KR20070031961A - Methods related to a single nucleotide polymorphism of the g protein coupled receptor, gpr40 - Google Patents

Abstract

The present invention relates to genotyping methods, methods of treatment, diagnostic tests, and methods of characterizing drugs, which are associated with a single nucleotide polymorphism of the GPR40 gene.

Description

방법 METHOD RELATED TO A SINGLE NUCLEOTIDE POLYMORPHISM OF THE G PROTEIN COUPLED RECEPTOR, GPR40}

The present invention relates to genotyping methods, methods of treatment, diagnostic tests and kits, and methods of characterizing a drug associated with a single nucleotide polymorphism of the GPR40 gene.

Diabetes is a serious and sometimes fatal disease affecting 17 million Americans and 151 million people worldwide [Gadsby, R., "Epidermiology of diabetes", Adv. Drug Deliv. Rev. , 54 , 1165-1172, 2002]. Diabetes results from the body's failure to produce insulin or respond properly. While glucose is the major substrate that regulates insulin secretion, other nutrients, such as free fatty acids (FFA) and amino acids, can also regulate insulin secretion [Haber, EP, et al., "Pleiotropic effects of fatty acids on pancreatic β-cells". , J. Cell Physiol ., 194 , 1-12, 2002].

There are two main types of diabetes. In type I diabetes, insulin-producing pancreatic β-cells are destroyed by the body's own immune system causing insulin deficiency. Type II diabetes results from loss of sensitivity by human cells to insulin action and the pancreas' inadequate release of insulin. The incidence of type II diabetes is increasing and is particularly prevalent in certain risky populations in the United States [Gadsby, R., "Epidermiology of diabetes", Adv. Drug Deliv. Rev. , 54 , 1165-1172, 2002].

Metabolic syndrome is a group of metabolic abnormalities that increase the risk of developing diabetes and cardiovascular disease [Park, YW et al., "The Metabolic Syndrome.Prevalence and associated risk factor findings in the US population from the Third National and Nutrition Examination Survey, 1988-1994 ", Arch. Intern. Med ., 163 , 427-436, 2003]. Abnormalities, including obesity, insulin resistance, dyslipidemia and hypertension, have been reported to be key components of metabolic syndrome [Reilly, MP and Rader, DJ, "The Metabolic Syndrome. More than the sum of its parts?", Circulation , 108 , 1546-1551, 2003; Park, YW et al., "The Metabolic Syndrome.Prevalence and associated risk factor findings in the US population from the Third National and Nutrition Examination Survey, 1988-1994", Arch. Intern. Med ., 163 , 427-436, 2003; Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adult, "Executive Summary of the Third Report of the national Cholesterol Education Program (NCEP)", JAMA 285 , 2486-2497, 2001].

Obesity has been determined to be a key factor in the metabolic syndrome. Reduction of overweight has been suggested to minimize the risk associated with metabolic syndrome [Shirai, K., "Obesity as the core of the metabolic syndrome and the management of coronary heart disease", Curr. Med. Res. Opin ., 20 , 295-304, 2004].

G-protein coupled receptors (GPCRs) are membrane proteins of the epidermis characterized by the presence of seven transmembrane α-helix fragments (referred to as TM1 to TM7) that connect alternating intracellular and extracellular loops [Gether] , U., "Uncovering molecular mechanisms involved in activation of G protein coupled receptors, Endocr. Rev. , 21 , 90-113, 2000]. GPCRs are signaling molecules such as hormones, neurotransmitters and local mediators, and intracellular enzymes. , Mediate signaling between ion channels and transporters (Johnson, GL and Dhanasekaran, N., "The G-protein family and their interaction with receptors", Endocr. Rev. , 10 , 317-331, 1989).

Sawzdargo, M. et al., “A cluster of four novel human G protein-coupled receptor genes occurring in close proximity to CD22 gene on chromosome 19q13.1”, Biochem. Boiphys. Res. Commun ., 239 , 543-547, 1997 discloses the identification of the GPCR gene GPR 40, located on chromosome 19q13.1, encoding a 300 amino acid protein and identified by GenBank® Accession No. AF024687. .

Haga, H., et al., "Gene-based SNP discovery as part of the Japanese Millennium Genome Project: identification of 190,562 genetic variations in the human genome", J. Hum. Genet ., 47 , 605-610, 2002] shows that nucleotide 632 of the coding sequence (corresponding to nucleotide 642 of the sequence of Genbank Accession No. AF024687) is guanine (G) or adenosine (A) followed by amino acid 211. 190,562 genetic variants of the human genome are disclosed, including a single nucleotide polymorphism of the GPR40 gene, which may be arginine or histidine. The single nucleotide polymorphism was identified with the accession number rs2301151 in the National Center for Biotechnology Information (NCBI) dbSNP database.

International Patent Application Publication No. WO 02/057783 and Briscoe, CP, et al., “The orphan G protein-coupled receptor GPR40 is activated by medium and long chain fatty acids”, J. Biol. Chem ., 278 , 11303-11311, 2003 disclose that GPR40 is specifically expressed in pancreatic β-cells as well as in the brains of humans, mice and rats. In addition, WO 02/057783 and Briscoe et al. Have identified medium and long chain saturated and unsaturated fatty acid ligands of GPR40 from experiments measuring calcium ion concentrations.

Kotarsky, K. et al., “A human cell surface receptor activated by free fatty acids and thiazolidinedione drugs”, Biochem. Biophys. Res. Commun ., 301 , 406-410, 2003 disclose that GPR40 is activated not only by a series of fatty acids but also by antidiabetic thiazolidinedione drugs, as measured by a receptor system bound to the GPR40 receptor.

Itoh, Y., et al., "Free fatty acids regulate insulin secretion from pancreatic β cells through GPR40", Nature , 173-176, 2003, suggest that binding and activation of GPR40 by free fatty acids increases insulin secretion. Is starting.

International Patent Application Publication No. WO 04/072650 discloses nucleic acid sequences and amino acid sequences of human GPR40, and hematological, peripheral and central nervous system disorders, gastrointestinal, respiratory, metabolic, cancer, cardiovascular and urinary diseases in mammals. Inhibition of the treatment is disclosed.

Summary of the Invention

The invention relates, in part, to a genotyping method comprising determining an amino acid encoded by the GPR40 gene of a human subject at amino acid 211. In a preferred embodiment, an amino acid that is encoded at amino acid 211 of the two alleles of the GPR40 gene is determined. In another preferred embodiment, the genotyping method comprises determining whether amino acid 211 is not histidine. In another preferred embodiment, the genotyping method comprises determining whether amino acid 211 is arginine. In another preferred embodiment, the genotyping method comprises determining whether amino acid 211 is histidine. In another preferred aspect, the method for genotyping comprises separating nucleic acids from a human subject; Amplifying a contiguous sequence of said nucleic acid comprising the nucleotide of GPR40 encoding amino acid 211 or its complementary sequence; Treating the contiguous sequence with a nucleotide probe that enables determination of an amino acid encoded at amino acid 211 of GPR40 of the subject; Determining whether the probe hybridizes to the contiguous sequence under very stringent conditions. In a preferred embodiment, the probe comprises SEQ ID NO: 9 or SEQ ID NO: 9 having the length of about 13 to about 35 nucleotides and comprising a nucleotide or complementary nucleotide sequence thereof that binds to a detectable label and encodes amino acid 211. Consists of contiguous portions of the nucleotide sequence of ten.

Another aspect of the invention determines an amino acid encoded by the GPR40 gene of a human subject at amino acid 211, wherein at least one allele of the GPR40 gene, which is encoded amino acid 211 is not histidine and preferably arginine, preferably Provided is a method of treatment comprising treating said subject with a therapeutically effective amount of a medicament for the treatment or prevention of a disease or pathological condition mediated by having both alleles. In a preferred embodiment, the agent is an insulin, insulin secretion stimulating sulfonylurea compound, glycogen phosphorylase inhibitor, biguanide hepatic glucose excretion inhibitor, alpha-glucosidase inhibitor, protein tyrosine phosphatase-1B inhibitor, dipept Thidyl peptidase IV inhibitors, glycogen synthase kinase-3 beta inhibitors, peroxysome growth factor-activated receptor gamma agonists, glucagon receptor antagonists, selective serotonin reuptake inhibitors, 3-hydroxy-3-methyl Glutaryl coenzyme A reductase inhibitors, gamma-aminobutyric acid agonists, angiotensin converting enzyme inhibitors, angiotensin-II receptor antagonists, phosphodiesterase type 5 inhibitors, sorbitol dehydrogenase inhibitors and aldose reductase inhibitors Selected from pharmaceutically acceptable prodrugs thereof or pharmaceutically acceptable salts thereof . In another preferred embodiment, the agent is an anti-obesity agent, preferably an apolipoprotein-B secretion / microsomal triglyceride transport protein inhibitor, an 11β-hydroxy steroid dehydrogenase-1 inhibitor, peptide YY _3-36 , Analogue of peptide YY _3-36 , cannabinoid antagonist, MCR-4 agonist, cholecystokinin-A agonist, monoamine reuptake inhibitor, sympathomimetic, β ₃ adrenergic receptor agonist, dopamine agonist, black Species-stimulating hormone receptor analogs, 5HT2c agonists, melanin enrichment hormone antagonists, leptin, leptin analogs, leptin receptor agonists, galanin antagonists, lipase inhibitors, appetite suppressants, neuropeptide-Y receptor antagonists, thyroid-like drugs , Dehydroepiandrosterone, analog of dehydroepiandrosterone, glucocorticoid receptor agonist, glue Corticoid receptor antagonists, orexin receptor antagonists, glucagon-like peptide-1 receptor agonists, ciliary nerve growth factor, human aguti-related proteins, ghrelin receptor antagonists, histamine 3 receptor antagonists, histamine 3 receptor reverse agonists and A therapeutic agent for obesity selected from neuromedin U receptor agonists.

Another aspect of the invention relates to a kit comprising a nucleotide probe that enables the determination of an amino acid encoded at amino acid 211 of the GPR40 gene of a human subject. In a preferred embodiment, the probe enables determination of whether amino acid 211 of the GPR40 gene is histidine. In another embodiment, the probe enables determination of whether amino acid 211 of the GPR40 gene is not histidine, preferably arginine. In another preferred aspect, the probe comprises SEQ ID NO: 9 or a sequence of about 13 to about 35 nucleotides in length and comprises a nucleotide or a complementary nucleotide sequence thereof that binds a detectable label and encodes amino acids 211 Consisting of contiguous portions of the nucleotide sequence of No. 10. In another preferred aspect, the kit further comprises a first polymerase chain reaction nucleotide primer complementary to a nucleotide sequence underlying a nucleotide encoding amino acid 211 of the GPR40 gene, and a nucleotide encoding amino acid 211. A second polymerase chain reaction primer complementary to the nucleotide sequence above; Or a first primer complementary to a nucleotide sequence above nucleotides encoding amino acid 211, and a second polymerase chain reaction primer complementary to a nucleotide sequence below nucleotides encoding amino acid 211. . In another preferred aspect, the kit also includes a container.

Another aspect of the invention measures competitive inhibition by a test agent of GPR40 ligand binding to a first human GPR40 receptor whose amino acid 211 is histidine, and a GPR40 ligand to a second human GPR40 receptor whose amino acid 211 is not histidine A method of characterizing a medicament, comprising measuring competitive inhibition by said compound of binding. In a preferred embodiment, amino acid 211 of the second receptor is arginine. In another preferred aspect, the method also includes comparing the competitive inhibition by the compound of the binding of the first receptor and the second receptor.

Another aspect of the invention measures activation by a test agent of a first human GPR40 receptor whose amino acid 211 is histidine and measures activation by a test agent of a second human GPR40 receptor whose amino acid 211 is not histidine It relates to a method of characterizing a medicament. In a preferred embodiment, amino acid 211 of the second receptor is arginine. In another preferred aspect, the method also includes comparing the activation by the compound of the first receptor and the second receptor.

Another aspect of the invention measures the inhibition by a test agent of activation by an activator of GPR40 of a first human GPR40 receptor whose amino acid 211 is histidine, wherein the amino acid 211 is not histidine and is preferably arginine. 2 A method of characterizing a medicament comprising measuring inhibition by a medicament of activation of a human GPR40 receptor by an activator of GPR40. In another preferred aspect, the method also includes comparing the inhibition of activation by the compound of the first receptor and the second receptor.

FIG. 1 illustrates the activation of GPR40 by a thiazolidinedione compound compared to HEK 293 cells that do not express GPR40 as measured by intracellular calcium ion release in HEK 293 cells that express GPR40.

Figure 2 shows the comparison of activation by thiazolidinedione compounds of GPR40 with histidine versus arginine at amino acid 211 as measured by intracellular calcium ion release in HEK 293 cells expressing GPR40.

FIG. 3 has 95% confidence limits (dashed lines) and individual data points for each genotype of GPR40 in a population of 2465 subjects (1332 men and 857 women), comparing body mass index versus likelihood of having diabetes The logistic regression curve (continuous line) is shown. Data points on the top of the plot represent body mass index of individuals with diabetes. Data points at the bottom of the plot represent body mass index of individuals without diabetes.

4 shows a restriction map of the expression vector used to prepare HEK 293 cells expressing GPR40.

Figure 5 shows the nucleotides encoding amino acid 211 as arginine and the nucleotide and amino acid sequence encoding human GPR40 (SEQ ID NOs: 1 and 2, respectively), with the amino acid 211 highlighted.

FIG. 6 shows the nucleotides encoding amino acid 211 as histidine and the nucleotide and amino acid sequences encoding human GPR40 (SEQ ID NOs: 3 and 4, respectively), with amino acids 211 highlighted.

7 shows the sequences of PCR primers and PCR probes that can be used to amplify and detect sequences containing the GPR40 allele. 7 also shows the nucleotide sequence encoding amino acid 211 as histidine (SEQ ID NO: 9) or arginine (SEQ ID NO: 10).

8 shows the sequence of an expression vector construct containing a GPR40 allele encoding histidine at amino acid 211.

The terms used herein have their usual meaning in the art. However, for the sake of clarity and convenience of the invention, the meanings of the specific terms and phrases used in the specification, including examples and appended claims, are shown below.

The term “drug (s)” is mediated by having one or more alleles of the GPR40 gene, preferably both alleles, wherein amino acid 211 is further described herein, which is not histidine and is preferably arginine. By substances, and compounds, including peptides, for the treatment or prevention of a disease or pathological condition.

In the context of the GPR40 gene the term “amino acid amino acid 211” refers to amino acid residue 211 in the sequence of Genbank Accession No. AF024687, also shown in SEQ ID NOs: 2 and 4.

The term “very stringent conditions” means washing conditions for 1 hour at 68 ° C. in the presence of about 0.2 × SSC and about 0.1% SDS. Useful variations of these washing conditions will be readily apparent to those of ordinary skill in the art. In general, the stringency of the hybridization is expressed in part with respect to the temperature at which the washing step is carried out. In general, the wash temperature is selected to be about 5-20 ° C. below the thermal melting point (T _m ) for the particular sequence at the defined ionic strength and pH. T _m is the temperature (at defined ionic strength and pH) at which 50% of the target sequence hybridizes to a fully matched probe. Equations for calculating T _m and conditions for nucleic acid hybridization are known and found in Volume 2, Chapter 9 of Sambrook et al., Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Press, Plainview NY, 1989. can see.

The term "diabetes" means diabetes as recognized by those skilled in the art. The term includes type I diabetes, also known as insulin dependent diabetes mellitus (IDDM), and type II diabetes, also known as non-insulin dependent diabetes mellitus (NIDDM). The World Health Organization (WHO) has set the standard for diagnosing diabetes (WHO 1980/85 Technical Report Series No. 646/727). Diabetes is also defined by the National Diabetes Data Group (see National Diabetes Data Group: Classification and diagnosis of diabetes mellitus and other categories of glucose intolerance, Diabetes , 28 : 1039-1044, 1979). However, more recently, for example, guidelines have been published by the American Diabetes Association to enable the diagnosis of type I and type II diabetes (see, eg, ADA, "Standards of medical care in diabetes (Clinical Practice Recommendations 2004) ", Diabetes Care , 27 (Suppl 1), S15-S35, 2004).

The term “therapeutically effective amount” means (i) treating or preventing a particular disease, disorder or disorder; (ii) alleviate, ameliorate or eliminate one or more symptoms of a particular disease, disorder or disorder; Or (iii) an amount of a medicament that prevents or delays the onset of one or more symptoms of a particular disease, disorder or disorder. The therapeutically effective amount can be determined on a personal basis by those of ordinary skill in the art, based on the present disclosure, and at least in part, the species of the subject, the physique of the subject, the type of delivery system used, and the disease. It will be based on consideration of the type of administration as progressing.

The expression “pharmaceutically acceptable salts” optionally includes both pharmaceutically acceptable acid addition salts and pharmaceutically acceptable cationic salts. Pharmaceutically acceptable cationic salts include alkali metal salts (such as sodium and potassium), alkaline earth metal salts (such as calcium and magnesium), aluminum salts, ammonium salts, and organic amines such as benzatin ( N, N'-dibenzylethylenediamine), choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine), benethamine (N-benzylphenylethylamine), diethylamine, pipe Salts with razin, tromethamine (2-amino-2-hydroxymethyl-1,3-propanediol) and procaine, but are not limited thereto. The expression “pharmaceutically acceptable acid addition salts” includes hydrochloride, hydrobromide, sulfate, hydrogen sulfate, phosphate, hydrogen phosphate, dihydrogen phosphate, acetate, succinate, citrate, methanesulfonate (mesylate) and p-toluenesulfonate (tosylate) salts, including but not limited to. Particularly preferred salts are sodium salts. The description of a compound shown herein, including the phrase "its prodrug or pharmaceutically acceptable salt thereof" or a substantially similar phrase, refers to a pharmaceutically acceptable salt of the applicable compound and a pharmaceutically acceptable salt of said prodrug. It is meant to include both salts.

The term “prodrug” means a compound that is converted in vivo to provide a compound of the invention. The conversion can occur by various mechanisms, for example, through blood hydrolysis. A review of the use of prodrugs is described in Higuchi, T. and Stella, W., "Pro-drugs as Novel Delivery Systems", Vol. 14 of the ACS Symposium Series, and Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987. For example, when a compound of the present invention contains a carboxylic acid functional group, the prodrug may comprise an ester resulting from the substitution of a hydrogen atom of an acid group with a group such as: (C ₁ -C ₈ ) alkyl, (C ₂ -C ₁₂ ) alkanoyloxymethyl, 1- (alkanoyloxy) ethyl with 4 to 9 carbon atoms, 1-methyl-1- (alkanoyloxy) -ethyl with 5 to 10 carbon atoms, 3 to 6 Alkoxycarbonyloxymethyl having 4 carbon atoms, 1- (alkoxycarbonyloxy) ethyl having 4 to 7 carbon atoms, 1-methyl-1- (alkoxycarbonyloxy) ethyl having 5 to 8 carbon atoms, 3 to N- (alkoxycarbonyl) aminomethyl with 9 carbon atoms, 1- (N- (alkoxycarbonyl) amino) ethyl, 4-phthalidyl, 4-crotonolactonyl, gamma with 4 to 10 carbon atoms - lactone by beauty-4-yl, di _{-N, N- (C 1 -C 2} ) alkylamino (C ₂ -C ₃₎ alkyl (e.g., b--dimethylaminoethyl), carbazole One - (C ₁ -C ₂₎ alkyl, N, N- di (C ₁ -C ₂₎ alkyl-carbamoyl) - (C ₁ -C ₂₎ alkyl and piperidino-, pyrrolidino-or morpholino (C ₂ -C ₃ ) alkyl. Similarly, when a compound of the present invention comprises an alcoholic functional group, the prodrug may be produced with the hydrogen atoms of the alcohol group substituted by the following groups: (C ₁ -C ₆ ) alkanoyloxymethyl, 1-(( C ₁ -C ₆ ) alkanoyloxy) ethyl, 1-methyl-1-((C ₁ -C ₆ ) alkanoyloxy) ethyl, (C ₁ -C ₆ ) alkoxycarbonyloxymethyl, N- (C ₁ -C ₆ ) alkoxycarbonylaminomethyl, succinoyl, (C ₁ -C ₆ ) alkanoyl, α-amino (C ₁ -C ₄ ) alkanoyl, arylacyl and α-aminoacyl, or α-aminoacyl -α-aminoacyl, wherein each α-aminoacyl group is independently a natural L-amino acid, P (O) (OH) ₂ , -P (O) (O (C ₁ -C ₆ ) alkyl) ₂ or Lycosyl (a radical resulting from the removal of hydroxyl groups in the hemiacetal form of carbohydrates). If the compound of the present invention comprises an amine functional group, the prodrug may be produced in which the hydrogen atoms of the amine group are substituted with the following groups: RX-carbonyl, RXO-carbonyl, NRXRX'-carbonyl (where RX And RX 'are each independently (C ₁ -C ₁₀ ) alkyl, (C ₃ -C ₇ ) cycloalkyl, benzyl, or RX-carbonyl is natural α-aminoacyl or natural α-aminoacyl-natural α- Aminoacyl), -C (OH) C (O) OYX, wherein YX is H, (C ₁ -C ₆ ) alkyl or benzyl), -C (OYX ₀ ) YX ₁ , wherein YX ₀ is ( C ₁ -C ₄ ) alkyl, YX ₁ is (C ₁ -C ₆ ) alkyl, carboxy (C ₁ -C ₆ ) alkyl, amino (C ₁ -C ₄ ) alkyl or mono-N- or di-N, N- (C ₁ -C _{6) alkyl-amino-alkyl), -C (YX 2) YX} 3 ( wherein, YX ₂ is H or methyl, YX ₃ is mono or di -N- -N, N- (C ₁ -C ₆ ) alkylamino, morpholino, piperidin-1-yl or pyrrolidin-1-yl).

"Nucleotide sequence" and "polynucleotide" refer to DNA or RNA in single- or double-stranded form. The term "complementary nucleotide sequence" refers to guanidine nucleotides (G) and cytidine nucleotides (C) and adenosine nucleotides (A) and thymine, except for RNA where T is substituted with uridine nucleotide (U) and U binds to A A nucleotide sequence that anneals (binds) to another nucleotide sequence upon binding to a Dean nucleotide (T).

Abbreviations used herein have their conventional meaning in the art. However, for clarity and convenience of the invention, the meanings of the specific abbreviations are indicated as follows: “° C.” means degrees Celsius; "CO ₂ " means carbon dioxide; "DMEM" refers to Dulbecco's modified Eagle's medium; "DNA" means deoxyribonucleic acid; "FBS" refers to fetal bovine serum; "g" means gram; "HEPES" means 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid; "kg" means kilograms; "mg" means milligrams; "mL" means milliliters; "mM" means millimoles; "Μl" means microliters; "μM" means micromolar; "ng" means nanograms; "nm" means nanometers; "nM" means nanomolar; "PCR" means polymerase chain reaction; "Pen / Sterp" refers to a mixture containing penicillin and streptomycin; "RNA" refers to ribonucleic acid; "RPM" means revolutions per minute; "xg" refers to gravity multiples.

The following amino acid abbreviations are used herein: A-alanine; T-threonine; V-valine; C-cysteine; L-leucine; Y-tyrosine; I-isoleucine; N-asparagine; P-proline; Q-glutamine; F-phenylalanine; D-aspartic acid; W-tryptophan; E-glutamic acid; M-methionine; K-lysine; G-glycine; R-arginine; S-serine; H-histidine.

The GPCR gene, GPR40, identified with GenBank Accession No. AF024687, encodes a 300 amino acid protein [Sawzdargo, M. et al., "A cluster of four novel human G protein-coupled receptor genes occurring in close proximity to CD22 gene on chromosome 19q13.1 ", Biochem. Biophys. Res. Commun ., 239 , 543-547, 1997]. Highly expressed GPR40 in pancreatic islet cells has been associated with insulin secretion regulation and has therefore been suggested as a target for the treatment of diabetes [Briscoe, CP, et al., “The orphan G protein-coupled receptor GPR40 is activated by medium and long chain fatty acids. acids ", J. Biol. Chem ., 278 , 11303-11311, 2003; Itoh, Y., et al., "Free fatty acids regulate insulin secretion from pancreatic β cells through GPR40", Nature , 173-176, 2003]. Kotarsky, K. et al., “A human cell surface receptor activated by free fatty acids and thiazolidinedione drugs”, Biochem. Biophys. Res. Commun ., 301 , 406-410, 2003] suggest that GPR40 mediates the response to thiazolidinedione-type diabetic drugs.

As in other GPCRs, GPR40 is involved in delivering extracellular signals intracellularly. Activation of GPR40 induces intracellular calcium release through pathways involving G _q protein [Briscoe, CP, et al., "The orphan G protein-coupled receptor GPR40 is activated by medium and long chain fatty acids ", J. Biol. Chem ., 278 , 11303-11311, 2003. The resulting calcium efflux is useful for detecting GPR40 activation by methods known to those skilled in the art based on the present disclosure (see, eg, Kotarsky, K. et al., “A human cell surface receptor activated by free fatty acids and thiazolidinedione drugs ", Biochem. Biophys. Res. Commun ., 301 , 406-410, 2003; Briscoe, CP, et al.," The orphan G protein-coupled receptor GPR40 is activated by medium and long chain fatty acids ", J. Biol. Chem ., 278 , 11303-11311, 2003).

Thiazolidinediones and certain fatty acids are known to activate GPR40 [Kotarsky, K. et al., "A human cell surface receptor activated by free fatty acids and thiazolidinedione drugs", Biochem. Biophys. Res. Commun ., 301 , 406-410, 2003; Briscoe, CP, et al., "The orphan G protein-coupled receptor GPR40 is activated by medium and long chain fatty acids", J. Biol. Chem ., 278 , 11303-11311, 2003. As illustrated in FIG. 1, in the present invention, thiazolidinedione compounds stimulate intracellular calcium ion release in cells expressing GPR40 as compared to cells that do not express GPR40.

The present invention is based, in part, on the discovery that single nucleotide polymorphisms affect the activation of GPR40 by, for example, thiazolidinedioone-type drugs. The polymorphism occurring at 632 nucleotides of the GPR40 coding sequence (corresponding to nucleotide 642 in the sequence of Genbank Accession No. AF024687) involves replacing A with a nucleotide other than A, preferably G, from histidine at residue 211. Amino acid substitutions other than histidine, preferably arginine, result in amino acid substitutions. The nucleotide and amino acid sequences of GPR40, wherein amino acid 211 is arginine, are shown in SEQ ID NOs: 1 and 2, respectively. The nucleotide and amino acid sequences of GPR40, wherein amino acid 211 is histidine, are shown in SEQ ID NOs: 3 and 4, respectively.

As illustrated in FIG. 2, 632 nucleotides of GPR40 show a higher response to, for example, thiazolidinedione activation compared to cells with A nucleotides at position 632. FIG. 2 shows the results of a cell-based assay measuring intracellular calcium ions in cells with G to A nucleotides at position 632.

In addition, the present invention is based, in part, on the discovery that individuals with G in nucleotide 642, particularly those with G in both alleles, have a higher risk of diabetes compared to individuals with the nucleotide A. . As illustrated in FIG. 3, subjects having a heterozygous or homozygous nucleotide at nucleotide 642 for a given body mass index have a significantly lower chance of developing type II diabetes than subjects having the nucleotide homozygous G.

The present invention provides methods for characterizing and treating or preventing a disease or pathological condition associated with having one or more alleles of GPR40, preferably both alleles, wherein the encoded amino acid 211 is not histidine and is preferably arginine. It includes. Genotyping of subjects in the methods of the invention can be performed by methods known to those skilled in the art based on the present disclosure. Exemplary methods are described, for example, in Chapter 2 of Dracopoli et al., Current Protocols in Human genetics , John Wiley & Sons, Hoboken, NJ, 2004.

Genotyping can be performed using appropriate tissues of the subject, as known to those skilled in the art, based on the present disclosure. In a preferred embodiment, the DNA for genotyping is isolated from the whole blood sample by procedures known to those skilled in the art based on the present disclosure. The procedure involves a variety of commercially available kits, such as Puregene® DNA Separation Kit (Gentra Systems, Inc., Minneapolis, Minn.), DNA separation for blood Kit (Cat. No. 2-032-805, Roche Diagnostics Corp.), GenomicPrep® Blood DNA Isolation Kit (Cat. No. 27-5236-01, Amersham Bioscience Corporation (Amersham Biosciences Corp.), Piscataway, NJ, PAXgene Blood DNA Kit (Catalog No. 761133, QIAGEN Inc., Valencia, CA), GNOME, Registered Whole blood DNA isolation kit (Cat. No. 2011-600, Qbiogene, Inc., Carlsbad, Calif.), And Wizard (G) DNA DNA Purification Kit (Cat. No. A1120) , Pro The U. S. can be carried out using (Promega U.S.), Madison, WI).

Nucleic acid regions containing a single nucleotide polymorphism can be amplified by PCR techniques using, for example, sense and anti-sense primers and detection probes. However, ligase chain reactions (see, eg, Abravaya, K. et al., Nucleic Acids Research , 23 , 675-682, 1995), branched DNA signal amplification (see, for example, Jrdea, MS et al., AIDS , 7 (supp. 2), S11-514, 1993), isothermal nucleic acid sequence based amplification (NASBA) (see, eg, Kivitts, T. et al., J. Virological , Methods 35 , 273-286, 1991), and other self-sustained sequence replication assays, may also be used.

Methods for making PCR primers and probes are known in the art and are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Press, Plainview NY, 1989; Ausubel et al., Current Protocols in Molecular Biology , John Wiley & Sons, Hoboken, NJ, 1994; and Innis et al., PCR Protocols, A Guide to Methods and Applications , Academic Press, San Diego CA, 1990. PCR primer pairs can be obtained from known sequences using, for example, computer programs designed for the same purpose as primers (version 0.5, 1991, Whitehead Institute for Biomedical Research, Cambridge, Mass.). Can be induced.

Oligonucleotides for use as primers can be selected using software known in the art for this purpose. For example, Oligo® version 6 software (Molecular Biology Insights, Inc., Cascade, Colorado, www.oligo.net ) has less than 100 Useful for the selection of each PCR primer pair of nucleotides. A similar primer selection program inserted additional features in the extended doses. For example, the PrimeOU Primer Screening Program (available to the public at the Genome Center at the University of Texas South West Medical Center in Dallas, Texas, ftp://ftp.genome.ou.edu/pub/programs/primou src) .tar ) can select specific primers from megabase pair sequences, and are therefore useful for designing primers over genome-region ranges. Primer 3, Version 0.9, Primer Screening Program (available to the public at the Whitehead Institute / MIT Center for Genome Research, Cambridge, Mass., Http://www-genome.wi.mit.edu/genome software / other / primer3. html ) allows the user to enter a "mispriming library" in which the sequence avoided as the primer binding site is user-specific. Primer 3 is particularly useful for the selection of oligonucleotides for microarrays (source code for the following two primer selection programs can also be obtained from their respective sources and can be modified to meet the specific needs of the user). PrimeGen program (available to the public at the UK Human Genome Mapping Project Resource Center in Cambridge, UK, http://www.hgmp.mrc.ac.uk/Registered/ Option / primegen.html ) allows the selection of primers that hybridize to either the maximal or minimal conserved regions of the aligned nucleic acid sequence by designing primers based on multiple sequence sequences. Thus, the program is useful for the identification of both unique and conserved oligonucleotides and polynucleotide fragments. Other oligonucleotide selection methods will be apparent to those skilled in the art based on the present disclosure.

Preferably, the length of the sequence used for the probe is minimized using a minor groove binder (MBG) bound to the probe sequence to identify two SNPs. MGB is described, for example, in US Pat. Nos. 5,801,155; 6,084,102; 6,426,408; 6,312,894; And 6,683,173, which are known to those skilled in the art.

In a preferred embodiment, Primer Express® version 1.4 software (Applied Biosystems, Foster City, CA) is used to design PCR primers and probes for the practice of the present invention. Exemplary primers include CTTGGCCATCACAGCCTTCT (SEQ ID NO: 5) for forward primers and CCACGTTGGAGGCGTTGT (SEQ ID NO: 6) for reverse primers. The probes were FAM (TM) and Big (VIC (R) dye-labeled Tagman (TM) MGB probes with SEQ ID NOs: 6FAM-CACTGGCCCACTCT and VIC-CACTGGCCCGCTC (SEQ ID NOs: 7 and 8, respectively). (Applied Biosystems). The isolated DNA of the subject is treated with PCR reagents, including primers and probes, and exposed to repeated thermal cycles to advance the PCR reaction. Detection of SNPs can be performed using, for example, a fluorescent base sequence detection system such as the ABI PRISM® 7900HT sequence detection system (AME Bioscience, Tored, Norway). Can be.

The invention also includes a diagnostic test kit for determining whether a subject has a nucleotide, preferably A, but not A at 632 of the GPR40 coding sequence. For example, the kit may comprise a reagent such as a labeled or labelable nucleic acid probe capable of detecting whether the subject has A or G at 632 of the GPR40 coding sequence. The probe is preferably less than 35 nucleotides in length, more preferably less than 25 nucleotides, even more preferably from 13 to 25 nucleotides in length. The kit may also include reagents for amplifying a nucleic acid region containing nucleotide 632, eg, a PCR primer. The reagent may be packaged in a suitable container. The kit may also include instructions for using the kit to detect whether the subject has A or G at 632 of the GPR40 coding sequence.

The present invention activates GPR40 by comparing activation by a test agent of a first human GPR40 receptor whose amino acid 211 is not histidine but preferably arginine with activation by a test agent of a second human GPR40 receptor whose amino acid 211 is histidine It provides a method for identifying a test agent to make. Any suitable method of measuring the activation of GPR40 may be used in the practice of the present invention. Such methods are known or will be apparent to those skilled in the art based on the present disclosure. For example, in a preferred embodiment, the method is sensitive to calcium in a fluorometric imaging reader system (ie FLIPR®) that measures calcium outflow following activation of G protein coupled receptor mediated through G _q protein. It involves the use of dyes (see, e.g., Chambers, C. et al., "Measuring Intracellular Calcium Fluxes in High Throughput Mode", Combinatorial Screening and High Throughput Screening , 6 , 355-362, 2003). Other methods known in the art include calcium binding proteins [Miyawaki, A. et al., "Fluorescent indicators for Ca2 + based on green fluorescent proteins and calmodulin", Nature , 388 , 882-887, 1997; Kain, SR, "Green fluorescent protein (GFP): applications in cell-based assays for drug discovery", Drug Discov. Today , 4 , 304-312, 1999] and calcium sensitive luciferase [Button, D. and Brownstein, M., "Aequorin-expressing mammalian cell lines used to report Ca2 + mobilization", Cell Calcium , 14 , 663-671, 1997 ; Stables, J. et al., "A bioluminescent assay for agonist activity at potentially any G-protein-coupled receptor", Anal. Biochem ., 252 , 115-126, 1997].

The present invention also provides for the activation of GPR40 by comparing the inhibition of activation by the test agent of the first human GPR40 receptor whose amino acid 211 is histidine to the inhibition of the second human GPR40 receptor whose amino acid 211 is not histidine, preferably arginine. It provides a method for identifying a test agent that inhibits. The method may utilize the activation assay described above using known activators of GPR40 in combination with test agents. The resulting detectable signal is compared to the signal expected when using a known activator alone. Kotarsky, K. et al., “A human cell surface receptor activated by free fatty acids and thiazolidinedione drugs”, Biochem. Biophys. Res. Commun ., 301 , 406-410, 2003; Briscoe, CP, et al., "The orphan G protein-coupled receptor GPR40 is activated by medium and long chain fatty acids", J. Biol. Chem ., 278 , 11303-11311, 2003 and International Patent Application Publication No. WO 02/057783, various GPR40 activators are known in the art and can be used in the process.

The present invention also provides a competitive inhibition of a test agent of known GPR40 ligand binding to a first human GPR40 receptor whose amino acid 211 is histidine, for a second human GPR40 receptor whose amino acid 211 is not histidine and preferably arginine. Methods for identifying test agents that bind GPR40 by comparison with competitive inhibition by the compound of known ligand binding. Such ligands can be labeled with a detectable label as known in the art, including, for example, radioisotopes, fluorescent dyes, and enzymes. Kotarsky, K. et al., “A human cell surface receptor activated by free fatty acids and thiazolidinedione drugs”, Biochem. Biophys. Res. Commun ., 301 , 406-410, 2003; Briscoe, CP, et al., "The orphan G protein-coupled receptor GPR40 is activated by medium and long chain fatty acids", J. Biol. Chem ., 278 , 11303-11311, 2003 and International Patent Application Publication No. WO 02/057783, ligands of various GPR40 are known in the art and can be used in the method.

Those skilled in the art will appreciate, based on the present disclosure, the comparison of activation, inhibition of activation and inhibition of binding of the methods of identifying the test agents of the present invention can be performed by any of a variety of methods. For example, measurement of activation and inhibition of activation of GPR40 can be performed using fluorometry or colorimetric methods (eg FLIPR) to measure calcium efflux. Thus, activation of GPR40 can be accurately quantified by this method using, for example, a photodetector device providing numerical values. The values obtained from these measurements can be compared by automated or manual methods. For example, in particular the actual method by which a large number of values were obtained would include comparing the values using a computerized system by methods known to those skilled in the art.

The invention also encompasses a method of treating or preventing a disease or pathological condition associated with having one or more alleles of GPR40, preferably both alleles, wherein amino acid 211 is not histidine and is preferably arginine. do. Methods of identifying such diseases or conditions are known to those skilled in the art or will be apparent based on the present disclosure. For example, FIG. 3 illustrates the results of a study comparing genotypes of subjects with type II diabetes and those without diabetes. To those skilled in the art, using methods similar to those described in FIGS. 3 and 5, one or more alleles, preferably two, of GPR40 in which the encoded amino acid 211 is not histidine and is preferably arginine It will be apparent that other diseases or conditions associated with having alleles can be identified.

In one aspect of the invention, the disease or pathological condition mediated by having one or more alleles, preferably both alleles, of GPR40, wherein the encoded amino acid 211 is not histidine and is preferably arginine is diabetes, preferably Preferably type II diabetes. In another embodiment, the disease or condition is a complication associated with diabetes. Such complications include atherosclerosis, diabetic cardiomyopathy, cataracts, foot ulcers, diabetic megaangiopathy, diabetic microangiopathy, diabetic nephropathy, diabetic retinopathy and diabetic neuropathy. In another embodiment, the disease or condition is any risk factor associated with metabolic syndrome, including obesity, hypertension, insulin resistance, type II diabetes and dyslipidemia.

The present invention provides a medicament for the treatment or prevention of a disease or pathological condition mediated by having one or more alleles, preferably both alleles, of the GPR40 gene wherein amino acid 211 is not histidine and is preferably arginine. It provides a method of treatment associated with the administration of (defined herein as "drug"). Such agents may be readily apparent to those skilled in the art based on the present disclosure, or may be readily identified by methods known in the art. For example, the drug may include insulin, insulin secretion stimulating sulfonylurea compound, glycogen phosphorylase inhibitor (GPI), biguanide hepatic glucose excretion inhibitor, thiazolidinedioone antidiabetic compound, alpha-glucose Cease Inhibitors, Protein Tyrosine Phosphatase-1B (PTP-1B) Inhibitors, Dipeptidyl Peptidase IV (DPPIV) Inhibitors, Glycogen Synthase Kinase-3 Beta (GSK-3β) Inhibitors, Peroxysome Growth Factors- Activated receptor gamma (PPARγ) agonist, glucagon receptor antagonist, selective serotonin reuptake inhibitor (SSRI), 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor (statin), γ-aminobutyric acid ( GABA) agonists, angiotensin converting enzyme (ACE) inhibitors, angiotensin-II (A-II) receptor antagonists, phosphodiesterase type 5 (PDE-5) inhibitors, sorbitol dehydrogenase inhibitors (SDI) and eggs DOS reductase inhibitors (A RI) may be included.

The medicament may comprise any protein tyrosine phosphatase-1B (PTP-1B) inhibitor. The term protein tyrosine phosphatase-1B inhibitor refers to any compound that inhibits the enzyme protein tyrosine phosphatase-1B. PTP-1B is thought to inhibit insulin's ability to reduce blood sugar levels.

Exemplary PTP-1B inhibitors, assays for identifying such inhibitors, and preferred dosages and administration methods are disclosed in the following US patents, international patent publications, and other publications: US 6,251,936, US 6,221,902, US 6,057,316, US 6,001,867, US 5,753,687, WO 01/46203, WO 01/46204, WO 01/46206, WO 01/17516, WO 00/53583, WO 99/58518, WO 99/61435, WO 99/58521, WO 99/58522 , WO 99/58514, WO 99/58520, WO 99/58519, WO 99/58511, WO 99/61410, WO 99/15529, Malamas, MS, et al., "New azolidinediones as inhibitors of protein tyrosine phosphatase 1B with antihyperglycemic properties ", J. Med. Chem ., 43 , 995-1010, 2000A; Bleasdale, JE, et al., "Small molecule peptidomimetics containing a novel phosphotyrosine bioisostere inhibit protein tyrosine phosphatase 1B and augment insulin action", Biochemistry , 40 , 5642-5654, 2001; Wrobel, J. et al., "PTP1B inhibition and antihyperglycemic activity in the ob / ob mouse model of novel 11-arylbenzo [b] naphtho [2,3-d] furans and 11-arylbenzo [b] naphtho [2,3 -d] thiophenes ", J. Med. Chem ., 42 : 3199-3202, 1999; Malamas, MS, et al., "Novel benzofuran and benzothiophene biphenyls as inhibitors of protein tyrosine phosphatase 1B with antihyperglycemic properties", J. Med. Chem ., 43 , 1293-1310, 2000B; Wang, Q. et al., "Naphthalenebis [alpha, alpha-difluoromethylene phosphonates] as potent inhibitors of protein tyrosine phosphatases", Bioorg. Med. Chem. Lett ., 8 , 345-350, 1998; Andersen, HS et al., "2- (oxalylamino) -benzoic acid is a general, competitive inhibitor of protein-tyrosine phosphatases", J. Biol. Chem ., 275 , 7101-7108, 2000; Iversen, LF et al., "Structure-based design of a low molecular weight, nonphosphorus, nonpeptide, and highly selective inhibitor of protein-tyrosine phophatase 1B", J. Biol. Chem ., 275 , 10300-10307, 2000; Wrobel, J. et al., "Synthesis and PTP1B inhibition of novel 4-aryl-1-oxa-9-thiacyclopenta [b] fluorenes", Bioorg. & Med. Chem. Lett ., 10 , 1535-1538, 2000; and Kees, KL, et al., "New potent antihyperglycemic agents in db / db mice: synthesis and structure-activity relationship studies of (4-substituted benzyl) (trifluoromethyl) pyrazoles and -pyrazolones", J. Med. Chem., 39, 3920-3928, 1996].

The medicament may comprise any glucagon receptor antagonist. The term glucagon receptor antagonist refers to any compound that antagonizes glucagon receptors and inhibits the release of glucose induced by glucagon binding to glucagon receptors. The antagonism is readily measured by those skilled in the art according to assays known to those skilled in the art.

Exemplary glucagon receptor antagonists, assays for identifying such antagonists, and preferred dosages and methods of administration are disclosed in the following US patents, international patent applications and other publications: US 6,218,431, US 5,138,090, WO 98 / 04528, WO 99/01423, WO 00/39088, WO 00/69810, WO 98/21957, WO 98/22109, WO 98/22108, WO 97/16442, Livingston et al., Diabetes , 48 Suppl. 1, 0862, 1999; Madsen, P. et al., "Discovery and structure-activity relationship of the first non-peptide competitive human glucagon receptor antagonists", J. Med. Chem ., 41 , 5150-5157, 1998; de Laszlo, SE et al., "Potent, orally absorbed glucagon receptor antagonists", Bioorg. & Med. Chem. Lett ., 9 , 641-646, 1999; Chang, LL et al., "Substituted imidazoles as glucagon receptor antagonists", Bioorg. & Med. Chem. Lett., 11, 2549-2553, 2001; Cascieri MA et al., "Characterization of a novel, non-peptidyl antagonist of the human glucagone receptor", J. Biol. Chem ., 274 , 8694-8697, 1999; Ling, A. et al., "Identification of alkylidene hydrazides as glucagon receptor antagonists", J. Med. Chem ., 44 , 3141-3149, 2001; and Guillon, J. et al., European Journal of Medicinal Chemistry , 33 , 293-308, 1998].

The medicament may comprise any glycogen synthase kinase-3 beta (GSK-3β) antagonist. Exemplary GSK-3β antagonists, assays for identifying such antagonists, and preferred dosages and methods of administration are disclosed in the following US patents, international patent applications and other publications: US 6,057,286, WO 01/56567 , WO 01/09106, WO 01/49709, WO 01/44246, WO 01/44206, WO 01/42224, WO 00/21927, WO 00/38675, WO 99/65897, WO 98/16528, Coghlan, MP et al., "Substituted imidazoles as glucagon receptor antagonists", Bioorg. & Med. Chem. Lett ., 9 , 641-646, 2000; Smith, DG et al., "3-Anilino-4-arylmaleimides: potent and selective inhibitors of glycogen synthase kinase-3 (GSK-3)", Bioorg. & Med. Chem. Lett ., 11 , 635-639, 2001; Cross, DA et al., "Selective samll-molecule inhibits of glycogen synthase kinase-3 activity protect primary neurones from death", Journal of Neurochemistry , 77 , 94-102, 2001; and Lochhead, PA et al., "Inhibition of GSK-3 selectively reduces glucose-6-phosphatase and phosphatase and phosphoenolypyruvate carboxykinase gene expression", Diabetes , 50 , 937-946, 2001].

The medicament of the present invention may comprise an alpha-glucosidase inhibitor. Any alpha-glucosidase inhibitor can be used as a medicament in the present invention. Exemplary alpha-glucosidase inhibitors include acarbose (also known as Precose®) and myglytol (also known as Glyset®), and these alpha-glucosidase inhibitors Analogs, derivatives, prodrugs and pharmaceutically acceptable salts thereof.

The medicament may include an insulin secretion stimulation sulfonylurea compound. Any insulin secretion stimulating sulfonylurea compound can be used as a medicament in the present invention. Exemplary insulin secretion stimulating sulfonylurea compounds include glyphide, also known as glucotrol® and glucotrol XL®, glymepyride (also known as Amaryl®), glyce Bulides and chlorpropamides (also known as Diabinese®), and analogs, derivatives, prodrugs and pharmaceutically acceptable salts thereof. Preferred insulin secretion stimulating sulfonylurea compounds are glyphides.

The medicament may include a biguanide hepatic glucose excretion inhibitor. Any biguanide hepatic glucose excretion inhibitor can be used as a medicament in the practice of the present invention. Exemplary biguanides are metformin, also known as Glucophage®.

The medicament may include PPARγ agonists, including thiazolidinediones and non-thiazolidinediones compounds. PPARγ agonists increase insulin sensitivity in tissues important for insulin action, such as adipose tissue, skeletal muscle and liver.

Any PPARγ agonist can be used as a medicament in the practice of the present invention. Exemplary PPARγ agonists include the following US patents: US 4,340,605; US 4,342,771; US 4,367,234; US 4,617,312; Those described in US 4, 687,777 and US 4,703,052, and analogs, derivatives, prodrugs and pharmaceutically acceptable salts thereof. Preferred PPARγ agonists include daglitazone, cyglitazone, englitazone, pioglitazone, also known as Actos®, and rosiglitazone, also known as Avandia® and BRL-49653. The PPARγ agonist is preferably in an amount ranging from about 0.1 to about 100 mg / day, preferably from about 0.1 to about 50 mg / day, for the average subject, depending on the thiazolidinedione antidiabetic compound and the route of administration. Administration in single or divided doses. However, some change in dosage will necessarily occur depending on the condition of the subject being treated. In either case, the individual in charge of administration will determine the appropriate dose for the individual subject.

The medicament may include any dipeptidyl peptidase IV (DPP IV) inhibitor. The term DPP IV inhibitor refers to any compound that inhibits the enzyme dipeptidyl peptidase. Such inhibition is readily determined by those skilled in the art according to assays as disclosed in International Patent Application Publication No. WO 98/19998.

Exemplary DPP IV inhibitors include US Pat. Nos. 6,124,305, US 6,110,949 and US 6,124,305, International Patent Application Publication Nos. WO 01/34594, WO 99/61431, WO 98/19998, WO 97/40832 and WO 95/15309, and literature Augustinns, KJL et al., Eur. J. Med. Chem ., 32 , 301-309, 1997, and analogs, derivatives, prodrugs and pharmaceutically acceptable salts thereof.

Preferred dosages and methods of administration are as provided in WO 01/34594 and WO 98/19998. Some variation in dosage may necessarily occur depending on the condition of the subject being treated. In either case, the individual in charge of administration will determine the appropriate dose for the individual subject.

The medicament may also include any selective serotonin reuptake inhibitor (SSRI). The term selective serotonin reuptake inhibitor refers to a compound that inhibits reuptake of serotonin by afferent neurons. Such inhibition is readily determined by those skilled in the art according to standard assays as disclosed in US 4,536,518 and other US patents cited in the following paragraphs.

Preferred SSRIs that can be used according to the invention include pemoxetine, which may be prepared as described in US 3,912,743; Fluoxetine, which may be prepared as described in US 4,314,081; Fluvoxamine, which may be prepared as described in US 4,085,225; Indalpin, which may be prepared as described in US 4,064,255; Indeloxazine, which may be prepared as described in US 4,109,088; Milnacipran, which may be prepared as described in US 4,478,836; Paroxetine, which may be prepared as described in US 3,912,743 or US 4,007,196; Sertraline, which may be prepared as described in US Pat. No. 4,536,518; Sibutramine, which may be prepared as described in US 4,929,629; And gimeldine, which may be prepared as described in US Pat. No. 3,928,369. Fluoxetine is also known as Prozac®. Sertraline hydrochloride, also known as Zoloft®, can be prepared as shown in US Pat. No. 4,536,518. Sibutramine is also known as Meridia®. SSRIs that can be used as medicaments include analogs, derivatives, prodrugs, and pharmaceutically acceptable salts of the foregoing SSRIs.

SSRIs are preferably administered in single or divided doses in an amount ranging from about 0.01 to about 500 mg / kg / day, preferably from about 10 to about 300 mg / day, for the average subject, depending on the SSRI and route of administration. . However, some change in dosage will necessarily occur depending on the condition of the subject being treated. In either case, the individual in charge of administration will determine the appropriate dose for the individual subject.

The agent may also include any 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor (statin). The term 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor is a pharmaceutical compound that inhibits enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase. Say The enzyme is involved in the conversion of HMG-CoA to mevalonate, one of the stages of cholesterol biosynthesis. Such inhibition is readily determined according to standard assays known to those skilled in the art.

Preferred statins which can be used according to the invention include atorvastatin disclosed in US Pat. No. 4,681,893, atorvastatin calcium disclosed in US 5,273,995, cerivastatin disclosed in US 5,502,199, dalvastatin disclosed in European Patent Application Publication No. 738,510 A2. , Fludostatin disclosed in European Patent Application Publication No. 363,934 A1, fluvastatin disclosed in US 4,739,073, lovastatin disclosed in US 4,231,938, mevastatin disclosed in US 3,983,140, pravastatin disclosed in US 4,346,227, US 4,444,784 Simvastatin disclosed in US Pat. No. 4,448,784 and belostatin disclosed in US Pat. No. 4,450,171. Particularly preferred 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors include atorvastatin, lovastatin also known as Lipotor®, lovastatin, also known as Mevacor®, pravacol ( Pravastatin, also known as Pravachol®, and simvastatin, also known as Zocor®; And analogs, derivatives, prodrugs and pharmaceutically acceptable salts thereof.

The statin is preferably in single or divided doses in an amount ranging from about 0.1 to about 1000 mg / kg / day, preferably from about 1 to about 200 mg / kg / day, for the average subject, depending on the statin and the route of administration. Administered. However, some change in dosage will necessarily occur depending on the condition of the subject being treated. In either case, the individual in charge of administration will determine the appropriate dose for the individual subject.

The medicament may include any angiotensin converting enzyme (ACE) inhibitor. The term angiotensin converting enzyme inhibitor refers to a pharmaceutical compound that inhibits angiotensin converting enzyme activity. ACE is involved in the conversion of angiotensin I to angiotensin II, a vasoconstrictor. The activity of an ACE inhibitor can be readily determined by methods known to those skilled in the art, including any standard assays described in the patents listed below.

Preferred ACE inhibitors include: alacepril disclosed in US Pat. No. 4,248,883; Benazepril as disclosed in US 4,410,520; Captopril disclosed in US 4,046,889 and 4,105,776; Seronapril, disclosed in US Pat. No. 4,452,790; Delapril disclosed in US 4,385,051; Enalapril, disclosed in US 4,374,829; Posinopril as disclosed in US 4,337,201; Imadapril as disclosed in US Pat. No. 4,508,727; Lisinopril as disclosed in US 4,555,502; Moexipril as disclosed in US 4,344,949; Mobeltopril, disclosed in Belgian Patent No. 893,553; Perindopril disclosed in US 4,508,729; Quinapril, disclosed in US 4,344,949; Ramipril, disclosed in US Pat. No. 4,587,258; Spirapril, disclosed in US 4,470,972; Temocapryl disclosed in US 4,699,905; And transdolapril, disclosed in US Pat. No. 4,933,361; And analogs, derivatives, prodrugs and pharmaceutically acceptable salts thereof.

The ACE inhibitor is preferably in single or divided doses in an amount ranging from about 0.01 to about 500 mg / kg / day, preferably from about 10 to about 300 mg / day, for the average subject, depending on the ACE inhibitor and the route of administration. Administered. However, some change in dosage will necessarily occur depending on the condition of the subject being treated. In either case, the individual in charge of administration will determine the appropriate dose for the individual subject.

The medicament may comprise any angiotensin-II receptor (A-II) antagonist. The term angiotensin-II receptor antagonist refers to a pharmaceutical compound that blocks the vasoconstrictor effect of angiotensin II by blocking the binding of angiotensin II to the AT ₁ receptor found in many tissues (eg, vascular smooth muscle, adrenal gland). The activity of the A-II antagonist can be readily determined by methods known to those skilled in the art, including any standard assays described in the patents listed below.

Preferred A-II antagonists include: candesartan, which may be prepared as disclosed in US Pat. No. 5,196,444; Eprosartan, which may be prepared as disclosed in US Pat. No. 5,185,351; Irbesartan, which may be prepared as disclosed in US 5,270,317; Losartan, which may be prepared as disclosed in US 5,138,069; And valsartan, which may be prepared as disclosed in US Pat. No. 5,399,578; And analogs, derivatives, prodrugs and pharmaceutically acceptable salts thereof. More preferred angiotensin-II receptor antagonists are losartan, irbesartan and valsartan.

The A-II antagonist is preferably in an amount ranging from about 0.01 to about 500 mg / kg / day, preferably from about 10 to about 300 mg / day, for the average subject, depending on the A-II antagonist and route of administration. In single or divided doses. However, some change in dosage will necessarily occur depending on the condition of the subject being treated. In either case, the individual in charge of administration will determine the appropriate dose for the individual subject.

The medicament may comprise any γ-aminobutyric acid (GABA) agonist. The term γ-aminobutyric acid agonist refers to a pharmaceutical compound that binds to GABA receptors in the central nervous system of a mammal. GABA is a major inhibitory neurotransmitter in the central nervous system of mammals. The activity of GABA agonists is described in Janssens de Verebeke, P. et al., Biochem. Pharmacol ., 31 , 2257-2261, 1982; Loscher, W., Biochem. Pharmacol ., 31 , 837-842, 1982; and / or Phillips, N. et al., Biochem. Pharmacol ., 31 , 2257-2261, 1982, including procedures disclosed in the art, can be readily determined by methods known to those skilled in the art.

Preferred GABA agonists include the following: molar moles, which may be prepared as disclosed in US Pat. No. 3,242,190; Procarbide, which may be prepared as disclosed in US Pat. No. 4,094,992; Rilusol, which may be prepared as disclosed in US Pat. No. 4,370,338; Baclofen, which may be prepared as disclosed in US 3,471,548; Gabapentin (Neurontin®), which may be prepared as disclosed in US Pat. No. 4,024,175; Biivathrin, which may be prepared as disclosed in US Pat. No. 3,960,927; Tiagabine (Gabitril®), which may be prepared as disclosed in US Pat. No. 5,010,090; Lamotrigine (Lamictal®), which may be prepared as disclosed in US Pat. No. 4,602,017; Pregabalin, which may be prepared as disclosed in US Pat. No. 6,028,214; Phenytoin (Dilantin®), which may be prepared as disclosed in US 2,409,754; Carbamazepine (Tegretol®), which may be prepared as disclosed in US 2,948,718; and Topiramate (Topamax®), which may be prepared as disclosed in US 4,513,006; And analogs, derivatives, prodrugs and pharmaceutically acceptable salts of said GABA agonists.

In general, according to the present invention, the GABA agonist used as a medicament will be administered in a single or divided dose in a dosage of about 4 to about 60 mg / kg body weight of the subject to be treated per day. However, some change in dosage will necessarily occur depending on the condition of the subject being treated. In either case, the individual in charge of administration will determine the appropriate dose for the individual subject. In particular, when used as a GABA agonist in the present invention, pregabalin is administered at about 300 to about 1200 mg per day; Gabapentin will be administered at about 600 to about 3600 mg per day.

The medicament may comprise any glycogen phosphorylase inhibitor (GPI). The term glycogen phosphorylase inhibitor refers to any substance or compound, or any mixture of substances and / or compounds, that reduces, delays or excludes the enzymatic action of glycogen phosphorylase. This action is readily determined by those skilled in the art according to standard assays as described in US Pat. No. 5,988,463. US 5,988,463, International Patent Application Publication Nos. WO 96/39384 and WO 96/39385 illustrate GPIs which may be medicaments.

The GPI is preferably in single or divided doses in an amount ranging from about 0.005 to about 50 mg / kg / day, preferably from about 0.1 to about 15 mg / kg / day, for the average subject, depending on the GPI and route of administration. Administered. However, some change in dosage will necessarily occur depending on the condition of the subject being treated. In either case, the individual in charge of administration will determine the appropriate dose for the individual subject.

The medicament may include any sorbitol dehydrogenase inhibitor (SDI). The term sorbitol dehydrogenase inhibitor refers to any substance or compound, or any mixture of substances and / or compounds, that reduces, delays or excludes the enzymatic action of sorbitol dehydrogenase. Sorbitol dehydrogenase promotes the oxidation of sorbitol to fructose.

Exemplary SDIs include those disclosed in commonly assigned US 5,728,704, US 5,866,578 and International Patent Application Publication No. WO 00/59510; And analogs, derivatives, prodrugs and pharmaceutically acceptable salts thereof.

SDI is preferably in single or divided doses in an amount ranging from about 0.001 to about 100 mg / kg / day, preferably from about 0.01 to about 10 mg / kg / day, for the average subject, depending on the SDI and route of administration. Administered. However, some change in dosage will necessarily occur depending on the condition of the subject being treated. In either case, the individual in charge of administration will determine the appropriate dose for the individual subject.

The medicament may include any phosphodiesterase type 5 (PDE-5) inhibitor. The term phosphodiesterase type 5 inhibitor is any substance or compound, or substance and / or compound that reduces, delays or excludes the enzymatic action of cyclic guanosine monophosphate (c-GMP) -specific PDE-5. Refers to any mixture. This action is readily determined by those skilled in the art according to assays as described in International Patent Application Publication No. WO 00/24745.

The following patent documents illustrate phosphodiesterase type 5 inhibitors that can be used as medicaments of the present invention and refer to methods of making such phosphodiesterase type 5 (PDE-5) inhibitors: Patent Application Publication No. WO 00/24745; International Patent Application Publication No. WO 94/28902; European Patent Application Publication No. 0463756A1; European Patent Application Publication No. 0526004A1 and European Patent Application Publication No. 0201188A2. Preferred phosphodiesterase type 5 inhibitors include sildenafil (preferably sildenafil citrate, also known as Viagra®), which may be prepared as shown in US 5,250,534 and 5,955,611; Tadalafil (also known as Cialis®), which may be prepared as shown in US 5,859,006; And vardinafil (also known as Levitra®), which may be prepared as shown in US Pat. No. 6,362,178. Exemplary PDE-5 inhibitors also include analogs, derivatives, prodrugs and pharmaceutically acceptable salts of the PDE-5 inhibitors listed above.

The PDE-5 inhibitor is preferably single or divided in an amount ranging from about 5 to about 500 mg / day, preferably from about 10 to about 250 mg / day, for the average subject, depending on the PDE-5 inhibitor and the route of administration. Administered in doses. However, some change in dosage will necessarily occur depending on the condition of the subject being treated. In either case, the individual in charge of administration will determine the appropriate dose for the individual subject.

The medicament may include any aldose reductase inhibitor. The term aldose reductase inhibitor refers to a compound that inhibits the bioconversion of glucose to sorbitol promoted by the enzyme aldose reductase. Exemplary aldose reductase inhibitors include fornalats disclosed in US Pat. No. 4,251,528; Tolesatt disclosed in US Pat. No. 4,600,724; Epalestat, disclosed in US 4,464,382, 4,791,126 and 4,831045; Genaresstat disclosed in US 4,734,419 and 4,883,800; Zopolestat, disclosed in US 4,939,140; And preferred aldose reductase inhibitors disclosed in US Pat. No. 6,579,879. Exemplary aldose reductase inhibitors also include analogs, derivatives, prodrugs and pharmaceutically acceptable salts of the aldose reductase inhibitors listed above.

The amount of aldose reductase inhibitor administered per dose, and the interval between administrations, depends on the aldose reductase inhibitor used, the type of pharmaceutical composition used, the nature of the subject being treated and, if present, the severity of the disease being treated. Will be different. The aldose reductase inhibitor preferably ranges from about 0.001 to about 1000 mg / kg / day, preferably from about 0.01 to about 500 mg / kg / day, for the average subject, depending on the aldose reductase inhibitor and the route of administration. In single or divided doses. However, some change in dosage will necessarily occur depending on the condition of the subject being treated. In either case, the individual in charge of administration will determine the appropriate dose for the individual subject. Preferred aldose reductase inhibitors are zopolestats, which are preferably administered at a dosage of 250 to 500 mg / day.

The medicament may also include the following anti-obesity agents: apolipoprotein-B secretion / microsomal triglyceride carrier protein (apo-B / MTP) inhibitors, 11β-hydroxy steroid dehydrogenase-1 (11β) -HSD type 1) inhibitors, peptide YY _3-36 or analogues thereof, cannabinoid antagonists (e.g. CB-1 antagonists, e.g. limonabant and purine described in US Patent Publication No. 2004/0092520) Compounds), MCR-4 agonists, cholecystokinin-A (CCK-A) agonists, monoamine reuptake inhibitors (eg sibutramine), sympathomimetic agents, β ₃ adrenergic receptor agonists, dopamine agonists (eg For example, bromocriptine), melanoma-stimulating hormone receptor analogs, 5HT2c agonists, melanin enriched hormone antagonists, leptin (OB protein), leptin analogs, leptin receptor agonists, galanin antagonists, lipase inhibitors (eg For example , Tetrahydrolipstatin, ie orlistat), anorexia antagonists (eg bombesin agonists), neuropeptide-Y receptor antagonists (eg NPY Y5 receptor antagonists, eg US 6,566,367). , 6,649,624, 6,638,942, 6,605,720, 6,495,559, 6,462,053, 6,388,077, 6,335,345 and 6,326,375, US Patent Publication Nos. 2002/0151456 and 2003/036652, and PCT Publication Nos. WO 03/010175, WO 03/082190 and WO 02/048152 Spiro compounds), thyroid-like drugs, dehydroepiandrosterone or analogues thereof, glucocorticoid receptor agonists or antagonists, orexin receptor antagonists, glucagon-like peptide-1 receptor agonists, ciliary nerve growth factor ( For example, Regeneron Pharmaceuticals, Inc. of Tarrytown, NY, and Procter & Gamble Co. of Cincinnati, Ohio. Stand in the evil Soka commercially available (Axokine, TM)), human agouti-related protein (AGRP), ghrelin receptor antagonists, histamine 3 receptor antagonists or inverse agonists, neuromedin U receptor agonists and the like. Other anti-obesity agents are known or will be readily apparent to those of ordinary skill in the art in light of the present disclosure. Orlistat (prepared as described in US 5,274,143, 5,420,305 and 5,540,917), sibutramine (prepared as described in US 4,929,629), bromocriptine (US 3,752,814 and 3,752,888), ephedrine, leptin, pseudoephedrine, limonavan (Prepared as described in US 5,624,941), peptide YY _3-36 or analogues thereof (prepared as described in US Patent Publication No. 2002/0141985 and PCT Publication No. WO 03/027637), and NPY Y5 receptors Antiobesity agents selected from the group consisting of antagonist 2-oxo-N- (5-phenylpyrazinyl) spiro- [isobenzofuran-1 (3H), 4'-piperidine] -1'-carboxamide desirable. Another preferred NPY Y5 receptor antagonist is 3-oxo-N- (5-phenyl-2-pyrazinyl) -spiro [isobenzofuran-1 (3H), 4'-piperidine] -1'-carboxamide ; 3-oxo-N- (7-trifluoromethylpyrido [3,2-b] pyridin-2-yl) -spiro- [isobenzofuran-1 (3H), 4'-piperidine] -1 '-Carboxamide; N- [5- (3-fluorophenyl) -2-pyrimidinyl] -3-oxospyro- [isobenzofuran-1 (3H), [4'-piperidine] -1'-carboxamide ; Trans-3'-oxo-N- (5-phenyl-2-pyrimidinyl)] spiro [cyclohexane-1,1 '(3'H) -isobenzofuran] -4-carboxamide; Trans-3'-oxo-N- [1- (3-quinolyl) -4-imidazolyl] spiro [cyclohexane-1,1 '(3'H) -isobenzofuran] -4-carbox Amides; Trans-3-oxo-N- (5-phenyl-2-pyrazinyl) spiro [4-azaiso-benzofuran-1 (3H), 1'-cyclohexane] -4'-carboxamide; Trans-N- [5- (3-fluorophenyl) -2-pyrimidinyl] -3-oxospyro [5-azaisobenzofuran-1 (3H), 1'-cyclohexane] -4'- Carboxamide; Trans-N- [5- (2-fluorophenyl) -2-pyrimidinyl] -3-oxospyro [5-azaisobenzofuran-1 (3H), 1'-cyclohexane] -4'- Carboxamide; Trans-N- [1- (3,5-difluorophenyl) -4-imidazolyl] -3-oxospyro [7-azaisobenzofuran-1 (3H), 1'-cyclohexane]- 4'-carboxamide; Trans-3-oxo-N- (1-phenyl-4-pyrazole) spiro [4-azaisobenzofuran-1 (3H), 1'-cyclohexane] -4'-carboxamide; Trans-N- [1- (2-fluorophenyl) -3-pyrazolyl] -3-oxospyro [6-azaisobenzofuran-1 (3H), 1'-cyclohexane] -4'-ca Voxamides; Trans-3-oxo-N- (1-phenyl-3-pyrazolyl) spiro [6-azaisobenzofuran-1 (3H), 1'-cyclohexane] -4'-carboxamide; Trans-3-oxo-N- (2-phenyl-1,2,3-triazol-4-yl) spiro [6-azaisobenzofuran-1 (3H), 1'-cyclohexane] -4 ' -Carboxamides, including those described in PCT Publication No. WO 03/082190, and pharmaceutically acceptable salts and prodrugs thereof. Preferably, the compounds of the present invention and combination therapies are administered in conjunction with exercise and appropriate diet.

Agents are used alone or in combination with one or more other agents in the methods of the present invention. A medicament may be administered alone or in combination with one or more pharmaceutically acceptable carriers, diluents or fillers. Pharmaceutical compositions containing medicaments can be easily administered in a variety of dosage forms such as tablets, powders, lozenges, syrups, injections and the like. The pharmaceutical composition may, if very desired, contain additional ingredients such as perfumes, binders, excipients and the like. For oral administration, tablets containing various excipients such as sodium citrate, calcium carbonate and calcium diphosphate, together with binders such as polyvinylpyrrolidone, sucrose, gelatin and acacia, can be used for various disintegrants, for example It can be used with starch, alginic acid and certain complex silicates. In addition, lubricants such as magnesium stearate, sodium lauryl sulfate and talc are often useful for tableting purposes. Solid compositions of a similar type may also be used as fillers in filled soft and hard gelatin capsules. Preferred materials for this use include lactose or lactose and high molecular weight polyethylene glycols. When aqueous suspensions or elixirs are preferred for oral administration, the medicaments therein are various sweetening or flavoring agents, coloring materials or dyes, and, if desired, together with diluents such as water, ethanol, propylene glycol, glycerin or various mixtures thereof. Can be mixed with emulsifiers or suspending agents.

For parenteral administration, solutions of medicaments useful in the present invention in sesame or peanut oil, aqueous propylene glycol or sterile aqueous solutions can be used. The aqueous solution should be properly buffered if necessary, and the liquid diluent should first be isotonic with sufficient saline or glucose. Such specific aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this regard, the sterile aqueous media used can all be readily obtained by standard techniques known to those skilled in the art. Methods of preparing various pharmaceutical compositions having specific amounts of active ingredients are known to those skilled in the art or will be apparent in light of the present disclosure. For example, methods for preparing pharmaceutical compositions are described in Gennaro, AR, "Remington: The Science and Practice of Pharmacy", Lippincott Williams & Wilkins, Philadelphia, PA, 20th Edition, 2003.

The disclosures of all patents, applications, publications and documents cited herein, including pamphlets and technical bulletins, are incorporated herein by reference in their entirety. It is believed that one skilled in the art can, based on the description of the invention, including examples, drawings and appended claims, utilize the invention to its fullest extent.

It is believed that one skilled in the art can, using the description of the invention including the examples, the sequence table and the appended claims, utilize the present invention to its fullest extent. The following examples are merely to illustrate the practice of the invention and should not be considered as limiting the rest of the disclosure in any way whatsoever.

Example 1

GPR40 expressing 293 cells

Expression vector constructs containing the GPR40 gene allele were prepared based on the vector, pIRESpuro (Catalog No. 6031-1, BD Biosciences, Franklin Lakes, NJ). One such expression vector construct has a sequence of SEQ ID NO: 11 and a restriction enzyme map shown in FIG. 4. Human fetal kidney (HEK) 293 cells transformed with adenovirus 5 (available from ATCC, catalog number CRL-1573) were transferred to DMEM (Cat. No. 11995-065, Invitrogen, Carlsbad, CA), 10% FBS. (Catalog No. 16140-071, Invitrogen) and 100 units pen / strep (Catalog No. 15140-122, Invitrogen). Once the cells reached 80% confluence, they were expressed using a GPR40 containing expression vector using Fugene-6 reagent (Roche Diagnostics Corp., Indianapolis, Indiana). Transfection. After 48 hours of incubation, the medium was aspirated and fresh medium containing DMEM, 10% FBS, 100 unit pen / strep and 1 μg / mL of puromycin was added. At week 4, cloning cylinders (Sigma-Aldrich Co., St. Louis, MO) were used to select stable clones with puromycin resistance delivered by the pIRESpuro vector. These colonies were further amplified for 4 weeks and fast growing colonies were selected to obtain HEK 293 / GPR40 cells for use in the FLIPR assay of Example 2.

Example 2

GPR40 FLIPR Method

On day 1, HEK 293 / GPR40 cells and control HEK 293 cells prepared according to Example 1 were added poly-D- with a clear bottom, adding 1 μg / mL of puromycin to HEK 293 / GPR40 cells only. 10,000 cells per well (3.3 × per well) in growth medium containing DMEM, 10% FBS and pen / strep (100 μg / mL) in lysine-coated 384-well black plates (Cat. No. 354663, BD Biosciences) 30 μl of 10 ⁵ cells / mL solution were plated and incubated at 37 ° C. under 5% CO ₂ . On day 2, remove the medium from each well and replace with 30 μl of serum-free medium (containing DMEM and pen / strep only, FBS free), with 1 μg / mL of puromycin HEK 293 / GPR40 Only cells were added. On day 3, a dye vial of the FLIPR® Ca 3 Analysis Kit (Cat. No. R8091, Molecular Devices, Sunnyvale, CA) was analyzed with 11 mL Hank buffered saline solution (FLIPR®) analysis. Components of the kit), and 225 μl of freshly prepared 250 mM probeneside stock [5N NaOH (1 mL), probeneside (0.74 g) (Cat. No. P8761, Sigma-Aldrich Company) and 1 × FLIPR buffer (9 mL) (pH 7.4, NaCl (84.68 g) per liter, anhydrous D (+) glucose (18.02 g), MgSO ₄ * 7H ₂ O (1.2 g), KCl (3.73 g), HEPES (23.8 g) and Containing from 10 × FLIPR buffer containing CaCl ₂ (1.11 g). Fatty acids and other GPR40 ligand test compounds were diluted in FLIPR / DMSO (1 × FLIPR buffer containing 0.25% DMSO) at a concentration of 0.05-100 μM. 30 μl of dye was added to each plate well and the plate was incubated at 37 ° C. and 5% CO ₂ for 1 hour. Calcium effluent was measured using a FLIPR plate reader (Molecula device). Test compound solutions were injected into each well at a rate of 10 μl per second (15 μl per well). The exposure was done in just 2 seconds for 0.4 seconds per exposure for 90 exposures.

Example 3

DNA isolation and purification

DNA was isolated and purified from whole blood samples using a Purified® DNA Separation Kit (Gentra Systems, Inc., Minneapolis, Minn.) According to the manufacturer's instructions. Alternatively, whole blood samples by mixing thawed whole blood (1 mL) in an RBC lysate (Gentra Systems, Inc.), for example, in a 3: 1 ratio (ie, 3 mL RBC lysates to 1 mL whole blood). Erythrocyte cells can be lysed from and mixed. The solution was incubated for 10 minutes at room temperature. After incubation, the solution was centrifuged at 1,800 × g for 10 minutes. The supernatant was decanted from the tube and the resulting pellet was resuspended in the residual supernatant. Cell lysate (1 mL) (Gentra Systems, Inc.) was then added, for example, in a volume equivalent to the initial whole blood volume. RNase A solution (5 μL) (Gentra Systems, Inc.) was added to the cell lysate and the tube was incubated at 37 ° C. for 15 minutes. The sample was then cooled to room temperature, protein precipitation solution (333 μl) (Gentra Systems, Inc.) was added to the cell lysate and the mixture was vortexed at high speed for at least 20 seconds to mix. The mixture was then centrifuged at 1,800 × g for 10 minutes to obtain dense protein pellets. The resulting DNA-containing supernatant was poured into a clean 15 mL centrifuge tube containing 10 mL of 100% isopropanol (2-propanol). Samples were mixed by gently inverting the tube (about 40-60 times) until DNA precipitated. If no DNA was visible, 7 μl of glycogen (20 μg / mL) per mL of whole blood (original starting material) was added and incubated for 15 minutes at room temperature. The sample was then centrifuged at 1,800 × g for 3 minutes to obtain small white DNA pellets. The supernatant was decanted and the tube drained to the absorbent site. 10 mL 70% ethanol was added and the tube was inverted several times to wash the DNA pellet. Samples were centrifuged at 1,800 × g for 1 minute. The supernatant was then decanted and the tubes turned upside down and air-dried.

Example 4

Allele identification

Primers and Tagman® MGB probes (Applied Biosystems) were designed using Software Primer Express Version 1.5 (Applied Biosystems). 2.5 μl of 2 × Tagman® Universal PCR Master Mix (without AmpErase), 200 nM of each probe, 900 nM forward and reverse primers and 5 μl of water to make 5 μl PCR reactions were performed by volume. Alternatively, primers and probes were obtained as TaqMan Assays-on Demand® (Applied Biosystems) and 2.5 μL of 2 × Tagman® Universal PCR Master Mix (AmpErase free). ), And 5 μl of 2.25 μl of water and 0.25 μl of 20 × assay mix. 5 μl reaction mixture was added to about 10 ng of dried DNA in a well of a 96 well plate. The plate was placed on a titration shaker and shaken vigorously for 10 minutes and then simply rotated at 1000 RPM. The heat cycle was performed in a GeneAmp® PCR System 9700 (Applied Biosystems) with a double 384-well sample block module using the following heat cycle conditions: 10 minutes at 95 ° C., 92 ° C. At 15 seconds and 40 minutes at 60 ° C. Fluorescent signals were detected using an ABI Prism 7900HT sequence detector (Applied Biosystems) according to the manufacturer's instructions. Data was analyzed using Sequence Detection System (SDS) software version 2.1 (Applied Biosystems).

Example 5

Variant Study on GPR40 in Patients with Type II Diabetes

Of the 2465 subjects analyzed, 1332 were male and 857 were female. Allele frequency was assessed by counting the presence of a particular allele of GPR40, then dividing by twice the number of subjects and counting the two alleles provided by each subject. Genotype frequency was assessed by counting the presence of a particular genotype and dividing by the number of subjects.

references

                         SEQUENCE LISTING <110> Pfizer Inc.        Houseknecht, Karen L.        Banerjee, Poulabi   <120> RNA BIOASSAY <130> PC26139A <150> US 60 / 584,686 <151> 2004-06-30 <160> 11 <170> PatentIn version 3.3 <210> 1 <211> 900 <212> DNA <213> Homo sapiens <400> 1 atggacctgc ccccgcagct ctccttcggc ctctatgtgg ccgcctttgc gctgggcttc 60 ccgctcaacg tcctggccat ccgaggcgcg acggcccacg cccggctccg tctcacccct 120 agcctggtct acgccctgaa cctgggctgc tccgacctgc tgctgacagt ctctctgccc 180 ctgaaggcgg tggaggcgct agcctccggg gcctggcctc tgccggcctc gctgtgcccc 240 gtcttcgcgg tggcccactt cttcccactc tatgccggcg ggggcttcct ggccgccctg 300 agtgcaggcc gctacctggg agcagccttc cccttgggct accaagcctt ccggaggccg 360 tgctattcct ggggggtgtg cgcggccatc tgggccctcg tcctgtgtca cctgggtctg 420 gtctttgggt tggaggctcc aggaggctgg ctggaccaca gcaacacctc cctgggcatc 480 aacacaccgg tcaacggctc tccggtctgc ctggaggcct gggacccggc ctctgccggc 540 ccggcccgct tcagcctctc tctcctgctc ttttttctgc ccttggccat cacagccttc 600 tgctacgtgg gctgcctccg ggcactggcc cgctccggcc tgacgcacag gcggaagctg 660 cgggccgcct gggtggccgg cggggccctc ctcacgctgc tgctctgcgt aggaccctac 720 aacgcctcca acgtggccag cttcctgtac cccaatctag gaggctcctg gcggaagctg 780 gggctcatca cgggtgcctg gagtgtggtg cttaatccgc tggtgaccgg ttacttggga 840 aggggtcctg gcctgaagac agtgtgtgcg gcaagaacgc aagggggcaa gtcccagaag 900 <210> 2 <211> 300 <212> PRT <213> Homo sapiens <400> 2 Met Asp Leu Pro Pro Gln Leu Ser Phe Gly Leu Tyr Val Ala Ala Phe 1 5 10 15 Ala Leu Gly Phe Pro Leu Asn Val Leu Ala Ile Arg Gly Ala Thr Ala             20 25 30 His Ala Arg Leu Arg Leu Thr Pro Ser Leu Val Tyr Ala Leu Asn Leu         35 40 45 Gly Cys Ser Asp Leu Leu Leu Thr Val Ser Leu Pro Leu Lys Ala Val     50 55 60 Glu Ala Leu Ala Ser Gly Ala Trp Pro Leu Pro Ala Ser Leu Cys Pro 65 70 75 80 Val Phe Ala Val Ala His Phe Phe Pro Leu Tyr Ala Gly Gly Gly Phe                 85 90 95 Leu Ala Ala Leu Ser Ala Gly Arg Tyr Leu Gly Ala Ala Phe Pro Leu             100 105 110 Gly Tyr Gln Ala Phe Arg Arg Pro Cys Tyr Ser Trp Gly Val Cys Ala         115 120 125 Ala Ile Trp Ala Leu Val Leu Cys His Leu Gly Leu Val Phe Gly Leu     130 135 140 Glu Ala Pro Gly Gly Trp Leu Asp His Ser Asn Thr Ser Leu Gly Ile 145 150 155 160 Asn Thr Pro Val Asn Gly Ser Pro Val Cys Leu Glu Ala Trp Asp Pro                 165 170 175 Ala Ser Ala Gly Pro Ala Arg Phe Ser Leu Ser Leu Leu Leu Phe Phe             180 185 190 Leu Pro Leu Ala Ile Thr Ala Phe Cys Tyr Val Gly Cys Leu Arg Ala         195 200 205 Leu Ala Arg Ser Gly Leu Thr His Arg Arg Lys Leu Arg Ala Ala Trp     210 215 220 Val Ala Gly Gly Ala Leu Leu Thr Leu Leu Leu Cys Val Gly Pro Tyr 225 230 235 240 Asn Ala Ser Asn Val Ala Ser Phe Leu Tyr Pro Asn Leu Gly Gly Ser                 245 250 255 Trp Arg Lys Leu Gly Leu Ile Thr Gly Ala Trp Ser Val Val Leu Asn             260 265 270 Pro Leu Val Thr Gly Tyr Leu Gly Arg Gly Pro Gly Leu Lys Thr Val         275 280 285 Cys Ala Ala Arg Thr Gln Gly Gly Lys Ser Gln Lys     290 295 300 <210> 3 <211> 900 <212> DNA <213> Homo sapiens <400> 3 atggacctgc ccccgcagct ctccttcggc ctctatgtgg ccgcctttgc gctgggcttc 60 ccgctcaacg tcctggccat ccgaggcgcg acggcccacg cccggctccg tctcacccct 120 agcctggtct acgccctgaa cctgggctgc tccgacctgc tgctgacagt ctctctgccc 180 ctgaaggcgg tggaggcgct agcctccggg gcctggcctc tgccggcctc gctgtgcccc 240 gtcttcgcgg tggcccactt cttcccactc tatgccggcg ggggcttcct ggccgccctg 300 agtgcaggcc gctacctggg agcagccttc cccttgggct accaagcctt ccggaggccg 360 tgctattcct ggggggtgtg cgcggccatc tgggccctcg tcctgtgtca cctgggtctg 420 gtctttgggt tggaggctcc aggaggctgg ctggaccaca gcaacacctc cctgggcatc 480 aacacaccgg tcaacggctc tccggtctgc ctggaggcct gggacccggc ctctgccggc 540 ccggcccgct tcagcctctc tctcctgctc ttttttctgc ccttggccat cacagccttc 600 tgctacgtgg gctgcctccg ggcactggcc cactccggcc tgacgcacag gcggaagctg 660 cgggccgcct gggtggccgg cggggccctc ctcacgctgc tgctctgcgt aggaccctac 720 aacgcctcca acgtggccag cttcctgtac cccaatctag gaggctcctg gcggaagctg 780 gggctcatca cgggtgcctg gagtgtggtg cttaatccgc tggtgaccgg ttacttggga 840 aggggtcctg gcctgaagac agtgtgtgcg gcaagaacgc aagggggcaa gtcccagaag 900 <210> 4 <211> 300 <212> PRT <213> Homo sapiens <400> 4 Met Asp Leu Pro Pro Gln Leu Ser Phe Gly Leu Tyr Val Ala Ala Phe 1 5 10 15 Ala Leu Gly Phe Pro Leu Asn Val Leu Ala Ile Arg Gly Ala Thr Ala             20 25 30 His Ala Arg Leu Arg Leu Thr Pro Ser Leu Val Tyr Ala Leu Asn Leu         35 40 45 Gly Cys Ser Asp Leu Leu Leu Thr Val Ser Leu Pro Leu Lys Ala Val     50 55 60 Glu Ala Leu Ala Ser Gly Ala Trp Pro Leu Pro Ala Ser Leu Cys Pro 65 70 75 80 Val Phe Ala Val Ala His Phe Phe Pro Leu Tyr Ala Gly Gly Gly Phe                 85 90 95 Leu Ala Ala Leu Ser Ala Gly Arg Tyr Leu Gly Ala Ala Phe Pro Leu             100 105 110 Gly Tyr Gln Ala Phe Arg Arg Pro Cys Tyr Ser Trp Gly Val Cys Ala         115 120 125 Ala Ile Trp Ala Leu Val Leu Cys His Leu Gly Leu Val Phe Gly Leu     130 135 140 Glu Ala Pro Gly Gly Trp Leu Asp His Ser Asn Thr Ser Leu Gly Ile 145 150 155 160 Asn Thr Pro Val Asn Gly Ser Pro Val Cys Leu Glu Ala Trp Asp Pro                 165 170 175 Ala Ser Ala Gly Pro Ala Arg Phe Ser Leu Ser Leu Leu Leu Phe Phe             180 185 190 Leu Pro Leu Ala Ile Thr Ala Phe Cys Tyr Val Gly Cys Leu Arg Ala         195 200 205 Leu Ala His Ser Gly Leu Thr His Arg Arg Lys Leu Arg Ala Ala Trp     210 215 220 Val Ala Gly Gly Ala Leu Leu Thr Leu Leu Leu Cys Val Gly Pro Tyr 225 230 235 240 Asn Ala Ser Asn Val Ala Ser Phe Leu Tyr Pro Asn Leu Gly Gly Ser                 245 250 255 Trp Arg Lys Leu Gly Leu Ile Thr Gly Ala Trp Ser Val Val Leu Asn             260 265 270 Pro Leu Val Thr Gly Tyr Leu Gly Arg Gly Pro Gly Leu Lys Thr Val         275 280 285 Cys Ala Ala Arg Thr Gln Gly Gly Lys Ser Gln Lys     290 295 300 <210> 5 <211> 20 <212> DNA <213> Homo sapiens <400> 5 cttggccatc acagccttct 20 <210> 6 <211> 18 <212> DNA <213> Homo sapiens <400> 6 ccacgttgga ggcgttgt 18 <210> 7 <211> 14 <212> DNA <213> Homo sapiens <400> 7 cactggccca ctct 14 <210> 8 <211> 13 <212> DNA <213> Homo sapiens <400> 8 cactggcccg ctc 13 <210> 9 <211> 71 <212> DNA <213> Homo sapiens <400> 9 cttctgctac gtgggctgcc tccgggcact ggcccgctcc ggcctgacgc acaggcggaa 60 gctgcgggcc g 71 <210> 10 <211> 71 <212> DNA <213> Homo sapiens <400> 10 cttctgctac gtgggctgcc tccgggcact ggcccactcc ggcctgacgc acaggcggaa 60 gctgcgggcc g 71 <210> 11 <211> 6201 <212> DNA <213> Expression Vector <400> 11 gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 60 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420 attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 480 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 900 gtttatcaca agtttgtaca aaaaagcagc tggcgccgga accaattcag tcgactggat 960 ccggtaccga attcgctaga gccttgctgg ccggcgcccc accatggacc tgcccccgca 1020 gctctccttc ggcctctatg tggccgcctt tgcgctgggc ttcccgctca acgtcctggc 1080 catccgaggc gcgacggccc acgcccggct ccgtctcacc cctagcctgg tctacgccct 1140 gaacctgggc tgctccgacc tgctgctgac agtctctctg cccctgaagg cggtggaggc 1200 gctagcctcc ggggcctggc ctctgccggc ctcgctgtgc cccgtcttcg cggtggccca 1260 cttcttccca ctctatgccg gcgggggctt cctggccgcc ctgagtgcag gccgctacct 1320 gggagcagcc ttccccttgg gctaccaagc cttccggagg ccgtgctatt cctggggggt 1380 gtgtgcggcc atctgggccc tcgtcctgtg tcacctgggt ctggtctttg ggttggaggc 1440 tccaggaggc tggctggacc acagcaacac ctccctgggc atcaacacac cggtcaacgg 1500 ctctccggtc tgcctggagg cctgggaccc ggcctctgcc ggcccggccc gcttcagcct 1560 ctctctcctg ctcttttttc tgcccttggc catcacagcc ttctgctacg tgggctgcct 1620 ccgggcactg gcccactccg gcctgacgca caggcggaag ctgcgggccg cctgggtggc 1680 cggcggggcc ctcctcacgc tgctgctctg cgtaggaccc tacaacgcct ccaacgtggc 1740 cagcttcctg taccccaatc taggaggctc ctggcggaag ctggggctca tcacgggtgc 1800 ctggagtgtg gtgcttaatc cgctggtgac cggttacttg ggaaggggtc ctggcctgaa 1860 gacagtgtgt gcggcaagaa cgcaaggggg caagtcccag aagtaactcg agggccaact 1920 aggcctagcc gaattcgcgg ccgcactcga gatatctaga cccagctttc ttgtacaaag 1980 tggtaagatc cgcggccgca tagataactg atccagtgtg ctggaattaa ttcgctgtct 2040 gcgagggcca gctgttgggg tgagtactcc ctctcaaaag cgggcatgac ttctgcgcta 2100 agattgtcag tttccaaaaa cgaggaggat ttgatattca cctggcccgc ggtgatgcct 2160 ttgagggtgg ccgcgtccat ctggtcagaa aagacaatct ttttgttgtc aagcttgagg 2220 tgtggcaggc ttgagatctg gccatacact tgagtgacaa tgacatccac tttgcctttc 2280 tctccacagg tgtccactcc caggtccaac tgcaggtcga gcatgcatct agggcggcca 2340 attccgcccc tctccctccc ccccccctaa cgttactggc cgaagccgct tggaataagg 2400 ccggtgtgcg tttgtctata tgtgattttc caccatattg ccgtcttttg gcaatgtgag 2460 ggcccggaaa cctggccctg tcttcttgac gagcattcct aggggtcttt cccctctcgc 2520 caaaggaatg caaggtctgt tgaatgtcgt gaaggaagca gttcctctgg aagcttcttg 2580 aagacaaaca acgtctgtag cgaccctttg caggcagcgg aaccccccac ctggcgacag 2640 gtgcctctgc ggccaaaagc cacgtgtata agatacacct gcaaaggcgg cacaacccca 2700 gtgccacgtt gtgagttgga tagttgtgga aagagtcaaa tggctctcct caagcgtatt 2760 caacaagggg ctgaaggatg cccagaaggt accccattgt atgggatctg atctggggcc 2820 tcggtgcaca tgctttacat gtgtttagtc gaggttaaaa aaacgtctag gccccccgaa 2880 ccacggggac gtggttttcc tttgaaaaac acgatgataa gcttgccaca acccacaagg 2940 agacgacctt ccatgaccga gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc 3000 ccccgggccg tacgcaccct cgccgccgcg ttcgccgact accccgccac gcgccacacc 3060 gtcgacccgg accgccacat cgagcgggtc accgagctgc aagaactctt cctcacgcgc 3120 gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg 3180 accacgccgg agagcgtcga agcgggggcg gtgttcgccg agatcggccc gcgcatggcc 3240 gagttgagcg gttcccggct ggccgcgcag caacagatgg aaggcctcct ggcgccgcac 3300 cggcccaagg agcccgcgtg gttcctggcc accgtcggcg tctcgcccga ccaccagggc 3360 aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg cgccggggtg 3420 cccgccttcc tggagacctc cgcgccccgc aacctcccct tctacgagcg gctcggcttc 3480 accgtcaccg ccgacgtcga gtgcccgaag gaccgcgcga cctggtgcat gacccgcaag 3540 cccggtgcct gacgcccgcc ccacgacccg cagcgcccga ccgaaaggag cgcacgaccc 3600 catggctccg accgaagccg acccgggcgg ccccgccgac cccgcacccg cccccgaggc 3660 ccaccgactc tagagctcgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg 3720 ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt 3780 cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg 3840 gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg 3900 atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctcg agtgcattct 3960 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct 4020 agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc 4080 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga 4140 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg 4200 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg 4260 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg 4320 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga 4380 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg 4440 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag 4500 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc 4560 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg 4620 ggaagcgtgg cgctttctca atgctcacgc tgtaggtatc tcagttcggt gtaggtcgtt 4680 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc 4740 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc 4800 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg 4860 tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca 4920 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc 4980 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat 5040 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt 5100 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt 5160 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc 5220 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc 5280 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata 5340 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg 5400 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc 5460 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct 5520 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa 5580 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt 5640 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca 5700 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac 5760 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca 5820 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt 5880 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc 5940 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca 6000 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata 6060 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc 6120 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc 6180 cgaaaagtgc cacctgacgt c 6201  

Claims (15)

A method of genotyping comprising determining an amino acid encoded by the GPR40 gene of a human subject at amino acid 211. The method of claim 1, A method of genotyping, further comprising determining an amino acid encoded at amino acid 211 by two alleles of the GPR40 gene of a human subject. The method of claim 1, And determining whether the GPR40 gene of the human subject encodes amino acid 211 but not histidine. The method of claim 1, And determining whether the GPR40 gene of the human subject encodes amino acid 211, which is arginine. The method of claim 1, And determining if the GPR40 gene of the human subject encodes amino acid 211, which is histidine. The method of claim 1, Isolating nucleic acids from a human subject; Amplifying a contiguous sequence of said nucleic acid comprising the nucleotide of GPR40 encoding amino acid 211 or its complementary sequence; Treating the contiguous sequence with a nucleotide probe that enables determination of an amino acid encoded at amino acid 211 of the GPR40 gene of the subject; And determining if said probe hybridizes to said contiguous sequence under very stringent conditions. The method of claim 6, A genotyping method capable of determining whether amino acid 211 of the GPR40 gene is histidine by a probe. The method of claim 6, A genotyping method capable of determining whether amino acid 211 of the GPR40 gene is arginine by a probe. The method of claim 6, A genotyping method comprising a contiguous portion of the nucleotide sequence of SEQ ID NO: 9, wherein the probe has a length of about 13 to about 35 nucleotides and comprises a nucleotide or a complementary nucleotide sequence thereof that encodes a detectable label and encodes amino acid 211 . The method of claim 6, A genotyping method consisting of contiguous portions of the nucleotide sequence of SEQ ID NO: 10, wherein the probe has a length of about 13 to about 35 nucleotides, and binds to a detectable label and comprises a nucleotide encoding amino acid 211 or a complementary nucleotide sequence thereof . Determining an amino acid encoded by the GPR40 gene of a human subject at amino acid 211; A method of treatment comprising treating the subject with a therapeutically effective amount of a medicament for the treatment or prevention of a disease or pathological condition mediated by amino acid 211 having one or more alleles of the GPR40 gene other than histidine . The method of claim 11, The agent is a thiazolidinedione antidiabetic compound, a pharmaceutically acceptable prodrug thereof, or a pharmaceutically acceptable salt thereof. The method of claim 11, The agent is a dipeptidyl peptidase IV inhibitor, a pharmaceutically acceptable prodrug thereof, or a pharmaceutically acceptable salt thereof. A kit comprising a nucleotide probe that enables determination of an amino acid encoded at amino acid 211 of the GPR40 gene of a human subject. Measuring competitive inhibition by the test agent of GPR40 ligand binding to a first human GPR40 receptor whose amino acid 211 is histidine; Measuring competitive inhibition by said compound of GPR40 ligand binding to a second human GPR40 receptor wherein amino acid 211 is not histidine, How to characterize a drug.

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