Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving oxidoreductase
Reexamination Certificate
1994-07-19
2003-09-16
Nashed, Nashaat T. (Department: 1652)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving oxidoreductase
C435S029000, C435S069100, C435S069700, C435S189000, C435S254210, C435S320100, C536S023200, C536S023400
Reexamination Certificate
active
06620593
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a method for evaluation of the safety of a chemical compound using recombinant yeasts expressing human cytochrome P450.
BACKGROUND OF THE INVENTION
The cytochrome P450 is an enzyme catalyzing the mono-oxygenation of a substance in the human liver.
It is known that recombinant human cells expressing heterogeneous human cytochrome P450 species have been used for determination of metabolisms and toxicities of chemical substances. However, this method is unsatisfactory as a method of evaluation of the safety of chemical compounds partly because the kinds of the human cytochrome P450 species expressed by the cells and the levels of the expression are so limited that the amount of metabolite obtained is not enough for determination of the metabolism and toxicity, and partly because it requires not only a high density culture technique but a high cultivation cost. Accordingly, there has been a great demand for developing an advantageous method.
SUMMARY OF THE INVENTION
As a result of the extensive study, the present inventors have found that yeasts are particularly suitable as hosts for production of human cytochrome P450 and yeast NADPH-P450 reductase to be used in vitro determination of metabolisms and toxicities of chemical substances because yeasts grow so rapidly and can stably express both the human cytochrome P450 and yeast NADPH-P450 reductase at high expression levels to provide sufficient amounts of the metabolites in a short period of time, thereby enabling a precise and quick analysis of the metabolites.
Moreover, they have also found that, despite that there are a considerable number of human cytochrome P450 molecular species, the human metabolic system for chemical compounds can be reproduced in vitro when at least four human cytochrome P450 molecular species, i.e., human cytochrome P450 1A2, P450 2C9, P450 2E1 and P450 3A4, are combined.
Thus, the present invention provides a method for evaluation of the safety of a chemical compound, which comprises the steps of:
(a) reacting a chemical compound with recombinant yeast cells expressing, or in other words producing, human cytochrome P450 molecular species P450 1A2, P450 2C9, P450 2E1 and P450 3A4 together with a yeast NADPH-P450 reductase, which may be in the form of a fused enzyme with each of said human cytochrome P450 molecular species, or with the cell free extracts of the yeast cells; and
(b) analyzing the resulting metabolite to determine the safety of the compound.
The present invention further provides a method for determination of the human metabolite of a chemical compound, which comprises the steps of:
(a) reacting a chemical compound with recombinant yeast cells producing human cytochrome P450 molecular species P450 1A2, P450 2C9, P450 2E1 and P450 3A4 together with a yeast NADPH-P450 reductase, which may be in the form of a fused enzyme with each of said human cytochrome P450 molecular species, or with cell free extracts of the yeast cells; and
(b) identifying the resulting metabolite.
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Hayashi Koji
Kaneko Hideo
Komai Koichiro
Nakatsuka Iwao
Sakaki Toshiyuki
Moore William W.
Nashed Nashaat T.
Sumitomo Chemical Company Limited
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