WO2009125851A1

WO2009125851A1 - Method for detection of effectiveness of phenylalanine derivative-type compound in diabetes patient

Info

Publication number: WO2009125851A1
Application number: PCT/JP2009/057395
Authority: WO
Inventors: 義光山▲崎▼; 健遅野井
Original assignee: 株式会社サインポスト; アステラス製薬株式会社
Priority date: 2008-04-11
Filing date: 2009-04-10
Publication date: 2009-10-15
Also published as: JP2011125218A

Abstract

Disclosed is a method for selecting and identifying a diabetes patient for who a phenylalanine derivative-type compound (e.g., nateglinide) is effective based on the gene polymorphism associated with the effectiveness of a therapeutic agent for diabetes comprising the compound as an active ingredient. Also disclosed is a substance which is effective for the treatment of diabetes. The method is achieved by employing, as an indicator, a matter that a diabetes patient of interest has a genotype shown in 'GENOTYPES (1)' with respect to at least one gene selected from the genes (1) to (3).

Description

Phenylalanine derivative-based drug efficacy detection method for diabetic patients

The present invention relates to a method for detecting the effectiveness of an antidiabetic agent comprising a phenylalanine derivative-based drug as an active ingredient for individual diabetic patients based on genetic information of the diabetic patients. The present invention also relates to a therapeutic agent for diabetes comprising a phenylalanine derivative-based drug used as an active ingredient for a specific diabetic patient having a unique genetic polymorphism.

The above-described method of the present invention provides effective and accurate treatment guidelines for treating and improving diabetes to diabetic patients and medical professionals. As a result of selecting therapeutic agents suitable for individual patients, It is useful in that effective and accurate treatment can be performed and the mental, physical and economic burden caused by invalid treatment can be avoided.

According to the 2002 diabetes fact-finding survey by the Ministry of Health, Labor and Welfare (Japan), the number of diabetic patients in Japan is approximately 7.4 million, including the reserve arm that is considered “a person who cannot be denied the possibility of diabetes”. It is estimated that it is about 16.2 million people, one in 6.3 people. The increase in the number of diabetics is said to be greatly affected by changes in dietary habits and aging, and the number of people including the reserves increased by 2.5 million compared to the survey five years ago. Excluding diabetes, it is estimated that the number of people who are strongly suspected will reach 10.8 million.

The main pillars of diabetes treatment are diet, exercise, and medication. However, only 50% of people who are strongly suspected of having diabetes are being treated. In addition, there are not a few diabetic patients who once stop treatment after starting treatment, which is thought to lead to an increase in complications. In addition, there are reports that only about 30% of diabetic patients who have already undergone drug treatment have maintained a good state, and a more effective drug selection method is required.

On the other hand, according to the “National Health Care Expenditure” published by the Ministry of Health, Labor and Welfare (Japan), the medical cost of diabetes in FY 2004 was 1,116.8 billion yen, which would exceed 2 trillion yen in 20 years It is predicted. Diabetes health care costs are on the rise worldwide, and the International Diabetes Federation (IDF) announced in 2003 the global health care costs for adult diabetes was $ 153 billion, and in 2025 that amount would be 213 billion Is estimated to increase to $ 396 billion. Medical costs are particularly high because of complications of diabetes that require long-term treatment (heart disease, cerebrovascular disease, kidney disorders, foot disorders, etc.). There are also reports that account for about 74% of the cost of treating the disease.

In order to prevent such complications, it is necessary to maintain HbA _1c (hemoglobin A _1c ) for less than 6.5% for a long time by appropriate treatment selection.

Oral preparations (oral diabetes drugs) currently used for the treatment of diabetes can be roughly classified into the following three types according to their mechanism of action:
(A) insulin secretagogue (sulfonylurea, phenylalanine derivative),
(B) a drug (alpha glucosidase inhibitor) that delays the absorption of food and releases the sugar toxicity caused by hyperglycemia,
(C) Insulin resistance improving drug (biguanide, thiazolidine drug).

However, when these antidiabetic drugs are administered to diabetic patients, patients such as those who show high efficacy (good responder), those who are poorly effective (poor responder), or those who do not show any effect (non responder) There are various responses, and it is recognized that there is a need to accurately select and prescribe drugs suitable for individual patients. However, there is no objective selection criterion for determining the type and dosage of insulin secretagogues, and the current situation is that it depends heavily on the empirical judgment of doctors. In addition, depending on the type of drug, hypoglycemia and side effects on the cardiovascular system have been pointed out. Therefore, the establishment of a system for prescribing appropriate antidiabetic drugs according to patients is extremely important in medicine. In particular, in recent years, based on the philosophy of Evidence Based Medicine (EBM), in addition to doctors' expertise, experience, and technology, evidence based on the latest and best medical technology confirmed by scientific methods In addition, the most effective and safe medical care for individual patients is required, and therefore, effective patient groups for the administration of various antidiabetic drugs are based on scientific evidence. It is important to clarify. By doing so, the patient's compliance can be improved, an effective therapeutic effect can be obtained, and the medical expenses can be efficiently operated.

Recently, it has been reported that the development of diabetes involves not only non-genetic factors such as lifestyle including food, but also some genetic factors. For example, Adiponectin (G276T) (Non-patent document 1), PGC-1 (G1302A) (Non-patent document 2), beta3-Adrenergic Receptor (Trp64Arg) (Non-patent document 3) ), Resistin (C-420G) (Non-patent Document 4), etc. have been identified, and their polymorphisms [for example, SNPs (single nucleotide polymorphisms)] and diabetes have been reported. . In addition, it has been reported that there are differences between individuals and side effects in the expression of drug effects due to differences in gene polymorphisms that are directly involved in the mechanism of action of specific drugs (for example, Non-Patent Documents 5-6). However, it is not known that genetic polymorphisms of genes other than diabetes-related genes reported in these reports serve as selection indicators for evaluating the effectiveness of various antidiabetic drugs in advance. There is no idea yet to select an effective anti-diabetic drug suitable for the patient based on the genetic information.

The present invention provides a gene polymorphism related to the effectiveness of an antidiabetic agent comprising a phenylalanine derivative drug as an active ingredient among oral diabetes drugs. Based on this information, the phenylalanine derivative drug is effectively used. The purpose is to select and determine diabetic patients who respond.

In other words, an object of the present invention is to provide a method for detecting and determining in advance in advance the effectiveness of a therapeutic agent for diabetes comprising the above-described phenylalanine derivative-based drug as an active ingredient for diabetic patients.

Another object of the present invention is to provide a therapeutic agent for diabetes suitable for a diabetic patient having a specific gene polymorphism, and to provide a method for treating diabetes suitable for the patient.

The inventors of the present invention have been diligently studying to achieve the above-mentioned object. As a result, gene polymorphisms defining drug responsiveness for diabetic patients, particularly gene polymorphisms defining responsiveness to phenylalanine derivative drugs. Succeeded in identifying. Specifically, the present inventors, among diabetic patients, (1) skeletal muscle glycogen synthase (hereinafter simply referred to as “GYS1”) gene (Genbank Accession No. BC002617) position 260 Patients with G (guanine) as a homozygote (GG) or heterozygote (AG or GA); (2) Soluble epoxide hydrolase (hereinafter referred to simply as “EPHX2”) gene (Genbank Accession No . (BC011628) patients with A (adenine) at position 860 as homozygote (AA) or heterozygote (AG or GA); (3) C at position -159 of the CD14 gene (Genbank Accession No. AF097335) It has been found that patients having cytosine) as a homozygote (CC) or heterozygote (CT or TC) have a markedly improved diabetes-improving effect with a phenylalanine derivative.

Based on these findings, in each individual with diabetes, the GENOTYPE of any of the genes (gene polymorphisms) (1) to (3) above is examined in vitro, and it is confirmed that the GENOTYPE is a specific TYPE. By doing so, it is possible to determine in advance the administration effectiveness of a therapeutic agent for diabetes containing a phenylalanine derivative drug as an active ingredient, and as a result, the therapeutic agent can be selected and determined as an effective therapeutic agent for diabetes for the patient. What can be done (in other words, it is possible to determine whether or not the diabetic patient is effective in administering a therapeutic agent containing a phenylalanine derivative drug), and the patient whose administration effectiveness has been determined (effective administration) The present invention is completed with the conviction that effective and accurate treatment of diabetes can be achieved by selectively administering the therapeutic agent to It led to.

That is, the present invention has the following aspects.

I. A method for detecting the administration effectiveness of an antidiabetic agent comprising a phenylalanine derivative as an active ingredient in diabetic patients
I-1. A method for detecting in vitro the effectiveness of administration of a therapeutic agent for diabetes comprising a phenylalanine derivative-based drug as an active ingredient for a diabetic patient, wherein the diabetic patient has at least one of the genes (1) to (3) A method characterized by having the following GENOTYPE (1) as an index:

I-2. The method described in I-1, having the following steps:
(A) a step of detecting GENOTYPE in a polymorphism of at least one gene described in (1) to (3) above for a biological sample of a diabetic patient;
(B) A step of identifying whether the GENOTYPE in the polymorphism of at least one gene described in (1) to (3) is the GENOTYPE (1) described in I-1.

I-3. The method according to I-2, further comprising the following step (c):
(c) Based on the result of (b) above, GENOTYPE in the polymorphism of at least one gene described in (1) to (3) above is described in (1) to (3) of I-1 above A step of determining that administration of a therapeutic agent for diabetes comprising a phenylalanine derivative-based drug as an active ingredient is effective for the treatment of the diabetic patient when the GENOTYPE (1) matches.

I-4. The phenylalanine derivative is at least one selected from the group consisting of nateglinide, mitiglinide, repaglinide, and pharmaceutically acceptable salts thereof, I-1 to I- 3. The method according to any one of 3.

II. Antidiabetic agent comprising phenylalanine derivative as active ingredient
II-1. Diabetes treatment comprising a phenylalanine derivative-based drug as an active ingredient, which is administered to a diabetic patient whose at least one gene of any one of (1) to (3) is confirmed to have the following GENOTYPE Agent:

II-2. The diabetes therapeutic agent according to II-1, wherein the phenylalanine derivative is nateglinide, mitiglinide, repaglinide, or a pharmaceutically acceptable salt thereof.

III. Diabetes treatment method for specific diabetic patients
III-1. A step of administering a therapeutic agent for diabetes comprising a phenylalanine derivative-based drug as an active ingredient to a diabetic patient who has been confirmed to have the following GENOTYPE (1) for at least one gene of any one of (1) to (3) A method for treating diabetes having:

III-2. The above-mentioned phenylalanine derivative drug is at least one selected from the group consisting of nateglinide, mitiglinide, repaglinide, and pharmaceutically acceptable salts thereof, described in III-1 How to treat diabetes.

IV. Use of phenylalanine derivatives for certain diabetics
IV-1. Use of a phenylalanine derivative-based drug for the manufacture of a therapeutic drug for a diabetic patient who has been confirmed to have the following GENOTYPE (1) for at least one gene of (1) to (3):

IV-2. Described in IV-1, wherein the phenylalanine derivative is at least one selected from the group consisting of nateglinide, mitiglinide, repaglinide, and pharmaceutically acceptable salts thereof. use.

IV-3. A phenylalanine derivative-based drug for treating a diabetic patient who is confirmed to have the following GENOTYPE (1) for at least one gene of any one of (1) to (3):

IV-4. (IV-3), wherein the phenylalanine derivative is at least one selected from the group consisting of nateglinide, mitiglinide, repaglinide, and pharmaceutically acceptable salts thereof. Phenylalanine derivative drugs to be described.

According to the present invention, the GENOTYPE of at least one gene polymorphism of any one of (1) GYS1 (A260G), (2) EPHX2 (G860A), and (3) CD14 (T-159C) is examined for a diabetic patient. Thus, the effectiveness of the diabetic patient with respect to the phenylalanine derivative can be detected or determined in advance in vitro. In addition, for patients who are judged to be effective, administration of diabetes treatments containing phenylalanine derivative drugs as active ingredients enables accurate and effective treatment of diabetes according to individual patients. It becomes. This, on the other hand, reduces the burden of treatment costs (medical costs) caused by the treatment that is accurate and ineffective for the patient, reduces physical and mental distress due to possible side effects, and delays treatment. It is also effective in preventing diabetes progression and complications.

It is a flowchart which shows the determination method of the effectiveness of a diabetes therapeutic agent.

(1) Explanation of terms Unless otherwise specified, “gene” in this specification has double-stranded DNA including human genomic DNA, single-stranded DNA (sense strand), and a sequence complementary to the sense strand. Both single-stranded DNA (antisense strand) and fragments thereof are included, and “gene” in the present specification distinguishes a regulatory region, a coding region, an exon, and an intron unless otherwise specified. It shall be shown without doing.

(1) GYS1 (A260G), (2) EPHX2 (G860A), and (3) CD14 (T-159C) gene polymorphism information (base sequence, chromosome position, etc.) and gene polymorphism described in this specification The position information of the mold is based on the following documents.

(1) GYS1 (A260G)
Shimomura H, et al. A missense mutation of the muscle glycogen synthase gene (M416V) is associated with insulin resistance in the Japanese population. Diabetologia. 1997 Aug; 40 (8): 947-952.

(2) EPHX2 (G860A)
Ohtoshi K, et al. Association of soluble epoxide hydrolase gene polymorphism with insulin resistance in type 2 diabetic patients. Biochem Biophys Res Commun. 2005 May 27; 331 (1): 347-350.

(3) CD14 (T-159C)
Fernandez-Real JM, et al. CD14 monocyte receptor, involved in the inflammatory cascade, and insulin sensitivity. J Clin Endocrinol Metab. 2003 Apr; 88 (4): 1780-1784.
Unkelbach K, et al., A new promoter polymorphism in the gene of lipopolysaccharide receptor CD14 is associated with expired myocardial infarction in patients with low atherosclerotic risk profile. Arterioscler Thromb Vasc Biol. 1999 Apr; 19 (4): 932-8.

In this specification, (1) “GYS1 (A260G)” means that the human GYS1 gene (Genbank Accession No. BC002617) has a gene polymorphism in which the 260th base of the nucleotide sequence is A or G. means. Similarly, (2) “EPHX2 (G860A)” means that the human EPHX2 gene (Genbank Accession No. BC011628) has a gene polymorphism in which the 860th base of the nucleotide sequence is G or A: (3 ) “CD14 (T-159C)” means that the human CD14 gene (Genbank Accession No. AF097335) has a gene polymorphism in which the -159th base of the nucleotide sequence is T or C, respectively. . The SNP position relating to each gene is a position indicated by “+1” as the transcription start point of each gene. That is, the 260th base in the base sequence of the GYS1 gene means the 260th base downstream from the transcription start point of the GYS1 gene. Similarly, the 860th base in the base sequence of the EPHX2 gene refers to the EPHX2 gene transcription start point. It means the 860th base downstream. Further, the −159th base in the base sequence of the CD14 gene means the 159th base upstream from the transcription start point of the CD14 gene, that is, the promoter region −159th base of the CD14 gene.

“Gene polymorphism” means the diversity of genes in which two or more types of alleles exist at one locus in the same population. Specifically, it indicates a mutation of a gene that exists at a certain frequency or more in a certain population. The gene mutation referred to here is not limited to the region transcribed as RNA, but includes mutations in all DNAs that can be identified on the human genome including regulatory regions such as promoters and enhancers. 99.9% of human genomic DNA is common among individuals, and the remaining 0.1% is responsible for such diversity, and is involved in individual differences in susceptibility to specific diseases, responsiveness to drugs and environmental factors. obtain. A genetic polymorphism does not always make a difference in phenotype. The SNP (single nucleotide polymorphism) targeted by the present invention is also a kind of genetic polymorphism.

The “diabetes” targeted in the present invention is mainly type 2 diabetes (insulin-independent diabetes). According to the guidelines of the Japan Diabetes Society (1999), if the blood glucose level is 200 mg / dL or higher, the fasting blood glucose level is 126 mg / dL or higher, and the 75 g oral glucose tolerance test 2 hour value is 200 mg / dL or higher It can be judged as diabetes. In addition, if the above criteria are met twice on a different day, or if there is a characteristic symptom of diabetes even after one check, HbA _1c (hemoglobin A _1c ) is 6.5% or more, or diabetic retinopathy If there is, it can be judged as diabetes. In the blood glucose control index, the HbA _1c value is regarded as important, and the main determination is made based on this. The HbA _1c value is an index that reflects the average blood glucose level of the patient over the past 1 to 2 months, and is the most important index of blood glucose control status with little variation in the value of one patient.

(2) Method for detecting the administration effectiveness of a therapeutic agent for diabetes containing a phenylalanine derivative-based drug as an active ingredient for diabetic patients The present invention relates to the effectiveness of administration of a therapeutic agent for diabetes containing a phenylalanine derivative-based drug as an active ingredient for diabetic patients. Provide a method of detecting

This method can be carried out by in vitro detection of GENOTYPE in at least one gene polymorphism of any one of the following (1) to (3) for a biological sample of a diabetic patient.

Among the genes shown in (1) to (3) above, (1) GYS1 gene will be described as an example. The method of the present invention uses the biological sample of a diabetic patient as a material, and position 260 of the GYS1 gene [GYS1 ( A260G)]) to detect GG homozygote, AG or GA heterozygote, or AA homozygote, and based on the description in Table 6 above, In the case of a homozygote or an AG or GA heterozygote, it can be determined in advance that administration of a phenylalanine derivative-based drug is effective for the treatment of the diabetic patient. Conversely, when GYS1 (A260G) is neither a GG homozygote nor an AG or GA heterozygote (ie, AA homozygote), a phenylalanine derivative is administered to treat the diabetic patient. Is not very effective and can be determined not to be the first choice.

Similarly, in the case of targeting the genes (2) to (3) as well, based on the GENOTYPE described in Table 6 above, the administration effectiveness of phenylalanine derivative drugs can be detected in advance in vitro in diabetic patients. .

Specifically, regarding (2) the EPHX2 gene, the method of the present invention uses a biological sample of a diabetic patient as a material, detects the GENOTYPE at position 860 of the EPHX2 gene [EPHX2 (G860A)], and homozygotes for AA A method of identifying heterozygotes for AG, GA, or homozygotes for GG. Based on the above description, in the case of AA homozygote or AG or GA heterozygote, it can be determined in advance that administration of a phenylalanine derivative is effective for the treatment of the diabetic patient. . Conversely, in the case of a GG homozygote, it can be determined that administration of a phenylalanine derivative-based drug is not very effective for the treatment of the diabetic patient and is not a first option.

Further, (3) with respect to the CD14 gene, the method of the present invention uses a biological sample of a diabetic patient as a material to detect the GENOTYPE at position -159 (CD14 (T-159C)) of the promoter region of the CD14 gene, and A method of identifying another homozygote, CT or TC heterozygote, or TT homozygote. Based on the above description, in the case of CC homozygote or CT or TC heterozygote, it can be determined in advance that the administration of a phenylalanine derivative is effective for the treatment of the diabetic patient. . On the other hand, in the case of a TT homozygote, it can be determined that administration of a phenylalanine derivative is not very effective in treating the diabetic patient and does not become the first option.

These genes (1) to (3) (gene polymorphisms) are gene markers indicating the effectiveness of phenylalanine derivative drugs (sensitivity to phenylalanine derivative drugs) in diabetic patients. The gene marker can be suitably used for diabetic patients to determine whether administration of phenylalanine derivative drugs is effective for the treatment of diabetes. If the target patient gene (gene polymorphism) matches the GENOTYPE (1) shown in Table 6 above, the patient is responsive (sensitive) to the phenylalanine derivative drug, that is, the phenylalanine derivative drug. Means that the patient is effective in treating diabetes.

The phenylalanine derivative drug targeted here will be described in detail in (3) below. Preferable phenylalanine derivative drugs include nateglinide, mitiglinide, repaglinide and pharmaceutically acceptable salts thereof. In addition, these drugs are administered in an effective amount in one or a combination of two or more as necessary to diabetic patients determined to be responsive (sensitive) to phenylalanine derivative drugs by the method of the present invention. can do.

Specifically, genotype detection is performed by (i) performing PCR in a region containing at least one gene polymorphism described in (1) to (3) on the subject's genomic DNA, and using the SSCP method. (Ii) The subject is genomic DNA of the subject, PCR is performed in the region containing the gene polymorphism of at least one gene described in (1) to (3), and the restriction enzyme cleavage pattern for the PCR product (Iii) a method in which the PCR product is directly sequenced and sequenced, and (iv) a genomic DNA of a subject subject to at least one gene described in (1) to (3) Using oligonucleotides that hybridize to regions containing genetic polymorphisms as probes and hybridizing with individual DNA, the ASO (allele specific oligonucleotide) method, (v) Inheritance of at least one gene described in The oligonucleotide that hybridizes to a region containing the polymorphism used as a probe, and a method of detecting in a mass spectrometer or the like, can be carried out by using a known method.

More specifically, the method of the present invention can be carried out by performing the following steps (a) and (b):
(A) a step of detecting GENOTYPE in a genetic polymorphism of at least one gene described in (1) to (3) for a biological sample of a diabetic patient;
(B) A step of identifying whether the GENOTYPE in the polymorphism of at least one gene described in (1) to (3) is GENOTYPE (1) described in Table 6 above.

Note that the step (a) can be performed specifically on genomic DNA extracted from a biological sample of a diabetic patient.

In these steps, when the GENOTYPE in the polymorphism of at least one gene described in (1) to (3) matches the GENOTYPE (1) described in Table 6 above, the diabetes that provided the genomic DNA sample It can be judged that administration of phenylalanine derivative drugs is effective for treating diabetes in patients. Conversely, if it does not match GENOTYPE (1) listed in the above table, the phenylalanine derivative is not very effective for treatment, and can give the option of considering administration of other therapeutic agents for diabetes.

Such a determination step is not an essential step of the method of the present invention, but such a determination step can optionally be included as the following step (c):
(c) Based on the result of (b) above, the GENOTYPE in the polymorphism of at least one gene described in (1) to (3) of diabetic patients matches the GENOTYPE (1) described in Table 6 above. If it is determined that the administration of a phenylalanine derivative is effective for the treatment of the diabetic patient, and conversely does not match the GENOTYPE (1) described in Table 6 above, A step of judging that administration of a phenylalanine derivative is not very effective.

That is, according to the method of the present invention, it is possible to determine the presence or absence of administration effectiveness of a phenylalanine derivative-based drug for diabetic patients. This decision can be made even by a specialist by determining whether the genetic polymorphisms of the above (1) to (3) genes in diabetes patients fall under the GENOTYPE (1) shown in Table 6 above. Extremely difficult decisions can be made automatically / mechanically.

Note that the above steps (a) and (b) are known methods (for example, Bruce, et al., Geneme Analysis / A laboratory Manual (vol.4), Cold Spring Harbor Laboratory, NY., (1999)). Can be used.

Specific examples of the biological sample to be processed in step (a) include genomic DNA as described above. Such genomic DNA can be any cell (including cultured cells, excluding germ cells), tissue (including cultured tissue), or body fluid (eg, blood, saliva, lymph, airway mucosa, semen, isolated from a diabetic patient. , Sweat, urine, etc.) can be prepared as a material. Preferably it is blood or urine. Incidentally, extraction of the genomic DNA is not limited to the above method, methods well known in the art (e.g., Sambrook J. et al,.. "Molecular Cloning:. A Laboratry Manual (2 nd Ed)" Cold Spring Harbor Laboratory, NY) and commercially available DNA extraction kits can be used.

In step (b), the base located at the gene polymorphic site of at least one gene of (1) to (3) is identified from the extract containing human genomic DNA obtained as described above. As a method of identifying the target base, in addition to the method of directly determining the gene sequence of the corresponding region, if the polymorphic sequence is a restriction enzyme recognition site, the difference in restriction enzyme cleavage pattern is used to change GENOTYPE. Method of determination (hereinafter referred to as RFLP), method based on hybridization using polymorphic-specific probes (for example, a specific probe is stuck on a chip, glass slide, nylon membrane, and hybridization intensity for those probes) Determine the type of polymorphism by detecting the difference between them, or specify the efficiency of specific probe hybridization, and identify the GENOTYPE by detecting the amount of probe that polymerase degrades during template double-stranded amplification Fluorescence emitted by a certain type of double-strand-specific fluorescent dyes can be obtained by following the change in temperature. A method of detecting the temperature difference of the polymorphism, thereby identifying the polymorphism, attaching a complementary sequence to both ends of the polymorphic site-specific oligo probe, and depending on the temperature, the corresponding probe creates a secondary structure within the self molecule, There is a method of specifying a genotype using a difference of whether it hybridizes to a target region. In addition, a method in which a base-extension reaction is performed by polymerase from a template-specific primer and a base incorporated into a polymorphic site at that time is identified (by using dideoxynucleotide, each is fluorescently labeled, and each fluorescence is Detection method, method of detecting incorporated dideoxynucleotide by mass spectrometry), and further using template-specific primers followed by the presence or absence of base pairs complementary to the mutation site or non-complementary base pairs. There is a method to make it recognize.

Hereinafter, well-known representative methods for detecting gene polymorphisms are listed below, but the present invention is not limited thereto.

(a) RFLP (restriction fragment length polymorphism) method, (b) PCR-SSCP method (single-stranded DNA higher-order structure polymorphism analysis) [Biotechniques, 16, 296-297 (1994), and Biotechniques, 21 , 510-514 (1996)], (c) ASO (Allele Specific Oligonucleotide) hybridization method [Clin. Chim. Acta, 189, 153-157 (1990)], (d) Direct sequencing method [Biotechniques, 11, 246 -249 (1991)], (e) ARMS (Amplification Refracting Mutation System) method (Nuc. Acids. Res., 19, 3561-3567 (1991); Nuc. Acids. Res., 20, 4831-4837 (1992) ],
(F) Denaturing Gradient Gel Electrophoresis (DGGE) method [Biotechniques, 27, 1016-1018 (1999)], (g) RNase A cleavage method [DNA Cell. Biol., 14, 87- 94 (1995)], (h) Chemical cleavage method [Biotechniques, 21, 216-218 (1996)], (i) DOL (Dye-labeled Oligonucleotide Ligation) method [Genome Res., 8, 549-556 (1998) ], (J) TaqMan-PCR method [Genet. Anal., 14, 143-149 (1999); J. Clin. Microbiol., 34, 2933-2936 (1996)], (k) Invader method [Science, 5109 , 778-783 (1993); J. Biol. Chem., 30, 21387-21394 (1999); Nat. Biotechnol., 17, 292-296 (1999)], (l) MALDI-TOF / MS method (Matrix Assisted Laser Desorption-time of Flight / Mass Spectrometry (Genome Res., 7, 378-388 (1997); Eur. J. Clin. Chem. Clin. Biochem., 35, 545-548 (1997)), ( m) TDI (Template-directed Dye-terminator Incorporation) method [Proc. Natl. Acad. Sci. USA, 94, 10756-10761 (1997)], (n) Molecular Beacons method [N at. Biotechnol., 1, p49-53 (1998); gene medicine, 4, p46-48 (2000)], (o) Dynamic Allele-Specific Hybridization (DASH) method [Nat Biotechnol., 1.p.87-88 (1999); Gene Medicine, 4, 47-48 (2000)], (p) Padlock Probe method [Nat. Genet., 3, p225-232 (1998); gene medicine, 4, p50-51 (2000)], (q) UCAN method (see Takara Shuzo Co., Ltd. home page (http://www.takara.co.jp)), (r) DNA Chip or DNA microarray ("SNP gene polymorphism strategy" Kenichi Matsubara, Yoshiyuki Tsuji, Nakayama Shoten, p128-135), (s) ECA method [Anal. Chem., 72, 1334-1341, (2000)].

The above are typical gene polymorphism detection methods, but the method of the present invention is not limited to these, and other known or future gene polymorphism detection methods can be widely used. Moreover, when detecting the gene polymorphism of the present invention, these gene polymorphism detection methods may be used singly or in combination of two or more.

In the case where a mutation occurs in the encoded amino acid based on the difference in the genetic polymorphisms of (1) to (3), the method of the present invention is not limited to the method of directly detecting and identifying the GENOTYPE of the gene. A method of detecting the amino acid sequence of a protein that is an expression product of a gene and identifying the presence or absence of an amino acid mutation can also be employed. For example, the GYS1 gene has a mutation in the 416th amino acid (Met) of exon 10 based on the above gene polymorphism (Met416Val) (Diabetologia. 1997 Aug; 40 (8): 947-52.) The EPHX2 gene is known to have a mutation at the 287th amino acid (Arg) based on the above polymorphism (Arg287Gln) (Circulation. 2004 Jan 27; 109 (3): 335-9. Epub 2004 Jan19 .).

Probes and primers, for example, for detecting and distinguishing gene polymorphisms of the above (1) to (3), for each diabetic patient, select whether or not the patient is a person who is effectively administered a phenylalanine derivative. Can be used as a tool for

The steps (b) and (c) of the method of the present invention can also be performed by electronic information processing. A method using the electronic information processing will be described with reference to the flowchart shown in FIG. Each processing described below includes a CPU, a memory, a recording device (for example, a hard disk), an input device (for example, a keyboard, a mouse, a flexible disk drive, a CD-ROM drive), a display device (for example, a liquid crystal display), and a communication device (for example). For example, the description will be made assuming that a computer equipped with a network board) is used. That is, the processing target data is input via the input device and the communication device and recorded in the recording device, and the CPU executes processing on the data read from the recording device using the memory as a work area. The intermediate result of the process and the final result are recorded in a predetermined area of the recording unit as necessary.

First, in step S1, the input of genetic information relating to the target diabetic patient is accepted, and the input data is recorded in the recording device. Here, the gene information includes at least one gene polymorphism (GENOTYPE) information selected from GYS1 (A260G), EPHX2 (G860A), and CD14 (T-590C). Specifically, it is a pair of text data representing a gene polymorphism name and text data representing its GENOTYPE. The gene information may be input as a code. For example, a code (text data) assigned to each gene polymorphism name or GENOTYPE may be input. The genetic information may be input via a keyboard, may be input via a read drive from a state recorded on a recording medium, or may be input via a communication line. Furthermore, the genetic information may be input by being selected from a plurality of candidates displayed on the display device, or may be input using other known methods.

In step S2, the genetic polymorphism information table is read from the recording unit. Table 7 shows an example of a table recorded in advance in the recording device.

As will be described later, this is a table of genetic polymorphism information indicating the administration effectiveness of a therapeutic agent for diabetes containing phenylalanine derivative drugs, particularly nateglinide as an active ingredient. In the table of Table 7, genes (gene polymorphisms) and GENOTYPE are described. In this case, the gene (gene polymorphism) and GENOTYPE correspond to the gene polymorphism information. In other words, it is assumed that the data shown in Table 7 and the data indicating the corresponding relationship are recorded as text data, for example, in the recording device.

In step S3, it is determined whether the genetic polymorphism information in the table read in step S2 is included in the patient genetic information input in step S1, and the result is temporarily stored in a predetermined area of the memory. Record. For example, in Table 7, “GYS1 (A260G)” and “GG or AG” in the first row are read, the gene (gene polymorphism) is “GYS1 (A260G)”, and GENOTYPE is “GG or AG”. It is determined whether the genetic polymorphism information is included in the patient genetic information. If it is included, for example, “1” is set in a flag corresponding to the genetic polymorphism information in the first row (assumed that the initial value is set to “0” on the memory). If not included, do nothing for the flag and read “EPHX2 (G860A)” and “AA or AG” in the second row, and if they are included in the patient's genetic information as well If it is included, “1” is set in the flag corresponding to the gene polymorphism information in the second row. If not included, the following genetic polymorphism information is similarly processed. In this way, whether or not the genetic polymorphism information in the table of Table 7 is included in the genetic information of the diabetic patient is temporarily recorded as a flag, for example.

Here, it may be determined whether all the genetic polymorphism information in Table 7 is included in the patient genetic information, but at least one genetic polymorphism information is included in the patient genetic information. If so, the process may move to step S4 without processing the remaining genetic polymorphism information.

In step S4, according to the determination result in step S3, whether or not the therapeutic agent for diabetes containing a phenylalanine derivative-based drug as an active ingredient is effective for the patient is displayed (for example, displayed on a display device). For example, if at least one of the above flag values is “1”, it indicates that it is valid, and if all the flag values are “0”, it indicates that it is not valid.

Through the above processing, the effectiveness of administering a therapeutic agent for diabetes containing a phenylalanine derivative-based drug as an active ingredient to the patient can be determined in advance according to the genetic information possessed by the specific diabetic patient.

Therefore, according to the present invention, tailor-made treatment for each diabetic patient becomes possible. Further, the present invention is to select a therapeutic agent for diabetes containing a phenylalanine derivative as an active ingredient as an effective drug for a specific diabetic patient having GENOTYPE (1) from a plurality of therapeutic agents for diabetes. It can also be used effectively.

Note that the table used for the determination is not limited to the table shown in Table 7, but may be any table including the gene (gene polymorphism) and GENOTYPE information shown in Table 7.

Also, here, the embodiment of the present invention has been described as processing performed using electronic data corresponding to a table according to the flowchart shown in FIG. 1, but the present invention is not limited to this. It is only necessary to use the table shown in the table or a table containing genetic polymorphism information equivalent to this (both are not limited to electronic data). It is also possible to determine whether the described gene (gene polymorphism) and GENOTYPE are included, and to determine the effectiveness of a therapeutic agent for diabetes containing a phenylalanine derivative as an active ingredient for the patient.

(3) Diabetes therapeutic agent comprising phenylalanine derivative-based drug as active ingredient The diabetes therapeutic agent provided by the present invention has at least one gene (gene polymorphism) of any of the following (1) to (3) shown below as GENOTYPE: (1) A drug containing a phenylalanine derivative as an active ingredient, which is effective for diabetic patients. The drug is administered to a diabetic patient in which at least one of the following genes (1) to (3) is confirmed to have the following GENOTYPE (1): .

A diabetic patient to be treated by the therapeutic agent for diabetes of the present invention has been confirmed to have the above-mentioned GENOTYPE (1) for at least one gene among the above (1) to (3) by genetic testing The patient may be a patient having the above-described GENOTYPE (1) for any two or more genes. Here, the genetic test means a test for examining a genetic polymorphism of at least one gene described in the above (1) to (3). Specifically, an inspection method having the steps (a) and (b) described in the above (2), and an inspection method further having the step (c) in addition to the steps (a) and (b) are given. be able to.

The phenylalanine derivative-based drug which is an active ingredient of the therapeutic agent for diabetes of the present invention suppresses an ATP-dependent K channel composed of a sulfonylurea receptor (SUR1) and an inward rectifier K ⁺ channel in pancreatic β cells. It is a drug that promotes insulin secretion by allowing extracellular calcium to flow into the cell membrane. The therapeutic agent for diabetes of the present invention is not particularly limited as long as it contains, as an active ingredient, a phenylalanine derivative based on such an action mechanism that induces insulin secretion and lowers blood glucose.

Note that the phenylalanine derivative drugs targeted here include the compounds represented by the following general formula (I) and related compounds:

(Wherein R ₁ is a hydrogen atom, an alkyl group having 1 to 5 carbon atoms, an aryl group having 6 to 12 carbon atoms, or an aralkyl group having 6 to 12 carbon atoms; R ₂ may have a substituent) , An aryl group having 6 to 12 carbon atoms, a hetero 5- or 6-membered ring, a cycloalkyl group, or a cycloalkenyl group; R ₃ is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms; A is —N— or —CH Means-).

In the compound represented by the general formula (I), the alkyl group having 1 to 5 carbon atoms represented by R ₁ in the formula (I) is a methyl group, an ethyl group, an n-propyl group, an isopropyl group, or an n-butyl group. Or a sec-butyl group; an aryl group having 6 to 12 carbon atoms is a phenyl group, a tolyl group or a naphthyl group; and an aralkyl group having 6 to 12 carbon atoms is preferably a benzyl group:
In the formula (I), the aryl group having 6 to 12 carbon atoms represented by R ₂ is a phenyl group, a naphthyl group or an indanyl group; the hetero 5-membered ring is a 2-benzofuranyl group; the hetero 6-membered ring Is a quinolinyl group and a pyridyl group; a cycloalkyl group is a cyclohexyl group and a cyclopentyl group; or a cycloalkenyl group is a 1-cyclohexenyl group, a 2-cyclohexenyl group, a 3-cyclohexenyl group, a 1-cyclopentenyl group, A 2-cyclopentenyl group can be mentioned. Any of these groups can have one or more substituents.

Substituents include halogen atoms (for example, fluorine atoms or chlorine atoms), alkyl groups having 1 to 5 carbon atoms (for example, methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, sec-butyl group) ), An alkenyl group having 1 to 5 carbon atoms (for example, ethenyl group, propenyl group, butenyl group), an alkyloxy group having 1 to 5 carbon atoms (for example, methoxy group, ethoxy group), an alkyloxy group having 1 to 5 carbon atoms An alkyl group having 1 to 5 carbon atoms (eg, methoxymethyl group, 1-ethoxyethyl group) substituted with a group, an alkylene group having 1 to 5 carbon atoms substituted with an alkyloxy group having 1 to 5 carbon atoms (eg, , A methoxyethylene group), and an alkylidene group represented by the following formula.

In the formula (I), the alkyl group having 1 to 6 carbon atoms represented by R ₃ is preferably the same as the alkyl group described for R ₁ .

In the formula (I), A is —N— (nitrogen atom) or —CH— (methylene group in which one hydrogen atom is substituted with R ₃ ), and these can be arbitrarily selected. The compound (I) in which A is —N— can also be defined as a D-phenylalanine derivative, and the compound (I) in which A is —CH— can be defined as a benzyl succinic acid derivative.

The phenylalanine derivative-based drug referred to in the present invention includes hydrates and pharmaceutically acceptable salts of the compound represented by the above formula (I). Examples of the salt herein include alkali metal salts such as sodium and potassium, alkaline earth metal salts such as calcium and magnesium; non-ammonium such as ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, and ethylamine. Toxic ammonium, quaternary ammonium and amine cations can be exemplified.

Preferred as a phenylalanine derivative-based drug is a compound corresponding to a D-phenylalanine derivative, and nateglinide represented by the following formula (1) [trade name “Starsys Tablets” (Astellas Pharma, etc.):

Moreover, it is a compound corresponding to a benzyl succinic acid derivative, and mitiglinide represented by the following formula (2) or its calcium hydrate [trade name “Glufast” (Kissei Pharmaceutical, etc.)]:

Can be mentioned.

Moreover, as a phenylalanine derivative-type drug preferable as an analog of the phenylalanine derivative, repaglinide (repaglinide) represented by the following formula (3) [“SMP-508” (Dainippon Sumitomo Pharma Co., Ltd.), trade name “NovoNorm”: Novo Nordisk Can be mentioned:

The anti-diabetic agent of the present invention only needs to contain these phenylalanine derivative drugs in an effective amount that exerts a therapeutic effect on diabetes, and as long as other components, such as pharmaceutically acceptable carriers and additives, are used. May be contained. The therapeutic agent for diabetes of the present invention can usually be administered orally and is in a form suitable for their administration (for example, tablets, pills, powders, solutions, suspensions, emulsions, granules, capsules, etc.). Can be taken.

As the pharmaceutically acceptable carrier that can be blended in the antidiabetic agent, those commonly used in the art can be widely used depending on the above various administration forms. For example, a filler, a bulking agent, a binder, a moistening agent, a disintegrating agent, a surfactant, a lubricant, a buffering agent, a chelating agent, a pH adjusting agent, a surfactant and the like can be mentioned. In addition, stabilizers, bactericides, coloring agents, preservatives, fragrances, flavoring agents, sweetening agents, and the like can be blended with the antidiabetic agent of the present invention as necessary.

In addition, the therapeutic agent for diabetes of the present invention can be prepared in the form of a sustained-release preparation or a sustained-release preparation containing a phenylalanine derivative drug as an active ingredient according to a known method.

The amount of the phenylalanine derivative-based drug to be contained in the therapeutic agent for diabetes of the present invention is not particularly limited and is appropriately selected from a wide range, but is usually preferably about 1 to 70% by weight.

There are no particular restrictions on the dosage and mode of administration of the therapeutic agent for diabetes of the present invention. For example, it depends on the type of phenylalanine derivative-based drug to be blended as an active ingredient, various pharmaceutical forms, patient age, sex and other conditions, and the degree of disease. Administered by the method. For example, nateglinide is administered 90 to 120 mg at a time, 3 times a day immediately before each meal (within 10 minutes before meal); mitiglinide calcium hydrate is administered at a dose of 10 mg, 3 times a day, immediately before each meal (within 10 minutes before a meal). It is recommended to do.

(4) Diabetes Treatment Method for Specific Diabetes Patients In the present invention, it is confirmed that at least one gene (gene polymorphism) of any of the following (1) to (3) is the following GENOTYPE (1) The present invention relates to a diabetic treatment method performed for diabetic patients. Specifically, the method is intended for patients with type 2 diabetes who have undergone genetic testing and have been confirmed that at least one of the following genes (1) to (3) has GENOTYPE (1). This can be carried out by administering a drug containing a phenylalanine derivative drug as an active ingredient to a patient.

The diabetic patient who is the target of the treatment method of the present invention is a patient who has undergone genetic testing and has been confirmed to have the above-mentioned GENOTYPE (1) for at least one of the genes (1) to (3). What is necessary is just to be a patient who has said GENOTYPE (1) about these arbitrary two or more genes. Here, the genetic test means a test for examining a genetic polymorphism of at least one gene described in the above (1) to (3). Specifically, an inspection method having the steps (a) and (b) described in the above (2), and an inspection method further having the step (c) in addition to the steps (a) and (b) are given. be able to.

The types of phenylalanine derivative drugs to be administered to the patient and the administration method thereof are as described in (3) above.

In the treatment method of the present invention, phenylalanine is applied to a patient who has been subjected to a genetic test in advance and has been confirmed to have the GENOTYPE (1) for at least one of the genes (1) to (3). In addition to the method of administering a therapeutic agent for diabetes containing a derivative drug as an active ingredient, the above-described genetic test is performed on patients with type 2 diabetes prior to the administration of the phenylalanine derivative drug, and the above (1) to (3) A method including a step of detecting and selecting a patient having the above-described GENOTYPE (1) for at least one gene may be used.

Hereinafter, the present invention will be described more specifically by experimental examples.
In 1320 type 2 diabetic patients with consent, the environmental factors and genetic factors considered to be related to the degree of diabetes, drugs administered, and diabetic complications were investigated. Of these, 136 patients (82 males) were administered a therapeutic agent for diabetes (trade name “Starsys Tablets”: Astellas Pharma Inc.) containing a phenylalanine derivative (nateglide), a type 2 diabetes drug. Example, 54 women). Each patient's age (years), disease duration (years), and average BMI (kg / m ² ) are 63.2 (± 8.0) years, 11.4 (± 6.8) years, and 22.9 (± 2.7) kg, respectively. / m ² (standard deviation in parentheses).

For these 136 type 2 diabetic patients (phenylalanine derivative-based drug recipients), GYS1 gene, EPHX2 gene, and CD14 gene polymorphisms GENOTYPE and hemoglobin A1c (HbA1c) were measured. Based on the results, each patient was classified into GENOTYPE (1) and (2) for each gene shown in Table 10, and further classified into a group with HbA1c of 6.5% or more and a group with less than 6.5%. By the way, if HbA1c is reduced to less than 6.5%, it is recognized that the administration of phenylalanine derivatives is effective in the treatment of diabetes.

In addition, in order to show the efficacy of phenylalanine derivative-based drug administration, GENOTYPE (1) and GENOTYPE (2) for each gene, and based on the result of dividing this into four parts in total, HbA1c ≧ 6.5% and HbA1c <6.5% Independence was tested by Fisher's exact probability calculation. The results are also shown in Table 10. When P <0.05, there is a significant difference. Specifically, the percentage of patients with GENOTYPE (1) that are controlled to have HbA1c less than 6.5% is significant compared to patients with GENOTYPE (2). It means that there were many.

From Table 10, the following can be seen for the gene polymorphism of each gene.

(1) GYS (A260G)
Patients with type 2 diabetes whose GENOTYPE is GENOTYPE (1) (ie, GG or AG or GA), the percentage of patients whose HbA _1c value was reduced to less than 6.5% by administering phenylalanine derivatives Is significantly more than type 2 diabetic patients with GENOTYPE (2) (ie AA). That is, the phenylalanine derivative drug is more effective for the treatment of diabetes in patients with type 2 diabetes whose GYS (A260G) is GENOTYPE (2).

(2) EPHX2 (G860A)
Patients with type 2 diabetes whose GENOTYPE is GENOTYPE (1) (ie, AA, AG, or GA), the percentage of patients whose HbA _1c value was reduced to less than 6.5% by administering phenylalanine derivatives was GENOTYPE Is significantly higher than type 2 diabetic patients with GENOTYPE (2) (ie GG). That is, the phenylalanine derivative-based drug is more effective in treating diabetes in patients with type 2 diabetes whose EPHX2 (G860A) is GENOTYPE (2).

(3) CD14 (T-159C)
Patients with type 2 diabetes whose GENOTYPE is GENOTYPE (1) (ie, CC, CT, or TC), the percentage of patients whose HbA _1c value was reduced to less than 6.5% by administering phenylalanine derivatives Is significantly higher than type 2 diabetic patients with GENOTYPE (2) (ie TT). That is, the phenylalanine derivative drug is more effective for the treatment of diabetes in patients with type 2 diabetes whose CD14 (T-159C) is GENOTYPE (2).

Claims

A method for detecting in vitro the effectiveness of administration of a therapeutic agent for diabetes comprising a phenylalanine derivative-based drug as an active ingredient for a diabetic patient, wherein the diabetic patient has at least one of the genes (1) to (3) A method characterized by having the following GENOTYPE (1) as an index:
The method according to claim 1, comprising the following steps:
(A) a step of detecting GENOTYPE in a genetic polymorphism of at least one gene described in (1) to (3) for a biological sample of a diabetic patient;
(B) A step of identifying whether the GENOTYPE in the polymorphism of at least one gene described in (1) to (3) is the following GENOTYPE (1)
The method according to claim 2, further comprising the following step (c):
(c) Based on the result of (b) above, when the GENOTYPE in the polymorphism of at least one gene described in (1) to (3) matches GENOTYPE (1), A step of determining that administration of an antidiabetic agent comprising a phenylalanine derivative-based drug as an active ingredient is effective for treatment.
The phenylalanine derivative-based drug is at least one selected from the group consisting of nateglinide, mitiglinide, repaglinide, and pharmaceutically acceptable salts thereof. Method.
A phenylalanine derivative-based drug, which is administered to a diabetic patient who has been confirmed to have the following GENOTYPE (1) for at least one gene of any one of (1) to (3) as an active ingredient Diabetes treatment:
6. The phenylalanine derivative-based drug is at least one selected from the group consisting of nateglinide, mitiglinide, repaglinide, and pharmaceutically acceptable salts thereof. Antidiabetic agent.
A step of administering a therapeutic agent for diabetes comprising a phenylalanine derivative-based drug as an active ingredient to a diabetic patient who has been confirmed to have the following GENOTYPE (1) for at least one gene of any one of (1) to (3) A method for treating diabetes having:
The phenylalanine derivative-based drug is at least one selected from the group consisting of nateglinide, mitiglinide, repaglinide, and pharmaceutically acceptable salts thereof. How to treat diabetes.
Use of a phenylalanine derivative-based drug for the manufacture of a therapeutic drug for a diabetic patient who has been confirmed to have the following GENOTYPE (1) for at least one gene of (1) to (3):
The phenylalanine derivative-based drug is at least one selected from the group consisting of nateglinide, mitiglinide, repaglinide, and pharmaceutically acceptable salts thereof. use.