WO2006052007A1 - Glycemic controlling agent - Google Patents

Abstract

It is intended to provide a glycemic controlling agent which promotes or inhibits glucose intake via the P2Y2 receptor in the skeletal muscle to thereby control the blood glucose level.

Description

 Technical specification

The present invention relates to a blood glucose regulator. Specifically, the present invention relates to an agent that regulates blood glucose by promoting or inhibiting glucose uptake through P 2 Y ₂ receptors in skeletal muscle. Background art

Skeletal muscle is a high-density tissue that accounts for about 40% of body weight. It is positioned as one of the main organs of glucose metabolism. The increase in blood glucose concentration (blood glucose level) due to meals is quickly processed by increasing glucose (sugar) uptake activity in skeletal muscle due to insulin action. Therefore, glucose uptake activity in skeletal muscle is said to directly affect blood glucose level, and elucidation of its regulatory function is an important issue in diabetes research. Glucose uptake in skeletal muscle is performed by a protein called glucose transporter (G 1 uTransporter: G LUT) that penetrates the cell membrane 12 times. GLUT forms a family of highly homologous proteins, and the presence of GLUT 1 to GLUT 7 has been reported. In skeletal muscle, the presence of GL UT 1, GL UT 3, GLUT 4, and G LUT 5 has been reported so far. In skeletal muscle, G LUT 1 and GL UT 4 are functionally expressed, and G LU T 1 is normally localized to the cell membrane and is thought to be involved in basic sugar uptake. On the other hand, G LUT 4 is usually present in the intracellular microsomal fraction, but it moves to the cell membrane (translocation) and functions to incorporate extracellular sugar into the cell. It has been. Stimulation of skeletal muscle such as insulin and muscle contraction is known to translocate GL UT 4 and increase the rate of sugar uptake (Nesheret a 1., Am. J. Physio, 24 9: 02 2 6-2 3 2, 1 9 8 5). The sugar uptake rate due to the maximum stimulation of insulin and the sugar uptake rate due to muscle contraction have an additive relationship, suggesting that insulin and muscle yield increase sugar uptake rates through different pathways. ing. In other words, in skeletal muscle, sugar uptake via GLU T 4 translocated to the cell membrane is thought to occur via both insulin-dependent and independent pathways. Therefore, drugs that directly act on skeletal muscle and promote glucose uptake have few side effects, and can be expected to have a wide range of hypoglycemic effects in any pathological condition of insulin secretion deficiency and insulin resistance. It was thought to be a new diabetes drug.

In general cultured cells, as an insulin-independent signal, Gq signal via GPCR (G-protein-coupled receptor) promotes GLUT 4 membrane translocation and sugar uptake. There is a report (K ishieta, J. B iol. C he m., 2 71: 2 5 6 1— 2 6 5 6 8, 1 9 9 6). GPCR is a seven-transmembrane receptor that performs intracellular signal transduction through the activation of a coupled G protein (G protein: guaninenu c-'leotide—binding protein) and when the activity of the receptor. (Agonist) is a physiologically active substance such as various hormones, neurotransmitters, etc. G-proteins that are conjugated to receptors are activated by binding of these ligands to specific receptors. The activated G protein activates further downstream signal transduction pathways and transmits information G protein conjugated to GP CR is a trimeric protein and its α subunit Depending on the type of signal, the signal to be transmitted differs, and the subunit is classified into four families: G s, G i, G q, and G 12. The signal transmitted to the G q subunit is downstream. Phospholipase C Activated, it is known to cause intracellular calcium mobilization. 3 T 3 L 1 cells G LUT 4 Contact Yopi PAF p 1 ate 1 et- activatingfactor) When the receptor was strongly expressed, it was observed that GL UT 4 membrane translocation and sugar uptake were promoted by ligand addition. G LUT 4 membrane translocation and promotion of sugar uptake by PAF stimulation can be achieved by Gi inhibitors (IAP: I slet — activating protein), PI 3— kinase P inhibitors (W rtmannin), PKC inhibitors (PDB u: phorbo 1 1 2, 1 3-dibutyrate) was not inhibited. It is clear that the P AF receptor is conjugated to the G q and G i subunits. From the previous experiment using an inhibitor, the signal of G q is GLUT 4 membrane translocation and It was thought to promote sugar uptake.

P 2 Y ₂ is a GPCR (accession # BC 0 2 8 1 3 5) is one of the GPCR families belonging to AT P of the purine receptors P 2 Fuami Li one to ligand. In the P 2 family, an ion channel P 2 X family has also been identified. The P 2 Y ₂ receptor is a Gi- and G q-coupled GPCR and its ligands are UTP and ATP.

 Also, in mouse skeletal muscle-derived cells C 2 C 12, the promotion of sugar uptake by P 2 agonist (ATP, UTP, 2-Me S AT P) was observed, and C 2 C 12 cells were This promotion of sugar uptake has been found even when differentiated into duct cells (Kimetal., Achives of Blochemistry and Biophysics, 4 0 1: 2 0 5— 2 1 4, 2 0 0 2).

In addition, a report has been reported that P 2 Y ₂ receptor protein is localized and expressed in the neuromuscular junction of skeletal muscle (T ungeta 1., Mo 1. P harmacol., 6 6: 7 9 4— 8 0 6, 2 0 0 4) Disclosure of the invention

The present inventors have reported that GP CR specifically expressed in skeletal muscle is G LU We thought it could be a signal for T4 membrane translocation and promotion of sugar uptake. Ο 1 Expression of all GPCRs except for factoy GPCR Approximately 3 70 types of expression From the results of the analysis, GPCRs that were specifically highly expressed in human and mouse skeletal muscle were examined. As a result, P 2 Y ₂ was identified as a GPCR that is highly expressed in both human and mouse skeletal muscle.

Furthermore, we have found that m 2 Α expression level of Ρ 2 Υ ₂ receptor increases in myoblasts from myoblasts to myotube cells in C 2 C 12 cells. An increase in the mRNA expression level of the P 2 Y ₂ receptor accompanying this differentiation was also confirmed in rat skeletal muscle-derived cell L 6. Specific and high expression of P 2 Y ₂ receptor in skeletal muscle, Ρ 2 agonists promote glucose uptake in mouse myotubes, and insulin-independent GLUT 4 by G q signal This suggests that the P 2 Y ₂ receptor may be directly involved in sugar uptake in skeletal muscle. The present inventors have, G LUT 4 is strongly expressed stably expressed was the rat skeletal muscle-derived cells L 6 in [rho 2 Upsilon ₂ receptor, glucose uptake by AT P is a ligand of the [rho 2 Upsilon ₂ receptor As a result of the examination, it was confirmed that ATP, that is, P 2 Y ₂ agonist-specific promotion of sugar uptake was confirmed, and further research was conducted to complete the present invention.

 That is, the present invention

[1] Ρ 2 Υ _{2 A} hypoglycemic agent comprising a compound that promotes the activity of a receptor protein or a salt thereof or a salt thereof;

[2] Ρ 2 Υ _{2 A} hypoglycemic agent comprising a compound that promotes expression of a receptor protein or a salt thereof or a salt thereof;

[3] Ρ ₂ Υ 2 receptor protein or agent according to claim 1 or 2 wherein the salt is a skeletal muscle [rho 2 v2 receptor protein or a salt thereof;

 [4] The agent according to any one of claims 1 to 3, which is a prophylactic / therapeutic agent for diabetes, obesity or hyperlipidemia.

[5] Ρ 2 Υ _{2 A} blood glucose-elevating agent comprising a compound that inhibits the activity of a receptor protein or a salt thereof or a salt thereof; [6] A hyperglycemic agent comprising a compound or a salt thereof that inhibits expression of a gene for a P 2 Y ₂ receptor protein or a salt thereof;

[7] The agent according to claim 5 or 6, wherein the P 2 Y ₂ receptor protein or a salt thereof is a skeletal muscle P 2 Υ 2 receptor protein or a salt thereof;

 [8] The agent according to any one of claims 5 to 7, which is a prophylactic / therapeutic agent for hypoglycemia;

[9] Ρ 2 Υ ₂ receptor protein or salt thereof is a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, or a partial peptide or salt thereof Agent according to claims 1 to 8;

[1 0] Ρ ₂ Υ 2 receptor protein or a salt thereof SEQ ID NO: agent of claims 1 to 8, wherein the protein or a salt thereof consisting § amino acid sequence represented by 1;

[1 1] Ρ 2 Υ ₂ Antihyperglycemic agent comprising a polynucleotide comprising a polynucleotide encoding a receptor protein or a partial peptide thereof; [1 2] Prevention of diabetes, obesity or hyperlipidemia · The agent according to claim 11, which is a therapeutic agent;

[1 3] Ρ 2 Υ _{2 The} polynucleotide encoding the receptor protein is a polynucleotide having the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 2. 1 Agent described in 2;

[1 4] Ρ 2 Υ _{2 An} antihyperglycemic agent comprising a receptor protein or a partial peptide thereof or a salt thereof;

 [1 5] The agent according to claim 14, which is a hypoglycemia prevention / treatment agent;

[1 6] Ρ 2 Υ ₂ A protein or its partial peptide, wherein the receptor protein or a salt thereof contains the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1. The agent according to any one of claims 14 to 15, which is a salt thereof;

[1 7] Ρ 2 Υ ₂ An agent for increasing blood glucose, comprising an antisense polynucleotide containing a base sequence complementary to or substantially complementary to the base sequence of a polynucleotide encoding a receptor protein or a part thereof ; [1 8] The agent according to claim 17, which is a hypoglycemia prevention / treatment agent;

[1 9] P 2 Y ₂ encoding receptor protein polynucleotide is SEQ ID NO: a nucleotide sequence the same or substantially the same base sequence represented by to 1 7 claims a polynucleotide having free 1 agent according to 8;

[2 0] Ρ 2 Υ _{2 An} antihyperglycemic agent comprising an antibody against a receptor protein or a salt thereof;

 [2 1] The agent according to claim 20, which is a prophylactic / therapeutic agent for hypoglycemia;

[2 2] Ρ 2 Υ ₂ A protein or its partial peptide or a peptide thereof, wherein the receptor protein or a salt thereof contains the amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1. The agent according to claim 20 which is a salt;

[2 3] Ρ 2 Υ ₂ Diagnostic agent for diabetes or hypoglycemia comprising an antibody against the receptor protein or a salt thereof;

[24] [rho diagnostic agent 2 Upsilon ₂ receptor protein comprising polynucleotides encoding diabetes or hypoglycemia;

[2 5] Ρ 2 Υ ₂ receptor protein or a salt thereof, a method for screening a compound for regulating blood glucose level or a salt thereof;

[2 6] The screening method according to claim 22, wherein the Ρ 2 Υ ₂ receptor protein or a salt thereof is a skeletal muscle Ρ 2 Υ ₂ receptor protein or a salt thereof;

[2 7] Ρ 2 Υ ₂ receptor protein or 'or a salt thereof, a protein containing the amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, or a partial peptide thereof, or The screening method according to claim 25, which is a salt thereof;

[2 8] Ρ 2 Υ ₂ screening kit for a compound that regulates blood glucose level or a salt thereof, characterized by containing a receptor protein or a salt thereof;

[2 9] 方法 2 Υ _{2 A} method for screening a compound that regulates blood glucose or a salt thereof, characterized by using a polynucleotide encoding a receptor protein;

[3 0] Ρ 2 Υ. The polynucleotide encoding the receptor protein is SEQ ID NO: The screening method according to claim 29, which is a polynucleotide having the same or substantially the same nucleotide sequence as the nucleotide sequence represented by No. 2:

[3 1] characterized by containing a polynucleotide encoding a P 2 Y ₂ receptor protein ', a kit for screening a compound that regulates blood glucose or a salt thereof;

[3 _2] Ρ 2 Υ 2 which comprises using the receptor protein or a salt thereof, Ρ ₂ Υ 2 receptor protein or screening method Agonisu bets to salts thereof;

[3 3] Ρ 2 Υ ₂ receptor protein or salt thereof, characterized in that the antagonist screening method for Ρ 2 Υ ₂ receptor protein or salt thereof;

[3 4] (1) Ρ the 2 Upsilon ₂ receptor protein or a labeled ligand or a salt of a salt thereof, and when contacted with the protein or a salt thereof, (2) test compound and [rho 2 Upsilon ₂ receptor When a labeled ligand of a body protein or a salt thereof or a salt thereof is contacted with the protein or a salt thereof,

The screening method according to claim 32 or 33, wherein the amount of binding between the labeled ligand of the 2 ₂ receptor protein or a salt thereof or a salt thereof and the protein or the salt thereof is measured. ;

[3 5] (1) Ρ the 2 Upsilon ₂ receptor protein or a labeled ligand or a salt of a salt thereof, and when contacted with a cell or a cell membrane fraction containing the protein, (2) test compound the and [rho 2 Upsilon ₂ receptor protein or a labeled ligand or a salt of a salt thereof, in the case also cells containing the protein is in contact with the cell membrane fraction, [rho 2 Upsilon ₂ receptor protein or its A binding amount of a ligand labeled with a salt or a salt thereof and a cell containing the protein or a cell membrane fraction thereof is measured.

3 A screening method according to 2 or 3 3;

[36] (1) Ρ2) _{2 When a} receptor protein or a salt thereof or a salt thereof is contacted with a cell containing the protein or a cell membrane fraction thereof, and (2) Ρ2Υ. Receptor protein or its salt ligand or its salt It is characterized by measuring a cell stimulating activity via a P 2 Y ₂ receptor protein when a test compound is brought into contact with a cell containing the protein or its cell membrane fraction in the presence or absence. The screening method according to claim 3 2 or 3 3;

[37] (1) When a ligand of a Ρ 2 Υ ₂ receptor protein or a salt thereof or a salt thereof is brought into contact with a cell containing the protein or a cell membrane fraction thereof, and (2) 受 容 2 Υ ₂ receptor Intracellular Ca ²⁺ concentration or cells when a test compound is contacted with a cell containing the protein or a cell membrane fraction thereof in the presence or absence of a ligand of the body protein or salt thereof or the salt thereof A screening method according to claim 3 2 or 3 3, characterized in that c AMP production is measured;

[3 8] The screening method according to claim 3, wherein GLUT 4 is allowed to coexist with P 2 Y ₂ receptor protein or a salt thereof; [3 9] Skeletal muscle-derived cells Or the screening method according to claim 32, wherein skeletal muscle cells are used;

 [4 0] The screening method according to claim 32, wherein the ligand is U Τ Ρ or ATP;

[4 1] A method for lowering blood glucose characterized by promoting the activity of P 2 Y ₂ receptor protein or a salt thereof;

[4 2] Ρ 2 Υ _{2 A} method for lowering blood glucose, characterized by promoting expression of a receptor protein or a salt thereof;

[4 3] The method according to claim 41 or 42, wherein the Ρ 2 Υ ₂ receptor protein or a salt thereof is a skeletal muscle Ρ 2 Υ ₂ receptor protein or a salt thereof;

 [4 4] The method according to any one of claims 41 to 43, which is a method for preventing / treating diabetes, obesity or hyperlipidemia;

[4 5] A method for increasing blood glucose characterized by inhibiting the activity of Ρ 2 Υ ₂ receptor protein or a salt thereof;

[4 6] Ρ 2 Υ _{2 A} method for increasing blood glucose characterized by inhibiting expression of a receptor protein or a salt thereof; [4 7] The method of P 2 Y ₂ receptor protein or claim 4 5 or 4 6, wherein the salt is a P 2 Y ₂受有body蛋white matter or a salt thereof of skeletal muscle;

[4 8] The method according to claim 45, which is a method for the prevention and treatment of hypoglycemia;-[4 9] Υ ₂化合物 2 compound or salt thereof that promotes the activity of receptor protein or its salt A method for lowering blood glucose, comprising administering an effective amount of

[5 0] Ρ 2 Υ _{2 A} method for lowering blood glucose, comprising administering to a mammal an effective amount of a compound or a salt thereof that promotes expression of a receptor protein or a salt thereof;

[5 1] The method according to claim 49 or 50, wherein the Ρ 2 Υ ₂ receptor protein or a salt thereof is a skeletal muscle Ρ 2 Υ ₂ receptor protein or a salt thereof;

 [52] The method according to any one of claims 49 to 51, which is a method for preventing / treating diabetes, obesity or hyperlipidemia;

[5 3] Ρ 2 Υ _{2 A} method for increasing blood glucose, comprising administering to a mammal an effective amount of a compound that inhibits the activity of a receptor protein or a salt thereof or a salt thereof;

[5 4] Ρ 2 Υ _{2 A} method for increasing blood glucose, comprising administering to a mammal an effective amount of a compound or salt thereof that inhibits expression of a receptor protein or a salt thereof;

[5 5] Ρ ₂ Υ 2 receptor protein or method of claim 3 or 4, wherein the salt is a skeletal muscle [rho 2 Upsilon ₂ receptor蛋white matter or a salt thereof;

 [5 6] The method according to claims 5 3 to 5 5, which is a method for preventing and treating hypoglycemia;

[5 7] Use of a compound or a salt thereof that promotes the activity of Ρ 2 Υ ₂ receptor protein or a salt thereof for the manufacture of a hypoglycemic agent;

[5 8] Use of a compound or a salt thereof that promotes the expression of a gene of a Ρ 2 Υ ₂ receptor protein or a salt thereof for the manufacture of a hypoglycemic agent;

[5 9] Ρ ₂ Υ 2 receptor protein or a salt thereof is according to claim 5 7, or 5 8, wherein the skeletal muscle [rho 2 Upsilon ₂ receptor蛋white matter or a salt thereof; [60] The use according to any one of claims 57 to 59, wherein the hypoglycemic agent is a method for preventing / treating diabetes, obesity or hyperlipidemia;

[6 1] Use of a compound or a salt thereof that inhibits the activity of a P 2 Y ₂ receptor protein or a salt thereof for the production of a hyperglycemic agent;

[6 2] Use of a compound or a salt thereof that inhibits the expression of a gene of a Ρ 2 Υ ₂ receptor protein or a salt thereof for the manufacture of a blood glucose-elevating agent;

[6 3] The use according to claim 61 or 62, wherein the Ρ 2 Υ ₂ receptor protein or a salt thereof is a skeletal muscle Ρ 2 Υ ₂ receptor protein or a salt thereof;

 [6 4] The use according to any one of claims 61 to 63, wherein the hyperglycemic agent is a hypoglycemia prevention / treatment agent;

I will provide a. Brief Description of Drawings

 FIG. 1 shows the expression of mRN 高 in the top 40 GP CCR species in human or mouse skeletal muscle.

Figure 2 represents the expression distribution of P 2Y ₂ mRN Α.

FIG. 3 represents the expression of P 2 Y ₂ mRNA in 'myoblast differentiation'.

 FIG. 4 represents sugar uptake induced by ATP in rat myoblasts L 6 -S G 4 -81. BEST MODE FOR CARRYING OUT THE INVENTION

The P 2 Y ₂ receptor protein used in the present invention is a receptor protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1.

Ρ 2 Υ ₂ receptor protein can be used in any cell (eg, spleen cell, nerve, etc.) of, for example, human mammals (eg, guinea pigs, rats, mice, rabbits, pigs, hidges, bushes, monkeys, etc.). Cells, glial cells, knee β cells, knees Langerhans islets, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, endothelial cells, fibroblasts, fibrocytes, muscle cells, fat Cells, immune cells (eg, macrophages, T cells, B cells, natura 3 killer cells, mast cells, neutrophils, basophils, eosinophils, monocytes), megakaryocytes, synovial cells, cartilage Cells, bone cells, osteoblasts, osteoclasts, breast cells, hepatocytes or stromal cells, or progenitors of these cells, stem cells or cancer cells) or blood cells, or their Any tissue in which cells are present, for example, brain, brain regions (eg, olfactory bulb, cephalic nucleus, basal sphere, hippocampus, hypothalamus, hypothalamic nucleus, cerebral cortex, medulla, cerebellum, occipital lobe, frontal lobe , Temporal lobe, putamen, caudate nucleus, brain stain, substantia nigra), spinal cord, pituitary gland, stomach, knee, kidney, liver, reproductive gland, thyroid, gallbladder, bone marrow, adrenal gland, skin, muscle, lung , Digestive tract (eg, large intestine, small intestine), blood vessel, heart, thymus, spleen Proteins derived from submandibular gland, peripheral blood, peripheral blood cells, prostate gland, testis, testis, ovary, placenta, uterus, bone, joint, skeletal muscle, etc., or synthetic protein . In particular, the P 2 Y ₂ receptor protein is highly expressed in skeletal muscle.

The amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 is, for example, about 85% or more, preferably 90% or more, more preferably, the amino acid sequence represented by SEQ ID NO: 1. Examples include amino acid sequences having about 95% homology. Specifically, the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 is mouse P 2 Y ₂ (P 2 U) (Gen Bank Accession No. LI 4 7 5 1; SEQ ID NO: 3), rat P 2 Y ₂ (P 2 U) (Gen Bank Accession No. U 0 9 4 0 2; SEQ ID NO: 4), and the like.

 Examples of the protein containing the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 of the present invention include an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1. A protein having substantially the same activity as the protein consisting of the amino acid sequence represented by SEQ ID NO: 1 is preferred.

Examples of the activity of substantially the same quality include ligand binding activity and signal information transmission action. With substantially the same quality, their activity is sex Qualitatively the same quality. Therefore, the ligand binding activity and the activity such as Danal signal transduction are equivalent (eg, about 0.1 to 100 times, preferably about 0.5 to 2 times Q, more preferably Is preferably about 0.5 to 2 times), but the degree of these activities may be different in quantitative factors such as the molecular weight of protein.

 Measurement of activities such as ligand binding activity and signal signal transduction can be performed according to a method known per se, for example, it can be measured according to a screening method described later.

The P 2 Y ₂ receptor protein includes: a) 1 or 2 or more (preferably about 1 to 30 and more preferably 1 to 10) in the amino acid sequence represented by SEQ ID NO: 1. Degree, more preferably several (1-5) amino acid sequences lacking amino acids, b) 1 or more amino acid sequences represented by SEQ ID NO: 1 (preferably 1 to An amino acid sequence to which about 30 amino acids, more preferably about 1 to 10 amino acids, and more preferably several (1 to 5) amino acids are added; c) in the amino acid sequence represented by SEQ ID NO: 1 1 or 2 or more (preferably about 1 to 30 amino acids, more preferably about 1 to 10 amino acids, more preferably several (1 to 5)) amino acids substituted with other amino acids Sequence, or d) Proteins containing amino acid sequences that combine them are also used. I can.

In the present specification, the P 2 Y ₂ receptor protein has a left end at the heel end (amino end) and a right end at the C end (carboxyl end) in accordance with the convention of peptide labeling. Proteins including the amino acid sequence represented by SEQ ID NO: 1 Ρ 2 Υ ₂ receptor protein, the C-terminus is carboxyl group (one C Ο Η Η), carboxylate (one C Ο Ο—), amino (One CONH ₂ ) or ester (—COOR).

Here, as R in the ester, e.g., methyl, Echiru, n- propyl, isopropyl also properly n - C w alkyl group such as heptyl, if example embodiment, consequent opening pentyl, C ₃ _ ₈ consequent such hexyl consequent opening mouth Anorekinore group, for example, Fuweniru, 'alpha - C _6, such as naphthyl - ₁₂ Ariru group, for example, Ben Phenyl, phenethyl and other phenyl C ² alkyl groups or monomethyl naphthyl such as phenylmethyl C ₇ — ₁₄ aralkyl groups and other viva commonly used as an oral ester A royloxymethyl group is used. -'

When the P 2 Y ₂ receptor protein has a carboxyl group (or carboxylate) other than the C-terminus, a protein in which the carboxyl group is amidated or esterified is also used in the present invention. ₂ Included in receptor protein. As the ester in this case, for example, the above-mentioned C-terminal ester is used.

Further, [rho 2 Upsilon ₂ The receptor protein, in the protein described above, Amino group protecting groups Mechionin residues of Ν-terminus (e.g., C _chi _ ₆ such as formyl group, C ₂ _ ₆ Arukanoiru group such Asechiru That are protected with an acyl group, etc., a dartamyl group that is cleaved in vivo on the heel side, and a dartamine-oxidized amino acid, a substituent on the side chain of an amino acid in the molecule (for example, protection _ SH, Amino group, Lee imidazole group, fin d'groups, etc. Guanijino group) appropriate protecting groups (e.g., formyl group, etc. C w Ashiru group such as C ₂ _ ₆ Arukanoi Le group such Asechiru) Or a complex protein such as a so-called glycoprotein to which a sugar chain is bound.

The partial peptide of the P 2 Y ₂ receptor protein may be any partial peptide of the above-mentioned Ρ 2 Υ ₂ receptor protein S, for example, Ρ 2 Υ ₂ receptor Among protein molecules of body proteins, those that are exposed to the outside of the cell membrane and have substantially the same receptor binding activity are used.

Specifically, the partial peptide of Ρ 2 Υ ₂ receptor protein having the amino acid sequence represented by SEQ ID NO: 1 is an extracellular region (hydrophilic site) in the hydrophobic plot analysis. And a peptide containing the analyzed portion. A peptide partially containing a hydrophobic site can also be used. Peptides containing individual domains can also be used, but peptides that contain multiple domains simultaneously may be used. The number of amino acids in the partial peptide of the present invention is at least 20 or more, preferably 50 or more, more preferably 100 or more of the constituent amino acid sequences of the acceptor protein of the present invention described above. Peptides having the above amino acid sequence are preferred. ■.

 Here, “substantially the same receptor activity” has the same meaning as described above. “Substantially the same receptor activity” can be measured in the same manner as described above. In addition, the partial peptide used in the present invention lacks one or more (preferably about 1 to 10, more preferably several (1 to 5)) amino acids in the above amino acid sequence. Alternatively, one or more amino acids are added to the amino acid sequence (preferably about 1 to 20, more preferably about 1 to 10, more preferably several (1 to 5)). Or one or more (preferably about 1 to 10, more preferably several, more preferably about 1 to 5) amino acids in the amino acid sequence are substituted with other amino acids. You can do it.

In the partial peptide used in the present invention, the C-terminus is either a carboxyl group (one COOH), canolepoxylate (one COOO—), an amide (one CONH ₂ ) or an ester (one COOR). It ’s okay. In the case where the partial peptide used in the present invention has a carboxyl group (or carboxylate) in addition to the C-terminus, those in which the carboxyl group is amidated or esterified are also used in the present invention. Included in peptides. As the ester in this case, for example, the above C-terminal ester or the like is used.

Further, in the partial peptide used in the present invention, as in the above-described P 2 Y ₂ receptor protein, the amino acid group of the methionine residue at the heel end is protected with a protecting group, and the heel end side is in vivo. G 1 η cleaved by cleaved by pyroglutamine oxidation, substituents on the side chains of amino acids in the molecule are protected with appropriate protecting groups, or so-called sugar peptides with sugar chains attached Also includes complex peptides.

Ρ 2 used in the present invention. Of the receptor protein or its partial peptides Examples of the salt include physiologically acceptable salts with acids or bases, and physiologically acceptable acid addition salts are particularly preferable. Examples of such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid), or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, malein). Acid, succinic acid, tartaric acid, citrate, malic acid, succinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like are used.

The P 2 Y ₂ receptor protein or salt thereof used in the present invention can be produced from the above-described human mammalian cells or tissues by a known method for purifying receptor protein, and will be described later. It can also be produced by culturing a transformant containing DN 2 that codes for _{2 2} receptor protein. Moreover, it can also be produced according to the protein synthesis method described later or this.

 When producing human tissues or cells from mammals, homogenize the tissues or cells of human mammals, extract with acid, etc., and extract the extracted solution using reverse-phase chromatography or ion-exchange chromatography. It can be purified and isolated by combining chromatography such as chromatography.

For synthesizing the P 2 Y ₂ receptor protein or its partial peptide or its salt or its amide used in the present invention, a commercially available resin for protein synthesis can be used. Examples of such resins include black methyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, Ρ Α Μ resin, 4 -Hydroxymethylmethyl phenylamide methyl resin, polyacrylamide resin, 4_ (2 ', 4, dimethyoxyphenylhydroxymethyl) phenoxy resin, 4 (2', 4'dimethyoxyphenyru F moc (Aminoethyl) phenoxy resin and the like. Using such a resin, an amino acid having an α-amino group and a side chain functional group appropriately protected is condensed on the resin according to various known condensation methods according to the sequence of the target protein. Anti At the end of the reaction, the protein is cut out from the resin and at the same time, various protecting groups are removed, and an intramolecular disulfide bond forming reaction is performed in a highly diluted solution to obtain the target protein or its amide.

 Regarding the condensation of the above-mentioned protected amino acids, the ability to use various activating reagents that can be used for protein synthesis, in particular, calpositimides are preferred. Examples of carbopositimides include DCC, N, N′-diisopropylcarbodiimide, N-ethyl-N, mono (3-dimethylaminoprolyl) carbodiimide, and the like. For activation by these, the ability to add a protected amino acid directly to the resin together with a racemization-suppressing additive, such as HOB t, HOOB t, or a symmetric anhydride or HOB t ester is a HOOB t ester Can be added to the resin after activation of the protected amino acid in advance.

The solvent used for the activation of the protected amino acid and the condensation with the resin may be appropriately selected from solvents known to be usable for the protein condensation reaction. For example, N, N-dimethylformamide, N, N-dimethylacetamide, acid amides such as N-methylpyrrolidone, halogenated hydrocarbons such as methylene chloride and chlorohonolem, and trifnoreo oral ethanol Alcohols such as mono-ole, sulfoxides such as dimethyl sulfoxide, ethers such as pyridine, dioxane and tetrahydrofuran, -tolyls such as acetonitrile and propio-tolyl, methyl acetate and ethyl acetate Esters or appropriate mixtures thereof are used. The reaction temperature is appropriately selected from a range known to be used for protein bond forming reaction, and is usually selected appropriately from a range of about −20 ° C. to 50 ° C. Activated amino acid derivatives are usually used in excess of 1.5 to 4 times. As a result of the test using the ninhydrin reaction, if the condensation is insufficient, sufficient condensation can be performed by repeating the condensation reaction without removing the protecting group. If sufficient condensation cannot be obtained even after repeating the reaction, unreacted amino acid can be acetylated using acetic anhydride or acetyl imidazole. Examples of protecting groups for the amino group of the raw material include Z, Boc, tertiary pentinorexinoreboninore, isobonorenoreoxycarboninore, and 4-methoxypendinoreoxycanorepo. Nyl, C 1-1 Z, B 1-- Z, adamantyl 'oxycanolebonyl, trifnoleoloacetinore, phthaloidol, honoreminore, 2-12 trophenylsnofenenore, dipheninorephosphinothioinore, F moc Etc. are used.

 The carboxyl group is, for example, alkyl esterified (for example, methyl, ethinole, propyl, petitenole, tert-butinole, cyclopentinole, cyclohexyl, cycloheptinole, succulent cutinore, 2-adamantinore, etc. Linear, branched, or cyclic alkyl esterification), aralkyl esterenoyl (eg, benzenoreester, 4-12 trobenzinoreestenole, 4-methoxybenzenoreestenole, 4-clonal benzene) Noreestenole, benzhydryl esterification), phenacyl esterification, benzyloxycarbonyl hydrazide, tertiary butoxycarbonyl hydrazide, trityl hydrazide, and the like.

 The hydroxyl group of selenium can be protected by, for example, esterification or etherification. Examples of the group suitable for esterification include a group derived from carbonic acid such as a lower alkyl group such as acetyl group, an aryl group such as benzoyl group, a benzyloxycarbonyl group, and a ethoxycarbonyl group. Used. Examples of groups suitable for etherification include a benzyl group, a tetrahydrobiranyl group, and a t-butyl group.

Examples of the protecting group for the phenolic hydroxyl group of tyrosine include B z 1, C l ₂ — B zl, 2-nitrite benzyl, B r— Z, and tertiary butyl.

Examples of the protecting group for imidazole of histidine include Tos, 41-Methoxy-1, 2,3-, 6-trimethylbenzene sulphononiole, DNP, benzenoreoxymethyl, B um, B oc, T rt, F moc, etc. are used. Examples of the activated carboxyl group of the raw material include ^ "corresponding acid anhydride, azide, active ester [alcohol (eg, pentac oral phenol, 2, 4, 5 — trichlorophenol). , 2,4-dinitrophenol, cysomethino alcohol, norani trofenore, HONB, N-hydroxy succinide, N-hydroxy phthalenolide, ester with HOB t) etc. As the activated amino group, for example, a corresponding phosphate amide is used.

 Examples of methods for removing (eliminating) protecting groups include catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd-black or Pd-carbon, and anhydrous hydrogen fluoride and methanesulfone. Acid treatment with acid, 卜 rifroleoromethane sulfonic acid, trifluoroacetic acid, or a mixture of these, base treatment with diisopropylpropylamine, triethylamine, piperidine, piperazine, etc., and sodium in liquid ammonia Reduction with hum is also used. In general, the elimination reaction by the acid treatment is performed at a temperature of about 120 ° C. to 40 ° C. In the acid treatment, for example, anisole, phenol, thioanisole, methazolesole, norac Addition of cation scavengers such as resorenoles, dimethinolesnore, 1,4-butanedithionore, 1,2-ethanedithionore is effective. In addition, the 2,4-dinitophenyl phenyl group used as an imidazole protecting group for histidine was removed by thiophenol treatment, and the formyl group used as an indole protecting group for tryptophan was the above 1,2-ethanedithiol, 1, 4 In addition to deprotection by acid treatment in the presence of -butanedithiol, etc., it can also be removed by alkaline treatment with dilute sodium hydroxide solution or dilute ammonia.

 The protection of the functional group that should not be involved in the reaction of the raw material, the protecting group, the removal of the protecting group, the activation of the functional group involved in the reaction, etc. can be appropriately selected from known groups or known means.

As another method for obtaining an amide form of a protein, for example, first, the α-stroxyl group of a carboxyl terminal amino acid is amidated to be protected, After extending the peptide (protein) chain to the desired chain length on the non-group side, remove only the N-terminal _α -amino protecting group of the peptide chain and the C-terminal carboxyl protecting group. The removed protein is produced, and both proteins are condensed in a mixed solvent as described above. The details of the condensation reaction are the same as described above. After purifying the protected protein obtained by the condensation, all the protecting groups are removed by the above method to obtain the desired crude protein. This crude protein can be purified using various known purification means, and the main fraction can be freeze-dried to obtain an amide of the desired protein.

 In order to obtain an ester of a protein, for example, the a-carboxyl group of the carboxy terminal amino acid is condensed with a desired alcohol to form an amino acid ester, and then the ester of the desired protein is obtained in the same manner as the protein amide. Can be obtained.

The partial peptide of P 2 Y ₂ receptor protein or a salt thereof used in the present invention is produced according to a known peptide synthesis method or by cleaving Ρ 2 Υ ₂ receptor protein with an appropriate peptidase. be able to. As a peptide synthesis method, for example, either a solid phase synthesis method or a liquid phase synthesis method may be used. That, [rho 2 Upsilon ₂ receptor protein by condensing portion Bae flop tides or amino acids and the remaining portion may constitute, when the product Ru-which have a protecting group for the purposes of Bae by leaving a protective group Peptide can be manufactured. Examples of known condensation methods and protecting group removal include the methods described in a) to e) below.

 a) M. Bodanszky and M. A. Ondetti, Peptide Synthesis, Interscience Publishers, New York (1966)

 b) Schroeder Luebke, The Peptide Academic Press, New York (1965;)

 c) Nobuo Izumiya et al., Peptide Synthesis Fundamentals and Experiments, Maruzen Co., Ltd. (1975) d) Haruaki Yajima and Shunpei Sugawara, Biochemistry Experiment Course 1, Protein Chemistry IV, 205, (1977)

e) Supervised by Haruaki Yajima, Development of follow-up medicines XIV Peptide synthesis Hirokawa Shoten In addition, after the reaction, the partial peptide of the present invention can be purified and isolated by combining ordinary purification methods, for example, solvent extraction 'distillation' color chromatography / liquid chromatography / recrystallization. When the partial peptide obtained by the above method is a free form, it can be converted into an appropriate salt by a known method. Conversely, when it is obtained as a salt, it is released by a known method. Can be converted into a body.

The polynucleotide encoding the P 2 Y ₂ receptor protein used in the present invention contains the base sequence encoding D 2 ₂ receptor protein (D 2 or RNA, preferably DNA). Anything can be used. The polynucleotide is a DNA encoding a P 2 Y ₂ receptor protein, an RNA such as mRNA, and may be double-stranded or single-stranded. In the case of double-stranded DNA, double-stranded DNA, double-stranded RNA or DN A: RNA hybrid may be used. In the case of a single strand, it may be a sense strand (ie, a code strand) or an antisense strand (ie, a non-coding strand).

Examples of the DNA encoding the P 2 Y ₂ receptor protein used in the present invention include genomic DNA, genomic DNA library, cDNA derived from the cells / tissues described above, and cDNA library derived from the cells / tissues described above. Any of synthetic DNA may be used. The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. Further, it can also be directly amplified by reverse transcriptase polymerase chain reaction (hereinafter abbreviated as RT-PCR method) using a total RNA or mRNA fraction prepared from the above-mentioned cell tissue.

Specifically, D コ ー ド encoding the P 2 Y ₂ receptor protein used in the present invention is, for example, DNA containing a base sequence represented by SEQ ID NO: 2 or represented by SEQ ID NO: 2. A P 2 Y receptor protein consisting of an amino acid sequence represented by SEQ ID NO: 1, and a base sequence that is complementary to the base sequence and a base sequence that is hybridized under highly stringent conditions; Any DNA may be used as long as it encodes a receptor protein having substantially the same quality of activity (eg, ligand binding activity, signal signaling, etc.).

The DNA that can hybridize with the base sequence complementary to the base sequence represented by SEQ ID NO: 2 is, for example, about 85% or more, preferably about 90% or more, with the base sequence represented by SEQ ID NO: 2. More preferably, a DNA containing a nucleotide sequence having a homology of about 95% or more is used. Specifically, DNA that can be hybridized with a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 2 is mouse P 2 Y ₂ (P 2 U) (Gen Bank Acession No L 1 4 7 5 1; SEQ ID NO: 5), Rat P 2 Y ₂ (P 2 U) (Gen Bank Accession No. U 0 9 4 0 2; SEQ ID NO: 6) It is done.

 Hybridization can be performed according to a known method or a method similar thereto, for example, the method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). . Moreover, when using a commercially available library, it can be carried out according to the method described in the attached instruction manual. More preferably, it can be carried out according to highly stringent conditions.

 The high stringent conditions include, for example, a sodium concentration of about 19 to 40 mM, preferably about 19 to 20 mM, and a temperature of about 50 to 70 ° C, preferably about 60 to 6 Indicates conditions at 5 ° C. In particular, it is most preferable that the sodium concentration is about 19 mM and the temperature is about 65 ° C.

More specifically, D す る を encoding the human P 2 Y ₂ receptor protein containing the amino acid sequence represented by SEQ ID NO: 1 is DN A containing the base sequence represented by SEQ ID NO: 2. Etc. are used.

The polynucleotide containing a part of the base sequence of DN コ ー ド encoding the P 2 Y ₂ receptor protein used in the present invention includes not only the DNA encoding the partial peptide of the present invention described below. Including RNA Used to mean.

According to the present invention, an antisense polynucleotide (nucleic acid) capable of inhibiting the replication or expression of a P 2 Y ₂ receptor protein gene is used as a clone of DN コ ー ド encoding Ρ 2 Υ ₂ receptor protein. It is possible to design and synthesize based on the base sequence information obtained or determined. Such a polynucleotide (nucleic acid) can be hybridized with the RNA of the P 2 Y ₂ receptor protein gene and can inhibit the synthesis or function of the RNA, or the P 2 Y ₂ receptor protein. It is possible to regulate and control the expression of P 2 Y ₂ receptor protein gene through the interaction with RN 関 連 related to. Polynucleotides that can be specifically hybridized with polynucleotides that are complementary to selected sequences of Ν 2 Υ ₂ receptor proteins, R す る Α, and RNAs associated with P 2 Y ₂ receptor proteins are are useful for modulating 'controls the expression of [rho 2 Upsilon ₂ receptor protein gene in vivo Contact Yopi vitro, also useful for the treatment or diagnosis of diseases. The term “corresponding” means homologous to or complementary to a specific sequence of nucleotides, base sequences or nucleic acids including genes. “Corresponding” between a nucleotide, nucleotide sequence or nucleic acid and peptide (protein) usually refers to the amino acid of the peptide (protein) in the instruction derived from the sequence of the nucleotide (nucleic acid) or its complement. ing. Ρ 2 Υ ₂ receptor protein gene 5, end hairpin loop, 5 'end 6 — base pair' repeat, 5, end untranslated region, polypeptide translation initiation codon, protein coding region, ORF translation stop codon 3 'end untranslated region, 3' end palindromic region, opium 3, and end hairpin loop can be selected as preferred target regions, but any region within the P 2 Y ₂ receptor protein gene can be selected as target. sell. The relationship between the target nucleic acid and a polynucleotide that can be hybridized to be complementary to at least a part of the target region can be said to be “antisense J”. Polyoxyribonucleotides containing Doxyribose, Polyribonucleotides containing D-ribose, Purine or Pyridine Other types of polynucleotides that are N-glycosides of the midine base; tides, or other polymers with non-nucleotide backbones (eg, commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers) or other containing special linkages (However, the polymer contains a nucleotide having a configuration that allows attachment of a base as found in DNA or RNA). They can be double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, or even DNA: RNA hybrids, and unmodified polynucleotides (or unmodified oligonucleotides). In addition, those with known modifications, such as those with labels known in the art, capped, methylated, and one or more natural nucleotides in analogs Substituted, intramolecular nucleotide-modified, eg with uncharged bonds (eg methinorephosphonate, phosphotriestenole, phosphonoreamidate, carpamate, etc.), charged bond or sulfur-containing Have binding (eg phosphorothioate, phosphorodithioate, etc.), eg proteins (nucleases, nucleases' inhibitors, Toxins, antibodies, signal peptides, poly-L-lysine, etc.) and sugars (for example, monosaccharides), etc., and side-current groups (eg, acridine, psoralen, etc.) Those containing chelating compounds (eg metals, radioactive metals, boron, oxidizing metals, etc.), those containing alkylating agents, those with modified bonds (eg α-anomers) Type nucleic acid). As used herein, “nucleoside”, “nucleotide” and “nucleic acid” may include those containing not only purine and pyrimidine bases but also other modified heterocyclic bases. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles. Modified nucleotides and modified nucleotides may also be modified at the sugar moiety, eg, one or more hydroxyl groups are replaced by halogens, aliphatic groups, etc. Or may be converted to a functional group such as ether or amine. The antisense polynucleotide (nucleic acid) of the present invention is RNA, DNA, or modified nucleic acid (RNA, DNA). Specific examples of modified nucleic acids include, but are not limited to, nucleic acid sulfur derivatives, thiophosphate derivatives, and those that are resistant to degradation of polynucleoside amide nucleoside amides. It is not something. The antisense nucleic acid of the present invention can be preferably designed according to the following policy. That is, make the antisense nucleic acid more stable in the cell, increase the cell permeability of the antisense nucleic acid, increase the affinity for the target sense strand, and if it is toxic This makes the antisense nucleic acid less toxic.

 Many such modifications are known in the art, such as J. Kawakami et al., Pharra Tech Japan, Vol. 8, pp. 247, 1992; Vol. 8, pp. 395, 1992; ST Crooke et al. ed., Antisense Research and Applications, CRC Press, 1993.

The antisense nucleic acid of the present invention may be altered or contain modified sugars, bases and bonds, provided in special forms such as ribosomes, microspheres, applied by gene therapy, or added. Could be given in the form As such, it can be used in an additional form as a polycationic substance such as polylysine, which acts to neutralize the charge of the phosphate group skeleton, enhance the interaction with the cell membrane, or incorporate nucleic acid. Examples include lipids that increase the amount of crude water (for example, phospholipids, cholesterol, etc.). Preferred lipids to be added include cholestadiol and its derivatives (eg, cholesteryl chloroformate, cholesteric acid, etc.). These include the 3 'end or 5' of nucleic acids. 'Can be attached to the end and can be attached via a base, sugar, or intramolecular nucleoside bond. Other groups are specifically located at the 3' or 5 'end of the nucleic acid. It is a base for capping to prevent degradation by nucleases such as exonuclease and RNase. Can be mentioned. Examples of the group for capping include, but are not limited to, hydroxyl protecting groups known in the art, including glycols such as polyethylene glycol and tetraethylene glycol. . .

 The inhibitory activity of the antisense nucleic acid is examined using the transformant of the present invention, the in vivo or in vitro gene expression system of the present invention, or the in vivo or in vitro translation system of the G protein-coupled receptor protein. Can do. The nucleic acid can be applied to cells by various known methods.

The DN コ ー ド encoding the partial peptide of the P 2 Y ₂ receptor protein used in the present invention is any as long as it contains a base sequence encoding the partial peptide used in the present invention. May be. In addition, any of genomic DNA, genomic DNA library, cDNA derived from the above-described cell 'tissue, cDNA library derived from the above-described cell / tissue, and synthetic DNA may be used. The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. In addition, it can be directly amplified by RT_PCR using a mRNA prepared from the above-mentioned cell tissue.

Specifically, the DNA encoding the partial peptide of the P 2 Y ₂ receptor protein used in the present invention is, for example, (1) a partial base of D Ν を having the base sequence represented by SEQ ID NO: 2 (2) having a sequence, or (2) having a base sequence that is complementary to the base sequence represented by SEQ ID NO: 2 and a base sequence that hybridizes under high stringency conditions, and represented by SEQ ID NO: 1 A partial base sequence of DN コ ー ド that encodes a receptor protein having substantially the same activity as the Ρ 2 Υ ₂ receptor protein (eg, ligand binding activity, signal signal transduction, etc.). The DN 有 す る that has is used.

Examples of DNA that can be hybridized with a base sequence complementary to the base sequence represented by SEQ ID NO: 2 include, for example, about 85% or more of the base sequence represented by SEQ ID NO: 2, 'preferably about 90% Or more, more preferably about 95% DNA containing a base sequence having the above homology is used.

As a means of cloning DN Α that completely encodes P 2 Y ₂ receptor protein or its partial peptide used in the present invention, DN す る that completely encodes P 2 Y ₂ receptor protein or its partial peptide can be used. Ability to amplify by PCR using a synthetic DNA primer with a partial nucleotide sequence of DNA or a DNA that completely encodes a P 2 Y ₂ receptor protein or its partial peptide when DNA is incorporated into an appropriate vector. It can be selected by performing hybridization with DNA fragments corresponding to a part or all of the region or those labeled with synthetic DNA. The hybridization method can be carried out according to the method described in Molecular Cloning, 2nd u iambrook et al., Cold Spring Harbor Lao. Press, 1989). In addition, when using a commercially available library, it can be performed according to the method described in the attached instruction manual.

 Substitution of DNA base sequence can be done by using PCR or known kits such as Mu tan ™ -super Express Km (Takara Shuzo Co., Ltd.), Mu tan ™ Ichi K (Takara Shuzo Co., Ltd.), etc. It can be performed according to a known method such as DA-LAPCR method, Gapped duplex method, Kunkel method or the like, or a method analogous thereto.

D す る which encodes the cloned P 2 Y ₂ receptor protein can be used as it is depending on the purpose, or it can be digested with a restriction enzyme or added with a linker as desired. The DNA may have ATG as a translation initiation codon on the 5 ′ end side, and may have TAA, TGA, or TAG as a translation termination codon on the 3 ′ end side. These translation initiation codons and translation termination codons can also be added using an appropriate synthetic DNA adapter.

The expression vector for P 2 Y ₂ receptor protein is, for example: (ii) excising the desired DNA fragment from DNA encoding Ρ 2 Υ ₂ receptor protein, and (mouth) placing the DN Α fragment in an appropriate expression vector. Linked downstream of the promoter Can be manufactured.

 The vectors include plasmids derived from E. coli (eg, p BR 3 2 2, p BR 3 25, p UC 12 2, p UC 1 3), and plasmids derived from Bacillus subtilis (eg, p UB 110, p TP 5, p C 1 9 4.), plasmids derived from yeast (eg, p SHI 9, p SH 15), pacteriophages such as λ phage, animal winoles such as retro winoles, vaccinia winores In addition, p A l—11, p XT l, p R c ZCMV, p R c / RSV, pc DNA l / Neo, etc. are used.

 The promoter used in the present invention may be any promoter as long as it is suitable for the host used for gene expression. For example, when an animal cell is used as a host, S Ra promoter, S V 40 promoter, L TR promoter, CMV promoter, H S V -TK promoter and the like can be mentioned. Of these, it is preferable to use a CMV promoter, an SR promoter or the like.

 When the host is Escherichia, trp promoter, lac promoter, rec A promoter, LPL promoter, lpp promoter, etc. When the host is Bacillus, SPO promoter, SP When the host is an enzyme such as 02 promoter or pen P promoter, PH05 promoter, PGK promoter, GAP promoter, ADH promoter, etc. are preferable. When the host is an insect cell, a polyhedrin promoter, P10 promoter, etc. are preferred.

In addition to the above, expression vectors include enhancers, splicing signals, poly A addition signals, selectable markers, SV 40 replication origin (hereinafter sometimes abbreviated as SV 40 ori), etc. What is contained can be used. Is a selection marker one, for example, dihydrofolate reductase (hereinafter sometimes abbreviated as dhfr) gene [main source preparative Rekise preparative (MTX) resistance], ampicillin resistant gene (hereinafter, the Am p ^r Neomycin resistance gene (hereinafter, Neo ^r) May be abbreviated as G 4 18 resistance). In particular, when the dhfr gene is used as a selection marker using (J HO (dhfr-) cells, the target gene can also be selected by using a medium that does not contain thymidine. A signal sequence is added to the N-terminal side of the receptor protein of the present invention When the host is a bacterium belonging to the genus Escherichia, Pho A · signal sequence, Om p A · signal sequence, etc. If the host is an animal cell, such as α-amylase · signal sequence, subtilisin 'signal sequence, etc. if the host is yeast, MF a · signal sequence, SUC 2 · signal sequence, etc. Insulin signal sequence, ct-interferon signal sequence IJ, antibody molecule / signal sequence, etc. can be used.

A transformant can be produced by using the thus constructed vector containing DNΑ encoding the P 2 Y ₂ receptor protein of the present invention. As the host, for example, Escherichia bacteria, Bacillus bacteria, yeast, insect cells, insects, animal cells, etc. are used.

 A specific example of the genus Escherichia is Escherichia coli K 1 2 · D Η 1 [Procedures · The · National · The Power Demi-'op · Sciences · (Proc. Natl. Acad. Sci. USA), 60 0, 1 60 (1 9 6 8)], JM 1 0 3 [Nucleic Acids Research, 9卷, 3 0 9 (1 9 8 1)], JA 2 2 1 [Journal of Molecular Biology], — (Journal of Molecular Biology), 1 2 0 卷, 5 1 7 (1 9 7 8) ], HB 1 0 1 [Journal 'Ob' Molecular 'Biology, 4 1 卷, 4 5 9 (1 9 6 9)], C 6 0 0 [Genetics, 3 9 卷, 4 4 0 (1 9 5 4)] etc. are used.

Examples of Bacillus spp. Include Bacillus subtilis MI 1 1 4 [gene, 2 4 卷, 2 5 5 (1 9 8 3)], 2 0 7 — 2 1 'Journal of Biochemistry, 9 5 卷, 8 7 (1 9 8 4)], etc. are used. Examples of yeast include Saccharqmyces cerevisiae AH 2 2, AH 2 2 R- 'Ν A 8 7— 1 1 A, DKD— 5 D, 2 OB— 1 2, Schizosaccharomyces bomb ( Schizosaccharomyces pombe) NCYC 1 9 1 3, NCYC 2 0 3 6 and Pichia pastoris.

 As insect cells, for example, when the virus is Ac NPV, larvae-derived strains of larvae (Spodoptera frugiperda cell; Sf cells), MG 1 cells derived from the midgut of Trichoplusia ni, Trichoplusia ni High Five ™ Itoda vesicles derived from eggs, Hozuki moon sac from Mamestra brassicae, or cells derived from Estigmena acrea are used. When the virus is BmNPV, cocoon-derived cell lines (Bombyxmori N; B mN cells) are used. Examples of the S f cells include S f 9 cells (ATCCCRL 1711), S f 21 cells (hereinafter, Vaughn, JL et al., In Vivo, 13, 213-217, (1977). ) Etc. are used.

 As insects, for example, silkworm larvae are used [Maeda et al., Nature, 3 1 5 卷, 5 9 2 (1 9 8 5)].

 Examples of animal cells include monkey cell CO S-7, V ero, Chinese hamster cell CHO (hereinafter abbreviated as CHO cell), dhfr gene-deficient Chinese hamster cell C HO (hereinafter CHO (dhfr)) Abbreviated cells), mouse L cells, mouse At T-20, mouse myeloma cells, rat GH 3, human FL cells, and the like.

 To transform Escherichia spp., For example, Proc. Natl. Acad. Sci. USA, 6 Proc. Of the National 'Academy of' Sciense 'of' O ' 9 卷, 2 1 1 0 (1 9 7 2), Gene, 1 7 volumes, 1 0 7 (1 9 8 2), etc.

To transform Bacillus, for example, the method described in Molecular & General Genetics, Vol. 1 68, 1 1 1 (1 9 7 9), etc. may be used. Can To transform yeast, for example, Methods in Enzymology, 1 9 4 卷, 1 8 2— 1 8 7 (1 9 9 1) · National 'Academy ■ Ob'Sciences' of Ob The USA (Proc. Natl. Acad. Sci. USA), 7 5 卷, 1 9 2 9 (1 9 7 8) Can be done according to

 Insect cells or insects can be transformed, for example, according to the method described in Bio / Technology, 6, 47-55 (1988).

 To transform animal cells, for example, Cell Engineering Supplement 8 New Cell Engineering Experiment Protocol. 2 6 3 — 2 6 7 (1 9 9 5) (published by Shujunsha), Virology, 5 This can be done according to the method described in 2, 4 5 6 (1 9 7 3).

In this way, a transformant transformed with an expression vector containing DN Α encoding the P 2 Y ₂ receptor protein is obtained.

 When cultivating a transformant whose host is Escherichia or Bacillus, a liquid medium is suitable as a medium used for the culture, including a carbon source necessary for the growth of the transformant, Nitrogen sources, inorganic substances, etc. can be included. Examples of carbon sources include 'glucose, dextrin, soluble starch, sucrose, etc., and examples of nitrogen sources include ammonium salts, nitrates, corn stee' liquor, peptone, casein, meat extract, Examples of inorganic or organic substances and inorganic substances such as soybean meal and potato extract include calcium chloride, sodium dihydrogen phosphate, and magnesium chloride. In addition, yeast extract, vitamins, growth promoting factors and the like may be added. The pH of the medium is preferably about 5-8.

For example, M 9 medium containing glucose and casamino acids [Miller, Journal 'Ob'Exeper in Molecular Molecular Genes (Journal) of Experiments in Molecular Genetics), 4 3 1 — 4 3 3, Cold Spring Harbor Laboratory, New York 1 9 7 2]. If necessary, in order to make the promoter work efficiently, for example, a drug such as 3in ^^ or rilatalylic acid can be added.

 When the host is a bacterium belonging to the genus Escherichia, the culture is usually carried out at about 15 to 43 ° C for about 3 to 24 hours, and if necessary, aeration or agitation can be added.

 When the host is Bacillus, the culture is usually carried out at about 30 to 40 ° C for about 6 to 24 hours, and if necessary, aeration or agitation can be added.

 When cultivating a transformant whose host is yeast, examples of the culture medium include, but are not limited to, Burkholder I / J, medium [Bostian, KL, et al., Proc. · Sciences · The · USA (Proc. Natl. Acad. Sci. USA), 7 7 4, 4 5 0 5 (1 9 8 0)] and SD media containing 0.5% casamino acid [ Bitter, GA et al., Proc. Natl. Acad. Sci. USA, 8 1 卷, 5 3 3 0 (Proc. Natl. Acad. Sci. USA) 1 9 8 4)]. The pH of the medium is preferably adjusted to about 5-8. Incubate at approximately 20 ° C to 35 ° C for approximately 24 to 7 to 2 hours, with aeration and agitation as necessary.

 When culturing a transformant whose host is an insect cell or insect, Grace's Insect Medium (Grace, TCC, Nature, 195, 788 (1962)) was used as the medium. What added an additive such as% urine serum as appropriate is used. The pH of the medium is preferably adjusted to about 6.2 to 6.4. Incubate at about 27 ° C for about 3 to 5 days, and add air or agitation as necessary.

When cultivating a transformant whose host is an animal cell, the medium may be, for example, a MEM medium containing about 5 to 20% fetal bovine serum [Science, 1 2 2 卷, 5 0 1 (1 9 5 2)], DMEM medium [Virology, 8 卷, 3 9 6 (1 9 5 9)], R PM I 1 6 4 0 medium [Journal of 'The' American 'Medical' Association (The Journal of the American Medical Association) 1 9 9 卷, 5 1 9 (1 9 6 7)], 1 9 9 Medium [Proceeding of the Society (Proceeding of the Society) for the Biological Medicine), 7 3 卷, 1 (1 9 5 0)]. 11 is preferably about 6-8. Cultivation is usually performed at about 30 ° C to 40 ° C for about 15 to 60 hours, with aeration and agitation as necessary.

As described above, the P 2 Y ₂ receptor protein of the present invention can be produced in the cell, in the cell membrane or outside of the transformant.

In the [rho 2 Upsilon ₂ receptor protein used in the present invention is separated and purified from the culture described above, for example, can be carried out by the following method.

In [rho 2 Upsilon ₂ receptor protein is extracted culture or cells or found to be used in the present invention, after cultivation, collected bacteria or cells by a known method, suspended in a suitable buffer solution, super waves, fungus body § Rui such as by lysozyme and / or freeze-thaw then disrupted cells, a method of obtaining a crude extract of more [rho 2 Upsilon ₂ receptor protein to a centrifugal separation or filtration is appropriately used. The buffer solution may contain a protein denaturing agent such as urea or guanidine hydrochloride, or a surfactant such as Triton (registered trademark) X-100. When the P 2 Y ₂ receptor protein is secreted into the culture solution, after completion of the culture, the cells or cells are separated from the supernatant by a known method, and the supernatant is collected.

Purification of the 上清₂ receptor protein contained in the culture supernatant or the extract thus obtained can be performed by appropriately combining known separation and purification methods. These known separation and purification methods include mainly molecular weights such as methods that utilize solubility, such as salting-out solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyatalylamide gel electrophoresis. Method using difference in charge, method using difference in charge such as ion exchange chromatography, method using specific newness such as affinity chromatography, difference in hydrophobicity such as reversed-phase high-performance liquid chromatography And a method using the difference in isoelectric point such as isoelectric focusing. '· When the P 2 Y ₂ receptor protein obtained in this way is obtained in free form, it can be converted to a salt by a known method or a method analogous thereto, and conversely In some cases, it can be converted to a free form or other salt by a known method or a method analogous thereto.

In addition, the 産生 2 を₂ receptor protein produced by the recombinant may be arbitrarily modified or partially removed by allowing an appropriate protein-modifying enzyme to act before or after purification. You can also. Examples of protein modifying enzymes include trypsin, chymotribsin, arginyl endopeptidase, protein kinase, and glycosidase.

The activity of the _{2 2} receptor protein produced in this way can be measured by a binding experiment with a labeled ligand and an enzyme immunoassay using a specific antibody.

Antibodies against [rho 2 Upsilon ₂ receptor protein used in the present invention, if an antibody capable of recognizing the need is [rho 2 Upsilon ₂ receptor protein use in the present invention, polyclonal antibodies, be any of the monoclonal antibodies Good.

The antibody against the _{2 2} receptor protein used in the present invention can be produced according to a known antibody or antiserum production method using the _{2 2} receptor protein as an antigen.

 [Preparation of monoclonal antibodies]

 (a) Preparation of monoclonal antibody-producing cells

The P 2 Y ₂ receptor protein used in the present invention is administered to a mammal at a site where antibody production is possible by administration, together with a carrier or a diluent. In order to enhance the antibody-producing ability upon administration, complete Freund adjuvant or incomplete Freund adjuvant may be administered. Administration is usually once every 2 to 6 weeks, for a total of 2 to 10 times. Examples of mammals to be used include monkeys, rabbits, dogs, guinea pigs, mice, rats, hidges, and goats, but mouse rats are preferably used.

When producing monoclonal antibody-producing cells, the temperature of the immunized antigen Select individuals with antibody titers from blood animals such as mice, collect spleen or lymph nodes 2-5 days after the last immunization, and fuse antibody-producing cells contained in them with myeloma cells Thus, a monoclonal antibody-producing hybridoma can be prepared. The antibody titer in the antiserum can be measured, for example, by reacting the labeled receptor protein described below with the antiserum and then measuring the activity of the labeling agent bound to the antibody. The fusion operation can be carried out according to known methods, for example, the method of Kohler and Milstein [Nature, 2 56 6, pp. 4 95 (1 97 5)]. Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus, but preferably PEG is used.

Examples of myeloma cells include NS-1, P 3 U 1 and SP 2/0, and P 3 U 1 is preferably used. The preferred ratio between the number of antibody producing cells (spleen cells) and the number of myeloma cells is about 1: 1 to 20 _: 1, and PEG (preferably P EG 100 00 to PEG 60 00) Is added at a concentration of about 10 to 80%, and cell fusion is carried out efficiently by incubating at about 20 to 4 ° C, preferably about 30 to 37 ° C for about 1 to 10 minutes. it can.

 Various methods can be used for screening monoclonal antibody-producing hybridomas. For example, a solid phase (eg, a microplate) on which a receptor protein antigen is adsorbed directly or with a carrier is used. Add the supernatant, and then add anti-immunoglobulin antibody labeled with a radioactive substance or enzyme (if the cells used for cell fusion are mice, use anti-mouse immunoglobulin antibody) or protein A. To detect monoclonal antibodies bound to the phase, add the supernatant of the hybridoma culture to the solid phase adsorbed with anti-immunoglobulin antibody or protein A, add the receptor protein labeled with a radioactive substance or enzyme, Examples thereof include a method for detecting a monoclonal antibody bound to a solid phase.

'' Monoclonal antibody selection should be done according to known or similar methods. Usually, it can be carried out in an animal cell medium supplemented with HAT (hypoxanthine, aminopterin, thymidine). As a selection and breeding medium, any medium can be used as long as it can grow hyperprideoma. For example, RPMI 16 40 medium containing 1 to 20%, preferably 10 to 20% fetal bovine serum :! A GIT medium (Wako Pure Chemical Industries, Ltd.) containing ~ 10% fetal bovine serum or a serum-free medium for culturing Hypridoma (SFM-101, Nissui Pharmaceutical Co., Ltd.) can be used. The culture temperature is usually 20 to 40 ° C, preferably about 37 ° C. The culture time is usually 5 days to 3 weeks, preferably 1 to 2 weeks. Culture can usually be performed under 5% carbon dioxide. The antibody titer of the hybridoma culture supernatant can be measured in the same manner as the antibody titer in the antiserum described above.

 (b) Purification of monoclonal antibody

 Monoclonal antibodies can be separated and purified by immunoglobulin separation and purification methods (eg, salting-out method, alcohol precipitation method, isoelectric precipitation method, electrophoresis method, ion exchanger). (For example, DEAE) adsorption / desorption method, ultracentrifugation method, gel filtration method, antigen binding solid phase or active adsorbent such as protein A or protein G Specific purification method to obtain

 [Preparation of polyclonal antibodies]

The polyclonal antibody of the present invention can be produced by a known method or a method analogous thereto. For example, a complex of an immunizing antigen (P 2 Y ₂ receptor protein antigen) and a carrier protein is prepared, and a mammal is immunized in the same manner as in the production of the monoclonal antibody described above. It can be produced by collecting an antibody-containing substance against the Ρ2 蛋白₂ receptor protein used in the invention and separating and purifying the antibody.

Regarding the complex of immunizing antigen and carrier protein used to immunize mammals, the type of carrier protein and the mixing ratio of carrier and hapten are the antibodies against the hapten immunized by cross-linking the carrier. However, any ratio can be cross-linked as long as it can be efficiently processed. For example, rosin serum albumin, ushisailogrobulin, keyhole limpet, mosianin, etc. Then, a method is used in which the hapten 1 is coupled at a ratio of about 0.1 to 20 and preferably about 1 to 5.

 In addition, various condensing agents can be used for hapten and carrier force printing, such as active aldehyde reagent containing aldehyde aldehyde, maleimide active ester, thiol group, and dithiobilidyl group. Used.

 The condensation product is administered to a warm-blooded animal either at the site where antibody production is possible, or with a carrier or diluent. In order to enhance the antibody-producing ability upon administration, complete Freund's adjuvant or incomplete Freund's adjuvant may be administered. Administration can be performed about once every 2 to 6 weeks, usually about 3 to 10 times in total.

 Polyclonal antibodies can be collected from blood, ascites, etc. of mammals immunized by the above method, preferably blood.

 The polyclonal antibody titer in the antiserum can be measured in the same manner as the above-described measurement of the antibody titer in the serum. The separation and purification of the polyclonal antibody can be performed according to the same method for separating and purifying the immunoglobulin as the above-described separation and purification of the monoclonal antibody.

More specifically, as an antibody against [rho 2 Upsilon ₂ receptor protein used in the present invention, for example, [rho 2 Upsilon ₂ receptor protein C-terminal peptide or antibody that recognizes a salt thereof (in particular, monoclonal Antibody).

In addition, the 2 ₂ receptor protein used in the present invention is highly expressed particularly in skeletal muscle.

Accordingly, as used in the present invention [rho 2 Upsilon ₂ receptor protein, Ρ ₂ Υ 2 DNA encoding the receptor protein, P 2 Y ₂ antibody against the receptor protein, P 2 Y ₂ D to the receptor protein for co one de Antisense D Α Α against Ν を has the following uses.

'(1)' Hypoglycemic agent The DNA encoding the P 2 Y ₂ 'receptor protein used in the present invention can be used as a medicine such as _{λ or} a hypoglycemic agent.

For example, there is a patient who cannot expect physiological actions such as UT Ρ or ATP ligand (deficiency of P 2 Y ₂ receptor protein) due to decreased Ρ 2 Υ ₂ receptor protein in vivo A) administering a P 2 Y ₂ receptor protein to the patient and supplementing the amount of the _{2 2} protein; b) (ii) DN ₂ encoding the P 2 Y ₂ receptor protein Or (mouth) after inserting D Ν コ ー ド encoding the P 2 Y ₂ receptor protein used in the present invention into a target cell and expressing it, By transplanting cells into the patient, the amount of P 2 Y ₂ receptor protein in the patient's body can be increased, and the action of the ligand can be fully exerted. That is, the DNA encoding the Ρ2 蛋白₂ receptor protein used in the present invention is useful as a safe and low-toxic hypoglycemic agent.

Specifically, D Nyuarufa encoding [rho 2 Upsilon ₂ receptor protein used in the present invention are, for example, diabetes mellitus, impaired glucose tolerance, ketosis, acidosis, diabetic neuropathy, diabetic nephropathy, diabetic Retinopathy, hyperlipidemia, obesity, metabolic syndrome, sexual dysfunction, skin disease, arthropathy, osteopenia, arteriosclerosis, blood plug disease, dyspepsia, memory learning disorder, etc. preferably used for diabetes be able to. Diabetes includes insulin-dependent (type I) diabetes and non-insulin-dependent (type II) diabetes.

When the DN ₂ encoding the _{2 2} receptor protein used in the present invention is used as the above-mentioned hypoglycemic agent, it can be formulated according to conventional means.

On the other hand, when the DNA encoding the P 2 Y ₂ receptor protein is used as the above-mentioned hypoglycemic agent, the DNA alone or retrovirus vector, adenovirus vector, adenovirus associated virus vector After inserting into an appropriate vector such as, it can be carried out according to conventional means. The DNA can be used as it is or with an auxiliary agent for promoting intake. Therefore, it can be administered. ₍₄₎ For example, DNA encoding the P 2 Y ₂ receptor protein used in the present invention is orally administered as a tablet, capsule, elixir, mic mouth capsule, etc. It can be used parenterally in the form of sterile solutions with water or other pharmaceutically acceptable liquids or injections such as suspensions. For example, known carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, etc. that are physiologically recognized as D ₂コ ー ド encoding the Ρ 2 Υ ₂ receptor protein used in the present invention And can be prepared by mixing in the unit dosage forms required for accepted formulation practice. The amount of active ingredient in these preparations is such that an appropriate volume within the indicated range can be obtained.

Additives that can be incorporated into tablets, force-pellants, etc. include, for example, binders such as gelatin, corn starch, tragacanth, gum arabic, excipients such as crystalline cellulose, corn starch, gelatin For example, a swelling agent such as alginic acid, a lubricant such as magnesium stearate, a sweetening agent such as sucrose, lactose or saccharine, a flavoring agent such as peppermint, squid mono oil or tea. When the dosage unit form is a capsule, a liquid carrier such as fats and oils can be further contained in the above type of material. Sterile compositions for injection should be treated according to normal formulation practices such as dissolving or suspending active substances in vehicles such as water for injection, naturally occurring vegetable oils such as sesame oil, coconut oil, etc. Can do. As an aqueous solution for injection, for example, physiological saline, isotonic solutions containing glucose and other adjuvants (for example, D-sorbitol, D-mannitol, sodium chloride, etc.) are used. Soluble solubilizers such as alcohol (eg, ethanol), polyalcohol (eg, propylene glycol, polyethylene glycol), nonionic surfactants (eg, polysorbate 80 TM, HCO- 50) You can use it together. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and may be used in combination with solubilizing agents such as benzyl benzoate and benzyl alcohol. Examples of the hypoglycemic agent include, for example, a buffer (for example, phosphate buffer, sodium acetate buffer), a soothing agent (for example, benzalkonium chloride, hydrochloride pro-in), and a stabilizer (for example, , Human serum albumin, polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants, etc. The prepared injection solution is usually filled in a suitable ampoule.

 The preparations obtained in this way are safe and have low toxicity. For example, for human mammals (eg rats, mice, rabbits, hidges, pigs, tusks, cats, dogs, monkeys, etc.) Can be administered.

The dose of DNA encoding the P 2 Y ₂ receptor protein used in the present invention varies depending on the administration target, target organ, symptoms, administration method, etc. In the case of 60 kg, it is about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg per day. When administered parenterally, the single dose varies depending on the subject of administration, target organ, symptom, administration method, etc. For example, in the form of injection, for example, diabetic patients (60 kg) ), About 0.1 to 30 mg per day, preferably about 0.1 to 20 mg, more preferably about 0.1 to: LO mg is administered by intravenous injection. It is convenient to do. In the case of other animals, an amount converted per 60 kg can be administered.

 (2) Glycemic agent

P 2 Y ₂ receptor protein or its partial base peptide used in the present invention, Anchisensuporinukureochidoma for DN Alpha encoding [rho 2 Upsilon ₂ receptor protein used in the present invention for the P 2 Y ₂ receptor protein The antibody can be used as a medicine such as a clot raising agent.

For example, [rho 2 Upsilon when there are excessive patients of ₂ receptor protein, a) Ρ ₂ Υ 2 antibody to the receptor protein itself or P 2 Y ₂ receptor protein is administered to the patient the [rho 2 Upsilon ₂ receptor B) an antisense polynucleotide against DN コ ー ド that encodes the P 2 Y ₂ receptor protein. By suppressing the expression of D Ν コ ー ド encoding P 2 Y ₂ receptor protein when administered to patients, the amount of P 2 Y ₂ receptor protein in the patient's body is decreased, and receptor function is normalized. can do. That is, antisense polynucleotides against Α 2 Υ ₂ receptor protein or partial peptides, DN コ ー ド encoding Ρ 2 Υ ₂ receptor protein, or antibodies to P 2 Y ₂ receptor protein are safe and low toxic. It is useful as a blood glucose raising agent.

Specifically, the present invention is used in [rho 2 Upsilon ₂ receptor protein or its parts partial peptide, antisense poly quinuclidine Reochi de or P for DN Alpha encoding [rho 2 Upsilon ₂ receptor protein used in the present invention An antibody against 2 Y ₂ receptor protein can be used, for example, for hypoglycemia.

When using antibodies to DNA encoding the [rho 2 Upsilon ₂ receptor protein for antisense polynucleotide or [rho 2 Upsilon ₂ receptor protein as the hyperglycemic agent may be formulated in a conventional manner.

On the other hand, when the antisense polynucleotide against D Α コ ー ド encoding Ρ 2 Υ ₂ receptor protein is used as the above-mentioned blood glucose-elevating agent, the DNA alone, retrovirus vector, adenovirus vector, adenovirus association is used. After insertion into an appropriate vector such as an eted virus vector, it can be performed according to conventional means. DNA can be administered as is or with an adjunct to facilitate uptake through a catheter, such as a gene gun or a high-mouth gel catheter.

For example, P 2 Y ₂ antibody to the receptor protein antisense polynucleotide or P 2 Y ₂ receptor protein to DN Alpha encoding are tablets which may be sugar coated as necessary, capsules, elixirs, microcapsules Le It can be used orally as an agent, or parenterally in the form of a sterile solution with water or other pharmaceutically acceptable liquid, or an injection such as a suspension. For example, a known carrier, flavoring agent, and enhancer that is physiologically recognized as an antisense polynucleotide for D 2 コ ー that encodes a 2 ₂ receptor protein or an antibody for P 2 ₂ receptor protein used in the present invention. Generally accepted with form, vehicle, preservatives, stabilizers, binders, etc. Can be made by mixing in the unit dosage form required for the implementation of the formulated product. The amount of active ingredient in these preparations is such that an appropriate volume within the indicated range can be obtained.

Additives that can be mixed into tablets, capsules, etc. include, for example, binders such as gelatin, corn starch, tragacanth, gum arabic, excipients such as crystalline cellulose, corn starch, For example, a swelling agent such as gelatin or alginic acid, a lubricant such as magnesium stearate, a sweetening agent such as sucrose, lactose or saccharin, a flavoring agent such as peppermint, cocoa oil or chili is used. When the dosage unit form is a capsule, a liquid carrier such as fats and oils can be further contained in the above type of material. Sterile compositions for injection should be treated according to normal pharmaceutical practice, such as dissolving or suspending active substances in vehicles such as water for injection, naturally produced vegetable oils such as sesame oil and coconut oil. Can do. As an aqueous solution for injection, for example, physiological saline, isotonic solutions containing glucose and other adjuvants (for example, D-sorbitol, D-mannitol, sodium chloride, etc.) are used. Dissolving aids, for example, alcohol (eg, ethanol), polyalcohol (eg, propylene dallicol, polyethylene glycol), nonionic surfactant (eg, polysorbate 80 ^TM , HCO- 50) Do it anyway. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and they may be used in combination with solubilizing agents such as benzyl benzoate and benzyl alcohol. Examples of the blood glucose-elevating agent include a buffer (for example, phosphate buffer, sodium acetate buffer), a soothing agent (for example, benzalkonium chloride, hydrochloride pro-in), and a stabilizer (for example, , Human serum albumin, polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants, etc. The prepared injection solution is usually filled in a suitable ampoule.

The preparations obtained in this way are safe and low toxic. For example, humans such as mammals (eg rat, mouse, rabbit, hidge, pig, ushi, Cats, dogs, monkeys, etc.). , Antisense polynucleotide against DN コ ー ド encoding P 2 Y ₂ receptor protein used in the present invention The dose varies depending on the administration subject, target organ, symptom, administration method, etc. In the case of administration, for example, in hypoglycemic patients (as 60 kg), generally about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1. 0 to 20 mg. When administered parenterally, the single dose varies depending on the subject of administration, target organ, symptom, administration method, etc. For example, in the form of injection, for example, hypoglycemic patients (60 kg) In this case, about 0.1 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day is administered by intravenous injection. Is convenient. In the case of other animals, an amount converted per 6 O kg can be administered.

The dose of the antibody against the P 2 Y ₂ receptor protein used in the present invention varies depending on the administration subject, target organ, symptom, administration method, etc., but in the case of oral administration, generally, for example, hypoglycemia In a patient (as 60 kg), it is about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg per day. When administered parenterally, the single dose varies depending on the subject of administration, target organ, symptoms, administration method, etc. For example, in the form of an injection, for example, a hypoglycemic patient (60 (in kg), about 0.1 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg is administered intravenously per day. Is convenient. In the case of other animals, an amount converted per 60 kg can be administered.

The dose of the P 2 Y ₂ receptor protein used in the present invention varies depending on the administration subject, target organ, symptom, administration method, and the like. However, in the case of oral administration, generally, for example, patients with hypoglycemia (6 (As 0 kg) is about 0.1 to 1: OO mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg per day. If administered parenterally, dosing once The amount varies depending on the administration subject, target organ, symptom, administration method, etc. For example, in the form of an injection, it is usually about 0.00 per day in a hypoglycemic patient (as 60 kg). About 1 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 9 mg; It is convenient to administer ~ 10 mg by intravenous injection. In the case of other animals, an amount converted per 60 kg can be administered.

 (3) Diabetes or hypoglycemia diagnostic agent

The DN コ ー ド encoding the P 2 Y ₂ receptor protein used in the present invention can be used as a prop for humans or mammals (eg rat, mouse, rabbit, hidge, pig, ushi, Cats, dogs, monkeys, etc.) can detect an abnormality (gene abnormality) in DNA or mRNA encoding the P 2 Y ₂ receptor protein or its partial peptide used in the present invention. It is useful as a genetic diagnostic agent for damage, mutation or reduction of expression of DNA or mRNA, increase of DNA or mRNA, or excessive expression.

The above-described genetic diagnosis using the DNA encoding the P 2 Y ₂ receptor protein used in the present invention is, for example, the known Northern hybridization using the PCR-SSCP method (Genomics, No. 1). 5 卷, 8 7 4-8 7 9 (1 9 8 9), Proceedings of the National Academy, Op The National, Power Demi 1 Ob, Sciences 'Ob' USA of Sciences of the United States oi America;, pp. 86, pp. 2 706-2 770 (1 9 8 9)).

For example, when a decrease in the expression of the P 2 Y ₂ receptor protein used in the present invention is detected by Northern hyperpridition, for example, it is related to dysfunction of the Ρ 2 Υ ₂ receptor protein used in the present invention. It can be diagnosed that the disease is highly likely or is likely to be affected in the future. In addition, when overexpression of the Ρ 2 Υ ₂ receptor protein used in the present invention is detected by Northern hyperprecipitation, for example, it is used in the present invention. It can be diagnosed that the disease is likely caused by overexpression of the P 2 Y ₂ receptor protein, or is likely to be affected in the future.

Examples of diseases associated with dysfunction of Ρ 2 Υ ₂ receptor protein used in the present invention include diabetes, impaired glucose tolerance, keto-cissis, acidosis, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, Examples include hyperlipidemia, obesity, metabolic syndrome, sexual dysfunction, skin disease, arthropathy, osteopenia, arteriosclerosis, thrombotic disease, dyspepsia, memory learning disorder. Further, diseases caused by overexpression of [rho 2 Upsilon ₂ receptor proteins are found used in the present invention include, for example, hypoglycemia.

The antibodies of the present invention is capable of specifically recognizing the [rho 2 Upsilon ₂ receptor protein, quantification of [rho 2 Upsilon ₂ receptor protein in a test fluid, especially, for quantification by Sanditsuchi immunoassay can do.

 That is, the present invention

(i) The antibody of the present invention is competitively reacted with a test solution and a labeled P 2 Y ₂ receptor protein, and the labeled Ρ 2 Υ ₂ receptor protein bound to the antibody is A method for quantifying Ρ 2 Υ ₂ receptor protein in a test solution, characterized by measuring a ratio; and

(ii) After reacting the test solution with the antibody of the present invention insolubilized on the carrier and the labeled another antibody of the present invention simultaneously or successively, the activity of the labeling agent on the insolubilized carrier is measured. A method for quantifying P 2 Y ₂ receptor protein in a test solution is provided.

In the quantification method of (ii) above, one antibody is an antibody that recognizes the heel end of the P 2 Y ₂ receptor protein and the other antibody is an antibody that reacts with the C terminus of the Ρ 2 Υ ₂ receptor protein. It is desirable that

It is also possible to perform the detection by the [rho 2 Upsilon ₂ can perform quantification of [rho 2 Upsilon ₂ receptor protein with monoclonal antibodies to the receptor protein Ho force tissue staining or the like. For these purposes, the antibody molecule itself may be used, or the F (ab ′) ₂ , Fab, or Fab fraction of the antibody molecule may be used. The method for quantifying the P 2 Y ₂ receptor protein using the antibody of the present invention is not particularly limited, and an antibody corresponding to the amount of antigen in the solution to be measured (for example, Ρ 2 Υ ₂ receptor protein mass). Any method can be used as long as the amount of antigen or antibody-antigen complex is detected by chemical or physical means, and this is calculated from a standard curve prepared using a standard solution containing a known amount of antigen. The measurement method described above may be used. For example, a nephrometry, a competition method, an immunometric method and a sandwich method are preferably used, but the sandwich method described below is particularly preferable in terms of sensitivity and specificity.

Examples of the labeling agent used in the measurement method using the labeling substance include radioisotopes, enzymes, fluorescent substances, and luminescent substances. As the radioisotope, for example, [ ¹²⁵ I], [ ¹³¹ I], [ ³ H], [ ¹⁴ C] and the like are used. As the enzyme, a stable enzyme having a large specific activity is preferable. For example, / 3-galactosidase, _darcosidase, alkaline phosphatase, peroxidase, phosphonate dehydrogenase and the like are used. As the fluorescent material, for example, fluorescamine, fluorescein isothiocyanate and the like are used. As the luminescent substance, for example, luminol, luminol derivatives, luciferin, lucigenin and the like are used. Furthermore, a piotin-avidin system can be used for the binding between the antibody or antigen and the labeling agent.

Further, when a decrease in the concentration of ₂ 2 Υ ₂ receptor protein is detected by quantifying the concentration of P 2 · Y ₂ receptor protein using the antibody of the present invention, for example, 受 容 2 Υ ₂ receptor It can be diagnosed that the disease is related to dysfunction of body protein, or is likely to be affected in the future. Further, when increasing concentrations of [rho 2 Upsilon ₂ receptor protein is detected, for example, is a disease caused by overexpression of [rho 2 Upsilon ₂ receptor protein, or likely to suffer future Can be diagnosed.

The diseases associated with dysfunction of the Ρ 2 Υ ₂ receptor protein of the present invention include: diabetes, glucose tolerance, ketosis, acidosis, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy Hyperlipidemia, obesity, metabolic syndrome, sexual function Disorders, skin diseases, arthropathy, osteopenia, arteriosclerosis, thrombotic diseases, dyspepsia, memory learning disorders, etc. Moreover, examples of the disease caused by overexpression of the P 2 Y ₂ receptor protein of the present invention include hypoglycemia.

 (4) Screening method for compounds that regulate blood glucose levels

Disk Riyungu of compounds that alter the expression level of [rho 2 Upsilon ₂ receptor protein used in the present invention,

(i) A certain period of time (30 to 24 hours before, preferably 30 minutes before giving a drug or physical stress to normal or disease model non-human mammals) ~ 1 to 2 hours ago, more preferably 1 hour to 6 hours before) or after a certain time (30 minutes to 3 days, preferably 1 hour to 2 days later, more preferably 1 hour to 24 hours later) ), Or the test compound is administered at the same time as the drug or physical stress, and after a certain period of time after administration (30 minutes to 3 days, preferably 1 hour to 2 days, more preferably 1 hour to 2 4 hours later), the amount of mRNA in the P 2 Y ₂ receptor protein contained in the cells can be quantified and analyzed.

(ii) When the transformant is cultured according to a conventional method, the test compound is mixed in the medium and cultured for a certain period of time (after 1 day to 7 days, preferably after 1 day to 3 days, more preferably after 2 days to 3 days later), it can be carried out by quantifying and analyzing the amount of mRN of the P 2 Y ₂ receptor protein contained in the transformant. The compound obtained by using the screening method of the present invention or a salt thereof is a compound having an action of changing the expression level of the P 2 Y ₂ receptor protein used in the present invention. Specifically, by increasing the expression amount of (I) [rho 2 Upsilon ₂ receptor protein, [rho 2 Upsilon mediated cell stimulating activities ₂ receptor protein (e.g., Arakidon acid release, Asechirukorin release, intracellular C a ²⁺ release, cell Activity that promotes or suppresses intracellular cAMP generation, intracellular cGMP generation, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activation, pH reduction, etc. (Mouth) A compound that attenuates the cell-stimulating activity by decreasing the expression level of the P 2 Y ₂ receptor protein. Examples of the compound include peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, and the like. These compounds may be novel compounds or known compounds.

Therefore, the compound that changes the expression level of the P 2 Y ₂ receptor protein of the present invention obtained by the above screening method can be used for a disease associated with dysfunction of Ρ 2 Υ ₂ receptor protein. .

Specifically, the expression level of [rho 2 Upsilon ₂ receptor protein of the present invention is increased, a compound that enhances the cell stimulating activity, against diseases associated with functional insufficiency of [rho 2 Upsilon ₂ receptor protein of the present invention It is useful as a hypoglycemic agent.

The expression level of [rho 2 Upsilon ₂ receptor protein of the present invention reduces, compounds that decrease the cell-stimulating activity is safe and low toxic for diseases caused by overexpression of [rho 2 Upsilon ₂ receptor protein of the present invention It is useful as a blood glucose raising agent.

The diseases associated with dysfunction of the [rho 2 Upsilon ₂ receptor protein of the present invention, diabetes, impaired glucose tolerance, ketosis, acidosis, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, high Examples include lipemia, obesity, metabolic syndrome, sexual dysfunction, skin disease, arthropathy, osteopenia, arteriosclerosis, thrombotic disease, dyspepsia, memory learning disorder. Further, diseases caused by overexpression of [rho 2 Upsilon ₂ receptor protein of the present invention include, for example, hypoglycemia. Examples of salts of the compounds include physiologically acceptable acids (eg, inorganic acids, organic acids, etc.) and bases (eg, alkali metals such as sodium and potassium; alkaline earth metals such as calcium), etc. And physiologically acceptable acid addition salts are particularly preferred. Examples of such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid). Salts with acid, succinic acid, tartaric acid, citrate, malic acid, succinic acid, benzoic acid, methane sulfonic acid, benzene sulfonic acid, etc.).

 Hereinafter, the same salt is used.

When the compound obtained by using the screening method of the present invention or a salt thereof is used as a pharmaceutical composition, it can be formulated according to conventional means. For example, the compound may be used orally as tablets, capsules, elixirs, microcapsules, etc. with sugar coating as required, or with water or other pharmaceutically acceptable liquids. It can be used parenterally in the form of an injectable solution such as a sterile solution or suspension. For example, the unit required for the practice of a generally accepted formulation together with known physiologically recognized carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, etc. It can be produced by mixing in the form. The amount of active ingredient in these preparations is such that an appropriate volume within the indicated range is obtained.

Examples of additives that can be mixed into tablets and capsules include gelatin, corn starch, tragacanth, binders such as gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, and alginic acid. For example, a leavening agent such as magnesium stearate, a sweetening agent such as sucrose, lactose or saccharin, a flavoring agent such as peppermint, cocoa oil, or cherry is used. When the dosage unit form is a capsule, a liquid carrier such as fats and oils can be further contained in the above type of material. Sterile compositions for injection should be treated according to normal formulation practices such as dissolving or suspending active substances in vehicles such as water for injection, naturally occurring vegetable oils such as sesame oil, coconut oil, etc. Can do. As an aqueous solution for injection, for example, physiological saline, isotonic solutions containing glucose and other adjuvants (for example, D-sorbitol, D-mannitol, sodium chloride, etc.) are used. Suitable solubilizers such as alcohol (eg, ethanol), polyalcohol (eg, propylene glycol, polyethylene dallicol), nonionic surfactants (eg, polysorbate 80 ^TM , HCO-50), etc. You may use together. Examples of the oily liquid include sesame oil and soybean oil, which may be used in combination with a solubilizing agent such as benzyl benzoate or benzyl alcohol. In addition, the pharmaceutical composition includes, for example, a buffer (for example, a phosphate buffer, Sodium acetate buffer), soothing agent (eg, benzalkonium chloride, hydrochloride pro-in, etc.), stabilizer (eg, human serum albumin, polyethylene glycol, etc.), preserved IJ (eg, benzyl alcohol, phenol) Etc.), and may be blended with an antioxidant. The prepared injection solution is usually filled in a suitable ampoule.

 The preparations obtained in this way are safe and low toxic. For example, they can be used in human mammals (eg rats, mice, rabbits, hidges, pigs, tusks, cats, dogs, monkeys, etc.). Can be administered.

 The dose of the compound or a salt thereof varies depending on the administration subject, target organ, symptom, administration method, etc., but in the case of oral administration, for example, in diabetic patients (as 6 O kg), About 0. per day! ˜10 O mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg. When administered parenterally, the single dose varies depending on the subject of administration, subject organ, symptoms, administration method, etc. For example, in the form of injections, for example, diabetic patients (as 60 kg) , About 0.1 to 30 mg per day, preferably about 0.1:! To 20 mg, more preferably about 0.1 to 10 mg is administered by intravenous injection. Is convenient. In the case of other animals, an amount converted per 60 kg can be administered.

 (5) Agonist Top / Antagonist Screening Method

In order to screen a compound that changes the binding property between the ligand and the P 2 Y ₂ receptor protein used in the present invention, for example, an appropriate Ρ 2 Υ ₂ receptor protein fraction and a label are used. Ligand is needed.

[Rho Examples 2 Upsilon ₂ receptor protein fraction, native [rho 2 Upsilon ₂ receptor protein fraction component forceヽor recombinant having an activity equivalent and it [rho 2 Upsilon like _receptor protein fraction is desirable. Here, the equivalent activity indicates an equivalent ligand binding activity, a signal information transmission action, and the like.

As the labeled ligand, a labeled ligand, a labeled ligand analog compound, or the like is used. For example ^[3 H], ^[125 I], ^[14 C], ^[35 S] A ligand labeled with such as is used. The _¾ specifically, in performing the disk re one Eng compounds that alter the binding property between a ligand and P 2 Y ₂ receptor protein, cells or cell containing first [rho 2 Upsilon ₂ receptor protein membrane Prepare Ρ 2 Υ ₂ receptor protein preparation by suspending aliquots in a buffer suitable for screening. Any buffer can be used as long as it does not inhibit the binding of ρ 2 ₂ receptor protein such as ρ 〜 4 to 10 (preferably ρ Η 6 to 8) phosphate buffer or Tris monohydrochloride buffer. But you can. In order to reduce non-specific binding, surfactants such as CHAP S, Tween -80 ™ (Kaoichi Atlas), digitonin, and deoxycholate can be added to the buffer. Furthermore, protease inhibitors such as PMS F, leupeptin, E-64 (manufactured by Peptide Institute) and pepstatin can be added for the purpose of suppressing degradation of receptors and ligands by protease. A fixed amount (5 0 00 cpm to 5 OOOOO cpm) of labeled ligand is added to 0.01 ml to 10 ml of the receptor solution, and simultaneously 1 0— ⁴ M to 1 0_ ^1Q M test compound Coexist. Prepare a reaction tube with a large excess of unlabeled ligand to determine the amount of non-specific binding (NSB). The reaction is carried out at about 0 to 50 ° C., preferably about 4 to 37 ° C. for about 20 minutes to 24 hours, preferably about 30 minutes to 3 hours. After the reaction, filter with a glass fiber filter, etc., wash with an appropriate amount of the same buffer, and then measure the radioactivity remaining on the glass fiber filter with a liquid scintillation counter or γ-force counter. When there is no antagonistic substance (Β0) minus the non-specific binding amount (ΝSB) (BO—NSB) is 100%, the specific binding amount (B—NSB) is For example, a test compound that is 50% or less can be selected as a candidate substance capable of competitive inhibition.

Another method for screening for a compound that changes the binding property between the ligand and the P 2 Y ₂ receptor protein is, for example, cell stimulating activity via the Ρ 2 Υ ₂ receptor protein (eg, arachidon). Acid release, acetylcholine release, intracellular ^{Ca2 +} release, intracellular cAMP generation, intracellular cGMP generation, wild boar Tall phosphate production, cell membrane potential fluctuation, phosphorylation of intracellular protein, — fos activation, activity to promote or suppress pH reduction, etc. Especially, intracellular Ca ²⁺ concentration increasing activity, intracellular c The measurement is carried out using a known method or a commercially available measurement kit. Specifically, first, cells containing the P 2 Y ₂ receptor protein of the present invention are cultured on a multiwall plate or the like. Before performing screening, replace with a fresh medium or an appropriate buffer that is not toxic to cells, add test compounds, etc., and incubate for a certain period of time. Quantify the resulting product according to the respective method. If it is difficult to produce a substance (for example, C ²⁺ , c AMP, etc.) that serves as an index of cell stimulating activity using a degrading enzyme contained in the cell, an inhibitor for the degrading enzyme is added to perform the assay. It's okay. Further, the activity such as cAMP production inhibition can be detected as a production inhibitory action on cells whose basic production amount has been increased with forskolin or the like.

In order to measure cell stimulating activity and perform screening, cells expressing an appropriate P 2 Y ₂ receptor protein are required. The cells expressing [rho 2 Upsilon ₂ receptor protein, cells having a native-type [rho 2 Upsilon ₂ receptor protein strains, such as the above recombinant [rho 2 Upsilon ₂ cell lines expressing the receptor protein is desirable.

 Examples of test compounds include peptides, proteins, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, and animal tissue extracts. These compounds are novel compounds. It may be a known compound.

As the test compound, [rho 2 Upsilon ₂ receptor based on the protein atomic positions coordinate and ligand binding pocket of the active site of, compounds I designed to bind to the ligand binding pocket is preferably used. Measurement of [rho 2 Upsilon ₂ receptor protein located atomic coordinates and ligand binding pocket Bok of the active site of the leaves in be conducted using a known method or a method analogous thereto. The specific evaluation method of whether the compound that changes the binding property between the ligand and the P 2 Y ₂ receptor protein of the present invention is an agonist or an antagonist is the following (i) or (ii) Just follow.

(i) After performing the above screening method to obtain a compound or a salt thereof that changes the binding property between the ligand and the P 2 Y ₂ receptor protein (in particular, inhibits binding), It is measured whether the salt has the cell stimulating activity described above. Specifically, it is possible to confirm with Agonisu bets subscription-learning process against the above-mentioned [rho 2 Upsilon ₂ receptor protein, a compound or a salt thereof having Said sub胞刺stimulation activity is an Agonisu DOO The compound having no cell stimulating activity or a salt thereof is an antagonist.

(ii) (a) A test compound is contacted with a cell containing the P 2 Y ₂ receptor protein of the present invention, and the above-mentioned cell stimulating activity is measured. Test compounds that have the cell-stimulating activity is Agonisu preparative for [rho 2 Upsilon ₂ receptor protein.

(b) P 2 Y ₂ receptor protein compounds that activate (e.g., ligand) to activate the when contacted to the cells containing the [rho 2 Upsilon ₂ receptor protein, the [rho 2 Upsilon ₂ receptor protein compounds and the test compounds was measured cell stimulating activity when contacted with a cell containing a [rho 2 Upsilon ₂ receptor protein and compared. [Rho 2 Upsilon ₂ receptor protein test compound capable of decline the cell stimulating activity induced by the compound that activates the are antagonistic preparative for [rho 2 Upsilon ₂ receptor protein.

 (6) Screening kit

Or compounds that alter the binding property between the ligand and the [rho 2 Upsilon ₂ receptor protein kit for screening a salt thereof, [rho 2 Upsilon ₂ receptor protein, cells containing [rho 2 Upsilon ₂ receptor protein or [rho, Such as those containing a membrane fraction of cells containing _{2 2} receptor proteins.

 Examples of the screening kit of the present invention include the following.

 1. Reagent for screening

a) Measurement buffer and washing buffer Hanks' Balanced Salt Solution (manufactured by Gibco) plus 0.05% urushi serum albumin (manufactured by Sigma).

Sterilize by filtration through a 0.45 μπι filter and store at 4 ° C or prepare at the time of use. -b) P 2 Y ₂ receptor protein preparation

CHO cells expressing the [rho 2 Upsilon ₂ receptor protein used in the present invention, and passaged 1 2 well plates at 5 X 1 0 ⁵ cells / well, 3 7. C, 5% C 0 ₂ , 95% air cultured for 2 days.

 c) Labeled ligand

Store commercially available ligand aqueous solution labeled with [ ³ H], [ ¹²⁵ I], [ ¹⁴ C], [ ³⁵ S], etc. at 4 ° C or 120 ° C for measurement at the time of use. Dilute to 1 μΜ with buffer.

 d) Ligand standard solution

 Dissolve the ligand to 1 mM with P B S containing 0.1% ushi serum alpmin (manufactured by Shidama) and store at 120 ° C.

 2. Measurement method

a) After washing the P 2 Y ₂ receptor protein-expressing CHO cells cultured in a 1-well tissue culture plate twice with 1 ml of the measurement buffer, 490 1 of the measurement buffer was added. Add to each hole.

^{b) 1 0- 3 ~ 1 0} 4 ° after test compound solution 5 1 added Micromax, labeled ligand 5 zeta 1 was added and reacted at room temperature for 1 hour. To determine the amount of non-specific binding, add 5 μ 1 of 10–3 リ ガ ン ド ligand instead of the test compound. c) Remove the reaction solution and wash 3 times with 1 ml of washing buffer. Dissolve the labeled ligand bound to the cells with 0.2 NN a OH- l% SDS and mix with 4 ml of liquid scintillator A (Wako Pure Chemical Industries, Ltd.).

 d) Measure radioactivity using a liquid scintillation counter (manufactured by Beckman) and calculate the Percent Maximum Binding (PMB) using the following formula.

 PMB = [(B— N S B) / (BO-N S B)] X 1 0 0

PMB: Percent Maximum Binding B: Value when the sample is added

 N S B: Non-spec i f i c B inding

 B 0: Maximum binding amount

The compound obtained by using the screening method or the screening kit of the present invention or a salt thereof is a compound having an action of changing the binding property between the ligand and the P 2 Y ₂ receptor protein. (I) a compound having a cell stimulating activity via a G protein-coupled receptor (so-called agonist for Ρ 2 Υ ₂ receptor protein), (mouth) a compound not having the cell stimulating activity (so-called, antagonists g) for [rho 2 Upsilon ₂ receptor proteins, binding force between (c) a compound that potentiates the binding affinity of the ligand and the [rho 2 Upsilon ₂ receptor protein, or (d) ligands and [rho 2 Upsilon ₂ receptor protein Use a compound that reduces.

 Examples of the compound include peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, and the like. These compounds may be novel compounds or known compounds.

The compounds may be filed with compounds designed based on the position of the atomic coordinates Oyopi ligand binding Boke' bets of the active site of the above-mentioned [rho 2 Upsilon ₂ receptor protein.

Compound or its enhance binding force between Agonisu bets or ligand and [rho 2 Upsilon ₂ receptor protein for disk re one Jung method or subscription one obtained using a Jung for kit [rho 2 Upsilon ₂ receptor protein of the present invention Salts, for example, diabetes, impaired glucose tolerance, ketosis, acidosis, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, hyperlipidemia, obesity, metabolic syndrome, sexual dysfunction, skin diseases, joints It can be used for diseases such as symptom, osteopenia, arteriosclerosis, thrombotic disease, indigestion, memory learning disorder, etc., and is preferably useful as a hypoglycemic agent for diabetes.

Furthermore, antagonists bets or Li ligand and [rho 2 Upsilon respect is [rho 2 Upsilon ₂ receptor protein obtained using the disk Li one Jung method or kit for screening of the present invention. A compound or its compound that decreases the binding power to the receptor protein This salt is useful, for example, as a blood glucose raising agent.

The compound obtained by using the screening method or the screening kit of the present invention or a salt thereof is a compound or a salt thereof that changes the expression level of the P 2 Y ₂ receptor protein. Drugs such as other diabetes treatment agents, diabetic complication treatment agents, hyperlipidemia treatment agents, antihypertensive agents, anti-obesity agents, diuretics, chemotherapeutic agents, immunotherapy agents (hereinafter abbreviated as combination drugs) Can be used in combination. At this time, the administration time of the compound obtained by using the screening method or the screening kit of the present invention or a salt thereof and a concomitant drug is not limited, and these are administered simultaneously to the administration subject. May be administered after a time lag. The dose of the concomitant drug can be appropriately selected based on the clinically used dose. Further, the compounding ratio of the compound obtained by using the screening method or screening kit of the present invention or a salt thereof and a concomitant drug should be appropriately selected depending on the administration subject, administration route, target disease, symptom, combination, etc. Can do. For example, when the administration subject is a human, to Agonisu sheet 1 part by weight In example embodiment, a combination drug 0.0 may Re Re for 1 to 1 0 0 parts by _Q

 When the compound obtained by using the screening method or screening kit of the present invention or a salt thereof is used as the above pharmaceutical composition, it can be formulated according to conventional means.

For example, the compound is sterilized orally as tablets, capsules, elixirs, microcapsules or the like with sugar coating as required, or with water or other pharmaceutically acceptable liquids. It can be used parenterally in the form of injections such as solutions or suspensions. For example, the compound in a unit dosage form generally required for practicing recognized formulations along with known physiologically recognized carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, etc. It can be produced by mixing. The amount of active ingredient in these preparations should be such that an appropriate volume within the indicated range is obtained. Additives that can be mixed into glazes, force-pellants, etc. include, for example, binders such as gelatin, corn starch, tragacanth, arabic gum, excipients such as crystalline cellulose, corn starch , Gelatinizing agents such as alginic acid, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharine, flavoring agents such as peppermint, cocoa oil or tea Used. When the dosage unit form is a capsule, a liquid carrier such as fats and oils can be further contained in the above type of material. Sterile compositions for injection should be treated according to normal pharmaceutical practice, such as dissolving or suspending active substances in vehicles such as water for injection, naturally occurring vegetable oils such as sesame oil, coconut oil, etc. be able to. As an aqueous solution for injection, for example, physiological saline, isotonic solutions containing buducose and other adjuvants (for example, D-sorbitol, D-mannitol, sodium chloride, etc.) are used. Suitable solubilizers such as alcohol (eg, ethanol), polyalcohol (eg, propylene glycol, polyethylene glycol), nonionic surfactants (eg, polysorbate 80 ™, HCO—50) You may use together. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and may be used in combination with solubilizing agents such as benzyl benzoate and benzyl alcohol. In addition, the above pharmaceutical composition includes, for example, a buffer (for example, phosphate buffer, sodium acetate buffer), a soothing agent (for example, benzalkonium chloride, hydrochloride pro-in, etc.), a stabilizer ( For example, human serum albumin, polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants, etc. may be added. The prepared injection solution is usually filled in a suitable ampoule.

 The preparations obtained in this way are safe and low toxic. For example, they can be used in human mammals (eg rats, mice, rabbits, hidges, pigs, tusks, cats, dogs, monkeys, etc.). Can be administered.

The dose of the compound or a salt thereof varies depending on the administration subject, target organ, symptom, administration method, and the like. In sick patients (as 60 kg), about 0.1 to 10 O mg per day, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg. When administered parenterally,, the single dose varies depending on the subject of administration, subject organ, symptom, administration method, etc. For example, in the form of an injection, for example, diabetic patients (as 6 O kg ), About 0.1 to 30 mg per day, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg is administered by intravenous injection. Is convenient. In the case of other animals, an amount converted per 60 kg can be administered.

 In this specification and drawings, when bases, amino acids, etc. are indicated by abbreviations, they are based on abbreviations by IUPAC—IUB Commission on Biochemical Nomenclature or conventional abbreviations in the field, and examples are as follows. In addition, when there are optical isomers for amino acids, L form is indicated unless otherwise specified.

 D N A: Deoxyribonucleic acid

 c D N A: complementary deoxyliponucleic acid

 A: Adenine

 T: Chimin

 G: Guanin

 C: cytosine

 U: Uracil

 R N A ·: Ribonucleic acid

 mRN A: Messenger Ribonucleic acid

 d ATP: Deoxyanosine triphosphate

 d T T P: De-xitimidine triphosphate

 d G T P: Deoxyguanosine triphosphate

 d C T P: Texicytidine triphosphate

 ATP: adenosine triphosphate

EDTA: ethylenediamine tetraacetic acid SDS: sodium dodecyl sulfate

 G 1 y: Glycine

 A l a: Alanine

 V a 1: Norin.

Leu: Leucine

 I 1 e

 Ser: Serine

 T hr

 C y s: Sistine

M e t: Methionine

 G 1 u: Glutamic acid

 A s p: Aspartic acid

 L y s: Lysine

 A r g: Arginine

H i s: Histidine

 P h e: Felanuaranin

 T y r: tyrosine

 T r p: Tribute fan

 P ro: Proline

A s n: Asparagine

 G i n: Gnoretamine

p G 1 u: pyroglutamic acid

 *: Corresponds to a stoppage

M e: methyl group

E t: Ethyl group

 B u: Puchinore group

 P h: Phenylore group

TC: thiazolidine-4 (R) monocarboxamide group In addition, the substituents, protecting groups and reagents frequently used in the present specification are described below. Expressed with a number.

 T o s ρ-Toluenesulfonyl '

 C H O Honoremi Nore

 B z 1 benzyl-

C 1 ₂ B z 1 2, 6—Dichlorobenzene

 B o m Benzenoreoxymethinore

 Z Penzino Leoxy Canole Boninole

 C 1 1 Z 2—Black-headed Penzinoreoxycanole Boninole

 B r-Z 2—Bromobenzyl'oxycanolonitrile / re

 B o c t

 D N P Gini Trofenore

 T r t trityl

 B um t

 F m o c N _ 9—Funoreleninoreme Toxicanoreboninole

 HO B t 1 -hi droxybenz riazonole

 HO O B t 3, 4-Dihi Draw 3-Hydroxy 1 4-Oxo 1

 1, 2, 3—Benzotriazine

 H O N B ： 1-Hydroxy-5-norolebornene_2, 3-dicanolevoxy imido

 DCCN, N'-hydroxyhexylcarbodiimide The SEQ ID NOs in the sequence listing of the present specification indicate the following sequences.

 [SEQ ID NO: 1]

The amino acid sequence of the human P 2 Y ₂ receptor protein used in the present invention is shown.

 [SEQ ID NO: 2]

₂ shows the base sequence of cDNA encoding the human 2-2 receptor protein used in the present invention.

 [SEQ ID NO: 3]

The amino acid sequence of mouse P 2 Y ₂ is shown.

[SEQ ID NO: 4] The amino acid sequence of rat P 2 Y ₂ is shown.

 [SEQ ID NO: 5]

The base sequence of c D D コ ー ド encoding mouse マ ウ ス 2 Υ ₂ is shown.

[SEQ ID NO: 6]-Shows the base sequence of c D Α コ ー ド encoding rat P 2 Y ₂ .

 [SEQ ID NO: 7]

2 shows the base sequence of the upstream primer used in Example 2 for amplifying the human P 2 Y ₂ region.

 [SEQ ID NO: 8]

2 shows the base sequence of the downstream primer used in Example 2 for amplifying the human 2 ₂ region.

 [SEQ ID NO: 9]

Used in Example 2, shows the base sequence of the probe for detecting amplification of human [rho 2 Upsilon ₂ region. '

 [SEQ ID NO: 1 0],

2 shows the base sequence of an upstream primer used in Example 2 for amplifying the mouse 2 ₂ region.

 [SEQ ID NO: 1 1]

2 shows the base sequence of the downstream primer used in Example 2 for amplifying the mouse 2 ₂ region.

 [SEQ ID NO: 1 2]

2 shows the base sequence of a probe used in Example 2 for detecting amplification of the mouse 2 ₂ region.

 [SEQ ID NO: 1 3]

The base sequence of the upstream primer used in Example 4 for amplifying the rat _{2 2} region is shown.

 [SEQ ID NO: 1 4]

The base sequence of the downstream primer used in Example 4 for amplifying the rat _{2 2} region is shown. [SEQ ID NO: 15] 'This shows the base sequence of the probe used in Example 4 for detecting the amplification of the rat P 2 Y ₂ region. Example

 The present invention will be described in more detail with reference to examples below, but these do not limit the scope of the present invention. Example 1

 (Measurement of c AMP production activity in L 6 1 S G 4 1 8 1 1)

 G L U T 4 expressing L 6 cells (L 6 — S G 4— 8 1 1) [J o u r n a l

0 f B i o 1 o g i c a l C h e m i st r y (1 9 9 3) v o 1.

2 6 8 P P. 2 2 1 1 9-2 2 1 2 6] is 10% F B S (I n v i t r o g en n), 5 0 u n i t s / mL P e n i c i l l i n G

S o d i m, 50 μg / m L S t e e p t o m y c i n s u 1 f ate (I n v i t r o g e n) mammalian cell culture basal medium D u 1 b e c c os M

(In n Vitrogen). 1 x 10 ⁴ per well 5 to 10% FBS, 50 units / m 1 penicill

Prepared with M inimum Essentia 1 M edium (Invitrogen), a white cell culture medium containing 1 n G sodium, 50 / _i g ml ml S trepto my cinsu 1 fate, white 9 6 one we 1 1 tissuecultureplate ( C Orning company to this seeding, 3 7 ° C, 5% C_〇 was over晚cultured ₂ conditions. P 2 Y ₂ expression plasmid pc DNA 3 · l H ygro ( +) / h P 2 Y 2 Oyo Pc control DNA DNA 3. IH ygro (+) [Invitrogen; # V 8 7 0 2 0] is a transfer reagent, Lipofectamine 2 0 0 0 (Invitrogen was used according to the attached protocol) L 6 — SG 4— 8 1 Introduced into 3 7 ° (:, After 4 hours at 5% C_〇 ₂ conditions, 1 washed with the medium used during the seeding and 1 2 hour incubation at 3 7 ° C, 5% C_〇 ₂ conditions. After washing once with Mimim Essential Essential Medium containing 0.1% fatty acidfree BSA (Sigma) 3 7 in this medium. C and 5% C 0 ₂ conditions were cultured for 4 hours. 1 1 6 mM N a C l containing fattyacidfree of BSA the (S IgM a Inc.) 0. 0 5%, 4. 7 mM KC 1, 1. 2 mM KH 2 P 0 4, 1. 2 m MM g S_〇 ₄ , 2 5 mM N a HC 0 3, 2 4 mM HE PES, 2. washed once with 5 mM C a C 1 ₂ buffer (0. 0 5% BSAKRBH), was replaced with the buffer solution. ATP is prepared with KR BH to a concentration of 10 times the final concentration. Add 10 ml per well, and add 3 μL. And 3 0 minutes cultured in 5% C0 ₂ conditions.

1 m C i / m 1 [H] 2—D eoxy— D—glucose (Perkin Elmer) containing 100 mM 2-D eoxy—D—glucose prepared with 0.05% BSA KR BH Then, 20 ml per well was added and cultured at 37 ° C. and 5% CO ₂ for 1 hour. The plate was washed 3 times with cold PBS, and 20 μl of EtOH was added per well. Add nightly scintillation cocktail (O pti P hase 'Super Mix', W a 1 lac) at 100 ml per well, shake with plate mixer for 2 minutes, and scintillation counter T op Count ( (Packard). As a result, it was detected that P 2 Y ₂ —transient expression L 6—SG 4—81 1 1 significantly affected ATP concentration-dependent uptake of 2-D eoxy—D-g 1 ucose into cells. Example 2

 (Comprehensive expression analysis of arbitrary receptors)

In this example, the TaqMan method is used to belong to a family of target G protein-coupled receptors, tyrosine kinases, ion channels, etc. in any human and mouse RNA sample. The presence of gene-derived mRNA and the production amount thereof were quantified, and the expression levels of genes belonging to the family, such as target G protein-coupled receptor, tyrosine phosphorylase receptor, and ion channel, were analyzed. In this example, we analyze the expression of genes belonging to the family, such as .360 target G protein-coupled receptors, tyrosine phosphatase receptors, and ion channels, using a 38-well plate. . Examples of target GPCR genes include ORL; Ml; M2; M3; M4; M5; Al; A2A; A2B; A3; A1A; aIB; aID; a2A; a2B; a2C; β1; j32 3; ATI; AT2; BB1; BB2; bb3; Bl; B2; CBl; CB2; CCRl; CCR2; CCR3; CCR4; CCR5; CCR6; CCR7; CCR8; CCR9; CCR10; CXCRl; CXCR2; CXCR3; CXCR4; CXCR5 CX3CR1; XCRl; C3a; C5a; fMLP; CCKl; CCK2; CRF1; CRF2; Dl; D2; D3; D4; D5; ETA; ETB; GAL1; GAL2; GAL3; mglul; mglu2; mglu3; mglu7; mglu7; raglu8; FSH; LSH; TSH; Hi; H2; H3; H4; 5-HT1A; 5-HT1B; 5-HT1D; 5-htlB; 5-htlF; 5-HT2A; 5-HT2F; 5- 5-HT5; 5-HT5; 5-HT5; 5-HT7; BLT: CysLTl; CysLT2; edgl; edg2; edg3; edg4; MCI; MC2; MC3; MC4; MC5; MTl; MT2; MT3; Yl; Y2; Y4; Y5; Y6; NTS1; NTS2; DOP; KOP; MOP; NOP; P2Y1; P2Y2; P2Y4; P2Y6; P2Y11; P2Y12; PPAR-a; PPAR-β; PPAR- FP; IP; TP; EP1; EP2; EP3; EP4; PARI; PAR2; PAR3; PAR4; sstl; sst2; sst3; sst4; s ST5; NKl; NK2; NK3; TRHl; TRH2; VPACl; VPAC2; PAC1; Via; Vlb; V2; 0T; Na + channel (Type I; Type 11 / Type IIA; Type III; SCLll / NaG; PN1; NaCh6 NaDRG; SkMl / ^ u 1, SkM2); K + channel (Kv; EAG; KQT; IRK; R0MK; GIRK; KATP, etc.); C a 2+ channel (alG; a IE; a IS: a 1C; a ID; a IB; 1A; IP3; ryanodine receptor; C 1 -channel (GABAA; GABAC; glycine receptor; C1C0; ClCl; CFTR, etc.); non-selective cation channel (nAChR; 5-HT3; NMDA; AMPA; P2XATP; CNG, etc.)

 Amplification was performed using the following samples.

As a human sample, total RNA derived from adult normal skeletal muscle (C 1 ontech) was purchased. The mouse sample was C 5 7 B LZ 6 right femoral skeletal muscle removed, Total I NA was prepared according to the manual of I sogen (Nitsubon Gene). As a positive control, a serially diluted solution of synthesized cDNA of the amplification region was used.

The amplification of the region of human P 2 Y ₂ mRN was performed using the upstream primer (SEQ ID NO: 7) and the downstream primer (SEQ ID NO: 8). Detection was performed using a probe (SEQ ID NO: 9) (Applied Biosystems Japan Co., Ltd.).

Amplification of the mouse P 2 Y ₂ mRNA region was performed using an upstream primer (SEQ ID NO: 10) and a downstream primer (SEQ ID NO: 11). Detection was performed using a probe (SEQ ID NO: 1 2) (Applied Biosystems Japan Co., Ltd.).

 In order to enable detection in the TaqMan format, the probe has a 5 'end with a fluorescein-type fluorescent dye (FAM: reporter) and a 3' end with a rhodamine-type fluorescent dye (TAMRA: It was labeled with Quenchia. When the labeled probe was not hybridized, the fluorescence of the reporter was suppressed due to the movement of fluorescence resonance energy. In order to prevent extension of the probe by DNA polymerase during amplification, the 3 'end of the probe was synthesized with a phosphate block.

 c For DNA synthesis, reverse transcription reaction was performed on 40 μg of the RNA described above using random primers. Use reverse transcriptase Superscript II (Invitrogen), react according to the attached protocol, precipitate with ethanol, and add 4 Ο / l TE (10 mM Tris—HC 1, 1 mM) Dissolved in EDTA pH 8.0).

 'Amplification and quantification were performed as follows.

The synthesized cDNA was dissolved in TE and diluted to 10 ng 1 in TE containing 50 μg / m 1 yeast tRNA. For diluted cDNA solution 2.5 μ1 (equivalent to 25 ng RNA), TaqMan (registered trademark) Universal PCR Master Mix (Applied Biosystems Japan Co., Ltd.) was used as the amplification reaction reagent. The total amount of the reaction solution was adjusted. Each The final concentration of lymer and probe was in accordance with the manual.

 TaqMan (registered trademark) PCR was performed with the ABI PRISM (registered trademark) 7900HT Sequence Detection System (Applied Biosystems Japan Co., Ltd.), and the temperature cycle used was: TaqMan (registered trademark). Universal PCR Master Mix (Applied) DBiosystems Japan) manual. For quantitative TaqMan analysis of amplified products, 7900HT SDS software (Applied Biosystems Japan Co., Ltd.) was used, and the number of copies per sample was calculated by the absolute quantification method.

 In the same manner, the amount of gene mRNA belonging to the family such as other target G protein-coupled receptors, tyrosin oxidase type receptors, and ion channels was quantified. Then, by comparing the production of all target G protein-coupled receptors, tyrosine phosphatase receptors, ion channels, and other Λ-transduced mRNAs, normal human and mouse normal skeletons are compared. Each target G protein-coupled receptor, tyrosine phosphatase receptor, ion channel, and other gene expression levels in muscle were analyzed.

 Figure 1 shows the analysis results.

In human skeletal muscle, P 2 Y ₂ was the fourth highest expression level in all target GPCRs measured. The expression level was about 1500 copies in lng total RNA. In mouse skeletal muscle, P 2 Y ₂ showed the third highest expression level in all target GPCRs measured. The expression level was about 400 copies in 1 ng of total RNA. Example 3

(P 2 Y ₂ tissue distribution)

As human samples, all RNAs derived from various human tissues (C 1 ontech and Biochain) were purchased. For mouse samples, each tissue was extracted from C 5 7 BL 6 forces, and total RNA was prepared according to the manual of I sogen (Nitsubon Gene). c For DNA synthesis, 40 μg of RNA was subjected to reverse transcription using random primers. The reverse transcriptase Super Script II (Invitrogen) was used, reacted according to the attached protocol, ethanol precipitated and dissolved in 400 μI Τ. The synthesized cD Α 溶解 was dissolved in Τ 、 and diluted to 10 ng / μ 1 in TE containing 50 μg Zm 1 yeast tRNA. Use TaqMan (registered trademark) Universal PCR Master Mix (Applied Biosystems Japan Co., Ltd.) as the amplification reaction reagent for the diluted cD Ν Α solution 2.5 μ1 (equivalent to 25 ηg RNA). The total reaction volume was 15 μl. The same primers and probes as those used in Example 1 were used for quantification. The final concentration of each primer and probe was in accordance with the manual.

 TaqMan (registered trademark) PCR was performed with ABI PRISM (registered trademark) 7900HT Sequence Detection System (Applied Biosystems Japan Co., Ltd.), and the temperature cycle used was TaqMan (registered trademark) Universal PCR Master Mix (Applied Biotechnology). System Japan Co., Ltd.) manual. For quantitative TaqMan analysis of amplified products, the number of copies per sample was calculated by absolute quantification using 7900HT SDS software (Applied Biosystems Japan Co., Ltd.).

 The result is shown in figure 2.

In humans, the highest level of expression was found in skeletal muscle, and in mice, the fourth highest level of expression was found in skeletal muscle following myocardium, trachea, and ileum. P 2 Y ₂ was shown to be relatively highly expressed in skeletal muscle in both human and mouse. Example 4

(Expression analysis of Ρ 2 Υ ₂ in skeletal muscle cell lines)

Both cells of mouse skeletal muscle cell line C 2 C 12 (AT CC) and rat skeletal muscle cell line L 6 (distributed from JC Lawrence Jr) are 10% FBS ^ lnvitrogen, 50 units / mL Penicillin G Sodium, 5 The cells were cultured in Dulbecco's Modified Eagle Medium (Invitrogen) containing 0 g / m1 Streptomycin sulfate (Invitrogen). C 2 C 12 was differentiated by replacing it with Dulbe <Sco, s Modified Eagle Medium containing 2% horse serum (ICN). L 6 promoted differentiation by replacing 2% FBS with-Minimum Essential Medium Invitrogen. The cells at each stage of differentiation were removed with 0.25% trypsin I mM EDTA (Invitrogen), and the number of cells was measured. Then, all RNAs were prepared according to the manual of I sogen (Nitsubon Gene). .

 In cDNA synthesis, reverse transcription was performed on the RNA 4 O / g described above using random primers. Reverse transcriptase Su p e r S c r i p t

Using 1 I (Invitrogen), the reaction was carried out according to the attached protocol, ethanol precipitation was carried out, and dissolution was carried out in 400 ml. The synthesized cDNA is dissolved in Τ 、 and dissolved in TE containing 50 μg Zm 1 of yeast tRNA.

Diluted to l. For diluted cDNA solution 2.5 μ1 (equivalent to 25 ng RNA), use TaqMan (registered trademark) Universal PCR Master Mix (Applied Biosystems Japan) as the amplification reaction reagent. A total reaction volume of μ 1 was prepared. The primers and probes used for quantification are the same as those used in Example 1 for mice, and the upstream primers (SEQ ID NO: 13) and downstream primers (SEQ ID NO: 14) are used for rats. It was. Detection was performed using a probe (sequence number: 15). The final concentration of each primer and probe was in accordance with Magyuannole.

TaqMan (registered trademark) PCR was performed with ABI PRISM (registered trademark) 7900HT Sequence Detection System (Applied Biosystems Japan Co., Ltd.), and the temperature cycle used was TaqMan (registered trademark) Universal PCR Master Mix (Applied Dubai). System Japan Co., Ltd.) Quantitative TaqMan analysis of amplification products was tested by the absolute quantification method using 7900HT SDS software (Applied Biosystems Japan Co., Ltd.). The number of copies per fee was calculated.

 The results are shown in Figure 3.

In both C 2 C 12 and L 6, the expression of P 2 Y ₂ increased with the differentiation from myoblasts to myotubes. -Example 5

 (Measurement of sugar uptake in L 6-S G 4-8 1 1)

G LUT 4-expressing L 6 cells (L 6— SG 4— 8 1 1) are 10% FBS (I η Vitogen), 50 units / mL Penicillin G Sodium, 50 μg / m 1 Streptomycin sulfate ( Invitrogen) and cultivated in Dulbecco's Modified Eagle Medium (Invitrogen). 10% FBS (Invitrogen), 50 units / mL Penicillin G Sodium, 50 μg / m 1 Streptomycin sulfate to make 1 XI 0 ⁴ per well, Minimum Essential Medium ( prepared in I Nvitrogen Co.) were seeded in white 96-well tissue culture plate (C orning Inc.), was over晚cultured in 3 7 ° C, 5% C 0 2 conditions. P 2Y ₂ expression plasmid pc DNA 3.1 / P 2 Y ₂ and control pc DNA 3.1 were prepared using the transfection reagent Lipofectamine 2000 (Invitrogen) according to the attached protocol L 6—SG 4— Introduced in 8 1 1. 3 7 ° C, 5% C_〇 After 4 hours at ₂ conditions, washed once with the medium used during the seeding and nutrient 1 2 hour culture at 3 7 ° C, 5% C0 2 conditions. Fatty acid-free BSA (Sigma) 0.1. /. It washed once with Minimum Essential Medium containing, and cultured for 4 hours under 3 7 ° C, 5% C_〇 _two conditions on this medium. 116mM NaCl, 4.7mM KCl, 1.2mM KH ₂ P0 ₄ , 1.2raM MgS0 ₄ , 25mM NaHC0 ₃ , 24mM HEPES, 2.5mM CaCl, buffer solution (0.05%) containing 0.5% fatty acid-free BSA (Sigma) After washing once with (% BSA KRBH), this buffer was replaced. AT P is prepared in KR B 'H at 1 0-fold concentration of the final concentration, was added 1 by 0 per Uweru, 3 7 ° C, 5% C0 2 The culture was performed for 30 minutes under the conditions. ,

1 μ Ci / ml [3H] 2 - Deoxy- D_glucose lOOuM 2 including (PerkinElmer Inc.) - Deoxy_D- glucose was prepared with 0.05% BSA KRBH, was added 2 by 0 mu 1 per Ueru, 3 7 ^P C, 5 The cells were cultured for 1 hour under% C 0 ₂ conditions. The plate was washed 3 times with chilled PBS, and ethanol was added at a rate of 201 per tool. Liquid scintillation cocktail (OptiPhase 'SuperMix', Wallac) was added at 100 µl per well, shaken with a plate mixer for 2 minutes, and measured with a scintillation counter TopCount (Packard).

As a result, as shown in FIG. 4, P ₂ Y 2 - over expression L 6 - SG 4 - takes Write-to 8 1 1 a significant ATP concentration-dependent 2-deoxy D-glucose in the cell Was detected. Industrial applicability

The present invention provides a useful blood glucose regulator. P 2 Y ₂ agonists can be a novel drug target with a wide range of hypoglycemic effects in diabetics with both insulin secretion deficiency and insulin resistance.

Claims

Range _s claims

I. A hypoglycemic agent comprising a compound that promotes the activity of a P 2 Y ₂ receptor protein or a salt thereof, or a salt thereof. .

2. Ρ 2 Υ _{2 A} hypoglycemic agent comprising a compound that promotes the expression of a receptor protein or a salt thereof or a salt thereof.

3. The agent according to claim 1 or 2, wherein the Ρ 2 Υ ₂ receptor protein or a salt thereof is a skeletal muscle Ρ 2 Υ ₂ receptor protein or a salt thereof.

 4. The agent according to any one of claims 1 to 3, which is a prophylactic / therapeutic agent for diabetes, obesity or hyperlipidemia.

5. Ρ ₂ Υ 2 receptor protein or hyperglycemic agent comprising the compound or its salt that inhibits the activity of the salt.

6. Ρ 2 Υ _{2 A} blood glucose-elevating agent comprising a compound that inhibits the expression of a receptor protein or a salt thereof or a salt thereof.

7. Ρ ₂ Υ 2 receptor protein or agent according to claim 5 or 6 wherein the salt is a skeletal muscle [rho 2 Upsilon ₂ receptor protein or a salt thereof.

 8. The agent according to any one of claims 5 to 7, which is a prophylactic / therapeutic agent for hypoglycemia.

9. Ρ 2 部分₂ receptor protein or a salt thereof is a protein or partial peptide or a salt thereof containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1. The agent according to any one of claims 1 to 8.

9. The agent according to any one of claims 1 to 8, wherein the 10 ₂ acceptor protein or a salt thereof is a protein comprising the amino acid sequence represented by SEQ ID NO: 1 or a salt thereof.

II. A hypoglycemic agent comprising a polynucleotide comprising a polynucleotide encoding a Ρ 2 Υ ₂ receptor protein or a partial peptide thereof.

 1 2. The agent according to claim 1, which is a preventive or therapeutic agent for diabetes, obesity or hyperlipidemia.

. 1 3 [rho 2 Upsilon ₂ receptor protein encoding polynucleotide of SEQ ID NO:. The same or substantially the nucleotide sequence represented by 2 to contain identical nucleotide sequence 'Ru poly claim 1 1 is the nucleotide Or 12. The agent according to 2.

1 4. A hyperglycemic agent comprising P 2 Y ₂ receptor protein or a partial peptide thereof or; ^ thereof.

 1 5. The agent according to claim 14, which is a prophylactic / therapeutic agent for hypoglycemia.

1 6. A P 2 Y ₂ receptor protein or a salt thereof is a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, or a partial peptide or a salt thereof The agent according to claims 14 to 15.

1-7. A blood glucose-elevating agent comprising an antisense polynucleotide comprising a base sequence complementary to or substantially complementary to the base sequence of a polynucleotide encoding the receptor protein ₂ or 2 or a part thereof.

 1 8. The agent according to claim 17, which is a prophylactic / therapeutic agent for hypoglycemia.

. 1 9 [rho 2 Upsilon ₂ receptor protein encoding polynucleotide of SEQ ID NO: identical or substantially the nucleotide sequence represented by a polynucleotide you contain identical nucleotide sequence according to claim 1 7 to 1 8. The agent according to 8.

2 0. Ρ 2 Υ _{2 A} blood glucose increasing agent comprising an antibody against a receptor protein or a salt thereof.

 21. The agent according to claim 20, which is a hypoglycemia prevention / treatment agent.

2 2. Ρ 2 Υ _{2 The} receptor protein or a salt thereof is a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, or a partial peptide or a salt thereof. The agent according to claim 20 to 21.

2 3. Ρ 2 Υ ₂ A diagnostic agent for diabetes or hypoglycemia comprising an antibody against the receptor protein or a salt thereof.

2 4. A diagnostic agent for diabetes or hypoglycemia comprising a polynucleotide encoding a receptor protein ₂ .

₂ 5. Ρ 2 Υ 2 receptor protein or the use of a salt thereof, wherein the compound or subscription-learning method salt thereof to modulate the blood sugar level.

2 6. [Rho 2 Upsilon ₂ receptor protein or subscription-learning method according to claim 2 wherein the salt is a skeletal muscle [rho 2 Upsilon ₂ receptor proteins' quality or a salt thereof.

2 7. A protein or partial peptide or salt thereof in which the P 2 Y ₂ receptor protein or a salt thereof contains the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1. The screening method according to claim 25. ·-2 8. A kit for screening a compound or a salt thereof that regulates blood glucose, characterized by containing a P 2 Y ₂ receptor protein or a salt thereof.

₂ 9. Ρ 2 Υ 2 receptor protein Mochiiruko polynucleotides encoding, characterized by capital, compound or scree Jung method salt thereof to regulate blood sugar.

3 0. [rho 2 Upsilon ₂ receptor protein encoding polynucleotide of SEQ ID NO: of claim 2 9, wherein a polynucleotide you containing a base sequence identical or substantially identical to the nucleotide sequence represented by Screening method.

3 1. A screening kit for a compound that regulates blood glucose or a salt thereof, comprising a polynucleotide that encodes a receptor protein ₂ .

3 2. Ρ 2 Υ ₂ Receptor protein or its salt is used, Ρ

2 ス ク リ ー ニ ン グ Method for screening agonists for ₂ receptor proteins or salts thereof.

3 3. Ρ ₂ Υ 2 receptor, wherein the protein or the use of a salt thereof, Ρ ₂ Υ 2 receptor protein or antagonists bets screening-learning method to salts thereof. ,

3 4. (1) [rho a 2 Upsilon ₂ receptor protein or labeled Rigandoma other salt thereof salt thereof, and when contacted with the protein or a salt thereof, (2) test of compound and [rho 2 Upsilon ₂ receptor protein or labeled Rigandoma other salt thereof and a salt thereof, when in contact with the protein or a salt thereof, [rho 2 Upsilon ₂ receptor protein or a labeled ligand or a salt of salt thereof The screening method according to claim 32, wherein the binding amount of the protein or a salt thereof is measured.

'3 5. (1) Ρ 2 Υ. Labeled ligand of receptor protein or its salt Or when a salt thereof is brought into contact with a cell containing the protein or a cell membrane fraction thereof, and (2) a labeled ligand of the test compound and the P 2 Y ₂ receptor protein or a salt thereof or a salt thereof. , When contacted with a cell containing the protein or a cell membrane fraction thereof, a labeled ligand of the の 2 Υ ₂ receptor protein or a salt thereof or a salt thereof, and a cell containing the protein or a cell membrane fraction thereof The screening method according to claim 32, wherein the binding amount to the minute is measured.

3 6. (1) Ρ 2 Υ ₂ receptor protein or a salt thereof, or a cell membrane fraction containing the protein, or (2) Ρ 2 Υ ₂ receptor When a test compound is brought into contact with a cell containing the protein or a cell membrane fraction thereof in the presence or absence of a ligand of the protein or a salt thereof or a salt thereof, the protein mediated by the Ρ 2 Υ ₂ receptor protein The screening method according to claim 32, wherein the cell stimulating activity is measured.

3 7. (1) Ρ 2 Υ ₂ receptor protein or a salt thereof, or a cell membrane fraction containing the protein, or (2) Ρ 2 Υ ₂ receptor Intracellular C a ²⁺ concentration or intracellular c when a test compound is contacted with a cell containing the protein or a cell membrane fraction thereof in the presence or absence of a ligand of the protein or a salt thereof or a salt thereof The screening method according to claim 32, wherein AMP production is measured.

3. The screening method according to claim 32, wherein GLUT 4 is allowed to coexist in the P 2 Y ₂ receptor protein or a salt thereof.

 3. The screening method according to claim 32, wherein skeletal muscle-derived cells or skeletal muscle cells are used.

 40. The screening method according to claim 32, wherein the ligand is UTP or ΑΤ Ρ.

4 1. A method of lowering blood glucose characterized by promoting the activity of Ρ 2 Υ ₂ receptor protein or its salt.

4 2. A method for lowering blood glucose, characterized by promoting expression of a gene for a P 2 Y ₂ receptor protein or a salt thereof.

4. The method according to claim 41 or 42, wherein the P 2 Y ₂ receptor protein or a salt thereof is a skeletal muscle Υ ₂ Υ ₂ receptor protein or a salt thereof.

 4. The method according to any one of claims 41 to 43, which is a method for preventing / treating diabetes, obesity or hyperlipidemia.

4 5. A method for increasing blood glucose, characterized by inhibiting the activity of Ρ 2 Υ ₂ receptor protein or a salt thereof.

4 6. Ρ ₂ Υ 2 receptor protein or hyperglycemic method characterized by inhibiting the expression of a gene of a salt thereof.

4 7. Ρ ₂ Υ 2 receptor protein or claim 4 5 or 4 6 A method according salt thereof is skeletal muscle [rho 2 Upsilon ₂ receptor protein or a salt thereof.

 The method according to any one of claims 45 to 47, which is a method for preventing and treating hypoglycemia.

4 9. Ρ ₂ Υ 2 receptor protein or a compound or hypoglycemic method characterized by an effective amount of a salt is administered to a mammal to facilitate the activity of the salt.

5 0. Ρ ₂ Υ 2 receptor protein or a compound or hypoglycemic method the administration of an effective amount of a salt to a mammal to facilitate the expression of a gene of a salt thereof.

5 1. Ρ ₂ Υ 2 receptor protein or Claim 4 9 or 5 0 The method according salt thereof is skeletal muscle [rho 2 Upsilon ₂ receptor protein or a salt thereof.

 5. The method according to any one of claims 49 to 51, which is a method for preventing and / or treating diabetes, obesity or scabemia.

5 3. Ρ ₂ Υ 2 receptor protein or a compound or hyperglycemic method characterized by an effective amount of a salt is administered to a mammal to inhibit the activity of the salt. 5 4. Ρ ₂ Υ 2 receptor protein or a compound or glucose Rise method characterized by an effective amount of a salt is administered to a mammal to inhibit gene expression of a salt thereof.

5 5. [Rho 2 Upsilon ₂ receptor protein or claim 3 or 4 The method according its salt skeletal muscle [rho 2 Upsilon ₂ receptor protein or a salt thereof.

'5 6. The method according to claim 53, which is a method for preventing and treating hypoglycemia.

5 7. Use of a compound or a salt thereof that promotes the activity of a P 2 Y ₂ receptor protein or a salt thereof for the production of a hypoglycemic agent.

5 8. The use of a compound or its. Of salt that promotes the expression of the gene of [rho 2 Upsilon ₂ receptor protein or a salt thereof for the manufacture of a hypoglycemic agent.

5. The use according to claim 57 or 58, wherein the Ρ 2 Υ ₂ receptor protein or a salt thereof is a skeletal muscle Ρ 2 Υ ₂ receptor protein or a salt thereof.

 6. The use according to claim 57, wherein the hypoglycemic agent is a method for preventing / treating diabetes, obesity or hyperlipidemia.

6 1. Use of a compound or a salt thereof that inhibits the activity of Ρ 2 Υ ₂ receptor protein or a salt thereof for the production of a blood glucose raising agent.

6 2. Use of a compound or its salt that inhibits the expression of genes in [rho 2 Upsilon ₂ receptor protein or a salt thereof for the manufacture of a hyperglycemic agent.

6 3. [rho 2 Upsilon ₂ receptor protein or the use of claim 6 1 or 6 wherein the salt is a skeletal muscle [rho 2 Upsilon ₂ receptor protein or a salt thereof.

6. The use according to any one of claims 61 to 63, wherein the hyperglycemic agent is a prophylactic / therapeutic agent for hypoglycemia.