JP2008019169A - Novel ppar regulator and its screening method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a screening method of a medicine for a more efficient treatment of diabetes and a novel compound for treating and preventing PPAR-related diseases. <P>SOLUTION: A method for identifying a compound that exhibits at least one activity selected from the group consisting of an insulin secretion activity of a pancreas β cell and a peripheral insulin sensitivity-promoting activity and is capable of regulating a peroxisome proliferator activator receptor (PPAR) is provided. The method comprises (A) a step for providing candidate compounds, (B) a step for measuring at least one activity selected from the group consisting of the insulin secretion activity of a pancreas β cell and the peripheral insulin sensitivity-promoting activity of the candidate compounds, (C) a step for subjecting the candidate compounds to an assay for measuring the PPAR activity, and (D) a step for identifying a substance which is judged to have the activity in each of the steps B and C as a lead compound. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to the diagnosis, treatment or prevention of diseases or disorders associated with PPAR genes such as diabetes. More particularly, the present invention relates to screening for drugs for diagnosis, treatment or prevention of diseases associated with the PPAR gene. The present invention also provides the drug itself obtained thereby.

  The hyperglycemia seen in type 2 diabetes is caused by multiple defects in both insulin secretion from pancreatic beta cells and insulin sensitivity of peripheral tissues (eg, liver, muscle and fat). Sulfonylurea (SU) agents have played a central role in pharmacotherapy of type 2 diabetes as effective oral hypoglycemic agents. A sulfonylurea agent first stimulates insulin secretion by binding to a sulfonylurea receptor (SUR) on the plasma membrane of pancreatic β cells (Non-patent Document 1: Gribble, FM, and Ashcroft, FM (2000) Metabolism 49, 3-6).

  In addition to such pancreatic effects, sulfonylureas (including glimepiride and glibenclamide) have a direct effect on fat, and sulfonylureas can induce insulin in 3T3-L1 cells or isolated adipocytes. Several reports have pointed out that it enhances or mimics its action. However, the molecular target leading to extrapancreatic action has not yet been determined.

  Peroxisome proliferator-activated receptor γ (PPARγ) is one of the important transcription factors that regulate glucose and lipid metabolism and lipogenesis (Non-patent Document 2: Wu, Z. et al. (1998) J. Biol. Clin. Invest. 101, 22-32; Non-patent document 3: Tontonoz, P. et al. (1994) Genes Dev. 8, 1224-1234; Non-patent document 4: Tontonoz, P., Hu, E., and Spiegelman, BM (1994) Cell 79, 1147-1156). Thiazolidinedione (TZD) is another hypoglycemic agent that improves peripheral insulin resistance. Thiazolidinedione is an effective PPARγ agonist, and most of its pharmacological actions are thought to be mediated by activation of PPARγ in adipocytes (Non-Patent Document 5: Lehmann, JM et al ( 1995) J. Biol. Chem. 270, 12953-12956; Non-Patent Document 6: Spiegelman, BM (1998) Diabetes 47, 507-514).

  For the treatment of diabetes, it is preferable to both enhance the action of insulin (ie, increase sensitivity) and stimulate insulin secretion (activate pancreatic β cells). However, a compound having both activities has not been known.

Merck describes a new regulator of PPAR. However, this compound has been described for use in combination with other diabetes therapeutic agents (eg, sulfonylurea agents). However, there is no description of a compound that both enhances the action of insulin (that is, increases sensitivity) and stimulates insulin secretion (activates pancreatic β cells) (Patent Document 1). ).
Gribble, FM, and Ashcroft, FM (2000) Metabolism 49, 3-6 Wu, Z. et al. (1998) J. Clin. Invest. 101,22-32 Tontonoz, P. et al. (1994) Genes Dev. 8, 1224-1234 Tontonoz, P., Hu, E., and Spiegelman, BM (1994) Cell 79, 1147-1156 Lehmann, JM et al (1995) J. Biol. Chem. 270, 12953-12956 Spiegelman, BM (1998) Diabetes 47, 507-514 WO2004 / 066963

  Therefore, an object of the present invention is to provide a method for screening a drug for treating diabetes more efficiently and a new compound for treating and preventing a disease associated with PPAR.

  The above problems have been solved by the present inventors by finding that a sulfonylurea agent unexpectedly has a PPAR regulating effect.

  Sulfonylurea agents (including glimepiride and glibenclamide) are widely used oral hypoglycemic agents, mainly by binding to the sulfonylurea receptor on the plasma membrane of pancreatic beta cells, Stimulates insulin secretion. Thiazolidinediones (thiazolidinediones) (eg, pioglitazone and rosiglitazone) are hypoglycemic agents that effectively improve peripheral insulin resistance through activation of peroxisome proliferator-activated receptor gamma (PPARγ). In the present invention, the inventors have found that glimepiride specifically induces PPARγ transcriptional activity in a luciferase reporter assay. Glimepiride is a coactivator of DRIP205 (abbreviation for vitamin D receptor-interacting protein 205; accession number AF283812; available from Dr. David J. Mangelsdorf (University of Texas Southwestern Medical Center, Dallas, TX)). Presser (N-CoR and SMRT. Abbreviation for nuclear receptor corepressor (N-CoR; accession number NM_006311) and silencing mediator for retinoid and thyroid hormone receptors (SMRT; for example, accession number AF113003). Enhanced dissociation of J. Mangelsdorf (available from the University of Texas Southwestern Medical Center, Dallas, TX). Furthermore, glimepiride directly bound to PPARγ in a manner that competes with rosiglitazone, a proven ligand for PPARγ. Furthermore, in 3T3-L1 adipocytes, glimepiride stimulates the transcriptional activity of gene promoters containing PPAR responsive elements (PPRE) and confers changes in mRNA levels of PPARγ target genes including aP2, leptin, and adiponectin It was. Finally, glimepiride induced adipocyte differentiation in 3T3-F442A cells. The 3T3-F442A cells were found to differentiate in a PPARγ agonist dependent manner. Interestingly, most of the effects observed with glimepiride were also reproduced with glibenclamide. These data indicate that glimepiride and glibenclamide (both belonging to the sulfonylurea agent) are agonists for PPARγ, but their ability was 12-20% of the maximum reached by pioglitazone . Our observation that glimepiride and glibenclamide were able to act not only on sulfonylurea receptors but also on PPARγ, development of ideal antidiabetic agents that enhance insulin secretion in pancreatic β cells and insulin sensitivity in peripheral sites Clues can be given.

  In this study, we studied the direct effect of sulfonylureas on PPARγ. In one embodiment, the inventors have found that glimepiride (which is one of the sulfonylurea agents) specifically induces PPARγ transcriptional activity. Furthermore, glimepiride was found to bind directly to PPARγ in a competitive manner with rosiglitazone. Furthermore, glimepiride altered the mRNA levels of PPARγ target genes in 3T3-L1 adipocytes and induced adipocyte differentiation in 3T3-F422A cells. Interestingly, most of the effects observed with glimepiride were also reproduced with glibenclamide. These data strongly suggest that both glimepiride and glibenclamide are agonists for PPARγ. Our observation that glimepiride and glibenclamide were able to act not only on mSUR but also on PPARγ suggests that an ideal anti-diabetic agent that enhances insulin secretion in pancreatic β-cells and peripheral insulin sensitivity Development clues can be given.

Accordingly, the present invention provides the following.
(1) A peroxisome proliferator activated receptor (PPAR) modulator comprising a sulfonylurea agent.
(2) The sulfonylurea agent is

Where R ₁ and R ₂ are each independently alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted Cycloalkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, carbocyclic group, substituted carbocyclic group, heterocyclic group, substituted heterocyclic group, halogen, hydroxy, substituted hydroxy, thiol, Substituted thiol, cyano, nitro, amino, substituted amino, carboxy, substituted carboxy, carbamoyl, acyl, substituted acyl, acylamino, thiocarboxy, amide, substituted carbonyl, substituted thiocarbonyl, Substituted sulfonyl and substituted sulfini Having a substituent selected from the group consisting of, regulator of claim 1.
(3) The PPAR regulator according to item 1, wherein the sulfonylurea agent is selected from the group consisting of glimepiride, glibenclamide, tolbutamide, chlorpropamide, acetohexamide and gliclazide.
(4) The PPAR regulator according to item 1, wherein the sulfonylurea agent is glimepiride or glibenclamide.
(5) The PPAR regulator according to Item 1, wherein the regulation is PPAR activation.
(6) The PPAR modulating agent according to item 1, further comprising at least one activity selected from the group consisting of insulin secreting activity and insulin sensitivity activity of pancreatic β cells.
(7) The PPAR modulating agent according to Item 1, wherein the PPAR modulating activity is at least 10% of thiazolidinedione (thiazolidinedione).
(8) The PPAR modulator according to Item 7, wherein the thiazolidinedione is pioglitazone.
(9) The PPAR modulator according to Item 1, wherein the PPAR is PPARγ.
(10) The PPAR is an amino acid sequence represented by SEQ ID NO: 2 selected from the group consisting of SEQ ID NO: 2 (NP_035276), SEQ ID NO: 4 (NP_619726), SEQ ID NO: 6 (NP_037256), and SEQ ID NO: 8 (AAB87480) 2. The PPAR modulating agent according to item 1, comprising a variant sequence comprising one or several substitutions, additions or deletions.
(11) The above-mentioned PPAR-modulating agent is adipocyte differentiation; diabetes; hyperglycinemia ;; low glucose tolerance; insulin resistance; obesity; steatosis; dyslipidemia; Hypercholesterolemia; Low HDL level; High LDL level; Atherosclerosis; Vascular stenosis; Irritable bowel syndrome; Inflammatory bowel disease; Crohn's disease; Intestinal ulcer; Inflammatory disease; Pancreatitis; PPAR modulators used for the treatment of diseases selected from the group consisting of sexually transmitted diseases; retinopathy; psoriasis; metabolic syndrome; ovarian hyperandrogenism.
(12) A method for identifying a compound having at least one activity selected from the group consisting of pancreatic β-cell insulin secretory activity and activity that promotes peripheral insulin sensitivity and having the ability to modulate PPAR. The above method is:
A) providing a candidate compound;
B) Measuring at least one activity selected from the group consisting of insulin secretory activity of pancreatic β cells and peripheral insulin sensitivity for the candidate compound;
C) subjecting the candidate compound to an assay for measuring the activity of PPAR; and D) identifying the substance determined to be active in each of Step B and Step C as a lead compound;
Including the method.
(13) The method according to item 12, wherein the candidate compound includes a sulfonylurea agent.
(14) The candidate compound is

Where R ₁ and R ₂ are each independently alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted Cycloalkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, carbocyclic group, substituted carbocyclic group, heterocyclic group, substituted heterocyclic group, halogen, hydroxy, substituted hydroxy, thiol, Substituted thiol, cyano, nitro, amino, substituted amino, carboxy, substituted carboxy, carbamoyl, acyl, substituted acyl, acylamino, thiocarboxy, amide, substituted carbonyl, substituted thiocarbonyl, Substituted sulfonyl and substituted sulfini The method according to claim 12 having a substituent selected from the group consisting of.
(15) The method according to item 12, wherein the modulation is activation of PPAR.
(16) The method according to item 12, wherein the compound has both the insulin secretory activity and the insulin sensitivity promoting activity of the pancreatic β cell.
(17) The method according to item 12, wherein the PPAR modulating activity is measured in comparison with thiazolidinedione (thiazolidinedione).
(18) The method according to item 12, wherein the PPAR is PPARγ.
(19) The PPAR is an amino acid sequence represented by SEQ ID NO: 2 selected from the group consisting of SEQ ID NO: 2 (NP_035276), SEQ ID NO: 4 (NP_619726), SEQ ID NO: 6 (NP_037256) and SEQ ID NO: 8 (AAB87480) 13. A method according to item 12, comprising a variant sequence comprising one or several substitutions, additions or deletions.
(20) The method according to item 12, wherein the step C) includes a step of observing whether the candidate compound is provided together with a sulfonylurea agent or a thiazolidinedione agent and competes.
(21) The method according to item 12, wherein the assay for measuring PPAR activity is a transcriptional activity assay.
(22) The insulin secretion activity of the pancreatic β cells is measured by directly measuring insulin secreted from the cells (J Pharmacol Exp Ther. 2004 Sep; 310 (3): 1273-80.), Item 12 The method described in 1.
(23) The method according to item 12, wherein the activity of promoting peripheral insulin sensitivity is measured by an insulin tolerance test.
(24) The activity of the PPAR is determined by determining the activity of the candidate compound in a system comprising a nucleic acid construct comprising a nucleic acid sequence encoding a reporter operably linked to a transcription factor recognition sequence and the selected PPAR. And when the expression of the reporter is enhanced, the candidate compound is determined to be an agonist of the PPAR, and when the expression of the reporter is decreased, the candidate compound is determined to be an antagonist of the PPAR. 13. The method according to item 12, measured by
(25) A method according to item 24, wherein the reporter is luciferase.
(26) A method according to item 24, wherein the system is a cell.
(27) The method according to item 12, wherein a protein containing a C-terminal activation function-2 (AF-2) domain is used as a target in the step C.
(28) The method according to item 27, wherein the target comprises the amino acid sequence PLLQEIYKDLY (SEQ ID NO: 9).
(29) The method according to item 12, wherein the interaction between the PPAR cofactor and PPAR is observed in the step C.
(30) The cofactor of the PPAR is selected from the group consisting of DRIP 205 (vitamin D receptor-interacting protein 205), N-CoR (nuclear receptor corepressor) and SMRT (silencing mediator for retinoid and thyroid hormone receptor), Item 29 The method described in 1.
(31) A system for identifying a compound having at least one activity selected from the group consisting of insulin secretory activity of pancreatic β cells and activity that promotes peripheral insulin sensitivity, and having the ability to modulate PPAR And the above system:
A) Candidate compound;
B) Means for measuring at least one activity selected from the group consisting of pancreatic β-cell insulin secretory activity and peripheral insulin sensitivity activity for the candidate compound;
C) means for subjecting the candidate compound to an assay for measuring the activity of PPAR; and D) means for identifying a substance determined to be active by each of means B and C as a lead compound,
A system comprising:
(32) A method for treating or preventing a disease associated with PPAR comprising the steps of:
A) a step of administering a sulfonylurea agent to a subject;
Including the method.
(33) A method according to item 32, wherein the sulfonylurea agent is selected from the group consisting of glimepiride, glibenclamide, tolbutamide, chlorpropamide, acetohexamide and gliclazide.
(34) Use of a sulfonylurea agent in the manufacture of a medicament for treating or preventing a disease associated with PPAR.
(35) The use according to item 34, wherein the sulfonylurea agent is selected from the group consisting of glimepiride, glibenclamide, tolbutamide, chlorpropamide, acetohexamide and gliclazide.

  Preferred embodiments of the present invention will be described below. However, those skilled in the art can appropriately implement the embodiments and the like from the description of the present invention and the accompanying drawings, as well as well-known and common techniques in the field, In addition to the effects of the invention, it should be recognized that the functions and effects of the present invention can be easily understood.

  The present invention provides a new screening method for the search for compounds for more efficient treatment or prevention of diabetes. The present invention also provides a new category of drugs related to PPARs.

  The invention will now be described by way of example, with reference to the accompanying drawings, where appropriate. Throughout this specification, it should be understood that the singular forms also include the plural concept unless specifically stated otherwise. Therefore, it should be understood that the expression of the singular includes the concept of the plural unless specifically stated otherwise. In addition, it is to be understood that the terms used in the present specification are used in the meaning normally used in the art unless otherwise specified. Thus, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.

  The embodiments provided below are provided for a better understanding of the present invention, and it is understood that the scope of the present invention should not be limited to the following description. Therefore, it is obvious that those skilled in the art can make appropriate modifications within the scope of the present invention with reference to the description in the present specification.

(Definition)
(disease)
In the present specification, “diabetes” is used in the same meaning as used in the art, and refers to a metabolic disease exhibiting persistent hyperglycemia / diabetes. Insulin dependence (type I) and insulin independence (type II). The former has rapid onset and severe symptoms and requires insulin administration. The latter is slow and does not necessarily require insulin administration.

  As used herein, “a disease associated with PPAR” refers to any disease related to adipocyte differentiation; diabetes; hyperglycinemia; low glucose tolerance; insulin resistance; obesity; dyslipidemia); hyperlipidemia; hypertriglyceridemia; hypercholesterolemia; low HDL levels; high LDL levels; atherosclerosis; vascular stenosis; irritable bowel syndrome; inflammatory bowel disease; Inflammatory diseases; pancreatitis; abdominal obesity; neurodegenerative diseases; retinopathy; psoriasis; metabolic syndrome;

  As used herein, “related to insulin metabolism” refers to any factor related to insulin secretion or glucose metabolism by insulin. Such a gene is typically shown in FIG. 5c. FIG. 5c shows genes involved in insulin receptor signaling in adipocytes.

  As used herein, “diagnosis” refers to identifying various parameters related to the type of disease, type and degree of disorder, physical condition, etc. in the subject, Determining responsiveness prediction, pathological change prediction or cause.

  As used herein, “treatment” refers to preventing a disease or disorder from deteriorating, preferably maintaining the status quo, more preferably reducing, when a certain disease or disorder becomes such a condition. Preferably, it means that it is reduced.

  As used herein, “prophylaxis” or “prevention” refers to treating a disease or disorder so that such a condition does not occur before such a condition is triggered. Thus, for a disease or disorder, this includes preventing such a condition, delaying, and preventing exacerbations. It is understood that the method of the present invention can also be used for prevention because it regenerates lung tissue.

  As used herein, “treatment” refers to any medical action performed on a certain disease, disorder or condition, and includes actions performed to contribute to diagnosis, treatment, prevention, prognosis and the like. Preferably, the procedure should be performed by a health care professional with some national license, such as a doctor, nurse or the like. It should be noted that even if the action worsens the course of the controversial disease and is detrimental to the patient, the action itself is still a “treatment”.

  In the present specification, the “instruction” is a description of a method of using the present invention or a method of diagnosis for a doctor, a person who performs administration such as a subject, and a person who diagnoses (which may be the subject). is there. When a diagnostic agent or the like is provided as a kit, instructions can be attached to instruct how to use the instruction. This instruction describes a word for instructing a procedure for administering the diagnostic agent or medicine of the present invention. This instruction is prepared in accordance with the format prescribed by the national supervisory authority (for example, the Ministry of Health, Labor and Welfare in Japan and the Food and Drug Administration (FDA) in the United States, etc. in the United States) where the present invention is implemented, and is approved by the supervisory authority. It is clearly stated that it has been received. The instruction sheet is a so-called package insert, and is usually provided as a paper medium, but is not limited thereto. For example, the instruction sheet is an electronic medium (for example, a homepage (web site) provided on the Internet, an electronic medium, etc. Mail, SMS, PDF document, etc.) can also be provided.

(Biochemistry)
(General technology)
Molecular biological techniques, biochemical techniques, and microbiological techniques used in this specification are well known and commonly used in the art, and are described in, for example, Sambrook J. et al. et al. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor and its 3rd Ed. (2001); Ausubel, F .; M.M. (1987). Current Protocols in Molecular Biology, Greene Pub. Associate ES and Wiley-Interscience; Ausubel, F .; M.M. (1989). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Innis, M .; A. (1990). PCR Protocols: A Guide to Methods and Applications, Academic Press; Ausubel, F .; M.M. (1992). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Ausubel, F .; M.M. (1995). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Innis, M .; A. et al. (1995). PCR Strategies, Academic Press; Ausubel, F .; M.M. (1999). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, and annual updates; Sninsky. J. et al. et al. (1999). PCR Applications: Protocols for Functional Genomics, Academic Press, a separate volume of experimental medicine “Gene Transfer & Expression Analysis Experimental Method” Yodosha, 1997, etc., which are all relevant in this specification (may be all) Is incorporated by reference.

  For DNA synthesis technology and nucleic acid chemistry for producing artificially synthesized genes, see, for example, Gait, M. et al. J. et al. (1985). Oligonucleotide Synthesis: A Practical Approach, IRLPpress; Gait, M .; J. et al. (1990). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein, F .; (1991). Oligonucleotides and Analogues: A Practical Approac, IRL Press; Adams, R .; L. etal. (1992). The Biochemistry of the Nucleic Acids, Chapman &Hall; Shabarova, Z. et al. et al. (1994). Advanced Organic Chemistry of Nucleic Acids, Weinheim; Blackburn, G .; M.M. et al. (1996). Nucleic Acids in Chemistry and Biology, Oxford University Press; Hermanson, G .; T.A. (I996). Bioconjugate Technologies, Academic Press, etc., which are incorporated herein by reference in the relevant part.

  The PPAR, adiponectin promoter, and fragments and variants thereof used in the present invention can be produced using transgenic animal production techniques and genetic engineering techniques.

(Description of PPAR)
As used herein, the term “PPAR” is an abbreviation for “peroxisome proliferator-activator receptor” and refers to one of the nuclear receptors. PPARα is represented by the sequences shown in SEQ ID NOs: 1 to 8 (SEQ ID NO: 1-2 (NP_035276), SEQ ID NO: 3-4 (NP_619726), SEQ ID NO: 5-6 (NP_037256) and SEQ ID NO: 7-8 (AAB87480), Mouse, human, rat, monkey nucleic acid sequence (odd number) and amino acid sequence (even number)), respectively. In the present invention, it is understood that variants of the above sequences also fall within the scope of this term as long as they have similar activity. PPAR is a kind of nuclear receptor, and refers to a molecule that initiates a biological reaction in the nucleus by binding to a ligand. Usually, the nuclear receptor is present in the nucleus, but also has a property of moving into the nucleus when a biological reaction is initiated. Nuclear receptors usually form complexes when bound to bind to DNA and act as transcription factors. Here, biological reactions of nuclear receptors typically include transcriptional regulation (for example, promotion, suppression, initiation, or termination of transcription, typically, promotion of transcription, but are not limited thereto). ). Typically, nuclear receptors are naturally present in the cytoplasm when not stimulated by a ligand, and when bound to a ligand, translocate into the nucleus and transmit the stimulus into the nucleus. A molecule. Alternatively, a nuclear receptor initiates a biological reaction in the nucleus by binding to a ligand without changing its localization.

  In the present specification, a “corresponding” amino acid has or is predicted to have the same action as a predetermined amino acid in a protein or polypeptide as a reference for comparison in a protein molecule or polypeptide molecule. For example, in the nuclear receptor, the domain corresponding to the A domain is a region having a transcription promoting function, and the domain corresponding to the B domain is a region having a transcription promoting function, / B domain. The domain corresponding to the C domain is a region having an action of DNA binding, and usually there are two characteristic Zn finger structures found in DNA binding proteins. The domain corresponding to the D domain is a region existing between the C domain and the E · F domain, and the domain corresponding to the E · F domain is a region having an action of binding to a ligand (hormone). Also called binding domain. This ligand binding domain (E / F domain) has a similar three-dimensional structure consisting of 12 α helices. The domain corresponding to the F domain is a region existing on the C-terminal side from the E domain. In the case of an enzyme molecule, it refers to an amino acid that exists at the same position in the active site and contributes similarly to the catalytic activity. Corresponding residues in PPARs can be identified by performing an alignment. In the present specification, it is understood that domains A to F in nuclear receptors other than those listed above correspond to domains in the range of amino acid positions corresponding to the first amino acid and the last amino acid in the above specific examples. Is done.

  Herein, the A to F domains can be determined based on definitions known in the art. For example, such a definition is described in Robinson-Rechavi M et al., J Cell Sci. 2003 Feb 15; 116 (Pt 4): 585-586 and the literature cited therein can be referred to.

  As used herein, the “biological activity” of PPAR is the activity provided by the nuclear receptor when the nuclear receptor is bound to the ligand, and is described, for example, immediately before in the present specification. Such activities include, but are not limited to, PPARγ transcription promotion, gluconeogenesis promotion, and the like. An agonist of a nuclear receptor (eg, PPARγ) induces at least one part or all of the biological activity of the nuclear receptor by binding to the nuclear receptor.

A typical example of PPAR is PPARγ, the sequence of which is shown in SEQ ID NOs: 1 to 8 (odd numbers indicate nucleic acid sequences, even numbers indicate amino acid sequences corresponding thereto; mouse, human, rat and monkey, respectively). It has a sequence or a modified sequence thereof. Typically, a PPAR can be a molecule such as:
(A) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2, 4, 6 or 8 or a fragment thereof;
(B) in the amino acid sequence of SEQ ID NO: 2, 4, 6 or 8, at least one amino acid has at least one mutation selected from the group consisting of substitution, addition and deletion, and biological A polypeptide having activity;
(C) a polypeptide encoded by a splice variant or allelic variant of the base sequence set forth in SEQ ID NO: 1, 3, 5 or 7;
(D) a polypeptide that is a species homologue of the amino acid sequence set forth in SEQ ID NO: 2, 4, 6 or 8;
(E) a polypeptide having an amino acid sequence with at least 70% identity to any one of the polypeptides of (a)-(d) and having biological activity; or (f) (a ) Having an amino acid sequence encoded by a polynucleotide that hybridizes under stringent hybridization conditions with a polynucleotide encoding any one of the polypeptides of (d) and having biological activity; A polypeptide;
including.

  As used herein, modulation of the biological function of a PPAR protein is achieved by contacting an inhibitor to the PPAR protein or introducing a mutation that reduces the function of the PPAR (eg, by gene therapy). be able to.

  The biological function of PPAR protein is determined by ELISA method, Northern blot analysis or quantitative PCR method, biochemical analysis method by measuring cell proliferation or differentiation, and the substance to be examined or the form of the biological extract Although it is possible to measure by performing microscopic analysis on the formation, it is not limited to these.

  As used herein, the terms “protein”, “polypeptide”, “oligopeptide” and “peptide” are used interchangeably herein to refer to polymers of amino acids of any length and variants thereof. Say. This polymer may be linear, branched, or cyclic. The amino acid may be natural or non-natural and may be a modified amino acid. The term also includes what can be assembled into a complex of multiple polypeptide chains. The term also encompasses natural or artificially modified amino acid polymers. Such modifications include, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or any other manipulation or modification (eg, conjugation with a labeling component). This definition also includes, for example, polypeptides containing one or more analogs of amino acids (eg, including unnatural amino acids, etc.), peptide-like compounds (eg, peptoids) and other modifications known in the art. Is included. When used in the composition of the present invention, the “protein” is preferably a protein that is compatible with the host in which the composition is to be used, as long as it can be treated to be compatible with that host. Any protein may be used. Whether a protein is compatible with a host, or can be treated to be compatible with a host, transplants the protein into the host and suppresses side reactions such as immune rejection as needed. It can be determined by observing whether or not it has settled in the host. Typically, a protein having the above-mentioned compatibility can include, but is not limited to, a protein derived from the host.

  As used herein, an “isolated” biological factor (eg, a nucleic acid or protein, etc.) refers to other biological factors within the cells of the organism in which it is naturally occurring (eg, When it is a nucleic acid, it is substantially separated from a non-nucleic acid and a nucleic acid containing a nucleic acid sequence other than the target nucleic acid; Or it is purified. “Isolated” nucleic acids and proteins include nucleic acids and proteins purified by standard purification methods. Thus, isolated nucleic acids and proteins include chemically synthesized nucleic acids and proteins.

  As used herein, a “purified” biological agent (such as a nucleic acid or protein) refers to a product from which at least part of the factors naturally associated with the biological agent has been removed. Thus, typically the purity of the biological agent in a purified biological agent is higher (ie, enriched) than the state in which the biological agent is normally present.

  The biomolecule used in the present invention can be collected from a living body or chemically synthesized by a method known to those skilled in the art. For example, for proteins, a synthesis method using an automated solid phase peptide synthesizer is described by: Stewart, J. et al. M.M. et al. (1984). Solid Phase Peptide Synthesis, Pierce Chemical LCo. Grant, G .; A. (1992). Synthetic Peptides: A User's Guide, W.M. H. Freeman; Bodanszky, M .; (1993). Principles of Peptide Synthesis, Springer-Verlag; Bodanzky, M .; et al. (1994). The Practice of Peptide Synthesis, Springer-Verlag; B. (1997). Phase Peptide Synthesis, Academic Press; Pennington, M .; W. et al. (1994). Peptide Synthesis Protocols, Humana Press; B. (1997). Solid-Phase Peptide Synthesis, Academic Press. Other molecules can also be synthesized using techniques well known in the art.

  In the present specification, “homology” of biomolecules (for example, nucleic acid sequences encoding amino acids such as PPAR, amino acid sequences, etc.) refers to the degree of identity of two or more sequences to each other when they have comparable sequences. Say. Therefore, the higher the homology between two sequences, the higher the identity or similarity between those sequences. Whether two sequences have homology can be determined by direct comparison of the sequences or, in the case of nucleic acids, hybridization methods under stringent conditions. When two sequences are directly compared, the sequences between the sequences are typically at least 50% identical, preferably at least 70% identical, more preferably at least 80%, 90%, 95% , 96%, 97%, 98% or 99% are identical, the genes have homology. In the present specification, “similarity” of biomolecules (for example, nucleic acid sequences, amino acid sequences, etc.) refers to two or more gene sequences when conservative substitutions are regarded as positive (identical) in the above homology. , Refers to the degree of identity to each other. Thus, if there is a conservative substitution, identity and similarity differ depending on the presence of the conservative substitution. When there is no conservative substitution, identity and similarity indicate the same numerical value. In the present invention, those having such high identity or similarity may also be useful.

  In this specification, the comparison of similarity, identity and homology between amino acid sequences and base sequences is calculated using default parameters using BLAST, which is a sequence analysis tool. The identity search can be performed, for example, using NCBI's BLAST 2.2.9 (issued 2004.5.12). In the present specification, the identity value usually refers to a value when the above BLAST is used and aligned under default conditions. However, if a higher value is obtained by changing the parameter, the highest value is the identity value. When identity is evaluated in a plurality of areas, the highest value among them is set as the identity value.

  In the present specification, the “amino acid” may be natural or non-natural. “Derivative amino acid” or “amino acid analog” refers to an amino acid that is different from a naturally occurring amino acid but has the same function as the original amino acid. Such derivative amino acids and amino acid analogs are well known in the art.

  The term “natural amino acid” refers to the L-isomer of a natural amino acid. Natural amino acids are glycine, alanine, valine, leucine, isoleucine, serine, methionine, threonine, phenylalanine, tyrosine, tryptophan, cysteine, proline, histidine, aspartic acid, asparagine, glutamic acid, glutamine, γ-carboxyglutamic acid, arginine, ornithine , And lysine. Unless otherwise indicated, all amino acids referred to herein are L-forms, but forms using D-form amino acids are also within the scope of the present invention.

  As used herein, “amino acid variant” refers to a molecule that is not a natural amino acid but is similar to the physical properties and / or functions of a natural amino acid. Examples of amino acid modifications include those in which an alkyl group, a halo group, a nitro group, or the like is bonded to phenylalanine benzyl side chain (para-position, meta-position, ortho-position, etc.), ethionine, canavanine, 2-methylglutamine and the like. It is done. In the present invention, it is understood that amino acid variants may include unnatural amino acids and amino acid mimetics.

  As used herein, “unnatural amino acid” means an amino acid that is not normally found naturally in proteins. Examples of non-natural amino acids include norleucine, para-nitrophenylalanine, homophenylalanine, para-fluorophenylalanine, 3-amino-2-benzylpropionic acid, homo-arginine D-form or L-form and D-phenylalanine.

  In the present specification, the “corresponding” amino acid or nucleic acid has or has the same action as a predetermined amino acid in a reference polypeptide or polynucleotide in a polypeptide molecule or polynucleotide molecule, respectively. In particular, in the case of an enzyme molecule, it means an amino acid that is present at the same position in the active site and contributes to the catalytic activity in the same way. For example, an antisense molecule of a polynucleotide can be a similar part in an ortholog corresponding to a particular part of the antisense molecule. In the case of the peptide of the present invention, a specific sequence in the human cell growth factor is used. In the specific sequence in the cell growth factor of other species of animals, the portion corresponding to the peptide of the present invention corresponds to the “corresponding amino acid”. Is understood.

  In the present specification, the “corresponding” gene refers to a gene having, or expected to have, the same action as that of a predetermined gene in a species as a reference for comparison in a certain species. When there are a plurality of genes having the same, those having the same origin evolutionarily. Thus, the corresponding gene of a gene can be an ortholog of that gene. For example, the gene corresponding to mouse PPAR is human PPAR.

  In the present specification, the “fragment” refers to a polypeptide or polynucleotide having a sequence length of 1 to n−1 with respect to a full-length polypeptide or polynucleotide (length is n). The length of the fragment can be appropriately changed according to the purpose. For example, the lower limit of the length is 3, 4, 5, 6, 7, 8, 9, 10, Examples include 15, 20, 25, 30, 40, 50 and more amino acids, and lengths expressed in integers not specifically listed here (eg, 11 etc.) are also suitable as lower limits. obtain. In the case of polynucleotides, examples include 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100 and more nucleotides. Non-integer lengths (eg, 11 etc.) may also be appropriate as a lower limit. In the present invention, when a polypeptide or a polynucleotide is used as a biomolecule, such a fragment is also used in the same manner as the full-length one as long as a desired purpose (for example, a cell attracting effect) is achieved. It is understood that it can be done.

  In the present specification, the lengths of polypeptides and polynucleotides can be represented by the number of amino acids or nucleic acids, respectively, as described above, but the above numbers are not absolute, and the upper limit is not limited as long as they have the same function. Alternatively, the above-mentioned number as the lower limit is intended to include the upper and lower numbers (or, for example, up and down 10%) of the number. In order to express such intention, in this specification, “about” may be added before the number. However, it should be understood herein that the presence or absence of “about” does not affect the interpretation of the value.

  As used herein, “biological activity” refers to an activity that a certain factor (eg, polypeptide or protein) may have in vivo, and includes an activity that exhibits various functions. For example, when a certain factor is an antisense molecule, its biological activity includes binding to a nucleic acid molecule of interest, thereby suppressing expression. For example, when a factor is an enzyme, the biological activity includes the enzyme activity. In another example, when an agent is a ligand, the ligand includes binding to the corresponding receptor. Such biological activity can be measured by techniques well known in the art.

  As used herein, the terms “polynucleotide”, “oligonucleotide”, and “nucleic acid” are used interchangeably herein and refer to a polymer of nucleotides of any length. The term also includes “oligonucleotide derivatives” or “polynucleotide derivatives”. “Oligonucleotide derivatives” or “polynucleotide derivatives” refer to oligonucleotides or polynucleotides that include derivatives of nucleotides or that have unusual linkages between nucleotides, and are used interchangeably. Specific examples of such oligonucleotides include, for example, 2′-O-methyl-ribonucleotides, oligonucleotide derivatives in which phosphodiester bonds in oligonucleotides are converted to phosphorothioate bonds, and phosphodiester bonds in oligonucleotides. Derivative converted to N3′-P5 ′ phosphoramidate bond, oligonucleotide derivative in which ribose and phosphodiester bond in oligonucleotide are converted to peptide nucleic acid bond, uracil in oligonucleotide is C− Oligonucleotide derivatives substituted with 5-propynyluracil, oligonucleotide derivatives wherein uracil in oligonucleotide is substituted with C-5 thiazole uracil, cytosine in oligonucleotide is C-5 propynylcytosine Substituted oligonucleotide derivatives, oligonucleotide derivatives in which the cytosine in the oligonucleotide is replaced with phenoxazine-modified cytosine, oligonucleotide derivatives in which the ribose in DNA is replaced with 2'-O-propylribose Examples thereof include oligonucleotide derivatives in which the ribose in the oligonucleotide is substituted with 2′-methoxyethoxyribose. Unless otherwise indicated, a particular nucleic acid sequence may also be conservatively modified (eg, degenerate codon substitutes) and complementary sequences, as well as those explicitly indicated. Is contemplated. Specifically, a degenerate codon substitute creates a sequence in which the third position of one or more selected (or all) codons is replaced with a mixed base and / or deoxyinosine residue. (Batzer et al., Nucleic Acid Res. 19: 5081 (1991); Oht sulfonylurea ka et al., J. Biol. Chem. 260: 2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8). : 91-98 (1994)).

  As used herein, “nucleotide” refers to a nucleoside in which the sugar moiety is a phosphate ester, and includes DNA, RNA, etc., and may be natural or non-natural. Here, nucleoside refers to a compound in which a base and a sugar form an N-glycoside bond. “Nucleotide derivative” or “nucleotide analog” refers to a substance that is different from a naturally occurring nucleotide but has a function similar to that of the original nucleotide. Such derivative nucleotides and nucleotide analogs are well known in the art. Examples of such derivative nucleotides and nucleotide analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNA). . DNA includes cDNA, genomic DNA, and synthetic DNA.

  In the present specification, the “corresponding” gene refers to a gene having, or expected to have, the same action as that of a predetermined gene in a species as a reference for comparison in a certain species. When there are a plurality of genes having the same, those having the same origin evolutionarily. Thus, the corresponding gene of a gene can be an ortholog of that gene. Therefore, a region corresponding to a region having promoter activity in the PPAR gene or the human adiponectin gene can be found in other animals (mouse, rat, pig, cow, etc.). Such corresponding genes, or promoter regions, can be identified using techniques well known in the art based on the disclosure herein. Thus, for example, for a corresponding gene in an animal, the sequence database of that animal (eg, mouse, rat) is searched using the sequence of the gene (eg, human adiponectin promoter) serving as a reference for the corresponding gene as a query sequence. Can be found by:

  In the present specification, the “corresponding” amino acid or nucleic acid has or has the same action as a predetermined amino acid in a reference polypeptide or polynucleotide in a polypeptide molecule or polynucleotide molecule, respectively. In particular, in the case of an enzyme molecule, it means an amino acid that is present at the same position in the active site and contributes to the catalytic activity in the same way. For example, an antisense molecule of a polynucleotide can be a similar part in an ortholog corresponding to a particular part of the antisense molecule. In the case of the promoter of the present invention, a specific sequence in the promoter is used. In the specific sequence in the promoter of an animal of another species, the portion corresponding to the nucleotide sequence of the present invention corresponds to the “corresponding nucleotide”. Is understood.

  In the present specification, the “variant” refers to a substance in which a part of the original substance such as a polypeptide or polynucleotide is changed. Such variants include substitutional variants, addition variants, deletion variants, truncated variants, allelic variants, and the like. Such a variant can also be used as the cell growth factor of the present invention as long as the desired purpose can be achieved. An allele refers to genetic variants that belong to the same locus and are distinguished from each other. Therefore, an “allelic variant” refers to a variant having an allelic relationship with a certain gene. Such allelic variants usually have the same or very similar sequence as their corresponding alleles, usually have nearly the same biological activity, but rarely have different biological activities. May have. “Species homologue or homolog” means homology (preferably at least 60% homology, more preferably at least 80%, at a certain amino acid level or nucleotide level within a certain species. 85% or higher, 90% or higher, 95% or higher homology). The method for obtaining such species homologues will be apparent from the description herein. “Ortholog” is also called an orthologous gene, which is a gene derived from speciation from a common ancestor with two genes. For example, taking the hemoglobin gene family with multiple gene structures as an example, the human and mouse alpha hemoglobin genes are orthologs, but the human alpha and beta hemoglobin genes are paralogs (genes generated by gene duplication). . Orthologs are useful for estimating molecular phylogenetic trees. Orthologs of the present invention can also be useful in the present invention, since orthologs can usually perform the same function as the original species in another species.

  As used herein, “conservative (modified) variants” applies to both amino acid and nucleic acid sequences. Conservatively modified variants with respect to a particular nucleic acid sequence refer to nucleic acids that encode the same or essentially the same amino acid sequence, and are essentially identical if the nucleic acid does not encode an amino acid sequence. An array. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide.

  Such a nucleic acid can be obtained by a well-known PCR method, and can also be chemically synthesized. These methods may be combined with, for example, a site-specific displacement induction method or a hybridization method.

  As used herein, “substitution, addition or deletion” of a polypeptide or polynucleotide is a substitution of an amino acid or a substitute thereof, or a nucleotide or a substitute thereof, respectively, with respect to the original polypeptide or polynucleotide. , Adding or removing. Such substitution, addition, or deletion techniques are well known in the art, and examples of such techniques include site-directed mutagenesis techniques. There may be any number of substitutions, additions or deletions, as long as it is one or more, and such numbers may be used for the desired function (eg, angiogenesis, cell regeneration) in a variant having that substitution, addition or deletion. Etc.) as long as it is retained. For example, such a number can be one or several, and preferably can be within 20%, within 10%, or less than 100, less than 50, less than 25, etc. of the total length.

  Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical LNomenclature Commission. Nucleotides may also be referred to by a generally recognized one letter code.

The character codes are as follows.
3 letter code of amino acid 1 letter code Meaning Ala A Alanine Cys C Cysteine Asp D Aspartic acid Glu E Glutamic acid Phe F Phenylalanine Gly G Glycine His H Histidine Ile I Isoleucine Lys K Lysine Leu L Leucine Met M N Glutamine Arg R Arginine Ser S Serine Thr T Threonine Val V Valine Trp W Tryptophan Tyr Y Tyrosine Asx Asparagine or Aspartate Glx Glutamine or Glutamate Xaa Unknown or other amino acids.

Base symbol Meaning
a adenine g guanine c cytosine t thymine u uracil r guanine or adenine purine y thymine / uracil or cytosine pyrimidine m adenine or cytosine amino group k guanine or thymine / uracil keto group s guanine or cytosine w adenine or thymine / uracil b guanine or cytosine Thymine / uracil d adenine or guanine or thymine / uracil h adenine or cytosine or thymine / uracil v adenine or guanine or cytosine n adenine or guanine or cytosine or thymine / uracil, unknown or other base.

  In the present specification, when referring to a gene, “vector” or “recombinant vector” refers to a vector capable of transferring a target polynucleotide sequence to a target cell. Examples of such vectors include those that can autonomously replicate in host cells such as prokaryotic cells, yeast, animal cells, plant cells, insect cells, animal individuals and plant individuals, or can be integrated into chromosomes. . Among vectors, a vector suitable for cloning is called “cloning vector”. Such cloning vectors usually contain multiple cloning sites that contain multiple restriction enzyme sites. Such restriction enzyme sites and multiple cloning sites are well known in the art, and those skilled in the art can appropriately select and use them according to the purpose. Such techniques are described in the literature described herein (eg, Sambrook et al., Supra).

  As used herein, “expression vector” refers to a nucleic acid sequence in which various regulatory elements are operably linked in a host cell in addition to a structural gene and a promoter that regulates its expression. Regulatory elements can preferably include terminators, selectable markers such as drug resistance genes, and enhancers. It is well known to those skilled in the art that the type of expression vector of an organism (eg, animal) and the type of regulatory elements used can vary depending on the host cell.

“Recombinant vectors” for prokaryotic cells that can be used in the present invention include pcDNA3 (+), pBluescript-SK (+/−), pGEM-T, pEF-BOS, pEGFP, pHAT, pUC18, pFT-DEST ^™ 42GATEWAY ( Invitrogen) and the like.

  Examples of “recombinant vectors” for animal cells that can be used in the present invention include pcDNAI / Amp, pcDNAI, pCDM8 (all available from Funakoshi), pAGE107 [JP-A-3-229 (Invitrogen), pAGE103 [J. Biochem. , 101, 1307 (1987)], pAMo, pAMoA [J. Biol. Chem. , 268, 22782-2787 (1993)], a retroviral expression vector based on murine stem cell virus (MSCV), pEF-BOS, pEGFP, and the like.

  As used herein, “terminator” is a sequence that is located downstream of a region encoding a protein of a gene and is involved in termination of transcription when DNA is transcribed into mRNA, and addition of a poly A sequence. It is known that the terminator is involved in the stability of mRNA and affects the expression level of the gene.

  In this specification, “promoter” refers to a region on DNA that determines the transcription start site of a gene and directly regulates its frequency, and is usually a base sequence that initiates transcription upon binding of RNA polymerase. . Therefore, in this specification, a part having a promoter function of a gene is referred to as a “promoter part”. Since the promoter region is usually a region within about 2 kbp upstream of the first exon of the putative protein coding region, if the protein coding region in the genomic base sequence is predicted using DNA analysis software, The promoter region can be deduced. The putative promoter region varies for each structural gene, but is usually upstream of the structural gene, but is not limited thereto, and may be downstream of the structural gene. Preferably, the putative promoter region is present within about 2 kbp upstream from the first exon translation start point. In the present invention, a region about 3.6 kb upstream from the translation start point was identified as a promoter region, and this region was found to provide high expression ability and high specificity in adipocytes.

  As used herein, “enhancer” refers to a sequence used to increase the expression efficiency of a target gene. Such enhancers are well known in the art. A plurality of enhancers may be used, but one may be used or may not be used.

  As used herein, “operably linked” refers to a transcriptional translational regulatory sequence (eg, promoter, enhancer, etc.) or a translational regulatory sequence that has the expression (operation) of a desired sequence. That means. In order for a promoter to be operably linked to a gene, the promoter is usually placed immediately upstream of the gene, but need not necessarily be adjacent.

  In this specification, any technique may be used for introducing a nucleic acid molecule into a cell, and examples thereof include transformation, transduction, and transfection. Such a technique for introducing a nucleic acid molecule is well known in the art and commonly used. For example, Ausubel F. et al. A. (1988), Current Protocols in Molecular Biology, Wiley, New York, NY; Sambrook J et al. (1987) Molecular Cloning: A Laboratory Manual, 2nd Ed. And its third edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, a separate volume of experimental medicine “Gene Transfer & Expression Analysis Experimental Method” Yodosha, 1997, and the like. Gene transfer can be confirmed using the methods described herein, such as Northern blot, Western blot analysis, or other well-known conventional techniques.

  Moreover, as a method for introducing a vector, any of the above-described methods for introducing DNA into cells can be used. For example, transfection, transduction, transformation, etc. (for example, calcium phosphate method, liposome method, DEAE dextran method, electroporation method, method using particle gun (gene gun), etc.).

  As used herein, “transformant” refers to all or part of a living organism such as a cell produced by transformation. Examples of the transformant include prokaryotic cells, yeast, animal cells, plant cells, insect cells and the like. A transformant is also referred to as a transformed cell, transformed tissue, transformed host, etc., depending on the subject. The cell used in the present invention may be a transformant.

  When prokaryotic cells are used in genetic manipulation or the like in the present invention, prokaryotic cells include, for example, prokaryotic cells belonging to the genus Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Pseudomonas, etc. Escherichia coli XL1-Blue, Escherichia coli XL2-Blue, Escherichia coli DH1 are exemplified.

  As used herein, animal cells include mouse myeloma cells, rat myeloma cells, mouse hybridoma cells, CHO cells that are Chinese hamster cells, BHK cells, African green monkey kidney cells, and human leukemia cells. HBT5637 (Japanese Patent Laid-Open No. 63-299), human colon cancer cell line, and the like. Mouse myeloma cells such as ps20 and NSO, rat myeloma cells such as YB2 / 0, human fetal kidney cells such as HEK293 (ATCC: CRL-1573), human leukemia cells such as BALL-1, and Africa COS-1, COS-7 as the kidney monkey cell, HCT-15 as the human colon cancer cell line, human neuroblastoma SK-N-SH, SK-N-SH-5Y, mouse neuroblastoma Neuro2A, etc. Is exemplified.

  As used herein, any method for introducing DNA can be used as a method for introducing a recombinant vector. For example, calcium chloride method, electroporation method [Methods Enzymol. , 194, 182 (1990)], lipofection method, spheroplast method [Proc. Natl. Acad. Sci. USA, 84, 1929 (1978)], lithium acetate method [J. Bacteriol. , 153, 163 (1983)], Proc. Natl. Acad. Sci. USA, 75, 1929 (1978) and the like.

  As used herein, retroviral infection methods are well known in the art, as described, for example, in Current Protocols in Molecular Biology supra (particularly Units 9.9-9.14). Then, the embryonic stem cells are made into a single-cell suspension, and then together with the culture supernatant of virus-producing cells (packaging cell line = packaging cell lines). A sufficient amount of infected cells can be obtained by co-culture for 2 hours.

  The transient expression of Cre enzyme used in the method for removing the genome or the locus used in the present specification, DNA mapping on the chromosome, etc. are described in the cell engineering separate experiment protocol series “FISH experiment protocol human genome”. From analysis to chromosome / gene diagnosis "Kenichi Matsubara, Hiroshi Yoshikawa, Shujunsha (Tokyo), etc. are well known in the field.

  As used herein, the term “biomolecule” refers to a molecule related to a living body and an aggregate thereof. In this specification, “living body” refers to a biological organism, and includes, but is not limited to, animals, plants, fungi, viruses and the like. The biomolecule includes a molecule extracted from a living body and an aggregate thereof, but is not limited thereto, and any molecule and aggregate that can affect the living body fall within the definition of a biomolecule. Therefore, a small molecule that can be used as a pharmaceutical (eg, a small molecule ligand) also falls within the definition of a biomolecule as long as an effect on the living body can be intended. Such biomolecules include proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (eg, DNA such as cDNA, genomic DNA, RNA such as mRNA), polysaccharides. , Oligosaccharides, lipids, small molecules (eg, hormones, ligands, signaling substances, small organic molecules, etc.), complex molecules thereof, and aggregates thereof (eg, extracellular matrix, fibers, etc.). It is not limited to them. As used herein, a biomolecule is intended to include any agent that interacts with PPAR.

  As used herein, “in vivo” or “in vivo” refers to the inside of a living body. In a particular context, “in vivo” refers to the location where the target tissue or organ is to be placed. When screening is performed, in vivo screening can be performed.

  As used herein, “in vitro” refers to a state in which a part of a living body is removed or released “in vitro” (eg, in a test tube) for various research purposes. A term that contrasts with in vivo. When screening is performed, in vitro screening can be performed.

  As used herein, “pharmaceutically acceptable carrier” refers to a substance that is used when producing a pharmaceutical or veterinary drug and does not adversely affect the active ingredient. Such pharmaceutically acceptable carriers include, for example, antioxidants, preservatives, colorants, flavors, and diluents, emulsifiers, suspending agents, solvents, fillers, bulking agents, buffering agents, delivery agents. Examples include, but are not limited to, vehicles, diluents, excipients and / or agricultural or pharmaceutical adjuvants.

  Methods for producing pharmaceuticals are well known in the art, and the pharmaceuticals of the present invention can be prepared as required by physiologically acceptable carriers, excipients or stabilizers (Japanese Pharmacopoeia 14th edition or the latest edition thereof). , Remington's Pharmaceuticals LSciences, 18th Edition, AR Gennaro, ed., Mack Publishing Company, 1990, etc.) and lyophilized by mixing with a cell composition having the desired degree of purity. Although it can be prepared and stored in a state, it is preferably stored in a suitable storage solution.

  Examples of the pharmaceutically acceptable carrier contained in the medicament of the present invention include any substance known in the art. Pharmaceutically acceptable carriers that can be used in the present invention include antioxidants, preservatives, colorants, flavors, and diluents, emulsifiers, suspending agents, solvents, fillers, bulking agents, buffering agents, Delivery vehicles, diluents, excipients and / or pharmaceutical adjuvants include but are not limited to them. Typically, the medicament of the present invention is administered in the form of a composition comprising a support and a peptide or variant thereof together with one or more physiologically acceptable carriers, excipients or diluents. For example, a suitable vehicle can be water for injection, physiological solution, or artificial cerebrospinal fluid, which can be supplemented with other materials common to compositions for transplantation.

  Exemplary suitable carriers include neutral buffered saline or saline mixed with serum albumin. Preferably, the product is formulated as a lyophilizer using a suitable excipient (eg, sucrose). Other standard carriers, diluents and excipients may be included as desired. Other exemplary compositions include pH 7.0-8.5 Tris buffer or pH 4.0-5.5 acetate buffer, which may further include sorbitol or a suitable substitute thereof. .

  Acceptable carriers, excipients or stabilizers used herein are non-toxic to the recipient and are preferably inert at the dosages and concentrations used and Phosphates, citrates, or other organic acids; antioxidants (eg, ascorbic acid); low molecular weight polypeptides; proteins (eg, serum albumin, gelatin, or immunoglobulins); hydrophilic polymers ( Amino acids (eg, glycine, glutamine, asparagine, arginine, or lysine); monosaccharides, disaccharides and other carbohydrates (including glucose, mannose, or dextrin); chelating agents (eg, EDTA); sugar alcohols (Eg mannitol or sorbitol ; Salt-forming counterions (such as sodium); and / or non-ionic surface-active agents (e.g., Tween, Pluronic (pluronic) or polyethylene glycol (PEG)).

  In a preferred embodiment, the drug or variant thereof used in the medicament of the present invention is advantageously derived from a living body intended for treatment, but is not limited thereto. Such a peptide of the same origin as the host or a variant thereof is considered advantageous because it is considered that there is almost no immune reaction or the like. However, the origin of the peptide or a variant thereof is not particularly limited because it is considered that an immune reaction usually does not occur if it is purified.

  In the present specification, the “instruction” is a description of a method of using the present invention or a method of diagnosis for a doctor, a person who performs administration such as a subject, and a person who diagnoses (which may be the subject). is there. When a diagnostic agent or the like is provided as a kit, instructions can be attached to instruct how to use the instruction. This instruction describes a word for instructing a procedure for administering the diagnostic agent or medicine of the present invention. This instruction is prepared in accordance with the format prescribed by the national supervisory authority (for example, the Ministry of Health, Labor and Welfare in Japan and the Food and Drug Administration (FDA) in the United States, etc. in the United States) where the present invention is implemented, and is approved by the supervisory authority. It is clearly stated that it has been received. The instruction sheet is a so-called package insert, and is usually provided in a paper medium, but is not limited thereto. For example, an electronic medium (for example, a home page provided on the Internet (web) Site), e-mail, SMS, PDF document, etc.).

  In certain embodiments, the present invention can be used in combination with gene therapy. Gene therapy refers to a therapy performed by administering to a subject a nucleic acid that is expressed or expressible. In this embodiment of the invention, the nucleic acid produces the encoded protein that mediates a therapeutic effect.

  Any method for gene therapy available in the art can be used in accordance with the present invention. Exemplary methods are described in GoldspielLet al., A general review of methods of gene therapy. , Clinica LP Pharmacy 12: 488-505 (1993); Wu and Wu, Biotherapy 3: 87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32: 573-596 (1993); Mulligan, Science 260: 926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62: 191-217 (1993); May, TIBTECH 11 (5): 155-215 (1993). Commonly known recombinant DNA techniques used in gene therapy are described in Ausubel et al. (Eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); and Kriegler, Gene TransferMand Transfer and ExpLandoral. , Stockton Press, NY (1990).

(Transcriptional activity assay)
As used herein, “transcriptional activity assay” refers to any assay for testing whether or not a certain region has transcriptional activity. In this assay, the influence of a chemical on a series of actions that binds to a receptor in a cell and activates transcription from a gene of interest to a protein is evaluated. For example, luciferase can be exemplified as a protein, but is not limited thereto. PPARγ is exemplified as the target gene. A GAL4 binding region is exemplified as a transcription factor recognition sequence upstream of the DNA encoding the reporter gene. Examples of transcriptional regulators include PPARγ binding domain (PPARγLBD) fused with GAL4 DNA binding domain (GAL4DBD). In this method, first, cultured cells into which a DNA encoding a target nuclear receptor and a DNA encoding a reporter gene are introduced are prepared. A chemical substance is added to the culture medium containing the cultured cells and cultured, and the fluorescence intensity and the like are measured. Here, it is evaluated whether a series of actions from the binding to the receptor to the protein synthesis from the chemical substance acts like a hormone. In this method, it is possible to evaluate whether a chemical substance acts as an agonist or an antagonist by allowing a target hormone and a chemical substance to coexist. Furthermore, in this method, it is possible to quickly process a large amount of sample using a high-throughput apparatus that is a robot system.

  Accordingly, in the present invention, such a transcription activity assay can be used in carrying out a method for determining whether a candidate compound is an agonist or antagonist of a nuclear receptor. In this method, after providing a candidate compound, it is determined whether or not the candidate compound is covalently bound to cysteine in the nuclear receptor, and the candidate compound determined to be covalently bound is selected and interacted with the nuclear receptor. Candidate compounds are exposed in a system (eg, a cell) comprising a nucleic acid construct comprising a nucleic acid sequence encoding a reporter operably linked to a nucleic acid sequence comprising a region of action and the selected nuclear receptor. Here, when the expression of the reporter is enhanced, the candidate compound is determined as an agonist of the nuclear receptor, and when the expression of the reporter decreases, the candidate compound is determined as an antagonist of the nuclear receptor.

  In the present specification, any reporter may be used as long as expression can be confirmed, but luciferase is typically used. It is because observation is easy because of chemiluminescence. In addition to this, for example, a fluorescent protein or the like can be used, but is not limited thereto.

  It will be appreciated that the system utilized in the transcriptional activity assay used in the present invention can be a cell, but is not limited thereto, and a cell-free system can also be used.

  Examples of the transcription factor recognition sequence include GAL4 DNA (Type CM, Klausing K., Methods Mol Med. 2003; 85: 175-83), LexA DBD (Chemistry and Biology, vol 10, no. 7, pp. 584-585 (Jury, 2003)) and the like, but is not limited thereto. It will be appreciated that such transcription factor recognition sequences can be used with any nuclear receptor. For other transcription factor recognition sequences, using this system of the present invention, a substance known to be an agonist was used in place of the candidate compound, and a transcription factor recognition sequence candidate sequence was used in place of the GAL4 DNA. It can be appreciated that any sequence in which the assay is run and a positive response by an agonist is seen in the assay can be used as a transcription factor recognition sequence.

(organic chemistry)
The present invention was partially completed by the discovery that sulfonylureas have the ability to modulate PPARs.

  In the present specification, the “sulfonylurea agent”

(In the formula, R ₁ and R _{2 each} represent an arbitrary substituent) are those having a structure in which urea is bonded to urea. The sulfonylurea has long been used as a substitute for insulin because of its hypoglycemic effect by stimulating insulin secretion. Examples of the sulfonylurea agent include, but are not limited to, glimepiride, glibenclamide, tolbutamide, chlorpropamide, acetohexamide, and gliclazide.

As used herein, “alkyl” refers to a monovalent group formed by loss of one hydrogen atom from an aliphatic hydrocarbon (alkane) such as methane, ethane, or propane, and is generally represented by C _n H _{2n + 1} −. Where n is a positive integer. Alkyl can be linear or branched. In the present specification, the “substituted alkyl” refers to an alkyl in which H of the alkyl is substituted with a substituent specified below. Specific examples thereof include C1-C2 alkyl, C1-C3 alkyl, C1-C4 alkyl, C1-C5 alkyl, C1-C6 alkyl, C1-C7 alkyl, C1-C8 alkyl, C1-C9 alkyl, C1-C10 alkyl. C1-C11 alkyl or C1-C12 alkyl, C1-C2 substituted alkyl, C1-C3 substituted alkyl, C1-C4 substituted alkyl, C1-C5 substituted alkyl, C1-C6 substituted alkyl C1-C7 substituted alkyl, C1-C8 substituted alkyl, C1-C9 substituted alkyl, C1-C10 substituted alkyl, C1-C11 substituted alkyl or C1-C12 substituted alkyl obtain. Here, for example, C1-C10 alkyl means linear or branched alkyl having 1 to 10 carbon atoms, methyl (CH _3- ), ethyl (C ₂ H _5- ), n-propyl. _{(CH 3 CH 2 CH 2 -} ), isopropyl _{((CH 3) 2 CH -} ), n- butyl _{(CH 3 CH 2 CH 2 CH} 2 -), n- pentyl _{(CH 3 CH 2 CH 2 CH} 2 CH 2 -), n-hexyl _{(CH 3 CH 2 CH 2 CH} 2 CH 2 CH 2 -), n- heptyl _{(CH 3 CH 2 CH 2 CH} 2 CH 2 CH 2 CH 2 -), n- octyl _(CH 3 CH _{2 CH 2 CH 2 CH 2 CH} 2 CH 2 CH 2 -), n- nonyl _{(CH 3 CH 2 CH 2 CH} 2 CH 2 CH 2 CH 2 CH 2 CH 2 -), n- decyl _(CH 3 CH 2 CH ₂ C _{H 2 CH 2 CH 2 CH 2} CH 2 CH 2 CH 2 -), - C (CH 3) 2 CH 2 CH 2 CH (CH 3) 2, -CH 2 CH (CH 3) such as ₂ are exemplified. Further, for example, C1-C10 substituted alkyl refers to C1-C10 alkyl, in which one or more hydrogen atoms are substituted with a substituent.

  As used herein, “optionally substituted alkyl” means that either “alkyl” or “substituted alkyl” as defined above may be used.

As used herein, “alkylene” refers to a divalent group formed by losing two hydrogen atoms from an aliphatic hydrocarbon (alkane) such as methylene, ethylene, propylene, and is generally represented by —C _n H _2n —. Where n is a positive integer. The alkylene can be straight or branched. As used herein, “substituted alkylene” refers to an alkylene in which H of alkylene is substituted with a substituent specified below. Specific examples thereof include C1-C2 alkylene, C1-C3 alkylene, C1-C4 alkylene, C1-C5 alkylene, C1-C6 alkylene, C1-C7 alkylene, C1-C8 alkylene, C1-C9 alkylene, C1-C10 alkylene. C1-C11 alkylene or C1-C12 alkylene, C1-C2 substituted alkylene, C1-C3 substituted alkylene, C1-C4 substituted alkylene, C1-C5 substituted alkylene, C1-C6 substituted alkylene C1-C7 substituted alkylene, C1-C8 substituted alkylene, C1-C9 substituted alkylene, C1-C10 substituted alkylene, C1-C11 substituted alkylene or C1-C12 substituted alkylene obtain. Here, for example, C1-C10 alkylene means linear or branched alkylene having 1 to 10 carbon atoms, methylene (—CH ₂ —), ethylene (—C ₂ H ₄ —), n - propylene _{(-CH 2 CH 2 CH 2 -} ), isopropylene _{(- (CH 3) 2 C} -), n- butylene _{(-CH 2 CH 2 CH 2 CH} 2 -), n- pentylene _(-CH 2 CH _{2 CH 2 CH 2 CH 2 -} ), n- hexylene _{(-CH 2 CH 2 CH 2 CH} 2 CH 2 CH 2 -), n- heptylene _{(-CH 2 CH 2 CH 2 CH} 2 CH 2 CH 2 CH 2 - ), N-octylene (—CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ —), n-nonylene (—CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -), n-decylene _{(-CH 2 CH 2 CH 2 CH} 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 -), - CH 2 C (CH 3) 2 - and the like are exemplified. Further, for example, C1-C10 substituted alkylene refers to C1-C10 alkylene, in which one or more hydrogen atoms are substituted with a substituent. In the present specification, “alkylene” may contain one or more atoms selected from an oxygen atom and a sulfur atom.

  As used herein, “optionally substituted alkylene” means that either “alkylene” or “substituted alkylene” as defined above may be used.

  As used herein, “cycloalkyl” refers to alkyl having a cyclic structure. “Substituted cycloalkyl” refers to a cycloalkyl in which H of the cycloalkyl is substituted by the substituent specified below. Specific examples include C3-C4 cycloalkyl, C3-C5 cycloalkyl, C3-C6 cycloalkyl, C3-C7 cycloalkyl, C3-C8 cycloalkyl, C3-C9 cycloalkyl, C3-C10 cycloalkyl, C3-C11. Cycloalkyl, C3-C12 cycloalkyl, C3-C4 substituted cycloalkyl, C3-C5 substituted cycloalkyl, C3-C6 substituted cycloalkyl, C3-C7 substituted cycloalkyl, C3-C8 substituted Cycloalkyl, C3-C9 substituted cycloalkyl, C3-C10 substituted cycloalkyl, C3-C11 substituted cycloalkyl or C3-C12 substituted cycloalkyl. For example, cycloalkyl is exemplified by cyclopropyl, cyclohexyl and the like.

  In the present specification, the “optionally substituted cycloalkyl” means either “cycloalkyl” or “substituted cycloalkyl” as defined above.

In the present specification, “alkenyl” refers to a monovalent group produced by losing one hydrogen atom from an aliphatic hydrocarbon having one double bond in the molecule, and is generally represented by C _n H _2n−1 —. (Where n is a positive integer greater than or equal to 2). “Substituted alkenyl” refers to alkenyl in which H of alkenyl is substituted by a substituent specified below. Specific examples include C2-C3 alkenyl, C2-C4 alkenyl, C2-C5 alkenyl, C2-C6 alkenyl, C2-C7 alkenyl, C2-C8 alkenyl, C2-C9 alkenyl, C2-C10 alkenyl, C2-C11 alkenyl or C2-C12 alkenyl, C2-C3 substituted alkenyl, C2-C4 substituted alkenyl, C2-C5 substituted alkenyl, C2-C6 substituted alkenyl, C2-C7 substituted alkenyl, C2-C8 substituted Alkenyl, C2-C9 substituted alkenyl, C2-C10 substituted alkenyl, C2-C11 substituted alkenyl or C2-C12 substituted alkenyl. Here, for example, is C2~C10 alkyl means a straight or branched alkenyl containing 2 to 10 carbon atoms, vinyl _(CH 2 = CH-), allyl _{(CH 2 = CHCH 2 -)} , CH ₃ CH═CH— and the like are exemplified. Also, for example, C2-C10 substituted alkenyl refers to C2-C10 alkenyl, in which one or more hydrogen atoms are substituted with substituents.

  As used herein, “optionally substituted alkenyl” means that either “alkenyl” or “substituted alkenyl” as defined above may be used.

As used herein, “alkenylene” refers to a divalent group formed by losing two hydrogen atoms from an aliphatic hydrocarbon having one double bond in the molecule, and is generally —C _n H _2n-2 —. (Where n is a positive integer greater than or equal to 2). “Substituted alkenylene” refers to alkenylene in which H of alkenylene is substituted by the substituent specified below. Specific examples include C2-C25 alkenylene or C2-C25 substituted alkenylene, especially C2-C3 alkenylene, C2-C4 alkenylene, C2-C5 alkenylene, C2-C6 alkenylene, C2-C7 alkenylene, C2 -C8 alkenylene, C2-C9 alkenylene, C2-C10 alkenylene, C2-C11 alkenylene or C2-C12 alkenylene, C2-C3-substituted alkenylene, C2-C4-substituted alkenylene, C2-C5-substituted alkenylene, C2- C6-substituted alkenylene, C2-C7 substituted alkenylene, C2-C8 substituted alkenylene, C2-C9 substituted alkenylene, C2-C10 substituted alkenylene, C2-C11 substituted alkenylene or C2~C12 substituted alkenylene are preferred. Here, for example, is C2~C10 alkyl means a straight or branched alkenylene including 2-10 carbon _{atoms, -CH = CH -, - CH} = CHCH 2 -, - (CH 3) C = CH- and the like are exemplified. Further, for example, C2-C10 substituted alkenylene refers to C2-C10 alkenylene, in which one or more hydrogen atoms are substituted with substituents. As used herein, “alkenylene” may contain one or more atoms selected from an oxygen atom and a sulfur atom.

  As used herein, “optionally substituted alkenylene” means that either “alkenylene” or “substituted alkenylene” as defined above may be used.

  As used herein, “cycloalkenyl” refers to alkenyl having a cyclic structure. “Substituted cycloalkenyl” refers to cycloalkenyl in which H of cycloalkenyl is substituted by the substituent specified below. Specific examples include C3-C4 cycloalkenyl, C3-C5 cycloalkenyl, C3-C6 cycloalkenyl, C3-C7 cycloalkenyl, C3-C8 cycloalkenyl, C3-C9 cycloalkenyl, C3-C10 cycloalkenyl, C3-C11. Cycloalkenyl, C3-C12 cycloalkenyl, C3-C4 substituted cycloalkenyl, C3-C5 substituted cycloalkenyl, C3-C6 substituted cycloalkenyl, C3-C7 substituted cycloalkenyl, C3-C8 substituted Cycloalkenyl, C3-C9 substituted cycloalkenyl, C3-C10 substituted cycloalkenyl, C3-C11 substituted cycloalkenyl or C3-C12 substituted cycloalkenyl. For example, preferable cycloalkenyl includes 1-cyclopentenyl, 2-cyclohexenyl and the like.

  In the present specification, “optionally substituted cycloalkenyl” means either “cycloalkenyl” or “substituted cycloalkenyl” as defined above.

In the present specification, “alkynyl” refers to a monovalent group formed by losing one hydrogen atom from an aliphatic hydrocarbon having one triple bond in the molecule, such as acetylene, and is generally C _n H _{2n −3} − (where n is a positive integer of 2 or more). “Substituted alkynyl” refers to alkynyl in which H of alkynyl is substituted by the substituent specified below. Specific examples include C2-C3 alkynyl, C2-C4 alkynyl, C2-C5 alkynyl, C2-C6 alkynyl, C2-C7 alkynyl, C2-C8 alkynyl, C2-C9 alkynyl, C2-C10 alkynyl, C2-C11 alkynyl, C2-C12 alkynyl, C2-C3 substituted alkynyl, C2-C4 substituted alkynyl, C2-C5 substituted alkynyl, C2-C6 substituted alkynyl, C2-C7 substituted alkynyl, C2-C8 substituted Alkynyl, C2-C9 substituted alkynyl, C2-C10 substituted alkynyl, C2-C11 substituted alkynyl or C2-C12 substituted alkynyl. Here, for example, C2-C10 alkynyl means, for example, straight-chain or branched alkynyl containing 2 to 10 carbon atoms, and includes ethynyl (CH≡C-), 1-propynyl (CH ₃ C≡C -) Etc. are exemplified. Further, for example, C2-C10 substituted alkynyl refers to C2-C10 alkynyl, in which one or more hydrogen atoms are substituted with a substituent.

  As used herein, “optionally substituted alkynyl” means that either “alkynyl” or “substituted alkynyl” as defined above may be used.

As used herein, “alkoxy” refers to a monovalent group generated by loss of a hydrogen atom of a hydroxy group of an alcohol, and is generally represented by C _n H _{2n + 1} O— (where n is 1 or more). Is an integer). “Substituted alkoxy” refers to alkoxy in which H of alkoxy is substituted by a substituent specified below. Specific examples include C1-C2 alkoxy, C1-C3 alkoxy, C1-C4 alkoxy, C1-C5 alkoxy, C1-C6 alkoxy, C1-C7 alkoxy, C1-C8 alkoxy, C1-C9 alkoxy, C1-C10 alkoxy, C1-C11 alkoxy, C1-C12 alkoxy, C1-C2-substituted alkoxy, C1-C3-substituted alkoxy, C1-C4-substituted alkoxy, C1-C5-substituted alkoxy, C1-C6-substituted alkoxy, C1-C7 substituted alkoxy, C1-C8 substituted alkoxy, C1-C9 substituted alkoxy, C1-C10 substituted alkoxy, C1-C11 substituted alkoxy or C1-C12 substituted alkoxy . Here, for example, C1-C10 alkoxy means linear or branched alkoxy containing 1 to 10 carbon atoms, and includes methoxy (CH ₃ O—), ethoxy (C ₂ H ₅ O—), An example is n-propoxy (CH ₃ CH ₂ CH ₂ O—).

  In the present specification, “optionally substituted alkoxy” means either “alkoxy” or “substituted alkoxy” as defined above.

  As used herein, “heterocycle (group)” refers to a group having a cyclic structure including carbon and heteroatoms. Here, the hetero atom is selected from the group consisting of O, S and N, and may be the same or different, and may be contained in one or more than one. Heterocyclic groups can be aromatic or non-aromatic and can be monocyclic or polycyclic. The heterocyclic group may be substituted.

  In the present specification, the “optionally substituted heterocycle (group)” may be either the “heterocycle (group)” or “substituted heterocycle (group)” defined above. Means.

  As used herein, “alcohol” refers to an organic compound in which one or more hydrogen atoms of an aliphatic hydrocarbon are substituted with a hydroxyl group. In the present specification, it is also expressed as ROH. Here, R is an alkyl group. Preferably R may be C1-C6 alkyl. Examples of the alcohol include, but are not limited to, methanol, ethanol, 1-propanol, 2-propanol and the like.

  In this specification, the “carbocyclic group” is a group containing a cyclic structure containing only carbon, and the above-mentioned “cycloalkyl”, “substituted cycloalkyl”, “cycloalkenyl”, “substituted cyclo A group other than “alkenyl”. The carbocyclic group can be aromatic or non-aromatic and can be monocyclic or polycyclic. The “substituted carbocyclic group” refers to a carbocyclic group in which H of the carbocyclic group is substituted with a substituent specified below. Specific examples include C3-C4 carbocyclic group, C3-C5 carbocyclic group, C3-C6 carbocyclic group, C3-C7 carbocyclic group, C3-C8 carbocyclic group, C3-C9 carbocyclic group, C3-C10. Carbocyclic group, C3-C11 carbocyclic group, C3-C12 carbocyclic group, C3-C4-substituted carbocyclic group, C3-C5-substituted carbocyclic group, C3-C6-substituted carbocyclic group, C3- C7 substituted carbocyclic group, C3-C8 substituted carbocyclic group, C3-C9 substituted carbocyclic group, C3-C10 substituted carbocyclic group, C3-C11 substituted carbocyclic group or C3- It can be a C12 substituted carbocyclic group. The carbocyclic group can also be a C4-C7 carbocyclic group or a C4-C7 substituted carbocyclic group. Examples of the carbocyclic group include those in which one hydrogen atom is deleted from the phenyl group. Here, the hydrogen deletion position may be any position chemically possible, and may be on an aromatic ring or a non-aromatic ring.

  In the present specification, the “optionally substituted carbocyclic group” means that any of the above-defined “carbocyclic group” or “substituted carbocyclic group” may be used.

  As used herein, “heterocyclic group” refers to a group having a cyclic structure including carbon and heteroatoms. Here, the hetero atom is selected from the group consisting of O, S and N, and may be the same or different, and may be contained in one or more than one. Heterocyclic groups can be aromatic or non-aromatic and can be monocyclic or polycyclic. The “substituted heterocyclic group” refers to a heterocyclic group in which H of the heterocyclic group is substituted with a substituent specified below. Specific examples include C3-C4 carbocyclic group, C3-C5 carbocyclic group, C3-C6 carbocyclic group, C3-C7 carbocyclic group, C3-C8 carbocyclic group, C3-C9 carbocyclic group, C3-C10. Carbocyclic group, C3-C11 carbocyclic group, C3-C12 carbocyclic group, C3-C4-substituted carbocyclic group, C3-C5-substituted carbocyclic group, C3-C6-substituted carbocyclic group, C3- C7 substituted carbocyclic group, C3-C8 substituted carbocyclic group, C3-C9 substituted carbocyclic group, C3-C10 substituted carbocyclic group, C3-C11 substituted carbocyclic group or C3- One or more carbon atoms of the C12 substituted carbocyclic group may be substituted with a heteroatom. Heterocyclic groups can also be those in which one or more heteroatoms are substituted for the carbon atoms of a C4-C7 carbocyclic group or a C4-C7 substituted carbocyclic group. Examples of the heterocyclic group include thienyl group, pyrrolyl group, furyl group, imidazolyl group, and pyridyl group. The hydrogen deletion position may be any chemically possible position, and may be on an aromatic ring or a non-aromatic ring.

  In this specification, a carbocyclic group or heterocyclic group may be substituted with a divalent substituent in addition to being substituted with a monovalent substituent as defined below. Such divalent substitutions can be oxo substitutions (= O) or thioxo substitutions (= S).

  In this specification, “halogen” refers to a monovalent group of an element such as fluorine (F), chlorine (Cl), bromine (Br), iodine (I) belonging to Group 7B of the periodic table.

  As used herein, “hydroxy” refers to a group represented by —OH. “Substituted hydroxy” refers to a hydroxy in which H is substituted with a substituent as defined below.

  In the present specification, the “thiol” is a group (mercapto group) in which an oxygen atom of a hydroxy group is substituted with a sulfur atom, and is represented by —SH. “Substituted thiol” refers to a group in which H of mercapto is substituted with a substituent defined below.

As used herein, “cyano” refers to a group represented by —CN. “Nitro” refers to a group represented by —NO ₂ . “Amino” refers to a group represented by —NH ₂ . “Substituted amino” refers to amino substituted with H as defined below.

  In this specification, “carboxy” refers to a group represented by —COOH. “Substituted carboxy” refers to a carboxy H substituted with a substituent as defined below.

  As used herein, “thiocarboxy” refers to a group in which an oxygen atom of a carboxy group is substituted with a sulfur atom, and may be represented by —C (═S) OH, —C (═O) SH, or —CSSH. “Substituted thiocarboxy” refers to thiocarboxy H substituted with the substituents defined below.

As used herein, “acyl” refers to a monovalent group formed by removing OH from a carboxylic acid. Representative examples of the acyl group include acetyl (CH ₃ CO—), benzoyl (C ₆ H ₅ CO—), and the like. “Substituted acyl” refers to an acyl hydrogen substituted with a substituent as defined below.

In the present specification, “amide” is a group obtained by substituting hydrogen of ammonia with an acid group (acyl group), and is preferably represented by —CONH ₂ . “Substituted amide” refers to a substituted amide.

  As used herein, “carbonyl” refers to a generic term for a substance including — (C═O) — which is a characteristic group of aldehyde and ketone. “Substituted carbonyl” means a carbonyl group substituted with a substituent selected below.

  In this specification, “thiocarbonyl” is a group in which an oxygen atom in carbonyl is substituted with a sulfur atom, and includes a characteristic group — (C═S) —. Thiocarbonyl includes thioketones and thioaldehydes. “Substituted thiocarbonyl” means thiocarbonyl substituted with a substituent selected below.

As used herein, “sulfonyl” refers to a generic term for a substance including a characteristic group —SO ₂ —. “Substituted sulfonyl” means a sulfonyl substituted with a substituent selected below.

  As used herein, “sulfinyl” is a generic term for a substance including a characteristic group —SO—. “Substituted sulfinyl” means sulfinyl substituted with a substituent selected below.

  As used herein, “aryl” refers to a group formed by leaving one hydrogen atom bonded to an aromatic hydrocarbon ring, and is included in the present specification as a carbocyclic group.

  In the present specification, unless otherwise specified, substitution refers to replacement of one or more hydrogen atoms in an organic compound or substituent with another atom or atomic group. One hydrogen atom can be removed and substituted with a monovalent substituent, and two hydrogen atoms can be removed and substituted with a divalent substituent.

  In the present specification, unless otherwise specified, substitution refers to replacement of one or more hydrogen atoms in an organic compound or substituent with another atom or atomic group. One hydrogen atom can be removed and substituted with a monovalent substituent, and two hydrogen atoms can be removed and substituted with a divalent substituent.

  Examples of the substituent in the present invention include alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, alkoxy, carbocyclic group, heterocyclic group, halogen, hydroxy, thiol, cyano, nitro, amino, carboxy, carbamoyl, acyl, acylamino, Examples include, but are not limited to, thiocarboxy, amide, substituted carbonyl, substituted thiocarbonyl, substituted sulfonyl, or substituted sulfinyl. Such a substituent can be appropriately used in the present invention when designing an amino acid.

  Preferably, when a plurality of substituents are present, each may be independently a hydrogen atom or alkyl, but not all of the plurality of substituents are hydrogen atoms. More preferably, independently when there are multiple substituents, each may be independently selected from the group consisting of hydrogen and C1-C6 alkyl. The substituents may all have a substituent other than hydrogen, but preferably have at least one hydrogen, more preferably 2 to n (where n is the number of substituents) hydrogen. obtain. It may be preferred that the number of hydrogens in the substituent is large. This is because a large substituent group or a polar substituent group may have a hindrance to the effect of the present invention (particularly, interaction with an aldehyde group). Accordingly, the substituent other than hydrogen may preferably be C1-C6 alkyl, C1-C5 alkyl, C1-C4 alkyl, C1-C3 alkyl, C1-C2 alkyl, methyl and the like. However, since the effect of the present invention may be enhanced, it may be preferable to have a large substituent.

  In this specification, C1, C2,. . . Cn represents the number of carbon atoms. Therefore, C1 is used to represent a substituent having 1 carbon.

  In this specification, “optical isomer” refers to one or a pair of a pair of compounds that have a mirror-image relationship with respect to a crystal or a molecular structure and cannot be superposed. It is a form of stereoisomer, and the other properties are the same, but only the optical rotation is different.

  As used herein, “protection reaction” refers to a reaction in which a protecting group such as Boc is added to a functional group desired to be protected. By protecting the functional group with the protecting group, the reaction of the functional group with higher reactivity can be suppressed, and only the functional group with lower reactivity can be reacted. The protection reaction can be performed by, for example, a dehydration reaction.

  As used herein, “deprotection reaction” refers to a reaction for removing a protecting group such as Boc. Examples of the deprotection reaction include a reaction such as a reduction reaction using Pd / C. The deprotection reaction can be performed, for example, by hydrolysis.

  In this specification, examples of the “protecting group” include a fluorenylmethoxycarbonyl (Fmoc) group, an acetyl group, a benzyl group, a benzoyl group, a t-butoxycarbonyl group, a t-butyldimethyl group, a silyl group, and a trimethylsilylethyl group. , N-phthalimidyl group, trimethylsilylethyloxycarbonyl group, 2-nitro-4,5-dimethoxybenzyl group, 2-nitro-4,5-dimethoxybenzyloxycarbonyl group, carbamate group and the like can be mentioned as typical protecting groups. .

  In each method of the present invention, the target product is a contaminant (unreacted weight loss, by-product, solvent, etc.) from the reaction solution, and a method commonly used in the art (for example, extraction, distillation, washing, concentration). , Precipitation, filtration, drying, etc.) followed by a combination of post-treatment methods commonly used in the art (eg, adsorption, elution, distillation, precipitation, precipitation, chromatography, etc.).

(screening)
As used herein, “screening” refers to selecting a target such as an organism, cell, or substance having a specific target property from a large number of populations by a specific operation / evaluation method. For screening, an agent (eg, antibody), polypeptide or nucleic acid molecule of the invention can be used. For screening, a system using real substances such as in vitro and in vivo may be used, or a library generated using an in silico (system using a computer) system may be used. In the present invention, it is understood that compounds obtained by screening having a desired activity are also encompassed within the scope of the present invention. The present invention also contemplates providing computer modeling drugs, diagnostic agents, therapeutic agents, and the like based on the disclosure of the present invention.

  As used herein, “candidate compound” refers to a compound that is a candidate for the purpose of screening when used in screening. Such a compound may be any factor. Candidate compounds include, for example, proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (including, for example, DNA such as cDNA, genomic DNA, RNA such as mRNA), polysaccharides, Oligosaccharides, lipids, small organic molecules (for example, hormones, ligands, signaling substances, small organic molecules, molecules synthesized by combinatorial chemistry, small molecules that can be used as pharmaceuticals (for example, small molecule ligands), etc.) However, it is not limited to them. A candidate compound is also referred to as a candidate drug when the candidate is a drug. Such a set of candidate compounds is also called a library.

  As used herein, “compound species” refers to a single compound having a desired property, such as having a specific target activity in a certain group of compounds. For example, if a compound is specified in a collection of compounds that modulate activity, such a compound may be referred to as a compound species. In this specification, it is also simply referred to as a compound.

  Therefore, the “library” in the present specification refers to a certain collection of compounds for screening. The library may be a collection of compounds having similar properties or a collection of random compounds. Preferably, a set of compounds predicted to have similar properties is used, but is not limited thereto. The compound library used in the present invention can be prepared or obtained by any means including, but not limited to, combinatorial chemistry techniques, fermentation methods, plant and cell extraction procedures, etc. Can do. Methods for creating combinatorial libraries are well known in the art. For example, E.I. R. Felder, Chimia 1994, 48, 512-541; Gallop et al. Med. Chem. 1994, 37, 1233-1251; A. Houghten, Trends Genet. 1993, 9, 235-239; Houghten et al., Nature 1991, 354, 84-86; Lam et al., Nature 1991, 354, 82-84; Carell et al., Chem. Biol. 1995, 3, 171-183; Madden et al., Perspectives in Drug Discovery and Design 2, 269-282; Cwirla et al., Biochemistry 1990, 87, 6378-6382; Brenner et al., Proc. Natl. Acad. Sci. USA 1992, 89, 5381-5383; Gordon et al. Med. Chem. 1994, 37, 1385-1401; Lebl et al., Biopolymers 1995, 37 177-198; and references cited therein. These references are incorporated herein by reference in their entirety.

  As used herein, “modulation” means that when referring to PPAR, its biological activity is altered.

  As used herein, “inhibition” refers to the reduction or elimination of biological activity when referring to PPAR.

  As used herein, “promoting” when referring to PPAR means that a state occurs from a state in which the biological activity is increased or absent.

  As used herein, “biological test” refers to determining whether a compound has a certain activity using an actual biological reaction. Biological tests include in vitro and in vivo tests. Biological testing is therefore an opposite concept to in silico.

  As used herein, “evaluation”, when used in screening, refers to determining whether a candidate compound meets the requirements of such an indicator with respect to an indicator (eg, pharmacological activity). Such evaluation can be performed using methods known in the art and can be performed in silico (using a computer) or wet (performing an actual biological assay).

  In the screening technique of the present invention, a screening hit can be confirmed by an assay using a genetic technique.

  In the present specification, “expression” of a gene product such as a gene, polynucleotide, or polypeptide means that the gene (usually in a DNA form) or the like undergoes a certain action in vivo to become another form. Preferably, a gene, polynucleotide or the like is transcribed and translated into a polypeptide form, but transcription to produce mRNA can also be a form of expression. In another embodiment, such polypeptide forms may have undergone post-translational processing (eg, leader sequence excision).

  In the present specification, the expression "specifically expresses" a gene means that the gene is expressed at a different level (preferably higher) at a specific site or time of an organism than other sites or times. . “Specific expression” may be expressed only at a certain site (for example, a specific site such as a diseased site) or may be expressed at other sites. The expression specifically preferably means that it is expressed only at a certain site. Such specific expression can be realized by utilizing the characteristics of antigen-presenting cells. Therefore, the pharmaceutical screening in the present invention can also be performed by confirming the specific expression of a specific index.

  The expression “only” in a cell in the present specification means that a gene is expressed only in that cell and is not substantially expressed in cells of other species. Therefore, expression only in a certain cell includes specific expression in that cell. In this case, it is active in adipocytes and there is little non-specific expression in other cells.

  As used herein, “activity” refers to an activity of a function that a gene or gene product originally intends to exhibit when a gene or gene product such as a nucleic acid or protein is referred to. Examples of such activity include, but are not limited to, PPAR, adipocyte differentiation, regulation of sugar metabolism, regulation of lipid metabolism, and the like.

  As used herein, “detection” or “quantification” of gene expression (eg, mRNA expression, polypeptide expression) can be accomplished using appropriate methods, including, for example, mRNA measurement and immunoassay methods. Examples of molecular biological measurement methods include Northern blotting, dot blotting, and PCR. Examples of the immunological measurement method include an ELISA method, an RIA method, a fluorescent antibody method, a Western blot method, an immunohistochemical staining method and the like using a microtiter plate as necessary. Examples of the quantitative method include an ELISA method and an RIA method. It can also be performed by a gene analysis method using an array (eg, DNA array, protein array). The DNA array is widely outlined in (edited by Shujunsha, separate volume of cell engineering "DNA microarray and latest PCR method"). For protein arrays, see Nat Genet. 2002 Dec; 32 suppl: 526-32. Examples of gene expression analysis methods include, but are not limited to, RT-PCR, RACE method, SSCP method, immunoprecipitation method, two-hybrid system, in vitro translation and the like. Such further analysis methods are described in, for example, Genome Analysis Experimental Method / Yusuke Nakamura Lab Manual, Editing / Yusuke Nakamura Yodosha (2002), etc., all of which are incorporated herein by reference. Is done.

  In the present specification, the “expression level” refers to an amount at which a polypeptide or mRNA is expressed in a target cell or the like. Such expression level is evaluated by any appropriate method including immunoassay methods such as ELISA method, RIA method, fluorescent antibody method, Western blot method, and immunohistochemical staining method using the antibody of the present invention. The expression level of the polypeptide of the present invention at the protein level or the polypeptide of the present invention evaluated by any appropriate method including molecular biological measurement methods such as Northern blotting, dot blotting, and PCR. The expression level at the mRNA level can be mentioned. “Change in expression level” means an increase in the expression level at the protein level or mRNA level of the polypeptide of the present invention evaluated by any appropriate method including the above immunological measurement method or molecular biological measurement method. Or it means to decrease.

  As used herein, the term “upstream” refers to a position from a particular reference point toward the 5 ′ end of a polynucleotide.

  As used herein, the term “downstream” refers to a position from a particular reference point toward the 3 ′ end of a polynucleotide.

  As used herein, the expressions “base pair” and “Watson & Crick base pair” are used interchangeably herein and, like those found in double-stranded DNA, the adenine residue is 2 Based on the identity of the sequence that binds to thymine or uracil residues by three hydrogen bonds and the cytosine and guanine residues by three hydrogen bonds, nucleotides that can hydrogen bond to each other are shown (Stryer, L , Biochemistry, 4th edition, 1995).

  As used herein, the terms “complementary” or “complement” are used herein to refer to a sequence of a polynucleotide that allows the entire complementary region to directly form a Watson & Crick base pair with another specific polynucleotide. Show. For purposes of the present invention, a first polynucleotide is considered complementary to a second polynucleotide when each base of the first polynucleotide is paired with its complementary base. Complementary bases are generally A and T (or A and U) or C and G. In the present specification, the term “complement” is used as a synonym for “complementary polynucleotide”, “complementary nucleic acid” and “complementary nucleotide sequence”. These terms apply to a pair of polynucleotides based solely on their sequence, and do not apply to the particular set in which the two polynucleotides are effectively in a combined state.

(Insulin sensitivity enhancement)
Below, the effect which supports the usefulness as an insulin resistance improving agent of this invention or an insulin sensitivity enhancer is demonstrated with an example of an assay. As an example, the glucose clamp method is one method for evaluating insulin sensitivity of peripheral tissues. At present, this glucose clamp method is said to be able to evaluate sensitivity most accurately. In this method, in order to keep blood insulin level at a constant level, glucose solution is injected while monitoring blood glucose level under continuous infusion of insulin, and blood glucose level is kept at fasting blood glucose level. A glucose infusion rate as a glucose metabolism rate is used as an index of insulin sensitivity.
The test result can be expressed as an average value ± standard error. The significance test is performed by a t-test. For example, when the p value is smaller than 0.05, it can be determined that the significance is significant.
Test plan example 1: 20 patients with type 2 diabetes are randomly assigned to 2 groups of 10 people each. Adjustments are made between the test group and placebo group so that there are no differences in age, sex, body mass index, oral hypoglycemic medication, fasting blood glucose, and background factors such as hemoglobin _A1c . All patients will not receive insulin. These patients, for example, are hospitalized at least two weeks prior to the study and are fed a weight maintenance diet containing at least 200 grams of carbohydrate per day.
Patients in the test group are orally administered with the candidate compound three times a day for a week for a week. Patients in the placebo group are orally administered a similar placebo tablet 3 times daily for 1 week. Prior to treatment and on the last treatment day, blood is drawn from the patient's vascular catheter on a fasting into a tube containing 1.2 mg EDTA and 400 KIU (Athylor, Bayer, Germany). Plasma collected by centrifugation was stored at −40 ° C. until analysis. Blood glucose, fructosamine and hemoglobin A _1c are measured, for example, by the glucose oxidase method, the NBT (NitroBlue Tetrazol chloride) method and the HPLC (High Performance Liquid Chromatography) method, respectively. The amount of insulin in plasma and the amount of C peptide in urine were measured using commercially available kits, insulin-III (Boehringer Mannheim, Germany) and C peptide kit (Daiichi Kagaku, Japan), respectively. The amount of C peptide in urine is calculated as an average value over 2 days.
All patients in both the test group and the placebo group undergo a glucose tolerance test in which 0.2 g of glucose per kg of body weight is intravenously administered before and on the last treatment day, and blood glucose and insulin secretion are measured over time.
Further, in some groups (for example, 5 patients) in both groups, a hyperinsulinic-normoglycemic-clamp (hereinafter referred to as “glucose clamp method”) test was performed before treatment and on the last treatment day. In addition, the total amount of insulin receptor and the amount of tyrosine autophosphorylation per 300 μl of erythrocytes are measured by ELISA (enzyme-linked immunosorbent assay) to evaluate insulin sensitivity. Measurement of the insulin receptor by ELISA is performed according to the method described in Diabetes, 43, 274-280 (1994).

In the glucose clamp test, insulin was infused at a rate of 0.8 mU / kg / min for 4 hours to suppress hepatic glucose production, and the plasma insulin concentration was maintained at 100 μU / ml (600 pmol / L). The glucose solution is injected so that the blood glucose level is maintained at 95 mg / dl (5.32 mmol / L). The glucose infusion rate for 2 to 4 hours after the start of insulin infusion is measured and used as the glucose metabolism rate.
Next, “insulin sensitivity enhancing action” which is one of the actions of the compound of the present invention will be described.
That is, the strength of the insulin sensitivity enhancing action can be evaluated by the blood glucose lowering rate when the drug is administered to the KKAy mouse. KKAy mice are diabetic model animals exhibiting insulin resistance (Japanese clinical volume 60, extra number 8, 38-44, (2002)), and sulfonylurea hypoglycemia that is a therapeutic agent for type 2 diabetes based on insulin secretion promoting action. Agents are known to be ineffective (Medical Pharmacy, Vol. 24, No. 3, 131-136, (1990)). In a hypoglycemic test by oral administration using KKAy mice, when the blood glucose level of KKAy mice is suppressed by about 45%, the blood glucose level of normal mice is almost the same, so the blood glucose lowering rate may be 40-50% preferable.
The measurement of the hypoglycemic rate by the oral administration hypoglycemic test using KKAy mice can be carried out by a known method, and preferred methods are shown below.

For example, 6 mice of 11 weeks old male mice (KKAy / Ta) are used as a group for the test. As a control, blood is collected from the tail to measure the blood glucose level before treatment. After blood collection, the biguanide derivative is dissolved in 0.5% CMC-Na (Sodium Carboxymethyl Cellulose) solution at an appropriate concentration and orally administered at a dose of 10 mL / kg. In addition, a mouse to which only the solvent is administered is prepared as a control. One hour, 2 hours, 4 hours, and 6 hours after drug administration, blood is collected from the tail to measure the blood glucose level. The blood glucose level is measured using Glucose CII-Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.).
Moreover, the blood glucose lowering rate is obtained by the following formula.

Hypoglycemic rate (%) = [(AUC of blood glucose level of control group−AUC of blood glucose level of compound administration group) / AUC of blood glucose level of control group] × 100
Here, the AUC of blood glucose level represents an area up to 6 hours after drug administration, with a glucose value of 0 as a baseline in a graph plotting changes in blood glucose level after drug administration against time. Specifically, A = blood glucose level before drug administration, B = blood glucose level 1 hour after drug administration, C = blood glucose level 2 hours after drug administration, D = blood glucose level 4 hours after drug administration, E = drug When the blood glucose level 6 hours after administration, the AUC of the blood glucose level is represented by the following formula:
AUC of blood glucose level = 1 × ((A + B) / 2) + 1 × ((B + C) / 2) + 2 × ((C + D) / 2) + 2 × ((D + E) / 2)
Can be obtained from

  When this blood glucose lowering rate is about 45%, the blood glucose level falls to a level almost equal to that of normal mice.

  In addition, the strength of the insulin sensitivity enhancing action is that when a drug is administered to a db / db mouse (Japanese clinical volume 60, extra number 8, 38-44, (2002)) which is a diabetes model animal showing insulin resistance. It can also be evaluated by the blood glucose lowering rate. In the glucose tolerance test by oral administration using db / db mice, when the blood glucose level of db / db mice is suppressed by about 50%, it becomes almost the same as that of normal mice. It is preferable that it is 50% or more.

  Measurement of the blood glucose lowering rate by an oral administration glucose tolerance test using db / db mice can be performed by a known method, and preferred methods are shown below. That is, first, a female mouse (C57BLKS / J-m + / + Lepr <db> (db / db)) 11 to 17 weeks old is fasted for 18 to 24 hours. At this time, 5 to 6 animals are used as a group for the test. As a control, blood is collected from the tail to measure the blood glucose level before treatment. After blood collection, the biguanide derivative is dissolved in phosphate buffered saline at an appropriate concentration and administered subcutaneously at a dose of 5 ml / kg. In addition, a mouse to which only the solvent is administered is prepared as a control. Furthermore, 30 minutes after administration of the compound or solvent, glucose is orally administered at a dose of 3 g / 6 ml / kg, and an oral glucose tolerance test is performed. Blood samples are collected from the tail 30 minutes, 1 hour and 2 hours after glucose administration to measure blood glucose levels. The blood glucose level is measured using a new blood sugar test (Roche Diagnostics Co., Ltd.) or glucose CII-Test Wako (Wako Pure Chemical Industries, Ltd.).

  And the blood glucose lowering rate is calculated | required from the following formula | equation.

Hypoglycemic rate (%) = [(AUC of blood glucose increase value of solvent administration group−AUC of blood glucose increase value of compound administration group) / AUC of blood glucose increase value of solvent administration group] × 100
Here, the AUC of the blood glucose elevation value is a graph in which the blood glucose level change after glucose administration is plotted against time, and the area of the increased portion up to 2 hours after glucose administration with the blood glucose level before glucose administration as the baseline. To express. Specifically, when A = blood glucose level before glucose administration, B = blood glucose level 30 minutes after glucose administration, C = blood glucose level 1 hour after glucose administration, D = blood glucose level 2 hours after glucose administration, The AUC for elevated blood glucose is given by the following formula:
AUC of blood glucose increase value = 0.5 × ((A + B) / 2−A) + 0.5 × ((B + C) / 2−A) + 1 × ((C + D) / 2−A)
Can be obtained from

  In addition, the blood glucose lowering rate and the blood lactic acid level in the oral glucose tolerance test can be measured by a known method, and the former measurement can be performed by the above-described method. Moreover, the latter measurement can be suitably performed by the following method. That is, first, a female mouse (C57BLKS / J-m + / + Lepr <db> (db / db)) 11 to 17 weeks old is fasted for 18 to 24 hours. At this time, 5 to 6 animals are used as a group for the test. As a control, blood is collected from the tail to measure the blood lactate level before treatment. After blood collection, the biguanide derivative is dissolved in phosphate buffered saline at an appropriate concentration and administered subcutaneously at a dose of 5 ml / kg. In addition, a mouse to which only the solvent is administered is prepared as a control. Furthermore, 30 minutes after administration of the compound or solvent, glucose is orally administered at a dose of 3 g / 6 ml / kg, and an oral glucose tolerance test is performed. Blood samples are collected from the tail to measure blood lactic acid levels 30 minutes, 1 hour and 2 hours after glucose administration. The blood lactic acid level can be measured using “Asuka Sigma” (manufactured by Sigma Diagnostics).

And the blood lactate level increase rate is expressed by the following formula:
Rate of increase in blood lactate level (%) = [(AUC of blood lactate level of compound administration group−AUC of blood lactate level of solvent administration group) / AUC of blood lactate level of solvent administration group] × 100
It is requested from.

Here, AUC of blood lactic acid level represents an area up to 2 hours after glucose administration in a graph in which changes in blood lactic acid level after glucose administration are plotted against time. Specifically, E = blood lactate value before glucose administration, F = blood lactate value 30 minutes after glucose administration, G = blood lactate value 1 hour after glucose administration, H = 2 hours after glucose administration When the blood lactate level is used, the AUC of the blood lactate level is expressed by the following formula:
AUC of blood lactate level = 0.5 × (E + F) /2+0.5× (F + G) / 2 + 1 × (G + H) / 2
Can be obtained from

(Modified design)
In this specification, the variant molecule is designed by analyzing the amino acid sequence and the three-dimensional structure of a pre-mutation protein or polypeptide molecule (for example, a wild-type molecule (eg, PPAR)). Appropriate to predict properties (eg, catalytic activity, interaction with other molecules, etc.) and provide desired property modifications (eg, improved catalytic activity, improved protein stability, etc.) This is done by calculating amino acid mutations. The design method is preferably performed using a computer. Examples of computer programs used in such a design method include the following as mentioned in this specification: As a program for analyzing the structure, DENZO (Mac) is a program for processing X-ray diffraction data. Science); PHASES (Univ. Of Pennsylvania, PA, USA) as a processing program for determining the phase; Program DM (CCP4 package, SERC); 3D graphics as a program for improving the initial phase Program O (Uppsala University, Uppsala, Sweden) as a program for the above; XPLOR (Yale University, CT, USA) as a three-dimensional structure refinement program; and a mutagenesis model As a program for the ring, Swiss-PDBViewer.

  In the present invention, based on the disclosure of the present invention, it is also intended to provide a drug (for example, an inhibitor, an activator, etc.) by computer modeling.

  In the present invention, a computer program for performing computer modeling can also be used in the process of selecting fragments or chemicals. Examples of such programs include the following.

  1. GRID (P. J. Goodford, “A Computational Procedure for Determining Favorable Binding Sites on Biologically Implanted Macromolecules., 198-49. GRID is available from Oxford University, Oxford, UK.

  2. MCSS (A. Miraranker et al., “Functional Map of Binding Bindings: A Multiple Copy Simulaneous Method”) Proteins: Structure, Function 29, and 29, 29. MCSS is available from Molecular Simulations, San Diego, CA.

  3. AUTODOCK (DS Goodsell et al., “Automated Docking of Proteins to Proteins by Simulated Annealing”, Proteins: Structure, Function, and Genetics, p. 19, 19202). AUTODOCK is available from Scripts Research Institute, La Jolla, CA.

  4). DOCK (ID Kuntz et al., “A Geometric Approach to Macromolecule-Ligand Interactions”, J. Mol. Biol., 161, 269-288 (1982)). DOCK is available from the University of California, San Francisco, CA.

  Once the appropriate candidate compounds (compound species) are selected, they can be assembled into a single compound or complex with PPAR. Prior to construction, a visual inspection of each fragment's relevance can be performed on a three-dimensional image displayed on the computer screen in relation to the structural coordinates of the PPAR or its modulating agent. Following this, Quanta or Sybyl [Tripos Associates, St. Manual model building using software such as Louis, MO] is performed.

  Useful programs that can be used in linking individual chemicals include:

  1. CAVEAT (P.A.Bartlett et al.,.. "CAVEAT: A Program to Facilitate the Structure-Derived Design of Biologically Active Molecules" (Molecular Recognition in Chemical and Biological Problems, Special Pub, Royal Chem.Soc, 78,182-196 (1989)); G, Lauri, and PA Bartlett, “CAVEEAT: a Program to Facility the Design of Organic Molecules”, J. Compute. Aided Mol. Des., 8, 51-66. 1994)). CAVEAT is available from the University of California, Berkeley, CA.

  2. A 3D database system such as ISIS (MDL Information Systems, San Leandro, CA). This field is C. Martin, “3D Database Searching in Drug Design”, J. Am. Med. Chem. 35, 2145-2154 (1992).

  3. HOOK (M.B. Eisen et al., “HOOK: A Program for Finding Novel Molecular Architecture, Saturity the Chemical. Sequential Refining of Bioc. (1994)). HOOK is available from Molecular Simulations, San Diego, CA.

  Instead of proceeding to build one chemical at a time as a PPAR inhibitor, etc. in a stepwise manner as described above, inhibitory or other PPAR binding compounds use or require an empty binding site. It can be designed globally or de novo, such as by including several known inhibitor moieties accordingly. Examples of many new ligand design methods include, but are not limited to, the following.

  1. LUDI (H.-J. Bohm, "The Computer Program LUDI: A New Method for the De no Design of Enzyme Inhibitors," J. Comp. Aid. Molec. 61-92, 61-92). LUDI is available from Molecular Simulations Incorporated, San Diego, CA.

  2. LEGEND (Y. Nishibata et al., Tetrahedron, 47, 8985 (1991)). LEGEND is available from Molecular Simulations Incorporated, San Diego, CA.

  3. LeapFrog (available from Tripos Associates, St. Louis, MO).

  4). SPROUT (V. Gillet et al., “SPROUT: A Program for Structure Generation”, J. Comput. Aided Mol. Design, 7, 127-153 (1993)). SPROUT is available from University of Lees, UK.

  Other molecular modeling techniques can also be used in accordance with the present invention [see, eg, N. C. Cohen et al., “Molecular Modeling Software and Methods for Medicinal Chemistry”, J. Am. Med. Chem. 33, 883-894 (1990); A. Navia and M.M. A. See also Murcko, “The Use of Structural Information in Drug Design,” Current Opinions in Structural Biology, 2, 202-210 (1992); M.M. Balbes et al., "A Perspective of Modern Methods in Computer-Aided Drug Design," Reviews in Computational Chemistry, vol. 5, pp.3, K. B. Lipkwitz and D. B. 1994)); C. Guida, “Software For Structure-Based Drug Design”, Curr. Opin. Struct. Biology. 4, 777-781 (1994)].

  Once a compound is designed or selected by the methods described above, the efficiency with which the substance can bind to PPAR can be tested and optimized by computational evaluation. For example, an effective PPAR inhibitor should preferably exhibit a relatively small energy difference between its bound and free states (ie, a small deformation energy of binding). A PPAR inhibitor can interact with the binding pocket in two or more conformations that are similar in total binding energy. In these cases, the deformation energy of binding is considered to be the difference between the energy of the free material and the average energy of these conformations observed when the inhibitor binds to the protein.

  The substance designed or selected to bind to the PPAR can be further optimized by calculation so that in its bound state, there is preferably no electrostatic repulsive interaction with the target enzyme and surrounding water molecules. Such non-complementary electrostatic interactions include charge-charge repulsive interactions, dipole-dipole repulsive interactions and charge-dipole repulsive interactions.

Specific computer software is available in the field of assessing compound deformation energy and electrostatic interactions. Examples of programs designed for such use include: Gaussian 94, revision C (MJ Frisch, Gaussian, Inc., Pittsburgh, PA 1995); AMBER, version 4.1 ( P. A. Kollman, University of California at San Francisco, 1995); QUANTA / CHARMM (Molecular Simulations, Inc., San Diego, CA 1995); ); DelPhi (Molecular Simulations, Inc., S) n Diego, CA 1995); and AMSOL (Quantum Chemistry Program Exchange, Indiana University). These programs may be executed using, for example, a Silicon Graphics workstation (eg, Indigo ² with “IMPACT” graphics). Other hardware systems and software packages are also known to those skilled in the art.

  Another approach possible with the present invention is the computational screening of small molecule databases for chemicals or compounds that can bind in whole or in part to PPARs. In this screening, the quality of such a substance's fit to the binding site can be determined by either geometric complementarity or estimated interaction energy [E. C. Meng et al. Comp. Chem. 16, 505-524 (1992)].

  In the present specification, “search” refers to finding another nucleobase sequence having a specific function and / or property using a nucleobase sequence electronically or biologically or by other methods. Say. As an electronic search, BLAST (Altschul et al., J. Mol. Biol. 215: 403-410 (1990)), FASTA (Pearson & Lipman, Proc. Natl. Acad. Sci., USA 85: 2444-). 2448 (1988)), the Smith and Waterman method (Smith and Waterman, J. Mol. Biol. 147: 195-197 (1981)), and the Needleman and Wunsch method (Needleman and Wunsch, J. Mol. Biol. 48:44:44). -453 (1970)) and the like. Biological searches include stringent hybridization, macroarrays with genomic DNA affixed to nylon membranes, microarrays affixed to glass plates (microarray assays), PCR, and in situ hybridization. It is not limited to. In the present specification, it is intended that the PPAR used in the present invention should include a corresponding gene identified by such an electronic search or biological search.

  As used herein, the percentage of “identity”, “homology” and “similarity” of sequences (such as amino acids or nucleic acids) is determined by comparing two sequences that are optimally aligned in a comparison window. . Here, the reference sequence for optimal alignment of the two sequences (a gap may occur if the other sequences contain additions, in the portion of the polynucleotide or polypeptide sequence within the comparison window, Reference sequences herein shall include no additions or deletions (ie, gaps) as compared to the reference). Find the number of match positions by determining the number of positions where the same nucleobase or amino acid residue is found in both sequences, and divide the number of match positions by the total number of positions in the comparison window. Is multiplied by 100 to calculate the percent identity. When used in a search, homology is assessed using an appropriate one from a variety of sequence comparison algorithms and programs well known in the art. Such algorithms and programs include TBLASTN, BLASTP, FASTA, TFASTA and CLUSTALW (Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85 (8): 2444-2448, Altschul et al., 1990 ,. J. Mol.Biol.215 (3): 403-410, Thompson et al., 1994, Nucleic Acids Res.22 (2): 4673-4680, Higgins et al., 1996, Methods Enzymol.266: 383-402. Altschul et al., 1990, J. Mol.Biol.215 (3): 403-410, Altschul et al. 1993, Nature Genetics 3: 266-272), but is not limited thereto. In particularly preferred embodiments, the Basic Local Alignment Tool (BLAST) (eg, Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87: 2267-2268, Altschul et al., 1990, well known in the prior art. J. Mol. Biol. 215: 403-410, Altschul et al., 1993, Nature Genetics 3: 266-272, Altschul et al., 1997, Nuc. Acids Res.25: 3389-3402). Used to assess protein and nucleic acid sequence homology. In particular, a comparison or search can be achieved by performing the following operations using five dedicated BLAST programs.

(1) Compare amino acid query sequence with protein sequence database in BLASTP and BLAST3;
(2) Compare nucleotide query sequence with nucleotide sequence database at BLASTN;
(3) Comparison of a conceptual translation product obtained by converting a nucleotide query sequence (both strands) with BLASTX in six reading frames with a protein sequence database;
(4) Compare protein query sequence with TBLASTN to nucleotide sequence database converted in all six reading frames (both strands);
(5) Comparison of nucleotide query sequence converted by TBLASTX in 6 reading frames with nucleotide sequence database converted in 6 reading frames.

  The BLAST program identifies similar segments called “high-score segment pairs” between an amino acid query sequence or nucleic acid query sequence, and preferably a test sequence obtained from a protein sequence database or nucleic acid sequence database. Thus, a homologous sequence is identified. High score segment pairs are preferably identified (ie, aligned) by a scoring matrix well known in the art. Preferably, a BLOSUM62 matrix (Gonnet et al., 1992, Science 256: 1443-1445, Henikoff and Henikoff, 1993, Proteins 17: 49-61) is used as the scoring matrix. Although not as preferred as this matrix, a PAM or PAM250 matrix can also be used (see, for example, Schwartz and Dayhoff, eds., 1978, Matrixes for Detection Distance Relationships: Atlas of Protein Sequence and Stance and Stench. thing). The BLAST program evaluates the statistical significance of all identified high score segment pairs and preferably selects segments that meet a user-defined threshold level of significance, such as a user-specific homology rate. It is preferred to evaluate the statistical significance of high score segment pairs using Karlin's formula for statistical significance (see Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87: 2267-2268). thing).

  As used herein, a nucleic acid molecule may have a portion of its nucleic acid sequence deleted or otherwise as long as the expressed polypeptide has substantially the same activity as the native polypeptide. It may be substituted with a base, or another nucleic acid sequence may be partially inserted. Alternatively, another nucleic acid may be bound to the 5 'end and / or the 3' end. Further, it may be a nucleic acid molecule that hybridizes under stringent conditions and has substantially the same function (promoter activity in the present invention).

  As used herein, “compound” means any identifiable chemical or molecule, including small molecules, peptides, proteins, sugars, nucleotides, or nucleic acids, including: Without limitation, such compounds can be natural or synthetic.

  In the present specification, the “small organic molecule” means an organic molecule having a relatively small molecular weight. Usually, a low molecular weight organic material has a molecular weight of about 1000 or less, but may have a higher molecular weight. The organic small molecule can be synthesized usually by using a method known in the art or a combination thereof. Such small organic molecules may be produced by living organisms. Examples of small organic molecules include hormones, ligands, signal transmitters, small organic molecules, molecules synthesized by combinatorial chemistry, and small molecules that can be used as pharmaceuticals (for example, small molecule ligands). It is not limited.

  The cells used in the present invention include any organism as long as it has an adipocyte (for example, mammals (for example, single pores, marsupials, rodents, wings, wings, meats, Cells derived from carnivores, long noses, odd-hoofed animals, even-hoofed animals, rodents, scales, sea cattle, cetaceans, primates, rodents, rabbits, etc.)) . In one embodiment, cells derived from primates (eg, chimpanzees, Japanese monkeys, humans), particularly cells derived from humans, are used, but are not limited thereto. Such cells can also be adherent cells including adipocytes, suspension cells, tissue-forming cells, mixtures thereof, and the like.

  As used herein, “tissue” refers to a population of cells having substantially the same function and / or morphology in a multicellular organism. A “tissue” usually has the same origin, but even cell populations with different origins can be referred to as a tissue if they have the same function and / or morphology. Usually, tissue constitutes part of an organ. Animal tissues are classified into epithelial tissues, connective tissues, muscle tissues, nerve tissues, etc. based on morphological, functional or developmental basis. Particularly in the present invention, adipose tissue is used as the object.

  In the present invention, when an organ is a target, such an organ may be any organ, and the tissue or cell targeted by the present invention may be derived from any organ or organ of an organism. In the present specification, the term “organ” or “organ” is used interchangeably, and a function of an organism individual is localized and operated in a specific part of the individual, and that part has morphological independence. Is a structure. In general, in a multicellular organism (eg, animal, plant), an organ is composed of several tissues having a specific spatial arrangement, and the tissue is composed of many cells. Such organs or organs include organs or organs associated with adipose tissue. In one embodiment, examples of the organ targeted by the present invention include, but are not limited to, internal organs (eg, abdomen, liver, etc.) having adipose tissue.

  As used herein, “organism” refers to a form of an organism that can exist as a single individual that can exist as a living organism.

  As used herein, “adipocyte” refers to a cell that is a major component of adipose tissue. The characteristics of this cell include interspersed tissues, or a large amount of fat that forms adipose tissue as a group along the running of capillaries as one of the loose connective tissues. Intracellular fat first appears as several microdroplets, gradually grows, fuses together, and finally occupies most of the cell body. Intracellular fat is easily detected by Sudan III or osmium tetroxide. Adipocytes include white fat cells and brown fat cells. Methods for identifying adipocytes are known in the art, and examples include, but are not limited to, detection of fat, expression of adipocyte differentiation markers, expression of adipocyte-specific cytokines, and the like.

  In this specification, “adipose tissue” is a type of connective tissue, which is characteristic in that it stores lipids, and also has various other important functions. Adipose tissue accumulates fat. Adipocytes in adipose tissue are surrounded by lattice fibers, and capillaries are densely distributed between the cells. A fat body is referred to when an almost constant mass or tufted adipose tissue is formed independently of other tissues. Adipose tissue develops in, for example, the abdomen, skeletal muscles, buttocks, chest, and internal organs.

  In the present invention, adipocytes used when a gene is introduced so as to be specifically expressed or suppressed in fat are derived from various organisms (for example, human, mouse, rat, rabbit, guinea pig, pig, cow, etc.). It may be an isolated cultured cell or a cell existing in a living body.

  As used herein, “fat” refers to a glycerin ester of a fatty acid that is solid at room temperature, and may be used interchangeably with adipose tissue or adipocyte herein.

  Means for “specific delivery” to a certain tissue, cell, etc. can be realized using DDS technology known in the art. Examples of such a specific delivery method to fat include, for example, a method in which an antibody specific for a surface marker specifically expressed in fat is bound to the inhibitor of the present invention, and a fat cell-specific promoter is used. Although the expression control etc. which were used can be mentioned, it is not limited to them. Such a technique is described in, for example, New Drug Delivery System, Supervision: Tsuneji Nagai CMC, 2000, and the like.

  In the present specification, the “effective amount for diagnosis, prevention, treatment or prognosis” refers to an amount which is recognized as being medically effective in diagnosis, prevention, treatment (or treatment) or prognosis, respectively. Such amounts can be determined by one skilled in the art using techniques well known in the art, taking into account various parameters.

  When the peptide of the present invention is used as a medicine, such a composition may further contain a pharmaceutically acceptable carrier and the like. Examples of the pharmaceutically acceptable carrier contained in the medicament of the present invention include any substance known in the art.

  The carriers used herein are preferably pharmaceutically acceptable, and such carriers include antioxidants, preservatives, colorants, flavors, and diluents, emulsifiers, suspensions. Examples include, but are not limited to, agents, solvents, fillers, bulking agents, buffering agents, delivery vehicles, diluents, excipients and / or pharmaceutical adjuvants. Typically, the medicament of the present invention is administered in the form of a composition comprising the peptide of the present invention, or a variant or derivative thereof, together with one or more physiologically acceptable carriers, excipients or diluents. Is done. For example, a suitable vehicle can be water for injection, physiological solution, or artificial cerebrospinal fluid, which can be supplemented with other materials common to compositions for parenteral delivery. .

  Acceptable carriers, excipients or stabilizers used herein are non-toxic to the recipient and are preferably inert at the dosages and concentrations used, for example Phosphate, citrate, or other organic acids; ascorbic acid, α-tocopherol; low molecular weight polypeptide; protein (eg, serum albumin, gelatin or immunoglobulin); hydrophilic polymer (eg, polyvinylpyrrolidone); amino acid (Eg, glycine, glutamine, asparagine, arginine or lysine); monosaccharides, disaccharides and other carbohydrates (including glucose, mannose, or dextrin); chelating agents (eg, EDTA); sugar alcohols (eg, mannitol or sorbitol) Salt formation vs. Io (E.g., sodium); and / or non-ionic surface-active agents (e.g., Tween, Pluronic (pluronic) or polyethylene glycol (PEG)) but the like are not limited thereto.

  Further exemplary suitable carriers include neutral buffered saline or saline mixed with serum albumin. Preferably, the product is formulated as a lyophilizer using a suitable excipient (eg, sucrose). Other standard carriers, diluents and excipients may be included as desired. Other exemplary compositions include pH 7.0-8.5 Tris buffer or pH 4.0-5.5 acetate buffer, which may further include sorbitol or a suitable substitute thereof. .

  The general preparation method of the pharmaceutical composition of this invention is shown below. In addition, veterinary drug compositions, quasi drugs, marine drug compositions, food compositions, cosmetic compositions and the like can also be produced by known preparation methods.

  The polypeptide, polynucleotide and the like of the present invention are mixed with a pharmaceutically acceptable carrier, and are solid preparations such as tablets, capsules, granules, powders, powders, suppositories, or syrups, injections, suspensions. It can be administered orally or parenterally as a liquid preparation such as an agent, solution or spray. As described above, the pharmaceutically acceptable carrier includes an excipient, a lubricant, a binder, a disintegrant, a disintegration inhibitor, an absorption enhancer, an adsorbent, a humectant, a solubilizing agent in a solid preparation, Stabilizers, solvents in liquid preparations, solubilizers, suspending agents, tonicity agents, buffers, soothing agents and the like can be mentioned. In addition, formulation additives such as preservatives, antioxidants, colorants, sweeteners and the like can be used as necessary. Moreover, it is also possible to mix | blend substances other than the polynucleotide of this invention, polypeptide, etc. with the composition of this invention. Parenteral routes of administration include, but are not limited to, intravenous injection, intramuscular injection, nasal, rectal, vaginal and transdermal.

  Examples of the excipient in the solid preparation include glucose, lactose, sucrose, D-mannitol, crystalline cellulose, starch, calcium carbonate, light anhydrous silicic acid, sodium chloride, kaolin and urea.

  Examples of the lubricant in the solid preparation include, but are not limited to, magnesium stearate, calcium stearate, boric acid powder, colloidal silicic acid, talc, and polyethylene glycol.

  Examples of the binder in the solid preparation include water, ethanol, propanol, sucrose, D-mannitol, crystalline cellulose, dextrin, methylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, carboxymethylcellulose, starch solution, gelatin solution, polyvinylpyrrolidone, calcium phosphate , Potassium phosphate, shellac and the like.

  Examples of disintegrants in solid preparations include starch, carboxymethyl cellulose, carboxymethyl cellulose calcium, agar powder, laminaran powder, croscarmellose sodium, sodium carboxymethyl starch, sodium alginate, sodium hydrogen carbonate, calcium carbonate, polyoxyethylene sorbitan fatty acid Examples include, but are not limited to, esters, sodium lauryl sulfate, starch, stearic acid monoglyceride, lactose, and calcium calcium glycolate.

  Preferable examples of the disintegration inhibitor in the solid preparation include, but are not limited to, hydrogenated oil, sucrose, stearin, cocoa butter, and hardened oil.

  Examples of the absorption enhancer in the solid preparation include, but are not limited to, quaternary ammonium bases and sodium lauryl sulfate.

  Examples of the adsorbent in the solid preparation include, but are not limited to, starch, lactose, kaolin, bentonite and colloidal silicic acid.

  Examples of the humectant in the solid preparation include, but are not limited to, glycerin and starch.

  Examples of the solubilizer in the solid preparation include, but are not limited to, arginine, glutamic acid, aspartic acid and the like.

  Examples of the stabilizer in the solid preparation include, but are not limited to, human serum albumin and lactose.

  When preparing tablets, pills and the like as solid preparations, they may be coated with a film of a gastric or enteric substance (sucrose, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose phthalate, etc.) as necessary. Tablets include tablets with ordinary coatings as necessary, such as sugar-coated tablets, gelatin-encapsulated tablets, enteric-coated tablets, film-coated tablets or double tablets, and multilayer tablets. Capsules include hard capsules and soft capsules. When forming into the form of a suppository, for example, higher alcohols, higher alcohol esters, semi-synthetic glycerides and the like can be added in addition to the additives listed above, but are not limited thereto.

  Preferable examples of the solvent in the liquid preparation include water for injection, alcohol, propylene glycol, macrogol, sesame oil and corn oil.

  Preferable examples of the solubilizer in the liquid preparation include polyethylene glycol, propylene glycol, D-mannitol, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate and the like. It is not limited to.

  Preferred examples of the suspending agent in the liquid preparation include surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, glyceryl monostearate, such as polyvinyl Examples include, but are not limited to, hydrophilic polymers such as alcohol, polyvinylpyrrolidone, sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, hydroxyethylcellulose, and hydroxypropylcellulose.

  Preferred examples of the isotonic agent in the liquid preparation include, but are not limited to, sodium chloride, glycerin, D-mannitol and the like.

  Preferable examples of the buffer in the liquid preparation include, but are not limited to, buffers such as phosphate, acetate, carbonate and citrate.

  Preferable examples of the soothing agent in the liquid preparation include, but are not limited to, benzyl alcohol, benzalkonium chloride, procaine hydrochloride and the like.

  Preferred examples of the preservative in the liquid preparation include, but are not limited to, paraoxybenzoates, chlorobutanol, benzyl alcohol, 2-phenylethyl alcohol, dehydroacetic acid, sorbic acid and the like.

  Preferred examples of the antioxidant in the liquid preparation include, but are not limited to, sulfite, ascorbic acid, α-tocopherol and cysteine.

  When preparing as an injection, it is preferable that the solution and suspension are sterilized and isotonic with blood. Usually, these are sterilized by filtration using a bacteria retention filter or the like, blending with a bactericidal agent, or irradiation. Furthermore, after these treatments, solidify by freeze-drying or other methods, and add sterile water or sterile diluent for injection (lidocaine hydrochloride aqueous solution, physiological saline, aqueous glucose solution, ethanol, or a mixed solution thereof) just before use. May be.

  Furthermore, if necessary, the pharmaceutical composition may contain coloring agents, preservatives, flavors, flavoring agents, sweeteners, and the like, as well as other agents.

  The medicament of the present invention may be a physiologically acceptable carrier, excipient or stabilizer as necessary (Japanese Pharmacopoeia 14th edition, its supplement or its latest edition, Remington's Pharmaceutical Sciences, 18th Edition, A R. Gennaro, ed., Mack Publishing Company, 1990, etc.) and a sugar chain composition having a desired degree of purity, and prepared and stored in the form of a lyophilized cake or aqueous solution. Can be done.

  The amount of the composition of the present invention to be administered depends on the purpose of use, target disease (type, severity, etc.), patient age, weight, sex, medical history, cell morphology or type, etc. Can be easily determined. The frequency with which the treatment method of the present invention is applied to a subject (or patient) also depends on the purpose of use, target disease (type, severity, etc.), patient age, weight, sex, medical history, treatment course, etc. In view of this, it can be easily determined by those skilled in the art. Examples of the frequency include administration every day to once every several months (for example, once a week to once a month). It is preferable to administer once a week to once a month while observing the course.

  As used herein, “patient” refers to an organism to which the treatment of the present invention is applied, and is also referred to as “subject” or “subject”. The patient may preferably be a human.

  The present invention provides methods of treatment, inhibition and prevention by administration of an effective amount of a composition of the present invention to a patient. In preferred aspects, the compositions of the present invention can be substantially purified (eg, substantially free of substances that limit their effects or cause undesirable side effects).

  In another embodiment, the compound or composition can be delivered encapsulated in vesicles, particularly liposomes (Langer, Science 249: 1527-1533 (1990); Treat et al., Liposomes in the Therapy of Infectious). Disesease and Cancer, Lopez-Berstein and Fidler (eds.), Liss, New York, pages 353-365 (1989); see Lopez-Berstein, pages 317-327; see broadly the same book).

  In yet another embodiment, the compound or composition can be delivered in a controlled sustained release system.

  As used herein, “administering” means giving a pharmaceutical composition of the present invention or the like or a pharmaceutical composition containing the same to a host intended for treatment alone or in combination with other therapeutic agents. The combinations can be administered, for example, either as a mixture simultaneously, separately but simultaneously or in parallel; or sequentially. This includes presentation where the combined drugs are administered together as a therapeutic mixture, and the procedure where the combined drugs are administered separately but simultaneously (eg, through separate mucosa to the same individual) Including. “Combination” administration further includes the separate administration of one of the compounds or agents given first, followed by the second.

  Administration of the medicament in the present invention may be performed using any technique, but it is advantageous to use needleless injection. This is because administration can be performed without undue burden on the patient.

  Here, the needleless syringe in the present invention means that the drug component is injected subcutaneously, more preferably subcutaneously, into the skin by moving the piston by the gas pressure or the elasticity of the elastic member without using the injection needle, and spraying the drug solution onto the skin. Means a medical device to be administered.

Specifically, for example, SHIMAJET ^TM (manufactured by Shimadzu Corporation), Medi-Jector Vision ^TM (manufactured by Elite medical), PenJet ^TM (manufactured by PenJet), etc. are commercially available. Yes.

(Adipose tissue-specific gene transfer / deficiency)
AP2 promoter-operated Cre transgenic mice have been extensively used to introduce or delete genes specifically in adipose tissue (Imai, T. et al., Proc. Natl. Acad. Sci. USA. 98, 224-228 (2001), Bluher, M. et al., Developmental Cell 3, 25-38 (2002) and Barlow, C. et al., Nuc. Acid Res. 25, 2543-2545 (1997)). However, the aP2 promoter is activated in macrophages and adipocytes (Fu, Y. et al., Atherosclerosis 165, 259-269 (2002)). Therefore, in aP2-Cre mice, some amount of Cre transgene expression occurs in other tissues, including macrophages (eg, spleen, lung and lymph nodes) (Boord, JB, Fazio, S. et al.). And Linton, MF Curr. Opin. Lipidol. 13, 141-7 (2002)). Furthermore, recent studies have revealed that macrophages in stored fat play an important role in the development of obesity-related metabolic diseases (Boord, JB, Fazio, S. and Linton, MF. Curr. Opin. Lipidol. 13, 141-7 (2002)). Based on these backgrounds, Cre transgenic mice that express the Cre gene in an adipocyte-specific manner are eagerly desired.

  Adiponectin / ACRP30 is a fat-derived hormone that we have identified by screening for fat-specific genes in a human cDNA project (Maeda, K. et al., Biochem. Biophys. Res. Commun. 221, 286- 289 (1996)). Adiponectin was first discovered as adipose most abundant gene 1 (apM1), which is very highly expressed in adipocytes. Adiponectin mRNA is expressed exclusively in human, monkey and mouse differentiated adipocytes and adipose tissue (Maeda, N. et al., Nature Med. 8, 731-737 (2002)). In other types of cells including macrophages, adiponectin mRNA expression is not detected. Therefore, the present inventors considered that the adiponectin promoter is ideal for obtaining a transgenic mouse that is completely adipocyte-specific.

The present inventors produced Cre transgenic mice that acted on adiponectin promoters and crossed them with PPAR ^{fold / flox} mice. Adiponectin-Cre / PPAR ^{fold / flox} lacked PPAR expression in a tissue-specific manner and showed a stimulatory effect of Akt signaling in adipose tissue, resulting in increased insulin sensitivity. Interestingly, fat PPAR deficiency significantly induces mitochondrial biosynthesis in stored fat, increases energy expenditure, and these changes are significant in body weight and fat weight despite increased feeding Accompanied by a decrease. Our physiological experiments also suggest that PPAR not only negatively regulates insulin-mediated signaling but also suppresses heat production by down-regulating mitochondria-related genes in adipocytes did. Our data indicate that inhibition of PPAR in adipocytes is a therapeutic strategy for treating obesity and type 2 diabetes by enhancing energy expenditure and insulin sensitivity.

(Fat specific promoter)
As a result of examining the promoter region of adiponectin, the present inventors have heretofore expressed specifically in adipocytes and non-specific expression in other cells from a region from the promoter region start point to about 2 kb upstream. Has succeeded in obtaining an adipose tissue-specific promoter (adiponectin 2 kb promoter) that is almost free from adenosine. (Shimmura et al., Diabetes 52: 1655-1663 (2003)). Furthermore, as a result of cloning and analysis of a DNA sequence of 3600 bases from the promoter region starting point, the adiponectin promoter region has a very high homology between human and mouse. It has been discovered that it has activity over the 2 kb promoter and promoter activity with adipocyte specificity. The adiponectin 3.6 kb promoter of the present invention also surpasses the AP-2 promoter not only in its specificity but also in its activity. In the present invention, such a promoter that induces expression specifically in fat (preferably only in fat) has been isolated.

Therefore, the present invention
(A) a polynucleotide comprising the sequence shown in SEQ ID NO: 10, or a fragment or modification thereof containing at least a part of any of the sequences from position 1 to position 1691 of SEQ ID NO: 10 and having adipocyte-specific promoter activity body;
(B) a polynucleotide having 80% homology with (a) and having adipocyte-specific promoter activity; and (c) under stringent conditions with the polynucleotide of (a) or (b) A polynucleotide that hybridizes and has adipocyte-specific promoter activity;
A polynucleotide selected from the group consisting of can be used.

  In one embodiment, the polynucleotide used in the present invention consists of the sequence shown in SEQ ID NO: 10.

  The present invention provides a vector comprising the polynucleotide. In one embodiment, a gene to be expressed in adipocytes is operably linked to the vector of the present invention. Here, the vector of the present invention may further contain a poly A sequence on the 3 'side of the gene.

  The present invention further provides an adipocyte-specific Cre expression vector comprising the above-mentioned polynucleotide and a Cre recombinase gene operably linked to the polynucleotide. In one embodiment, the vector of the present invention is a virus-derived vector (eg, an adenovirus-derived vector or a retrovirus-derived vector). Preferably, the vector further comprises a poly A sequence.

  The present invention provides an adipocyte into which the vector is introduced; an adipose tissue containing the adipocyte; a non-human animal (eg, mouse, rat, rabbit, guinea pig, etc.) containing the adipocyte.

  In another aspect, the present invention is a method for producing adipocytes that specifically and highly express useful genes, the step of introducing a vector used in the present invention into adipocytes, and the expression of the genes A method comprising the steps of: In another aspect, the present invention provides a method for specifically producing a target gene in an adipocyte, the step of introducing a vector used in the present invention into an adipocyte, and the step of expressing the gene. Including a method. Here, in one embodiment, the expression of the gene is suppressed in cells other than adipocytes (for example, human cells, mouse cells, rat cells, rabbit cells, and guinea pig cells, preferably human cells).

  In another aspect, the present invention relates to a method for producing a transgenic non-human animal (eg, mouse, rat, rabbit, guinea pig, etc., preferably a mouse, which comprises introducing the above-described adipocyte-specific Cre expression vector. And a transgenic non-human animal produced by this method, the invention also requires high expression of genes in adipocytes, but fibroblasts, epidermal cells, stromal systems The present invention relates to the use of the above polynucleotides for treating diseases in which expression of the gene in cells or macrophages is not desired.

  As used herein, “adiponectin” refers to a protein identified as a secreted protein produced in adipocytes or a gene encoding the same. Adiponectin has Acrp30 (adipocyte complement-related protein-related protein of 30 kDa), gelatin-binding protein (also known as Gelatin-binding protein-1), Adipose most bundant gene-1 The gene names are known as APM1, ACRP30, and GBP28. The mRNA is induced 100 times or more in the process of adipocyte differentiation. Adiponectin is also an abundant serum protein that results in energy, homeostasis and obesity. To date, it is known that there is a function of increasing the effect of insulin and lowering the blood sugar level. Since adiponectin is structurally very similar to TNFα and becomes unregulated in various obese forms of animals such as humans and mice, it has been studied as an important molecule that affects energy and homeostasis. Yes.

  Adiponectin is an animal adipose tissue-specific protein newly isolated from human adipose tissue by Maeda et al. In 1996, and its amino acid sequence has also been clarified. (Maeda K, et al. Biochem. Biophys. Res. Commun. 221: 286 (1996)). A substance called ACRP30 (Scherer PE et al. J. Biol. Chem. 270: 26746-26749 (1995)) cloned from mouse 3T3-F442A cells by another research group was almost the same as adiponectin. It is considered a substance. This adiponectin is present not only in adipose tissue but also in blood, and is present in normal human blood at a high concentration of 5 to 10 μg / ml (Arita Y et al, Biochem. Biophys. Res. Commun. 257: 79-83 (1999)). In addition, this adiponectin is paradoxically decreased in blood concentration as obesity progresses.

  As for the action of this adiponectin, several research groups have so far suppressed the proliferation of vascular smooth muscle and cell migration, anti-arteriosclerosis, monocyte and macrophage activation, anti-inflammatory and hepatic stellate cell activity. Suppression of cell formation, suppression of extracellular matrix production, suppression of hepatic stellate cell proliferation, promotion of hepatocyte proliferation, etc. have been clarified, but the full effect is still unclear.

  Adiponectin is also known as a collagen-like factor specific to human adipose tissue. The gene is known to be a protein composed of 244 amino acids. The nucleic acid and amino acid sequences of human adiponectin protein are shown in SEQ ID NOs: 11 and 12. In examining the physiological effects of human adiponectin, the present inventors have already found that human adiponectin suppresses the growth of monocyte-type cells and B-cell type cells, and as a proliferation inhibitor of these blood cell-type cells. It was shown to be effective. As described above, a phenomenon in which leukocyte cells such as monocytes, macrophages, and neutrophils proliferate and accumulate during inflammation is observed. Monocytes whose growth is suppressed by adiponectin are cells that play an important role in such cellular biodefense reactions and become macrophages that carry phagocytosis when mature, while interleukin-1, tumor necrosis factor Produces inflammatory cytokines such as α. Monocytes play such an important role in the process of inflammatory reaction, and adiponectin can be used as an anti-inflammatory agent by suppressing monocyte proliferation and preventing accumulation.

  Adiponectin is a protein that is produced in adipose tissue of animals including humans and is also present in large amounts in blood. As for human adiponectin, one purified from the cDNA encoding this by a gene recombination method has been obtained (Arita, Y. et al. Biochem. Biophys. Res. Commun. 257, 79-83 ( 1999)). As described above, mouse-derived ACRP30 is also highly purified and can be obtained on the market.

As used herein, “biological activity of adiponectin” includes at least one function of adiponectin, such as vascular endothelial function improving action, vascular smooth muscle growth inhibitory action, macrophage foaming inhibitory action, inflammatory cytokine secretion Examples include, but are not limited to, an inhibitory action and a muscle cell uptake promoting action. Such biological activity can be measured by [ ³ H] thymidine incorporation into vascular smooth muscle cells, macrophage culture supernatant TNFα secretion, muscle cell [ ³ H] deoxyglucose uptake, and the like.

  The promoter of the present invention is also very useful for studying adipocyte function in vivo. For example, by using the promoter of the present invention in combination with the Cre-loxP system, it is possible to analyze the function of adipocytes more accurately. The Cre-loxP system is an expression control system using a Cre recombinase and a loxP sequence that is a recognition sequence thereof. Cre recombinase recognizes the loxP sequence and cuts out a gene sandwiched between the loxP sequences. For the Cre-loxP system, see, for example, Sternberg et al., Proc Natl Acad Sci USA 75: 5594-5598 (1978); Mack et al., Nucleic Acids Res 20: 4451-4455 (1992); Hoess et al., J Mol Biol 216: 873. -882 (1990); Dymecki et al., Gene targeting-a practical approach. 2nd Ed. Oxford University Press, Oxford, pp. 37-99 (2000); and Torres et al., Oxford University Press, Oxford (1997). As described above, the promoter of the present invention has a high activity in adipocytes and has substantially no non-specificity in cells other than adipocytes. Therefore, a Cre-loxP system targeting only adipocytes is constructed. can do. This makes it possible to perform in vivo functional analysis of adipocytes, which was difficult in vitro, and contribute to further progress in functional analysis of adipocytes. Accordingly, in one aspect, the present invention provides an adipose tissue-specific Cre expression vector. In one embodiment, the vector is a virus-derived vector. The virus-derived vector is, for example, a retrovirus or adenovirus-derived vector. The vectors of the present invention may also include a poly A sequence 3 'to the Cre gene.

  The present invention also provides a method for producing a transgenic animal using the promoter of the present invention, and a transgenic animal produced by this method.

  Various methods are known in the art for the production of transgenic animals. For example, in the case of producing a transgenic mouse, a general production technique is described in International Publication WO01 / 13150 (Ludwig Inst. Cancer Res.). US Pat. No. 4,873,191 (Wagner et al.) Teaches a mammal with exogenous DNA, obtained by microinjection of DNA into a mammalian zygote. Furthermore, research has been conducted on methods for efficiently creating mutants such as animals and plants by inducing translocation of genetic elements (transposons) into endogenous DNA, or by further translocating it to cause structural changes in the DNA. Has been. It has become possible to introduce and add specific genes to chromosomes using transposons.

  In addition to this, various methods for producing transgenic animals include, for example, M.P. Markkula et al., Rev. Reprod. 1, 97-106 (1996); T.A. Wall et al. Dairy Sci. 80, 2213-2224 (1997); C. Dalton et al., Adv. Exp. Med. Biol. , 411, 419-428 (1997); Lubon et al., Transfus. Med. Rev. , 10, 131-143 (1996), but is not limited thereto.

  In recent years, it has become possible to produce transgenic (including knockout and knockin) animals via homologous recombination of embryonic stem (ES) cells. In higher organisms, for example, efficient selection of recombinants is accomplished by positive selection using a neomycin resistance gene and negative selection using an HSV thymidine kinase gene or diphtheria toxin gene. Selection of homologous recombinants is also performed by PCR or Southern blotting. That is, a targeting vector in which a part of the target gene is replaced with a neomycin resistance gene for positive selection and the like, and a HSVTK gene for negative selection is connected to the end of the target gene is prepared and introduced into ES cells by electroporation. And in the presence of ganciclovir, the resulting colonies are isolated and homologous recombinants are further selected by PCR or Southern blot.

  In this way, by replacing or destroying an endogenous target gene and producing a transgenic (target gene recombination) mouse having a mutation whose function has been lost or altered, the mutation is applied only to the target gene. Since it is introduced, it is useful for analyzing its gene function.

  After selecting a desired homologous recombinant, the obtained recombinant ES cell is mixed with a normal embryo by a scutellum injection method or an assembly chimera method to produce a chimeric mouse of ES cells and a host embryo. In the blastocyst injection method, ES cells are injected into a blastocyst with a glass pipette. In the assembly chimera method, an ES cell mass and an 8-cell embryo from which the zona pellucida has been removed are adhered. A blastocyst into which an ES cell has been introduced is transplanted into the uterus of a surrogate mother who is pseudopregnant to obtain a chimeric mouse. Since ES cells have totipotency, they can be differentiated into all kinds of cells including germ cells in vivo. When a chimeric mouse having germ cells derived from ES cells is mated with a normal mouse, mice having heterologous ES cell chromosomes are obtained, and when these mice are mated, a transgenic mouse having homologous ES cell modified chromosomes is obtained. It is done. In order to obtain a transgenic mouse having a modified chromosome homozygously from the obtained chimeric mouse, a male chimeric mouse and a female wild type mouse are mated to produce F1 generation heterozygous mice. The heterozygous mice are crossed to select F2 generation homozygous mice. Whether or not a desired gene mutation has been introduced in each generation of F1 and F2 is determined using methods commonly used in the art such as Southern blotting, PCR, and decoding of base sequences, as in the case of recombinant ES cell assays. Can be analyzed.

  Furthermore, in recent years, for the purpose of selectively analyzing various gene functions, attention has been paid to a technique using the above-described Cre-loxP site-specific recombination in combination. In transgenic mice using Cre-loxP, a neomycin resistance gene is introduced at a position where the expression of the target gene is not inhibited, and a targeting vector having the loxP sequence inserted so as to sandwich an exon is introduced into an ES cell, and then a homologous recombinant Is isolated. A chimeric mouse is obtained from the isolated clone, and a genetically modified mouse is prepared. Next, when this mouse is mated with a tissue-specific expression of the site-specific recombination enzyme Cre derived from the P1 phage of E. coli, the gene is disrupted only in the tissue expressing Cre (here, Cre specifically recognizes the loxP sequence (34 bp) and causes recombination with the sequence between the two loxP sequences, which is destroyed). Cre can be expressed in adults by mating with a transgenic mouse having a Cre gene linked to a tissue-specific promoter, or using a viral vector having a Cre gene. As described above, since the promoter of the present invention highly expresses a gene in an adipocyte-specific manner, Cre can be expressed in an adipocyte-specific manner by using the promoter of the present invention. Therefore, it is possible to destroy or mutate the target gene only in adipocytes without affecting other tissues. In this way, it is considered that the importance of this greatly contributes to the functional analysis of adipocytes, which has been attracting increasing attention in recent years. Even in other organisms, a transgenic organism can be produced using a similar mechanism.

  As described above, the present invention provides a novel adipocyte-specific promoter derived from adiponectin. Including the polynucleotide shown in SEQ ID NO: 10 (human) or SEQ ID NO: 13 (mouse) used in the promoter of the present invention. The present invention also encompasses a polynucleotide that hybridizes with the polynucleotide shown in SEQ ID NO: 10 or SEQ ID NO: 13 under stringent conditions and has adipocyte-specific promoter activity. Stringent conditions are already explained in the definition section. In the present specification, adiponectin promoter sequences such as SEQ ID NO: 10 (human) and SEQ ID NO: 13 (mouse) are provided. A part of these sequences is used as a promoter, and a technique well known in the art. Sequences from other species that can be obtained using are also intended to be included for use in the present invention. Thus, the polynucleotide used in the present invention does not depend on the species from which it originated.

  The present invention can also utilize a fragment or variant of the polynucleotide shown in SEQ ID NO: 10, SEQ ID NO: 13, etc. having adipocyte-specific promoter activity. Examples of fragments or variants of the invention include, for example, those with truncated ends or those with one or more base additions or deletions in the polynucleotide sequence. Whether the fragment or variant of the present invention has an activity equivalent to (or higher than) the promoter sequence shown in SEQ ID NO: 10, SEQ ID NO: 13, etc. can be determined by various methods known to those skilled in the art. Examples of the method for measuring promoter activity include, but are not limited to, a method using a reporter gene. Reporter genes include, but are not limited to, luciferase genes. When the luciferase gene is used as a reporter, a vector containing the fragment or variant of the present invention and the luciferase gene is prepared, a cell is transfected with this vector, and the expressed luciferase is released by lysis of the cell. Promoter activity can be measured by adding a luciferase substrate to the sample and measuring the resulting luminescence with a luminometer. Whether the fragment or variant has adipocyte-specific activity may be determined by conducting the same experiment using adipocytes and other cells and comparing the promoter activity. Using these techniques, those skilled in the art can select the promoter fragment or promoter variant of the present invention having adipocyte-specific promoter activity without undue experimentation. Accordingly, it is understood that such fragments or variants are also included in the scope of the present invention.

  In the present specification, “adipocyte-specific promoter activity” means that when the activity is measured by any method as described above, only a vector that does not contain a promoter is used in cells other than adipocytes. It means that the activity is substantially the same as or lower than that of (ie, control), but has a detectably high activity in adipocytes.

  As demonstrated in the following examples, the promoter of the present invention not only specifically and highly expresses in adipocytes, but also has a surprising activity of suppressing gene expression in cells other than adipocytes. . Such an activity was not found in the conventionally used AP-2 promoter.

  Accordingly, in one aspect, the present invention relates to a method for specifically and highly expressing a target gene in adipocytes. This method includes a step of introducing the vector of the present invention into an adipocyte and a step of expressing the gene. The meaning of this specificity includes suppression of expression in cells other than adipocytes. The present invention also relates to a method for producing adipocytes that specifically and highly express useful genes. This method includes a step of introducing the vector of the present invention into an adipocyte and a step of expressing the gene.

  The present invention also provides a vector comprising the promoter of the present invention. In one embodiment, the vector of the present invention comprises a promoter of the present invention and a gene operably linked to the promoter. This gene is a gene that is desired to be specifically expressed in adipocytes. In a preferred embodiment, the gene that is desired to be specifically expressed in adipocytes is an adipocytokine gene (particularly an adiponectin gene). The vector of the present invention may also contain a poly A sequence 3 'to the gene of interest.

  The present invention further provides an adipocyte comprising the promoter of the present invention. As used herein, the term “adipocyte” refers to the major components that make up adipose tissue. Adipose tissue is a kind of connective tissue and is characteristic in that it stores lipids. However, as described in the background section of the invention, it is a tissue having various other important functions. The adipocytes into which the vector of the present invention is introduced may be cultured cells isolated from various organisms (for example, humans, mice, rats, rabbits, guinea pigs, pigs, cows, etc.) or in vivo. It may be an existing cell. In one embodiment, the promoter of the present invention is introduced into adipocytes in the form of a vector. Alternatively, such cells can be identified using screening techniques well known in the art, using a portion of the promoter of the present invention as a probe in a sample containing cells.

  For example, when animal cells are used, the culture medium for culturing the cells of the present invention includes RPMI1640 medium (The Journal of the American Medical Association, 199, 519 (1967)), Eagle's MEM medium (Science, 122). , 501 (1952)), DMEM medium (Virology, 8, 396 (1959)), 199 medium (Proceedings of the Society for the Biological Medicine, 73, 1 (1950)), or fetal bovine serum or the like was added to these media A medium or the like is used.

The culture is usually performed for 1 to 7 days under conditions such as pH 6 to 8, 25 to 40 ° C., and 5% CO ₂ . Moreover, you may add antibiotics, such as kanamycin, penicillin, streptomycin, to a culture medium as needed during culture | cultivation.

  The present invention relates to adipose tissue containing the promoter of the present invention. The promoter of the present invention is preferably operably linked to a specific gene (for example, adiponectin).

  The present invention also relates to a non-human animal comprising the promoter of the present invention. The promoter of the present invention is preferably operably linked to a specific gene (for example, adiponectin). Examples of non-human animals include, but are not limited to, mice, rats, rabbits, guinea pigs, cows, pigs, and the like.

  Since the promoter of the present invention has a very high specificity for adipocytes, it is very useful when a target gene is desired to be expressed specifically in adipocytes. For example, as noted in the background section of the invention, reduced adiponectin levels are associated with obesity-related metabolic disorders such as insulin resistant diabetes and atherosclerosis. Therefore, the promoter of the present invention is very useful for introducing these genes into adiponectin gene adipocytes for high expression in adipocytes in order to treat these disorders. Therefore, the present invention relates to a method for specifically expressing a target gene in adipocytes. The method of the present invention includes the step of introducing a vector containing the promoter of the present invention and the gene of interest into adipocytes. The invention further relates to a method for treating or preventing a disease or disorder resulting from down-regulation of gene expression in adipocytes. This method includes the step of introducing into a fat cell a vector comprising the promoter of the present invention and a down-regulated gene. For example, the gene is adiponectin and the disease to be treated or prevented is an obesity related disease (eg, insulin resistant diabetes and atherosclerosis) as described above. The invention also relates to the use of a promoter or vector of the invention in the manufacture of a medicament for treating a disease as described above. In particular, the above diseases are diseases in which high gene expression in adipocytes is desired, but expression of the gene in cells other than adipocytes is undesirable. Such diseases include, but are not limited to, cancer and the like. Such diseases can be treated by specifically expressing factors that suppress cancer in adipocytes. The transcription factor SREBP-1a, which is highly expressed in the liver, causes fatty liver due to significant lipid accumulation, but even when highly expressed in fat, excessive accumulation of lipid does not occur. (Shimomura et al. J Biol Chem. 274 (42): 30028-32, 1999., Shimomura et al. J. Biol. Chem. 273; 35299-306, 1998., Horton JD, Shimomura I, et al. J Biol Chem. 278: 36652-60).

(Description of Preferred Embodiment)
Hereinafter, preferred embodiments of the present invention will be described. The embodiments provided below are provided for a better understanding of the present invention, and it is understood that the scope of the present invention should not be limited to the following description. Therefore, it is obvious that those skilled in the art can make appropriate modifications within the scope of the present invention with reference to the description in the present specification.

(New PPAR regulator)
In one aspect, the present invention provides a PPAR modulator comprising a sulfonylurea agent. Here, the sulfonylurea agent is typically

Where R ₁ and R ₂ are each independently alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted Cycloalkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, carbocyclic group, substituted carbocyclic group, heterocyclic group, substituted heterocyclic group, halogen, hydroxy, substituted hydroxy, thiol, Substituted thiol, cyano, nitro, amino, substituted amino, carboxy, substituted carboxy, carbamoyl, acyl, substituted acyl, acylamino, thiocarboxy, amide, substituted carbonyl, substituted thiocarbonyl, Substituted sulfonyl and substituted sulfini Having a substituent selected from the group consisting of.

  In the present invention, typical sulfonylurea agents include, but are not limited to, glimepiride, glibenclamide, tolbutamide, chlorpropamide, acetohexamide and gliclazide.

  In preferred embodiments, the sulfonylurea agent may advantageously use glimepiride or glibenclamide. This is because these sulfonylurea agents have more effective PPAR modulating activity.

  The PPAR modulator of the present invention may be an activator or an inhibitor. Preferably, it can be an activator.

  In one embodiment, the PPAR modulator of the present invention may further have at least one activity selected from the group consisting of insulin secretory activity and insulin sensitivity activity of pancreatic β cells.

  In the present invention, the PPAR modulating activity can be determined by comparison with a thiazolidinedione agent (for example, pioglitazone, rosiglitazone, etc .; preferably pioglitazone). The PPAR modulators of the present invention may typically have an activity of at least 10%, more preferably at least 15% of the thiazolinedione agent.

  The receptor targeted by the PPAR modulating agent of the present invention may be of any subtype, but preferably it is advantageous to target PPARγ.

  Here, the PPAR targeted by the present invention is an amino acid sequence represented by SEQ ID NO: selected from the group consisting of SEQ ID NOs: 2, 4, 6 and 8, or a variant thereof (for example, one or several substitutions, additions) Or a deletion).

  In a preferred embodiment, the PPAR targeted by the present invention preferably contains at least a C-terminal activation function-2 (AF-2) domain. In one embodiment, the PPAR targeted by the present invention preferably comprises the amino acid sequence PLLQEIYKDLY (SEQ ID NO: 9). This is because it was confirmed that the binding activity disappeared when this sequence was deleted.

  In one embodiment, in observing the binding between a PPAR targeted by the present invention and a candidate compound, the interaction between the PPAR cofactor and the PPAR can be observed. This is because it became clear that the sulfonylurea agent affects the interaction between the PPAR cofactor and PPAR.

  In certain embodiments, the PPAR modulating agent of the present invention comprises diabetes (especially type 2); hyperglycinemia; hypoglycemic tolerance; insulin resistance; obesity; steatosis; dyslipidemia; Hypertriglyceridemia; hypercholesterolemia; low HDL level; high LDL level; atherosclerosis; vascular stenosis; irritable bowel syndrome; inflammatory bowel disease; Crohn's disease; It can be used for the treatment or prevention of diseases selected from the group consisting of abdominal obesity; neurodegenerative disease; retinopathy; psoriasis; metabolic syndrome;

(PPAR screening method)
In another aspect, the present invention relates to a compound having at least one activity selected from the group consisting of pancreatic β-cell insulin secretory activity and activity that promotes peripheral insulin sensitivity, and the ability to modulate PPAR A method of identifying is provided. The method comprises: A) providing a candidate compound; B) measuring at least one activity selected from the group consisting of pancreatic β-cell insulin secretory activity and peripheral insulin sensitivity activity for the candidate compound And C) subjecting the candidate compound to an assay for measuring the activity of PPAR; and D) identifying a substance determined to be active in each of the B and C steps as a lead compound. To do.

  In one embodiment, candidate compounds targeted by the present invention include a sulfonylurea agent. As the sulfonylurea agent, any of those described above in the present specification can be used.

  In one embodiment, the subject of the screening method of the present invention may be a compound having a PPAR activation action or inhibitory action, and preferably a compound having an activation action.

  In one embodiment, the compound targeted by the screening method of the present invention preferably has both the insulin secretory activity of pancreatic β cells and the activity of promoting insulin sensitivity. Such activity measurement can be performed by using an arbitrary method as described above in this specification.

  In one embodiment, in the screening method of the present invention, PPAR modulating activity is measured relative to thiazolidinedione. In this case, they may be compared competitively or different experimental results may be compared.

  In one embodiment, the PPAR targeted by the screening method of the present invention may be PPARγ, but is not limited thereto. Such a PPAR may comprise the amino acid sequence shown in SEQ ID NO: selected from the group consisting of SEQ ID NO: 2, 4, 6, and 8, or a partial sequence thereof.

  In one embodiment, in the present invention, PPAR activity may include the step of observing whether a candidate compound is provided and competes with a sulfonylurea agent or thiazolidinedione agent.

  In one embodiment, the assay for measuring PPAR activity can utilize a transcriptional activity assay. Such transcription activity assays are described herein and are exemplified in the examples.

  In one embodiment, the insulin secretory activity of pancreatic β cells can be measured by directly measuring insulin secreted from the cells (J Pharmacol Exp Ther. 2004 Sep; 310 (3): 1273-80.).

  In one embodiment, the activity that promotes peripheral insulin sensitivity can be measured by the glucose clamp method, the insulin tolerance test.

  In a preferred embodiment, the activity of the PPAR is measured in a system comprising a nucleic acid construct comprising a nucleic acid sequence encoding a reporter operably linked to a transcription factor recognition sequence and the selected PPAR. Determining if said reporter expression is enhanced, said candidate compound is determined to be an agonist of said PPAR, and if said reporter expression is decreased, said candidate compound is determined to be an antagonist of said PPAR; Measured by process. Such methods are known in the art and are exemplified herein. Reporters that can be used here include luciferase. As the system, for example, a cell-free system or a cell can be used.

  In one embodiment, the target PPAR can be a protein comprising a C-terminal activation function-2 (AF-2) domain, preferably the target is the amino acid sequence PLLQEIYKDLY (SEQ ID NO: 9). Can be included.

  In a preferred embodiment, in this PPAR assay, the interaction of the PPAR cofactor and PPAR is observed.

  Here, the cofactor of this PPAR is a factor selected from the group consisting of DRIP 205 (vitamin D receptor-interacting protein 205), N-CoR (nuclear receptor corepressor) and SMRT (silencing mediator for retinoid and thyroid hormone receptor). obtain.

  In another embodiment, the present invention has at least one activity selected from the group consisting of insulin secretory activity of pancreatic β cells and activity that promotes peripheral insulin sensitivity and has the ability to modulate PPAR A system for identifying compounds is provided. The system measures: at least one activity selected from the group consisting of: A) a candidate compound as needed; B) an insulin secretory activity of pancreatic beta cells and an activity that promotes peripheral insulin sensitivity. Means; C) means for subjecting the candidate compound to an assay for measuring the activity of PPAR; and D) means for identifying a substance determined to be active by each of means B and C as a lead compound. Provide a system. It can be understood that this system can take any form that can be taken in the screening method.

(Treatment and prevention methods)
In another aspect, the present invention provides a method for treating or preventing a disease associated with PPAR comprising A) administering a sulfonylurea agent to a subject. Here, PPAR-related diseases include, for example, diabetes (especially type 2); hyperglycinemia; low glucose tolerance; insulin resistance; obesity; steatosis; dyslipidemia; Hypertriglyceridemia; hypercholesterolemia; low HDL level; high LDL level; atherosclerosis; vascular stenosis; irritable bowel syndrome; inflammatory bowel disease; Crohn's disease; Can include but is not limited to obesity; neurodegenerative disease; retinopathy; psoriasis; metabolic syndrome; Treatment or prevention methods may use methods known in the art and are exemplified herein.

  In one embodiment, as the sulfonylurea agent, one selected from the group consisting of glimepiride, glibenclamide, tolbutamide, chlorpropamide, acetohexamide, and gliclazide can be used.

(use)
In another aspect, the present invention provides the use of a sulfonylurea agent in the manufacture of a medicament for treating or preventing a disease associated with PPAR. Diabetes (especially type 2); hyperglycinemia; hypoglucose tolerance; insulin resistance; obesity; steatosis; dyslipidemia; hyperlipidemia; hypertriglyceridemia; HDL level; high LDL level; atherosclerosis; vascular stenosis; irritable bowel syndrome; inflammatory bowel disease; Crohn's disease; intestinal ulcer; inflammatory disease; pancreatitis; Syndrome; can include ovarian hyperandrogenism. As a method for preparing a medicament, a method known in the art can be used, and exemplified in the present specification.

  In one embodiment, as the sulfonylurea agent, one selected from the group consisting of glimepiride, glibenclamide, tolbutamide, chlorpropamide, acetohexamide, and gliclazide can be used. It will be understood that other specific forms may take any specific form shown elsewhere in the specification.

  References such as scientific literature, patents and patent applications cited herein are hereby incorporated by reference in their entirety to the same extent as if each was specifically described.

  The present invention has been described with reference to the preferred embodiments for easy understanding. In the following, the present invention will be described based on examples, but the above description and the following examples are provided only for the purpose of illustration, not for the purpose of limiting the present invention. Accordingly, the scope of the present invention is not limited to the embodiments or examples specifically described in this specification, but is limited only by the scope of the claims.

  The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the following examples. The reagents used in the following examples were those commercially available from Sigma (St. Louis, USA), Wako Pure Chemical (Osaka, Japan), etc., with exceptions. Animals were handled in accordance with the standards stipulated by Osaka University School of Medicine. A method for producing an expression vector used in the present invention will be described with a specific example. It should be noted that it is easy for those skilled in the art to replace the components such as the starting plasmid and promoter used in such examples with equivalent ones.

  First, in this section, experimental methods and materials used in the examples are described.

(Experimental method)
(material)
[ ³ H] rosiglitazone (ART1231; specific activity was 50 Ci / mmol) was purchased from American Radiolabeled Chemical (St Louis, MO). Pioglitazone is a courtesy of Takeda Pharmaceutical Company Limited (Osaka). Glimepiride is a courtesy of Aventis Pharma (Tokyo). Glibenclamide, tolbutamide, chlorpropamide, and gliclazide were purchased from Sigma Aldrich Japan Co., Ltd. (Tokyo).

(Plasmid)
Expression plasmid (pCMX-GAL4) encoding GAL4 (SEQ ID NO: 14-15), expression plasmid encoding GAL4-mouse PPARα chimeric protein (SEQ ID NO: 16-17) (pCMX-GAL4-mPPARα), GAL4-mouse PPARδ chimera Expression plasmid (pCMX-GAL4-mPPARδ) encoding protein (SEQ ID NO: 18-19), expression plasmid (pCMX-GAL4-mPPARγ) encoding GAL4-mouse PPARγ chimeric protein (SEQ ID NO: 20-21), full-length mouse PPARγ (In SEQ ID NO: 1-2, the sequence after amino acid 31 corresponding to isoform 1 (MVDTEMPFWPTNFGISSVDLSVMEDHSHSFDIKPFTTVDFSSISAPHYEDIPFTRADPMVADYKYDLKLQEYQSAIKVEPASPPYYSEKTQLYNRPHEEPSNSLMAIECRVCGDKASGFHYGVHACEGCKDRFRNKLQRIY ShierueibuijiemuesueichienueiaiaruefujiaruemuPikyueiikeiikeieruerueiiaiesuesudiaidikyueruenuPiiesueidieruarueierueikeieichieruwaidiesuwaiaikeiesuefuPierutikeieikeieiarueiaierutijikeititidikeiesuPiefubuiaiwaidiemuenuesueruemuemujiidikeiaikeiefukeieichiaitiPierukyuikyuesukeiibuieiaiaruaiefukyujishikyuefuaruesubuiieibuikyuiaitiiwaieikeienuaiPijiefuaienuerudieruenudikyubuitieruerukeiwaijibuieichiiaiaiwaitiemuerueiesueruemuenukeidijibuieruaiesuijikyujiefuemutiaruiefuerukeienueruarukeiPiefujidiefuemuiPikeiefuiefueibuikeiefuenueieruierudidiesudierueiaiefuaieibuiaiaieruesujidiaruPijierueruenubuikeiPiaiidiaikyudienueruerukyueieruierukyuerukeieruenueichiPiiesuesukyueruefueikeibuierukyukeiemutidieruarukyuaibuitiieichibuikyueruLHVIKKTETDMSLHPLLQEIYKDLY) encoding expression plasmid (pCMX-mPPARγ), VP16 (SEQ ID NO: 22-23) encoding expression plasmid (pCMX-VP16), VP16- murine PPARγ chimeric protein (SEQ ID NO: 24-25) encoding expression plasmid (PCMX-VP16-mPPARγ) and an expression plasmid (pCMX-β-gal) encoding β-galactosidase (SEQ ID NO: 26-27) were obtained from Dr. David Mangelsdorf (University of Texas Southwester). n Medical Center, Dallas, TX) (see, eg, Willy, PJ, and Mangelsdorf, DJ (1997) Genes Dev. 11, 289-298). A mouse ΔPPARγ mutant construct lacking 11 amino acids (PLLQEIYKDLY (SEQ ID NO: 9)) of the C-terminal activation function-2 (AF-2) domain (pCMX-ΔmPPARγ; ΔPPARγ variant is the PPARγ isoform 1 described above. The last 11 amino acids (PLLQEIYKDLY) have been deleted from the amino acid sequence (SEQ ID NO: 28-29), and the sequences to be deleted were deleted. Expression plasmid (pCMX-GAL4-DRIP205) encoding GAL4-vitamin D receptor binding protein (DRIP) 205 (SEQ ID NO: 30-31) and GAL4-nuclear receptor corepressor (N-CoR) (SEQ ID NO: 32-33) The encoding expression plasmid (pCMX-GAL4-N-CoR) has been described in the literature (Kaneko, E. et al (2003) J. Biol. Chem. 278, 36091-36098 for pCMX-GAL4-DRIP205, and Adachi, R., et al (2004) Mol. Endocrinol. 18, 43-52) for pCMX-GAL4-N-CoR. The nuclear receptor interaction domain of DRIP205 (amino acids 578-728) (SEQ ID NOs: 34-35) and the nuclear receptor interaction domain of N-CoR (amino acids 1990-2416) (SEQ ID NOs: 36-37) are the GAL4 DNA binding domain (sequence No. 38-39). Using the GAL4-responsive MH100 (UAS) × 4-tk-LUC reporter (SEQ ID NO: 40-41) and the PPAR-responsive PPRE × 3-tk-LUC reporter (SEQ ID NO: 42-43), the GAL4-chimeric receptor and PPAR (Willy, PJ, and Mangelsdorf, DJ (1997) Genes Dev. 11, 289-298 (GAL4-chimeric receptor) Kliewer, SA, et al (1994) Proc. Natl. Acad. Sci. USA 91,7355-7359 (PPAR)). Human adipocyte promoter wild-type luciferase reporter plasmid [p (-908) / LUC wt] (SEQ ID NO: 44) and PPRE mutant luciferase reporter plasmid [p (-908) / LUC PPRE mut] (SEQ ID NO: 45) are available from literature. (Iwaki, M., et al (2003) Diabetes 52, 1655-1663).

(Co-transfection assay in HEK293 cells)
Human embryonic kidney (HEK) 293 cells (ATCC (American Type Culture Collection) -available) were maintained in Dulbecco's Modified Eagle Medium (DMEM) containing 5% fetal bovine serum (FBS). Transfection was performed by the calcium phosphate coprecipitation assay (Lu, TT et al (2000) Mol. Cell 6, 507-515) as described in the literature. Ligand compounds were added 8 hours after transfection. Cells were harvested 16-20 hours later for luciferase and β-galactosidase assays. Typically, DNA co-transfection experiments were performed using 50 ng reporter plasmid, 20 ng pCM-β-gal (Dr. David J. Mangelsdorf (University of Texas Southwestern Medical Center, Dallas, TX) courtesy of pGEM carrier DNA (Nishizawa, H. et al (2002) J) containing 15 ng of each of the reporter expression plasmid and / or cofactor expression plasmid, plus a total of 150 ng of DNA. Biol. Chem. 277, 1586-1592). Luciferase data was normalized to the endogenous β-galactosidase control (Nishizawa, H. et al (2002) J. Biol. Chem. 277, 1586-1592).

(Ligand binding assay)
2 μg glutathione S-transferase (GST) —full length human PPARγ2 ((Y13467; also known as p53 regulatory protein, RB18Aprotein) and 40 nM [ ³ H] rosiglitazone, 100 μL of 10 mM Tris-HCl (pH 8.0), 50 mM KCl, Incubated in 10 mM dithiothreitol (DTT) and 4% glycerol for 24 hours at 4 ° C. For competitive binding assays, pioglitazone, glimepiride or glibenclamide was added to the reaction, Micro Spin G-25 column (Amersham Biosciences). The bound ligand was separated from the free ligand by centrifugation at 1. The radioactivity was measured with a Wallac 1409 liquid scintillation counter (Wallac, Turku, Finland). Each assay was performed in duplicate.

(GST pull-down assay)
The nuclear receptor interaction domain of DRIP205 (amino acids 578 to 728 of SEQ ID NO: 35) was cloned into the GST fusion vector pGEX-4T1 (Amersham Biosciences). GST-DRIP205 fusion protein was expressed in BL21 DE3 cells (Promega). ³⁵ S-labeled full-length mouse PPARγ was generated using TNT Quick Coupled Translation / Translation System (Promega). Approximately 2 μg of GST-DRIP205 was bound to glutathione-Sepharose beads (Amersham Biosciences) and 20 mM Tris-HCl (pH 7.9), 180 mM KCl, 0.2 mM EDTA, 0.05% Nonidet P-40, 0. Equilibrated in binding buffer containing 5 mM phenylmethanesulfonyl fluoride (PMSF), 1 mM DTT and 3.5% bovine serum albumin (BSA). The bound GST protein was then incubated with labeled mPPARγ and ligand for 1.5 hours at 4 ° C. After binding, with a wash buffer containing 20 mM Tris-HCl (pH 7.9), 180 mM KCl, 0.2 mM EDTA, 0.1% Nonidet P-40, 0.5 mM PMSF, 1 mM DTT and 3.5% BSA Washed 5 times, resuspended in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer and loaded onto a 10% SDS-polyacrylamide gel. After electrophoresis, bound proteins were imaged by autoradiography and quantified using a BAS 2500 system (Fujifilm, Tokyo, Japan).

(Transfection studies in 3T3-L1 adipocytes)
Murine 3T3-L1 preadipocytes (available from ATCC) were cultured and induced to differentiate as described in the literature (Iwaki, M. et al (2003) Diabetes 52, 1655-1663). On day 4 after induction of differentiation, the medium of 3T3-L1 cells was replaced with OPTI-MEM (Invitrogen, Tokyo, Japan) and as described in the literature (Iwaki, M. et al (2003) Diabetes 52, 1655-1663), LipofectAMINE 2000 reagent (Invitrogen) was used to transfect the cells with a reporter plasmid containing the PPREx3-tk-LUC reporter or the human adiponectin promoter. Luciferase reporter assay was performed 20 hours after drug treatment using Luciferase Assay System (Promega). Luciferase values were normalized with the internal β-galactosidase control and expressed as relative luciferase activity.

(Quantitative reverse transcription-PCR) Total RNA was extracted using super (Nacalai Tesque, Kyoto, Japan). A first strand of cDNA was synthesized from total RNA using Thermoscript RT (Invitrogen) and oligo dT primers. Real-time quantitative PCR amplification was performed with LightCycler (Roche Diagnostics) using LightCycler-FastStart DNA Master SYBR Green I (Roche Diagnostics, Tokyo). The primer set was as follows:
Mouse aP2, 5′-CCG CAG ACG ACA GGA-3 ′ (SEQ ID NO: 46) and 5′-CTC ATG CCC TTT CAT AAA CT-3 ′ (SEQ ID NO: 47);
Mouse leptin, 5′-GAT GGA CCA GAC TCT GGC AG-3 ′ (SEQ ID NO: 48) and 5′-AGA GTG AGG CTT CCA GGA CG-3 ′ (SEQ ID NO: 49);
Mouse adiponectin, 5′-GAT GGC AGA GAT GGC ACT CC-3 ′ (SEQ ID NO: 50) and 5′-CTT GCC AGT GCT GCC GTC AT-3 ′ (SEQ ID NO: 51);
Mouse cyclophilin, 5′-CAG ACG CCA CTG TCG CTT T-3 ′ (SEQ ID NO: 52) and 5′-TGT CTT TGG AAC TTT GTC TGC AA-3 ′ (SEQ ID NO: 53).

  mRNA levels were normalized to the amount of cyclophilin mRNA and expressed in arbitrary units.

(Analysis of adiponectin secretion in 3T3-L1 adipocytes)
On day 7 after differentiation induction, pioglitazone, glimepiride or glibenclamide was added to the medium for 48 hours. The amount of adiponectin secreted into the culture medium was measured with a mouse / rat adiponectin enzyme-linked immunosorbent assay (ELISA) kit (Otsuka Pharmaceutical, Tokyo, Japan).

(Adipocyte differentiation assay)
Mouse 3T3-F442A preadipocytes (special courtesy of Dr. Hiroshi Sakagami (Kobe University)) were maintained in DMEM containing 10% FBS. For differentiation, cells (3 days after reaching confluence) were incubated with or without compounds in DMEM mobilized with 10% FBS containing 1 μg / mL insulin. Differentiated adipocytes were fixed and stained with Oil Red O (Nacalai Tesque, Kyoto) to visualize the lipids.

(Statistical analysis)
Data are expressed as mean ± SE. Differences were analyzed by Dunnett test. Dunnett's test is a method of comparing one group designated as a control group with another group, and can be used even if a significant difference is not recognized by analysis of variance among multiple groups. It is possible to test without limiting the number of data of each group, whether it is variance or normal distribution. In this example, P <0.05 was considered statistically significant.

(Example 1: Effect of sulfonylurea on PPARγ)
In this example, the present inventors clarified the effect on PPARγ transcription activity using glimepiride and glibenclamide as sulfonylurea agents. In this example, a reporter assay was performed in HEK293 cells, which are non-adipocytes, using a GAL4-PPARγ and a GAL4-responsive luciferase reporter according to the experimental procedure described above.

  The ligand binding domain of PPARγ was fused to the DNA binding domain of the yeast transcription factor GAL4. Since the reporter used in this example is activated only by the exogenous GAL4-chimeric reporter, the effect of the endogenous reporter is eliminated. Interestingly, the inventors found that glimepiride and glibenclamide induced GAL4-PPARγ transcriptional activity at a dose of 10 μM. However, at 10 μM, the other sulfonylureas (tolbutamide, chlorpropamide, and gliclazide) did not detect PPARγ activation as much as glimepiride and glibenclamide (FIG. 1A). Therefore, when the activity of these other sulfonylurea agents was further increased and the activity was detected, it was found that the transcription activity of PPARγ was induced at 100 μM. Therefore, it was revealed that sulfonylurea agents generally induce the transcriptional activity of PPARγ (FIG. 1A). However, the activity seems to vary depending on the type of sulfonylurea agent. Therefore, it has been found that there is a possibility of improving PPARγ activity by developing a novel sulfonylurea agent.

  Next, the inventors examined the concentration-dependent activation of GAL4-PPARγ by glimepiride and glibenclamide in HEK293 cells. As shown in FIG. 1B, treatment with glimepiride or glibenclamide resulted in a concentration-dependent activation of GAL4-PPARγ. Glimepiride and glibenclamide activated GAL4-PPARγ to the maximum levels of 12% and 20% activated by pioglitazone, respectively.

  Thus, sulfonylureas have been shown to be partial agonists of PPAR.

(Example 2: PPAR subtype specificity of sulfonylurea)
In this example, in order to examine PPAR subtype selectivity of glimepiride, we performed a reporter assay using GAL-PPAR in HEK293 cells. Wy14643, GW501516, and pioglitazone (these are known as specific ligands for each PPAR subtype (Hsu, MH et al (2001) J. Biol. Chem. 276, 27950-27958; Oliver, WRet al (2001) Proc. Natl. Acad. Sci. USA 98, 5306-5311; Spiegelman, BM (1998) Diabetes 47, 507-514) activated GAL-PPARα, GAL-PPARδ and PPARγ, respectively (Fig. 1C) Interestingly, glimepiride activates GAL4-PPARγ, but the effect of glimepiride on the transcriptional activity of GAL4-chimeric reporters RXRα, LXRα, LXRβ, and FXR was investigated. It had no effect on the reporter.

  Thus, glimepiride was found to be specific for PPAR.

(Example 3: Binding mode of sulfonylurea agent and PPAR)
Next, in this example, to clarify whether glimepiride and pioglitazone bind directly to PPARγ, we used GST-full-length PPARγ and [ ³ H] rosiglitazone. Competitive binding assays were performed.

When competitive binding assays were performed according to the experimental procedure described above, replacement of [ ³ H] rosiglitazone by pioglitazone was observed. Its IC ₅₀ value was 2.0 μM (FIG. 1D). Replacement with glimepiride and pioglitazone was also concentration dependent, and IC ₅₀ values were 24 μM and 96 μM, respectively (FIG. 1D).

  These data indicate that glimepiride and pioglitazone activate PPARγ by direct binding and are partial agonists for PPARγ.

(Example 4: Effect on interaction between cofactor of sulfonylurea agent and PPAR)
Next, in this example, it was examined whether glimepiride which is a sulfonylurea agent increases the interaction between cofactor and PPARγ.

  This interaction experiment was performed according to the above experimental procedure. Nuclear receptors undergo a conformational change when bound to a ligand, and this change results in AF-2 domain-dependent dissociation of co-repressors and recruitment of coactivators (Glass, CK, and Rosenfeld, MG (2000 ) Genes Dev. 14, 121-141). We investigated the effect of glimepiride on PPARγ AF-2 deletion mutants by co-transfection of full-length PPARγ or mutant PPARγ (ΔAF-2) and PPRE × 3-tk-LUC reporter. Glimepiride as well as pioglitazone activated full-length PPARγ. However, glimepiride-dependent activation of PPARγ was completely abolished by cleavage of the AF-2 domain (FIG. 2A).

  Next, we have PPARγ and coactivator DRIP205 (also known as PPAR binding protein (PBP). Zhu, Y. et al (1997) J. Biol. Chem. 272, 25500-25506. Yang, W., Rachez, C., and Freedman, LP (2000) Mol. Cell. Biol. 20,8008-8017), or the effect of glimepiride on the interaction with the corepressor N-CoR. According to the mammalian two-hybrid assay in HEK293 cells. These assays are for detecting ligand-dependent cofactor mobilization or dissociation, full length PPARγ fused to the transactivation domain of herpesvirus VP16 protein, and DRIP205 or NF fused to the DNA binding domain of GAL4. -Performed using the nuclear receptor interaction domain of CoR (Makishima, M. et al (1999) Science 284, 1362-1365). Briefly, the two-hybrid system is a mammalian cell-based system that performs quantification with luciferase. It is an effective method that can detect protein-protein interactions in vivo using the modular domain found in transcription factors. When the target protein X and protein Y interact with each other, the transcriptional activation domain (VP16) and the DNA binding domain (GAL4) associate to promote the transcriptional activity of the reporter gene.

  As a result, both pioglitazone and glimepiride significantly increased transcriptional activity due to co-transfection of VP16-PPARγ, GAL4-DRIP205 and GAL4-responsive LUC reporter (FIG. 2B). These results indicate that both glimepiride and pioglitazone induce PPARγ association with DRIP205. On the other hand, co-transfection of VP16-PPARγ and GAL4-N-CoR resulted in reporter activation in the absence of ligand. In the presence of pioglitazone or glimepiride, reporter activity was significantly suppressed (FIG. 2C). Similar pioglitazone or glimepiride also showed an inhibitory effect on the interaction between PPARγ and the corepressor SMRT.

  These results indicate that sulfonylureas such as glimepiride induce dissociation from PPARγ corepressors N-CoR or SMRT.

Furthermore, we evaluated the direct effect of glimepiride on the interaction of full-length PPARγ with DRIP205 using a GST pull-down assay. As shown in FIG. 2D, ³⁵ S-labeled PPARγ bound to GST-DRIP205, and this binding was enhanced by pioglitazone (lane 8). Similarly, glimepiride induced the interaction of labeled PPARγ with GST-DRIP205 in a dose-dependent manner (FIG. 3D, lanes 9-11). No binding between GST alone and labeled PPARγ was observed either in the presence or absence of ligand (FIG. 2D, lanes 2-6). These data strongly suggest that direct binding of glimepiride to PPARγ induces association with PPARγ coactivator DRIP205.

  Therefore, it was revealed that glimepiride, which is a sulfonylurea agent, has a direct effect on the interaction between the cofactor and PPARγ.

(Example 5: Effect of sulfonylurea agent on PPAR-dependent transcriptional activity)
Next, in this example, it was examined whether a sulfonylurea agent contributes to PPAR-dependent transcriptional activity in cells such as 3T3-L1 adipocytes.

  In this example, glimepiride and glibenclamide were used as the sulfonylurea agents.

  To study the effects of glimepiride and glibenclamide on PPAR-dependent transactivation in adipocytes, we used a luciferase reporter assay using PPRE × 3-tk-LUC in 3T3-L1 adipocytes. Carried out. Glimepiride significantly induced PPEE reporter activity in adipocytes in a dose-dependent manner (FIG. 3A). Glibenclamide also activated the PPRE reporter in adipocytes in a dose-dependent manner as effectively as glimepiride (FIG. 3A). These results suggest that both glimepiride and glibenclamide activate endogenous PPARγ in adipocytes. The inventors then examined the direct effect of glimepiride on adiponectin gene transcription by a reporter assay in 3T3-L1 adipocytes. We have previously demonstrated that the expression of the adiponectin gene is upregulated by PPARγ activation (Iwaki, M. et al (2003) Diabetes 52, 1655-1663). As shown in FIG. 3B, glimepiride as well as pioglitazone increased the transcriptional activity of the wild type adiponectin promoter in adipocytes. As previously reported (Iwaki, M. et al (2003) Diabetes 52, 1655-1663), transfection of the PPRE mutant adiponectin promoter significantly reduces transcriptional activity in the basal condition. And no further induction by treatment with pioglitazone was observed. Interestingly, glimepiride-dependent activation of the adiponectin promoter was also completely abolished by mutations in PPRE (FIG. 3B). These data suggest that glimepiride activates the adiponectin promoter through a PPRE-dependent mechanism in adipocytes.

  Therefore, it became clear that sulfonylurea induces PPAR-dependent transcriptional activity in 3T3-L1 adipocytes.

(Example 6: Effect of sulfonylurea agent on adiponectin production in adipocytes)
Next, in this example, it was observed whether there was an effect on the production of adiponectin and leptin as an effect of the sulfonylurea downstream of PPAR.

  The inventors examined the effect of glimepiride on mRNA expression of aP2 and leptin, which are known PPARγ target genes, in differentiated 3T3-L1 adipocytes. As reported in the literature (Perrey, S. et al (2001) Metabolism 50, 36-40; Kallen, CB, and Lazar, MA (1996) Proc. Natl. Acad. Sci. USA 93, 5793-5796) Pioglitazone significantly increased aP2 mRNA levels and decreased leptin mRNA levels in adipocytes (FIGS. 4A and 4B). Similarly, glimepiride significantly altered both aP2 and leptin mRNA levels (FIGS. 4A and 4B). Next, the inventors investigated the effect of glimepiride on adiponectin mRNA expression and secretion in differentiated 3T3-L1 adipocytes. As reported in the known literature (Maeda, N. et al (2001) Diabetes 50, 2094-2099), pioglitazone increased both mRNA expression and secretion of adiponectin (FIGS. 4C and 4D). Interestingly, treatment with glimepiride significantly increased adiponectin mRNA levels in adipocytes (FIG. 4C). Furthermore, glimepiride stimulated adiponectin secretion into the medium in a dose-dependent manner (FIG. 4D). Furthermore, the present inventors examined the secretion of adiponectin in adipocytes treated with glibenclamide. Glibenclamide increased adiponectin secretion similar to glimepiride (FIG. 4D). These results suggest that both glimepiride and glibenclamide increase the production of adiponectin, an insulin sensitizing hormone, through PPARγ activation in adipocytes.

  As a result, it was revealed that glimepiride and glibenclamide increase adiponectin production in adipocytes. Glimepiride decreased the gene expression of leptin in adipocytes. It is effective for hyperleptinemia associated with obesity.

(Example 7: Effect of sulfonylurea on adipocyte differentiation)
Next, the effect of sulfonylurea on cell differentiation was examined.

  Traditional PPARγ agonists are known to promote the conversion of preadipocytes to mature adipocytes (Tontonoz, P., Hu, E., and Spiegelman, BM (1994) Cell 79, 1147-1156). . In this example, it was investigated whether sulfonylureas such as glimepiride and glibenclamide have such PPAR agonist activity. The present inventors examined the effects of glimepiride and glibenclamide on adipocyte differentiation in 3T3-F422A preadipocytes (courtesy of Dr. Hiroshi Sakagami (Kobe University)). 3T3-F442A preadipocytes are known to exhibit PPARγ agonist-dependent adipocyte differentiation (Wright, H. M. et al (2000) J. Biol. Chem. 275, 1873-1877). Incubation with glimepiride as well as pioglitazone significantly stimulated adipocyte differentiation, as shown by fat staining with Oil red O (FIG. 5A). Fat deposition due to glibenclamide was observed, and fat deposition was also observed in glimepiride. Furthermore, the present inventors examined the effects of glimepiride and glibenclamide in the induction of adipocyte differentiation marker genes, aP2 and adiponectin. Both glimepiride and glibenclamide significantly induced mRNA expression of these genes in 3T3-F442A cells (FIGS. 5B and 5C), similar to pioglitazone. These results suggest that both glimepiride and glibenclamide stimulate adipocyte differentiation through PPARγ activation.

(Summary)
In the present invention, the present inventors have found at least the following 1) to 5):
1) Glimepiride induced PPARγ transcriptional activity in HEK293 cells;
2) Glimepiride increased recruitment of coactivator DRIP205 and dissociation of corepressors (eg, N-CoR and SMRT);
3) Glimepiride bound directly to PPARγ in a manner that competes with rosiglitazone;
4) Glimepiride stimulated the transcriptional activity of gene promoters including PPER and altered mRNA levels of PPARγ target genes in 3T3 adipocytes; and
5) Glimepiride induced adipocyte differentiation in 3T3-F442A cells.

  Interestingly, most of the effects observed with glimepiride were also reproduced with pioglitazone. These data indicate that glimepiride and glipenclamide (both of which belong to the sulfonylurea antidiabetic drugs) act as agonists for PPARγ, but their ability was 12-20% of pioglitazone. Our results provide a novel aspect of sulfonylureas that are PPARγ agonists.

  Several compounds have been reported as partial agonists for PPARγ ((1) Oberfield, JL et al (1999) Proc. Natl. Acad. Sci. USA 96, 6102-6106; (2) Kurosaki, E. et al. al (2003) Biochem. Pharmacol. 65, 795-805; (3) Schupp, M. et al (2004) Circulation 109, 2054-2057; (4) Benson, SC et al (2004) Hypertension 43, 993-1002 ). For example, GW0072

Is one of those partial agonists and has the same ability as a full agonist in the dissociation of the corepressor N-CoR. However, the recruitment of coactivators (eg CBP and SRC-1) is limited compared to thiazolidinediones (Oberfield, JL et al (1999) Proc. Natl. Acad. Sci. USA 96, 6102-6106). ). We have observed that glimepiride induces mobilization of DRIP205 and dissociation of both N-CoR and SMRT, similar to pioglitazone. Thus, suppression of the maximum level of PPARγ transactivation by glimepiride and glibenclamide may be due to dysfunction of recruitment of other coactivators other than DRIP205.

  Previous clinical studies have shown that plasma adiponectin levels are increased in type 2 diabetes (Tsunekawa, T. et al (2003) Diabetes Care 26, 285-289). We have previously reported that PPARγ agonists increase plasma adiponectin levels in humans (Maeda, N. et al (2001) Diabetes 50, 2094-2099). Thus, increasing the effect of glimepiride on plasma adiponectin in human subjects can be explained in part by PPARγ agonist activity.

  Hyperglycemia in type 2 diabetes is the result of multiple defects in both insulin secretion from pancreatic beta cells and insulin sensitivity in peripheral tissues. Thus, it has been considered important not only to increase plasma insulin levels but also to improve insulin sensitivity in order to develop a pharmacological approach to type 2 diabetes. Sulfonylurea agents have been widely used so far because it was believed that sulfonylurea agents effectively reduce blood glucose by stimulating pancreatic insulin secretion. In contrast, thiazolidinediones, PPARγ agonists, exhibit potent hypoglycemic effects by improving peripheral insulin resistance.

  The doses of glimepiride and pioglitazone used in our experiments are higher than the doses required to induce insulin secretion from the islets of Langerhans previously used to exert PPARγ agonist activity. it was high. However, the inventor's finding that both glimepiride and glibenclamide can act not only on sulfonylurea receptors (SUR) but also on PPARγ is the ideal anti-diabetic agent, namely insulin secretion from pancreatic β cells and It provides an important clue to developing anti-diabetes that can enhance both insulin sensitivity.

(Example 8: Screening)
In this example, a substance that may enhance the activity of PPAR is screened using a reporter gene assay.

(Reporter Gene Assay)
(Method)
Phosphate buffered saline (PBS; 10 mM phosphate; 150 mM sodium chloride, pH 7.2 to 7.3.)
Luciferase reporter assay kit:
Lysis solution: 25 mM Tris (7.5), 2 mM dithiothreitol (DTT), 10% glycerol, 1% Triton X-100; Luciferin mix: 20 mM Tricine / 1.07 mM (MgCO ₃ ) ₄ Mg (OH) can be used _{2 -5H 2 O / 2.67mMMgSO 4 /0.1mM} EDTA / 33.3mM DTT / 270μM coenzyme (coenzyme) a / 470μM luciferin / 530μMATP (Nippon Gene of PicaGene (Code No.PGK-L100) ).

(Adipocytes used)
White adipocytes: 3T3-L1, 3T3-F442A, Ob1771
Brown adipocytes: HIB1B
Primary cultured cells of white and brown adipocytes Others: C3H 10T1 / 2 (Because it is a multipotent cell, it may become an adipocyte)
(Culture medium)
These cells are appropriately cultured in Dulbecco's modified Eagle medium using 10% fetal calf serum or the like.

(Luciferase)
Luciferase is an enzyme that produces fluorescence (about 560 nm). A plasmid containing a nucleic acid sequence (SEQ ID NO: 1) encoding PPAR and a reporter plasmid (luciferase reporter) are forcibly expressed in the cell simultaneously, and the reporter activity is measured. Gene expression can be measured by measuring fluorescence due to expression of the luciferase gene product. Here, the transfection and the like can be performed according to the technique described in the above (3T3-L1 adipocyte transfection study).

(Method)
(1) Transfect each of the cells using the plasmid.
(2) The cells are cultured for 48 to 72 hours.
(3) Remove the culture supernatant.
(4) Wash wells or dishes used for culture.
(5) Lysis of cells (in case of 24-well plate, lysis solution 200 μl / well).
(6) Transfer the lysate to the Eppendorf tube.
(7) Enucleation (centrifugation 15 Krpm, 3 minutes, 4 ° C.).
(8) Transfer supernatant to another Eppendorf tube (9) Insert sample into liquid scintillation counter.
(10) Add 0.5 μl of sample supernatant to the luciferin mix.
(11) Measure the count due to fluorescence.

  From the fluorescence thus obtained, the candidate compound can measure suppression or enhancement of the function of PPAR.

(Example 9: Automation)
The screening performed in Example 8 can be automated using a robot. In this case, for example, a system using a microplate can be constructed using Beckman Coulter's Biomek series, or a system can be constructed using Zymark's Staccato Mini-System series.

  The lead compound thus obtained can be used for animal experiments. Alternatively, other compounds can be designed based on such lead compounds.

(Example 10: Animal experiment)
The compounds that were found to activate PPAR in Examples 8 and 9 were administered to animals such as mice, rats, and monkeys to reduce the amount of PPAR mRNA expression or protein specifically in adipose tissue The compound to be screened is screened.

  Such a screening would actually differentiate adipocytes in animals; diabetes; hyperglycinemia; low glucose tolerance; insulin resistance; obesity; steatosis; dyslipidemia; Hypercholesterolemia; Low HDL level; High LDL level; Atherosclerosis; Vascular stenosis; Irritable bowel syndrome; Inflammatory bowel disease; Crohn's disease; Intestinal ulcer; Inflammatory disease; Substances that affect diseases such as sexually transmitted diseases; retinopathy; psoriasis; metabolic syndrome; ovarian hyperandrogenies can be screened.

(Example 11: Clinical trial in human subjects)
Of the substances that actually had a test effect in Example 10, those that were not toxic were administered to human subjects to observe the effect on obesity. Here, clinical trials are conducted using energy metabolism, body weight, fat mass, body fat percentage and the like as indices. Thereby, it can be determined whether the compound identified as the lead compound in Example 10 actually has an effect on the disease.

  As mentioned above, although this invention has been illustrated using preferable embodiment of this invention, it is understood that the scope of this invention should be construed only by the claims. Patents, patent applications, and documents cited herein should be incorporated by reference in their entirety, as if the contents themselves were specifically described herein. Understood.

  The present invention has the utility of being able to treat PPAR-related diseases using targets and screening methods thereby useful in the diagnosis, treatment and prevention of PPAR-related diseases, and medicaments identified by the methods. The present invention has also shown that sulfonylureas are effective for such PPAR-related diseases. Thus, the present invention is useful in developing a drug for effective treatment of diabetes.

FIG. 1 shows that glimepiride and glibenclamide are partial agonists for PPARγ. A: Effect of sulfonylurea on the transcriptional activity of PPARγ. HEK293 cells were transfected with GAL4-mouse PPARγ (mPPARγ) and MH100 (UAS) × 4-tk-LUC reporters and treated with vehicle control (cont) or with sulfonylureas. Each of the sulfonylureas was 10 μM and was glimepiride (gmp), glibenclamide (gbc), tolbutamide (tb), chlorpropamide (cp), and gliclazide (gc). Luciferase values were normalized by the endogenous β-galactosidase control and expressed as relative luciferase activity. Values are mean ± SE (n = 3). B: PPARγ concentration-dependent activation by pioglitazone. HEK293 cells were cotransfected with GAL-mPPARγ and MH100 (UAS) × 4-tk-LUC reporter and treated with the indicated doses using pioglitazone (pio), glimepiride, glibenclamide. Luciferase activity values were normalized by β-galactosidase activity and expressed as multiples of that relative to the vehicle control. The value is an average value ± SE (n = 3). C: PPAR subtype selectivity of glimepiride. HEK293 cells were transfected with GAL4, GAL4 mouse PPARγ, GAL4-mouse PPARδ, or GAL4-mPPARγ in combination with MH100 (UAS) × 4-tk-LUC reporter, and vehicle control, each PPAR subtype Or a ligand for 10 μM glimepiride (PPARα ligand, 10 μM Wy 14643; PPARδ ligand, 10 μM GW 501516; PPARγ ligand, 1 μM pioglitazone). Luciferase was normalized by β-galactosidase activity and expressed as relative luciferase activity. The value is an average value ± SE (n = 3). D: Dose response curve of replacement of [ ³ H] rosiglitazone binding to PPARγ by pioglitazone. Competitive binding assays were performed as described in the experimental procedure. Data show the average of duplicate points. FIG. 1A is a graph showing whether a sulfonylurea agent is effective at a higher concentration than that shown in FIG. The experimental conditions are all as described in FIG. 1 and the experimental procedure except for the concentrations used. FIG. 2 shows that glimepiride affects the interaction of PPARγ with cofactors. A: AF-2-dependent activation of PPARγ by glimepiride. HEK293 cells were co-transfected with CMX control vector (cont), CMX-mPPARγ or CMX-ΔmPPARγ and PPRE × 3-tk-LUC and treated with 1 μM pioglitazone (pio) or 10 μM glimepiride (gmp) . Luciferase values were normalized by the endogenous β-galactosidase control and expressed as relative luciferase activity. Values are mean ± SE (n = 3). B and C: Effect of glimepiride on the interaction between PPARγ and cofactor in cell-based systems. Mammalian two-hybrid analysis was performed in HEK 293 cells by using GAL4-DRIP205 or GAL4-N-CoR in combination with VP16 or VP16-mPPARγ and MH100 (UAS) × 4-tk-LUC. . Cells were then treated with 1 μM pioglitazone or 10 μM glimepiride. Luciferase values were normalized with β-galactosidase and expressed as relative luciferase activity. The value is an average value ± SE (n = 3). D: Direct effect of glimepiride on the interaction between PPARγ and DRIP205 in a cell-free system. The GST pull-down assay was performed as described in “Experimental Procedures”. In vitro translation ³⁵ S-mouse PPARγ was detected as a result of interaction with GST-DRIP205 in the presence of pioglitazone or glimepiride. FIG. 3 shows that glimepiride and glibenclamide induce PPAR-dependent transactivation in 3T3 adipocytes. A: Effect of glimepiride and glibenclamide on PPARγ-dependent transcriptional activity in 3T3-L1 adipocytes. Differentiated 3T3-L1 cells were transfected with the PPRE × 3-tk-LUC reporter plasmid as described in “Experimental procedures”. Following transfection, cells were treated with vehicle control (cont), 1 μM pioglitazone (pio) for 20 hours, or at concentrations indicated with glimepiride (gmp) or glibenclamide (gbc). Luciferase values were normalized with β-galactosidase and expressed as relative luciferase activity. The value is an average value ± SE (n = 4). * P <0.05 compared to the control group. B: Effect of glimepiride on adiponectin promoter activity. Differentiated 3T3-L1 cells were isolated from human adiponectin promoter-luciferase reporter plasmid, wild type [p (−908) / LUC wt] or PPRE mutant reporter [p (−908) / Transfected with LUC PPRE mut]. Following transfection, cells were treated with control vehicle (cont), 1 μM pioglitazone or 10 μM glimepiride for 20 hours. Luciferase values were normalized by β-galactosidase and expressed as relative luciferase activity values. Luciferase values were normalized with β-galactosidase and expressed as relative luciferase activity. The value is an average value ± SE (n = 3). * P <0.05 is compared with the control group. FIG. 4 shows that glimepiride and glibenclamide enhance adiponectin production in 3T3-L1 adipocytes. A, B, and C are the effects of glimepiride on mRNA expression of the PPARγ target gene. Seven days after induction of differentiation, 3T3-L1 adipocytes were treated with vehicle control (cont), 10 μM pioglitazone (pio) or 20 μM glimepiride (gmp) for 24 hours. Total RNA was subjected to real-time quantitative RT-PCR analysis for mRNA expression of these genes. aP2, leptin, and adiponectin mRNA levels were normalized to the amount of cyclophilin mRNA. The value is an average value ± SE (n = 3). * P <0.05 compared to the control group. D: Effect of glimepiride and glibenclamide on adiponectin secretion. Differentiated 3T3-L1 cells were treated with vehicle control, 10 μM pioglitazone, the indicated concentrations of glimepiride, or 25 μM glibenclamide (gbc) for 48 hours. An aliquot of this medium was subjected to ELISA to measure the amount of secreted adiponectin. The value is an average value ± SE (n = 3). * P <0.05 compared to control group. FIG. 5 shows that glimepiride and glibenclamide stimulate adipocyte differentiation. A: Effect of glimepiride on adipocyte differentiation in 3T3-F422A preadipocytes. After incubation for 9 days with vehicle control (cont), 1 μM pioglitazone (pio) or 10 μM glimepiride (gbc), cells were stained with Oil red O. B and C: Effect of glimepiride and glibenclamide on induction of adipocyte differentiation marker gene in 3T3-F442A adipocyte precursor cells. 3T3-F422A preadipocytes were incubated for 2 days with vehicle control, 1 μM pioglitazone, 10 μM glimepiride or 10 μM glibenclamide (gbc). Total RNA was extracted and subjected to real-time quantitative RT-PCR analysis for mRNA expression of the aP2 and adiponectin genes. The mRNA level was normalized relative to the amount of cyclophilin mRNA. The value is an average value ± SE (n = 3). ^* P <0.05 compared to control group.

(Explanation of sequence listing)
SEQ ID NO: 1 is the nucleic acid sequence of mouse PPARγ. (NM_011146, PPARγ isoform 2 is shown.)
SEQ ID NO: 2 is the amino acid sequence of mouse PPARγ. (NP_035276, PPARγ isoform 2 is shown.)
SEQ ID NO: 3 is the nucleic acid sequence of human PPARγ. (NM_138712)
SEQ ID NO: 4 is the amino acid sequence of human PPARγ. (NP_619726)
SEQ ID NO: 5 is the nucleic acid sequence of rat PPARγ. (NM_013124)
SEQ ID NO: 6 is the amino acid sequence of rat PPARγ. NP_037256)
SEQ ID NO: 7 is the nucleic acid sequence of monkey PPARγ. (AF033103)
SEQ ID NO: 8 is the amino acid sequence of monkey PPARγ. (AAB87480)
SEQ ID NO: 9 shows the amino acid sequence PLLQEIYKDLY, which is one of the binding essential sequences in PPAR.
SEQ ID NO: 10 is the nucleic acid sequence of the human adiponectin 3.6 kb promoter. (AF304467)
SEQ ID NO: 11 is the nucleic acid sequence of human adiponectin protein. (NM_004797)
SEQ ID NO: 12 is the amino acid sequence of human adiponectin protein. (NP_004788)
SEQ ID NO: 13 is the nucleic acid sequence of the mouse adiponectin promoter (Das, K. et al (2001) Biochem Biophys Res Commun. 280, 1120-1129, Seo JB et al (2004) J. Biol. Chem. 279, 22108). -22117 etc.).
SEQ ID NO: 14 is the nucleic acid sequence of GAL4. (X85976; corresponding to amino acids 1-147: atgaagctactgtcttctatcgaacaagcatgcgatatttgccgacttaaaaagctcaagtgctccaaagaaaaaccgaagtgcgccaagtgtctgaagaacaactgggagtgtcgctactctcccaaaaccaaaaggtctccgctgactagggcacatctgacagaagtggaatcaaggctagaaagactggaacagctatttctactgatttttcctcgagaagaccttgacatgattttgaaaatggattctttacaggatataaaagcattgttaacaggattatttgtacaagataatgtgaataaagatgccgtcacagatagattggcttcagtggagactgatatgcctctaacattgagacagcatagaataagtgcgacatcatcatcggaagagagtagtaacaaaggtcaaagacagttgactgtatcg)
SEQ ID NO: 15 is the amino acid sequence of GAL4. (X85976) (amino acids 1-147; MKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTKRSPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNKDAVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVS)
SEQ ID NO: 16 is the nucleic acid sequence of the GAL4-mouse PPARα chimeric protein. (Mouse PPARα, NM_011144)
SEQ ID NO: 17 is the amino acid sequence of the GAL4-mouse PPARα chimeric protein. (Mouse PPARα, NP_035274)
SEQ ID NO: 18 is the nucleic acid sequence of the GAL4-mouse PPARδ chimeric protein. (Mouse PPARδ, NM_011145)
SEQ ID NO: 19 is the amino acid sequence of the GAL4-mouse PPARδ chimeric protein. (Mouse PPARδ, NP_035275)
SEQ ID NO: 20 is the nucleic acid sequence of the GAL4-mouse PPARγ chimeric protein. (Mouse PPARγ, NM_011146)
SEQ ID NO: 21 is the amino acid sequence of the GAL4-mouse PPARγ chimeric protein. (Mouse PPARγ, NP_035276)
(SEQ ID NO: 16-21 is a fusion of the above-mentioned GAL4 sequence and each mouse PPAR ligand binding region. Kliewer, SA, et al (1994) Proc. Natl. Acad. Sci. USA 91,7355-7359, Umesono, K et al (1991) Cell 65, 1255-1266 etc.)
SEQ ID NO: 22 is the nucleic acid sequence of VP16. (Consisting of 78 amino acids)
(Gcccccccgaccgatgtcagcctgggggacgagctccacttagacggcgaggacgtggcgatggcgcatgccgacgcgctagacgatttcgatctggacatgttgggggcacgactccgccgtg
SEQ ID NO: 23 is the amino acid sequence of VP16. (78 amino acids)
(APPTDVSLGDELHLDGEDVAMAHADALDDFDLDMLGDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYGG)
SEQ ID NO: 24 is the nucleic acid sequence of the VP16-mouse PPARγ chimeric protein.
SEQ ID NO: 25 is the amino acid sequence of the VP16-mouse PPARγ chimeric protein.
SEQ ID NO: 26 is the nucleic acid sequence for β-galactosidase.
SEQ ID NO: 27 is the amino acid sequence of β-galactosidase.
(See SEQ ID NOs: 24-27 for Willy, PJ, and Mangelsdorf, DJ (1997) Genes Dev. 11, 289-298)
SEQ ID NO: 28 is the nucleic acid sequence of the mouse ΔPPARγ mutant construct. ()
SEQ ID NO: 29 is the amino acid sequence of the mouse ΔPPARγ mutant construct. (MVDTEMPFWPTNFGISSVDLSVMEDHSHSFDIKPFTTVDFSSISAPHYEDIPFTRADPMVADYKYDLKLQEYQSAIKVEPASPPYYSEKTQLYNRPHEEPSNSLMAIECRVCGDKASGFHYGVHACEGCKGFFRRTIRLKLIYDRCDLNCRIHKKSRNKCQYCRFQKCLAVGMSHNAIRFGRMPQAEKEKLLAEISSDIDQLNPESADLRALAKHLYDSYIKSFPLTKAKARAILTGKTTDKSPFVIYDMNSLMMGEDKIKFKHITPLQEQSKEVAIRIFQGCQFRSVEAVQEITEYAKNIPGFINLDLNDQVTLLKYGVHEIIYTMLASLMNKDGVLISEGQGFMTREFLKNLRKPFGDFMEPKFEFAVKFNALELDDSDLAIFIAVIILSGDRPGLLNVKPIEDIQDNLLQALELQLKLNHPESSQLFAKVLQKMTDLRQIVTEHVQLLHVIKKTETDMSLH)
SEQ ID NO: 30 is the nucleic acid sequence of GAL4-vitamin D receptor binding protein (DRIP) 205. It is a fusion of the above-mentioned GAL4 sequence and the nuclear receptor interaction domain (amino acids 578 to 728) of DRIP205 described later.
SEQ ID NO: 31 is the amino acid sequence of GAL4-vitamin D receptor binding protein (DRIP) 205. It is a fusion of the above-mentioned GAL4 sequence and the nuclear receptor interaction domain (amino acids 578 to 728) of DRIP205 described later.
SEQ ID NO: 32 is the nucleic acid sequence of the GAL4-nuclear receptor corepressor (N-CoR). It is a fusion of the GAL4 sequence described above and the N-CoR nuclear receptor interaction domain (amino acids 1990-2416) described below.
SEQ ID NO: 33 is the amino acid sequence of the GAL4-nuclear receptor corepressor (N-CoR). It is a fusion of the GAL4 sequence described above and the N-CoR nuclear receptor interaction domain (amino acids 1990-2416) described below.
SEQ ID NO: 34 is the nucleic acid sequence of the nuclear receptor interaction domain (amino acids 578-728) of DRIP205. (Y13467) (also known as p53 regulatory protein, RB18Aprotein).
SEQ ID NO: 35 is the amino acid sequence of the nuclear receptor interaction domain (amino acids 578-728) of DRIP205. (Y13467) (also known as p53 regulatory protein, RB18Aprotein).
SEQ ID NO: 36 is the nucleic acid sequence of the nuclear receptor interaction domain of N-CoR (amino acids 1990-2416). (U35312)
SEQ ID NO: 37 is the amino acid sequence of the N-CoR nuclear receptor interaction domain (amino acids 1990-2416). (U35312)
SEQ ID NO: 38 is the nucleic acid sequence of the GAL4 DNA binding domain. (See Willy, PJ, and Mangelsdorf, DJ (1997) Genes Dev. 11,289-298).
SEQ ID NO: 39 is the amino acid sequence of the GAL4 DNA binding domain. (See Willy, PJ, and Mangelsdorf, DJ (1997) Genes Dev. 11,289-298).
SEQ ID NO: 40 is the nucleic acid sequence of the GAL4-responsive MH100 (UAS) × 4-tk-LUC reporter. (See Willy, PJ, and Mangelsdorf, DJ (1997) Genes Dev. 11,289-298).
SEQ ID NO: 41 is the amino acid sequence of the GAL4-responsive MH100 (UAS) × 4-tk-LUC reporter. (See Willy, PJ, and Mangelsdorf, DJ (1997) Genes Dev. 11,289-298).
SEQ ID NO: 42 is the nucleic acid sequence of the PPAR-responsive PPRE × 3-tk-LUC reporter. (See, for example, Kliewer, SA, et al (1994) Proc. Natl. Acad. Sci. USA 91,7355-7359, Kliewer, SA, et al (1992) Nature (London) 358, 771-774).
SEQ ID NO: 43 is the amino acid sequence of the PPAR-responsive PPRE × 3-tk-LUC reporter. (See, for example, Kliewer, SA, et al (1994) Proc. Natl. Acad. Sci. USA 91,7355-7359, Kliewer, SA, et al (1992) Nature (London) 358, 771-774).
SEQ ID NO: 44 is the nucleic acid sequence of the wild type luciferase reporter plasmid of the human adipocyte promoter. See Iwaki, M., et al (2003) Diabetes 52, 1655-1663.
SEQ ID NO: 45 is the nucleic acid sequence of the PPRE mutant luciferase reporter plasmid [p (−908) / LUC PPRE mut]. See Iwaki, M., et al (2003) Diabetes 52, 1655-1663.
SEQ ID NO: 46 is a nucleic acid sequence in the forward direction of the mouse aP2 primer (mouse aP2, 5′-CCG CAG ACG ACA GGA-3 ′).
SEQ ID NO: 47 is a nucleic acid sequence in the reverse direction of the primer of mouse aP2 (5′-CTC ATG CCC TTT CAT AAA CT-3 ′).
SEQ ID NO: 48 is a nucleic acid sequence in the primer forward direction of mouse leptin (5′-GAT GGA CCA GAC TCT GGC AG-3 ′).
SEQ ID NO: 49 is a nucleic acid sequence in the reverse direction of the primer for mouse leptin (5′-AGA GTG AGG CTT CCA GGA CG-3 ′).
SEQ ID NO: 50 is a nucleic acid sequence in the primer forward direction of mouse adiponectin (5′-GAT GGC AGA GAT GGC ACT CC-3 ′).
SEQ ID NO: 51 is a nucleic acid sequence in the reverse direction of the primer of mouse adiponectin (5′-CTT GCC AGT GCT GCC GTC AT-3 ′).
SEQ ID NO: 52 is the nucleic acid sequence in the forward direction of the mouse cyclophilin primer (5′-CAG ACG CCA CTG TCG CTT T-3 ′).
SEQ ID NO: 53 is a nucleic acid sequence in the reverse direction of the primer of mouse cyclophilin (5′-TGT CTT TGG AAC TTT GTC TGC AA-3 ′).

Claims (35)

A peroxisome proliferator activated receptor (PPAR) modulator comprising a sulfonylurea agent. The sulfonylurea agent is

Where R ₁ and R ₂ are each independently alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted Cycloalkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, carbocyclic group, substituted carbocyclic group, heterocyclic group, substituted heterocyclic group, halogen, hydroxy, substituted hydroxy, thiol, Substituted thiol, cyano, nitro, amino, substituted amino, carboxy, substituted carboxy, carbamoyl, acyl, substituted acyl, acylamino, thiocarboxy, amide, substituted carbonyl, substituted thiocarbonyl, Substituted sulfonyl and substituted sulfini Having a substituent selected from the group consisting of, modulating agent according to claim 1. The PPAR regulator according to claim 1, wherein the sulfonylurea agent is selected from the group consisting of glimepiride, glibenclamide, tolbutamide, chlorpropamide, acetohexamide and gliclazide. The PPAR regulator according to claim 1, wherein the sulfonylurea agent is glimepiride or glibenclamide. The PPAR modulator according to claim 1, wherein the modulation is activation of PPAR. The PPAR modulator according to claim 1, further comprising at least one activity selected from the group consisting of insulin secretory activity of pancreatic β cells and activity that promotes insulin sensitivity. The PPAR modulator according to claim 1, wherein the PPAR modulator has at least 10% of thiazolidinedione (thiazolidinedione). The PPAR modulator according to claim 7, wherein the thiazolidinedione is pioglitazone. The PPAR regulator according to claim 1, wherein the PPAR is PPARγ. The PPAR is an amino acid sequence represented by SEQ ID NO: 1 selected from the group consisting of SEQ ID NO: 2 (NP_035276), SEQ ID NO: 4 (NP_619726), SEQ ID NO: 6 (NP_037256) and SEQ ID NO: 8 (AAB87480), or one or the number 2. The PPAR modulating agent of claim 1 comprising a variant sequence comprising one substitution, addition or deletion.   The PPAR modulators are: adipocyte differentiation; diabetes; hyperglycinemia ;; low glucose tolerance; insulin resistance; obesity; steatosis; dyslipidemia; hyperlipidemia; Hypercholesterolemia; Low HDL level; High LDL level; Atherosclerosis; Vascular stenosis; Irritable bowel syndrome; Inflammatory bowel disease; Crohn's disease; Intestinal ulcer; Inflammatory disease; Pancreatitis; PPAR modulators used for the treatment or prevention of a condition or disease selected from the group consisting of: psoriasis; metabolic syndrome; ovarian hyperandrogenism. A method for identifying a compound having at least one activity selected from the group consisting of insulin secretory activity of pancreatic β cells and activity that promotes peripheral insulin sensitivity, and having the ability to modulate PPAR, The method is:
A) providing a candidate compound;
B) measuring at least one activity selected from the group consisting of pancreatic β-cell insulin secretory activity and peripheral insulin sensitivity activity for the candidate compound;
C) subjecting the candidate compound to an assay for measuring the activity of PPAR; and D) identifying the substance determined to be active in each of steps B and C as a lead compound,
Including the method. The method of claim 12, wherein the candidate compound comprises a sulfonylurea agent. The candidate compound is

Where R ₁ and R ₂ are each independently alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted Cycloalkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, carbocyclic group, substituted carbocyclic group, heterocyclic group, substituted heterocyclic group, halogen, hydroxy, substituted hydroxy, thiol, Substituted thiol, cyano, nitro, amino, substituted amino, carboxy, substituted carboxy, carbamoyl, acyl, substituted acyl, acylamino, thiocarboxy, amide, substituted carbonyl, substituted thiocarbonyl, Substituted sulfonyl and substituted sulfini The method of claim 12, having a substituent selected from the group consisting of. 13. The method of claim 12, wherein the modulation is PPAR activation. 13. The method of claim 12, wherein the compound has both insulin secretory activity and insulin sensitivity promoting activity in the pancreatic beta cells. 13. The method of claim 12, wherein the PPAR modulating activity is measured relative to thiazolidinedione (TZD). The method of claim 12, wherein the PPAR is PPARγ. The PPAR is an amino acid sequence represented by SEQ ID NO: 1 selected from the group consisting of SEQ ID NO: 2 (NP_035276), SEQ ID NO: 4 (NP_619726), SEQ ID NO: 6 (NP_037256) and SEQ ID NO: 8 (AAB87480), or one or the number 13. A method according to claim 12, comprising a variant sequence comprising one substitution, addition or deletion. The method according to claim 12, wherein the step (C) includes the step of observing whether the candidate compound provides and competes with a sulfonylurea agent or a thiazolidinedione agent. 13. The method of claim 12, wherein the assay for measuring PPAR activity is a transcriptional activity assay. The method according to claim 12, wherein the insulin secretory activity of the pancreatic β cells is measured by directly measuring insulin secreted from the cells. The method according to claim 12, wherein the activity of promoting peripheral insulin sensitivity is measured by an insulin tolerance test. The activity of the PPAR is a step of determining the activity of the candidate compound in a system comprising a nucleic acid construct comprising a nucleic acid sequence encoding a reporter operably linked to a transcription factor recognition sequence and the selected PPAR. Wherein if the expression of the reporter is enhanced, the candidate compound is determined to be an agonist of the PPAR, and if the expression of the reporter is decreased, the candidate compound is determined to be an antagonist of the PPAR. The method according to claim 12. 25. The method of claim 24, wherein the reporter is luciferase. 25. The method of claim 24, wherein the system is a cell. The method according to claim 12, wherein a protein containing a C-terminal activation function-2 (AF-2) domain is used as a target in the step C. 28. The method of claim 27, wherein the target comprises the amino acid sequence PLLQEIYKDLY (SEQ ID NO: 9). 13. The method of claim 12, wherein in the step C, an interaction between a PPAR cofactor and a PPAR is observed. 30. The method of claim 29, wherein the PPAR cofactor is selected from the group consisting of DRIP 205, N-CoR and SMRT. A system for identifying a compound having at least one activity selected from the group consisting of insulin secretory activity of pancreatic β cells and activity that promotes peripheral insulin sensitivity, and having the ability to modulate PPAR. The system:
A) Candidate compound;
B) Means for measuring at least one activity selected from the group consisting of insulin secretory activity of pancreatic β cells and peripheral insulin sensitivity for the candidate compound;
C) means for subjecting the candidate compound to an assay for measuring the activity of PPAR; and D) means for identifying a substance determined to be active by each of means B and C as a lead compound;
A system comprising: A method for treating or preventing a disease associated with PPAR comprising:
A) a step of administering a sulfonylurea agent to a subject;
Including the method. 33. The method of claim 32, wherein the sulfonylurea agent is selected from the group consisting of glimepiride, glibenclamide, tolbutamide, chlorpropamide, acetohexamide and gliclazide.   Use of a sulfonylurea agent in the manufacture of a medicament for treating or preventing a disease associated with PPAR. 35. Use according to claim 34, wherein the sulfonylurea agent is selected from the group consisting of glimepiride, glibenclamide, tolbutamide, chlorpropamide, acetohexamide and gliclazide.