JP2011219375A - Use of ornithine aminotransferase enzyme activity inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a medicine and/or a method for treating diseases by clarify the new physiological role of OAT in a metabolic diseases such as diabetes and adjusting the function of the OAT, and to provide a method for detecting (diagnosing) a disease indexing the expression amount and/or enzymatic activity of the OAT.SOLUTION: The medicine containing the OAT enzyme activity inhibitor as an active ingredient exhibits an effect for lowering blood sugar by a single administration or a continuous administration, and is therefore singly effective as a medicine for preventing, improving or treating life style diseases including diabetes, or as a medicine for supplementing insulin treatment.

Description

The present invention relates to a medicine useful for treatment of lifestyle-related diseases and a method for screening a substance that is a candidate for the medicine, and further relates to a method for detecting (diagnosing) whether or not a subject suffers from lifestyle-related disease. More specifically, a lifestyle-related disease therapeutic agent containing a substance that inhibits the enzyme activity of OAT as an active ingredient, a screening method for the substance, and further, the subject suffers from lifestyle-related disease using the expression level of OAT as an index. The present invention relates to a method (diagnosis method) for detecting whether or not there is.

Lifestyle-related diseases are caused by “diseases related to the development and progression of lifestyle habits such as eating habits, exercise habits, rest, smoking, and drinking”, that is, due to inappropriate eating habits, lack of exercise, smoking, etc. This is a disease (metabolic syndrome) that occurs in the form of, for example, diabetes, stroke, heart disease, hyperlipidemia, hypertension and the like.
The treatment of lifestyle-related diseases, for which the number of patients has been increasing in recent years, is merely symptomatic treatment, and the drugs currently in use have not yet obtained sufficient therapeutic effects. It is craved in the medical field.
Further, in recent medical practice, it is desired to select a treatment method accurately according to the symptoms of individual patients as well as lifestyle-related diseases. In recent years, the need to improve quality of life (QOL) has been recognized. In particular, there is a strong demand for appropriate treatment according to the symptoms of individual patients, rather than treatment common to all people. Yes. In order to carry out such so-called tailor-made treatments, we must develop disease markers that accurately diagnose patient symptoms and their causes (genetic background) for individual diseases, and therapeutic drugs targeting the causative genes. The research we are aiming for is underway.

On the other hand, ornithine aminotransferase (ornithine ketoacid aminotransferase; EC 2.6.1.13, hereinafter referred to as OAT) is represented by the following reaction formula: L-glutamate 5-semialdehyde and ornithine (L-glutamate 5-semialdehyde) Ornithine) is an enzyme that catalyzes a reversible reaction. OAT is conserved in all eukaryotes and its existence is known from humans to Drosophila, yeast and plants.

The OAT gene is present on chromosome 10q26 in humans. For example, the base sequence of the human gene is known as GenBank Accession No. NM_000274, and the base sequence of the mouse gene is known as the sequence described in GenBank Accession No. NM_016978. The distribution of OAT in the living body is widely expressed in living bodies such as the liver, small intestine, tubules, and brain, but it is known that it is highly expressed in the retinal epithelium. The expression level in the retinal pigment epithelium is expressed in the liver. Is reported to be several times the activity in
Regarding OAT expression control, it is known that expression is increased by glucagon or a high protein diet, and that expression is inhibited by glucose (Non-patent Documents 1 and 2). It has also been reported as one of genes whose expression is controlled by insulin signals (Patent Document 1).

  Studies on the OAT protein have been made for a long time, and for example, there are reports extracted from rat liver and human liver. In addition, there is a report that human OAT protein is obtained using a recombinant E. coli. There have also been many reports on the purification of OAT protein, and X-ray crystal structure analysis of the purified protein has also been conducted. Regarding the X-ray crystal structure analysis of OAT protein, analysis of the binding mode between 5-fluoromethyl-L-ornithine (5-Fluoromethylornithine: 5FMOrn), which is an enzyme activity inhibitor, and human OAT protein has been reported. Examples of obtaining antibodies for OAT protein include antibodies to purified rat liver-derived protein (Non-patent Document 3). Thus, the OAT protein is a protein well known to those skilled in the art, both biochemically and structurally.

OAT as an enzyme catalyzes the reaction in the direction of ornithine production from P-5-C in neonates from the knowledge of KO mice, but conversely in the direction of production of P-5-C from ornithine in adults. It has been suggested to catalyze the reaction (Non-Patent Document 4). Also in humans, patients with mutations in the OAT gene have high ornithine levels in the serum, so that in adults as well as mice, the reaction is mainly in the direction in which P-5-C is produced from ornithine. (Non-Patent Document 5) In addition, in the neonatal period, in the mutant patient, not the ornithine but the ammonia level is high, so the production of ornithine is reduced by the same reaction as that of the newborn mouse, and the function of the urea cycle is reduced. (Non-Patent Document 6).
Regarding the relationship between OAT and disease state, it has been clarified that it is the causative gene of gyrate atrophy (hereinafter referred to as GA) in humans ("Human Mendelian Genetics" online, OMIM 258870) . It is known that an OAT-deficient mouse also exhibits a phenotype similar to that of human pathology as a GA model mouse (Non-patent Document 7). The cause of OAT deficiency in GA is thought to be the main cause of decreased proline supply from the results of in vitro studies. In other words, the decrease in proline synthesis from ornithine via OAT via P-5-C and the decrease in proline supply from glutamate due to the loss of OAT involved in P-5-C synthesis are considered as mechanisms. .
In addition, reports on the OAT gene as a gene related to the pathological condition include, for example, reports as one of nine genes that may be related to type 2 diabetes (Non-patent Document 8), angina pectoris, arrhythmia, etc. There is a report (Patent Document 2) as one of 15 modulators related to cardiovascular diseases. For details, see Non-Patent Document 8 by computer analysis combining 5 or 6 types from 7 types of gene analysis databases and data software: Methods GeneSeeker, eVOC system, DGP, PROSPECTR, SUSPECTS, G2D, POCUS. Nine genes including OAT have been identified as genes that are significantly expressed in type 2 diabetes. However, specifically, compared to healthy individuals, increased expression of OAT gene and OAT protein and increased OAT enzyme activity in biological samples (blood, cell suspension, cell extract, etc.) of diabetic patients have been reported. However, there is no mention that inhibition of OAT expression or inhibition of enzyme activity leads to improvement of the disease state. Patent Document 2 describes ornithine aminotransferase as Gene ID: 10183, and it shows that OAT gene expression is increased in patients with atherosclerosis, coronary artery disease, lipid abnormalities, and the like. Based on this, only reports that OAT is a gene related to cardiovascular diseases are described.

As a specific inhibitor for the enzyme activity of OAT, 5-Fluoromethylornithine (5FMOrn) is already known as a typical inhibitor. There are already many experimental reports using 5FMOrn. For example, it has been reported that ornithine content in tissues increases when administered to mice or chickens, but no abnormalities due to toxicity can be found in the eyes or other tissues. At this time, it has also been reported that OAT activity remains about 10-20% even when high concentration of 5FMOrn is administered.
In addition, 5-position substituted ornithine derivatives have already been technically disclosed as other reported OAT enzyme activity inhibitors.

In addition, although not specific, gabaculine as a pyridoxal phosphate-requiring enzyme inhibitor is also known to inhibit the enzyme activity of OAT. Or 4-aminobutyrate such as 4-aminohex-5-ynoic acid, 5-amino-1,3-cyclohexadienyl-carboxylic acid (gabaculine) and (S) -2-amino-4-amino-oxybutyric acid (L-Canaline) Analogs have also been reported to inhibit OAT activity.
Regarding the use of these compounds, the use of peripheral compounds of enzyme activity inhibitors for Alzheimer type dementia (use of enzyme activity inhibitors of ornithine aminotransferase in the treatment of Alzheimer's disease type dementia: (Patent Document 3)) is known. The mechanism of action is based on the enhancement of the liver cycle function in the liver by increasing the ornithine concentration by inhibiting the enzyme activity of OAT, and is considered to be useful for the treatment of temporary deficiency of ornithine.

  As described above, it has never been known that inhibition of enzyme activity of OAT is effective in improving sugar metabolism and treating lifestyle-related diseases such as metabolic syndrome.

US Patent Publication No. 2005085436 International Publication No.WO2003 / 039341 International Publication No. WO94 / 17795

Archives of Biochemistry and Biophysics Vol.259 p250-261 (1987) Archives of Biochemistry & Biophysics.Vol.237: 373-85 (1985) Archives of Biochemistry and Biophysics Vol.174 p262-272 (1976) Nature Genetics Vol.11 P185 (1995) Tohoku J. Exp. Med Vol.205 P335-342 (2005) J. Inherit.Metab.Dis.Vol.28 p673-679 (2005) Nature Genetics Vol.11 P185 (1995) Nucleic Acids Research (2006), Vol.34 p3067-3081 Biochemical Frontiers of Fluorine Chemistry, ACS Stnoisuyn Series 639, American Chemical Society, Washington DC. (1996) p196-212 Current Drug Targets Vol.1 p119-153 (2000)

  The problem to be solved by the present invention is to identify a new causative gene in a metabolic disease such as diabetes, thereby increasing the therapeutic agent for the disease caused by the gene, the expression level of the gene / protein and / or the enzyme activity of the protein. It is to provide a disease detection (diagnosis) method or the like as an index.

In order to solve the above-mentioned problems, the present inventors have intensively studied, and from the results of OAT gene expression analysis in the liver tissue of diabetic patients and healthy persons, OAT in the liver tissue and surrounding tissue tissues with the worsening of the pathological condition of diabetic patients. It was found that the gene expression was remarkably high compared to normal tissues. In addition, it was found that inhibition of OAT enzyme activity improves sugar metabolism, and it has been elucidated that OAT enzyme activity inhibitors are effective in the treatment of lifestyle-related diseases such as diabetes.
Specifically, it was clarified that blood glucose decreases when an inhibitor of OAT enzyme activity is administered to diabetic model mice. This indicates that OAT is effective in suppressing the increase in blood glucose in the diabetic state.
In addition, administration of an OAT enzyme activity inhibitor decreased the amount of drinking water associated with diabetes and markedly decreased the floor covering weight reflecting urine volume. This indicates that the OAT enzyme activity inhibitor not only lowers blood glucose but also reduces kidney urine filtration.
The present invention has been completed based on such findings.

That is, the present invention relates to the following [1] to [13]:
[1] A preventive, ameliorating, or therapeutic agent for lifestyle-related diseases, comprising a substance that inhibits the enzyme activity of OAT as an active ingredient.
[2] The agent for preventing, improving, or treating lifestyle-related diseases according to [1], wherein the substance that inhibits the enzyme activity of OAT is an ornithine derivative.
[3] The preventive, ameliorating, or therapeutic agent for lifestyle-related diseases according to [2], wherein the ornithine derivative is 5-fluoromethyl-L-ornithine.
[4] The agent for preventing, improving, or treating lifestyle-related diseases according to [1], wherein the substance that inhibits the enzyme activity of OAT is an antisense nucleotide or siRNA that inhibits the expression of the OAT gene.
[5] The agent for preventing, improving, or treating lifestyle-related diseases according to [1], wherein the substance that inhibits the enzyme activity of OAT is an antibody against OAT.
[6] The lifestyle according to [1] to [5], wherein the lifestyle-related disease is selected from the group consisting of diabetes, obesity, myocardial infarction, hypertrophic cardiomyopathy, congestive dilated cardiomyopathy, and myocardial fibrosis coronary artery disease Prevention, amelioration, or treatment of disease.
[7] A screening method for a candidate substance for an active ingredient of a preventive, ameliorating or therapeutic agent for lifestyle-related diseases, comprising the following steps (a), (b) and (c):
(A) contacting a test substance with an OAT protein and a substrate;
(B) a step of measuring the enzyme activity of OAT caused by the step (a) and comparing it with the enzyme activity of OAT when the test substance is not contacted; and (c) the comparison result of (b) above. A step of selecting a test substance that inhibits the enzyme activity of OAT based on the above.
[8] The screening method according to [7], wherein the OAT protein is human OAT, mouse OAT, or rat OAT.
[9] The screening method according to [7] or [8], wherein the lifestyle-related disease is selected from the group consisting of diabetes, obesity, myocardial infarction, hypertrophic cardiomyopathy, congestive dilated cardiomyopathy, and myocardial fibrosis coronary artery disease .
[10] A method for detecting lifestyle-related diseases comprising the following steps (a), (b), (c) and (d):
(A) collecting mRNA from a biological sample of a subject,
(B) a step of synthesizing cDNA from the mRNA obtained in (a) above,
(C) a step of measuring the expression level of the OAT gene using a primer encoding the OAT gene, and (d) a measurement result obtained for a healthy subject based on the measurement result of (c) above. In contrast, the step of determining that the patient is suffering from a lifestyle-related disease when the expression level of the OAT gene is increased.
[11] A method for detecting lifestyle-related diseases, comprising the following steps (a), (b), (c) and (d):
(A) contacting the subject's biological sample with the anti-OAT primary antibody;
(B) adding a labeled anti-OAT secondary antibody to the sample recognized by the antibody in (a),
(C) a step of adding a chromogenic labeling reagent to the sample recognized by the antibody in (b) and measuring the expression level of OAT protein, and (d) a measurement obtained on the subject based on the measurement result of (c) above Comparing the result with the measurement result obtained for a healthy person, and determining that the patient is suffering from a lifestyle-related disease when the protein expression level of OAT is increased,
[12] A method for detecting lifestyle-related diseases, comprising the following steps (a), (b), and (c):
(A) contacting the biological sample of the subject with the substrate;
(B) a step of measuring the enzyme activity of OAT caused by the step (a) above, and (c) a measurement obtained on the subject based on the measurement result of (b) above, a measurement obtained for a healthy person In contrast to the results, a step of determining that the patient is suffering from a lifestyle-related disease when the enzyme activity of OAT is increased.
[13] The lifestyle-related disease is selected from the group consisting of diabetes, obesity, myocardial infarction, hypertrophic cardiomyopathy, congestive dilated cardiomyopathy, and myocardial fibrosis coronary artery disease [10], [11], or [12] The detection method.

According to the present invention, it became clear that an OAT activity inhibitor can improve glucose metabolism by suppressing blood sugar increase in diabetic state, and prevent, ameliorate, and treat lifestyle-related diseases. It became possible to prevent, improve, and provide lifestyle-related diseases as ingredients.
Further, the present invention has revealed that the expression level of OAT gene and its expression product (protein, (poly) (oligo) peptide) and the enzyme activity of OAT are related to the pathology of lifestyle-related diseases. By using the expression level of OAT and / or the enzyme activity as an index, it has become possible to provide a detection (diagnosis) marker and a detection (diagnosis) method for lifestyle-related diseases including diabetes.

1 illustrates one embodiment of the present invention.
FIG. 1 is a graph showing the results of measuring sugar production in rat primary hepatocytes in the pharmacological test of Example 4. The comparison result of sugar production in Scramble RNA-introduced cells (Control) and two types of siRNA (siRNA1, siRNA4) -introduced cells for OAT is shown. Scramble represents a Scramble RNA-introduced cell (Control), OAT (1) represents a siRNA1-introduced cell, and OAT (4) represents a siRNA4-introduced cell. FBP (5) represents an siRNA-introduced cell for the gluconeogenic enzyme fructose bisphosphatase gene. FIG. 2 is a graph showing the results of measurement of OAT gene expression (a) and OAT enzyme activity (b) in livers derived from KKAy mice, which are diabetes model mice, in the pharmacological test of Example 5. In the expression (a) of the OAT gene, KK (10w) is the expression level of the OAT gene in hepatocytes of normal mice (KK / Ta Jcl), and KKAy (10w) is the hepatocytes of diabetic model mice (KKAy / Ta Jcl). Represents the expression level of the OAT gene. OAT activity (b) means OAT enzyme activity, KK / Ta is OAT enzyme activity in normal mouse (KK / Ta Jcl) hepatocytes, KKAy / Ta is diabetes model mouse (KKAy / Ta Jcl) Represents the enzymatic activity of OAT in hepatocytes. FIG. 3 is a graph showing the results of measurement of OAT gene expression (a) and enzyme activity (b) of OAT in the liver from db / db mice, which are diabetes model mice, in the pharmacological test of Example 6. In OAT gene expression (a), db / m is the expression level of the OAT gene in hepatocytes of normal mice (BKS.Cg-m + / + Leprdb / Jcl), and db / db is the diabetes model mouse (BKS.Cg- (+ Leprdb / + Leprdb / Jcl) represents the expression level of the OAT gene in hepatocytes. OAT activity (b) means the enzyme activity of OAT, db / m is the enzyme activity of OAT in hepatocytes of normal mice (BKS.Cg-m + / + Leprdb / Jcl), and db / db is a diabetes model mouse ( (BKS.Cg- + Leprdb / + Leprdb / Jcl) represents the enzyme activity of OAT in hepatocytes. FIG. 4 is a graph obtained by analyzing the expression levels of various genes per unit RNA amount using the Gene Chip system of Affymetrix by Ascenta (Gene Logics), which is a disease-related database of Example 7. (A) shows the comparison result of the expression level of the OAT gene in a patient who does not have diabetes and a patient with diabetes. Liver, Normal; Non-diabetes shows the results of patients without diabetes, and Liver, Normal; Diabetes shows the results of diabetic patients. (B) shows the comparison result of the expression level of the OAT gene in obese non-diabetic patients and diabetic patients. Liver, Normal; Obesity; shows the expression level of the OAT gene in obese non-diabetic patients, and Liver, Normal; Diabetes shows the results of diabetic patients. FIG. 5 (a) In the pharmacological test of Example 8, 5-FM-orunitine was intraperitoneally administered to db / db mice (BKS.Cg− + Leprdb / + Leprdb / Jcl) which are diabetes model mice, and 24 hours later. It is a graph which shows the result of having extract | collected the liver and measuring OAT enzyme activity. FIG. 5 (b) shows that 5-FM-orunitine was orally administered to db / db mice (BKS.Cg− + Leprdb / + Leprdb / Jcl), which are diabetes model mice, in the pharmacological test of Example 8, and the liver was 24 hours later. Is a graph showing the results of measuring OAT enzyme activity. Control was administered with physiological saline, Metformin was administered with Metformin 300 mg / kg, and 5-FM-orunitine was administered with 5 mg / kg (racemic 20 mg / kg). FIG. 6 shows that in the pharmacological test of Example 9, (a) an OAT enzyme activity inhibitor was administered intraperitoneally to db / db mice (BKS.Cg− + Leprdb / + Leprdb / Jcl) as diabetes model mice. It is a graph which shows the result of having measured the blood glucose (glucose amount) at the time. Shows blood glucose level (glucose level) over time after single intraperitoneal administration of Control (saline), metformin (300 mg / kg), 5-FM-ornithine (20 mg / kg (racemic)) , ◆ is physiological saline, ■ is Metformin (300 mg / kg), ▲ is 5-FM-ornithine (20 mg / kg (racemic)), × is the result of administering a mixture of Metformin and 5-FM-ornithine It is. FIG. 7 shows the blood glucose level (glucose level) at the timing of 6 hours from the start of administration when the test substance was administered intraperitoneally once in the pharmacological test of Example 9. ◆ indicates physiological saline, ■ indicates Metformin (300 mg / kg), ▲ indicates 5-FM-ornithine (20 mg / kg (racemic)), and x indicates a mixture of Metformin and 5-FM-ornithine To do. FIG. 8 shows the lactic acid value (lactose amount) over time when a test substance is administered intraperitoneally once in the pharmacological test of Example 9. ◆ is physiological saline, ■ is Metformin (300 mg / kg), ▲ is 5-FM-ornithine (20 mg / kg (racemic)), × is the result of administering a mixture of Metformin and 5-FM-ornithine is there. FIG. 9 shows the blood glucose when an OAT enzyme activity inhibitor was continuously administered intraperitoneally to db / db mice (BKS.Cg− + Leprdb / + Leprdb / Jcl), which are diabetes model mice, in the pharmacological test of Example 10. It is a graph which shows the result of having measured glucose amount. Saline for saline, Metformin (300 mg / kg) for Met, 5-FM-ornithine for FM-orn (20 mg / kg (racemic)), Met + FM for Metformin and 5-FM-ornithine Means something. FIG. 10 shows the body weight when an OAT enzyme activity inhibitor was continuously administered intraperitoneally to db / db mice (BKS.Cg− + Leprdb / + Leprdb / Jcl) as a diabetes model mouse in the pharmacological test of Example 10. The results of measuring a) and weight gain (b) are shown. ◆ is raw food (saline), ■ is Metformin (300 mg / kg), ▲ is 5-FM-orn (5-FM-ornithine 5 mg (20 mg / kg (racemic))), × is Met + Fmor (Metformin and 5-FM-ornithine) FIG. 11 shows 7 cages when OAT activity inhibitors were continuously administered intraperitoneally to db / db mice (BKS.Cg− + Leprdb / + Leprdb / Jcl) as diabetes model mice in the pharmacological test of Example 10. The amount of food intake per person, the amount of drinking water, and the increase in floor covering weight are measured, and the values corrected per day are shown. ◆ is raw (saline), ■ is Metformin (300 mg / kg), ▲ is 5-FM-orn (5-FM-ornithine (20 mg / kg (racemic))), × is Met + Fmor (Metformin and Means those administered 5-FM-ornithine). FIG. 12 shows the expression level of various genes per unit RNA amount analyzed by Ascenta (Gene Logics), which is a disease-related database of Example 11, using the Gene Chip system of Affymetrix. The comparison result in the heart of the expression level of the OAT gene in the patient who does not suffer from cardiovascular disease and the patient with cardiovascular disease is shown. The black bar on the right of each bar graph shows the results of patients who do not have cardiovascular disease, and the white bar on the left shows the results of patients with cardiovascular disease.

The present invention is described in detail below.
In this specification, the abbreviations of amino acids, (poly) peptides, (poly) nucleotides, etc. are defined by IUPAC-IUB [IUPAC-IUB Communication on Biological Nomenclature, Eur. J. Biochem., 138: 9 (1984). ], “Guidelines for the preparation of specifications including base sequences or amino acid sequences” (edited by the Japan Patent Office), and conventional symbols in the field.

When using the term “OAT gene” or “OAT DNA” in the present specification, unless otherwise stated, the human OAT gene (DNA) represented by a specific nucleotide sequence (SEQ ID NO: 1), its homologues and variants And a gene (DNA) encoding a derivative or the like. Specifically, the human OAT gene described in SEQ ID NO: 1 (GenBank Accession No. NM_000274), the mouse OAT gene described in SEQ ID NO: 3 (GenBank Accession No. NM_016978), and the rat described in SEQ ID NO: 5. Examples include the OAT gene (GenBank Accession No. NM_022521).
The “gene” or “DNA” is encoded not only by “gene” or “DNA” represented by a specific base sequence (SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5) but also by these. Also included are “genes” or “DNAs” that encode proteins (eg, homologues (orthologs, splice variants, etc.), variants and derivatives) that have biological functions equivalent to those of the protein. Specifically, the “gene” or “DNA” encoding the homologue, variant or derivative is the above-mentioned SEQ ID NO: 1 under the stringent conditions described in the following section (3-1). And “gene” or “DNA” having a base sequence that hybridizes with a complementary sequence of the specific base sequence of SEQ ID NO: 3 and SEQ ID NO: 5.
In the present specification, “polynucleotide” is used to include both RNA and DNA.

  When the term “OAT protein” or simply “OAT” is used herein, not only human OAT represented by the specific amino acid sequence (SEQ ID NO: 2) but also biological functions thereof are equivalent unless otherwise stated. As long as there is a certain limit, it is used to include homologues thereof (orthologs and splice variants), mutants, derivatives, matured forms, and amino acid modifications. Examples of orthologs include proteins from other species such as mice and rats that correspond to human proteins, and these were identified by HomoloGene (http://www.ncbi.nlm.nih.gov/HomoloGene/). It can be identified from the base sequence of the gene (SEQ ID NO: 1). Variants also include naturally occurring allelic variants, non-naturally occurring variants, and variants having amino acid sequences that have been altered by artificial deletion, substitution, addition or insertion. . Examples of the mutant include those that are at least 70%, preferably 80%, more preferably 95%, and still more preferably 97% homologous to a protein or (poly) peptide without mutation. The amino acid modifications include naturally occurring amino acid modifications and non-naturally occurring amino acid modifications, and include phosphorylated forms of amino acids. Specifically, human OAT having the amino acid sequence described in SEQ ID NO: 2 (GenBank Accession No. NP_000265), mouse OAT having the amino acid sequence described in SEQ ID NO: 4 (GenBank Accession No. NP_058674), and sequence And rat OAT having the amino acid sequence described in No. 6 (GenBank Accession No. NP_071966).

In the present specification, the OAT enzyme activity inhibitor includes a naturally occurring compound, a synthesized compound, a nucleic acid exhibiting an RNAi effect, and the like, a low molecular compound, a low molecular peptide, and a low molecular polynucleotide. These may have an appropriate salt or solvate form. In addition, nucleic acids such as antisense oligonucleotides for genes encoding OAT can be used for gene therapy, and such embodiments are also included in the present invention. OAT enzyme activity inhibitors in the present invention include ornithine derivatives, OAT antisense oligonucleotides, OAT siRNA, anti-OAT antibodies, and the like.
The ornithine derivatives used in the present invention include compounds derived from ornithine, pharmaceutically acceptable salts, optical isomers of these D or L conformations, and mixtures thereof. 5FMOrn is a particularly preferred ornithine derivative.

  Antisense oligonucleotide used in the present invention refers to DNA having a sequence complementary to a DNA coding sequence or a fragment DNA in a 5 ′ non-coding sequence or RNA corresponding to the DNA, and binds to DNA or RNA. It means something that regulates the expression of OAT. An antisense oligonucleotide can be produced, for example, as DNA based on the base sequence of a gene encoding an OAT protein, or can be produced as RNA by incorporating this DNA into an expression plasmid in the antisense orientation. The antisense oligonucleotide may be a sequence complementary to the DNA coding sequence of DNA consisting of a base sequence or a DNA fragment of any part of the 5 ′ non-coding sequence, preferably a transcription initiation site, a translation initiation site, It is desirable that the sequence is complementary to the 5 ′ untranslated region, the border region between exons and introns, or the 5 ′ CAP region. Preferable length of this antisense oligonucleotide is, for example, 5 to 200 bases, more preferably 10 to 50 bases, and particularly preferably 15 to 50 bases. When an antisense oligonucleotide is incorporated into an expression plasmid, the preferred length of the antisense oligonucleotide is 1000 bases or less, preferably 500 bases or less, more preferably 150 bases or less. After the antisense oligonucleotide is incorporated into an expression plasmid, it is introduced into a target cell according to a conventional method. The introduction can be performed by a method using a liposome or a recombinant virus. An antisense oligonucleotide expression plasmid can be prepared by ligation using a normal expression vector, downstream of the promoter and in the reverse direction, that is, so that the OAT gene is transcribed in the 3 'to 5' direction.

The “siRNA (short-interfering RNA) used in the invention” is a short double-stranded RNA having about 15 to 40 bases homologous to mRNA and protruding at both 3 ′ ends. siRNA can be synthesized using OAT cDNA as a template. The siRNA used in the present invention may have the same relationship as that of the above-mentioned antisense oligonucleotide with respect to the base sequence of OAT. When siRNA is introduced into a cell, it recognizes and cleaves the target mRNA in a sequence-specific manner, thereby inhibiting the expression of the target gene.
The “anti-OAT antibody” used in the present invention is useful as long as it shows an ability to inhibit OAT enzyme activity, whether it is an antibody capable of specifically recognizing OAT, or an antibody against OAT / substrate. is there. “Specifically recognizes” means binding to OAT. The antibody may be a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a single chain antibody, or a part of the above antibody having antigen binding properties such as a Fab fragment or a fragment generated by a Fab expression library. Further, an antibody can be obtained by a conventional method known to those skilled in the art using an OAT protein as an antigen, or a peptide fragment containing the epitope thereof. If antibodies are commercially available, they may be used. The anti-OAT antibody is useful as a therapeutic agent for lifestyle-related diseases, a screening method for detecting a drug that suppresses glucose production, which is a symptom of diabetes, and an immunological diagnosis.

The dosage form of a drug containing an OAT enzyme activity inhibitor as an active ingredient can be dissolved in water, powdered form, or can be used as it is, and can also be used as a formulation, food and drink, pharmaceutical It can mix | blend suitably. In general, it is dissolved or dispersed in a suitable liquid carrier, or mixed with a suitable powder carrier or adsorbed thereto, and in some cases, they are further mixed with an emulsifier, a dispersant, a suspension, Add a dressing, penetrating agent, wetting agent, stabilizer and the like, and use it as a preparation such as emulsion, oil, wettable powder, powder, tablet, capsule (including soft capsule), liquid. When used as a preparation, the amount of the compound used varies depending on the form of the preparation, but is preferably 0.01% by weight or more and 50% by weight or less. When the active ingredient in the present invention is used as a preventive or therapeutic agent for diabetes, it can contain a pharmaceutically acceptable carrier as described above. siRNA can be introduced directly into cells or incorporated into an siRNA expression vector ("RNAi Experimental Protocol" Yodosha). When administering antisense oligonucleotides, antisense oligonucleotide chemical modifications or siRNA as they are, they are mixed with stabilizers, buffers, solvents, etc., and then formulated with antibiotics, anti-inflammatory agents, anesthesia It may be administered simultaneously with drugs.
The dosage form is not particularly limited, and for example, oral preparations such as powders, fine granules, granules, tablets, capsules, suspensions, emulsions, syrups, extracts, or pills, or injections And parenteral preparations such as topical solutions, ointments, suppositories, topically applied creams, and eye drops. Of these, oral preparations are excipients, binders, disintegrants, surfactants, lubricants, fluidity promoters, diluents, preservatives, colorants, fragrances, stabilizers, humectants, preservatives. Or it can manufacture in accordance with a conventional method using antioxidant. Parenteral preparations can also be produced by conventional methods, and examples of the administration method include intramuscular injection, intravenous injection, transdermal administration, and rectal administration.

  The dose when the OAT enzyme activity inhibitor in the present invention is used for treatment of lifestyle-related diseases varies depending on symptoms, age, body weight, sex, etc., but preferably 1 μg / kg to 100 mg / kg per adult day. May be administered at 10 μg / kg to 20 mg / kg, more preferably 50 μg / kg to 5 mg / kg, orally or parenterally, every day or every few days to every few weeks. In order to avoid such frequent administration, it is also possible to prepare a sustained-release mini-pellet preparation and embed it near the affected area. Alternatively, it can be gradually and gradually administered to the affected area using an osmotic pump or the like. When administered in this manner, it exhibits an OAT enzyme activity inhibitory effect and can reduce symptoms of lifestyle diseases such as diabetes.

Examples of cells used for screening for OAT enzyme activity inhibitors include all cultured cells that express the OAT gene, regardless of whether they are endogenous or exogenous. The expression of the OAT gene can be easily confirmed by detecting the expression of these genes by a known Northern blot method or RT-PCR method. In addition, OAT enzyme activity is also measured by a method known to those skilled in the art, in which o-Aminobenzaldehyde reacts with 1-Pyrroline-5-carboxylate (P-5-C), which is a product of the OAT enzyme reaction, to develop color ( Journal of Biological Chemistry Vol.225 p825-834 (1957)), and other detection methods using radioactive substrates with higher sensitivity (Journal of Neurochemistry Vol.36 p501-505 (1981 You can also check with)). Measurement reports using this method include, for example, examination of the difference in activity between tissues in rats (Biochimica et Biophysica Acta Vol. 73 p222-231 (1963), Journal of Biological Chemistry Vol. 243 p3327-3332 (1968)) In addition, studies on activity in rat kidney and liver (Archives of Biochemistry and Biophysics Vol. 180 p472-479 (1977)) and reports on activity control using rat primary stem cells (Annals of the New York Academy of Sciences Vol. .349 p99-110 (1980), Archives of Biochemistry and Biophysics Vol.237 p373-385 (1985)), etc., for example, the following steps (a), (b), (c), and (d) And a method for screening a candidate substance for an OAT enzyme activity inhibitor.
(A) contacting a test substance with a biological sample and a substrate ornithine and a reaction solution (for example, 100 mM potassium phosphate buffer (pH 8.0), 0.1 mM PLP, 35 mM ornithine, 5 mM o-aminobezaldehyde),
(B) a step of detecting and quantifying the enzyme activity of OAT caused by the step (a) above by measuring absorption at 440 nm with an absorptiometer under conditions kept at 37 ° C.,
(C) comparing the enzyme activity of OAT when the test substance is not contacted with a biological sample and the activity of (b) above, and (d) the enzyme activity of OAT based on the comparison result of (c) above. A step of selecting a test substance to be inhibited.

  Specific examples of cells used in the screening include (A) liver tissue and liver-derived cells isolated and prepared from animal models of diabetes, and (B) cells into which the gene of the present invention has been introduced. Can do. Here, the animal model of the former (A) diabetes may be any animal model as long as it is known as an animal model of diabetes, and specifically, KKAy / TaJcl, BKS Cg- + Lepradb / + Leprdb / Jcr, etc. be able to. The latter (B) of the gene-introduced cell of the present invention includes, in addition to the former (A) cell, a host cell usually used for gene introduction, namely L-929 (derived from connective tissue, ATCC strain number CCL-1), C127I ( Breast cancer tissue-derived, ATCC strain number CRL-1616), Sp2 / 0-Ag14 (myeloma-derived, ATCC strain number CRL-1581), NIH3T3 (fetal tissue-derived, ATCC strain number CRL-1658) and other mouse-derived cells, rats Derived cells, BHK-21 (derived from Syrian hamster kidney tissue, ATCC strain number CCL-10), hamster-derived cells such as CHO-K1 (derived from Chinese hamster ovary, ATCC strain number CCL-61), COS1 (African green monkey kidney tissue) Origin, ATCC strain number CRL-1650), CV1 (derived from African green monkey kidney tissue, ATCC strain number CCL-70), Vero (derived from African green monkey kidney tissue, Sumitomo Dainippon Pharma Co., Ltd.), HeLa (uterus Cervical cancer-derived, Dainippon Sumitomo Pharma Co., Ltd.), 293 (fetal kidney-derived, ATCC strain number CRL-1573) and other human-derived cells, And insect-derived cells such as Sf9 (Invitrogen Corporation) and Sf21 (Invitrogen Corporation). Furthermore, the cells used in the screening method of the present invention include tissues that are aggregates of cells.

  In the screening method of the present invention, not only cells but also an enzyme solution containing OAT activity or a purified protein can be used.

Test substances (candidate substances) to be screened by the screening method of the present invention are not limited, but include nucleic acids (including antisense nucleotides of OAT gene), peptides, polypeptides, proteins, organic compounds, inorganic compounds, and appropriate salts thereof. Specifically, the screening of the present invention is performed by bringing these test substances or a sample (test sample) containing them into contact with the cells and / or tissues, enzyme solution, or purified protein. . Examples of such test samples include, but are not limited to, cell extracts containing test substances, gene library expression products, synthetic low-molecular compounds, synthetic peptides, natural compounds, and the like. In the screening of the present invention, the condition for bringing the test substance into contact with the cell or the enzyme protein is not particularly limited. However, when the test substance is brought into contact with the cell, the culture condition (temperature: 30) in which the cell does not die and can express the OAT gene. ℃ -40 ℃, pH: 6.5-8.5, Medium: William's Medium E, 10% FBS, 100 nM 3,3,5-triiodo-L-thyronine, 100 nM Dexamethasone, 1 nM Insulin, etc. (rat primary cultured hepatocytes In the case of contact with enzyme protein, the enzyme activity is not inactivated, for example, 100 mM potassium phosphate buffer (pH 8.0), 0.1 mM PLP, 35 mM ornithine, 5 mM o-aminobezaldehyde. Is preferred.
A substance selected by the screening method of the present invention can be positioned as an OAT enzyme activity inhibitor. These substances inhibit the onset and progression of diabetes by inhibiting the enzyme activity of OAT, and thus become potential candidates for drugs for preventing, ameliorating or treating diabetes.

  In the present specification, the “disease marker” refers to those used directly or indirectly to detect / diagnose the presence / absence, degree of morbidity, presence / absence or improvement of lifestyle-related diseases. For example, i) a poly-oligonucleotide capable of specifically recognizing and binding to an OAT gene, ii) an antibody capable of specifically recognizing and binding to an OAT protein, and iii) an OAT enzyme activity Can be mentioned. The diagnostic methods i), ii) and iii) are shown below.

i) Diagnosis method based on OAT gene expression Examples of samples used for diagnosis include biological samples (blood, cell suspension, cell extract) of a subject. (a) A step of extracting and preparing total RNA from the biological sample using RNeasy Mini Kit (QIAGEN), (b) TaqMan Reverse Transcription Reagents (Applied Biosystems) using the obtained total RNA as a template And (c) a pair of primers encoding the OAT (positive strand that binds to the cDNA (-strand), + Using the SYBR Green RT-PCR Reagents (Applied Biosystems), prepare an RT-PCR reaction solution according to the protocol using the ABI PRIME 7900 Sequence Detection System (Applied Biosystems). A step of detecting and quantifying the reaction product, (d) a step of preparing a correlation value between the expression level of the OAT gene and lifestyle-related diseases in advance using biological samples of healthy individuals and lifestyle-related diseases; e) Measurement results obtained for the subject using the correlation value described in (d) above as an index. A method of diagnosis comprising the step of determining that the subject is suffering from a lifestyle-related disease when the expression level of the OAT gene is increased in comparison with the measurement result obtained for a healthy person.

ii) Diagnosis method by OAT protein expression Examples of samples used for diagnosis include biological samples of subjects (blood, cell suspension, cell extract). A label such as an enzyme label, a chromogenic label, a radiolabel, or a luminescent label is bound to an OAT antibody or a secondary antibody, and this label can be detected or measured. Immunoassays that can be performed in the present invention include ELISA, Western blot, immunoprecipitation, slot or dot blot assay, immunohistochemical staining, radioimmunoassay (RIA), fluorescent immunoassay, immunoassay using avidin-biotin or streptavidin-biotin system However, it is not limited to these.
For example, as a diagnostic method by ELISA, measurement is performed according to a manual attached to an ELISA quantification kit (Quantikine: manufactured by R & D Systems), (a) a step of coating an anti-OAT primary antibody on a 96-well plate for ELISA, (B) The step of adding the prepared biological sample to the plate of (a), (c) The step of adding the avidin-anti-OAT secondary antibody to the plate of (c), (d) The middle plate of (c) The step of detecting and quantifying the production amount of OAT protein, and the step of (e) correlating the correlation between the production amount of OAT protein and lifestyle-related diseases in advance using biological samples of healthy individuals and patients with lifestyle-related diseases (F) Using the correlation value described in (e) as an index, the measurement result obtained for the subject is compared with the measurement result obtained for a healthy person, and the expression level of OAT protein is increased. If lifestyle-related diseases And a method of diagnosis including the step of determining that the patient is affected.

iii) Diagnosis method based on OAT enzyme activity Samples used for diagnosis include biological samples (blood, cell suspension, cell extract) of a subject. (A) mixing biological sample and substrate ornithine with a reaction solution (100 mM potassium phosphate buffer (pH 8.0), 0.1 mM PLP, 35 mM ornithine, 5 mM o-aminobezaldehyde),
(B) a step of detecting and quantifying the enzyme activity of OAT caused by the step (a) above by measuring absorption at 440 nm with an absorptiometer under conditions kept at 37 ° C.,
(C) a step of preparing in advance a correlation value between OAT enzyme activity and lifestyle-related disease using biological samples of healthy individuals and patients with lifestyle-related disease, (d) subject using the correlation value described in (c) above as an index Compared with the measurement results obtained for healthy subjects, the method includes a step of determining that the patient is suffering from a lifestyle-related disease when the value of OAT enzyme activity is increased. It is done.

Next, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

Example 1 (RT-PCR)
Total RNA was extracted from mouse liver tissue using RNeasy Mini Kit (QIAGEN). In addition, total RNA was prepared from rat primary cultured hepatocytes using QuickGene800 (Fujifilm) and RNA cultured cell kit S (Fujifilm). The obtained total RNA was subjected to cDNA synthesis with random primers using TaqMan Reverse Transcription Reagents (Applied Biosystems).
The RT-PCR method uses a pair of primers (the above cDNA (-strand) so that the nucleotide sequence region encoding OAT can be specifically amplified using cDNA prepared from RNA obtained from liver tissue or liver cells as a template. ) And the reverse chain that binds to the + strand are designed and synthesized in the usual manner. When quantifying mouse OAT, primers having the sequences of SEQ ID NOs: 7 and 8 were used. When quantifying rat OAT, primers having the sequences of SEQ ID NOs: 9 and 10 were used. Using the synthesized primers, prepare an RT-PCR reaction solution according to the protocol using SYBR Green RT-PCR Reagents (Applied Biosystems) and react with ABI PRIME 7900 Sequence Detection System (Applied Biosystems). The reaction product was detected and quantified.

Example 2 (Measurement of OAT activity)
To measure the OAT activity in the liver, add 4 times the amount of extraction buffer (100 mM KP (pH 8.0), 100 uM PLP, 150 mM KCl, 10 mM 2-ME) to the collected liver, and cool with ice. It was crushed with a Nizer (Ultra Turrax). After crushing, it was placed in an Eppendorf tube, centrifuged at 2000 rpm for 2 min, and the supernatant was used for activity measurement. 1 ml each of a reaction solution containing 20 mM α-ketoglutalate and a reaction solution not containing (100 mM potassium phosphate buffer (pH 8.0), 0.1 mM PLP, 35 mM ornithine, 5 mM o-aminobezaldehyde) were placed in an Eppendorf tube and kept at 37 ° C. in advance. 50 μl of sample was added, immediately stirred and kept at 37 ° C. After the reaction time, 50 ul of 100% TCA was added and stirred to stop the reaction. After standing at room temperature for 30 min, it was centrifuged at 15,000 rpm for 5 min, and the absorbance at 440 nm of the obtained supernatant was measured. The concentration obtained from the absorbance obtained from the difference in absorbance between the tube containing α-ketoglutalate and the tube not containing α-ketoglutalate was calculated using the extinction coefficient of reaction product 2.45 (mmol, cm, A440 nm).

Example 3 (Preparation of rat primary cultured hepatocytes)
Rat hepatocytes were isolated according to the collagenase perfusion method (J Cell Biol, 43: 506-520, 1969).
Wistar rats (male 8 weeks old; 264 g) were anesthetized by intraperitoneal injection of somnopentyl. After sterilization with EtOH, the portal vein was exposed, and the Sirflow Flash (22G) was stabbed, followed by perfusion with Liver Perfusion Medium (Gibco # 17701-038) pre-heated at 37 ° C with a peristaltic pump. The vein was cut. After exsanguination, perfusion was started with a collagenase solution prepared and kept warm as described below.
The collagenase solution was prepared as follows. To 400 μl of Liberase Blenzyme 2 solution (Roche 5 mg / ml), 400 μl of 10% BSA (Sigma A-6003) solution and 200 μl of 10% Trypsin Inhibitor (Sigma T-6522) were added and mixed. Add 2 ml of 10% BSA in advance and add 200 ml of solution (HANKS'BALANCED SALT powder (Sigma H-6136), HEPES 2.38 g / L, sodium bicarbonate 0.35 g / L) and stir gently. Used.
After sufficient perfusion with a collagenase solution and digestion, the digestion was stopped by switching to perfusion with Liver Perfusion Medium. Cut out the digested liver, wash buffer (HANKS 'BARANCED SALT (Sigma H-6136), 5% FBS (JRH Cat.No. 12176-500M), HEPES 2,38 g / L, Na bicarbonate 0.35 g / L, EGTA The cells were dispersed and collected in 0.19 g / L). After washing 5 times with wash buffer while passing through the mesh, the suspension was suspended in a medium (William's Medium E, 10% FBS, 100 nM 3,3,5-triiodo-L-thyronine, 100 nM Dexamethasone, 1 nM Insulin).

Example 4 (Introduction of siRNA)
siRNA was introduced using RNAiFect Transfection Reagent (QIAGEN). As siRNA, scramble siRNA (target sequence SEQ ID NO: 11) was used as a control, and two kinds of siRNA for rat OAT (rOAT1 target sequence SEQ ID NO: 12 and rOAT4 target sequence SEQ ID NO: 13) were used. Rat primary cultured hepatocytes were prepared at 5 × 10 ⁵ cells / ml (12-well plate) (William's Medium E, 10% FBS, 100 nM 3,3,5-triiodo-L-thyronine, 100 nM Dexamethasone, 1 nM Insulin) . 400 pmol of siRNA was suspended in 400 μl of EC-R buffer and stirred with vortex for 10 seconds. Next, 24 μl of RNAiFect reagent was added and stirred with vortex for 10 seconds. 200 μl each of the mixed solution was placed in 2 wells of Collagen coated 12 Well Plate coated with Type II Collagen and allowed to stand at room temperature for 15 min (more). The previously prepared rat primary hepatocytes were added in an amount of 600 μl / well (3 × 10 ⁵ cells / well). Three hours later, 60 μl of 10-fold concentration Pen./Str. (Invitrogen Gibco Penicillin-Streptomycin Cat. No. 15140-122) was added. After culturing for 24 hours, the medium was changed with WME + 10% FBS + 1 × Pen. / Str. + 1 nM insulin + 100 nMDexamethasone. After culturing for 24 hours, the medium (WME + 10% FBS + 1 × Pen. / Str. + 1 nM insulin + 1 nM Dexamethasone + 100 pM glucagon) was changed. After another 24 hours, the medium was changed in the same manner. Three days later, sugar production was measured. The cells were washed several times with D-PBS (-), and then D-MEM (Sigma Cat.No.D-5030 containing 0.1% fructose, 100 uM cAMP, 0.75 mMoleate, 10 mM ornithine in Sodium Bicarbonate (Sigma Cat.No. S-5761) (added 3.7 g / L) was added at 1 ml / well and cultured in a 37 ° C. CO ₂ incubator for 6 hours. After the culture, the culture supernatant was collected and quantified with a glucose CII test Wako assay reagent (Wako Pure Chemicals # 4399090). In addition, RNA was extracted from the cells after collecting the supernatant using RNA cultured cell kit S with QuickGene-800 (Fujifilm). Compared to Scramble siRNA-introduced cells, decreased sugar production was observed in cells into which two types of siRNA for OAT, rOAT1 and rOAT4, were introduced (FIG. 1).

Example 5 (Expression and activity in KKAy mouse liver)
Eight KKAy / Ta Jcl (Japan Claire ♂ 10 weeks 齡) and KK / Ta Jcl (Japan Claire ♂ 10) confirmed to be urine sugar of 500 mg / dl or more by urine sugar measurement with test paper Weekly) Livers were collected from 5 animals (average body weight 32.1 g), RNA was extracted, and RT-PCR was performed by the method described in Example 1. Further, OAT activity was measured from the same liver by the method described in Example 2. The expression of RNA was increased in KKAy / Ta Jcl, a diabetes model mouse (FIG. 2 (a)), and the increase in activity was also observed in the OAT activity (FIG. 2 (b)). .

Example 6 (expression and activity in db / db mouse liver)
BKS.Cg- + Leprdb / + Leprdb / Jcl (Japan Claire ♂ 10 weeks 齡) 3 (average weight 41.4 g) and BKS.Cg-m + / + Leprdb / Jcl (Japan Claire ♂ 10 weeks 齡) 3 (average) Liver was collected from 26.0 g of body weight, RNA was extracted, and RT-PCR was performed by the method described in Example 1. Further, OAT activity was measured from the same liver by the method described in Example 2. The expression of RNA was increased in BKS.Cg- + Leprdb / + Leprdb / Jcl, which is a model mouse for diabetes (FIG. 3 (a)), and the increase in activity was also observed in the OAT activity. (FIG. 3 (b)).

Example 7 (Expression in human liver)
Ascenta (Gene Logics), which is a disease-related database, analyzed the expression level of various genes per unit RNA using the Gene Chip system of Affymetrix, and the present inventors used this to analyze OAT The gene expression pattern in pathological and non-pathological tissues was analyzed. As a result, the expression level of OAT gene in the liver of diabetic patients was about 1.4 times higher than the expression level of OAT gene in the liver of non-diabetic patients. In addition, when this comparison was focused on non-diabetic and obese patients, the difference further widened (FIGS. 4 (a) and (b)). 4 (a) and 4 (b) both show OAT gene expression. (A) shows the comparison result of OAT gene expression levels in patients who cannot confirm diabetes and diabetic patients, and (b) is obese non-diabetic. The comparison result of the expression level of OAT gene in a patient and a diabetic patient is shown. It was found that more OAT genes were expressed in diabetic patients.

Example 8 (5-FM-ornithine ex vivo test)
BKS.Cg- + Leprdb / + Leprdb / Jcl (CLEA Japan ♂ 10 weeks 齡) (average body weight about 46 g) with saline (n = 3) and 5-FM-ornithine (racemate 20 mg / kg, n = 4) was administered intraperitoneally, and the liver was collected 24 hours later. The OAT activity was measured by the method described in Example 2 using the collected liver. In contrast to 1320 nmol / mg.hr in the control, it was 128 nmol / mg.hr in the 5-FM-ornithine administration group, and a clear inhibition of activity was observed (FIG. 5 (a)).
Similarly, BKS.Cg- + Leprdb / + Leprdb / Jcl (Japan Claire ♂ 40 weeks 齡) saline (n = 1) and 5-FM-ornithine (racemate 20 mg / kg, n = 2) were orally administered. The liver was collected 24 hours later. The OAT activity was measured by the method described in Example 2 using the collected liver. Clear inhibition of activity was observed in the 5-FM-ornithine administration group (FIG. 5 (b)).

Example 9 (5-FM-ornithine db / db single administration)
BKS.Cg- + Leprdb / + Leprdb / Jcl (CLEA Japan ♂ 9 weeks 齡) (average weight about 40g) with saline (n = 7), metformin (300mg / kg, n = 7), 5-FM- Ornithine (racemate 20 mg / kg, n = 7) was administered intraperitoneally. The bait was removed immediately after administration, and blood was collected from the tail vein 2, 4, and 6 hours later and treated with perchloric acid. After 6 hours, food was returned and normal breeding was continued, and blood was collected 22 hours later. Blood glucose was quantified with a glucose CII test Wako assay reagent (Wako Pure Chemical # 4399090) (FIG. 6). A significant (P <0.01) decrease in blood glucose of 190 mg / dL was observed in the 5-FM-ornithine administration group compared to 260 mg / dL in control of blood glucose 6 hours later. On the other hand, it was 146 mg / dL in the metformin administration group of the control group (FIG. 7).
Lactic acid levels of the obtained serum samples were measured using Determiner LA (Kyowa Medex). The metformin of the control group showed an increase in lactic acid level after 2 hours, whereas the 5-FM-ornithine administration group showed no increase in lactic acid level at any time (FIG. 8).

Example 10 (5-FM-ornithine db / db consecutive throw)
BKS.Cg- + Leprdb / + Leprdb / Jcl (Japan Claire ♂ 9 weeks 齡) (average body weight approx. 40 g) with saline (n = 7), metformin (300 mg / kg, n = 7) 5-FM- Ornithine (racemate 20 mg / kg, n = 7) was administered intraperitoneally. The food was removed from immediately after administration until 6 hours, and then the food was returned and normal breeding was continued. The same amount was administered every day from the next day. One week later, blood from the tail vein was collected and blood glucose was measured. A significant (P <0.01) decrease in blood glucose of 463 mg / dL was observed in the 5-FM-ornithine administration group versus 601 mg / dL in the control. On the other hand, it was 490 mg / dL in the metformin administration group of the control group (Fig. 9). There was no difference in weight change during the breeding period in any group (FIGS. 10 (a) and (b)), and no clear difference was observed in food consumption (FIG. 11 (a)). On the other hand, there was a clear decrease of about 30% in drinking water between the 5-FM-ornithine administration group and the metformin administration group (Fig. 11 (b)), and at the same time the floor covering weight change was also reduced by about 30%. (FIG. 11 (c)).

Example 11 (Expression in a human disease state sample)
Ascenta (Gene Logics), which is a disease-related database, analyzed the expression level of various genes per unit RNA using the Gene Chip system of Affymetrix, and the present inventors used this to analyze OAT The gene expression pattern in pathological and non-pathological tissues was analyzed. As a result, the expression level of the OAT gene in the heart of the cardiovascular disease patient was higher than the expression level of the OAT gene in the heart of the non-disease patient (FIG. 12).

According to the present invention, OAT gene expression, protein expression, further, enzyme activity is involved in the pathology of lifestyle-related diseases, and further, administration of an OAT enzyme activity inhibitor prevents or improves lifestyle-related diseases, It became clear that it could be treated.
Therefore, OAT enzyme activity inhibitors containing nucleic acids, antibodies, and low molecular weight compounds that inhibit OAT enzyme activity as active ingredients can be used as preventive, ameliorating, and therapeutic agents for lifestyle-related diseases, and OAT enzyme activity It is possible to screen for drugs that can prevent, ameliorate, and treat lifestyle-related diseases using the index as an index, and to detect (diagnose) lifestyle-related diseases using OAT enzyme activity as an index.
In addition, OAT enzyme activity inhibitors can be expected not only for humans but also for other animals as a preventive, ameliorative, and therapeutic agent for lifestyle-related diseases. In fact, in dogs and cats, insulin treatment is mainly used to treat diabetes, but effective low-molecular-weight therapeutic agents are not used, and drugs that inhibit OAT enzyme activity and have a hypoglycemic effect are insulin. It can also be expected as a therapeutic agent that complements treatment or as a single therapeutic agent.

SEQ ID NOs: 7 to 10: PCR primers SEQ ID NOs: 11 to 13: siRNA

Claims (13)

A preventive, ameliorating, or therapeutic agent for lifestyle-related diseases comprising a substance that inhibits the enzyme activity of OAT as an active ingredient. 2. The preventive, ameliorating, or therapeutic agent for lifestyle-related diseases according to claim 1, wherein the substance that inhibits the enzyme activity of OAT is an ornithine derivative. The preventive, ameliorating, or therapeutic agent for lifestyle-related diseases according to claim 2, wherein the ornithine derivative is 5-fluoromethyl-L-ornithine. 2. The preventive, ameliorating, or therapeutic agent for lifestyle-related diseases according to claim 1, wherein the substance that inhibits the enzyme activity of OAT is an antisense nucleotide or siRNA that inhibits the expression of the OAT gene. The agent for preventing, improving, or treating lifestyle-related diseases according to claim 1, wherein the substance that inhibits the enzyme activity of OAT is an antibody against OAT. 6. Prevention of lifestyle-related diseases according to claims 1 to 5, wherein the lifestyle-related diseases are selected from the group consisting of diabetes, obesity, myocardial infarction, hypertrophic cardiomyopathy, congestive dilated cardiomyopathy, and myocardial fibrosis coronary artery disease. , Improvement, or treatment. A method for screening a candidate substance for an active ingredient of a preventive, ameliorating, or therapeutic agent for lifestyle-related diseases, comprising the following steps (a), (b), and (c):
(A) contacting a test substance with an OAT protein and a substrate;
(B) a step of measuring the enzyme activity of OAT caused by the step (a) and comparing it with the enzyme activity of OAT when the test substance is not contacted; and (c) the comparison result of (b) above. A step of selecting a test substance that inhibits the enzyme activity of OAT based on the above. 8. The screening method according to claim 7, wherein the OAT protein is human OAT, mouse OAT, or rat OAT. The screening method according to claim 7 or 8, wherein the lifestyle-related disease is selected from the group consisting of diabetes, obesity, myocardial infarction, hypertrophic cardiomyopathy, congestive dilated cardiomyopathy, and myocardial fibrosis coronary artery disease. A method for detecting lifestyle-related diseases comprising the following steps (a), (b), (c), and (d):
(A) collecting mRNA from a biological sample of a subject,
(B) a step of synthesizing cDNA from the mRNA obtained in (a) above,
(C) a step of measuring the expression level of the OAT gene using a primer encoding the OAT gene, and (d) a measurement result obtained for a healthy subject based on the measurement result of (c) above. In contrast, the step of determining that the patient is suffering from a lifestyle-related disease when the expression level of the OAT gene is increased. A method for detecting lifestyle-related diseases comprising the following steps (a), (b), (c), and (d):
(A) contacting the subject's biological sample with the anti-OAT primary antibody;
(B) adding a labeled anti-OAT secondary antibody to the sample recognized by the antibody in (a),
(C) a step of adding a chromogenic labeling reagent to the sample recognized by the antibody in (b) and measuring the expression level of OAT protein, and (d) a measurement obtained on the subject based on the measurement result of (c) above A step of comparing the result with a measurement result obtained for a healthy person and determining that the patient is suffering from a lifestyle-related disease when the protein expression level of OAT is increased. A method for detecting lifestyle-related diseases comprising the following steps (a), (b), and (c):
(A) contacting the biological sample of the subject with the substrate;
(B) a step of measuring the enzyme activity of OAT caused by the step (a) above, and (c) a measurement obtained on the subject based on the measurement result of (b) above, a measurement obtained for a healthy person In contrast to the results, a step of determining that the patient is suffering from a lifestyle-related disease when the enzyme activity of OAT is increased. The detection method according to claim 10, 11 or 12, wherein the lifestyle-related disease is selected from the group consisting of diabetes, obesity, myocardial infarction, hypertrophic cardiomyopathy, congestive dilated cardiomyopathy, and myocardial fibrosis coronary artery disease.

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