Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Primate cell – per se
Reexamination Certificate
2001-10-25
2004-06-29
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Primate cell, per se
C435S189000, C536S023200
Reexamination Certificate
active
06756229
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the following features.
1. A cell line derived from human hepatic carcinoma capable of stably expressing human cytochromes P450.
2. (1) A method for analyzing an enzyme participating in the metabolism of a xenobiotic and/or an endogenous substrate, (2) a method for analyzing a metabolic pathway of a xenobiotic and/or an endogenous substrate, (3) a method for analyzing a chemical structure of the metabolite of a xenobiotic and/or an endogenous substrate, (4) a method for preparing the metabolite of a xenobiotic and/or an endogenous substrate, (5) a method for analyzing inhibition of the metabolizing enzyme for a xenobiotic and/or an endogenous substrate, (6) a method for analyzing an accelerated activity of the metabolizing enzyme for a xenobiotic and/or an endogenous substrate, (7) a method for analyzing expression of cytotoxicity by the metabolism of a xenobiotic and/or an endogenous substrate, (8) a method for analyzing expression of genotoxicity by the metabolism of a xenobiotic and/or an endogenous substrate, (9) a method for analyzing expression of carcinogenicity by the metabolism of a xenobiotic and/or endogenous substrate, (10) a method for analyzing mutagenicity by the metabolism of a xenobiotic and/or an endogenous substrate, (11) a method for analyzing expression of hepatotoxicity by the metabolism of a xenobiotic and/or an endogenous substrate, and (12) a method for analyzing a xenobiotic and/or an endogenous substrate that acts on the liver, each method comprising use of the cell line according to (1).
3. (1) A method for screening a substance capable of inhibiting a xenobiotic and/or an endogenous substrate, (2) a method for screening a substance capable of accelerating the activity of a metabolizing enzyme for a xenobiotic and/or an endogenous substrate, (3) a method for screening a substance capable of expressing cytotoxicity by the metabolism of a xenobiotic and/or an endogenous substrate, (4) a method for screening a substance capable of expressing genotoxicity by the metabolism of a xenobiotic and/or an endogenous substrate, (5) a method for screening a substance capable of expressing carcinogenicity by the metabolism of a xenobiotic and/or an endogenous substrate, (6) a method for screening a substance capable of expressing mutagenicity by the metabolism of a xenobiotic and/or an endogenous substrate, (7) a method for screening a substance capable of expressing hepatotoxicity by the metabolism of a xenobiotic and/or an endogenous substrate, (8) a method for screening a xenobiotic and/or an endogenous substrate which acts on the liver, and (9) a method for screening a substance capable of acquiring a new physiological activity or increasing or decreasing the inherent physiological activity, through the metabolism of a xenobiotic and/or an endogenous substrate, each method comprising use of the cell line according to 1.
4. A compound or its salt obtainable using the screening method according to 3.
BACKGROUND ART
Hepatocytes are known to have a great many physiological functions, all of which play a very important function in terms of the metabolism of xenobiotics and/or endogenous substrates such as drugs, food additives, environmental pollutants, industrial chemicals and the like. At the same time, the function of metabolizing xenobiotics and/or endogenous substrates might lead to inducing the inhibition of metabolizing enzymes for xenobiotics and/or endogenous substrates by xenobiotics and/or endogenous substrates, to accelerate the activity of metabolizing enzymes for xenobiotics and/or endogenous substrates, to express cytotoxicity by the metabolism of xenobiotics and/or endogenous substrates, to express genotoxicity by the metabolism of xenobiotics and/or endogenous substrates, to express carcinogenicity by the metabolism of xenobiotics and/or endogenous substrates, to express mutagenicity by the metabolism of xenobiotics and/or endogenous substrates, to express hepatotoxicity by the metabolism of xenobiotics and/or endogenous substrates, and so on. For these reasons, the function of xenobiotics and/or endogenous substrates has been widely studied. It is known that many enzymes are associated with the metabolism of xenobiotics and/or endogenous substrates referred to herein. Examples of such enzymes include UDP-glucuronosyltransferase, sulfotransferase, glutathione transferase, epoxy hydratase, N-acetyltransferase, flavin monooxygenase and cytochromes P450. Also, the presence of a cytochrome P450 reductase is crucial for expressing the enzymatic function of cytochromes P450. Of an array of these enzymes, cytochromes P450 play the most important role in the metabolism of xenobiotics and/or endogenous substrates. The term cytochromes P450 collectively refers to a class of enzymes including a great many molecular species. In the metabolism of xenobiotics and/or endogenous substrates in human liver, ten (10) species of CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4 are considered important. Also, these enzymes, which are distributed in human liver, are functionally different depending on species and hence, human-derived liver specimens are unable to be used as a stable test system. On the other hand, such a metabolic function of the liver involves a very strong specificity, i.e., differences in nature, depending on species, which makes it difficult to predict such diverse metabolic functions of human liver from experimental animals, e.g., rats. However, it is practically impossible to analyze these functions of interest in humans. For these reasons, human-derived cultured hepatocytes are considered useful not only in examining the function of human liver in a rapid, inexpensive, safe and accurate way provided in place of experimental animals, but also in producing a so-called artificial liver as a functional substitute for human liver. However, it is impossible to subculture normal human hepatocytes separated from tissues in vivo. Cells that can be established as a cell line often lack the differentiation capability possessed inherently and in most cases, do not exactly reflect the function of tissues to which the cell line originally belongs. A family of enzymes that metabolize xenobiotics and/or endogenous substrates especially in liver cells, among others, the family of cytochromes P450 molecular species loses its activity in an extremely short period of time in primary culture; any cell line that fully retains the property has not been found so far (J. Dich et al., Hepatology, 8, 39-45 (1988)). Thus, in light of the foregoing, there is an extensive need for hepatocytes that can retain the capability of metabolizing xenobiotics and/or endogenous substrates and can be incubated.
To date, however, no cultured cell line has been obtained as retaining the function associated with the metabolism of xenobiotics and/or endogenous substrates as in the liver. Particularly because the activity of cytochromes P450 is widely recognized to be rapidly lost in cultured cells, it has been hitherto attempted to stably express cytochromes P450 in the established cultured cells and by this, take over the metabolizing function of liver (M. Sawada et al., Mutation Research, 411, 19-43 (1998)). However, as stated above, the cell line for expression of cytochromes P450 should indispensably be derived from human liver cells. In addition, the activity of NADPH cytochromes P450 reductase is required for expressing the activity of cytochromes P450, requiring further expression of many more enzymes. Therefore, stable and safe reproduction of the metabolizing function in human liver should be in human-derived cultured hepatocytes that retain the activity of enzymes participating in the metabolism of cytochromes P450 as well as various other metabolisms.
As examples of the expression of cytochromes P450 in cells retaining the activity of various enzymes participating in metabolism, there are cases in which P450 was expressed in HepG2 cells using vaccinia virus (Methods in Enzymology, T. Aoyama et al
Asahi Satoru
Ikemoto Keiko
Nanba Masayoshi
Yoshitomi Sumie
Edwards & Angell LLP
Piffat Kathryn A.
Prouty Rebecca E.
Swope Sheridan
Takeda Chemical Industries Ltd.
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