JP5492365B2 - Human cell line stably expressing cytochrome P450 - Google Patents

Description

The present invention
1. a cell line derived from human liver cancer cells that stably express human cytochrome P450,
2. using the cell line (1) a method for analyzing xenobiotics and / or endogenous substrate metabolism, (2) a method for analyzing xenobiotics and / or endogenous substrate metabolic pathways, ( 3) Methods for analyzing the chemical structure of xenobiotics and / or endogenous substrate metabolites, (4) Methods for preparing xenobiotics and / or endogenous substrate metabolites, (5) Xenobiotics and / or endogenous substrate metabolizing enzymes Inhibition analysis method, (6) Xenobiotic and / or endogenous substrate metabolic enzyme activity promotion method, (7) Xenobiotic and / or endogenous substrate metabolism cytotoxicity expression analysis method, (8) Analysis method of genotoxicity due to xenobiotic and / or endogenous substrate metabolism, (9) Analysis method of carcinogenicity due to drug metabolism, (10) Mutagenicity due to xenobiotic and / or endogenous substrate metabolism Analysis method, (11) xenobiotics and / or The method analyzes the others hepatotoxicity expressed endogenous substrate metabolism (12) METHOD analysis of xenobiotics and / or endogenous substrates acting on the liver,
3. Use of the cell line (1) a method for searching for substances that inhibit xenobiotics and / or endogenous substrate metabolizing enzymes, (2) promote the activities of xenobiotics and / or endogenous substrate metabolizing enzymes (3) Searching for substances that express cytotoxicity due to xenobiotics and / or endogenous substrate metabolism, (4) Searching for substances that express genotoxicity due to xenobiotics and / or endogenous substrate metabolism (5) a method for searching for a substance that exhibits carcinogenicity by xenobiotics and / or endogenous substrate metabolism, (6) a method for searching for a substance that expresses mutagenicity by xenobiotics and / or endogenous substrate metabolism, (7) a method for searching for xenobiotics and / or substances that develop hepatotoxicity by endogenous substrate metabolism, (8) a method for searching for xenobiotics and / or endogenous substrates acting on the liver, (9) xenobiotics and / or For endogenous substrate metabolism A method for searching for a substance that acquires new physiological activity or increases or decreases its own physiological activity,
4. It relates to a compound or a salt thereof obtained by using the searching method.

Liver cells have a great number of physiological functions, among them very important functions for metabolism of xenobiotics and / or endogenous substrates such as drugs, food additives, environmental pollutants and chemical industrial products Plays. The function of metabolism of the xenobiotic and / or endogenous substrate is at the same time the inhibition of the xenobiotic and / or endogenous substrate metabolizing enzyme by the xenobiotic and / or endogenous substrate, the activity of the xenobiotic and / or endogenous substrate metabolizing enzyme. Expression of cytotoxicity by promotion, xenobiotic and / or endogenous substrate metabolism, expression of genotoxicity by xenobiotic and / or endogenous substrate metabolism, carcinogenic expression by xenobiotic and / or endogenous substrate metabolism, xenobiotic and There are cases where mutagenic expression due to endogenous substrate metabolism and / or hepatotoxicity expression due to xenobiotics and / or endogenous substrate metabolism may be brought about, and research is being conducted very widely. It is known that many enzymes are involved in the metabolism of xenobiotics and / or endogenous substrates. These include UDP-glucuronosyltransferase, sulfotransferase, glutathione transferase, epoxy hydratase, N-acetyltransferase, flavin monooxygenase and cytochrome P450. In addition, the presence of cytochrome P450 reductase is essential for cytochrome P450 to express its enzyme function. Among these enzymes, cytochrome P450 plays the most important role in the metabolism of xenobiotics and / or endogenous substrates. Cytochrome P450 is a collective term for a group of enzymes including a large number of molecular species. In the metabolism of xenobiotics and / or endogenous substrates in the human liver, CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, 10 types of CYP2D6, CYP2E1, and CYP3A4 are considered to be particularly important. In addition, since the enzymes distributed in the human liver have large individual differences, liver samples derived from humans cannot be used as a stable test system. On the other hand, the metabolic function of the liver has a very strong specificity, that is, a species difference depending on the species, and it is difficult to predict various metabolic functions in humans in laboratory animals such as rats. However, in many cases, it is impossible to analyze these examination items in an actual human. For these reasons, human cultured hepatocytes can be used not only to provide a method for quickly, inexpensively, safely and accurately examining liver functions in humans, but also to create so-called artificial livers that replace liver functions. Is considered to be possible. However, normal human hepatocytes isolated from living tissue cannot be subcultured. Cells that can be established as cell lines often do not have the original differentiation traits, and often do not accurately reflect the function of the tissue to which the cell line originally belonged. In particular, enzymes that metabolize xenobiotics and / or endogenous substrates in hepatocytes, especially those belonging to the cytochrome P450 molecular species, lose their activity in primary cultures in a very short time, and are found in cell lines. No one has been found that retains its properties sufficiently (J. Dich et.al., Hepatology, 8, 39-45 (1988)). From this point of view, hepatocytes that retain the metabolic ability of xenobiotics and / or endogenous substrates and can be cultured have been widely demanded.
However, until now, no cultured cell line has been obtained that retains functions related to xenobiotics and / or endogenous substrate metabolism in the same manner as the liver. In particular, it is widely recognized that the activity of cytochrome P450 is rapidly lost by cell culture. Therefore, it is intended to stably express cytochrome P450 in cultured cells and to replace the metabolic function of the liver. Attempts have been made in the past (M. Sawada et. Al., Mutation Research 411, 19-43 (1998)). However, for the reasons mentioned above, it is essential that the cell line that expresses cytochrome P450 is derived from human hepatocytes, and NADPH cytochrome P450 reductase activity is required for the expression of cytochrome P450 activity. Enzyme expression is required. Therefore, in order to stably and safely reproduce the metabolic function of human liver, it is necessary that the cell is a cultured human hepatocyte that retains not only cytochrome P450 but also the activities of enzymes involved in various metabolisms.
Examples of cytochrome P450 expression in cells retaining various enzyme activities involved in metabolism include HepG2 cells expressing P450 using vaccinia virus (Methods in Enzymology, T. Aoyama et.al in Methods in Enzymology). 260, pages 85-92 Academic Press (1991), MR Waterman, and CYP2E1 expressed in HepG2 cells (Y. Dai et al, Biochemistry 32, 6928-6937 1993). The former requires careful handling and is a practical barrier. In addition, the latter is an attempt for CYP2E1 alone, and no attempt has been made for many cytochrome P450s present in the liver. Therefore, if a cultured cell line that retains enzymes involved in metabolism of xenobiotics and / or endogenous substrates in the liver can be obtained, the cell line can be used to (1) xenobiotics and / or endogenous substrate metabolism. Analysis of enzymes involved, (2) Analysis of xenobiotics and / or endogenous substrate metabolic pathways, (3) Analysis of chemical structure of xenobiotics and / or endogenous substrate metabolites, (4) Xenobiotics and / or endogenous (5) analysis of xenobiotic and / or endogenous substrate metabolizing enzyme inhibition, (6) analysis of xenobiotic and / or endogenous substrate metabolizing enzyme activity, (7) xenobiotic And / or analysis of expression of cytotoxicity by endogenous substrate metabolism, (8) analysis of expression of genotoxicity by xenobiotic and / or endogenous substrate metabolism, (9) carcinogenesis by xenobiotic and / or endogenous substrate metabolism Sexual expression solution (10) Analysis of mutagenicity by xenobiotics and / or endogenous substrate metabolism, (11) Analysis of hepatotoxicity on xenobiotics and / or endogenous substrate metabolism, (12) Xenobiotics acting on the liver and In addition to enabling analysis of endogenous substrates, etc., the cell line can be used to (1) search for xenobiotics and / or substances that inhibit endogenous substrate metabolic enzymes, (2) xenobiotics and / or Or, search for substances that promote the activity of endogenous substrate-metabolizing enzymes, (3) search for xenobiotics and / or substances that express cytotoxicity by endogenous substrate metabolism, (4) by xenobiotics and / or endogenous substrate metabolism Search for substances that express genotoxicity, (5) Search for substances that express carcinogenicity by xenobiotic and / or endogenous substrate metabolism, (6) Express mutagenicity by xenobiotic and / or endogenous substrate metabolism (7) living body search (8) search for xenobiotics and / or endogenous substrates acting on the liver, (9) xenobiotics and / or endogenous substrate metabolism, It becomes possible to search for a substance that acquires a new physiological activity or increases or decreases its own physiological activity, and a specific compound or a salt thereof can be obtained by using the analysis method and / or the search method.

Problems to be solved by the invention

An object of the present invention is to provide a cultured cell line derived from human liver, and a cell line that stably expresses human cytochrome P450 CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4. There is a separate manufacturing.
These cells are (1) analysis of xenobiotics and / or endogenous substrate metabolism, (2) analysis of xenobiotics and / or endogenous substrate metabolic pathways, and (3) xenobiotic and / or endogenous substrate metabolism. Analysis of the chemical structure of the product, (4) Preparation of xenobiotics and / or endogenous substrate metabolites, (5) Analysis of inhibition of xenobiotics and / or endogenous substrate metabolizing enzymes, (6) Xenobiotics and / or endogenous (7) Analysis of cytotoxicity due to xenobiotic and / or endogenous substrate metabolism, (8) Analysis of genotoxicity due to xenobiotic and / or endogenous substrate metabolism , (9) analysis of carcinogenicity by xenobiotic and / or endogenous substrate metabolism, (10) analysis of mutagenicity by xenobiotic and / or endogenous substrate metabolism, (11) xenobiotic and / or endogenous Of hepatotoxicity in sex substrate metabolism Analysis, not only to allow (12) of xenobiotics and / or endogenous substrates acts on liver analysis and the like,
(1) Search for substances that inhibit xenobiotics and / or endogenous substrate metabolizing enzymes, (2) Search for substances that promote the activities of xenobiotics and / or endogenous substrate metabolizing enzymes, (3) Xenobiotics and / or Search for substances that express cytotoxicity due to endogenous substrate metabolism, (4) Search for substances that express genotoxicity due to xenobiotics and / or endogenous substrate metabolism, (5) Originate due to xenobiotics and / or endogenous substrate metabolism Search for substances that express cancer, (6) Search for substances that express mutagenicity by xenobiotic and / or endogenous substrate metabolism, (7) Express hepatotoxicity by xenobiotic and / or endogenous substrate metabolism Search for substances, (8) Search for xenobiotics and / or endogenous substrates that act on the liver, (9) Acquire new physiological activities or obtain their own physiological activities by metabolism of xenobiotics and / or endogenous substrates Increase or To allow the search, etc. of the weak substance using the analyzing method and / or the screening method makes it possible that a particular compound or its salts obtained.

Means for solving the problem

In light of the above-mentioned problems, the present inventors have conducted extensive research, and as a result, stably and highly expressed cytochrome P450 involved in human xenobiotics and / or endogenous substrate metabolism in human liver cancer-derived cell lines. As a result of establishing a stable transformant and conducting further research, the present invention has been completed.
That is, the present invention
(1) a human liver cancer-derived cell line that stably expresses human cytochrome P450,
(2) The cell line according to (1) above, wherein human cytochrome P450 stably expresses CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, P2C9, CYP2C19, CYP2D6, CYP2E1, or CYP3A4,
(3) The cultured cell line according to (1), wherein the human liver cancer cell is HepG2.
(4) Hepc / 1A1.4, Hepc / 1A2.9, Hepc / 2B6.68, Hepc / 2C8.46, Hepc / 2C9.1, Hepc / 2C19.12, Hepc / 2D6.39, Hepc / 2E1.3 -8 or Hepc / 3A4.5, the cell line according to (1) above,
(5) The cell line described in (1) above is used, (a) an enzyme involved in xenobiotic and / or endogenous substrate metabolism, (b) xenobiotic and / or endogenous substrate metabolic pathway, c) chemical structure of xenobiotics and / or endogenous substrate metabolites, (d) inhibition of xenobiotics and / or endogenous substrate metabolizing enzymes, (e) promotion of activities of xenobiotics and / or endogenous substrate metabolizing enzymes, (f) cytotoxicity due to xenobiotics and / or endogenous substrate metabolism, (g) genotoxicity due to xenobiotics and / or endogenous substrate metabolism, (h) carcinogenicity due to xenobiotics and / or endogenous substrate metabolism, ( i) mutagenicity due to xenobiotics and / or endogenous substrate metabolism, (j) hepatotoxicity due to xenobiotics and / or endogenous substrate metabolism, or (k) xenobiotics and / or endogenous substrates acting on the liver analysis method,
(6) A method for preparing a xenobiotic and / or endogenous substrate metabolite, characterized by using the cell line described in (1) above,
(7) A cell line described in (1) above, wherein (a) a substance that inhibits xenobiotics and / or endogenous substrate metabolizing enzymes, (b) xenobiotics and / or endogenous substrate metabolizing enzymes A substance that promotes activity, (c) a substance that exhibits cytotoxicity due to xenobiotics and / or endogenous substrate metabolism, (d) a substance that exhibits genotoxicity due to xenobiotics and / or endogenous substrate metabolism, (e) xenobiotics And / or a substance that develops carcinogenicity by endogenous substrate metabolism, (f) a substance that exhibits mutagenicity by xenobiotics and / or endogenous substrate metabolism, and (g) by xenobiotics and / or endogenous substrate metabolism. Substances that exhibit hepatotoxicity or (h) xenobiotics and / or endogenous substrates that act on the liver, (i) acquire new physiological activity or have their own physiological activity by metabolism of xenobiotics and / or endogenous substrates Increase Search method for a substance stomach to attenuate,

(8) A compound or salt thereof obtained using the method described in (7) above,
(9) A pharmaceutical composition comprising the compound or salt thereof according to (8) above,
(10) Use two or more cultured cell lines derived from human liver that stably express any one or more of CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 Features (a) enzymes involved in xenobiotic and / or endogenous substrate metabolism, (b) xenobiotic and / or endogenous substrate metabolic pathways, (c) chemical structures of xenobiotics and / or endogenous substrate metabolites , (D) inhibition of xenobiotics and / or endogenous substrate metabolizing enzymes, (e) promotion of activities of xenobiotics and / or endogenous substrate metabolizing enzymes, (f) cytotoxicity due to xenobiotics and / or endogenous substrate metabolism , (G) genotoxicity due to xenobiotics and / or endogenous substrate metabolism, (h) carcinogenicity due to xenobiotics and / or endogenous substrate metabolism, (i) mutagens due to xenobiotics and / or endogenous substrate metabolism. Sex, (j) xenobiotics and / or Is hepatotoxicity due to endogenous substrate metabolism, or (k) a method for analyzing xenobiotics and / or endogenous substrates acting on the liver,
(11) Use two or more types of cultured cell lines derived from human liver that stably express one or more of CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 A method for the preparation of the characteristic xenobiotics and / or endogenous substrate metabolites,
(12) Use two or more types of cultured cell lines derived from human liver that stably express one or more of CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4. (A) a substance that inhibits xenobiotics and / or endogenous substrate metabolizing enzymes, (b) a substance that promotes the activity of xenobiotics and / or endogenous substrate metabolizing enzymes, (c) xenobiotics and / or endogenous Substance that expresses cytotoxicity by sexual substrate metabolism, (d) Substance that expresses genotoxicity by xenobiotic and / or endogenous substrate metabolism, (e) Substance that expresses carcinogenicity by xenobiotic and / or endogenous substrate metabolism (F) a substance that exhibits mutagenicity by xenobiotics and / or endogenous substrate metabolism, (g) a substance that exhibits hepatotoxicity by xenobiotics and / or endogenous substrate metabolism, or (h) a living body that acts on the liver. Foreign objects and / or Is an endogenous substrate, (i) a method for searching for a substance that acquires a new physiological activity or increases or decreases its own physiological activity by xenobiotic and / or endogenous substrate metabolism,
(13) The present invention relates to a compound obtained by using the method described in (12) above or a salt thereof, and (14) a pharmaceutical composition containing the compound described in (13) above or a salt thereof.

In the present specification, xenobiotic means, for example, drugs, food additives, environmental pollutants, chemical products in general, and endogenous substrate means all substances existing in the living body. Among these, drug metabolism is preferably used for the metabolism of xenobiotics centering on drugs.
The human liver cancer cells to be used can be obtained by separating a cultured cell line derived from human liver cancer (preferably HepG2) from human liver cancer. Here, various separately separated genes encoding cytochrome P450 are stably expressed.
In order to stably express a DNA fragment encoding cytochrome P450, for example, a DNA fragment encoding individual cytochrome P450 is obtained and expressed under the control of an exogenous promoter. The nucleotide sequence of a DNA fragment encoding cytochrome P450 can be obtained from a public database. Based on this base sequence, a DNA fragment encoding cytochrome P450 can be separated by a known method such as a PCR method or a hybridization screening method. The DNA fragment thus obtained is introduced into a vector that provides a transformed strain that stably expresses a foreign gene in cultured mammalian cells, and a transformation vector is prepared. The prepared vector is introduced into hepatoma cells by a known method. Transformants are selected by examining the enzyme activity resulting from the expression of cytochrome P450 introduced into them, and excellent clones are selected. Furthermore, the stability of the property of the obtained clone can be confirmed by repeated cryopreservation.
Examples of the exogenous promoter include SRα promoter, SV40 promoter, LTR promoter, CMV promoter, HSV-TK promoter and the like.

“Stable expression of human cytochrome P450” means
It means that the expression of human cytochrome P450 is not transient, specifically, that the activity of cytochrome P450 is not lost by cell culture (passaging). In addition, cells expressing human cytochrome P450 are not only cytochrome P450 but also enzymes involved in various metabolisms (specifically, UDP-glucuronosyltransferase, sulfotransferase, glutathione transferase, epoxy hydratase, N-acetyl). Cells in which transferase, flavin monooxygenase, etc.) are functioning are preferred.
Examples of cytochrome P450 molecular species involved in liver xenobiotics and / or endogenous substrate metabolism include CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4. These enzymes not only carry out metabolic reactions of xenobiotics and / or endogenous substrates, but also inhibit xenobiotics and / or endogenous substrate metabolizing enzymes depending on the properties of the metabolites, xenobiotics and / or endogenous substrate metabolizing enzymes. Promotion of activity, expression of cytotoxicity by xenobiotic and / or endogenous substrate metabolism, expression of genotoxicity by xenobiotic and / or endogenous substrate metabolism, carcinogenic expression by xenobiotic and / or endogenous substrate metabolism, living body It causes mutagenic expression due to foreign substances and / or endogenous substrate metabolism, hepatotoxicity expression due to xenobiotics and / or endogenous substrate metabolism, and the like. However, functions involved in liver xenobiotics and / or endogenous substrate metabolism are not simply performed by cytochrome P450 alone but by UDP-glucuronosyltransferase, sulfotransferase, glutathione transferase, epoxy hydratase, N-acetyl. Relies on the joint action of various enzymes such as transferase, flavin monooxygenase and cytochrome P450 reductase.
Therefore, in order to reproduce the liver function by the expression of cytochrome P450, the cells must be at least UDP-glucuronosyltransferase, sulfotransferase, glutathione transferase, epoxy hydratase, N-acetyltransferase, flavin monooxygenase derived from human. It must be a cell in which the enzyme is functioning. One such cell is human liver cancer-derived cultured cell HepG2. HepG2 cells are known to function UDP-glucuronosyltransferase, sulfotransferase, glutathione transferase, epoxy hydratase, N-acetyltransferase, flavin monooxygenase and NADPH P450 reductase (J Rueff et. Al., Mutation Research, 353, 151-176 (1996) In view of the above, the present inventors have achieved rapid, inexpensive, safe and accurate human liver by stably expressing cytochrome P450 in HepG2. We succeeded in reproducing the function of.
Among them, Hepc / 3A4.5, Hepc / 2E1.3-8, Hepc / 2C9.1, Hepc / 2C8.46, Hepc / 1A2.9, Hepc / 1A1.4, Hepc / 2B6.68, Hepc / 2D6 .39, Hepc / 2A6L.9, Hepc / 2C19.12 and the like are preferably used.
Hepc / 3A4.5 is a CYP3A4 high activity expressing cell, Hepc / 2E1.3-8 is a CYP2E1 high activity expressing cell, Hepc / 2C9.1 is a CYP2C9 high activity expressing cell, Hepc / 2C8 .46 is a CYP2C8 high activity cell, Hepc / 1A2.9 is a CYP1A2 high activity cell, Hepc / 1A1.4 is a CYP1A1 high activity cell, Hepc / 2B6.68 is a CYP2B6 cell Hepc / 2D6.39 is a CYP2D6 high activity expression cell, Hepc / 2A6L.9 is a CYP2A6 high activity expression cell, and Hepc / 2C19.12 is a CYP2C19 high activity expression cell. is there.
Furthermore, the present invention uses a cultured cell line derived from human liver cancer that stably expresses the above-mentioned human cytochrome p450 (a) a method for analyzing an enzyme involved in xenobiotic and / or endogenous substrate metabolism (B) Xenobiotic and / or endogenous substrate metabolic pathway analysis method, (c) Xenobiotic and / or endogenous substrate metabolite chemical structure analysis method, (d) Xenobiotic and / or endogenous substrate metabolism (E) Xenobiotic and / or endogenous substrate metabolizing enzyme inhibition analysis method, (f) Xenobiotic and / or endogenous substrate metabolizing enzyme activity promotion method, (g) Xenobiotic And / or a method for analyzing the expression of cytotoxicity due to endogenous substrate metabolism, (h) a method for analyzing the expression of genotoxicity due to xenobiotic and / or endogenous substrate metabolism, (i) based on xenobiotic and / or endogenous substrate metabolism Departure (J) analysis method of mutagenicity by xenobiotic and / or endogenous substrate metabolism, (k) analysis method of hepatotoxicity expression by xenobiotic and / or endogenous substrate metabolism or (l ) It relates to a method for analyzing xenobiotics and / or endogenous substrates acting on the liver.

Each method described in the above (a) to (l) will be described below.
(a) Methods for analyzing xenobiotics and / or enzymes involved in endogenous substrate metabolism:
For example, xenobiotics and / or endogenous substrates by analyzing changes in the structure of xenobiotics and / or endogenous substrates due to exposure to a cultured cell line derived from human liver cancer that stably expresses cytochrome p450 as a test substance Analysis of enzymes involved in metabolism is possible (JL Napoli et al. Methods in Enzymology vol. 206 pp.491-501 Ed. By MR Waterman et al. Academic Press 1991, HK Kroemer et al. Methods in Enzymology vol. 272 pp.99- 198 Ed. By MR Waterman and Academic Press 1996). Specifically, xenobiotics and / or by analyzing changes in the structure of xenobiotics and / or endogenous substrates due to exposure to human liver cancer-derived cultured cell lines that stably express cytochrome p450 as a test substance Identification of enzymes involved in endogenous substrate metabolism, analysis of xenobiotics and / or changes in the structure of endogenous substrates due to exposure of test substances to cells, analysis of substrate specificity, etc. I can give you.
Examples of the test substance include peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, plasma, etc. These compounds are novel compounds. It may be a known compound.
(b) Xenobiotic and / or endogenous substrate metabolic pathway analysis method:
For example, xenobiotics and / or endogenous substrates by analyzing changes in the structure of xenobiotics and / or endogenous substrates due to exposure to a cultured cell line derived from human liver cancer that stably expresses cytochrome P450 as a test substance (JL Napoli et al. Methods in Enzymology vol. 206 pp.491-501 Ed. By MR Waterman et al. Academic Press 1991, HK Kroemer et al. Methods in Enzymology vol. 272 pp.99-198 Ed. by MR Waterman and Academic Press 1996).
As the test substance, those similar to the above are used.

(c) Method for analyzing the chemical structure of xenobiotics and / or endogenous substrate metabolites:
For example, by analyzing changes in the structure of xenobiotics and / or endogenous substrates caused by exposure to a cultured cell line derived from human liver cancer that stably expresses the test substance cytochrome P450, xenobiotics and / or endogenous Analyzes of chemical structures of metabolites of sexual substrates (JL Napoli et al. Methods in Enzymology vol. 206 pp.491-501 Ed. By MR Waterman et al. Academic Press 1991, HK Kroemer et al. Methods in Enzymology vol. 272 pp. 99-198 Ed. By MR Waterman et al. Academic Press 1996).
As the test substance, those similar to the above are used.
(d) Preparation of xenobiotics and / or endogenous substrate metabolites:
For example, a suitable method for collecting xenobiotics and / or endogenous substrate conversion substances (so-called metabolites) resulting from exposure of a test substance to a cultured cell line derived from human liver cancer that stably expresses cytochrome P450 It is possible to prepare metabolites of xenobiotics and / or endogenous substrates (JL Napoli et al. Methods in Enzymology vol. 206 pp.491-501 Ed. By MR Waterman et al. Academic Press 1991).
As the test substance, those similar to the above are used.
(e) Method for analyzing xenobiotic and / or endogenous substrate metabolic enzyme inhibition For example, xenobiotic and / or endogenous by exposing a test substance to a cultured cell line derived from human liver cancer that stably expresses cytochrome P450. It is possible to analyze the inhibition of the activity of sex substrate metabolizing enzymes (JL Napoli et al. Methods in Enzymology vol. 206 pp.491-501 Ed. By MR Waterman et al. Academic Press 1991). Specifically, it can be detected by inhibiting cytochrome P450 enzyme activity, decreasing the amount of protein, decreasing mRNA, and the like. As a detection method, known methods such as measurement of enzyme activities corresponding to various P450s, Western blotting corresponding to various P450 proteins, Northern hybridization corresponding to various P450 mRNAs, or RT-PCR methods can be used.
As the test substance, those similar to the above are used.

(f) Analytical method for promoting the activity of xenobiotics and / or endogenous substrate metabolizing enzymes:
For example, a test substance is exposed to a cultured cell line derived from human liver cancer that stably expresses cytochrome P450, and the enzyme activity of xenobiotics and / or endogenous substrate metabolism is increased, the amount of the enzyme is increased, or the gene encoding the enzyme It is possible to analyze the promotion of the activity of xenobiotics and / or endogenous substrate metabolizing enzymes by detecting an increase in the transcription amount of J. Rueff et al. (Mutation Research 353 (1996) 151-176). Specifically, it is possible by detecting an increase in cytochrome P450 enzyme activity, an increase in protein amount, and an increase in mRNA. As a detection method, known methods such as measurement of enzyme activities corresponding to various P450s, Western blotting corresponding to various P450 proteins, Northern hybridization corresponding to various P450 mRNAs, or RT-PCR methods can be used.
As the test substance, those similar to the above are used.
(g) Method for analyzing cytotoxicity due to xenobiotics and / or endogenous substrate metabolism:
For example, exposure to a cultured cell line derived from human liver cancer that stably expresses the test substance cytochrome P450 enables analysis of cytotoxicity due to metabolism of xenobiotics and / or endogenous substrates. Specifically, changes in cell morphology due to test substance exposure, changes in the number of living cells by known methods such as MTT assay, trypan blue staining or crystal violet staining, leakage of intracellular enzymes such as lactate dehydrogenase, It is analyzed by observing changes in cell surface structure or changes in intracellular enzymes (D. Wu et al. Journal of Biological Chemistry, 271, (1996) 23914-23919).
As the test substance, those similar to the above are used.

(h) Methods for analyzing genotoxicity due to xenobiotics and / or endogenous substrate metabolism:
For example, by exposing a test substance to a cultured cell line derived from human liver cancer that stably expresses cytochrome P450 and subjecting the cell to chromosomal aberration test, micronucleus test, etc., inheritance by xenobiotic and / or endogenous substrate metabolism Toxicity analysis is possible. In addition, after exposing the test substance to a cultured cell line derived from human liver cancer that stably expresses cytochrome P450, the test substance changed by the cells is evaluated by an appropriate evaluation system, whereby a chromosome abnormality test, (J. Rueff et al. Mutation Research 353 (1996) 151-176, ME McManus et al. Methods in Enzymology vol. 206 pp.501-508 Ed. By MR Waterman Other Academic Press 1991)).
As the test substance, those similar to the above are used.
(i) Analysis method of carcinogenicity by xenobiotic and / or endogenous substrate metabolism:
For example, a test substance is exposed to a cultured cell line derived from human liver cancer that stably expresses cytochrome P450, and the cell is subjected to chromosomal aberration test, DNA modification, etc. Cancer analysis is possible. Furthermore, after the test substance is exposed to a cultured cell line derived from human liver cancer that stably expresses cytochrome P450, analysis is performed by evaluating the test substance changed by the cell using a carcinogenesis evaluation system with an appropriate chemical substance. (J. Rueff et al. Mutation Research 353 (1996) 151-176, K. Kawajiri et al. Cytochromes P450 metabolic and toxicological aspects pp77-98 ed. By C. Ioannides CRC press (1996)).
As the test substance, those similar to the above are used.
(j) Methods for analyzing mutagenicity due to xenobiotics and / or endogenous substrate metabolism:
For example, depending on xenobiotics and / or endogenous substrate metabolism, the test substance is exposed to a cultured cell line derived from human liver cancer that stably expresses cytochrome P450, and the cells are subjected to a chromosomal aberration test, a micronucleus test, etc. Mutagenic analysis is possible. In addition, after exposing the test substance to a cultured cell line derived from human liver cancer that stably expresses cytochrome P450, the test substance changed by the cells is evaluated by an appropriate evaluation system, whereby a chromosome abnormality test, Analysis is possible by subjecting it to tests, reverse mutation tests, etc. (J. Rueff et al. Mutation Research 353 (1996) 151-176).
As the test substance, those similar to the above are used.

(k) Method for analyzing hepatotoxicity due to xenobiotics and / or endogenous substrate metabolism:
For example, by exposing the test substance to a cultured cell line derived from human liver cancer that stably expresses cytochrome P450 and observing the expression of cytotoxicity, or the test substance is human liver that stably expresses cytochrome P450. After exposure to cultured cell lines derived from cancer, the test substance changed by the cells is administered to other hepatocytes, liver sections, isolated livers, or experimental animals, and the resulting changes in cells, tissues, and living bodies are observed. It is possible to analyze hepatotoxicity due to xenobiotics and / or endogenous substrate metabolism.
As the test substance, those similar to the above are used.
(l) Methods for analyzing xenobiotics and / or endogenous substrates that act on the liver:
For example, by observing the expression of changes in cells by exposure to a cultured cell line derived from human liver cancer that stably expresses the test substance cytochrome P450, or the test substance is a human that stably expresses cytochrome P450 After exposure to a cultured cell line derived from liver cancer, the test substance changed by the cell is administered to other hepatocytes, liver sections, isolated liver, or experimental animals, and the changes in the cells, tissues, and organisms are observed. It is possible to analyze the expression of the action on the liver.
As the test substance, those similar to the above are used.
Furthermore, the present invention uses (A) a substance that inhibits xenobiotics and / or endogenous substrate metabolizing enzymes, characterized by using a cultured cell line derived from human liver cancer that stably expresses the above-mentioned human cytochrome p450. B) Substances that promote the activity of xenobiotics and / or endogenous substrate metabolizing enzymes, (C) Substances that exhibit cytotoxicity due to xenobiotics and / or endogenous substrate metabolism, (D) Xenobiotics and / or endogenous substrate metabolism (E) a substance that expresses carcinogenicity due to xenobiotics and / or endogenous substrate metabolism, (F) a substance that expresses mutagenicity due to xenobiotics and / or endogenous substrate metabolism, (G) a substance that exhibits hepatotoxicity due to xenobiotics and / or endogenous substrate metabolism or (H) xenobiotics and / or endogenous substrates that act on the liver, (I) xenobiotics and / or endogenous substrate metabolism. Provides a compound or a salt thereof obtained by the screening method and these searching method for a substance that increases or attenuate the physiological activity possessed by the acquisition or itself a new physiological activity.
(A) As a method for searching for substances that inhibit xenobiotics and / or endogenous substrate metabolizing enzymes, analysis according to the analysis method of xenobiotics and / or endogenous substrate metabolizing enzymes described in (e) above, for example, It is possible to select a test substance that causes inhibition of cytochrome P450 enzyme activity, decrease in protein amount, decrease in mRNA, etc. as a substance that inhibits xenobiotics and / or endogenous substrate metabolic enzymes.

(B) As a method for searching for a substance that promotes the activity of xenobiotics and / or endogenous substrate metabolizing enzymes, the method for analyzing the activity of xenobiotics and / or endogenous substrate metabolizing enzymes described in (f) above is used. It is possible to analyze and select, for example, a test substance that promotes cytochrome P450 enzyme activity, increases the amount of protein, increases mRNA, and the like as a substance that inhibits xenobiotics and / or endogenous substrate metabolic enzymes.
(C) As a method for searching for a substance that exhibits cytotoxicity due to xenobiotics and / or endogenous substrate metabolism, analysis is performed according to the method for analyzing cytotoxicity due to xenobiotics and / or endogenous substrate metabolism described in (g) above. For example, a test substance that causes a change in cell morphology, a change in the number of living cells, leakage of intracellular enzymes, a change in cell surface structure or a change in intracellular enzymes, etc. due to exposure of the test substance can be introduced as a xenobiotic and / or It can be selected as a substance that exhibits cytotoxicity by endogenous substrate metabolism.
(D) As a method for searching for a substance that exhibits genotoxicity due to xenobiotics and / or endogenous substrate metabolism, analysis according to the analysis method of genotoxicity due to xenobiotics and / or endogenous substrate metabolism described in (h) above, For example, a test substance that causes genotoxicity due to xenobiotics and / or endogenous substrate metabolism is selected as a substance that exhibits genotoxicity due to xenobiotics and / or endogenous substrate metabolism by being subjected to a chromosome aberration test, micronucleus test, etc. It is possible.
(E) As a method for searching for a substance that expresses carcinogenicity by xenobiotics and / or endogenous substrate metabolism, the method for analyzing carcinogenicity by xenobiotics and / or endogenous substrate metabolism described in (i) above is used. Therefore, the test substance that causes carcinogenicity due to xenobiotics and / or endogenous substrate metabolism is expressed by carcinogenicity due to xenobiotics and / or endogenous substrate metabolism by analyzing and subjecting to chromosomal aberration test, DNA modification, etc. It is possible to select as a substance to be performed.
(F) As a method for searching for a substance that exhibits mutagenicity by xenobiotic and / or endogenous substrate metabolism, the method for analyzing mutagenicity by xenobiotic and / or endogenous substrate metabolism described in (j) above The test substance that causes mutagenicity due to xenobiotics and / or endogenous substrate metabolism by subjecting it to, for example, chromosomal aberration test, micronucleus test, etc., is made mutagenic by xenobiotic and / or endogenous substrate metabolism. It is possible to select the substance to be expressed.
(G) As a method for searching for a substance that exhibits hepatotoxicity due to xenobiotics and / or endogenous substrate metabolism, analysis is performed according to the analysis method for hepatotoxicity due to xenobiotics and / or endogenous substrate metabolism described in (k) above. For example, after exposing a test substance to a cell, the test substance changed by the cell is administered to another hepatocyte, a liver section, an isolated liver, or an experimental animal, and a change in the cell, tissue, or living body is observed by that. Thus, a test substance that causes hepatotoxicity due to xenobiotics and / or endogenous substrate metabolism can be selected as a substance that exhibits hepatotoxicity due to xenobiotics and / or endogenous substrate metabolism.
(H) As a method for searching for xenobiotics and / or endogenous substrates acting on the liver, analysis is performed according to the analysis method for xenobiotics and / or endogenous substrates acting on the liver described in (l) above. For example, after exposing a test substance to a cell, the test substance changed by the cell is administered to another hepatocyte, a liver section, an isolated liver, or an experimental animal, and a change in the cell, tissue, or living body is observed by that. This makes it possible to search for xenobiotics and / or endogenous substrates that act on the liver.

(I) As a method of searching for substances (including so-called prodrugs) that acquire new physiological activity or increase or decrease its own physiological activity by xenobiotic and / or endogenous substrate metabolism, It is possible to search by analyzing according to the analysis method of the chemical structure of the xenobiotic and / or endogenous monster substrate metabolite described in the above and observing the physiological activity of the metabolite.
The compound or salt thereof obtained by the search method of (A) to (I) above is a compound or salt thereof selected from the test substance that brings about the above-mentioned actions and properties, etc., and relates to abnormal metabolism of liver xenobiotics Since it has a therapeutic / preventive effect on a disease (for example, liver dysfunction), it can be used as a pharmaceutical composition such as a safe and low-toxic therapeutic / preventive agent for the disease.
The compound obtained by the search method may form a salt. Examples of the salt of the compound include physiologically acceptable acids (eg, inorganic acids, organic acids), bases (eg, alkali metals) and the like. In particular, physiologically acceptable acid addition salts are preferred. Such salts include, for example, salts with inorganic acids (eg hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid). Acid, tartaric acid, citric acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like.
A pharmaceutical containing the compound obtained by the searching method or a salt thereof can be produced by a known production method or a method analogous thereto. Since the preparation thus obtained is safe and low toxic, it can be used, for example, in humans or mammals (eg, rats, mice, guinea pigs, rabbits, sheep, pigs, cows, horses, cats, dogs, monkeys, etc.). Can be administered.

The dose of the compound or a salt thereof varies depending on the target disease, administration subject, administration route, etc. For example, when the compound is administered orally for the purpose of treating liver dysfunction, it is generally used for adults (body weight 60 kg). As), about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg of the compound is administered per day. When administered parenterally, the single dose of the compound varies depending on the administration subject, target disease, etc. For example, for the purpose of treating liver dysfunction, the compound is usually administered in the form of an injection (60 kg). The compound is administered at about 0.01 to 30 mg per day, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg by intravenous injection per day. It is. In the case of other animals, an amount converted per 60 kg can be administered.
Specific examples of dosage forms of the above preparations include tablets (including sugar-coated tablets and film-coated tablets), pills, capsules (including microcapsules), granules, fine granules, powders, syrups, and emulsions. Suspensions, injections, inhalants, ointments and the like are used. These preparations are prepared according to conventional methods (for example, methods described in the Japanese Pharmacopoeia).
In the preparation, the content of the compound obtained by the above screening method or a salt thereof varies depending on the form of the preparation, but is usually 0.01 to 100% by weight, preferably 0.1 to 50%, based on the whole preparation. % By weight, more preferably about 0.5 to 20% by weight.
Specifically, the tablet production method is as follows: a pharmaceutical product as it is, and an excipient, a binder, a disintegrant or other suitable additive added and mixed evenly into granules by an appropriate method; Add a lubricant, etc., and compress or mold it, or directly compress and mold the drug as it is, or evenly mixed with excipients, binders, disintegrants or other appropriate additives. Alternatively, the granule prepared in advance can be produced as it is, or by adding an appropriate additive and mixing evenly and then compression molding. Moreover, this agent can add a coloring agent, a corrigent, etc. as needed. Furthermore, the agent can be coated with an appropriate coating agent.
Injectables are prepared in a certain amount by dissolving, suspending or emulsifying a certain amount of pharmaceuticals in water for injection, physiological saline, Ringer's solution, etc. in the case of aqueous solvents, and usually in vegetable oil in the case of non-aqueous solvents. Alternatively, a certain amount of medicine can be taken and sealed in an injection container.
As the oral pharmaceutical carrier, for example, substances commonly used in the pharmaceutical field such as starch, mannitol, crystalline cellulose, sodium carboxymethyl cellulose and the like are used. As the carrier for injection, for example, distilled water, physiological saline, glucose solution, infusion agent and the like are used. In addition, additives generally used for preparations can be appropriately added.

Furthermore, the present invention provides
(1) Use two or more types of cultured cell lines derived from human liver that stably express one or more of CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 Features (a) enzymes involved in xenobiotic and / or endogenous substrate metabolism, (b) xenobiotic and / or endogenous substrate metabolic pathways, (c) chemical structures of xenobiotics and / or endogenous substrate metabolites , (D) inhibition of xenobiotics and / or endogenous substrate metabolizing enzymes, (e) promotion of activities of xenobiotics and / or endogenous substrate metabolizing enzymes, (f) cytotoxicity due to xenobiotics and / or endogenous substrate metabolism , (G) genotoxicity due to xenobiotics and / or endogenous substrate metabolism, (h) carcinogenicity due to xenobiotics and / or endogenous substrate metabolism, (i) mutagens due to xenobiotics and / or endogenous substrate metabolism. Sex, (j) xenobiotics and / or Hepatotoxicity by endogenous substrate metabolism or (k) the method Analysis of xenobiotics and / or endogenous substrates acts on the liver,
(2) Use of two or more cultured cell lines derived from human liver that stably express one or more of CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 A method for the preparation of the characteristic xenobiotics and / or endogenous substrate metabolites,
(3) Use of two or more cultured cell lines derived from human liver that stably express one or more of CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 (A) a substance that inhibits xenobiotics and / or endogenous substrate metabolizing enzymes, (b) a substance that promotes the activity of xenobiotics and / or endogenous substrate metabolizing enzymes, (c) xenobiotics and / or endogenous Substance that expresses cytotoxicity by sexual substrate metabolism, (d) Substance that expresses genotoxicity by xenobiotic and / or endogenous substrate metabolism, (e) Substance that expresses carcinogenicity by xenobiotic and / or endogenous substrate metabolism (F) a substance that exhibits mutagenicity by xenobiotic and / or endogenous substrate metabolism, (g) a substance that exhibits hepatotoxicity by xenobiotic and / or endogenous substrate metabolism, (h) a living body that acts on the liver Foreign matter and / or inside Sex by substrate or (i) of xenobiotics and / or endogenous substrates metabolism searching method for a substance that increases or attenuate the physiological activity possessed by the acquisition or itself a new physiological activity,
(4) A compound or a salt thereof (pharmaceutical composition) obtained by using the method described in (3) above is also provided.

“(A) enzymes involved in xenobiotics and / or endogenous substrate metabolism, (b) xenobiotics and / or endogenous substrate metabolic pathways, (c) chemical structures of xenobiotics and / or endogenous substrate metabolites, ( d) inhibition of xenobiotics and / or endogenous substrate metabolizing enzymes, (e) promotion of the activity of xenobiotics and / or endogenous substrate metabolizing enzymes, (f) cytotoxicity due to xenobiotics and / or endogenous substrate metabolism, ( g) genotoxicity due to xenobiotics and / or endogenous substrate metabolism, (h) carcinogenicity due to xenobiotics and / or endogenous substrate metabolism, (i) mutagenicity due to xenobiotics and / or endogenous substrate metabolism, (j) hepatotoxicity due to xenobiotics and / or endogenous substrate metabolism, or (k) method for analyzing xenobiotics and / or endogenous substrates acting on the liver "," preparation of xenobiotics and / or endogenous substrate metabolites Method ”,“ (a) Xenobiotics and And / or substances that inhibit endogenous substrate metabolizing enzymes, (b) substances that promote the activity of xenobiotics and / or endogenous substrate metabolizing enzymes, (c) cytotoxicity due to xenobiotics and / or endogenous substrate metabolism (D) a substance that exhibits genotoxicity due to xenobiotics and / or endogenous substrate metabolism, (e) a substance that exhibits carcinogenicity due to xenobiotics and / or endogenous substrate metabolism, (f) xenobiotics and / or Or a substance that expresses mutagenicity by endogenous substrate metabolism, (g) a xenobiotic and / or a substance that expresses hepatotoxicity by endogenous substrate metabolism, (h) a xenobiotic and / or endogenous substrate that acts on the liver, or (i) a method for searching for a substance that acquires new physiological activity or increases or decreases its own physiological activity by xenobiotic and / or endogenous substrate metabolism "and" (a) xenobiotic and / or endogenous A substance that inhibits substrate metabolic enzymes, (b) a substance that promotes the activity of xenobiotics and / or endogenous substrate metabolic enzymes, (c) a substance that exhibits cytotoxicity due to xenobiotics and / or endogenous substrate metabolism, (d ) Xenobiotics and / or substances that exhibit genotoxicity by endogenous substrate metabolism, (e) Xenobiotics and / or substances that develop carcinogenicity by endogenous substrate metabolism, (f) Xenobiotics and / or endogenous substrate metabolism (G) Xenobiotic and / or endogenous substrate metabolism, (h) Xenobiotic and / or endogenous substrate acting on the liver, or (i) Xenobiotic And / or a compound or salt thereof (pharmaceutical composition) obtained by a method for searching for a substance that acquires a new physiological activity or increases or attenuates its own physiological activity by endogenous substrate metabolism. Used in the above and similar meanings.
"Use two or more cultured cell lines derived from human liver that stably express one or more of CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4" The above analysis method, preparation method, and search method are for CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4. In comparison, analysis, preparation, and search in a state closer to the living body are possible.
Moreover, when using 2 or more types of this cell line, you may use each cell line simultaneously, and may use each separately, and may compare each analysis, preparation, and search result.

  The cell lines obtained in the examples described below are Hepc / 3A4.5, Hepc / 2E1.3-8, Hepc / 2C9.1, Hepc / 2C8.46, Hepc / 1A2.9, Hepc / 1A1.4 in 1999. From February 10, 2000, 2-17-85 Jusohoncho, Yodogawa-ku, Osaka, Osaka Prefecture, and the Foundation and Fermentation Research Institute (IFO), deposit numbers IFO 50502, 50503, 50504, 50505, 50506, 50507, respectively, 2000 From April 12th, 1-1-3 Higashi 1-1, Tsukuba, Ibaraki Prefecture, Deposited with FERM BP-7120, FERM BP-7121, FERM BP-7122, FERM Deposited as BP-7123, FERM BP-7124, FERM BP-7125. Hepc / 2B6.68 and Hepc / 2D6.39 are deposit numbers IFO 50508 from February 15th, 1999, 2-17-85 Jusohoncho, Yodogawa-ku, Osaka, Osaka Prefecture, Fermentation Research Institute (IFO), respectively. , 50509, from April 12, 2000 to Tsukuba City East 1-1-3, Ibaraki Prefectural Government, Ministry of International Trade and Industry, Institute of Industrial Science and Technology (NIBH) as deposit numbers FERM BP-7126 and FERM BP-7127, respectively It has been deposited. Hepc / 2A6L.9 and Hepc / 2C19.12 are the deposit numbers IFO from February 15th, 1999, 2-17-85 Juzahoncho, Yodogawa-ku, Osaka, Osaka, Japan. 50511 and 50512, deposit numbers FERM BP-7128 and FERM BP-7129 from April 12, 2000 to Tsukuba City East 1-1-3, Tsukuba City, Ibaraki Prefecture, Institute of Industrial Science and Technology (NIBH) Has been deposited.

  Examples of the present invention will be described in detail below, but the present invention is not limited thereto. The gene manipulation techniques used were general methods such as Sambrook et al. (Molecular Cloning: A Laboratory Manual), Cold Spring Harbor Laboratory Press, etc. unless otherwise specified.

Example 1 Cloning of a DNA fragment encoding cytochrome P450 and preparation of an expression vector Cloning of a DNA fragment encoding human cytochrome P450 was performed by an established method from a complementary DNA (cDNA) library derived from human adult liver. Cloning was performed by a certain polymerase chain reaction (PCR) method. The cDNA sequence of human cytochrome P450 to be cloned is available from the gene bank database. Acceptance numbers in GeneBank are K03191 for CYP1A1, M55053 or M38504 for CYP1A2, M33318 or M33316 for CYP2A6, M29874 or J02864 for CYP2B6, M17397 or J03472 for CYP2C8, M61857 or J05326 for CYP2C9, M61854 or 0 for C062006 CYP2E1 is J02625 and CYP3A4 is J04449.
Each cloned cDNA was introduced into the pcDNA3.1 (+) vector (Invtrogen Co.) CMV (cytomegalovirus) enhancer promoter in the direction of the promoter's function, and 1A1 / pcDNA3 introduced with CYP1A1. .1 (+), 1A2 / pcDNA3.1 (+) with CYP1A2 introduced, 2A6 / pcDNA3.1 (+) with CYP2A6 introduced, 2B6 / pcDNA3.1 (+) with CYP2B6 introduced, 2C8 with CYP2C8 introduced /pcDNA3.1(+), 2C9 / pcDNA3.1 (+) with CYP2C9 introduced, 2C19 / pcDNA3.1 (+) with CYP2C19 introduced, 2D6 / pcDNA3.1 (+) with CYP2D6 introduced, CYP2E1 introduced 2E1 / pcDNA3.1 (+) and 3A4 / pcDNA3.1 (+) into which CYP3A4 was introduced were obtained.

Example 2 Selection of cells expressing cytochrome P450 high activity
HepG2 was maintained in DMEM (Dulbecco's Modofied Eagle's medium) medium containing 10% FCS (fetal calf serum) (Bio Whittaker). HepG2 is seeded in a 60 mm dish, cultured in a CO ₂ incubator until it is 50-60% confluent, and then ₂ μg of 1A1 / pcDNA3.1 (+), 1A2 / pcDNA3.1 using Lipofectamine reagent (GIBCO BRL) (+), 2A6 / pcDNA3.1 (+), 2B6 / pcDNA3.1 (+), 2C8 / pcDNA3.1 (+), 2C9 / pcDNA3.1 (+), 2C19 / pcDNA3.1 (+), 2D6 /pcDNA3.1(+), 2E1 / pcDNA3.1 (+), or 3A4 / pcDNA3.1 (+) were transfected. After culturing in DMEM medium containing 10% FCS for 2 days, the medium was replaced with 500 μg / ml G418 (GIBCO BRL), DMEM medium containing 10% FCS, and replaced with a new medium every 3 to 4 days, and a G418 resistant strain was cloned. . The obtained G418 resistant strain was maintained in a DMEM medium containing 200 μg / ml G418 (GIBCO BRL) and 10% FCS. The cytochrome P450 activity of each of the obtained G418-resistant strains was measured by the method described below, a cell line with high activity was measured, and highly active expressing cells were selected.

(1) Measurement of activity of CYP1A1 and CYP1A2 expressing cells and selection of highly active cells Ethoxyresorufin (Molecular Probes) was diluted to 2 mM with DMSO (dimethyl sulfoxide) (Wako Pure Chemical Industries). . Next, this was diluted to 500 μM with phenol red-free DMEM medium (GIBCO BRL) containing 2% FCS (Bio Whittaker).
CYP1A1 or CYP1A2-expressing cells were seeded in a 12-well plate (Falcon) and cultured in a CO ₂ incubator until confluent. After culturing, the medium was aspirated and the cells adhering to the plate were washed with phenol red-free DMEM medium, and then 500 μl ethoxyresorufin diluted as described above was added at 500 μl / well. After reacting at 37 ° C. in the dark, the reaction solution was recovered from each well. Add 1800 μl of methanol (Wako Pure Chemicals) to 300 μl of the reaction solution, centrifuge to remove insoluble substances, etc., and then measure the fluorescence intensity at an excitation wavelength of 550 nm and a fluorescence wavelength of 586 nm with a spectrofluorometer to quantify the generated resorufin . The standard substance of resorufin (Molecular Probes) used what was purchased from Moleclar Probes.
From the obtained CYP1A1 or CYP1A2 active expression strain, Hepc / 1A1.4 strain was obtained as a CYP1A1 high expression strain, and Hepc / 1A2.9 strain was obtained as a CYP1A2 high expression strain.

(2) Measurement of activity of cells expressing CYP2A6 and selection of cells expressing high activity Coumarin (Wako Pure Chemical Industries) was diluted with methanol (Wako Pure Chemical Industries) to 50 mM. Next, this was diluted with phenol red-free DMEM medium (GIBCO BRL) containing 2% FCS (Bio Whittaker) to 500 μM.
CYP2A6-expressing cells were seeded in a 12-well plate (Falcon) and cultured in a CO2 incubator until confluent. After the culture, the medium was aspirated and the cells adhering to the plate were washed with phenol red-free DMEM medium, and then 500 μl of coumarin diluted as described above was added at 500 μl / well. After reacting at 37 ° C., the reaction solution was recovered from each well. The reaction solution was diluted 10-fold with 0.1 M Tris-HCl (PH 7.4), the fluorescence intensity at an excitation wavelength of 390 nm and a fluorescence wavelength of 440 nm was measured with a spectrofluorometer, and the produced 7-hydroxycoumarin was quantified. The standard substance for 7-hydroxycoumarin was purchased from Extrasynthese.
Hepe / 2A6L.9 strain was obtained as a CYP2A6 high expression strain from the obtained CYP2A6 activity expression strain.

(3) Measurement of CYP2B6-expressing cell activity and selection of highly active cells
7-Ethoxycoumarin (Molecular Probes) was diluted to 10 mM with DMSO (Wako Pure Chemical Industries). Next, this was diluted with phenol red-free DMEM medium (GIBCO BRL) containing 2% FCS (Bio Whittaker) to 500 μM.
CYP2B6-expressing cells were seeded in 12-well plates (Falcon) and cultured in a CO2 incubator until confluent. After culturing, the medium was aspirated, and the cells adhering to the plate were washed with phenol red-free DMEM medium, and 500 μM 7-ethoxycoumarin diluted as described above was added at 500 μl / well. After reacting at 37 ° C., the reaction solution was recovered from each well. Dilute the reaction solution 10-fold with 0.1M Tris-HCl (PH 7.4), and measure the fluorescence intensity at excitation wavelength 390nm and fluorescence wavelength 440nm with a spectrofluorometer (Hitachi spectrofluorometer F-2000). The 7-hydroxycoumarin was quantified. The standard substance for 7-hydroxycoumarin was purchased from Extrasynthese.
Among the obtained CYP2B6 activity expression strains, Hepc / 2B6.68 strain was obtained as a CYP2B6 high expression strain.

(4) Measurement of activity of cells expressing CYP2C8 and selection of cells expressing high activity Taxol (ULTRAFINE chemicals) was diluted to 10 mM with methanol (Wako Pure Chemical Industries). Next, this was diluted to 30 μM with phenol red-free DMEM medium (GIBCO BRL) containing 2% FCS (Bio Whittaker).
CYP2C8-expressing cells were seeded in a 12-well plate (Falcon) and cultured in a CO2 incubator until confluent. After culturing, the medium was aspirated, and the cells adhering to the plate were washed with phenol red-free DMEM medium, and then 30 μM taxol diluted as described above was added at 500 μl / well and reacted at 37 ° C. The reaction solution was collected from each well, an equal volume of acetonitrile (Wako Pure Chemical Industries) was added and mixed, and then insoluble materials were removed by centrifugation. The 6α-hydroxypaclitaxel produced in the reaction solution was quantified by HPLC.
As the column, Capcell Pak C18 AG120 (5 μm, 4.6 mmφ × 250 mm, Shiseido) was used. As the mobile phase, 40% acetonitrile (HPLC reagent, Wako Pure Chemical Industries) was used. 40 μl of the reaction solution was injected and eluted with the mobile phase shown above at a flow rate of 1.0 ml / mim and a column temperature of 40 ° C. Taxol and 6α-hydroxypaclitaxel were detected at 230 nm (absorbance). As standard substances, 10 μM taxol and 40 μl of 5 μM 6α-hydroxypaclitaxel (Gentest) were injected.
Among the obtained CYP2C8 activity expression strains, Hepc / 2C8.46 strain was obtained as a CYP2C8 high expression strain.

(5) Measurement of activity of cells expressing CYP2C9 and selection of cells expressing high activity Tolbutamide (Research Biochemicals International) was diluted to 50 mM with methanol (Wako Pure Chemical Industries). Next, this was diluted with phenol red-free DMEM medium (GIBCO BRL) containing 2% FCS (Bio Whittaker) to 500 μM.
CYP2C9-expressing cells were seeded in a 12-well plate (Falcon) and cultured in a CO ₂ incubator until confluent. After culturing, the medium was aspirated, and the cells adhering to the plate were washed with phenol red-free DMEM medium, and then 500 μl tolbutamide diluted as described above was added at 500 μl / well and reacted at 37 ° C. The reaction solution was collected from each well, an equal volume of acetonitrile (Wako Pure Chemical Industries) was added and mixed, and then insoluble materials were removed by centrifugation. Hydroxytolbutamide generated in the reaction solution was quantified by HPLC.
As the column, Inertsil ODS-2 (5 μm, 4.6 mmφ × 150 mm, GL Science) was used. As the mobile phase, a mixture of 10 mM Acetate Buffer (pH 4.3) and acetonitrile (HPLC reagent, Wako Pure Chemical Industries, Ltd.) at 72:28 v / v was used. 40 μl of the reaction solution was injected and eluted using the mobile phase shown above at a flow rate of 1.0 ml / mim and a column temperature of 40 ° C. Tolbutamide and hydroxytolbutamide were detected at 230 nm (absorbance). As standard substances, 100 μM tolbutamide and 10 μM hydroxytolbutamide (Sumitomo Chemical Analysis Center) 40 μl were injected.
Among the obtained CYP2C9 active expression strains, Hepc / 2C9.1 strain was obtained as a CYP2C9 high expression strain.

(6) Measurement of CYP2C19-expressing cell activity and selection of highly active cells
(S) Mephenytoin (Sumitomo Chemical Analysis Center) was diluted to 10 mM with methanol (Wako Pure Chemical Industries). Next, this was diluted with phenol red-free DMEM medium (GIBCO BRL) containing 2% FCS (Bio Whittaker) to 100 μM.
CYP2C19-expressing cells were seeded in a 12-well plate (Falcon) and cultured in a CO ₂ incubator until confluent. After culturing, the medium was aspirated, and the cells adhering to the plate were washed with phenol red-free DMEM medium, and then 100 μM (S) mephenytoin diluted as described above was added at 500 μl / well and reacted at 37 ° C. The reaction solution was collected from each well, an equal volume of acetonitrile (Wako Pure Chemical Industries) was added and mixed, and then insoluble materials were removed by centrifugation. This was quantified for 4′-hydroxymephenytoin generated in the reaction solution by HPLC.
As the column, Capcell Pak C18 AG120 (5 μm, 4.6 mmφ × 250 mm, Shiseido) was used. As the mobile phase, a mixture of 0.05M KH ₂ PO ₄ (pH 4.0) and acetonitrile (HPLC reagent, Wako Pure Chemical Industries) at 74:26 v / v was used. 40 μl of the reaction solution was injected and eluted with the mobile phase shown above at a flow rate of 0.8 ml / mim and a column temperature of 40 ° C. (S) Mephenytoin and 4′-hydroxymephenytoin were detected at 204 nm (absorbance). As standard substances, 50 μM (S) mephenytoin and 40 μl of 5 μM 4′-hydroxymephenytoin (Sumitomo Chemical Analysis Center) were injected.
Among the obtained CYP2C19 active expression strains, Hepc / 2C19.12 strain was obtained as a CYP2C19 high expression strain.

(7) Measurement of activity of CYP2D6-expressing cells and selection of high-activity expressing cells Bufuralol (Sumitomo Chemical Analysis Center) was diluted to 20 mM with distilled water. Next, this was diluted to 200 μM with phenol red-free DMEM medium (GIBCO BRL) containing 2% FCS (Bio Whittaker).
CYP2D6-expressing cells were seeded in a 12-well plate (Falcon) and cultured in a CO ₂ incubator until confluent. After culturing, the medium was aspirated, and the cells adhering to the plate were washed with phenol red-free DMEM medium, and then 200 μM bufuralol diluted as described above was added at 500 μl / well and reacted at 37 ° C. The reaction solution was collected from each well, and 1′-hydroxybufuralol produced in the reaction solution was quantified by HPLC.
As a column, Inertsil ODS (5 μm, 4.6 mmφ × 250 mm, GL Science) was used. As the mobile phase, a 30% acetonitrile solution (HPLC reagent, Wako Pure Chemicals) containing 1 mM perchloric acid (Wako Pure Chemicals) was used. The reaction solution was diluted 100-fold with distilled water, 40 μl thereof was injected, and eluted with the mobile phase shown above at a flow rate of 1.0 ml / mim and a column temperature of 50 ° C. Bufulalol and hydroxybufuralol were detected at an excitation wavelength of 252 nm and a fluorescence wavelength of 302 nm. As standard substances, 40 μl of 100 pM bufuralol and 10 pM 1′-hydroxybufuralol (Sumitomo Chemical Analysis Center) were injected.
Among the obtained CYP2D6 activity expression strains, Hepc / 2D6.39 strain was obtained as a CYP2D6 high expression strain.

(8) Measurement of CYP2E1-expressing cell activity and selection of highly active expressing cells Paranitrophenol (Wako Pure Chemical Industries) was diluted to 2 mM with DMSO (Wako Pure Chemical Industries). Next, this was diluted with phenol red-free DMEM medium (GIBCO BRL) containing 2% FCS (Bio Whittaker) to 500 μM.
2E1-expressing cells were seeded in a 12-well plate (Falcon) and cultured in a CO ₂ incubator until confluent. After culturing, the medium was aspirated and the cells adhering to the plate were washed with phenol red-free DMEM medium, and then 500 μl of paranitrophenol diluted as described above was added at 500 μl / well. After reacting at 37 ° C., the reaction solution was recovered from each well. 50 μl of 2N NaOH (Wako Pure Chemical Industries, Ltd.) was added to 100 μl of the reaction solution, and insoluble substances and the like were removed by centrifugation. Then, absorption at 540 nm to 620 nm was measured, and the produced 4-nitrocatechol was quantified. The standard substance of 4-nitrocatechol used was purchased from Wako Pure Chemical.
Among the obtained CYP2E1 activity expression strains, Hepc / 2E1.3-8 strain was obtained as a CYP2E1 high expression strain.

(9) Measurement of activity of cells expressing CYP3A4 and selection of cells expressing high activity Testosterone (Wako Pure Chemical Industries) was diluted to 10 mM with methanol (Wako Pure Chemical Industries). Next, this was diluted to 100 μM with a phenol red-free DMEM medium (GIBCO BRL) containing 2% FCS (Bio Whittaker).
CYP3A4-expressing cells were seeded in a 12-well plate (Falcon) and cultured in a CO ₂ incubator until confluent. After culturing, the medium was aspirated, and the cells adhering to the plate were washed with phenol red-free DMEM medium, and then 500 μl / well of 100 μM testosterone diluted as described above was added and reacted at 37 ° C. The reaction solution was collected from each well, an equal volume of acetonitrile (Wako Pure Chemical Industries) was added and mixed, and then insoluble materials were removed by centrifugation. The 6β-hydroxytestosterone produced in the reaction solution was quantified by HPLC.
The column used was Capcell Pak C18 AG120 (5 μm, 4.6 mmφ × 250 mm, Shiseido). As solution A, 40% methanol (HPLC reagent, Wako Pure Chemical Industries) and 3.5% acetonitrile solution (HPLC reagent, Wako Pure Chemical Industries) were used, and as solution B, 40% methanol and 20% acetonitrile solution were used. A program was prepared so that 0 to 100% linear gradient of solution B was obtained from 0 to 20 minutes, solution B was 100% from 20 to 30 minutes, and solution A was 100% thereafter. 40 μl of the reaction solution was injected and eluted with the mobile phase shown above at a flow rate of 1.0 ml / mim and a column temperature of 40 ° C. Testosterone and 6β-droxytestosterone were detected at 254 nm (absorbance). As standard substances, 50 μM testosterone and 40 μl of 5 μM 6β-hydroxytestosterone (Sumitomo Chemical Analysis Center) were injected.
Among the obtained CYP3A4 activity expression strains, Hepc / 3A4.5 strain was obtained as a CYP3A4 high expression strain.

Example 3 Kinetic analysis of a cytochrome P450 high expression strain Each cytochrome P450 high expression strain obtained in Example 2 was reacted with various concentrations of the substrate according to the enzyme activity assay method described in Example 2. Line Weber-Burk plot was taken, and Km value and Vmax value were obtained from the X-axis and Y-axis intercepts. The results are shown in Table 1.

Example 4 Metabolic activation of acetaminophen by CYP2E1-expressing cells and cytotoxicity expression experimental method MTT assay
HepG2 or CYP2E1 expressing cells (Hepc / 2E1.3-8) cells (4 x10 ⁵ cells / ml) is, CO ₂ with acetaminophen diluted to a predetermined concentration in DMEM medium containing 5% FCS (Wako Pure Chemical) Cultured in an incubator. In an experiment for stopping glutathione conjugation, L-Buthionine [S, R] -Sulfoximine (BSO, Sigma) was added thereto to a final concentration of 100 μM. 3- (4,5-Dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazolium bromide (MTT, Sigma) solution prepared to 1 mg / ml in PBS (Flow) after culturing for 4 days 20 μl was added and incubated at 37 ° C. for 3 hours. Next, 100 μl of 10% SDS, 0.01N HCl solution was added to each well and incubated overnight at 37 ° C., and then the absorbance at 590 nm was measured. The results are shown in FIG.
2. LDH (lactate dehydrogenase) leakage measurement
Two plates were prepared with the same plate design as when the MTT assay was performed (for measuring LDH activity in the culture supernatant and for measuring LDH activity in the culture supernatant + LDH activity in the cells). After culturing in a CO ₂ incubator for 3 days, collect 10 μl of culture supernatant from each well of one plate (for measuring LDH activity in the culture supernatant), add it to another 96-well plate, and then add distilled water. 40 μl was added. On the other hand, 10 μl of 10% Triton X100 (Wako Pure Chemical Industries) was added to each well of the other plate (for measuring LDH activity in culture supernatant + LDH activity in cells), and incubated at 37 ° C for 45 minutes. . After centrifugation at 1500 rpm for 5 minutes, 10 μl of culture supernatant was collected from each well and added to another 96-well plate, and 40 μl of distilled water was further added. The LDH activity of each well of these plates was measured with Cytotox 96 Non-Radioactive Cytotoxicity Assay Kit (Promega). Multiscan MS-UV was used to measure the absorbance. LDH activity in the culture supernatant / (LDH activity in the culture supernatant + LDH activity in the cells) was defined as the LDH leakage rate. The results are shown in FIG.
Results (Fig. 1, Fig. 2)
When a large amount of acetaminophen is present, depletion of active sulfuric acid, which is the rate-limiting factor of sulfate conjugation, occurs. Glucuronidation is large in dose but has a limited reaction rate, so N-hydroxylation occurs by P450. The active intermediate N acetylbenzoquinone imine formed from N hydroxide is usually detoxified by glutathione conjugation. However, when glutathione is depleted, this active intermediate is known to covalently bind to liver macromolecules and cause hepatocyte necrosis (MJJ Ronis et al., Cytochromes P450 Metabolic and Toxicological Aspects, pages 211-240, supervised) C. Ioannides et al. CRC Press 1996).
Acetaminophen slightly leaked LDH from HepG2 and Hepe / 2E1.3-8 cells. When BSO coexists here and intracellular glutathione is depleted, the sensitivity to acetaminophen increases about 4 times in Hepc / 2E1.3-8 cells, and LDH leaks at a lower concentration in a concentration-dependent manner. I let you. HepG2 showed no coexistence effect of BSO (Fig. 2). On the other hand, acetaminophen slightly decreased the MTT activity of HepG2 and Hepc / 2E1.3-8 cells in the MTT method. When BSO was allowed to coexist, the sensitivity to acetaminophen increased in Hepc / 2E1.3-8 cells, and a concentration-dependent decrease in MTT activity was observed at a lower concentration (FIG. 1). CYP2E1 activity expressed in Hepc / 2E1.3-8 cells metabolizes acetaminophen, but metabolites are detoxified by glutathione conjugation. However, when glutathione is depleted by acting BSO 3, it is considered that cytotoxicity is exerted by the resulting metabolic intermediate. Hepc / 2E1.3-8 cells are shown to have not only CYP2E1 activity but also glutathione transferase activity.

Example 5 Experimental method of metabolic activation of benzanthracene by CYP1A1-expressing cells and expression of cytotoxicity
CYP1A1-expressing cells (Hepc / 1A1.4) (4 x 10 ⁵ cells / ml) are mixed with benzanthracene (Sigma) diluted with DMEM medium containing 5% FCS to a predetermined concentration in a CO2 incubator with DMEM medium containing 5% FCS. Incubated inside. After culturing for 4 days, 20 μl of MTT (Sigma) solution adjusted to 1 mg / ml with PBS (Flow) was added to each well and incubated at 37 ° C. for 3 hours. Next, 100 μl of 10% SDS, 0.01N HCl solution was added to each well and incubated overnight at 37 ° C., and then the absorbance at 590 nm was measured. The results are shown in FIG.
Results (Figure 3)
Benzanthracene is metabolized by CYP1A1 activity, and the resulting metabolic intermediates are known to cause cytotoxicity, carcinogenicity, and mutagenesis (K. Kawajiri et al. Cytochromes P450 Metabolic and Toxicological Aspects 77-97, Supervised by C. Ioannides et al. CRC Press 1996). Compared with HepG2, the concentration of benzanthracene was strongly decreased in Hepc / 1A1.4 cells depending on the concentration of benzanthracene. Benzanthracene is metabolized by CYP1A1 activity expressed in Hepc / 1A1.4 cells, and cytotoxicity is exerted by the resulting metabolic intermediate.

Example 6 Metabolic activation of cyclophosphamide by CYP2B6-expressing cells and cytotoxicity expression experimental method MTT assay
CYP2B6-expressing cells (Hepc / 2B6.68) (4 x 10 ⁵ cells / ml) were prepared in DMEM medium containing 5% FCS together with cyclophosphamide (Sigma) diluted to the prescribed concentration in DMEM medium containing 5% FCS. Cultivation was performed in a CO ₂ incubator. After culturing for 5 days, 20 μl of MTT (Sigma) solution prepared to 1 mg / ml with PBS (Flow) was added to each well and incubated at 37 ° C. for 3 hours. Next, 100 μl of 10% SDS, 0.01N HCl solution was added to each well and incubated overnight at 37 ° C., and then the absorbance at 590 nm was measured. The results are shown in FIG.
2. Measurement of LDH leakage
Two plates were prepared with the same plate design as when the MTT assay was performed (for measuring LDH activity in the culture supernatant and for measuring LDH activity in the culture supernatant + LDH activity in the cells). After culturing in a CO ₂ incubator for 4 days, collect 10 μl of culture supernatant from each well of one plate (for measuring LDH activity in the culture supernatant), add it to another 96-well plate, and further distill 40 μl of water was added. Meanwhile, 10 μl of 10% Triton X100 was added to each well of the other plate (for measuring LDH activity in culture supernatant + LDH activity in cells), and incubated at 37 ° C. for 45 minutes. After centrifugation at 1500 rpm for 5 minutes, 10 μl of the culture supernatant was collected from each well and added to another 96-well plate, and 40 μl of distilled water was further added. The LDH activity of each well of these plates was measured with Cytotox 96 Non-Radioactive Cytotoxicity Assay Kit (Promega). Multiscan MS-UV was used to measure the absorbance. LDH activity in the culture supernatant / (LDH activity in the culture supernatant + LDH activity in the cells) was defined as the LDH leakage rate. The results are shown in FIG.
Results (Figs. 4 and 5)
Cyclophosphamide is hydroxylated at the 4-position, and then phosphoramide and acrolein produced by non-enzymatic degradation act as alkylating agents, which are triggered by macromolecular components and covalent bonds in hepatocytes. It is thought to cause injury (KHThomas et al. Cancer. Research 53: 5629-5637, 1993). Cyclophosphamide leaked LDH concentration-dependently at concentrations up to 2 mM in Hepc / 2B6.68 cells, reaching a plateau at concentrations thereafter. Cyclophosphamide was slightly cytotoxic in HepG2 (FIG. 4). On the other hand, the MTT method also showed a decrease in MTT activity depending on the concentration of cyclophosphamide in Hepc / 2B6.68 cells (FIG. 5). Cyphosphamide was metabolized by CYP2B6 activity expressed in Hepc / 2B6.68 cells, indicating cytotoxicity by the resulting metabolic intermediate.

Example 7 Experimental method for analysis of inhibition of CYP3A4 activity
Hepc / 3A4.5 cells were seeded in a 12-well microplate and cultured until confluent. After washing twice with phenol red-free DMEM medium, various concentrations of ketoconazole (Biomol Research Lab.) Diluted with phenol red-free DMEM medium containing 500 μl of 2% FCS were added and incubated at 37 ° C for 4 hours. did. After washing twice with phenol red-free DMEM medium, 500 μl of testosterone at a final concentration of 100 μM diluted with phenol red-free DMEM medium containing 2% FCS was added. Incubate at 37 ° C for 1 hour and collect the supernatant. A sample was prepared by adding and mixing an equal volume of acetonitrile and then removing the insoluble material by centrifugation. In the obtained sample, 6β-hydroxytestosterone was quantified using HPLC by the method shown in Example 9, (9).
The experimental results are shown as relative values with the amount of 6β-hydroxytestosterone produced when no drug was added as 100% (FIG. 6).
Ketoconazole is known as a strong CYP3A4 inhibitor (SJ Baldwin et al. Xenobiotica 25: 261-270 1995). Depending on the ketoconazole concentration, CYP3A4 activity (testosterone 6β-hydroxylation activity) in Hepc / 3A4.5 cells was inhibited (FIG. 6). Ketoconazole had an IC ₅₀ of 0.3 μM or less.

Example 8 Analytical experiment method for CYP2E1 activity induction
Hepc / 2E1.3-8 cells (5 x 10 ⁵ cells / ml) are mixed with ethanol and DMSO (dimethyl sulfoxide) (Wako Pure Chemical Industries) diluted to a predetermined concentration in DMEM medium containing 5% FCS. The cells were seeded and cultured for 3 days in a CO ₂ incubator. The medium was aspirated and the cells adhering to the plate were washed with phenol red-free DMEM medium, and 500 μM paranitrophenol was added at 500 μl / well. After reacting at 37 ° C., the reaction solution was recovered from each well. After adding 50 μl of 2N NaOH (Wako Pure Chemicals) to 100 μl of the reaction solution and removing the insoluble substances by centrifugation, the absorption at 540 nm to 620 nm was measured with Multiscan MS-UV (Lab Systems). Nitrocatechol was quantified.
Experimental results (Fig. 7)
It has been shown that the addition of ethanol and DMSO (dimethyl sulfoxide) increases enzyme activity and induces intracellular CYP2E1 (MJJ Ronis et al. Cytochromes P450 Metabolic and Toxicological Aspects pages 211-239, supervised by C. Ioannides et al. CRC Press 1996), this was also shown in this example.

Effect of the invention

  A cultured cell line derived from human liver cancer that stably expresses human cytochrome P450 of the present invention comprises (a) an enzyme involved in xenobiotic and / or endogenous substrate metabolism, and (b) xenobiotic and / or endogenous substrate. Metabolic pathway, (c) chemical structure of xenobiotics and / or endogenous substrate metabolites, (d) inhibition of xenobiotics and / or endogenous substrate metabolizing enzymes, (e) xenobiotics and / or endogenous substrate metabolizing enzymes (F) xenobiotic and / or endogenous substrate metabolism, (g) xenobiotic and / or endogenous substrate metabolism, genotoxicity, (h) xenobiotic and / or endogenous substrate metabolism Cancerous, (i) mutagenicity due to xenobiotics and / or endogenous substrate metabolism, (j) hepatotoxicity due to xenobiotics and / or endogenous substrate metabolism, or (k) xenobiotics acting on the liver and / or Analysis of endogenous substrates, biological differences And / or for the preparation of endogenous substrate metabolites.

The result of the MTT test regarding the cytotoxic expression of acetaminophen shown in Example 4 is shown. In the figure, − ◯ − represents HepG2, −Δ− represents Hepc / 2E1.3-8, − ● − represents HepG2 + 100 μM BSO, and − ▲ − represents Hepc / 2E1.3-8 + 100 μM BS0. The result of the LDH leakage test regarding the cytotoxic expression of acetaminophen shown in Example 4 is shown. In the figure, − ◯ − indicates HepG2, −Δ− indicates Hepc / 2E1.3-8, − ● − indicates HepG2 + 100 μM BSO, and − ▲ − indicates Hepc / 2E1.3-8 + 100 μM BSO. The result of the test regarding the cytotoxic expression of benzanthracene shown in Example 5 is shown. In the figure, -O- indicates HepG2, and-●-indicates Hepc / 1A1.4. The result of the MTT test regarding the cytotoxic expression of the cyclophosphamide shown in Example 6 is shown. In the figure, -O- indicates HepG2, and-●-indicates Hepc / 2B6.68. The result of the LDH leakage test regarding the cytotoxic expression of cyclophosphamide shown in Example 6 is shown. In the figure, -O- indicates HepG2, and-●-indicates Hepc / 2B6.68. The result of the test regarding CYP3A4 activity inhibition of ketoconazole shown in Example 7 is shown. In the figure,-□-indicates ketoconazole. The result of the test regarding CYP2E1 activity induction shown in Example 8 is shown.

Claims (4)

A combination of cultured cell lines derived from two or more human liver cancer cells that stably express human cytochrome P450,
The human cytochrome P450 is selected from the group consisting of CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4;
The human liver cancer cell is HepG2,
The cultured cell lines are Hepc / 1A1.4 (FERM BP-7125), Hepc / 1A2.9 (FERM BP-7124), Hepc / 2A6L.9 (FERM BP-7128), Hepc / 2B6.68 (FERM BP -7126), Hepc / 2C8.46 (FERM BP-7123), Hepc / 2C9.1 (FERM BP-7122), Hepc / 2C19.12 (FERM BP-7129), Hepc / 2D6.39 (FERM BP-7127 And a combination of cultured cell lines selected from the group consisting of Hepc / 3A4.5 (FERM BP-7120). A combination of cell lines according to claim 1, characterized in that (a) an enzyme involved in xenobiotic and / or endogenous substrate metabolism, (b) a xenobiotic and / or endogenous substrate metabolic pathway, (c) Chemical structure of xenobiotics and / or endogenous substrate metabolites, (d) inhibition of xenobiotics and / or endogenous substrate metabolizing enzymes, (e) promotion of activities of xenobiotics and / or endogenous substrate metabolizing enzymes, (f ) Cytotoxicity due to xenobiotics and / or endogenous substrate metabolism, (g) genotoxicity due to xenobiotics and / or endogenous substrate metabolism, (h) carcinogenicity due to xenobiotics and / or endogenous substrate metabolism, (i) Mutagenicity due to xenobiotics and / or endogenous substrate metabolism, (j) hepatotoxicity due to xenobiotics and / or endogenous substrate metabolism, or (k) methods for analyzing xenobiotics and / or endogenous substrates acting on the liver . A method for preparing xenobiotics and / or endogenous substrate metabolites, comprising using the combination of cell lines according to claim 1. (A) a substance that inhibits xenobiotics and / or endogenous substrate-metabolizing enzymes, and (b) an activity of xenobiotics and / or endogenous substrate-metabolizing enzymes characterized in that the cell line combination according to claim 1 is used. A substance that promotes, (c) a substance that exhibits cytotoxicity by xenobiotic and / or endogenous substrate metabolism, (d) a substance that exhibits genotoxicity by xenobiotic and / or endogenous substrate metabolism, (e) xenobiotic and / or Or substances that develop carcinogenicity by endogenous substrate metabolism, (f) xenobiotics and / or substances that express mutagenicity by endogenous substrate metabolism, (g) hepatotoxicity by xenobiotics and / or endogenous substrate metabolism Substances that express, (h) xenobiotics and / or endogenous substrates acting on the liver, or (i) xenobiotics and / or endogenous substrate metabolism, acquire new physiological activities or increase their own physiological activities Or a method of searching for material to be attenuated.

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