JPWO2006077873A1 - Novel drug metabolizing enzyme of monkey and nucleic acid encoding it - Google Patents

Abstract

An object of the present invention is to provide a novel drug metabolizing enzyme unique to monkeys and a gene encoding the same. Another object of the present invention is to provide a method for evaluating the reliability and safety of metabolic tests using monkeys as model animals using the novel drug-metabolizing enzymes. The present invention relates to (a) a protein comprising the amino acid sequence set forth in SEQ ID NO: 2 or (b) one or several amino acids substituted, deleted, added and / or substituted in the amino acid sequence set forth in SEQ ID NO: 2. Provided is a protein that is a drug metabolizing enzyme and a nucleic acid that encodes it, comprising an inserted amino acid sequence, and further provides a method for evaluating a metabolic test using a monkey as a model animal, using the protein according to the present invention did.

Description

  The present invention relates to a novel drug-metabolizing enzyme specifically possessed by monkeys, a nucleic acid encoding the same, a method for evaluating a preclinical test using monkeys as model animals using the novel drug-metabolizing enzyme, and the like.

Traditionally, in the process of new drug development, the metabolic pattern is very similar to that of humans, so pharmacokinetic tests, drug effects tests, safety tests, etc., including metabolic tests using monkeys as model animals, have been widely conducted. . However, for some drugs, it has been reported that the metabolic pattern of monkeys may differ from that of humans (Non-Patent Documents 1 to 4).
An enzyme called cytochrome P450 (CYP) is deeply involved in drug metabolism. CYP forms a gene family, and the presence of more than 50 types of CYP genes has been reported so far in humans, and it is known that other mammals have many CYP genes. CYP is a heme protein coordinated with iron ions, and the structure of each CYP is very similar, but has different substrates and metabolic activities.
CYPs are classified based on amino acid sequence homology, and each CYP represents a family name (number), subfamily name (alphabet), and unique name (number) following CYP, for example, “CYP1A1”. Is specified by
It has been reported that the substrate of each CYP is different, and recently, the metabolic activity and the substrate specificity are different for each CYP gene polymorphism (for example, JP-A-2004-4; Patent Document 1). As one of the causes of individual differences, sex differences, age differences, species differences, and race differences seen in the onset of toxicity, research on molecular species and polymorphisms of CYP is underway.
In general, a compound metabolized by a CYP molecular species having a large individual difference is likely to have different drug efficacy and side effects depending on humans, and is considered undesirable as a pharmaceutical product. When developing such a compound as a pharmaceutical product, during clinical development, examine the polymorphism of the molecular species of the clinical trial candidate in advance, and identify specific polymorphisms that are effective and have few side effects It is necessary to take measures such as excluding candidates having polymorphisms that are likely to cause side effects, only for candidates having polymorphisms.
Japanese Patent Laid-Open No. 2004-4 Nartimatsu S. et al., Chem Biol Interact (2000) 127: 73-90 Sharer JE. Et al., Drug Metab Dispos (1995) 23: 1231-1241 Stevens JC. Et al., Drg Metab Dispos (1993) 21: 753-760 Weaver RJ. Et al., Xenobiotica (1999) 29: 467-482

When conducting metabolic tests using model animals, the significance of the test results can be evaluated more accurately by considering differences between human CYP and model animals CYP, polymorphisms between model animals, etc. It is thought that it can be utilized. For example, if there is a drug candidate compound that causes significant side effects in cynomolgus monkeys and there is a drug candidate compound that causes significant side effects in cynomolgus monkeys, it is found that the difference in metabolic patterns is due to the difference in CYP between humans and cynomolgus monkeys. If it can be confirmed that similar side effects do not occur, there is a possibility that the development of the compound as a human drug can be continued and promoted without wastefully stopping the development of the compound.
As described above, since the metabolic pattern of cynomolgus monkeys is very similar to that of humans, it has been considered that the activity of drug metabolizing enzymes is almost the same as that of humans. However, only 6 types of CYP cDNA of cynomolgus monkeys have been reported to GenBank so far, and little is known about differences in substrate from human CYP and differences among individual monkeys.
On the other hand, it has been revealed that there are actually drugs with different metabolic patterns between humans and cynomolgus monkeys.
Therefore, an object of the present invention is to find a monkey-specific drug-metabolizing enzyme and a gene encoding it that are not found in mammals other than monkeys.
Furthermore, the present invention provides a method for accurately evaluating and effectively utilizing the results of pharmacokinetic tests using monkeys as model animals by measuring the metabolic activity of drugs by the above-mentioned monkey-specific drug metabolizing enzymes. The purpose is to provide.

The present inventors have conducted research to solve the above-mentioned problems, and have determined the full-length base sequence of cDNA having a relatively high homology with the human CYP gene by screening a cynomolgus monkey liver cDNA library. We succeeded in detecting 21 cynomolgus monkey CYP genes. And in this, it discovered that the CYP2C gene with a low homology with a human gene compared with another CYP gene was contained, and this gene is expressed in the Old World monkey including the cynomolgus monkey, and the New World monkey. In addition, it was confirmed that no expression was observed in large apes including humans.
Furthermore, the novel CYP2C protein was expressed using E. coli, and it was clarified that there is a difference in metabolic activity against certain drugs between human CYP2C and other CYP2Cs of cynomolgus monkeys, and the present invention was completed. It was.
That is, the present invention
[1] The following protein (a) or (b):
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 2,
(B) a protein which is a drug-metabolizing enzyme comprising an amino acid sequence in which one or several amino acids are substituted, deleted, added and / or inserted in the amino acid sequence of SEQ ID NO: 2;
[2] A nucleic acid encoding the protein according to [1];
[3] From a base sequence complementary to the DNA consisting of the base sequence described in SEQ ID NO: 1 or SEQ ID NO: 3, or the DNA consisting of the base sequence described in SEQ ID NO: 1 or SEQ ID NO: 3 or a part thereof A monkey-derived DNA that hybridizes with a DNA sequence of
[4] A recombinant vector comprising the DNA according to [3];
[5] A transformant comprising the recombinant vector according to [4];
[6] The method for producing the protein according to [1], wherein the transformant according to [5] is cultured, and the protein according to [1] is purified from the culture;
[7] an antibody against the protein according to [1];
[8] When the metabolism of the test substance is different between a monkey and a mammal other than a monkey, the step A for contacting the protein according to [1] and the test substance, and the metabolism of the test substance, A method for evaluating a cause of different metabolism of the test substance, which comprises the step B of measuring the presence or absence of involvement of the protein according to [1];
[9] The method according to [8], wherein the protein according to [1] used in the step A is produced by the method for producing a protein according to [7];
[10] In the step B, when the protein according to [1] is involved in metabolism of the test substance, the difference in metabolism of the test substance between the monkey and the mammal other than the monkey is The method according to [8] or [9], wherein the evaluation is based on a difference in
[11] Whether or not the protein according to [1] is involved in the metabolism of the drug candidate compound when testing the metabolism, efficacy and / or safety of the drug candidate compound for humans using monkeys as model animals And the test result of metabolism, efficacy and / or safety of the drug candidate compound in a monkey as a model animal in the case of the involvement, the metabolism, efficacy and efficacy of the drug compound in humans. And / or a process D for predicting a difference from safety, and a method for evaluating metabolism, efficacy and / or safety testing;
[12] The method according to [11], wherein the step C is performed in vitro;
[13] A method for evaluating the feasibility of testing metabolism, drug efficacy and / or safety of a human drug candidate compound using a monkey as a model animal, the protein according to [1] and the drug Including the step E of contacting a candidate compound and the step F of measuring the degree of involvement of the protein according to [1] with respect to metabolism of the pharmaceutical compound, A negative assessment of testing as a model animal;
[14] A kit containing the protein according to [1], which is used for evaluation of metabolism, drug efficacy and / or safety of a human drug candidate compound using a monkey as a model animal.

  Since the protein and gene according to the present invention exist specifically in monkeys, whether the metabolic activity is specific to monkeys by measuring the influence of this protein on the metabolic activity of a certain drug, humans, etc. It is possible to predict what is common to other animals. Based on this prediction, it is possible to evaluate the pros and cons of using monkeys as model animals for metabolic tests of certain drugs.

Hereinafter, embodiments of the present invention will be described in detail.
The protein according to the present invention is a monkey-specific drug-metabolizing enzyme that is expressed in old world monkeys such as cynomolgus monkeys, rhesus monkeys, and African green monkeys, and in new world monkeys such as common marmoset. The amino acid sequence of a novel CYP2C protein identified from cynomolgus monkey (hereinafter referred to as “new mfCYP2C”) is shown in SEQ ID NO: 2 (lower part of FIG. 1).
The inventors found novel mfCYP2C by the following method.
First, a cynomolgus monkey liver cDNA library was screened to identify a clone having high homology to the human CYP gene, and the base sequence of the cDNA was determined. This identified 21 cynomolgus monkey CYP genes, including novel ones. Most of these CYP genes were 94-95% homologous to the corresponding human CYP gene, but only one gene with a maximum of 79% homology with human CYP2C cDNA was discovered. This was designated as a new mfCYP2C.
In order to confirm whether the new mfCYP2C is a monkey-specific gene or only a human homolog has not been identified so far, RT-PCR was performed using gene-specific primers, and other primates The presence or absence of novel mfCYP2C in was investigated. As a result, the new mfCYP2C gene is present in all primates excluding humans and great apes such as all measured Old World and New World monkeys, but not in humans and large apes such as chimpanzees and orangutans. confirmed. In addition, a blast search using human and chimpanzee genome databases did not reveal a novel mypCYP2C CYP2C gene having a homology of 90% or more. This also indicates that the protein of the present invention is monkey-specific. It was a result that supported it.
In addition, total RNA was extracted from each tissue-derived sample, and real-time RT-PCR was performed using a novel mfCYP2C gene-specific primer. The expression level of this gene was overwhelmingly large in the liver, and other CYP2C Has been confirmed to be expressed in small amounts in the heart, muscle, brain, testis, etc., which are not normally expressed.
Further, the ratio of the new mfCYP2C in the total CYP expressed in the cynomolgus monkey liver, calculated by dividing the copy number of each CYP gene in the cynomolgus monkey liver cDNA library by the copy number of all CYP genes, was 28.0. % And the difference in expression level between males and females was about twice.
The novel mfCYP2C can be expressed and obtained by inserting a novel mfCYP2C gene into an appropriate vector and transforming the host using this vector.
The present inventors have confirmed that the recombinant novel mfCYP2C protein thus obtained exhibits metabolic activity against Tolbutamide and testosterone, but not against taxol or S-mephenytoin. When a monkey-specific enzyme has metabolic activity for a certain drug, it is suggested that the metabolic pattern for the drug may be different between monkeys and humans.
As long as the protein according to the present invention is a drug-metabolizing enzyme and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 2, one or several amino acids in the amino acid sequence shown in SEQ ID NO: 2 Mutations such as substitution, deletion, addition and / or insertion of one (for example, 1 to 10, preferably 1 to 5) amino acids may occur. These mutations may occur naturally or may be artificially modified.
Here, the drug-metabolizing enzyme means an enzyme that changes the chemical structure of a drug taken or administered from outside the body in vivo.

The nucleic acid according to the present invention is a nucleic acid encoding the protein according to the present invention, regardless of whether it is DNA or RNA. As described above, it encodes a protein having an amino acid sequence in which a mutation such as substitution, deletion, addition and / or insertion of one or several amino acids in the amino acid sequence shown in SEQ ID NO: 2 has occurred. Nucleic acids to be used are also included in the present invention as long as the protein is a protein according to the present invention.
As an example of such a nucleic acid, the novel mfCYP2C cDNA is shown in SEQ ID NO: 1 (upper part of FIG. 1), and the full length of the new mfCYP2C gene is shown in SEQ ID NO: 3.
The nucleic acid according to the present invention hybridizes under stringent conditions with a DNA sequence consisting of a base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 or a base sequence complementary to the DNA consisting of a part thereof. Also included is a monkey-derived nucleic acid encoding a protein that is a drug-metabolizing enzyme. A nucleic acid that hybridizes under stringent conditions with a DNA sequence consisting of a base sequence complementary to the base sequence described in SEQ ID NO: 1 or SEQ ID NO: 3 or a part thereof is SEQ ID NO: 1 or sequence It is considered that there is a high possibility that the encoded protein has the same function as that of the base sequence described in No. 3 with high homology. The stringent conditions include, for example, 5% Denhardt's Solution (containing 0.1% Ficoll (Pharmacia), 0.1% polyvinylpyrrolidone, 0.1% bovine serum albumin), 0.5% SDS And a condition of washing at 65 ° C. in a 6 × SSC solution (1 × SSC is 0.15 M NaCl, 15 mM sodium citrate) containing 100 μg / ml salmon sperm DNA. Stringency can be controlled by salt concentration (ionic strength), temperature, etc. Under conditions of higher stringency, ie lower salt concentration and higher temperature, only DNA with sufficiently high homology will hybridize. Soybeans. Therefore, under such conditions, a DNA that hybridizes under stringent conditions with a DNA sequence consisting of a base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 or a base sequence complementary to the DNA consisting of a part thereof is used. The amino acid sequence of the encoded protein has high homology with the amino acid sequence set forth in SEQ ID NO: 2 and is considered to have the same function. A person skilled in the art can appropriately select stringent conditions by adjusting the temperature and the salt concentration.

The origin of the protein and nucleic acid according to the present invention is not limited as long as it is understood that they are included in the present invention by their amino acid sequence, base sequence and / or function, but it is preferably derived from monkeys, more preferably cynomolgus monkeys. From the Old World monkeys such as rhesus monkeys and African green monkeys, or from the New World monkeys such as common marmoset.
In the present specification, “monkey” means primates excluding large apes, including old world monkeys, new world monkeys, and original monkeys.

The present invention also provides a vector into which the DNA according to the present invention is inserted. The vector used in the present invention is not particularly limited as long as it stably retains the inserted DNA. For example, plasmids derived from E. coli (for example, pBR322, pBR325), plasmids derived from Bacillus subtilis (for example, pUB110, pTP5), yeast-derived plasmids (for example, pSH19, pSH15) and the like. When Escherichia coli is used as a host, bacteriophages such as λ phage system and M13 phage system are often used. When animal cells are used as hosts, viral DNAs such as SV40, papilloma virus, vaccinia virus, retrovirus, baculovirus and the like can be used as vectors.
Furthermore, a promoter sequence, an enhancer sequence, a Shine-Dalgarno sequence, a signal sequence, a poly A signal, and the like suitable for the host are appropriately selected so that the protein according to the present invention is efficiently expressed in the host and inserted into the vector. can do.
The DNA of the present invention can be inserted into the vector by, for example, cleaving the vector using a restriction enzyme and ligating the DNA to the cleavage site by ligase reaction.

The present invention also includes a transformant transformed with the vector according to the present invention. The transformant of the present invention can be obtained by selecting a host suitable for the vector into which the DNA of the present invention has been inserted and introducing the vector into this host. Examples of the host include Gram-negative bacteria such as Escherichia coli and Pseudomonas aeruginosa, Gram-positive bacteria such as Bacillus subtilis, actinomycetes, yeasts, filamentous fungi, animal and plant culture cells, and insect culture cells. Of these, Escherichia coli is preferred.
As a vector introduction method, when the host is Escherichia coli, the calcium chloride method, when Bacillus subtilis is the host, the competent cell method or the lithium acetate method, Bacillus subtilis, actinomycetes, yeast, etc., the protoplast method, As a method widely used for animal and plant cells, yeast, bacteria, etc., it can be appropriately selected from a calcium phosphate coprecipitation method, an electroporation method, a method of forming a complex with a polymer such as DEAE-dextran and polybrene. Lipofectamine (Invitrogen) that forms a complex with a liposome of a cationic lipid can also be used.

The present invention also includes a method for producing a protein, wherein the transformant is cultured to produce the protein according to the present invention. The culture of the transformant can be selected according to the characteristics of the host cell, the characteristics of the protein to be expressed, the characteristics of the promoter, etc. For example, a known medium such as MEM medium, DMEM medium, Williams E medium (Gibco) is used. can do. For example, when a plasmid having antibiotic resistance is used, expression of the target protein can be regulated by adding the antibiotic to the medium. When a lactose-dependent plasmid is used, IPTG is added to the medium. Addition can induce expression.
The protein according to the present invention, which is expressed by culturing the transformant thus obtained, can be purified using a conventional protein purification method. Depending on the expression system used, for example, various chromatography, It can be purified by a combination of external filtration, salting out, osmotic shock, sonication and the like. Examples of the chromatography method include ion exchange chromatography performed in an aqueous solution, gel filtration, hydrophobic chromatography, affinity chromatography, and reverse phase chromatography using an organic solvent. The present invention also includes proteins purified using these purification methods.
The expressed protein can be modified before or after purification using an appropriate protein modifying enzyme or the peptide can be partially removed, and these modified proteins are also included in the present invention. . Examples of protein modifying enzymes include trypsin, chymotrypsin, protein kinase and glucosidase. A person skilled in the art can easily change the expressed protein to a salt if it is in a released state, or to a released state if it is obtained as a salt.

The present invention also provides an antibody that binds to the protein of the present invention or a partial peptide of the protein. The antibody according to the present invention is a novel mfCYP2C protein, a protein functionally equivalent to the novel mfCYP2C (in the amino acid sequence shown in SEQ ID NO: 2, one or several amino acids are substituted, deleted, added and / or inserted) As long as it specifically binds to a protein that is a drug metabolizing enzyme, or a partial peptide thereof, and may be a polyclonal antibody or a monoclonal antibody. In addition to antibodies obtained by immunizing mammals other than humans with this protein, humanized antibodies and antibodies obtained by genetic recombination can also be used as long as they specifically bind to the protein of the present invention or a partial peptide thereof. It is included in the present invention. The antibody according to the present invention can be produced by a method known per se or a method analogous thereto, and typical methods are exemplified below.
First, a polyclonal antibody uses a novel mfCYP2C or a partial peptide thereof to immunize mammals, preferably rodents such as mice and rats, primates such as rabbits, monkeys, etc. Can be obtained by purifying through an affinity column to which the novel mfCYP2C or a partial peptide thereof is immobilized.
Monoclonal antibodies are also used to immunize mammals first, fuse antibody-producing cells obtained from these animals with highly proliferative myeloma cells, isolate individual fused cells, and test the desired antibody-producing ability. Thus, cells that produce only one kind of antibody molecule that specifically reacts with one epitope of the antigen can be selected and cultured by culturing the cells. A monoclonal antibody can also be obtained by a genetic engineering method by inserting a DNA encoding the amino acid sequence of a monoclonal antibody into a vector, introducing the vector into a host, and producing the vector. By immunizing a transgenic animal into which a human antibody gene has been introduced, human antibodies can also be produced, and the cDNA encoding the antigen-binding site of the mouse monoclonal antibody and the gene encoding the constant region of the human IgG gene are linked. It is also possible to produce chimeric antibody molecules by introducing them into B lymphocytes, and these antibodies are also included in the present invention. The antibody of the present invention may be a fragment or a modified antibody as long as it specifically recognizes and binds to the protein or partial peptide of the present invention. Examples of the antibody fragment include F (ab), F (ab ′) 2, Fc fragment, single chain Fv, and the like. Examples of the modified antibody include an antibody bound to a compound such as polyethylene glycol. It is done. The antibody of the present invention can be used for purification, detection and quantification of novel mfCYP2C, and can be an agonist or antagonist when it has an action of enhancing or suppressing the biological activity of novel mfCYP2C.

As described above, since the new mfCYP2C according to the present invention is a monkey-specific protein, the metabolic pattern of monkeys and large apes (humans, chimpanzees, orangutans, etc.) can be different for compounds in which the new mfCYP2C is involved in metabolism High nature.
Therefore, before conducting a metabolic test, drug efficacy test, or safety test for a drug candidate compound, in vitro, the monkey is tested as a model animal by confirming whether the new mfCYP2C is involved in the metabolism of the candidate compound. It is possible to evaluate whether or not to do. For example, if the novel mfCYP2C is deeply involved in metabolism, it is suggested that the metabolism of the candidate compound in humans may be significantly different from that in monkeys, and it is evaluated that it is not desirable to conduct studies using monkeys as model animals. . On the other hand, when the novel mfCYP2C is not involved in the metabolism of the candidate compound, it is suggested that the candidate compound may be metabolized by another metabolic enzyme having high homology with humans. It is considered possible to infer metabolism in humans using the results.
By using the new mfCYP2C in this way, it is possible to omit a useless test and speed up the test.

Furthermore, since no monkey CYP gene having a lower homology with human CYP than new mfCYP2C has been found so far, drugs with different metabolic patterns in humans and monkeys are involved in the metabolism of new mfCYP2C It is estimated that the possibility is high. Therefore, the cause of this difference in metabolic pattern is evaluated by contacting a drug with different metabolic patterns between human and monkey with new mfCYP2C, incubating under appropriate conditions, and measuring the involvement of new mfCYP2C in the drug metabolism. It is possible.
With this method, if it is found that the difference in metabolic pattern is due to the new mfCYP2C, the test can be continued and expedited without being stopped in vain due to the test results of monkeys as model animals. Is possible.

  The present invention further provides a kit containing a novel mfCYP2C, which is used for evaluation of metabolism, efficacy and / or safety of a human drug candidate compound using a monkey as a model animal. Such a kit may contain reagents, containers, devices and the like necessary for the novel mfCYP2C to metabolize the substrate. Such a kit can be used to estimate whether a test result similar to that obtained in a monkey model animal can be obtained in a human test, to investigate the cause of a test result that differs between a human and a monkey, Can be used to examine whether or not to test as a model animal.

[Tissue sample preparation and RNA extraction]
Tissue samples were taken from 6 cynomolgus monkeys (3 males and 3 females), 2 rhesus monkeys (male), and 2 common marmoset.
Orangutan and chimpanzee tissue samples were also prepared.
In order to prevent RNA degradation, all samples were frozen with liquid nitrogen immediately after collection, and ground and ground with a mortar and pestle in the presence of liquid nitrogen.
Subsequently, using Polytron PT1200E (Kinematica), the powdered tissue was homogenized in TRIzol (Invitrogen), and the total RNA was extracted according to the product instructions. (Integrity) was confirmed. The extracted total RNA was treated with DNase I (Takara) and then purified with the GenElute Mammalian Total RNA Mini Kit (Sigma-Aldrich).
On the other hand, COS1 cells (obtained from ATCC) as samples of African green monkeys and HepG2 cells (obtained from RIKEN) as samples of human hepatocytes were obtained by the method of Saito et al. (Saito et al., J Biol Chem, (2001) 276: 38010-38022). RNA extraction from the cells and subsequent DNase I treatment were performed using RNeasy Mini Kit (QIAGEN) according to the product instructions.
[Isolation of cynomolgus monkey CYP2C cDNA]
First, a clone with high homology to human CYP was identified by screening a cynomolgus monkey liver cDNA library. For the identified clones, the full length of the insert was amplified by PCR. PCR was performed in a total volume of 20 μl of the reaction solution including the following.
・ Primers (0.4μM each)
FW: 5'-TCAGTGGATGTTGCCTTTAC-3 '(SEQ ID NO: 4)
RV: 5'-TGTGGGAGGTTTTTTCTCTA-3 '(SEQ ID NO: 5)
・ DNTP (0.22μM)
・ MgCl ₂ (2mM)
・ AmpliTaq Gold Taq polymerase (Applied Biosystems) (1 unit)
As the reaction conditions, after the first denaturation was performed at 95 ° C. for 10 minutes, a cycle of 95 ° C. for 20 seconds, 55 ° C. for 20 seconds and 72 ° C. for 3 minutes was repeated 35 times, and the final extension was 72 ° C. for 10 minutes.
After analysis on a 1% agarose gel, the PCR product was purified and used for nucleotide sequence analysis with ABI Prism BigDye Terminator v3.0 Ready Reaction Cycle Sequencing Kit (Applied Biosystems), and then ABI-3730 DNA Analyzer (Applied Biosystems) Electrophoresis).
[Sequence analysis]
The obtained sequence data was imported into DNASIS Pro (Hitachi Software) for sequence analysis. After trimming the vector sequences, all data were visually reviewed to remove regions with inadequate sequence quality before each sequence was integrated.
The homology search was performed by the BLAST program (National Center for Biotechnology Information). To identify highly homologous CYP cDNA sequences, multiple alignments using Clustral W were performed on cDNA and amino acid sequences.
Among the 21 cynomolgus monkey CYPs found by the inventors, the results of multiple alignment of mfCYP2C9v1, mfCYP2C9v3 and new mfCYP2C, and mfCYP2C20, which is a known cynomolgus CYP gene, are shown in FIG. From the top, mfCYP2C20 (SEQ ID NO: 6), mfCYP2C9v1 (SEQ ID NO: 7), mfCYP2C9v3 (SEQ ID NO: 8), and new mfCYP2C (SEQ ID NO: 9).
The new mfCYP2C, mfCYP2C9v1, and mfCYP2C9v3 were named CYP2C76, CYP2C43, and CYP2C75, respectively, according to the Committee on Standardized P450 Nomenclature, and registered as accession numbers DQ074806, DQ074805, and DQ074807 in GenBank, respectively.
The nucleotide sequence (SEQ ID NO: 1) of the novel mfCYP2C cDNA is shown in FIG.
The novel mfCYP2C cDNA has an open reading frame of 489 amino acids, one residue less than all previously known human and monkey CYP2Cs. The amino acid sequence deduced from the base sequence includes the N-terminus, which has a very high hydrophobicity, a heme-binding region, six substrate recognition sites (Gotoh, (1992), J. Biol Chem 267: 83-90), and other It contained an amino acid sequence common to CYP2C molecules.
Using the cDNA base sequence, Blat search (UCSC Genome Bioinformatics) of mfCYP2C homologue was also performed on human and chimpanzee genomes.
The results of the homology search are shown in Table 1.

mfCYP2C9v1およびmfCYP2C9v3は、ヒトCYP2Cのアミノ酸に対して約92％の相同性を示し、これはCYP2C20等、公知のカニクイザルのCYP2C遺伝子と同じレベルであった。
しかしながら、新規mfCYP2Cのアミノ酸は、いずれのヒトCYP2Cのアミノ酸に対しても相同性が約71％しかなく、またGenBankデータベースにおいてもこれ以上の相同性を示すヒトcDNAは存在しなかった。
塩基配列の結果は、デフォルト・パラメータによるPHYLIPを使用して分子進化系統樹を描くためにも用いた。分子進化系統樹を図３に示す。この結果、新規mfCYP2C（CYP2C76）は、全てのヒトCYP2C及び他のカニクイザルCYP2Cから、離れたクレードに属することがわかった。
〔他の霊長類における新規mfCYP2Cに対応するcDNAのクローニング〕
そこで、新規mfCYP2Cが、サルに特異的に存在する遺伝子なのか、従来ヒトのホモログが見出されていないだけなのかを確認するため、いくつかの遺伝子特異的プライマーを用いてRT-PCRを行い、他の霊長類における、新規mfCYP2Cのorthologus遺伝子の有無を調べた。
他種のサルからの新規mfCYP2Cホモログは、肝（アカゲザル、コモン・マーモセット、オランウータンおよびチンパンジー）またはCOS1細胞（アフリカミドリザル）由来のRNAサンプルを用いて、RT-PCRにより同定した。RT反応は、M-MLV reverse transcriptase（Toyobo社）を用い、1μgの全RNAとオリゴ（dT）プライマーまたはランダムプライマーを使用して、37℃で1時間行った。得られたRT産物は、25倍に希釈してPCRに供した。ヒトについては、市販の肝RT産物（BD Biosciences社）を使用した。いくつかのプライマーペアを試したところ、アカゲザル、ミドリザルおよびコモン・マーモセットのCYP2CホモログのcDNAは、それぞれ以下の塩基配列を有するプライマーペアにより増幅することができた。
アカゲザル
FW: mfCYP2C (5rt2) 5'-CCCAGCAATGGATCTCTTCA-3' （配列番号：１０）
RV: mfCYP2C (3polyA2a) 5'-TGCCTAGACAGGTAGATAGGAGTG-3' （配列番号：１１）
アフリカミドリザル
FW: mfCYP2C (5rt2) 5'-CCCAGCAATGGATCTCTTCA-3' （配列番号：１０）
RV: mfCYP2C (3ex4b) 5'-GAAAAGTGGGATCACAGGGA-3' （配列番号：１２）
コモン・マーモセット
FW: mfCYP2C (5ex2a) 5'-GTATTTTCTGGCCGAGGGAG-3' （配列番号：１３）
RV: mfCYP2C (3ex4a) 5'-ACAGGGAACACAACCCAGAA-3' （配列番号：１４）
増幅は、KOD Plus DNA polymerase（Toyobo社）を使用してMJ Research thermal cycler（MJ Research社）によって行い、最初の変性を95℃2分で行った後、95℃20秒、55℃20秒、72℃2分のサイクルを30回繰り返し、最終伸長を72℃10分とした。
3'A-overhangを付加した後、PCR産物を、TOPO TA Cloning Kit（Invitrogen社）を用いてベクターにクローニングした。続いて、ABI Prism BigDye Terminator v3.0 Ready Reaction Cycle Sequencing Kit（Applied Biosystems社）を用いて、インサートの塩基配列解析を行い、ABI PRISM 3730 DNA Analyzer（Applied Biosystems社）により電気泳動を行った。
この結果、増幅産物は、旧世界ザル（カニクイザル、アカゲザル、アフリカミドリザル）および新世界ザル（コモン・マーモセット）には見られ、これらのサルが新規mfCYP2Cのホモログを有しており、互いに99％以上の相同性を有することが確認された。この結果は、これらのサルが進化系統的に近い種であることを示している。
結果を図４〜６に示す。図４および５はそれぞれ新規mfCYP2CのcDNAの塩基配列を示し、上段から、カニクイザル（配列番号：１５）、アカゲザル（配列番号：１６）、アフリカミドリザル（配列番号：１７）およびコモン・マーモセット（配列番号：１８）である。図６は新規mfCYP2Cタンパク質のアミノ酸配列を示し、上段から、カニクイザル（配列番号：２）、アカゲザル（配列番号：１９）、アフリカミドリザル（配列番号：２０）およびコモン・マーモセット（配列番号：２１）である。
一方、ヒトや、チンパンジーおよびオランウータン等の大型類人猿由来のサンプルからは検出可能なバンドが得られなかった（data not shown）。さらに、Blastによるヒトおよびチンパンジーのゲノム・データベース検索でも、新規mfCYP2Cに90％以上の相同性を有する遺伝子が発見されないことから、新規mfCYP2Cは、サル特異的であることが示された。
〔新規mfCYP2Cの遺伝子構造〕
エクソンとイントロンの構造を決定するため、4種類のmfCYP2C（mfCYP2C9v1、mfCYP2C9v3、CYP2C20および新規mfCYP2C)のcDNAの塩基配列を比較して、遺伝子特異的プライマーを各エクソン上に設計し、すべてのイントロンをlong PCRによって増幅した。PCRは、LA Taq PCR kit（Takara社）を使用し、5p moleのフォワードおよびリバースプライマー（表２）、0.5mMのdNTP、2mMのMgCl₂、1単位のLA Taq polymerase（Takara株式会社）を用いて全量20μlとして行った。

mfCYP2C9v1 and mfCYP2C9v3 showed about 92% homology to amino acids of human CYP2C, which was at the same level as CYP2C20 and other known cynomolgus monkey CYP2C genes.
However, the amino acid of the novel mfCYP2C has only about 71% homology with any human CYP2C amino acid, and there was no human cDNA exhibiting more homology in the GenBank database.
Base sequence results were also used to draw molecular evolutionary phylogenetic trees using PHYLIP with default parameters. The molecular evolutionary tree is shown in FIG. As a result, it was found that the novel mfCYP2C (CYP2C76) belongs to a clade distant from all human CYP2C and other cynomolgus CYP2C.
[Cloning of cDNA corresponding to novel mfCYP2C in other primates]
Therefore, RT-PCR was performed using several gene-specific primers in order to confirm whether the new mfCYP2C is a gene that is specifically present in monkeys or has not been found in human homologs. The presence of a novel mfCYP2C orthologus gene in other primates was examined.
Novel mfCYP2C homologs from other species of monkeys were identified by RT-PCR using RNA samples from liver (rhesus monkey, common marmoset, orangutan and chimpanzee) or COS1 cells (African green monkey). RT reaction was performed at 37 ° C. for 1 hour using M-MLV reverse transcriptase (Toyobo) using 1 μg of total RNA and oligo (dT) primer or random primer. The obtained RT product was diluted 25 times and subjected to PCR. For humans, a commercially available liver RT product (BD Biosciences) was used. When several primer pairs were tested, the cDNAs of rhesus monkey, green monkey, and common marmoset CYP2C homologs could be amplified by primer pairs having the following base sequences, respectively.
Rhesus monkey
FW: mfCYP2C (5rt2) 5'-CCCAGCAATGGATCTCTTCA-3 '(SEQ ID NO: 10)
RV: mfCYP2C (3polyA2a) 5'-TGCCTAGACAGGTAGATAGGAGTG-3 '(SEQ ID NO: 11)
African green monkey
FW: mfCYP2C (5rt2) 5'-CCCAGCAATGGATCTCTTCA-3 '(SEQ ID NO: 10)
RV: mfCYP2C (3ex4b) 5'-GAAAAGTGGGATCACAGGGA-3 '(SEQ ID NO: 12)
Common marmoset
FW: mfCYP2C (5ex2a) 5'-GTATTTTCTGGCCGAGGGAG-3 '(SEQ ID NO: 13)
RV: mfCYP2C (3ex4a) 5'-ACAGGGAACACAACCCAGAA-3 '(SEQ ID NO: 14)
Amplification was performed by MJ Research thermal cycler (MJ Research) using KOD Plus DNA polymerase (Toyobo). First denaturation was performed at 95 ° C for 2 minutes, followed by 95 ° C for 20 seconds, 55 ° C for 20 seconds, The cycle at 72 ° C. for 2 minutes was repeated 30 times, and the final extension was 72 ° C. for 10 minutes.
After adding 3′A-overhang, the PCR product was cloned into a vector using TOPO TA Cloning Kit (Invitrogen). Subsequently, the base sequence of the insert was analyzed using ABI Prism BigDye Terminator v3.0 Ready Reaction Cycle Sequencing Kit (Applied Biosystems), and electrophoresis was performed using ABI PRISM 3730 DNA Analyzer (Applied Biosystems).
As a result, amplification products were found in Old World monkeys (cynomolgus monkeys, rhesus monkeys, African green monkeys) and New World monkeys (common marmoset), and these monkeys have new mfCYP2C homologs, and more than 99% of each other. It was confirmed to have the homology of This result indicates that these monkeys are close evolutionary species.
The results are shown in FIGS. 4 and 5 show the cDNA sequences of the novel mfCYP2C cDNA. From the top, cynomolgus monkey (SEQ ID NO: 15), rhesus monkey (SEQ ID NO: 16), African green monkey (SEQ ID NO: 17), and common marmoset (SEQ ID NO: 17). : 18). FIG. 6 shows the amino acid sequence of the novel mfCYP2C protein. From the top, cynomolgus monkey (SEQ ID NO: 2), rhesus monkey (SEQ ID NO: 19), African green monkey (SEQ ID NO: 20) and common marmoset (SEQ ID NO: 21). is there.
On the other hand, no detectable band was obtained from samples from humans, large apes such as chimpanzees and orangutans (data not shown). Furthermore, human and chimpanzee genome database searches by Blast did not find a gene with 90% or more homology to the new mfCYP2C, indicating that the new mfCYP2C is monkey-specific.
[Genetic structure of novel mfCYP2C]
To determine the structure of exons and introns, compare the cDNA sequences of four types of mfCYP2C (mfCYP2C9v1, mfCYP2C9v3, CYP2C20, and novel mfCYP2C), design gene-specific primers on each exon, and configure all introns. Amplified by long PCR. PCR uses the LA Taq PCR kit (Takara Co.), forward and reverse primers (Table 2) of 5p mole, with 0.5mM of dNTPs, 2 mM of MgCl _2, 1 unit of LA Taq polymerase (Takara Co., Ltd.) The total volume was 20 μl.

PCRは、95℃2分で変性させてから、95℃20秒、55℃30秒、72℃5分のサイクルを35回繰り返し、最終伸長を72℃20分として行った。0.8％アガロースゲル上で電気泳動した後、PCR産物はゲル精製し、TOPO TAまたはXL cloning kit（Invitrogen社）を使用して、製品の指示書に従ってベクターにクローニングし、配列解析した。
配列解析は上述の方法に従って行い、initial sequencingには、以下のM13フォワードおよびリバースプライマーを用いた。
プライマー
FW: M13FW 5'-GTAAAACGACGGCCAG-3' （配列番号：３８）
RV: M13RV 5'-CAGGAAACAGCTATGAC-3' （配列番号：３９）
すべてのイントロンの塩基配列はプライマーウォーキング法により完全に決定され、当該配列は、DNASIS Pro（Hitachi Software社）により、各イントロンの全長に統合された。
この結果、新規mfCYP2Cは約19.6kbにおよび（配列番号：３）、すべてのヒトCYP2Cと同様に9つのエクソンを有する遺伝子であることが確認された（図７）。エクソンおよびイントロンのサイズは、それぞれ142-693bp、937-4307bpであった。表３に示すように、エクソン（小文字）−イントロン（大文字）の境界のほとんどは、GU-AC則に従っていたが（太字）、イントロン8の5'スプライス部位はGUがGCに置換されていた。

PCR was denatured at 95 ° C. for 2 minutes, and then a cycle of 95 ° C. for 20 seconds, 55 ° C. for 30 seconds, and 72 ° C. for 5 minutes was repeated 35 times, and the final extension was performed at 72 ° C. for 20 minutes. After electrophoresis on a 0.8% agarose gel, the PCR product was gel purified and cloned into a vector using TOPO TA or XL cloning kit (Invitrogen) according to product instructions and sequenced.
Sequence analysis was performed according to the method described above, and the following M13 forward and reverse primers were used for initial sequencing.
Primer
FW: M13FW 5'-GTAAAACGACGGCCAG-3 '(SEQ ID NO: 38)
RV: M13RV 5'-CAGGAAACAGCTATGAC-3 '(SEQ ID NO: 39)
The base sequences of all introns were completely determined by the primer walking method, and the sequences were integrated into the full length of each intron by DNASIS Pro (Hitachi Software).
As a result, it was confirmed that the novel mfCYP2C is a gene having about 19.6 kb (SEQ ID NO: 3) and having 9 exons as in all human CYP2C (FIG. 7). Exon and intron sizes were 142-693 bp and 937-4307 bp, respectively. As shown in Table 3, most of the exon (lowercase) -intron (uppercase) boundary followed the GU-AC rule (bold), but in the 5 ′ splice site of intron 8, GU was replaced with GC.

〔サルCYP2C遺伝子座のゲノム構造〕
新規mfCYP2Cの種特異性を確認するために、サルのCYP2C BACクローンを用いて、ゲノムにおける新規mfCYP2C遺伝子座を解析した。カニクイザルのBACライブラリーは入手不可能であったため、代わりにアカゲザルのBACライブラリーを使用した。
アカゲザルBACライブラリー（BACPAC）を、CYP2C9v3及び新規mfCYP2CのcDNAをプローブとしてスクリーニングし、CYP2Cを含むBACクローンを単離した。ライブラリー・フィルターとのハイブリダイゼーションは、BACPACの指示書に従って行い、プローブは、［α］³²P-dCTP（Amersham Biosciences社）の存在下で、RadPrime DNA labeling system（Invitrogen社）を用いて合成した。同定されたBACクローンをBACPACより入手し、BAC DNAをDNA PhasePrep^TM BAC DNA Kit（Sigma-Aldrich社）により精製した。各BAC DNAに含まれるCYP2C遺伝子を同定するため、精製されたDNAをテンプレートとして、AmpliTaq Gold DNA p
olymerase（Applied Biosystems社）及び各遺伝子の5'及び3'末端に特異的なプライマーを用いて、PCRを行った。
プライマーペアは、各遺伝子のエクソン1及び9で設計し、位置は、macaqueのcDNAとヒトのCYP2C遺伝子とを比較して決定した。相同性の高いCYP2C9v1及びCYP2C9v3では、設計したプライマーは遺伝子特異的な増幅パターンを示さなかった。そこで、macaqueゲノムデータを検索してこれらの遺伝子の遺伝子特異的indelを同定し、それらのindel（insertion/deletion）を認識するプライマーを設計した。
使用したプライマーを表４に示す。Grayらの方法（Gray IC et al., Genomics, (1995) 28:328-332）に従い、増幅パターンを用いてゲノムにおけるCYP2C遺伝子のアレンジメントを決定した。同様の目的で、BACPACの指示書に従い、DNAは、BAC end sequencing及びBamHIまたはEcoRIによる制限酵素マッピングにも用いた。

この実験の過程で、アカゲザルのゲノムの一部の配列が明らかにされ、ヒトCYP2C18遺伝子の塩基配列と非常に相同性が高い遺伝子が存在することがわかった。そこで、このCYP2C18様遺伝子に特異的なプライマーを設計して、BACクローンの増幅に使用した。
各遺伝子の増幅パターンから、5つのCYP2C遺伝子は、サルゲノムでは一つのクラスタ内に存在することがわかった（図８）。さらに、BAC end sequencing及び制限酵素マッピングにより、新規mfCYP2C（図中CYP2C76で表される）がそのクラスタの末端、即ち、ヒトゲノムにおけるCYP2Cクラスタとそれに隣接する遺伝子の間の領域に位置することが確認された。この結果は、新規mfCYP2C遺伝子が種特異的に存在することを強く裏付けるものである。
〔遺伝子発現の組織分布解析〕
新規mfCYP2C、CYP2C20、CYP2C9v1、及びCYP2C9v3の遺伝子発現の組織分布を決定するため、脳、肺、心臓、肝臓、腎臓、副腎、空腸、精巣、卵巣及び子宮由来のRNAを用いて、リアルタイムRT-PCRを行った。
各遺伝子に特異的なプライマー及びTaqMan（登録商標）MGBプローブは、Primer Express sftware（Applied Biosystems社）を用いて設計し、Applied Biosystems社で合成された。各プライマー及びプローブの塩基配列を表５に示す。

プローブは、5'末端をFAM fluorescence reporter dyeにより標識した。RT反応は、ランダムプライマーを用いて行い、得られた反応産物を25倍に希釈してPCRに供した。PCRは、全量25μlとしてTaqMan Universal PCR Master Mix（Applied Biosystems社）を用い、ABI Prism 7700 sequence detection system（Applied Biosystems社）により行った。各プライマーの最終濃度は、CYP2C20、及びCYP2C9v1は0.3μM、CYP2C9v3は0.9μM、新規mfCYP2Cは0.1μMとした。全CYP2Cに対し、プローブの最終濃度は0.25μMとした。サーマルサイクラーの条件は、50℃で2分、95℃で10分の後、95℃15秒、60℃1分を40サイクルとした。
すべてのCYP2C遺伝子に対し、アッセイの特異性の確認は、予想されたサイズの目的PCR産物が単一のバンドとしてゲル上にあらわれること、及び、標的遺伝子のcDNAプラスミドが、他のCYP2C遺伝子に対し少なくとも500倍増幅されることを確かめることにより、行った。各遺伝子の相対的な発現レベルは、Applied Byiosystems社製のキットにより、18SリボソームRNAレベルを測定することにより標準化した。各遺伝子について、少なくとも3回の増幅を行った。
結果を図９に示す。新規mfCYP2C（図中CYP2C76で表される）、肝臓における発現が圧倒的に多かったが、他の組織でも発現が見られた。また、追加の解析により、心臓、筋肉、脳等、通常P450が発現しない種々の組織においても新規mfCYP2Cの発現が見られ、また雄と雌では約2倍の発現量の差があることが認められた（図１０）。
〔新規mfCYP2C遺伝子のためのレポーター・プラスミドの構築および配列解析〕
Inverse PCRにより、5'フランキング領域を同定した。以下に、方法を説明する。まず、各カニクイザル由来のゲノムDNAの1μgをPstI存在下、37℃で一晩消化した。この生成物を、フェノール：クロロホルム（1：1）の溶媒で抽出精製した後、エタノール沈殿を行った。得られたDNAをLigation high（Toyobo社）を用いて16℃でセルフライゲーションさせた後、同様の方法で精製した。精製したDNAは、KOD plus DNA polymerase（Toyobo社）および新規mfCYP2C cDNA配列の5'末端をもとに設計した以下に示す2つのプライマーと共に、PCRに供した。
プライマー
FW: mfCYP2C (5inv3) 5'-TCCTCTCCCCGTTATTGGAA-3' （配列番号：８８）
RV: mfCYP2C (3inv2) 5'-CACCAGGATGATGAAGAGATCC-3' （配列番号：８９）
増幅したDNAを1％アガロースゲルでゲル精製した後、mfCYP2C（5inv3）を用いてシーケンスを行い、5'フランキング領域の最末端の塩基配列を決定した。この配列に基づき、2つのプライマーをさらに設計した。
プライマー
FW: mfCYP2C (5flk1aK) 5'-CGGGGTACCGCAGGCCAACATTCAAATTC-3'（配列番号：９０）
RV: mfCYP2C (3flk1aH) 5'-CCCAAGCTTGCTGGGCTCTTTGAAAAC-3'（配列番号：９１）
PCRは上述の方法で行い、PCR産物はpGL3-basicベクター（Promega社）にクローニングした。インサートの全長の塩基配列を決定した後、決定した配列では、TRANSFACを使用した調節エレメント探索を行い、構築物は、レポーターアッセイに用いた。
TRANSFACプログラムによる解析では、主要な肝臓の転写因子の結合部位が見出された（threshold score≧75.0）（図１１）。各結合部位の位置（location）は、転写開始部位をゼロとして示されている。
〔新規mfCYP2C遺伝子の5'フランキング領域のレポーターアッセイ〕
HepG2細胞を独立行政法人理化学研究所から購入し、Dulbecco's modified Eagle's Medium（日水製薬株式会社）に、10％のウシ胎児血清（Cambrex Bioscience Walkersville社）、非必須アミノ酸（ICN社）および1mMのピルビン酸ナトリウム（Invitrogen社）を添加した培地で、37℃5％CO₂供給下で培養した。12ウェルのプレートに、2×10⁵個/ウェルとなるように移した後、24時間インキュベートした。
次に、これらの細胞を、レポーター構築物（0.45μg）、pRL-SV40（0.05μg；Promega社；内部標準）により形質転換した（FuGENE6（Roche Diagnostics社）を使用）。アッセイの際、必要に応じて、肝臓の主要な転写因子であるHNF1α、HNF3βおよびHNF4αのoverexpression vectorを加えた。
48時間後、これらの細胞をリン酸緩衝生理食塩水で洗浄し、製品の指示書に従ってdual-luciferase assay（Promega社）に供した。
結果を図２２に示す。
すべての転写因子が新規mfCYP2Cの発現を著しく亢進させ、中でもHNF1αによる効果が最も大きかった。これらの結果は、新規mfCYP2C遺伝子の発現調節にこれらの転写因子が重要な機能を果たすことを示す。また、新規mfCYP2Cが、RT-PCRで解析した組織の中で、肝臓において高く発現していた結果と一致する。
〔E.Coliにおける4つのサルCYP2Cタンパク質の発現〕
新規mfCYP2C、mfCYP2C9v1、mfCYP2c9v3およびCYP2C20のcDNAを用いて、Iwataらの方法（Iwata, H. et al., Biochem Pharmacol (1998) 55:1315-1325）に従って各タンパク質を発現させた。タンパク質の発現レベルを上昇させるために、各cDNAのオープン・リーディング・フレーム（ORF）を、表６に示すプライマーとKOD Plus DNA polymerase（Toyobo社）を用いて増幅することにより、N末端の8残基を、ウシCYP17のN末端の8残基であるMALLLAVFに置換した（Barnes, HJ et al., Proc Natl Acad Sci USA (1991) 88:5597-5601）。

[Genomic structure of the monkey CYP2C locus]
To confirm the species specificity of the novel mfCYP2C, a novel mfCYP2C locus in the genome was analyzed using monkey CYP2C BAC clones. Since the cynomolgus monkey BAC library was not available, the rhesus monkey BAC library was used instead.
Rhesus monkey BAC library (BACPAC) was screened using CYP2C9v3 and novel mfCYP2C cDNA as probes, and a BAC clone containing CYP2C was isolated. Hybridization with the library filter was performed according to the instructions of BACPAC, and the probe was synthesized using RadPrime DNA labeling system (Invitrogen) in the presence of [α] ³² P-dCTP (Amersham Biosciences). . The identified BAC clone was obtained from BACPAC, and BAC DNA was purified by DNA PhasePrep ^™ BAC DNA Kit (Sigma-Aldrich). To identify the CYP2C gene contained in each BAC DNA, AmpliTaq Gold DNA p using purified DNA as a template
PCR was performed using olymerase (Applied Biosystems) and primers specific for the 5 ′ and 3 ′ ends of each gene.
The primer pair was designed with exons 1 and 9 of each gene, and the position was determined by comparing the macaque cDNA with the human CYP2C gene. For the highly homologous CYP2C9v1 and CYP2C9v3, the designed primers did not show a gene-specific amplification pattern. Therefore, we searched the macaque genome data to identify gene-specific indels of these genes and designed primers that recognize these indels (insertion / deletion).
The primers used are shown in Table 4. According to the method of Gray et al. (Gray IC et al., Genomics, (1995) 28: 328-332), the arrangement of CYP2C genes in the genome was determined using the amplification pattern. For the same purpose, DNA was also used for BAC end sequencing and restriction enzyme mapping with BamHI or EcoRI according to BACPAC instructions.

In the course of this experiment, the sequence of a part of the rhesus monkey genome was clarified, and it was found that there was a gene having a very high homology with the nucleotide sequence of the human CYP2C18 gene. Therefore, a primer specific to this CYP2C18-like gene was designed and used to amplify the BAC clone.
From the amplification pattern of each gene, it was found that five CYP2C genes exist in one cluster in the monkey genome (FIG. 8). In addition, BAC end sequencing and restriction enzyme mapping confirmed that the novel mfCYP2C (represented by CYP2C76 in the figure) is located at the end of the cluster, ie, the region between the CYP2C cluster in the human genome and its adjacent gene. It was. This result strongly supports the existence of a novel mfCYP2C gene in a species-specific manner.
[Tissue distribution analysis of gene expression]
Real-time RT-PCR using RNA from brain, lung, heart, liver, kidney, adrenal gland, jejunum, testis, ovary and uterus to determine the tissue distribution of gene expression of novel mfCYP2C, CYP2C20, CYP2C9v1, and CYP2C9v3 Went.
Primers specific to each gene and TaqMan® MGB probe were designed using Primer Express sftware (Applied Biosystems) and synthesized by Applied Biosystems. Table 5 shows the base sequences of each primer and probe.

The probe was labeled at the 5 ′ end with FAM fluorescence reporter dye. The RT reaction was performed using random primers, and the resulting reaction product was diluted 25 times and subjected to PCR. PCR was performed by ABI Prism 7700 sequence detection system (Applied Biosystems) using TaqMan Universal PCR Master Mix (Applied Biosystems) in a total volume of 25 μl. The final concentration of each primer was 0.3 μM for CYP2C20 and CYP2C9v1, 0.9 μM for CYP2C9v3, and 0.1 μM for new mfCYP2C. The final probe concentration was 0.25 μM for all CYP2C. The thermal cycler conditions were 50 ° C for 2 minutes, 95 ° C for 10 minutes, 95 ° C for 15 seconds, and 60 ° C for 1 minute for 40 cycles.
For all CYP2C genes, the specificity of the assay is confirmed by confirming that the target PCR product of the expected size appears on the gel as a single band, and that the target gene cDNA plasmid is relative to other CYP2C genes. This was done by making sure that it was amplified at least 500 times. The relative expression level of each gene was standardized by measuring 18S ribosomal RNA levels with a kit manufactured by Applied Byiosystems. At least three amplifications were performed for each gene.
The results are shown in FIG. New mfCYP2C (represented by CYP2C76 in the figure) and liver were overwhelmingly expressed, but expression was also observed in other tissues. In addition, additional analysis shows that new mfCYP2C is expressed in various tissues that do not normally express P450, such as the heart, muscle, and brain. (FIG. 10).
[Construction and sequence analysis of reporter plasmid for novel mfCYP2C gene]
The 5 ′ flanking region was identified by Inverse PCR. The method will be described below. First, 1 μg of genomic DNA derived from each cynomolgus monkey was digested overnight at 37 ° C. in the presence of PstI. This product was extracted and purified with a solvent of phenol: chloroform (1: 1), followed by ethanol precipitation. The obtained DNA was self-ligated at 16 ° C. using Ligation high (Toyobo) and then purified by the same method. The purified DNA was subjected to PCR together with KOD plus DNA polymerase (Toyobo) and the following two primers designed based on the 5 ′ end of the novel mfCYP2C cDNA sequence.
Primer
FW: mfCYP2C (5inv3) 5'-TCCTCTCCCCGTTATTGGAA-3 '(SEQ ID NO: 88)
RV: mfCYP2C (3inv2) 5'-CACCAGGATGATGAAGAGATCC-3 '(SEQ ID NO: 89)
The amplified DNA was gel purified on a 1% agarose gel and then sequenced using mfCYP2C (5inv3) to determine the most terminal nucleotide sequence of the 5 ′ flanking region. Two primers were further designed based on this sequence.
Primer
FW: mfCYP2C (5flk1aK) 5'-CGGGGTACCGCAGGCCAACATTCAAATTC-3 '(SEQ ID NO: 90)
RV: mfCYP2C (3flk1aH) 5'-CCCAAGCTTGCTGGGCTCTTTGAAAAC-3 '(SEQ ID NO: 91)
PCR was performed as described above, and the PCR product was cloned into the pGL3-basic vector (Promega). After determining the base sequence of the full length of the insert, the determined sequence was subjected to a regulatory element search using TRANSFAC, and the construct was used for a reporter assay.
Analysis by the TRANSFAC program found major liver transcription factor binding sites (threshold score ≧ 75.0) (FIG. 11). The location of each binding site is indicated with the transcription start site as zero.
[Reporter assay of 5 'flanking region of novel mfCYP2C gene]
HepG2 cells were purchased from RIKEN, Dulbecco's modified Eagle's Medium (Nissui Pharmaceutical Co., Ltd.), 10% fetal bovine serum (Cambrex Bioscience Walkersville), non-essential amino acids (ICN) and 1 mM pyrvin In a medium supplemented with sodium acid (Invitrogen), the cells were cultured at 37 ° C. under 5% CO ₂ supply. After transferring to a 12-well plate at 2 × 10 ⁵ cells / well, the plate was incubated for 24 hours.
These cells were then transformed with the reporter construct (0.45 μg), pRL-SV40 (0.05 μg; Promega; internal standard) (using FuGENE6 (Roche Diagnostics)). During the assay, overexpression vectors of HNF1α, HNF3β and HNF4α, which are the major transcription factors of the liver, were added as necessary.
After 48 hours, these cells were washed with phosphate buffered saline and subjected to dual-luciferase assay (Promega) according to the product instructions.
The results are shown in FIG.
All transcription factors markedly enhanced the expression of the novel mfCYP2C, with HNF1α being the most effective. These results indicate that these transcription factors play an important function in regulating the expression of a novel mfCYP2C gene. Moreover, it is in agreement with the result that novel mfCYP2C was highly expressed in the liver in the tissues analyzed by RT-PCR.
[Expression of four monkey CYP2C proteins in E. Coli]
The novel mfCYP2C, mfCYP2C9v1, mfCYP2c9v3 and CYP2C20 cDNAs were used to express each protein according to the method of Iwata et al. (Iwata, H. et al., Biochem Pharmacol (1998) 55: 1315-1325). In order to increase the protein expression level, the open reading frame (ORF) of each cDNA was amplified using the primers shown in Table 6 and KOD Plus DNA polymerase (Toyobo), thereby leaving 8 residues at the N-terminus. The group was replaced with MALLLAVF, the N-terminal 8 residue of bovine CYP17 (Barnes, HJ et al., Proc Natl Acad Sci USA (1991) 88: 5597-5601).

NdeI認識部位とXbaI認識部位をそれぞれ含むフォワードおよびリバースプライマーを用いることにより、PCR産物を、Iwataらが、ヒト・レダクターゼcDNAと共発現させるようにpCWベクター（Barnes, HJ., Methods Enzymol (1996) 272:3-14）をもとに作製したベクターに直接クローニングした。本発明者らは、この構築物をそれぞれのcDNAの由来に因んで、pCW-CYP2C20/OR、pCW-mfCYP2C9v1/OR、pCW-mfCYP2C9v2/ORおよびpCW-新規mfCYP2C/ORと名付けた。
DH5α competent cells（Invitrogen社）を形質転換した後、インサートの塩基配列と配向性をシーケンシングにより確認した。バクテリアは、100μg/mlのアンピシリンを含むLuria-bertani broth中で一晩培養した後、Iwataらの方法（1998）に従って調整した。Terrific Brothで100倍に希釈し、200μg/mlのアンピシリン存在下、30℃で6時間から12時間、OD₆₀₀が約0.6〜0.8になるまで培養した。
続いてイソプロピル-B-D-チオガラクトシド(IPTG)を最終濃度が1.5mMとなるように培養液に添加した。16時間から20時間後、公知の方法（Omura, T. et al., J Biol Chem (1964) 239:2379-2385）に従い、U-3000 spectrophotometer（Shimadzu社）を使用して、培養物のP450スペクトルを求めた。結果を図１３に示す。
P450の発現を確認した後、培養した細胞を採取し、細胞膜画分を調製した。そして、Iwataらの方法（1998）に従って、P450およびNAPDH-P450レダクターゼの濃度を測定した。
〔4つのサルCYP2Cの代謝活性〕
すべて組換えCYP2Cタンパク質を用いて、4つの基質、taxol、tolbutamide、S-mephenytoinおよびtestosteroneに対する新規mfCYP2C、CYP2C20、CYP2C9v1、及びCYP2C9v3の代謝活性を解析した。
反応混合物は、以下のように調製した。まず、¹⁴C-taxol (6μM)、¹⁴C-tolbutamide (100μM)、¹⁴C-testosterone (50μM)、¹⁴C-S-mephenytoin (50μM)と、サル肝ミクロゾーム若しくは精製した組換えCYP2Cとを、チューブに用意し、37℃で5分、インキュベートした。続いて、代謝反応を開始させるため、1.3mMのNADP+、3.3mMのグルコース-6-リン酸、0.4U/mlのグルコース-6-リン酸デヒドロゲナーゼ、および3.3mMの塩化マグネシウを溶解させた100mMリン酸ナトリウム緩衝液を含むNADPH再生系を添加した。最終濃度は、1mg protein/mL、または200pmol P450/mLであった。
taxolとtestosteroneについては37℃で15分、S-mephenytoinについては45分、tolbutamideについては60分インキュベートした後、等量のメタノールを加えて反応を停止させた。組換えmfCYP2Cタンパク質の場合は、いずれの化合物についてもインキュベーションは30分とした。反応終了後、サンプルは、遠心分離され、各アリコットは、乾燥するまで上清を蒸発させ、残渣を15μlのメタノールに溶解させた。
6α-hydroxytaxolと3-hydroxytaxolの分析は、藤野らの方法（Fujino, H. et al., J Chromatogr B Biomed Sci Appl (2001) 757:143-150）に従って行った。各上清2μlずつを、TLCプレート(Silicagel 60F254, 20x20 cm, Merck)にスポットし、展開溶媒（トルエン‐アセトン‐ギ酸(60:39:1,v/v/v)）と共に、溶媒蒸気で飽和させた水平なTLC展開槽に12cm展開した。
6β-hydroxytestosteroneの場合は、展開溶媒をジクロロメタン−アセトン（4:1,v/v）とし、16cm展開した。
4-hydroxytolbutamideは、Ludwigらの方法（Ludwig, E. et al., J Chromatogr B Biomed Sci Appl (1998) 707:347-350）に従って、展開溶媒をトルエン−アセトン−ギ酸(60:39:1,v/v/v)とし、10cm展開した。
S-mephenytoin 4-hydroxylase活性は、Shimadaらの方法（Shimada, T. et al., Anal Biochem (1985) 147:174-179）に従って測定し、展開溶媒をクロロホルム−メタノール−28％アンモニア(90:10:1,v/v/v)とし12cm展開した。
TLCプレートを乾燥し、phosphor imaging plate (IP)に接触させて12時間静置した。化合物と代謝産物の量の変化は、BAS-2500（富士フイルム社）を使用して測定した。放射性を示す代謝産物のRf値が、標識なしの標準に比較して明確に識別された。
結果を図１４に示す。レーン1はサルの肝ミクロゾーム、レーン2はCYP2C20、レーン3はCYP2C9v1、レーン4はCYP2C9v3、レーン5は新規mfCYP2Cである。
新規mfCYP2Cは、4つの基質のうち、tolbutamideとtestosteroneを代謝することが確認された。
これに対し、mfCYP2C20は、taxolを代謝したが、他の化合物に対しては代謝活性を有しなかった。また、mfCYP2C9v3はtolbutamide、S-mephenytoin、testosteroneを、mfCYP2C9v1は、S-mephenytoinとtestosteroneを代謝することが確認された。
新規mfCYP2C、mfCYP2C9v1、mfCYP2C9v3は、いずれもtestosteroneを基質とした代謝活性を示したが、代謝産物は同一ではなかった。
以上から、サル特異的な新規mfCYP2Cタンパク質が、ある種の薬物に対して代謝活性を示すことが確認された。このことは、新規mfCYP2Cがサルとそれ以外の霊長類における代謝パターンの相違の一因となっていることを示唆するものである。
〔抗新規mfCYP2C抗体の作製及びイムノブロッティング〕
新規mfCYP2Cに対するポリクローナル抗体は、このタンパク質の特異的なアミノ酸配列を有するペプチドを合成し、このペプチドでウサギを免疫することによって、NeoMPS社（SanDiego,CA）で産生された。まず、4つのmfCYP2Cの配列を比較することによって選択した特異的なアミノ酸配列をもとに以下の2つのペプチドを合成した。
（A） H-CQLNTKNISKSISMLA-NH₂ （配列番号：９８）
（B） H-CLYNAFPHLRVL-NH₂ （配列番号：９９）
合成したペプチドは精製し、C末端のシステイン残基のチオール基を介してKeyhole Limpet Hemocyanin（キャリアタンパク質）に結合させ、New Zealand White Rabbitを免疫し、ウサギ抗新規mfCYP2C抗体を得た（図１５）。
遺伝子組換えP450タンパク質として、サルCYP2CからCYP2C20、CYP2C9v1、CYP2C9v3、及び新規mfCYP2C、またヒトCYP2CからCYP2C8、CYP2C9、CYP2C18、及びCYP2C19（各1.0pmol）を10％SDSポリアクリルアミドゲルで電気泳動し、Hybond-P filter（Amersham Biosciences社）上に移行した。このフィルターを用いて、ウサギ抗新規mfCYP2C抗体（1:250）、及びホースラディッシュペルオキシダーゼにコンジュゲートされたロバ抗ウサギIgG抗体（SantaCruz Biotechnology社）を用いたイムノブロッティングを行った。製品の指示書に従い、ECL Western Blotting detection reagent（Amersham Biosciences社）により可視化したところ、抗体（Ａ）を使用した場合のみ、新規mfCYP2C（CYP2C76）に相当するバンドが1つのみ得られ、免疫特異性が確認された（図１６（Ａ））。
また、ヒト、チンパンジー、オランウータン、カニクイザル、及びアカゲザルの5種類の霊長類から調製した肝ミクロソームを使用した抗新規mfCYP2C（CYP2C76）抗体によるイムノブロッティングも行った。期待されたサイズのバンドが、カニクイザル及びアカゲザルのサンプルにおいてのみ確認され、新規mfCYP2C遺伝子の発現パターンと一致した結果が得られた（図１６（Ｂ））。
〔免疫組織化学〕
カニクイザルの肝臓の切片を用いて、標準的方法に従い、抗新規mfCYP2C抗体による免疫組織化学染色を行った。一次抗体は50倍に希釈し、切片に接触させて4℃で一晩放置した。結合した抗体を、EnVision+System及びliquid diaminobenzidine（いずれもDakoCytomation社）を用い、製品の指示書に従って検出した。スライドを、harris hematoxylinにより対照染色した。ネガティブ・コントロールとして、ウサギのpreimmune血清を一次抗体の代わりに使用した。免疫組織化学特異性を検証するために、抗体は、過剰量の新規mfCYP2C特異的ペプチド（0.05mg/ml）と共に4℃で一晩プレインキュベートし、一次抗体の代わりに使用した。この結果、肝細胞は強く染色されたが、胆管や血管の細胞は染色されなかった。ブロッキングペプチドまたは免疫前血清の存在下では、染色は見られず（図１７）、染色が新規mfCYP2Cに特異的であることが示唆された。

By using forward and reverse primers containing an NdeI recognition site and an XbaI recognition site, respectively, the PCR product can be co-expressed with the human reductase cDNA by the pCW vector (Barnes, HJ., Methods Enzymol (1996) 272: 3-14) and cloned directly into the vector prepared. We named this construct pCW-CYP2C20 / OR, pCW-mfCYP2C9v1 / OR, pCW-mfCYP2C9v2 / OR and pCW-new mfCYP2C / OR, due to the origin of the respective cDNAs.
After transforming DH5α competent cells (Invitrogen), the base sequence and orientation of the insert were confirmed by sequencing. Bacteria were cultured overnight in Luria-bertani broth containing 100 μg / ml ampicillin and then prepared according to the method of Iwata et al. (1998). The resultant was diluted 100-fold with Terrific Broth and cultured in the presence of 200 μg / ml ampicillin at 30 ° C. for 6 to 12 hours until the OD ₆₀₀ was about 0.6 to 0.8.
Subsequently, isopropyl-BD-thiogalactoside (IPTG) was added to the culture solution to a final concentration of 1.5 mM. After 16 to 20 hours, according to known methods (Omura, T. et al., J Biol Chem (1964) 239: 2379-2385), using a U-3000 spectrophotometer (Shimadzu), P450 of the culture The spectrum was determined. The results are shown in FIG.
After confirming the expression of P450, the cultured cells were collected and a cell membrane fraction was prepared. Then, the concentrations of P450 and NAPDH-P450 reductase were measured according to the method of Iwata et al. (1998).
[Metabolic activity of four monkeys CYP2C]
All the recombinant CYP2C proteins were used to analyze the metabolic activity of novel mfCYP2C, CYP2C20, CYP2C9v1, and CYP2C9v3 against four substrates, taxol, tolbutamide, S-mephenytoin and testosterone.
The reaction mixture was prepared as follows. First, ¹⁴ C-taxol (6 μM), ¹⁴ C-tolbutamide (100 μM), ¹⁴ C-testosterone (50 μM), ¹⁴ CS-mephenytoin (50 μM) and monkey liver microsome or purified recombinant CYP2C are prepared in a tube. And incubated at 37 ° C. for 5 minutes. Subsequently, 100 mM phosphorus in which 1.3 mM NADP +, 3.3 mM glucose-6-phosphate, 0.4 U / ml glucose-6-phosphate dehydrogenase, and 3.3 mM magnesium chloride were dissolved to initiate the metabolic reaction. NADPH regeneration system containing sodium acid buffer was added. The final concentration was 1 mg protein / mL or 200 pmol P450 / mL.
Taxol and testosterone were incubated at 37 ° C. for 15 minutes, S-mephenytoin for 45 minutes, and tolbutamide for 60 minutes, and then the reaction was stopped by adding an equal amount of methanol. In the case of recombinant mfCYP2C protein, the incubation time was 30 minutes for all compounds. At the end of the reaction, the sample was centrifuged and each aliquot was evaporated to dryness and the residue was dissolved in 15 μl of methanol.
Analysis of 6α-hydroxytaxol and 3-hydroxytaxol was performed according to the method of Fujino et al. (Fujino, H. et al., J Chromatogr B Biomed Sci Appl (2001) 757: 143-150). 2 μl of each supernatant is spotted on a TLC plate (Silicagel 60F254, 20x20 cm, Merck) and saturated with solvent vapor with developing solvent (toluene-acetone-formic acid (60: 39: 1, v / v / v)) It was developed 12 cm in a horizontal TLC development tank.
In the case of 6β-hydroxytestosterone, the developing solvent was dichloromethane-acetone (4: 1, v / v) and developed for 16 cm.
4-Hydroxytolbutamide was developed according to the method of Ludwig et al. (Ludwig, E. et al., J Chromatogr B Biomed Sci Appl (1998) 707: 347-350), and the developing solvent was toluene-acetone-formic acid (60: 39: 1, v / v / v) and developed 10 cm.
S-mephenytoin 4-hydroxylase activity was measured according to the method of Shimada et al. (Shimada, T. et al., Anal Biochem (1985) 147: 174-179), and the developing solvent was chloroform-methanol-28% ammonia (90: 10: 1, v / v / v) and developed 12 cm.
The TLC plate was dried and allowed to stand for 12 hours in contact with a phosphor imaging plate (IP). Changes in the amount of compound and metabolites were measured using BAS-2500 (Fujifilm). The Rf values for radioactive metabolites were clearly identified compared to unlabeled standards.
The results are shown in FIG. Lane 1 is monkey liver microsome, lane 2 is CYP2C20, lane 3 is CYP2C9v1, lane 4 is CYP2C9v3, and lane 5 is new mfCYP2C.
New mfCYP2C was confirmed to metabolize tolbutamide and testosterone among the four substrates.
In contrast, mfCYP2C20 metabolized taxol but did not have metabolic activity against other compounds. It was also confirmed that mfCYP2C9v3 metabolizes tolbutamide, S-mephenytoin and testosterone, and mfCYP2C9v1 metabolizes S-mephenytoin and testosterone.
The new mfCYP2C, mfCYP2C9v1, and mfCYP2C9v3 all showed metabolic activity using testosterone as a substrate, but the metabolites were not identical.
From the above, it was confirmed that the monkey-specific novel mfCYP2C protein exhibits metabolic activity against certain drugs. This suggests that the novel mfCYP2C contributes to differences in metabolic patterns between monkeys and other primates.
[Preparation and immunoblotting of anti-new mfCYP2C antibody]
A novel polyclonal antibody against mfCYP2C was produced at NeoMPS (San Diego, Calif.) By synthesizing a peptide having a specific amino acid sequence of this protein and immunizing a rabbit with this peptide. First, the following two peptides were synthesized based on specific amino acid sequences selected by comparing the sequences of four mfCYP2Cs.
(A) H-CQLNTKNISKSISMLA-NH ₂ (SEQ ID NO: 98)
(B) H-CLYNAFPHLRVL-NH ₂ (SEQ ID NO: 99)
The synthesized peptide was purified, bound to Keyhole Limpet Hemocyanin (carrier protein) via the thiol group of the C-terminal cysteine residue, and immunized with New Zealand White Rabbit to obtain a rabbit anti-new mfCYP2C antibody (FIG. 15). .
As a recombinant P450 protein, monkey CYP2C to CYP2C20, CYP2C9v1, CYP2C9v3, and novel mfCYP2C, and human CYP2C to CYP2C8, CYP2C9, CYP2C18, and CYP2C19 (each 1.0 pmol) were electrophoresed on a 10% SDS polyacrylamide gel, Hybond -P filter (Amersham Biosciences) was transferred. Using this filter, immunoblotting was performed using a rabbit anti-new mfCYP2C antibody (1: 250) and a donkey anti-rabbit IgG antibody conjugated to horseradish peroxidase (SantaCruz Biotechnology). When visualized with ECL Western Blotting detection reagent (Amersham Biosciences) according to the product instructions, only one band corresponding to the novel mfCYP2C (CYP2C76) was obtained only when the antibody (A) was used. Was confirmed (FIG. 16A).
In addition, immunoblotting with an anti-novel mfCYP2C (CYP2C76) antibody using liver microsomes prepared from five primates of human, chimpanzee, orangutan, cynomolgus monkey, and rhesus monkey was also performed. A band of the expected size was confirmed only in the cynomolgus monkey and rhesus monkey samples, and the results were consistent with the expression pattern of the novel mfCYP2C gene (FIG. 16 (B)).
[Immunohistochemistry]
Immunohistochemical staining with anti-novel mfCYP2C antibody was performed using a section of cynomolgus monkey liver according to standard methods. The primary antibody was diluted 50 times, contacted with the section and left at 4 ° C. overnight. The bound antibody was detected using EnVision + System and liquid diaminobenzidine (both from DakoCytomation) according to the product instructions. Slides were control stained with harris hematoxylin. As a negative control, rabbit preimmune serum was used in place of the primary antibody. To verify immunohistochemical specificity, antibodies were preincubated overnight at 4 ° C. with an excess of novel mfCYP2C specific peptide (0.05 mg / ml) and used in place of the primary antibody. As a result, hepatocytes were strongly stained, but bile duct and blood vessel cells were not stained. In the presence of blocking peptide or preimmune serum, no staining was seen (FIG. 17), suggesting that the staining is specific for the novel mfCYP2C.

The upper part of FIG. 1 shows the nucleotide sequence (SEQ ID NO: 1) of the novel mfCYP2C cDNA, and the lower part shows the amino acid sequence (SEQ ID NO: 2) of the novel mfCYP2C protein. A region that is estimated to be a heme coupling region is surrounded by a frame. The extension (281 bases) in the transcript variant with a long 3 'untranslated region is underlined by Alternative polyadenylation. The amino acid sequence drawn with a broken line is the sequence of the peptide used in producing the anti-new mfCYP2C antibody. The cDNA is 1666 bases long and contains an open reading frame of 489 amino acid residues. It is a multiple alignment of the amino acid sequences of four monkey CYP2C proteins. From the top, the amino acid sequences of CYP2C20 (SEQ ID NO: 11), mfCYP2C9v1 (SEQ ID NO: 12), mfCYP2C9v3 (SEQ ID NO: 13), and novel mfCYP2C (SEQ ID NO: 14) are shown. The new mfCYP2C has 489 amino acid residues and is one residue shorter than other monkey CYP2C proteins and human CYP2C proteins. Draw using CYP2C amino acid sequences of chicken (c), human (h), pig (pig), dog (dog), rabbit (rab), mouse (m), cynomolgus monkey (mf), rat (r) It is a molecular evolutionary tree. The sequence of the novel mfCYP2C has been shown to be more similar to that of other animal species than humans. It is a multiple alignment of cDNA of a novel mfCYP2C homolog in other monkeys. From the top, the base sequences of cynomolgus monkey (part of SEQ ID NO: 1), rhesus monkey (SEQ ID NO: 15), African green monkey (SEQ ID NO: 16), and common marmoset (SEQ ID NO: 17) are shown. Due to sequence differences, only rhesus monkeys were able to fully amplify the coding region, and exon 1-4 and exon 2-4 were amplified for African green monkey and common marmoset, respectively. Among the species, only a few residues differed and a homology close to 100% was seen. FIG. 15 is a continuation of FIG. 15 and shows multiple alignment of cDNAs of novel mfCYP2C homologues in other monkeys. It is the multiple alignment of the amino acid sequence of the protein of the novel mfCYP2C homolog in other monkeys. From the top, amino acid sequences of novel mfCYP2C protein homologues of cynomolgus monkey (SEQ ID NO: 2), rhesus monkey (SEQ ID NO: 18), African green monkey (SEQ ID NO: 19), and common marmoset (SEQ ID NO: 20) are shown. It is a novel mfCYP2C exon-intron structure determined by performing long-distance PCR using gene-specific primers designed on each exon and analyzing the sequence of all introns. The new mfCYP2C gene was confirmed to contain 9 exons, similar to the human CYP2C gene. The structure of the monkey CYP2C gene determined by PCR amplification pattern of CYP2C BAC clone, restriction enzyme mapping, and end-sequencing is shown. Note that CYP2C9v1 and CYP2C9v3 (represented as CYP2C43 and CYP2C75 in the figure, respectively) are highly homologous to each other and the positions on the genome could not be determined, so they are drawn in a tentative order. A new monkey-specific mfCYP2C (designated CYP2C76 in the figure) is located at the end of the CYP2C cluster, which is the region between genes in the corresponding region of the human genome. Dashed lines indicate BAC clones where no clear amplification occurred. The tissue distribution of monkey CYP2C gene expression is shown. Real-time RT-PCR was performed using total RNA from 10 tissue samples. All CYP2Cs had the highest expression in the liver, and the expression of the new mfCYP2C (CYP2C76) was most prominent. It is the result of measuring the difference in the expression level of novel mfCYP2C in males and females. The expression level of males was about twice that of females. DNA binding elements for the major liver-specific transcription factors (HNF1α, HNF3β, HNF4α) identified by the TRNASFAC program (threshold score ≧ 75.0). The position of each element is calculated with the transfer start point as zero. The approximately 1 kb 5 ′ flanking region was analyzed by luciferase assay. The assay was performed using the pGL-1 kb construct in HepG2 cells and transcription factors were added as needed. All factors, especially HNF1α, markedly enhanced the expression of novel mfCYP2C. It is CO-difference spectra confirming the expression of recombinant novel mfCYP2C protein in E. coli. A peak peculiar to P450 protein (450 nm) can be confirmed. The metabolic activity of monkey CYP2C expressed in E. coli is shown. Lanes 1-5 show monkey liver microsomes, CYP2C20, CYP2C9v1, CYP2C9v3, and novel mfCYP2C, respectively. It is an antibody produced by immunizing a rabbit with a synthetic peptide having the amino acid sequence shown in FIG. FIG. 25A shows an antibody obtained by using peptide H-CQLNTKNISKSISMLA-NH ₂ (SEQ ID NO: 29) as an antigen, and FIG. B shows an antibody obtained by using peptide H-CLYNAFPHLRVL-NH ₂ (SEQ ID NO: 30) as an antigen. A. The result of the immunoblotting using an anti- novel mfCYP2C antibody is shown. Eight types of monkey and human P450 were electrophoresed and transferred to a PVDF filter to bind an anti-new mfCYP2C antibody. Only the new mfCYP2C (CYP2C76) band could be detected. B. A novel mfCYP2C homologue in liver microsomes of multiple animals was searched for in the same manner as in A. PDI protein as a control was detected in all samples, whereas the new mfCYP2C protein was detected only in cynomolgus and rhesus monkeys. The result of the immunohistochemical dyeing | staining of the novel mfCYP2C protein in a liver is shown. (A) shows immunostaining with anti-novel mfCYP2C antibody, (B) shows the results when pre-treatment with peptide blocking is performed, and (C) shows the results when pre-immune staining is performed. Strong staining in hepatocytes was confirmed, but not in bile duct (arrow) and vein (triangle) epithelial cells. (B) and (C) hardly stained. The solid scale indicates 100 μm.

Claims (14)

The following protein (a) or (b).
(A) A protein comprising the amino acid sequence set forth in SEQ ID NO: 2.
(B) A protein which is a drug metabolizing enzyme, comprising an amino acid sequence in which one or several amino acids are substituted, deleted, added and / or inserted in the amino acid sequence set forth in SEQ ID NO: 2.   A nucleic acid encoding the protein of claim 1. DNA of the following (c) or (d).
(C) DNA comprising the base sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3,
(D) a drug-metabolizing enzyme that hybridizes under stringent conditions with a DNA sequence comprising a base sequence complementary to the DNA comprising the base sequence described in SEQ ID NO: 1 or SEQ ID NO: 3 or a part thereof, or a part thereof. A monkey-derived DNA encoding a protein.   A recombinant vector comprising the DNA according to claim 3.   A transformant comprising the recombinant vector according to claim 4.   The method for producing a protein according to claim 1, wherein the transformant according to claim 5 is cultured, and the protein according to claim 1 is purified from the culture.   An antibody against the protein of claim 1. When the metabolism of the test substance differs between monkeys and non-monkey mammals,
A step A of bringing the protein according to claim 1 into contact with the test substance;
Including the step B of measuring the presence or absence of the protein according to claim 1 with respect to metabolism of the test substance,
A method for evaluating a cause of different metabolism of the test substance.   The method according to claim 8, wherein the protein according to claim 1 used in the step A is produced by the method for producing a protein according to claim 6. In the step B, when the protein according to claim 1 is involved in metabolism of the test substance,
The method according to claim 8 or 9, wherein the metabolic difference of the test substance between the monkey and the mammal other than the monkey is evaluated to be due to a difference in species. When testing the metabolism, efficacy and / or safety of human drug candidates using monkeys as model animals,
Measuring the presence or absence of the involvement of the protein of claim 1 in the metabolism of the drug candidate compound; and
When the drug is involved, the drug, compound, and / or safety test results of the drug candidate compound using monkeys as model animals differ from the metabolism, drug efficacy and / or safety of the drug compound in humans. A method for evaluating metabolic, medicinal and / or safety tests, comprising:   The method according to claim 11, wherein Step C is performed in vitro. A method for evaluating the feasibility of testing metabolism, drug efficacy and / or safety of a human drug candidate compound using a monkey as a model animal,
A step E of contacting the protein of claim 1 with the drug candidate compound;
A step F for measuring the degree of involvement of the protein according to claim 1 with respect to metabolism of the pharmaceutical compound, and when the involvement is a predetermined degree or more, the monkey is tested as a model animal. A method that makes negative evaluations. The kit containing the protein according to claim 1, which is used for evaluation of metabolism, drug efficacy and / or safety of a human drug candidate compound using a monkey as a model animal.

Images