Title:  Human cell line showing stable expression of cytochromes P450

United States Patent:  6,756,229

Issued:  June 29, 2004

Inventors:  Nanba; Masayoshi (400-1, Syuku, Okayama-shi, Okayama, JP 700-0001); Asahi; Satoru (Toyonaka, JP); Yoshitomi; Sumie (Osaka, JP); Ikemoto; Keiko (Takarazuka, JP)

Assignee:  Takeda Chemical Industries, Ltd. (Osaka, JP); Nanba; Masayoshi (Okayama, JP)

Appl. No.:  009158

Filed:  October 25, 2001

PCT Filed:  April 27, 2000

PCT NO:  PCT/JP00/02763

PCT PUB.NO.:  WO00/65031

PCT PUB. Date:  November 2, 2000

Abstract

This invention relates to cell lines that are obtained using cultured cell lines derived from human liver as a host and that stably express a number of human cytochromes P450. The human liver-derived cultured cell lines of the present invention are useful in, for example, analyzing an enzyme participating in the metabolism of xenobiotics or endogenous substrates, because of their stable expression of human cytochromes P450 CYP1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2C9, 2D6, and 3A4.

DISCLOSURE OF THE INVENTION

An object of this invention is to provide a cultured cell line derived from human liver, thereby to separate and produce the cell line that can stably express human cytochromes P450 CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4.

These cells enable a practitioner of the invention to (1) analyze an enzyme participating in the metabolism of xenobiotics and/or endogenous substrates, (2) analyze a metabolic pathway of xenobiotics and/or endogenous substrates, (3) analyze a chemical structure of the metabolite of xenobiotics and/or endogenous substrates, (4) prepare the metabolite of xenobiotics and/or endogenous substrates, (5) analyze inhibition of the metabolizing enzyme for xenobiotics and/or endogenous substrates, (6) analyze an accelerated activity of the metabolizing enzyme for xenobiotics and/or endogenous substrates, (7) analyze expression of cytotoxicity by the metabolism of xenobiotics and/or endogenous substrates, (8) analyze expression of genotoxicity by the metabolism of xenobiotics and/or endogenous substrates, (9) analyze expression of carcinogenicity by the metabolism of xenobiotics and/or endogenous substrates, (10) analyze mutagenicity by the metabolism of xenobiotics and/or endogenous substrates, (11) analyze the expression of hepatotoxicity by the metabolism of xenobiotics and/or endogenous substrates, and (12) analyze xenobiotics and/or endogenous substrates that act on the liver. The cells further enable a practitioner of the invention to (1) screen a substance capable of inhibiting xenobiotics and/or endogenous substrates, (2) screen a substance capable of accelerating the activity of metabolizing enzymes for xenobiotics and/or endogenous substrates, (3) screen a substance capable of expressing cytotoxicity by the metabolism of xenobiotics and/or endogenous substrates, (4) screen a substance capable of expressing genotoxicity by the metabolism of xenobiotics and/or endogenous substrates, (5) screen a substance capable of expressing carcinogenicity by the metabolism of xenobiotics and/or endogenous substrates, (6) screen a substance capable of expressing mutagenicity by the metabolism of xenobiotics and/or endogenous substrates, (7) screen a substance capable of expressing hepatotoxicity by the metabolism of xenobiotics and/or endogenous substrates, (8) screen xenobiotics and/or endogenous substrates which act on the liver, and (9) screen a substance capable of acquiring a new physiological activity or increasing or decreasing the inherent physiological activity, through the metabolism of xenobiotics and/or endogenous substrates. Thus, particular compounds or salts thereof, etc. can be obtained, using the method for analysis and/or the method for screening.

In view of the foregoing problems, the present inventors have made extensive studies. As a result, they have established stable transformants capable of stably expressing cytochromes P450 in a human hepatocarcinoma-derived (or hepatic carcinoma-derived) cell line with an enhanced activity for participation in the metabolism of xenobiotics and/or endogenous substrates. The following further studies have resulted in accomplishing this invention.

That is, the present invention relates to the following features.

(1) A cell line derived from human hepatic carcinoma capable of stably expressing human cytochromes P450.

(2) The cell line according to (1), wherein human cytochromes P450 are capable of stably expressing CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 or CYP3A4.

(3) The cultured cell line according to (1), wherein the human hepatic carcinoma cell is HepG2.

(4) The cell line according to (1), which is 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.

(5) A method for analyzing (a) an enzyme participating in the metabolism of a xenobiotic and/or an endogenous substrate, (b) a metabolic pathway of a xenobiotic and/or an endogenous substrate, (c) a chemical structure of the metabolite of a xenobiotic and/or an endogenous substrate, (d) inhibition of the metabolizing enzyme for a xenobiotic and/or an endogenous substrate, (e) an accelerated activity of the metabolizing enzyme for a xenobiotic and/or an endogenous substrate. (f) cytotoxicity by the metabolism of a xenobiotic and/or an endogenous substrate. (g) genotoxicity by the metabolism of a xenobiotic and/or an endogenous substrate, (h) carcinogenicity by the metabolism of a xenobiotic and/or endogenous substrate, (i) mutagenicity by the metabolism of a xenobiotic and/or an endogenous substrate, (j) hepatotoxicity by the metabolism of a xenobiotic and/or an endogenous substrate, or (k) a xenobiotic and/or an endogenous substrate that acts on the liver.

(6) A method for preparing the metabolite of a xenobiotic and/or an endogenous substrate, which comprises using the cell line according to (1).

(7) A method for screening a substance which comprises using the cell line according to (1), wherein the substance is (a) a substance capable of inhibiting a xenobiotic and/or an endogenous substrate, (b) a substance capable of accelerating an activity of the metabolizing enzyme for a xenobiotic and/or an endogenous substrate, (c) a substance capable of expressing cytotoxicity by the metabolism of a xenobiotic and/or an endogenous substrate, (d) a substance capable of expressing genotoxicity by the metabolism of a xenobiotic and/or an endogenous substrate, (e) a substance capable of expressing carcinogenicity by the metabolism of a xenobiotic and/or an endogenous substrate, (f) a substance capable of expressing mutagenicity by the metabolism of a xenobiotic and/or an endogenous substrate, (g) a substance capable of expressing hepatotoxicity by the metabolism of a xenobiotic and/or an endogenous substrate, (h) a xenobiotic and/or an endogenous substrate which acts on the liver, or (i) 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.

(8) A compound or its salt which is obtainable using the method according to (7).

(9) A pharmaceutical composition comprising the compound or its salt according to (8).

(10) A method for analysis which comprises using at least two cultured cell lines derived from human liver capable of stably expressing at least one of CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4, said analysis being for (a) an enzyme that participates in the metabolism of a xenobiotic and/or an endogenous substrate, (b) a metabolic pathway of a xenobiotic and/or an endogenous substrate, (c) a chemical structure of the metabolite of a xenobiotic and/or an endogenous substrate, (d) inhibition of the metabolizing enzyme for a xenobiotic and/or an endogenous substrate, (e) an accelerated activity of the metabolizing enzyme for a xenobiotic and/or an endogenous substrate, (f) cytotoxicity by the metabolism of a xenobiotic and/or an endogenous substrate, (g) genotoxicity by the metabolism of a xenobiotic and/or an endogenous substrate, (h) carcinogenicity by the metabolism of a xenobiotic and/or endogenous substrate, (i) mutagenicity by the metabolism of a xenobiotic and/or an endogenous substrate, (j) hepatotoxicity by the metabolism of a xenobiotic and/or an endogenous substrate, or (k) a xenobiotic and/or an endogenous substrate that acts on the liver.

(11) A method for preparation of the metabolite of a xenobiotic and/or an endogenous substrate, which comprises using at least two cultured cell lines from human liver capable of stably expressing at least one of CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4.

(12) A method for screening a substance which comprises using at least two cultured cell lines from human liver capable of stably expressing at least one of CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4, said substance being (a) a substance capable of inhibiting the metabolizing enzyme for a xenobiotic and/or an endogenous substrate, (b) a substance capable of accelerating an activity of the metabolizing enzyme for a xenobiotic and/or an endogenous substrate, (c) a substance capable of expressing cytotoxicity by the metabolism of a xenobiotic and/or an endogenous substrate, (d) a substance capable of expressing genotoxicity by the metabolism of a xenobiotic and/or an endogenous substrate, (e) a substance capable of expressing carcinogenicity by the metabolism of a xenobiotic and/or an endogenous substrate, (f) a substance capable of expressing mutagenicity by the metabolism of a xenobiotic and/or an endogenous substrate, (g) a substance capable of expressing hepatotoxicity by the metabolism of a xenobiotic and/or an endogenous substrate, (h) a xenobiotic and/or an endogenous substrate which acts on the liver, or (i) 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.

(13) A compound or a salt thereof, which is obtainable using the method according to (12).

(14) A pharmaceutical compound comprising a compound or a salt thereof according to (13).

BEST MODE OF EMBODIMENT OF THE INVENTION

Throughout the specification, the term "xenobiotics or foreign matters in vivo" collectively refers to, e.g., medicaments, food additives, environmental pollutants, chemicals in general, etc. and the term "endogenous substrates" refers to all substances present in vivo. For the metabolism of xenobiotics, among others, medicaments or drugs as the main component, drug metabolism is preferably used.

The human hepatic carcinoma cells used can be collected by separating a human hepatic carcinoma-derived cultured cell line (preferably HepG2) from human hepatic carcinoma. Genes that encode various species of cytochromes P450 separately isolated are stably expressed in the human hepatic carcinoma cells.

In order to stably express DNA fragments encoding cytochromes P450, first, DNA fragments encoding, e.g., individual cytochromes P450 are obtained and placed under control of a foreign promoter for expression. The base sequences of DNA fragments encoding cytochromes P450 are available from public database. Based on the base sequences, a cytochromes P450-encoding DNA fragment can be isolated by publicly known methods including PCR, hybridization screening, etc. The DNA fragment thus obtained is inserted into a vector which produces transformants capable of stably expressing a foreign gene in a cultured mammalian cell, whereby a vector for transformation is produced. The resulting vector is transfected into hepatic carcinoma cells by publicly known methods. Transformants are selected by examining the enzyme activity induced by the expression of cytochromes P450 transformed therein, in order to select excellent clones. In addition, clones obtained can be confirmed with stability of their properties through repeated frozen storage.

Examples of the foreign promoter include SRa promoter, SV40 promoter, LTR promoter, CMV promoter and HSV-TK promoter.

The term "stably expressing human cytochromes P450" is used to mean that the expression of human cytochromes P450 is not transient and specifically, the activity of cytochromes P450 is not lost when cells are cultured (subcultured). The cells capable of expressing human cytochromes P450 are preferably cells in which not only cytochromes P450 but also enzymes associated with various aspects of metabolism (specifically, UDP-glucuronosyltransferase, sulfotransferase, glutathione transferase, epoxy hydratase, N-acetyltransferase, flavin monooxygenase, etc.) are capable of functioning.

Examples of the cytochromes P450 molecular species which participate in the metabolism of xenobiotics and/or endogenous substrates in liver include CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4. These enzymes not only catalyze the metabolism of xenobiotics and/or endogenous substrates but also induce, depending upon properties of their metabolites, the inhibition of metabolizing enzymes for xenobiotics and/or endogenous substrates, the acceleration of the activity of metabolizing enzymes for xenobiotics and/or endogenous substrates, the expression of cytotoxicity by the metabolism of xenobiotics and/or endogenous substrates, the expression of genotoxicity by the metabolism of xenobiotics and/or endogenous substrates, the expression of carcinogenicity by the metabolism of xenobiotics and/or endogenous substrates, the expression of mutagenicity by the metabolism of xenobiotics and/or endogenous substrates, the expression of hepatotoxicity by the metabolism of xenobiotics and/or endogenous substrates, and so on. However, these functions associated with the metabolism of xenobiotics and/or endogenous substrates are not only catalyzed by cytochromes P450 alone but are dependent on concerted actions with various enzymes such as UDP-glucuronosyltransferase, sulfotransferase, glutathione transferase, epoxy hydratase, N-acetyltransferase, flavin monooxygenase and cytochromes P450 reductase.

For reproducing the function of the liver by expressing cytochromes P450, the cells should thus be those capable of functioning at least, human-derived UDP-glucuronosyltransferase, sulfotransferase, glutathione transferase, epoxy hydratase, N-acetyltransferase or flavin monooxygenase in the cells. One of such cells is cultured cell HepG2 originating from human hepatic carcinoma. The HepG2 cell is known to be capable of functioning UDP-glucuronosyltransferase, sulfotransferase, glutathione transferase, epoxy hydratase, N-acetyltransferase, flavin monooxygenase and NADPH P450 reductase function in HepG2 (J. Rueffet al., Mutation Research, 353, 151-176 (1996). In light of the foregoing, the present inventors have succeeded in stably expressing cytochromes P450 in HepG2 in order to reproduce the function of human liver in a rapid, inexpensive, safe and accurate fashion.

In particular, preferred embodiments include 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, etc.

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 and Hepc/2C19.12 are highly active cells obtained by the expression of CYP3A4, CYP2E1, CYP2C9, CYP2C8, CYP1A2, CYP1A1, CYP2B6, CYP2D6, CYP2A6 and CYP2C19, respectively.

The present invention further relates to a method, which comprises using the aforesaid human hepatic carcinoma-derived cultured cell line capable of stably expressing human cytochromes P450, including (a) a method for analyzing an enzyme participating in the metabolism of a xenobiotic and/or an endogenous substrate, (b) a method for analyzing a metabolic pathway of a xenobiotic and/or an endogenous substrate, (c) a method for analyzing a chemical structure of the metabolite of a xenobiotic and/or an endogenous substrate, (d) a method for preparing the metabolite of a xenobiotic and/or an endogenous substrate, (e) a method for analyzing inhibition of the metabolizing enzyme for a xenobiotic and/or an endogenous substrate, (f) a method for analyzing an accelerated activity of the metabolizing enzyme for a xenobiotic and/or an endogenous substrate, (g) a method for analyzing expression of cytotoxicity by the metabolism of a xenobiotic and/or an endogenous substrate, (h) a method for analyzing expression of genotoxicity by the metabolism of a xenobiotic and/or an endogenous substrate, (i) a method for analyzing expression of carcinogenicity by the metabolism of a xenobiotic and/or endogenous substrate, (j) a method for analyzing mutagenicity by the metabolism of a xenobiotic and/or an endogenous substrate, (k) a method for analyzing expression of hepatotoxicity by the metabolism of a xenobiotic and/or an endogenous substrate, and (l) a method for analyzing a xenobiotic and/or an endogenous substrate that acts on the liver.

The respective methods described in (a) through (1) are described below.

(a) Method for Analyzing an Enzyme Participating in the Metabolism of Xenobiotics and/or Endogenous Substrates:

By analyzing a change in the structure of xenobiotics and/or endogenous substrates through exposure of a test specimen, e.g., to the human hepatic carcinoma-derived cultured cell line capable of stably expressing cytochromes P450, enzymes that participate in the metabolism of xenobiotics and/or endogenous substrates can be analyzed (J. L. Napoli et al., Methods in Enzymology, vol. 206, pp. 491-501, Ed. by M. R. Waterman et al., Academic Press, 1991; H. K. Kroemer et al., Methods in Enzymology, vol. 272, pp. 99-108, Ed. by M. R. Waterman et al., Academic Press, 1996). Specific examples include identification of an enzyme participating the metabolism of xenobiotics and/or endogenous substrates by analyzing a change in the structure of xenobiotics and/or endogenous substrates upon exposure of a test specimen to the human hepatic carcinoma-derived cultured cell line capable of stably expressing cytochromes P450. Other specific examples include analysis of the mechanism in an enzymatic reaction by analyzing a change in the structure of xenobiotics and/or endogenous substrates upon exposure of a test specimen of interest to the cell, and analysis of substrate specificity.

Examples of the test specimen to be tested include a peptide, a protein, a non-peptidic compound, a synthetic compound, a fermentation product, a cell extract, a plant extract, an animal tissue extract, plasma, etc. These substances may be either novel or publicly known ones.

(b) Method for Analyzing a Metabolic Pathway of Xenobiotics and/or Endogenous Substrates:

By analyzing a change in the structure of xenobiotics and/or endogenous substrates through exposure of a test specimen, e.g., to the human hepatic carcinoma-derived cultured cell line capable of stably expressing cytochromes P450, the metabolic pathway of xenobiotics and/or endogenous substrates can be analyzed (J. L. Napoli et al., Methods in Enzymology, vol. 206, pp. 491-501, Ed. by M. R. Waterman et al., Academic Press, 1991; H. K. Kroemeret al., Methods in Enzymology, vol. 272, pp. 99-108, Ed. by M. R. Waterman et al., Academic Press, 1996).

The examples of the test specimen given above apply also to the specimen to be tested here.

(c) Method for Analyzing a Chemical Structure of the Metabolite of Xenobiotics and/or Endogenous Substrates:

By analyzing a change in the structure of xenobiotics and/or endogenous substrates caused upon exposure of a test specimen. e.g., to the human hepatic carcinoma-derived cultured cell line capable of stably expressing cytochromes P450, the chemical structure of the metabolite of xenobiotics and/or endogenous substrates can be analyzed (J. L. Napoli et al., Methods in Enzymology, vol. 206, pp. 491-501, Ed. by M. R. Waterman et al., Academic Press, 1991; H. K. Kroemer et al., Methods in Enzymology, vol. 272, pp. 99-108, Ed. by M. R. Waterman et al., Academic Press, 1996).

The examples of the test specimen given above apply also to the specimen to be tested here.

(d) Method for Preparing the Metabolite of Xenobiotics and/or Endogenous Substrates:

By collecting the altered product (the so-called metabolite) from xenobiotics and/or endogenous substrates produced as a result of exposing a test specimen, e.g., to the human hepatic carcinoma-derived cultured cell line capable of stably expressing cytochromes P450 and purifying and isolating the product in an appropriate manner, the metabolite of xenobiotics and/or endogenous substrates can be prepared (J. L. Napoli et al., Methods in Enzymology, vol. 206, pp. 491-501, Ed. by M. R. Waterman et al., Academic Press, 1991).

The examples of the test specimen given above apply also to the specimen to be tested here.

(e) Method for Analyzing Inhibition of the Metabolizing Enzyme for Xenobiotics and/or Endogenous Substrates:

By exposing a test specimen, e.g., to the human hepatic carcinoma-derived cultured cell line capable of stably expressing cytochromes P450, the inhibition of a metabolizing enzyme for xenobiotics and/or endogenous substrates can be analyzed (J. L. Napoli et al., Methods in Enzymology, vol. 206, pp. 491-501, Ed. by M. R. Waterman et al., Academic Press, 1991). Specifically, the inhibition can be detected by the inhibition of cytochromes P450 enzyme activity, reduction in the amount of protein, decreased mRNA, etc. The detection may be made using publicly known methods, including an assay for enzyme activity corresponding to the respective members of P450, Western blotting corresponding to the respective P450 proteins, Northern hybridization corresponding to various P450 mRNAs, RT-PCR, etc.

The examples of the test specimen given above apply also to the specimen to be tested here.

(f) Method for Analyzing an Accelerated Activity of the Metabolizing Enzyme for Xenobiotics and/or Endogenous Substrates:

By exposing a test specimen, e.g., to the human hepatic carcinoma-derived cultured cell line capable of stably expressing cytochromes P450 and detecting the increased enzyme activity in the metabolism of xenobiotics and/or endogenous substrates, the increased amount of the enzyme or the increased amount of transcription in gene encoding the enzyme, the accelerated activity of the metabolizing enzyme for xenobiotics and/or endogenous substrates can be analyzed (J. Rueffet al., Mutation Research, 353 (1996) 151-176). Specifically, the accelerated activity can be analyzed by detecting the increased enzyme activity of cytochromes P450, the increased amount of protein or the increased mRNA. The detection may be made using publicly known methods, including Western blotting corresponding to the respective P450 proteins, Northern hybridization corresponding to various P450 mRNAs, RT-PCR, etc.

The examples of the test specimen given above apply also to the specimen to be tested here.

(g) Method for Analyzing Cytotoxicity by the Metabolism of Xenobiotics and/or Endogenous Substrates:

By exposing a test specimen, e.g., to the human hepatic carcinoma-derived cultured cell line capable of stably expressing cytochromes P450, the cytotoxicity caused by the metabolism of xenobiotics and/or endogenous substrates can be analyzed. Specifically, the cytotoxicity can be analyzed by observing a morphological change of the cell caused upon exposure of a test specimen; a change in viable cell count determined by publicly known methods including the MTT assay, Trypan Blue staining, Crystal Blue staining, etc.; leakage of intracellular enzyme such as lactose dehydrogenase; a change in structure of cells in the top layer; a change in intracellular enzyme, etc. (D. Wu, et al., Journal of Biological Chemistry, 271 (1996), 23914-23919).

The examples of the test specimen given above apply also to the specimen to be tested here.

(h) Method for Analyzing Expression of Genotoxicity by the Metabolism of Xenobiotics and/or Endogenous Substrates:

By exposing a test specimen, e.g., to the human hepatic carcinoma-derived cultured cell line capable of stably expressing cytochromes P450 and then subjecting the cells to the chromosomal aberration test or the micronucleus test, the genotoxicity caused by the metabolism of xenobiotics and/or endogenous substrates can be analyzed. The genotoxicity can also be analyzed by exposing a test specimen to the human hepatic carcinoma-derived cultured cell line capable of stably expressing cytochromes P450 and then subjecting the cells to the chromosomal aberration test, to the micronucleus test or to the reverse mutation test. This involves assessment of the test specimen altered by the cells in an appropriate assessment system (J. Rueffet al., Mutation Research, 353 (1996) 151-176; M. E. McManus et al., Methods in Enzymology, vol. 206, pp. 501-508, Ed by M. R. Waterman et al., Academic Press, 1991).

The examples of the test specimen given above apply also to the specimen to be tested here.

(i) Method for Analyzing Carcinogenicity by the Metabolism of a Xenobiotic and/or Endogenous Substrate:

By exposing a test specimen, e.g., to the human hepatic carcinoma-derived cultured cell line capable of stably expressing cytochromes P450 and then subjecting the cells to the chromosomal aberration test or to DNA modification, the carcinogenicity caused by the metabolism of xenobiotics and/or endogenous substrates can be analyzed. The carcinogenicity can also be analyzed by exposing a test specimen to the human hepatic carcinoma-derived cultured cell line capable of stably expressing cytochromes P450, followed by assessment of the test specimen altered by the cells in an appropriate system for evaluating carcinogenesis (J. Rueffet al., Mutation Research, 353 (1996) 151-176; K. Kawajiri, et al., Cytochromes, P450, Metabolic and Toxicological Aspects, pp. 77-98, ed. by C. Ioannides, CRC Press (1996)).

The examples of the test specimen given above apply also to the specimen to be tested here.

(j) Method for Analyzing Mutagenicity by the Metabolism of Xenobiotics and/or Endogenous Substrates:

By exposing a test specimen, e.g., to the human hepatic carcinoma-derived cultured cell line capable of stably expressing cytochromes P450 and then subjecting the cells to the chromosomal aberration test or the micronucleus test, the mutagenicity by the metabolism of xenobiotics and/or endogenous substrates can be analyzed. The mutagenicity can also be analyzed by exposing a test specimen to the human hepatic carcinoma-derived cultured cell line capable of stably expressing cytochromes P450 and then subjecting the cells to the chromosomal aberration test, the micronucleus test or the reverse mutation test, which involves assessment of the test specimen altered by the cells in an appropriate assessment system (J. Rueff et al., Mutation Research, 353 (1996) 151-176).

The examples of the test specimen given above apply also to the specimen to be tested here.

(k) Method for Analyzing Hepatotoxicity by the Metabolism of Xenobiotics and/or Endogenous Substrates:

The hepatotoxicity by the metabolism of xenobiotics and/or endogenous substrates can be analyzed by exposing a test specimen, e.g., to the human hepatic carcinoma-derived cultured cell line capable of stably expressing cytochromes P450 and then observing the expression of cytotoxicity. Alternatively, it can be analyzed by exposing a test specimen to the human hepatic carcinoma-derived cultured cell line capable of stably expressing cytochromes P450, administering the test specimen altered by the cells to other liver cells, liver slices or removed liver or to an experimental animal and then observing a change in cell or tissue or an in vivo change caused thereby.

The examples of the test specimen given above apply also to the specimen to be tested here.

(l) Method for Analyzing Xenobiotics and/or Endogenous Substrates that Act on the Liver:

The expression of the action on the liver can be analyzed either by exposing a test specimen, e.g., to the human hepatic carcinoma-derived cultured cell line capable of stably expressing cytochromes P450 and then observing the expression of a change in the cells caused, or by exposing a test specimen to the human hepatic carcinoma-derived cultured cell line capable of stably expressing cytochromes P450, administering the test specimen altered by the cells to other liver cells, liver slices or removed liver or to experimental animal and then observing a change in cell or tissue or an in vivo change caused thereby.

The examples of the test specimen given above apply also to the specimen to be tested here.

Furthermore, the present invention provides a method for screening a substance, which comprises using the human hepatic carcinoma-derived cultured cell line capable of stably expressing cytochromes P450, as well as a compound or its salt obtainable by the screening method, wherein the substance to be screened is (A) a substance capable of inhibiting the metabolizing enzyme for xenobiotics and/or endogenous substrates, (B) a substance capable of accelerating an activity of the metabolizing enzyme for xenobiotics and/or endogenous substrates, (C) a substance capable of expressing cytotoxicity by the metabolism of xenobiotics and/or endogenous substrates, (D) a substance capable of expressing genotoxicity by the metabolism of xenobiotics and/or endogenous substrates, (E) a substance capable of expressing carcinogenicity by the metabolism of xenobiotics and/or endogenous substrates, (F) a substance capable of expressing mutagenicity by the metabolism of xenobiotics and/or endogenous substrates, (G) a substance capable of expressing hepatotoxicity by the metabolism of xenobiotics and/or endogenous substrates, (H) xenobiotics and/or endogenous substrates which act on the liver, or (I) a substance capable of acquiring a new physiological activity or increasing or decreasing the inherent physiological activity, through the metabolism of xenobiotics and/or endogenous substrates.

(A) According to the method for screening a substance capable of inhibiting the metabolizing enzyme for xenobiotics and/or endogenous substrates, the inhibition is analyzed by the method described in (e) above for analyzing the inhibition of a metabolizing enzyme for xenobiotics and/or endogenous substrates, in which such a substance that inhibits the enzyme activity of cytochromes P450, reduces the amount of protein or reduces mRNA can be selected as the substance capable of inhibiting the enzyme activity for xenobiotics and/or endogenous substrates.

(B) According to the method for screening a substance capable of accelerating an activity of the metabolizing enzyme for xenobiotics and/or endogenous substrates, the acceleration is analyzed by the method described in (f) above for analyzing the acceleration of a metabolizing enzyme for xenobiotics and/or endogenous substrates, in which such a substance that accelerates the enzyme activity of cytochromes P450, increases the amount of protein or increases mRNA can be selected as the substance capable of inhibiting the enzyme activity for xenobiotics and/or endogenous substrates.

(C) According to the method for screening a substance capable of expressing cytotoxicity by the metabolism of xenobiotics and/or endogenous substrates, the cytotoxicity is analyzed by the method described in (g) above for analyzing the cytotoxicity by the metabolism of xenobiotics and/or endogenous substrates, in which such a substance that causes, e.g. a morphological change of the cell upon exposure of a test specimen, a change in viable cell count, leakage of intracellular enzyme, a change in structure of cells in the top layer, a change In intracellular enzyme. etc. can be selected as the substance capable of expressing the cytotoxicity by the metabolism of xenobiotics and/or endogenous substrates.

(D) According to the method for screening a substance capable of expressing genotoxicity by the metabolism of xenobiotics and/or endogenous substrates, the genotoxicity can be analyzed by the method described in (h) above for analyzing the genotoxicity by the metabolism of xenobiotics and/or endogenous substrates, in which such a test specimen that causes genotoxicity through the metabolism of xenobiotics and/or endogenous substrates by subjecting to, e.g., the chromosomal aberration test or the micronucleus test can be selected as a substance capable of expressing the genotoxicity by the metabolism of xenobiotics and/or endogenous substrates.

(E) According to the method for screening a substance capable of expressing carcinogenicity by the metabolism of xenobiotics and/or endogenous substrates, the carcinogenicity can be analyzed by the method described in (i) above for analyzing the carcinogenicity by the metabolism of xenobiotics and/or endogenous substrates, in which such a test specimen that causes carcinogenicity through the metabolism of xenobiotics and/or endogenous substrates by subjecting to, e.g., the chromosomal aberration test or the DNA modification can be selected as a substance capable of expressing the carcinogenicity by the metabolism of xenobiotics and/or endogenous substrates.

(F) According to the method for screening a substance capable of expressing mutagenicity by the metabolism of xenobiotics and/or endogenous substrates, the mutagenicity can be analyzed by the method described in (j) above for analyzing the mutagenicity by the metabolism of xenobiotics and/or endogenous substrates, in which such a is test specimen that causes mutagenicity through the metabolism of xenobiotics and/or endogenous substrates by subjecting to, e.g., the chromosomal aberration test or the micronucleus test can be selected as a substance capable of expressing the mutagenicity by the metabolism of xenobiotics and/or endogenous substrates.

(G) According to the method for screening a substance capable of expressing hepatotoxicity by the metabolism of xenobiotics and/or endogenous substrates, the hepatotoxicity can be analyzed following the method described in (k) above for analyzing the hepatotoxicity by the metabolism of xenobiotics and/or endogenous substrates, which involves, e.g., exposing a test specimen to cells, administering the test specimen altered by the cells to other liver cells, liver slices, removed liver or experimental animal, and observing a change in cells or tissues or a change in vivo. Such a test specimen that causes hepatotoxicity through the metabolism of xenobiotics and/or endogenous substrates can be selected as a substance capable of expressing the hepatotoxicity by the metabolism of xenobiotics and/or endogenous substrates.

(H) According to the method for screening xenobiotics and/or endogenous substrates which act on the liver, substances are analyzed by the method described in (l) above for analyzing xenobiotics and/or endogenous substrates which act on the liver, which involves, e.g., exposing a test specimen to a cell, administering the test specimen altered by the cell to other liver cells, liver slices, removed liver or experimental animal and then observing a change in cells or tissue or a change in vivo thereby. Thus, the xenobiotics and/or endogenous substrates which act on the liver can be screened.

(I) According to the method for screening a substance capable of acquiring a new physiological activity or increasing or decreasing the inherent physiological activity (including a so-called prodrug) through the metabolism of xenobiotics and/or endogenous substrates, the screening can be effected following the method described in (c) above for analyzing the chemical structure of the metabolite of xenobiotics and/or endogenous substrates and observing the physiological activity of the metabolite.

The compound or its salt that is obtainable by the screening methods described in (A) through (I) above is the compound or its salt selected from the test specimens that cause the activities, properties, etc. described above. Since these compounds are effective for the treatment and prevention of diseases (e.g., hepatic dysfunction) associated with metabolic aberration of xenobiotics in the liver, these compounds may be used as safe and low toxic pharmaceutical composition for the treatment and prevention of such diseases.

The compounds obtained by the screening methods above may be in the form of salts. As such salts of the compounds, there are salts with physiologically acceptable acids (e.g. inorganic acids or organic acids) or bases (e.g., alkaline metals), with physiologically acceptable acid additions salts being particularly preferred. Examples of such salts include salts with inorganic acids (e.g., hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) and salts with organic acids (e.g., acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid).

Pharmaceutical compositions comprising the compounds or salts thereof obtained by the screening methods may be manufactured by publicly known methods or those similar thereto. Since the pharmaceutical compositions thus obtained are safe and low toxic, the compositions can be administered, e.g., to human or another mammal (e.g., rat, mouse, guineapig, rabbit, sheep, swine, bovine, horse, cat, dog, monkey, etc.).

The dose of the compound or its salts may vary depending on target disease, subject to be administered, routes for administration, etc.; for the purpose of treating, e.g., hepatic dysfunction by oral administration of the compound, the dose is normally about 0.1 to about 100 mg, preferably about 1.0 to about 50 mg, more preferably about 1.0 to about 20 mg per day for adult (as 60 kg body weight). In parenteral administration, the single dose varies depending on subject to be administered, target disease, etc.; when administered in the form of an injection for the purpose of treating, e.g., hepatic dysfunction, it is advantageous to administer the compound intravenously at a daily dose of about 0.01 to about 30 mg, preferably about 0.1 to about 20 mg, more preferably about 0.1 to about 10 mg for adult (as 60 kg body weight). For other animal species, the corresponding dose as converted per 60 kg weight can be administered.

Specific examples of pharmaceutical preparations available for the form given above include tablets (including sugar coated tablets and film coated tablets), pills, capsules (including microcapsules), granules, fine granules, powders, syrup, emulsion, suspension, injections, inhalation, ointments, etc. These pharmaceutical preparations are prepared in a conventional manner (e.g., following the methods described in the Japanese Pharmacopoeia).

In the pharmaceutical preparation above, the amount of the compound or its salt obtained by the screening methods above may vary depending on the form of preparation but in general, is within a range of 0.01 to 100 wt %, preferably 0.1 to 50 wt %, more preferably 0.5 to 20 wt %. based on the total weight of the pharmaceutical preparation.

Specifically, tablets may be manufactured by subjecting drugs directly to compression molding, or by adding to the drugs an excipient, a binder, a disintegrator or other suitable additives, uniformly blending the resulting mixture, grinding to granules, adding a lubricant to the granules and then subjecting to compression molding. Alternatively, drugs may be subjected directly to compression molding; or an excipient, a binder, a disintegrator or other suitable additives are added to the drugs, uniformly blended and finally compression-molded. The pharmaceutical preparation may also be prepared by subjecting granules previously made directly to compression molding or after adding suitable additives to the granules and uniformly blending them. The pharmaceutical preparation may also be added with, if desired, a coloring agent, a corrigent, etc. Furthermore, the pharmaceutical preparation may also be coated with an appropriate coating agent.

To prepare injection, a predetermined amount of the medicament is dissolved, suspended or emulsified in water for injection, physiological saline, Ringer's fluid, etc., or usually in vegetable oil when using a non-aqueous solvent. Thus, the medicament is adjusted to a prescribed amount. Alternatively, injection may also be prepared by taking the medicament in a predetermined amount and sealing it into a container for injection.

Examples of carriers that may be used for oral preparations include those conventionally used in pharmaceutical preparations such as starch, mannitol, crystalline cellulose, carboxymethyl cellulose, etc. Examples of carriers for injection are distilled water, physiological saline, a glucose solution, fluid supplementation, etc. Other additives conventionally used for pharmaceutical preparations in general may also be added appropriately to the preparations.

The present invention further provides the following features.

(1) A method for analysis which comprises using at least two cultured cell lines derived from human liver capable of stably expressing at least one of CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4, said analysis being for (a) an enzyme that participates in the metabolism of xenobiotics and/or endogenous substrates, (b) a metabolic pathway of xenobiotics and/or endogenous substrates, (c) a chemical structure of the metabolite of xenobiotics and/or endogenous substrates, (d) inhibition of the metabolizing enzyme for xenobiotics and/or endogenous substrates, (e) an accelerated activity of the metabolizing enzyme for xenobiotics and/or endogenous substrates, (f) cytotoxicity by the metabolism of xenobiotics and/or endogenous substrates, (g) genotoxicity by the metabolism of xenobiotics and/or endogenous substrates, (h) carcinogenicity by the metabolism of a xenobiotic and/or endogenous substrate, (i) mutagenicity by the metabolism of xenobiotics and/or endogenous substrates, (j) hepatotoxicity by the metabolism of xenobiotics and/or endogenous substrates, or (k) xenobiotics and/or endogenous substrates that act on the liver.

(2) A method for preparation of the metabolite of xenobiotics and/or endogenous substrates, which comprises using at least two cultured cell lines from human liver capable of stably expressing at least one of CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4.

(3) A method for screening a substance which comprises using at least two cultured cell lines from human liver capable of stably expressing at least one of CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4, said substance being (a) a substance capable of inhibiting the metabolizing enzyme for xenobiotics and/or endogenous substrates, (b) a substance capable of accelerating an activity of the metabolizing enzyme for xenobiotics and/or endogenous substrates, (c) a substance capable of expressing cytotoxicity by the metabolism of xenobiotics and/or endogenous substrates, (d) a substance capable of expressing genotoxicity by the metabolism of xenobiotics and/or endogenous substrates, (e) a substance capable of expressing carcinogenicity by the metabolism of xenobiotics and/or endogenous substrates, (f) a substance capable of expressing mutagenicity by the metabolism of xenobiotics and/or endogenous substrates, (g) a substance capable of expressing hepatotoxicity by the metabolism of xenobiotics and/or endogenous substrates, (h) xenobiotics and/or endogenous substrates which act on the liver, or (i) a substance capable of acquiring a new physiological activity or increasing or decreasing the inherent physiological activity, through the metabolism of xenobiotics and/or endogenous substrates.

(4) A compound or a salt thereof (pharmaceutical composition), which is obtainable using the method according to (3).

The following terms are used to refer to the same meanings as given hereinabove: the term "a method for screening (a) an enzyme that participates in the metabolism of xenobiotics and/or endogenous substrates, (b) a metabolic pathway of xenobiotics and/or endogenous substrates, (c) a chemical structure of the metabolite of xenobiotics and/or endogenous substrates, (d) inhibition of the metabolizing enzyme for xenobiotics and/or endogenous substrates, (e) an accelerated activity of the metabolizing enzyme for xenobiotics and/or endogenous substrates, (f) cytotoxicity by the metabolism of xenobiotics and/or endogenous substrates, (g) genotoxicity by the metabolism of xenobiotics and/or endogenous substrates, (h) carcinogenicity by the metabolism of a xenobiotic and/or endogenous substrate, (i) mutagenicity by the metabolism of xenobiotics and/or endogenous substrates, (j) hepatotoxicity by the metabolism of xenobiotics and/or endogenous substrates, or (k) xenobiotics and/or endogenous substrates that act on the liver," the term "a method for preparation of the metabolite of xenobiotics and/or endogenous substrates," the term "a method for screening (a) a substance capable of inhibiting the metabolizing enzyme for xenobiotics and/or endogenous substrates, (b) a substance capable of accelerating an activity of the metabolizing enzyme for xenobiotics and/or endogenous substrates, (c) a substance capable of expressing cytotoxicity by the metabolism of xenobiotics and/or endogenous substrates, (d) a substance capable of expressing genotoxicity by the metabolism of xenobiotics and/or endogenous substrates, (e) a substance capable of expressing carcinogenicity by the metabolism of xenobiotics and/or endogenous substrates, (f) a substance capable of expressing mutagenicity by the metabolism of xenobiotics and/or endogenous substrates, (g) a substance capable of expressing hepatotoxicity by the metabolism of xenobiotics and/or endogenous substrates. (h) xenobiotics and/or endogenous substrates which act on the liver, or (i) a substance capable of acquiring a new physiological activity or increasing or decreasing the inherent physiological activity, through the metabolism of xenobiotics and/or endogenous substrates" as well as the terms "a compound or its salt (pharmaceutical composition), which is obtainable by the method for screening (a) a substance capable of inhibiting the metabolizing enzyme for xenobiotics and/or endogenous substrates, (b) a substance capable of accelerating an activity of the metabolizing enzyme for xenobiotics and/or endogenous substrates, (c) a substance capable of expressing cytotoxicity by the metabolism of xenobiotics and/or endogenous substrates, (d) a substance capable of expressing genotoxicity by the metabolism of xenobiotics and/or endogenous substrates, (e) a substance capable of expressing carcinogenicity by the metabolism of xenobiotics and/or endogenous substrates, (f) a substance capable of expressing mutagenicity by the metabolism of xenobiotics and/or endogenous substrates, (g) a substance capable of expressing hepatotoxicity by the metabolism of xenobiotics and/or endogenous substrates, (h) xenobiotics and/or endogenous substrates which acts on the liver, or (i) a substance capable of acquiring a new physiological activity or increasing or decreasing the inherent physiological activity, through the metabolism of xenobiotics and/or endogenous substrates."

The method for analysis, the method for preparation and the method for screening, described above, which comprises "using at least two cultured cell lines derived from human liver capable of stably expressing at least one of CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4" enable to achieve the analysis, preparation and screening in a way closer to in vivo, than in the case of using the cell line which expresses only one enzyme of CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4.

When two kinds or more of the cell lines are used, the respective cell lines may be used at the same time, or independently followed by comparison between the respective results in the analysis, preparation and screening.

The cell lines Hepc/3A4.5, Hepc/2E1.3-8, Hepc/2C9.1, Hepc/2C8.46, Hepc/1A2.9 and Hepc/1A1.4, which were obtained in EXAMPLES later described, have been deposited with Institute for Fermentation (IFO) at 2-17-85, Juso Honcho, Yodogawa-ku, Osaka, as deposit numbers IFO 50502, 50503, 50504, 50505, 50506 and 50507, respectively, since Feb. 10, 1999, and with the Ministry of International Trade and Industry, Agency of Industrial Science and Technology, National Institute of Bioscience and Human Technology (NIBH) at 1-1-3, Tsukuba Higashi, Tsukuba-shi, Ibaraki, as deposit numbers FERM BP-7120, FERM BP-7121, FERM BP-7122, FERM BP-7123, FERM BP-7124 and FERM BP-7125, respectively, since Apr. 12, 2000. Hepc/2B6.68 and Hepc/2D6.39 have been deposited with Institute for Fermentation (IFO) at 2-17-85, Juso Honcho, Yodogawa-ku, Osaka, as deposit numbers IFO 50508 and 50509, respectively, since Feb. 15, 1999. and with the Ministry of International Trade and Industry, Agency of Industrial Science and Technology, National Institute of Bioscience and Human Technology (NIBH) at 1-1-3, Tsukuba Higashi, Tsukuba-shi, Ibaraki, as deposit numbers FERM BP-7126 and FERM BP-7127, respectively, since Apr. 12, 2000. Hepc/2A6L.9 and Hepc/2C19.12 have been deposited with Institute for Fermentation (IFO) at 2-17-85, Juso Honcho, Yodogawa-ku. Osaka, as deposit numbers IFO 50511 and 50512, respectively, since Feb. 15, 1999, and with the Ministry of International Trade and Industry, Agency of Industrial Science and Technology, National Institute of Bioscience and Human Technology (NIBH) at 1-1-3, Tsukuba Higashi, Tsukuba-shi, Ibaraki, as deposit numbers FERM BP-7128 and FERM BP-7129, respectively, since Apr. 12, 2000.

Claim 1 of 1 Claim

What is claimed is:

1. An isolated cell line derived from a human hepatocarcinoma cell, which stably expresses human cytochrome P450, which is introduced by transfection, provided that when the human hepatocarcinoma cell is HepG2, the human cytochrome P450 is other than CYP2E 1, and wherein the cell line is Hepc/1A1.4, Hepc/1A2.9, Hepc/2B6.68, Hepc/2C8.46, Hepc/2C9.1, Hepc/2C19.12, Hepc/2D6.39, or Hepc/3A4.5.

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