidation, sulfation, acetylation and glutathione conjugation reactions. The cytochrome P450 (CYP) is major oxidative enzymes that localize in the endoplasmic reticulum, along with the phase II enzyme, glucuronosyltransferases. Many hydrophobic drugs are oxidized by cytocrome P450s and its product is conjugated by UGTs in the same microsomal membranes. These enzymes include a multiple forms. Therefore, the coordinated oxidation and glucuronidation from cells is an important determinant in the detoxication of many compounds in vivo. To better understand the inerplay between oxidation and glucuronidation, we are developing stable cell lines that express different combinations of P450 and UGT isoforms. In this project, we have constructed the yeast expression vectors, capabling expression both oxidative enzyme system, Cytochrome P450 Reductase (CPR)/CYP and conjugation enzyme, UGT. The yeast cells expressing both enzymes, CYP1 A1 and UGT1 A6 have been established. The yest microsomes showed the oxidation activity of 7 ethoxycoumarin (7-EC) to 7hydroxy coumarin (7-OHC) and subsequent glucuronidation reaction. This is the first drug metabolizing system, including both CYP system and UGT. By using this new multi-step drug metabolizing system, we can express different conbinations of CYP and UGT isoforms in yeast cell microsomes.
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