JP2010000055A - Method for predicting development of secondary failure in advance when sulfonylurea agent is administered, and examination kit, primer, and probe used for the same method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for pre-examining whether or not secondary failure is easily developed by administration of a sulfonylurea agent, and to provide an examination kit, a primer, and a probe used for the method. <P>SOLUTION: The method for predicting the development of the secondary failure in advance when the sulfonylurea agent represented by general formula (I): R<SB>1</SB>-SO<SB>2</SB>NHCONH-R<SB>2</SB>is administered includes collecting a gene from a subject; and detecting the presence or absence of at least one kind of polymorphism selected from among the group consisting of (A) 596C>T polymorphism of GPX1 gene; (B) IVS15-3T>C polymorphism of ABCC8 gene; (C) 79A>C polymorphism of HNF1A gene; (D) -1123A>G polymorphism of NFE2L2 gene; and (E) IVS15-29246C>T polymorphism of KCNQ1 gene. The examination kit, the primer, and the probe are used for the method. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for predicting the occurrence of secondary ineffectiveness in the case of administering a sulfonylurea agent, and a test kit, primer and probe used in the method, and more specifically, GPX1 gene, ABCC8 gene, HNF1A gene , A method for predicting the occurrence of secondary ineffectiveness when a sulfonylurea is administered by detecting the presence or absence of a polymorphism of the NFE2L2 gene or the KCNQ1 gene, a test kit used in the method, and the GPX1 gene And a primer and a probe for detecting the presence or absence of a polymorphism in the NFE2L2 gene.

  Diabetes is a progressive disease that cannot be cured once it develops, and is known to cause complications such as retinopathy, nephropathy, and neuropathy. In addition, arteriosclerosis associated with diabetes causes ischemic heart disease and cerebrovascular disorder, and has a profound effect on prognosis. Thus, diabetes not only significantly lowers the quality of life (QOL) of patients, but also imposes a heavy burden on society in terms of medical economics, and the number of patients is expected to increase in the future.

Currently, there are about 2.5 million diabetic patients who are continuously treated in Japan, including 18.7 million including the Diabetes Reserve Army. More than 95% of diabetes in Japan is type 2 diabetes. For the treatment of this type 2 diabetes, insulin secretion-promoting oral diabetes drugs that act on receptor complex molecules present on pancreatic β cells, especially sulfonylurea Agent (SU agent) is frequently used. However, even if this sulfonylurea drug is once effective, the effect of "secondary ineffectiveness" that eventually becomes ineffective occurs in more than 20% of patients with long-term use for several years. Is known and is a clinically significant problem (Non-Patent Documents 1 to 3).
Stryjek-Kaminska D. et al., Diabetes Res. Clin. Pract., 7: 149-154 (1989) Pontiroli AE et al., Diabetes Metab. Rev., 10: 31-43 (1994) Matthews DR et al. Diabet. Med., 15: 297-303 (1998)

  It is known that the hyperglycemia caused by the diminished medicinal effect causes a decrease in insulin secretory ability and insulin sensitivity due to the toxicity of sugar, resulting in a vicious circle in which they cause a further decrease in medicinal effect. In fact, in patients with secondary ineffectiveness, blood glucose levels are poorly controlled even after the introduction of insulin, and the odds ratio of retinopathy and neuropathy, which are complications of diabetes, is 3-4 times higher. This is a major factor that causes a decline in medical expenses and a rise in medical costs. For this reason, it is important to switch to treatments that focus on other drugs, such as early insulin introduction, for diabetic patients who are predicted to develop secondary ineffectiveness. It is strongly desired.

With recent advances in genetic analysis, it is known that individual differences in protein function in the living body and phenotypes based on this are defined by genetic factors. The mechanism of secondary ineffective expression is still unclear, but in the analysis of whites, 2911G> A (Gly971Arg) polymorphism of IRS1 gene encoding insulin receptor substrate-1 and sulfonylurea on pancreatic β cell membrane to form a receptor complex of agents, in humans with 67G> a (Glu23Lys) polymorphism KCNJ11 gene encoding K ⁺ channel (Kir6.2), with odds ratios of each secondary invalid expression 2.0 and 1.69 Yes, it has been reported that the risk of secondary ineffective expression increases (Non-Patent Document 4 and Non-Patent Document 5). However, when these polymorphisms were analyzed in Japanese, no significant difference was observed.
Sesti G. et al., Diabetes Care, 27: 1394-1398 (2004) Sesti G. et al., J. Clin. Endocrinol. Metab., 91: 2334-2339 (2006)

Oxidative stress due to hyperglycemia is considered as one of the mechanisms of secondary ineffectiveness. In fact, the increase in peroxide concentration in human islet cells due to hyperglycemia and the accompanying decrease in insulin secretion It has been reported (Non-Patent Document 6).
Tanaka Y. et al, Proc. Natl. Acad. Sci. USA, 99: 12363-12368 (2002)

On the other hand, hydrogen peroxide and organic hydroperoxide can be reduced to water and the corresponding alcohol using glutathione, and the expression of glutathione peroxidase (GPX1), which acts as an antioxidant stress-related enzyme, is increased by hyperglycemia treatment It has been reported (Non-Patent Documents 7 and 8). In addition, it has been reported that in pancreatic islets in which GPX1 is forcibly expressed, a decrease in insulin secretion due to hyperglycemia is suppressed (Non-Patent Document 6).
Brigelius-Flohe R., Biol. Chem., 387: 1329-1335 (2006) Laybutt DR et al., Diabetes, 51: 413-423 (2002)

  Therefore, the present inventors hypothesized that if the genetic polymorphism causes a decrease in GPX1 enzyme activity, it may lead to a decrease in insulin secretion and may also be associated with secondary ineffective expression. As a result of intensive research on the GPX1 gene, it is highly likely that patients with the relatively frequently occurring GPX1 gene haplotype * 2a are likely to develop secondary invalidity, and that the 596C> T polymorphism It was found useful as a tag for detecting haplotypes.

Furthermore, 3 genes that have been suggested to be associated with the onset of type 2 diabetes, namely ABCC8 gene encoding sulfonylurea receptor in pancreatic β cells (Non-patent document 9), pancreatic β cell proliferation and normal insulin HNF1A gene encoding the transcription factor HNF-1α involved in secretion (Non-patent Document 10) and the potassium channel that was found to be related to the onset of type 2 diabetes in Japanese by the present inventors KCNQ1 gene, and NFE2L2 gene (Non-patent Document 11) encoding the transcription factor Nrf2 that regulates the expression of various antioxidant stress genes, secondary ineffective expression that occurs when sulfonylurea is administered As a result of the analysis of these polymorphisms, it was found that diabetic patients with these polymorphisms were treated with sulfonylurea. The present invention was completed by finding that secondary invalidation is likely to occur.
Yokoi N. et al., Diabetes, 55: 2379-2386, 2006 Holmkvist J. et al., Diabetologia, 49: 2882-2891, 2006 Fukushima-Uesaka H. et al., Drug Metab. Pharmacokinet., 22: 212-219, 2007

Therefore, the first object of the present invention is to detect the presence or absence of a polymorphism of GPX1 gene, polymorphism of ABCC8 gene, polymorphism of HNF1A gene, polymorphism of NFE2L2 gene, or polymorphism of KCNQ1 gene. It is an object of the present invention to provide a method for examining in advance whether secondary ineffectiveness is likely to occur due to administration of a drug.
A second object of the present invention is to provide a primer used for detecting the presence or absence of a polymorphism in the GPX1 gene or a polymorphism in the NFE2L2 gene.
A third object of the present invention is to provide a probe used for detecting the presence or absence of a polymorphism in the GPX1 gene or a polymorphism in the NFE2L2 gene.

A fourth object of the present invention is to provide a test kit for detecting the presence or absence of a polymorphism in the GPX1 gene or a polymorphism in the NFE2L2 gene.
A fifth object of the present invention is to provide a test kit for inspecting in advance whether secondary invalidity is likely to occur due to administration of a sulfonylurea agent.

The above-mentioned objects of the present invention are methods for predicting in advance the occurrence of secondary ineffectiveness when a sulfonylurea agent represented by the following general formula (I) is administered,
The method is characterized by collecting a gene from a subject and detecting the presence or absence of at least one polymorphism selected from the group of genetic polymorphisms described in the following (A) to (E): It is achieved by a method for predicting the occurrence of secondary ineffectiveness when a sulfonylurea agent is administered in advance, a test kit used for the method, and a primer and probe for detecting the presence or absence of a polymorphism of the GPX1 gene and the NFE2L2 gene It was.
Formula (I)
R ₁ -SO ₂ NHCONH-R ₂ (I)
Here, R ₁ is a halogen, an alkyl group, an acetyl group, or a phenyl group optionally substituted with — (CH ₂ ) _n —NHCO—R ₃ , and R ₂ is optionally substituted with an alkyl group or an alkyl group. R ₃ is a group selected from carbocycles which do not contain an unsaturated bond and may contain a hetero atom, and R ₃ is an aryl group or heteroaryl group substituted with a halogen, an oxygen atom, an alkyl group or an alkoxy group. N is an integer from 1 to 5
(A) GPX1 gene: 596C> T polymorphism,
(B) ABCC8 gene: IVS15-3T> C polymorphism,
(C) HNF1A gene: 79A> C polymorphism,
(D) NFE2L2 gene: -1123A> G polymorphism,
(E) KCNQ1 gene: IVS15-29246C> T polymorphism

  According to the method of the present invention, since it is possible to easily detect whether or not secondary invalidity is likely to be expressed for each individual with diabetes by the polymorphic typing of the gene, based on the obtained results, For patients who are likely to develop ineffectiveness, it is possible to select treatments other than sulfonylureas, such as treatment with oral antidiabetics with other medicinal effects and early insulin introduction. . Therefore, the method of the present invention is extremely useful as a test method for drug selection for the purpose of ensuring the effectiveness of diabetes drug treatment.

In the present invention, the polymorphism to be detected in order to predict the occurrence of secondary ineffectiveness when the sulfonylurea agent is administered in advance is at least selected from the group of genetic polymorphisms described in (A) to (E) below. It is a kind of polymorphism.
(A) GPX1 gene: 596C> T polymorphism (rs1050450)
(B) ABCC8 gene: IVS15-3T> C polymorphism (rs1799854)
(C) HNF1A gene: 79A> C polymorphism (rs1169288)
(D) NFE2L2 gene: -1123A> G polymorphism
(E) KCNQ1 gene: IVS15-29246C> T polymorphism (rs2237892)

The GPX1 gene (NCBI, NW_921651.1) in the above polymorphism is located in 21.3 of the short arm of chromosome 3, and is composed of two exons, and uses glutathione with hydrogen peroxide and organic hydroperoxide. Thus, it is a gene encoding glutathione peroxidase (GPX1), which is an enzyme that reduces to water and the corresponding alcohol.
The ABCC8 gene (NCBI, NT_009237.17) is located at 15.1 of the short arm of chromosome 11 and is composed of 39 exons and is a gene encoding a receptor for sulfonylurea drugs on the pancreatic β cell membrane .

The HNF1A gene (NCBI, NT_009775.16) is located at 24.2 of the long arm of chromosome 12, and is composed of 10 exons, and is a transcription factor HNF involved in pancreatic β-cell proliferation and normal insulin secretion It is a gene encoding -1α and is also called TCF1 gene.
The NFE2L2 gene (NCBI, NT_005403.16) is located on the long arm 31 of chromosome 2 and is composed of 5 exons and encodes the transcription factor Nrf2 that controls the expression of antioxidant stress genes. It is a gene.

  The above KCNQ1 gene (NCBI, NT_009237.17) is located at 15.5 of the short arm of chromosome 11 and consists of 16 exons, and is related to the onset of type 2 diabetes in Japanese by the present inventors. It is a gene encoding a potassium channel that has been clarified.

In general, “polymorphism” is an individual difference of DNA sequences constituting a gene and means a state where the frequency is 1% or more. In the present invention, the occurrence frequency is a base having a frequency of less than 1%. The change of is treated as "polymorphism".
“Polymorphism” in the present invention is represented as “596C> T polymorphism”, for example. Here, the numbers represent the positions of bases counted 3 ′ from the adenine [A] of the translation initiation codon [ATG] of each gene, and the alphabets are bases, that is, adenine [A], thymine [T], guanine [G]. Represents cytosine [C].

  Therefore, the GPX1 gene polymorphism “596C> T polymorphism” is a cytosine [C], which is the 596th base counted from the adenine [A] of the translation initiation codon [ATG] of the GPX1 gene, in the downstream direction. Changed to [T]. The polymorphism of the ABCC8 gene, “IVS15-3T> C polymorphism”, is present on the 15th intron of the ABCC8 gene and is the third base thymine [T] counted upstream from the 5 ′ end of exon 16. ] Is changed to cytosine [C]. The polymorphism of the HNF1A gene, the 79A> C polymorphism, changes the 79th adenine [A] from the adenine [A] of the translation start codon [ATG] of the HNF1A gene to cytosine [C]. It is what. The NFE2L2 gene polymorphism, "-1123A> G polymorphism," is a translation of the translation start codon [ATG] from adenine [A] to the upstream 1123th base adenine [A] to guanine [G]. is there. (E) KCNQ1 gene polymorphism `` IVS15-29246C> T polymorphism '' is present on the 15th intron of KCNQ1 gene and is the 29246th base counted from the 5 'end of exon 16 in the upstream direction Cytosine [C] is changed to thymine [T].

In the present invention, the sulfonylurea agent includes a compound represented by the following general formula (I).
R ₁ -SO ₂ NHCONH-R ₂ (I)
Here, R ₁ is a halogen, an alkyl group, an acetyl group, or a phenyl group optionally substituted with — (CH ₂ ) _n —NHCO—R ₃ , and R ₂ is optionally substituted with an alkyl group or an alkyl group. A group selected from carbocycles which do not contain an unsaturated bond and may contain a hetero atom, R ₃ is an aryl group or a heteroaryl group substituted with a halogen, an oxygen atom, an alkyl group or an alkoxy group; n is an integer of 1-5.

In the present invention, in particular, R ₁ is appropriately substituted with a halogen such as chloro, an alkyl group having 1 to 3 carbon atoms such as a methyl group or an ethyl group, an acetyl group, or — (CH ₂ ) _n —NHCO—R _3. It is preferably a phenyl group. R ₂ is an alkyl group having 2 to 5 carbon atoms such as an ethyl group, a propyl group, or a butyl group, or an unsaturated group that is appropriately substituted with a methyl group or an ethyl group, such as O, N, and S. It is preferably a group selected from carbocyclic rings such as cyclohexyl group, pyrrolidine group, azabicyclooctyl group and the like, which may contain a hetero atom.
R ₃ is a halogen such as chloro, an alkyl group having 1 to 3 carbon atoms such as a methyl group or an ethyl group, an aryl group having 5 or 6 carbon atoms substituted with a methoxy group or an ethoxy group, or an oxygen atom, A heteroaryl group having 4 or 5 carbon atoms substituted with an alkyl group having 1 to 3 carbon atoms such as a methyl group or an ethyl group, a methoxy group, or an ethoxy group is preferable. Examples of the aryl group include a phenyl group, and examples of the heteroaryl group include a pyrroline group. Specific examples of R ₃ include a 2-methoxy-5-chlorophenyl group and a 2-oxo-3-ethyl-4-methylpyrroline group.

  Examples of the sulfonylurea represented by the general formula (I) include glimepiride, glibenclamide, gliclazide, tributamide, acetohexamide, chlorpropamide, glyclopyramide and the like.

  The method for detecting the presence or absence of a polymorphism in the present invention can be appropriately carried out by a known method. For example, GPX1 can be obtained from cells and nails of a subject's peripheral blood leukocytes, tissue cells such as pancreas, oral mucosa, hair and the like. It can be detected by preparing DNA or RNA containing the gene, ABCC8 gene, HNF1A gene, NFE2L2 gene and KCNQ1 gene and analyzing the single nucleotide polymorphism.

  As a method for analyzing the above single nucleotide polymorphism, a known analysis method can be used. For example, a direct sequencing method, a pyro sequencing method, a single-stranded DNA conformation polymorphism (SSCP) analysis, Restriction fragment length polymorphism (RFLP) analysis, allele-specific oligonucleotide (ASO) method, DNA chip / microarray method, matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF / MS) method, TaqMan method And an invader method.

  The direct sequencing method uses a primer that hybridizes to the gene of interest, amplifies the bases containing the polymorphism by PCR using the isolated DNA or RNA as a template, the die primer method, the dye terminator method, etc. In this method, the base sequence of the amplified DNA is determined by the cycle sequencing method (see Kwok PY and Duan S., Methods Mol. Biol. 212: 71-84 (2003)).

For example, using primers that hybridize to the GPX1 gene, ABCC8 gene, HNF1A gene, NFE2L2 gene, and KCNQ1 gene, using the isolated DNA as a template, PCR-amplified the region containing the polymorphism, and then the DNA amplified by the cycle sequencing method Can be determined. In addition, although the primer of this invention can also be used as said primer, this invention is not limited by these.
The DNA base sequence determined in this way is compared with a control to detect genetic polymorphisms of the GPX1, ABCC8, HNF1A, NFE2L2 and KCNQ1 genes.

  In general, since the sequences of the GPX1 gene, ABCC8 gene, HNF1A gene, NFE2L2 gene and KCNQ1 gene of a healthy person are considered normal, the above-mentioned `` compared to the control '' usually refers to the GPX1 gene of a healthy person, It means comparing with sequences of ABCC8 gene, HNF1A gene, NFE2L2 gene and KCNQ1 gene, respectively. In the present invention, the sequence of GPX1 gene (NCBI, NW — 921651.1) registered as a wild type in GenBank, the sequence of ABCC8 gene (NCBI, NT — 009237.17), the sequence of HNF1A gene (NCBI, NT — 009775.16), You may compare with the arrangement | sequence (NCBI, NT_005403.16) of NFE2L2 gene, and the arrangement | sequence (NCBI, NT_009237.17) of KCNQ1 gene, respectively.

  Pyrosequencing is an analysis method that uses a base extension reaction by DNA polymerase based on complementarity with the parent strand. Primers designed to sequence from one to several bases upstream of a single nucleotide polymorphism are used. This is a method of annealing, adding dNTPs to the system one by one, analyzing whether each base is extended, and determining the type of base at the site. In the detection of elongation, pyrophosphate released quantitatively from dNTPs when a base elongation reaction occurs is converted to ATP using adenosine-5'-phosphosulfate as a substrate and sulfurylase, and this ATP and luciferin are converted into substrates. The luciferase luminescence reaction generated as follows is detected by a CCD camera (see Langaee T. and Ronaghi M., Mutat Res. 573: 96-102 (2005)).

  For example, using primers that hybridize to the GPX1 gene, ABCC8 gene, HNF1A gene, NFE2L2 gene, and KCNQ1 gene, using the isolated DNA as a template, the region containing the polymorphism is PCR-amplified and then single-stranded, single nucleotides Primers designed to sequence from several bases upstream of the polymorphism are annealed. Next, dNTPs (dATP, dTTP, dCTP, dGTP) are added one by one and luciferase luminescence is measured. From the results, the DNA base sequence can be determined.

The base sequence of the DNA thus determined is compared with the control in the same manner as in the above direct sequencing method, and the polymorphisms of the GPX1, ABCC8, HNF1A, NFE2L2 and KCNQ1 genes are detected. .
In addition, although the primer of this invention can also be used as said primer, it is not limited to it.

  Single-stranded DNA conformation polymorphism (SSCP) analysis means that when a double-stranded DNA fragment is dissociated into single strands, each single strand forms a unique high-order structure depending on its base sequence. This is a method that uses properties. When this dissociated DNA strand is used for electrophoresis in a polyacrylamide gel that does not contain a denaturing agent, single-stranded DNA with the same strand length varies depending on the difference in each higher-order structure. Move to. The higher-order structure of this single-stranded DNA also changes by substitution, deletion, or insertion of a single base, and shows different mobility in polyacrylamide gel electrophoresis. Therefore, by detecting this change in mobility, it is possible to confirm that a mutation caused by point mutation, deletion, insertion or the like exists in the DNA fragment (Tahira T. et al., Methods Mol. Biol 212: 37-46 (2003)).

  Restriction fragment length polymorphism (RFLP) analysis is based on the fact that a site disappears or appears due to a mutation when the polymorphic sequence resulting from a point mutation is a site recognized by a specific restriction enzyme. Thus, this is a method for detecting a single nucleotide polymorphism (see Kim S. and Misra A., Annu Rev Biomed Eng. 9: 289-320 (2007)).

For example, analysis of the DNA containing the polymorphic site of GPX1 gene, ABCC8 gene, HNF1A gene, NFE2L2 gene and KCNQ1 gene using a specific restriction enzyme followed by Southern blot hybridization, etc. Can do.
The specific restriction enzyme is not particularly limited as long as it can detect mutations. For example, ApaI or Bpu1102I is used for analysis of GPX1 gene, DpnI is used for analysis of HNF1A gene, and ABCC8 gene is analyzed. For the analysis of PstI and KCNQ1 genes, SmaI and the like can be used.

The allele-specific oligonucleotide (ASO) method is the creation of an oligonucleotide containing a base sequence that is considered to have a mutation, and when hybridization is performed between this and the sample DNA, a mutation containing a single base substitution exists. If so, the efficiency of hybridization is reduced. This can be detected by Southern blotting or a method that utilizes the property of quenching by intercalating a special fluorescent reagent into the hybrid gap. Moreover, detection by the ribonuclease A mismatch cleavage method is also possible.
In the present invention, the probe of the present invention can be used as an oligonucleotide containing a base sequence considered to have the above mutation, but is not limited thereto.

The DNA chip / microarray method includes preparing a substrate containing a polymorphic site prepared from a subject and a substrate on which a nucleotide probe that hybridizes with the DNA is immobilized, and then bringing the DNA into contact with the substrate, This is a method for determining the presence or absence of the polymorphism by detecting the presence or absence of hybridization between the DNA sample and the nucleotide probe immobilized on the substrate, or the intensity difference thereof (Lindroos K. et al., Methods Mol. Biol. 212: 149-165 (2003) etc.).
The detection can be performed, for example, by previously labeling a DNA sample with a fluorescent dye or the like and reading the fluorescent signal with a scanner or the like.

In general, a DNA chip is composed of thousands of nucleotides printed on a substrate at high density. Usually, these DNAs are printed on the surface of an impermeable substrate. The surface layer of the substrate is generally glass, but a permeable film such as a nitrocellulose membrane can also be used as the surface layer of the substrate. As a substrate, not only a planar substrate but also a method using beads or the like is available.
In the present invention, the probe of the present invention can be used as a nucleotide probe that hybridizes with the above DNA, but is not limited thereto.

Matrix-assisted laser desorption / ionization-time-of-flight mass spectrometry (MALDI-TOF / MS) method is to prepare a primer having up to the base immediately before the polymorphic site of interest, which contains the polymorphic site After hybridizing with DNA, add multiple dideoxy bases corresponding to the polymorphic site, and further enzymatically extend one base, and analyze the molecular weight of the resulting single base extended primer by MALDI-TOF / MS It is a method to do. Since the molecular weight of the extended base is different, the molecular weight of the primer extended by one base is also different. This makes it possible to determine the base of the polymorphic site (see Storm N. et al., Methods Mol. Biol. 212: 241-262 (2003)).
In addition, although the primer of this invention can also be used as said primer, it is not limited to it.

The TaqMan method (registered trademark) refers to two TaqMan probes that are labeled with two different fluorescent dyes at the 5 'end and labeled with a quenching substance at the 3' end, and are complementary to the target polymorphic site base. PCR is performed using a primer designed to amplify a region containing the polymorphism and Taq DNA polymerase. Along with the PCR, the probe having a base complementary to the base of the polymorphic site is cleaved, the fluorescent dye is released from the quencher, and if the DNA is a homozygote, one type of fluorescence is detected and the heterozygote is detected. In the case of a conjugate, two types of fluorescence are detected simultaneously. The target polymorphism is determined based on the type and intensity of the fluorescence (see Livak KJ Methods Mol. Biol. 212: 129-147 (2003)).
In addition, although the primer of this invention can also be used as said primer, it is not limited to it.

The Invader (registered trademark) method is a method for detecting a polymorphism without using a PCR reaction, and a signal probe including a mismatch portion (flap sequence) and a polymorphic site sequence, and an invader oligo in which the polymorphic sites overlap. And a special enzyme called Cleavase (registered trademark) after binding to DNA having a polymorphic site, only when the base of the polymorphic site of the signal probe is complementary to the DNA sequence, The flap sequence is cleaved by this enzyme. The cleaved flap sequence acts as an invader oligo to the FRET probe (which has a fluorescent dye and a quencher) that generates a signal. As a result, the FRET probe is cleaved to release the quencher and the fluorescent dye and emit fluorescence. . The target polymorphism is determined based on the type and intensity of the fluorescence (see Lyamichev V. and Neri B. Methods Mol. Biol. 212: 229-240 (2003)).
In the present invention, the probe of the present invention can be used as an oligonucleotide containing a polymorphic site sequence, but is not limited thereto.

  If any one of the GPX1 gene, ABCC8 gene, HNF1A gene, NFE2L2 gene and KCNQ1 gene polymorphism is detected from the subject's cells by these methods, the subject is secondary ineffective when administered with a sulfonylurea agent. Is expected to occur. On the other hand, when it is not detected at all, it is predicted that secondary invalidation is unlikely to occur.

  When it is found by the method of the present invention that the subject tends to cause secondary ineffectiveness when administered with a sulfonylurea agent, the sulfonylurea agent is not administered, early insulin introduction, other oral diabetes drugs, for example, Therapeutic methods such as administration of biguanides such as buformin and metformin, α-glucosidase inhibitors such as acarbose and voglibose, and insulin sensitizers such as pioglitazone can be employed.

  Another embodiment of the present invention is a primer for detecting a polymorphism of the gene that amplifies a sequence portion containing the GPX1 gene, comprising SEQ ID NOs: 1, 3, 8-10, 12, 17-20, 23, A primer containing at least a part of the nucleotide sequence of 25 and 26, and a primer for detecting a polymorphism of the gene that amplifies a sequence portion containing the NFE2L2 gene, comprising SEQ ID NOs: 27, 28, 37, 42 and It is a primer comprising at least a part of the base sequence of 43. In particular, a primer containing at least a part of the base sequence of SEQ ID NOs: 20, 25, 26, 28, 42 and 43 is preferable as a primer for pyrosequencing.

  The primer in the present invention can be appropriately prepared by a known method. In addition, the primer of the present invention is preferably a nucleotide of about 10 to 30 bases, particularly about 15 to 25 bases, from the viewpoint of binding to the genome and its specificity, and the temperature of the extension reaction.

  The primer for detecting the polymorphism of the GPX1 gene is for amplifying the sequence portion containing the polymorphic base of the GPX1 gene, and SEQ ID NOs: 1, 3, 8 to 10, 12, 17 to 20, 23, The primer is not particularly limited as long as it is a primer including at least a part of the sequences of 25 and 26. Further, the primer for detecting the polymorphism of the NFE2L2 gene is for amplifying a sequence portion containing the polymorphic base of the NFE2L2 gene, and at least a part of SEQ ID NOs: 27, 28, 37, 42 and 43. There is no particular limitation as long as the primer includes a sequence.

  In the present invention, it is also possible to use a primer having a base sequence complementary to these primers, and the primer of the present invention can be used in the above-described method of the present invention.

Another embodiment of the present invention is a probe for detecting a polymorphism having a sequence portion containing a polymorphic base of GPX1 gene or NFE2L2 gene.
The probe in the present invention can be appropriately prepared by a known method, and is not particularly limited as long as it has a sequence portion containing a polymorphic base of GPX1 gene or NFE2L2 gene. Here, the sequence portion containing the GPX1 gene or NFE2L2 gene refers to a sequence portion of 10 to 50 bases containing these polymorphic bases, and examples thereof can include the base sequences of SEQ ID NOs: 44 and 45, respectively. . In particular, a sequence of about 15 to 25 bases is preferable from the viewpoint of specificity and binding property.

  In order to detect the 596C> T polymorphism of the GPX1 gene polymorphism, the 596th base counted in the downstream direction from the adenine [A] of the translation start codon [ATG] of the GPX1 gene contained in the probe of the present invention Is required to be cytosine [C] or thymine [T].

Further, in order to detect the -1123A> G polymorphism that is a polymorphism of the NFE2L2 gene, the 1123th base upstream from the adenine of the translation initiation codon of the NFE2L2 gene contained in the probe of the present invention is adenine [A] or It must be guanine [G].
In the present invention, it is also possible to use probes having a base sequence complementary to these probes.

  The probe of the present invention can be used in the above-described method of the present invention. Further, in order to facilitate detection, it can be appropriately labeled by a known method, and for example, it can be labeled with a radioisotope, an enzyme, a fluorescent dye or the like.

  Another aspect of the present invention is a test kit for detecting the presence or absence of the GPX1 gene polymorphism, which comprises a primer and / or a probe for detecting the GPX1 gene polymorphism. Another aspect of the present invention is a test kit for detecting the presence or absence of the polymorphism of the NFE2L2 gene, which has a primer and / or a probe for detecting the polymorphism of the NFE2L2 gene. These test kits are appropriately combined with the primer and / or probe of the present invention from among DNA extraction reagents, purification reagents, restriction enzymes, PCR amplification reagents, fluorescent dyes, luminescent reagents, coloring reagents, etc. It can be.

  In addition, another aspect of the present invention relates to a case where a sulfonylurea agent having at least one primer selected from the primers of the present invention and / or at least one probe selected from the probes of the present invention is administered. It is a test kit for predicting the occurrence of the next invalid in advance. In addition to the primer and / or probe of the present invention described above, a primer for amplifying sequences containing polymorphic bases of ABCC8 gene, HNF1A gene and KCNQ1 gene, sequencing primer and probe, DNA extraction reagent, purification reagent, A test kit can also be obtained by appropriately combining restriction enzymes, PCR amplification reagents, fluorescent dyes, luminescent reagents, coloring reagents, and the like.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further more concretely, this invention is not limited by these Examples.

<Detection of GPX1 gene polymorphism by direct sequencing>
GPX1 gene polymorphism was detected by direct sequencing using genomic DNA from 74 Japanese patients with type 2 diabetes, and haplotype estimation was performed based on the results. The primers used for direct sequencing are shown in [Table 1].
Of these, 32 cases were also used for the secondary invalid correlation analysis in Examples 2 and 3.

  In order to amplify the entire GPX1 gene region based on the genomic sequence (NCBI, NW — 921651.1), the forward primer and reverse primer for 1st PCR specific to GPX1 shown in [Table 1] above were designed. The first-stage PCR uses 1 μM each of the forward primer and reverse primer for 1st PCR, 0.025 units / μL of Z-Taq (Takara Bio Inc.), and 50 ng of genomic DNA in a total volume of 50 μL of GPX1 The full length of the gene was amplified. The PCR conditions in this case were [thermal denaturation 98 ° C., 5 seconds; annealing 55 ° C., 5 seconds; extension reaction 72 ° C., 190 seconds] × 30 cycles.

  Next, add 2 μL of Exonuclease I from the PCR Product Pre-Sequencing Kit (USB), 2 μL of Shrimp alkaline phosphatase, and 16 μL of purified water to 50 μL of the 1st PCR reaction solution, 15 minutes at 37 ° C, and 15 minutes at 80 ° C. After the treatment, it was cooled to 4 ° C.

  Next, in order to amplify the promoter and each exon region, a forward primer and a reverse primer for 2nd PCR having the sequences shown in [Table 1] above were designed. Using the 2nd PCR forward primer and reverse primer 0.5μM each and LA-Taq 0.05 units / μL (Takara Bio Inc.), including the promoter region or each exon region from the 1st PCR product of GPX1 gene (5μL) The DNA fragment was amplified in a total volume of 50 μL. PCR conditions at this time were heat denaturation 94 ° C., 5 minutes, [thermal denaturation 94 ° C., 30 seconds; annealing 55 ° C., 1 minute; extension reaction 72 ° C., 2 minutes] × 30 cycles, extension reaction 72 ° C., 7 minutes. It was.

  Subsequently, 5 μL of the 2nd PCR amplification product was added with 0.2 μL of PCR Product Pre-Sequencing Kit (USB), 0.2 μL of Shrimp alkaline phosphatase, and 1.6 μL of purified water to make a total of 7 μL. After 15 minutes at 80 ° C. and 15 minutes at 80 ° C., the mixture was cooled to 4 ° C.

  Next, primers for sequencing having the sequences shown in [Table 1] above were designed for the promoter and each exon region. For both strands of the 2nd PCR amplification product treated with PCR Product Pre-Sequencing Kit, ABI BigDye Terminator Cycle Sequencing Kit [Version 3.1] (Applied Biosystems) is directly used for sequencing. did.

More specifically, 1 μM of the above-described sequencing primer 1.6 μL, BigDye 2 μL, 5 × Sequencing buffer 3 μL, and purified water 7 μL are added to the total 7 μL of the 2nd PCR amplification product to make a total of 20.6 μL, [Thermal denaturation 96 ° C., 10 seconds; annealing 50 ° C., 5 seconds; extension reaction 60 ° C., 4 minutes] × 25 cycles, and then cooled to 4 ° C.
Excess dye was removed using DyeEx96 plate (Qiagen), and 20.6 μL of the total eluate was analyzed using ABI Prism 3730 DNA Analyzer (Applied Biosystems). At that time, the eluate was subjected to heat shock at 94 ° C. for 2 minutes.

  Thus, sequence analysis of the GPX1 gene region was performed on the genomic DNA of 74 Japanese type 2 diabetic patients, and a total of nine polymorphisms were found [Table 2]. Among these polymorphisms, there are two types that cause amino acid substitution, known 33_34insGCG (Alall_Alal2ins Ala) and 596C> T (Pro199Leu), and the frequency was 0.088. "33_34insGCG" has GCG (guanine-cytosine-guanine) inserted between the 33rd and 34th bases downstream from the adenine [A] of the translation start codon [ATG] of the GPX1 gene. "Alall_Alal2ins Ala" means that the alanine (Ala) is inserted between the 11th amino acid alanine (Ala) and the 12th amino acid alanine (Ala) from the N-terminal side of GPX1. Means that. “Pro199Leu” means that proline (Pro), which is the 199th amino acid from the N-terminal side of GPX1, is changed to leucine (Leu).

As a result of analysis using | D '| value and γ ² value by commercially available software SNPAlyze version 3.1 (Dynacom), strong linkage disequilibrium was observed over almost the entire analysis region, so the entire region was regarded as one block and the haplotype Analysis was performed. The | D '| and γ ² values were calculated based on the description in "Linkage analysis of the human genome" (J. Ott, translated by Takashi Gojobori, translated by Tokuichi Yasuda; published by Kodansha; pages 156-162). .
Haplotypes represent linkages between polymorphisms on chromosomes, and are known to show a higher correlation with phenotypes such as drug efficacy and side effects than when using a single polymorphism (Judson R et al., Pharmacogenomics, 1: 15-26 (2000)).

  In this example, estimation was performed by software SNPAlyze version 3.1 based on the Expectation-Maximization algorithm which is a standard method of haplotype estimation. The haplotype names are * 1 for those that do not have amino acid substitutions, * 2 for those that have amino acid substitutions, and subtypes are indicated by * numbers + lowercase alphabets [Table 2].

  Five * 1 subtypes and two * 2 subtypes were estimated. * 2 Haplotypes include the polymorphisms -649A> G, -46C> T, 33_34insGCG (Alall_Alal2ins Ala), and 596C> T (Pro199Leu), which are already known haplotypes (Hamanishi T. et al, Diabetes, 53: 2455-2460 (2004)). Among the estimated haplotypes, those with a frequency of 0.05 or more are * 1a, * 1b, * 2a, and 2 types [794 (* 185) as polymorphisms that serve as tags for classifying these 3 types A> G and 596C> T (Pro199Leu)] were selected. “* 185” represents a base located 185 bases downstream from guanine [G] of the translation termination codon [TAG].

<Typing of sulfonylurea long-term effective patient group and secondary ineffective patient group by pyrosequencing>
For the haplotype tag polymorphism selected in Example 1 with a frequency of 0.05 or more, we developed a typing system based on the pyrosequencing method, and used 278 Japanese sulfonylurea long-term effective patients (for more than 5 years using sulfonylurea, Effective) and 80 patients with secondary ineffectiveness (previously used sulfonylurea for more than 3 years and now shifted to insulin treatment). The background information and clinical information of these patients are shown in [Table 3]. In addition, as a patient who used the sulfonylurea agent, the patient who administered one or more types of sulfonylurea agents selected from glimepiride, glibenclamide, tolbutamide, and gliclazide was used.

It was confirmed that the frequency of neuropathy and retinopathy, which are complications of diabetes, was significantly higher in the secondary ineffective patient group (P value of Fisher's exact test with multiple comparison correction by Bonferroni method is p <0.005). Regarding the Bonferroni method, I refer to Naoyuki Kamatani, Statistics of Reality and Consciousness, p. 121, published by Yodosha.

  In the pyrosequencing method, primers were first designed based on the GPX1 genomic sequence (NCBI, NW — 921651.1). The designed primers are shown in [Table 4].

  In the PCR reaction, DNA was amplified in a total volume of 50 μL using 0.25 μM each of the amplification forward primer and reverse primer shown in [Table 4], LA-Taq 0.05 units / μL, and 25 ng of genomic DNA. . The PCR conditions were as follows: heat denaturation 94 ° C., 5 minutes, [thermal denaturation 94 ° C., 30 seconds; annealing 55 ° C., 45 seconds; extension reaction 72 ° C., 30 seconds] × 50 cycles, extension reaction 72 ° C., 5 Minutes.

  Subsequently, the PCR product was purified and single-stranded according to the conditions described in Saeki M. et al, Clin. Chem., 49: 1182-1185, (2003), and the above [Table 4] sequence was used. Primer (10 pmol) was added, treated at 95 ° C. for 2 minutes, and further cooled to hybridize. This was subjected to a mini-sequencing reaction using a pyro sequencer PSQ96MA apparatus (Biotage AB) and a Pyro Gold reagent kit (Biotage AB) to analyze the base of the polymorphic site.

<Haplotype analysis and correlation analysis between GPX1 haplotype and secondary ineffective expression>
Based on the results of the base analysis of the polymorphic site analyzed in Example 2, haplotype estimation was performed for each sulfonylurea long-term effective patient group and secondary ineffective patient by software SNPAlyze version 3.1, and the haplotype frequency was calculated. The results are shown in [Table 5].

  When the haplotype frequencies of the secondary ineffective patient group (80 cases) and the sulfonylurea long-term effective patient group (278 cases) were compared, the frequency of * 2a (0.125) in the secondary ineffective patient group (0.059) ) Was significantly higher (p = 0.028 after the multiple comparison correction by the Bonferroni method, odds ratio 2.3) ([Table 5] upper row). This significant difference was compared between the subgroups of the secondary ineffective patient group (42 cases) who transitioned to insulin treatment within 10 years of the start of sulfonylurea treatment and the sulfonylurea long-term effective patient group (130 cases) effective for 11 years or more. In the case (p = 0.0009 after correction, odds ratio 4.5) ([Table 5] lower row). On the other hand, there was no significant difference between the two groups in the frequency of the * 1b haplotype.

Finally, a logistic regression analysis was performed. The results are shown in [Table 6]. The logistic regression analysis was performed by a stepwise method using commercially available software JMP version 6 (SAS Institute Inc., Cary, NC, USA) and setting the p-value at the time of variable increase / decrease to 0.1.

First, in the analysis of sulfonylurea long-term effective patient group (278 cases) and secondary ineffective patient group (80 cases), the * 2a haplotype became a significant variable at p = 0.014. In addition to this, the starting age of sulfonylurea treatment (p <0.0001) and the duration of type 2 diabetes [period from onset] (P = 0.017) were significant variables. On the other hand, in the comparison between subgroups of the secondary ineffective patient group (42 cases) who transitioned to insulin treatment within 10 years from the start of sulfonylurea treatment and the sulfonylurea long-term effective patient group (130 cases) effective for more than 11 years, * 2a haplotype Only became a significant variable at p = 0.0009, and the above two patient background factors were not significant.
These results revealed that the * 2a haplotype of the GPX1 gene showed a significant correlation with secondary ineffective expression.

<Polymorphism analysis of ABCC8 gene, HNF1A gene and KCNQ1 gene by TaqMan method>
Using samples of patients who transitioned to insulin therapy within 10 years after the start of sulfonylurea treatment (42 patients with secondary ineffectiveness) and patients who were effective for 11 years or longer (130 patients with long-term sulfonylurea treatment), the ABCC8 gene TaqMan SNP Genotyping Assay Kit (Applied Biosystems) for IVS15-3T> C (rs1799854) polymorphism, HNF1A gene 79A> C (Ile27Leu, rs1169288) polymorphism, and KCNQ1 gene IVS15-29246C> T (rs2237892) polymorphism ) Polymorphism analysis. In addition, as a patient who used the sulfonylurea agent, the patient who administered one or more types of sulfonylurea agents selected from glimepiride, glibenclamide, tolbutamide, and gliclazide was used.
Specifically, 12.5 μL of TaqMan Universal PCR Master Mix (2X, Applied Biosystems), 1.25 μL of 20X TaqMan SNP Genotyping Mix, and 11.25 μL of sterilized purified water were mixed to 25 μL, and the PCR reaction was performed. PCR conditions were heat denaturation 95 ° C., 10 minutes, [thermal denaturation 92 ° C., 15 seconds; annealing and extension reaction 60 ° C., 1 minute] × 40 cycles. After completion of the PCR reaction, the fluorescence amount of the reaction solution was measured by ABI 7500 Real Time PCR System (Applied Biosystems), and polymorphism was determined from the fluorescence amount when each base was present.

<Polymorphism analysis of NFE2L2 gene by pyro-sequencing method>
Using the same sample as in Example 4, 6 haplotype tag polymorphisms of NFE2L2 gene [-1123A> G, -769G> A, -767G> A, -733C> A, 697C> T (Pro233Ser), 2229 (* 411 ) T> G] was analyzed by pyrosequencing.
Primers were designed based on the genomic sequence of NFE2L2 (NCBI, NT_005403.16). Primers used for pyro sequencing are shown in [Table 7].

  The PCR reaction was carried out in a system with a total volume of 50 μL using 0.2 μM each of the amplification forward primer and reverse primer shown in [Table 7], 0.02 units / μL of Ex-Taq (Takara Bio Inc.), and 20 ng of genomic DNA. DNA was amplified. For the -733C> A polymorphism analysis, LA-Taq 0.05 units / μL (Takara Bio Inc.) was used instead of Ex-Taq. The PCR conditions were as follows: thermal denaturation 94 ° C., 5 minutes, [thermal denaturation 94 ° C., 30 seconds; annealing 55 ° C., 45 seconds; extension reaction 72 ° C., 30 seconds] × 50 cycles, extension reaction 72 ° C., 5 Minutes.

  Subsequently, the PCR product was purified and single-stranded according to the conditions described in Saeki M. et al, Clin. Chem., 49: 1182-1185, (2003)). The sequence primer shown (10 pmol) was added, the mixture was treated at 95 ° C. for 2 minutes, and further cooled to allow hybridization. Next, using a Pyrosequencer PSQ96MA device (Biotage AB) and a Pyro Gold reagent kit (Biotage AB), a mini-sequencing reaction was performed to analyze the base of the polymorphic site.

<Correlation analysis and multivariate analysis between each gene polymorphism and secondary ineffective expression>
Analysis using the polymorphism allele model, dominant model, and inferior model of the polymorphism analyzed in Examples 4 and 5 was performed using commercially available SNPAlyze software version 7.0 (Dynacom).
The results of gene polymorphism analysis are shown in [Table 8]. ABCC8 gene IVS15-3T> C polymorphism, HNF1A gene 79A> C polymorphism (Ile27Leu), NFE2L2 gene -1123A> G polymorphism and 2229 (* 411) T> G polymorphism are allele model and dominant model The KCNQ1 IVS15-29246C> T polymorphism was a recessive model and showed a significant frequency difference (P <0.05). These results are all data before statistical multiplicity correction.

  The “allele model” is a method for comparing the number of major alleles and minor alleles (each individual has 2), and the “dominant model” is “homozygous major allele” and “heterozygous + minor allele” The “recessive model” is a method of comparing the numbers of “major allele homozygous + heterozygous” and “minor allele homozygous”.

Next, a logistic regression analysis was performed including the GPX1 * 2a haplotype and these five polymorphisms. For logistic regression analysis, as in Example 3, commercially available software JMP version 6 (SAS Institute Inc., Cary, NC, USA) was used, and the p-value at the time of variable increase / decrease was set to 0.1. Done by law.
The results of logistic regression analysis are shown in [Table 9]. Based on this result, we established a secondary ineffective prediction model containing 5 gene polymorphisms other than NFE2L2 2229 (* 411) T> G. In this case, patient background factors such as sex, age of sulfonylurea treatment onset and type 2 diabetes duration were not significant explanatory variables. Therefore, GPX1 * 2a haplotype [596C> T (Pro199Leu) polymorphism] and ABCC8 IVS15-3T> C, HNF1A 79A> C (Ile27Leu), NFE2L2 -1123A> G, KCNQ1 IVS15-29246C> T gene polymorphism However, it became clear that it was useful for the prediction of the secondary ineffective expression by a sulfonylurea agent.

  From these results, GPX1 gene 596C> T polymorphism, ABCC8 gene IVS15-3T> C polymorphism, HNF1A gene 79A> C polymorphism, NFE2L2 gene -1123A> G polymorphism and KCNQ1 gene IVS15-29246C It was confirmed that patients with> T polymorphism can be expected to be prone to secondary ineffectiveness when administered with sulfonylureas.

  According to the present invention, it is possible to predict whether or not secondary invalidity is likely to occur by examining a specific genetic polymorphism at the time of administration of a sulfonylurea agent, so that identification of a high-risk group and early insulin introduction for these patients In addition, it is possible to avoid secondary ineffectiveness by aggressive administration of oral and other oral diabetes drugs, and it is extremely effective from the viewpoint of reducing complications and maintaining patient QOL.

Claims (9)

A method for predicting in advance the occurrence of secondary ineffectiveness when a sulfonylurea agent represented by the following general formula (I) is administered,
The method is characterized by collecting a gene from a subject and detecting the presence or absence of at least one polymorphism selected from the group of genetic polymorphisms described in the following (A) to (E): A method for predicting the occurrence of secondary ineffectiveness when a sulfonylurea is administered;
Formula (I)
R ₁ -SO ₂ NHCONH-R ₂ (I)
Here, R ₁ is a halogen, an alkyl group, an acetyl group, or a phenyl group optionally substituted with — (CH ₂ ) _n —NHCO—R ₃ , and R ₂ is optionally substituted with an alkyl group or an alkyl group. R ₃ is a group selected from carbocycles which do not contain an unsaturated bond and may contain a hetero atom, and R ₃ is an aryl group or heteroaryl group substituted with a halogen, an oxygen atom, an alkyl group or an alkoxy group. And n is an integer from 1 to 5;
(A) GPX1 gene: 596C> T polymorphism,
(B) ABCC8 gene: IVS15-3T> C polymorphism,
(C) HNF1A gene: 79A> C polymorphism,
(D) NFE2L2 gene: -1123A> G polymorphism,
(E) KCNQ1 gene: IVS15-29246C> T polymorphism.   The method for predicting the occurrence of secondary ineffectiveness when the sulfonylurea agent according to claim 1 is administered, wherein the method for detecting the gene polymorphism is a direct sequencing method or a pyro sequencing method.   A primer for detecting a polymorphism of the GPX1 gene, which amplifies a sequence portion including the 596th base downstream from the adenine of the translation initiation codon of the GPX1 gene, which is SEQ ID NO: 1, 3, 8 to 10, 12, 17 A primer having a base sequence containing at least a part of -20, 23, 25 and 26, or a base sequence complementary to the base sequence.   A primer for detecting a polymorphism of the NFE2L2 gene, which amplifies a sequence portion including the 1123th base upstream from the translation start codon adenine of the NFE2L2 gene, comprising at least SEQ ID NOs: 27, 28, 37, 42 and 43 A primer having a partial base sequence or a base sequence complementary to the base sequence.   A probe for detecting a polymorphism of the GPX1 gene having a sequence portion containing the 596th base downstream from the adenine of the translation initiation codon of the GPX1 gene, or a base sequence complementary thereto.   A probe for detecting a polymorphism of the NFE2L2 gene having a sequence portion containing the 1123th base upstream from the translation start codon adenine of the NFE2L2 gene or a base sequence complementary thereto.   A test kit for detecting a polymorphism of the GPX1 gene, comprising the primer according to claim 3 and / or the probe according to claim 5.   A test kit for detecting a polymorphism of the NFE2L2 gene, comprising the primer according to claim 4 and / or the probe according to claim 6. A test kit for predicting in advance the occurrence of secondary ineffectiveness when a sulfonylurea agent represented by the following general formula (I) is administered, comprising at least one selected from the primers described in claims 3 to 4 Predicting the occurrence of secondary ineffectiveness when a sulfonylurea agent is administered, comprising at least one probe selected from a primer and / or a probe according to claims 5 to 6 Test kit for;
R ₁ -SO ₂ NHCONH-R ₂ (I)
Here, R ₁ is a halogen, an alkyl group, an acetyl group, or a phenyl group optionally substituted with — (CH ₂ ) _n —NHCO—R ₃ , and R ₂ is optionally substituted with an alkyl group or an alkyl group. R ₃ is a group selected from carbocycles which do not contain an unsaturated bond and may contain a hetero atom, and R ₃ is an aryl group or heteroaryl group substituted with a halogen, an oxygen atom, an alkyl group or an alkoxy group. And n is an integer from 1 to 5.