[go: up one dir, main page]

WO2004041301A1 - Antidiuretiques - Google Patents

Antidiuretiques

Info

Publication number
WO2004041301A1
WO2004041301A1 PCT/JP2003/014102 JP0314102W WO2004041301A1 WO 2004041301 A1 WO2004041301 A1 WO 2004041301A1 JP 0314102 W JP0314102 W JP 0314102W WO 2004041301 A1 WO2004041301 A1 WO 2004041301A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
amino acid
acid sequence
polypeptide
salt
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP2003/014102
Other languages
English (en)
Japanese (ja)
Inventor
Hirokazu Matsumoto
Koh Takagi
Masaaki Mori
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Takeda Pharmaceutical Co Ltd
Original Assignee
Takeda Pharmaceutical Co Ltd
Takeda Chemical Industries Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Takeda Pharmaceutical Co Ltd, Takeda Chemical Industries Ltd filed Critical Takeda Pharmaceutical Co Ltd
Priority to AU2003277553A priority Critical patent/AU2003277553A1/en
Publication of WO2004041301A1 publication Critical patent/WO2004041301A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/10—Drugs for disorders of the urinary system of the bladder
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/10—Drugs for disorders of the endocrine system of the posterior pituitary hormones, e.g. oxytocin, ADH
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/10—Antioedematous agents; Diuretics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/12—Antidiuretics, e.g. drugs for diabetes insipidus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention relates to an antidiuretic agent or a diuretic agent or a screening thereof.
  • the mechanism of urine production in animals is crucial for maintaining the homeostasis of body water and electrolyte metabolism, which accounts for 45-70% of total body weight. Accordingly, many diuretics and antidiuretics are used in the treatment setting based on various mechanisms of controlling urine production.
  • sulfanilamide or acetazolamide which is a carbonic anhydrase inhibitor that inhibits the Na + -H + exchange system in tubular cells
  • benzothiadiazine diuretics which are tubular reabsorption inhibitors
  • Na + / Cl— active in Henle's ascending leg Diuretics such as phenoxyacetic acid derivatives' or sulfamoylbenzoic acid derivatives, which eliminate the medulla osmotic gradient by suppressing transport, are known.
  • an antidiuretic there is vasopressin, an antidiuretic hormone, or a derivative thereof.
  • the present inventors have conducted intensive studies, and as a result, have found that the GPR8 ligand peptide described in WO01 / 98494 has an antidiuretic effect on rabbits, and further studies have been carried out. As a result, the present invention has been completed.
  • an antidiuretic agent comprising a polypeptide having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, or an amide thereof, an ester thereof or a salt thereof,
  • An antidiuretic agent characterized by using a polypeptide having an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1 or an amide thereof, an ester thereof or a salt thereof. Or a diuretic screening method,
  • (6) a protein having an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77 or SEQ ID NO: 79, or a partial peptide thereof Or a method of screening for an antidiuretic or diuretic, which comprises using a salt thereof.
  • An antidiuretic agent comprising a polypeptide having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, or a amide, an ester thereof, or a salt thereof. Or a diuretic screening kit,
  • a polynucleotide containing a polypeptide containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a polynucleotide containing a polynucleotide encoding the amide or ester thereof An antidiuretic or diuretic screening kit, characterized by the fact that
  • a method for suppressing urine production which comprises administering an effective amount;
  • Sequence identifier identical to the amino acid sequence represented by 1 A polypeptide containing one or substantially the same amino acid sequence or an amide or an ester or a salt thereof; (ii) a compound or a compound thereof which promotes the activity of the polypeptide or an amide or an ester thereof or a salt thereof A salt, or (iii) an effective amount of a polynucleotide encoding the polypeptide or its amide or ester thereof, which is characterized by polyuria, diabetes insipidus, hypernatrimuemia, metabolism Prevention and treatment of alkalosis, hypokalemia or Cushing syndrome,
  • Renal edema or an antidiuretic hormone characterized by administering an effective amount of an antisense polynucleotide containing a nucleotide sequence complementary or substantially complementary to a nucleotide sequence of DNA or a portion thereof.
  • SEQ ID NO: 1 for producing a prophylactic or therapeutic agent for polyuria, diabetes insipidus, hypernatremia, metabolic alkalosis, hypotension rheumatism or Cushing's syndrome
  • a polypeptide or an amide or ester thereof or a salt thereof containing the same or substantially the same amino acid sequence as the amino acid sequence (ii) promoting the activity of the polypeptide or the amide or ester thereof or a salt thereof (Iii) use of a compound or a salt thereof, or (iii) use of a polynucleotide encoding the polypeptide or an amide or an ester thereof;
  • a compound or a salt thereof that inhibits the activity of tide or an amide or an ester thereof or a salt thereof (ii) an antibody against the above-mentioned polypeptide or an amide or an ester thereof or a salt thereof, (iii) SEQ ID NO: 73 An antibody against a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 75, SEQ ID NO: 77 or SEQ ID NO: 79, a partial peptide thereof, or a salt thereof; (iv) an antisense polynucleotide containing a base sequence complementary to or substantially complementary to the base sequence of DNA encoding the above polypeptide, its amide, its ester, or its salt, or a part thereof, or V) Complementary or complementary to the nucleotide sequence of DNA encoding the above protein, its partial peptide, or a salt thereof Use of an antisense polynucleotide containing a substantially complementary nucleotide sequence or
  • (32) a polynucleotide having a base sequence represented by SEQ ID NO: 82 or SEQ ID NO: 84,
  • an antisense polynucleotide having a base sequence complementary to or substantially complementary to the polynucleotide of (30) or a part thereof,
  • an antisense polynucleotide having a base sequence complementary or substantially complementary to the polynucleotide according to (45) or a part thereof,
  • a polypeptide having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 is a human warm-blooded animal (eg, ' Guinea pig, rat, mouse, chick, egret, porcupine, sheep, pigeon, monkey, etc.
  • cells eg, retinal cells, hepatocytes, spleen cells, nerve cells, glial cells, kidney iS cells, bone marrow
  • Cells mesangial cells, Langer's cells, epidermal cells, epithelial cells, endothelial cells, fibroblasts, fiber cells, muscle cells, adipocytes, immune cells (eg, macrophages, T cells, B cells, natural killer cells) , Mast cells, neutrophils, basophils, eosinophils, monocytes), megakaryocytes, synovial cells, chondrocytes, bone cells, osteoblasts, osteoclasts, mammary cells, hepatocytes Kuha Stromal cells, or their precursors, stem cells or cancer cells, etc.) or any tissue where these cells are present, such as the brain, parts of the brain (eg, retina, olfactory bulb, amygdala, basal sphere of the cerebrum) , Hippocampus, thal
  • amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 is, for example, about 70% or more, preferably about 80% or more, preferably the amino acid sequence represented by SEQ ID NO: 1.
  • amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1 includes, in addition to the above amino acid sequence,
  • amino acid sequence obtained by adding 1 to 5 (preferably 1 to 3, more preferably 1 to 2, and more preferably 1) amino acids to the amino acid sequence represented by SEQ ID NO: 1 ,
  • amino acids in the amino acid sequence represented by SEQ ID NO: 1 are amino acids
  • polypeptide having an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 examples include, for example, a polypeptide having an amino acid sequence substantially the same as the amino acid sequence represented by the aforementioned SEQ ID NO: 1 Preferred is a polypeptide having substantially the same activity as the polypeptide having the amino acid sequence represented by SEQ ID NO: 1.
  • the substantially equivalent activity includes, for example, the activity of the polypeptide of the present invention (eg, antidiuretic action, etc.).
  • Substantially the same activity indicates that the activity is qualitatively (eg, physicochemically or pharmacologically) granular.
  • the measurement of the antidiuretic effect can be carried out according to a known method. For example, the method described in Modern Urine Chemistry (Bayer Corporation, New York, 1996) or a method based thereon, described in Examples below. It can be measured according to the method described above.
  • amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1 include, for example, SEQ ID NO: 21, SEQ ID NO: 2, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 , SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, Sequence SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58 or SEQ ID NO: 50
  • polypeptide of the present invention include, for example, a polypeptide having the amino acid sequence represented by SEQ ID NO: 1, a polypeptide having the amino acid sequence represented by SEQ ID NO: 2, and a polypeptide having the amino acid sequence represented by SEQ ID NO: 21
  • a polypeptide having an amino acid sequence represented by SEQ ID NO: 7; a polypeptide having an amino acid sequence represented by SEQ ID NO: 8; and a polypeptide having an amino acid sequence represented by SEQ ID NO: 9 A polypeptide having the amino acid sequence represented by SEQ ID NO: 10, a polypeptide having the amino acid sequence represented by SEQ ID NO: 11, a polypeptide having the amino acid sequence represented by SEQ ID NO: 11, Polypeptide having an amino acid sequence represented by SEQ ID NO: 24; polypeptide having an amino acid sequence represented by SEQ ID NO: 25; amino acid sequence represented by SEQ ID NO: 30
  • polypeptide of the present invention is used in a sense that it includes a precursor polypeptide of the polypeptide of the present invention.
  • the precursor polypeptide include, for example, a polypeptide characterized by containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 6.
  • amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 6 is about 80% or more, preferably about 90% or more, more preferably about 9% or more of the amino acid sequence represented by SEQ ID NO: 6.
  • Examples include amino acid sequences having a homology of 5% or more. .
  • amino acid sequence represented by SEQ ID NO: 6 is substantially the same as the above amino acid sequence
  • amino acids 1 to 15 (preferably 1 to 10, more preferably 1 to 5, and more preferably 1 to 3) amino acids are inserted into the amino acid sequence represented by SEQ ID NO: 6.
  • amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 6 include, for example, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 29 or SEQ ID NO: 35 And the like.
  • the precursor polypeptide include, for example, a polypeptide having an amino acid sequence represented by SEQ ID NO: 6, a polypeptide having an amino acid sequence represented by SEQ ID NO: 20; Having the amino acid sequence represented A polypeptide having an amino acid sequence represented by SEQ ID NO: 29; a polypeptide having an amino acid sequence represented by SEQ ID NO: 35; a polypeptide having an amino acid sequence represented by SEQ ID NO: 116; can give.
  • the receptor for the polypeptide of the present invention has a binding activity to the polypeptide of the present invention, and the polypeptide of the present invention stimulates the cell stimulating activity of cells expressing the receptor (for example, arachidonic acid).
  • the receptor for example, arachidonic acid.
  • (1) contains an amino acid sequence substantially identical to the amino acid sequence represented by GPR8 (SEQ ID NO: 73; Genomics, 28, 84-91, 1995) or SEQ ID NO: 73 Protein
  • mouse TGR26 SEQ ID NO: 77; WO 02/44368
  • GPR7 SEQ ID NO: : 79; Genomics, 28, 84-91, 1995
  • Egret GPR8 SEQ ID NO: 81
  • an amino acid sequence represented by SEQ ID NO: 81 A protein containing the same or substantially the same amino acid sequence
  • a protein having an amino acid sequence a protein having an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 81, a protein identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 83 Proteins containing the same amino acid sequence (hereinafter sometimes referred to as the receptor of the present invention) are human warm blood animals (eg, guinea pigs, rats, mice, chickens, egrets, bushes, and sheep).
  • human warm blood animals eg, guinea pigs, rats, mice, chickens, egrets, bushes, and sheep.
  • Cells eg, retinal cells, liver cells, spleen cells, nerve cells, glial cells, kidney i6 cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, endothelial cells
  • Fibroblasts fiber cells, muscle cells, adipocytes, immune cells (eg, macrophages, T cells, B cells, natural killer cells, fat cells, Neutrophils, basophils, eosinophils, monocytes), megakaryocytes, synovial cells, chondrocytes, osteocytes, osteoblasts, osteoclasts, breast cells, hepatocytes or stromal cells, or Progenitor cells, stem cells or cancer cells, etc.) or any tissue in which those cells reside, such as the brain, various parts of the brain (eg, retina, olfactory bulb, amygdala, basal cerebrum, hippocampus, thalamus, Hypothalamus
  • amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 73 is about 70% or more, preferably about 80% or more, the amino acid sequence represented by SEQ ID NO: 73 ⁇
  • amino acid sequence having about 90% or more homology is exemplified.
  • amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 75 for example, 85% or more, preferably about 90%, of the amino acid sequence represented by SEQ ID NO: 75 Amino acid sequence having at least about 95% homology Column.
  • amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 77 is, for example, 86% or more, preferably about 90% or more, more preferably the amino acid sequence represented by SEQ ID NO: 77 Include amino acid sequences having about 95% or more homology.
  • amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 79 about 70% or more, preferably about 80% or more, more preferably about 90% of the amino acid sequence represented by SEQ ID NO: 79 Amino acid sequences having the above homology are exemplified.
  • amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 81 is about 82% or more, preferably about 90% or more, more preferably about 95% of the amino acid sequence represented by SEQ ID NO: 73. Amino acid sequences having the above homology are exemplified.
  • amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 83 for example, 92% or more, preferably about 95% or more, more preferably the amino acid sequence represented by SEQ ID NO: 83 Include amino acid sequences having about 98% or more homology. '
  • a protein containing an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81 or SEQ ID NO: 83 For example, an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81 or SEQ ID NO: 83 Substantially the same as the protein having the amino acid sequence represented by SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81 or SEQ ID NO: 83 Proteins having activity are preferred.
  • the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81 or SEQ ID NO: 83 includes: (i) SEQ ID NO: No .: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81 or amino acid represented by SEQ ID NO: 83 An amino acid sequence in which 1 to 15 (preferably 1 to 10, more preferably 1 to 5, and more preferably 1 to 3) amino acids have been deleted from the acid sequence; (ii) sequence number : 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81 or 1 to 15 (preferably 1 to 15) in the amino acid sequence represented by SEQ ID NO: 83 (Iii) ⁇ SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: (10, more preferably
  • amino acid sequence represented by 1 to 15 (preferably 1 to 10, more preferably 1 to 5, Preferably 1 to 3) amino acids, (iv) SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, 1 to 15 (preferably 1 to 10, more preferably 1 to 5, and more preferably 1 to 3) in the amino acid sequence represented by SEQ ID NO: 81 or SEQ ID NO: 83 And (V) an amino acid sequence obtained by combining the above (i) to (iv).
  • Specific examples of the receptor of the present invention include, for example, a protein containing an amino acid sequence represented by SEQ ID NO: 73, a protein containing an amino acid sequence represented by SEQ ID NO: 75, A protein containing the amino acid sequence represented by SEQ ID NO: 77; a protein containing the amino acid sequence represented by SEQ ID NO: 79; a protein containing the amino acid sequence represented by SEQ ID NO: 81; A protein containing the amino acid sequence represented by No. 83 is used.
  • the partial peptide of the receptor for the polypeptide of the present invention may be any partial peptide that can be used in a method for screening a drug or the like described below.
  • a partial peptide having an ability to bind to the polypeptide of the present invention a partial peptide containing an amino acid sequence corresponding to an extracellular membrane region, and the like are used.
  • Peptides having an amino acid sequence of 20 or more, preferably 50 or more, more preferably 100 or more of the constituent amino acid sequences of the receptor of the present invention are preferred.
  • Specific examples include (a) a partial amino acid sequence consisting of the 1st (Met) to 123rd (Phe) amino acid residues or a part thereof in the amino acid sequence represented by SEQ ID NO: 73, Partial amino acid sequence consisting of the 31st (Asn) to 358th (Lys) amino acid residues or a part thereof, from the 548th (Tyr) to 59th (Arg) 'amino acid residues Or a partial amino acid sequence consisting of a partial amino acid sequence consisting of amino acid residues 843 (Ala) to 895 (lie) or one or more selected from the partial amino acid sequence A partial peptide containing a partial amino acid sequence; (b) a partial amino acid sequence consisting of the 1st (Met) to 85th (Asp) amino acid residues in the amino acid sequence represented by SEQ ID NO: 75, or one thereof; Or a portion consisting of the 2 '2nd (Cys) to 329th (Ala) amino acid residue
  • polypeptide, receptor or partial peptide thereof of the present invention the left end is the N-terminus (amino terminus) and the right end is the C-terminus (carboxyl terminus) according to the convention of peptide labeling.
  • R in the ester e.g., methyl, Echiru, n- propyl Le, alkyl groups such as isopropyl, n- butyl, cyclo pentyl, C 3 _ 8 cycloalkyl group such as cyclohexyl, for example, phenyl , such as ⁇ - naphthyl C 6 -
  • a pivaloyloxymethyl group commonly used as an oral ester is used.
  • the polypeptide, receptor or its partial peptide of the present invention When it has a poxyl group (or a carboxylate), a polypeptide in which the carboxyl group is amidated or esterified is also included in the polypeptide of the present invention.
  • the ester in this case, for example, the above-mentioned C-terminal ester and the like are used.
  • polypeptides of the present invention are those, which glutamine residue N-terminal region is cleaved in vivo to form pyroglutamic acid, the side chain of an intramolecular amino acid substituents (e.g.
  • ⁇ _H, - SH amino group, imidazole group, indole group, Guanijino group, etc.
  • protecting groups for example, c such Arukanoiru group such as a formyl group, Asechiru group, _ 6 Ashiru Or complex proteins such as so-called glycoproteins to which sugar chains are bound.
  • Salts of the polypeptide, receptor or partial peptide thereof of the present invention include salts with physiologically acceptable acids (eg, inorganic acids, organic acids) and bases (eg, alkali metal salts). And especially preferred are physiologically acceptable acid addition salts.
  • physiologically acceptable acids eg, inorganic acids, organic acids
  • bases eg, alkali metal salts
  • physiologically acceptable acid addition salts include, for example, 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, maleic acid, Salts with succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, etc. are used.
  • inorganic acids eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric
  • the polypeptide, the receptor or its partial peptide of the present invention can be produced from the above-mentioned human or warm-blooded animal cells or tissues by a known method for purifying polypeptides, or the polypeptide described below. It can also be produced by culturing a transformant transformed with DNA encoding the peptide. Also, it can be produced according to the peptide synthesis method described later. For example, it can be produced according to the method described in WO 01/98494, WO 0 274 368, and the like.
  • the human or mammalian tissues or cells are homogenized, and then extracted with an acid or the like. Purification and isolation can be achieved by a combination of chromatography such as mouth chromatography and ion exchange chromatography.
  • a commercially available resin for polypeptide synthesis can be generally used.
  • a resin include chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, 4-hydroxymethylmethylphenylacetamidomethyl resin, polyacrylamide resin, 4- (2 ', 4'-dimethoxyphenylhydroxymethyl) phenoxy resin, 4- (2', 4'-dimethoxyphenyl) Fmocaminoethyl) phenoxy resin and the like.
  • amino acids having a suitably protected amino group and side chain functional group are condensed on the resin in accordance with the sequence of the desired polypeptide according to various known condensation methods.
  • the polypeptide is cleaved from the resin, and at the same time, various protecting groups are removed.
  • an intramolecular disulfide bond formation reaction is carried out in a highly diluted solution, and the desired polypeptide, receptor, partial peptide or To obtain the amide form of
  • various activating reagents that can be used for the synthesis of polypeptides can be used, and carbodiimides are particularly preferable.
  • carbopimides examples include DCC, N, N'-diisopropyl carbopimide, N-ethyl-N '-(3-dimethylaminoprolyl) carbopimide, and the like. Activation by these involves the addition of a protected amino acid directly to the resin along with a racemization inhibitor additive (eg, H ⁇ B t, HOOB t), or a symmetrical acid anhydride or HOB t ester or H ⁇ B t ester Can be added to the resin after activation of the protected amino acid in advance.
  • a racemization inhibitor additive eg, H ⁇ B t, HOOB t
  • the solvent used for activating the protected amino acid or for condensing with the resin can be appropriately selected from solvents known to be usable for the polypeptide condensation reaction.
  • acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide, N-methylpyrrolidone, halogenated hydrocarbons such as methylene chloride, chloroform, and trifluoroethanol Alcohols such as dimethyls Sulfoxides such as rufoxide, ethers such as pyridine, dioxane, and tetrahydrofuran, nitriles such as acetonitrile and propionitrile, esters such as methyl acetate and ethyl acetate, or an appropriate mixture thereof are used.
  • the reaction temperature is appropriately selected from a range known to be usable for a polypeptide bond formation reaction, and is usually appropriately selected from a range of about ⁇ 20 ° C. to 50.
  • the activated amino acid derivative is usually used in a 1.5 to 4-fold excess.
  • Examples of the protecting group for the amino group of the starting material include Z, Boc, t-pentyloxycarbonyl, isoporonyloxycarbonyl, 4-methoxybenzyloxyl-ponyl, C11Z, Br_Z, a Damantyloxycarponyl, trifluoroacetyl, 'phthaloyl, formyl, 2-ditrophenylsulfenyl, diphenylphosphinothioyl, Fmoc and the like are used.
  • the lipoxyl group may be, for example, alkyl esterified (eg, methyl, ethyl, propyl, butyl, t-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl, etc.) Alkyl esterification), aralkyl esterification (for example, benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-cyclobenzyl ester, benzhydryl esterification), phenacyl esterification, benzyl ester It can be protected by xycarbonyl hydrazide, t-butoxycarbonyl hydrazide, trityl hydrazide, or the like.
  • alkyl esterified eg, methyl, ethyl, propyl, butyl, t-butyl, cyclopentyl, cycl
  • the hydroxyl group of serine can be protected, for example, by esterification or etherification.
  • esterification for example, a group derived from carbonic acid such as a lower (C ⁇ ) alkanol group such as an acetyl group, an aroyl group such as a benzoyl group, a benzyloxycarbonyl group, and an ethoxycarbonyl group is used.
  • groups suitable for etherification include, for example, a benzyl group, Tedrahydrovinylil group, tributyl group and the like.
  • the protecting group of the phenolic hydroxyl group of tyrosine for example, Bz 1, C 1 2 one B zl, 2-two Torobenjiru, B r- Z, such as t one-butyl is used.
  • protecting group for imidazole of histidine for example, Tos, 4-methoxy2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, Fmoc and the like are used.
  • Examples of the raw material in which the hydroxyl group of the raw material is activated include the corresponding acid anhydride, azide, active ester [alcohol (eg, pentachlorophenol, 2,4,5-trichloromouth phenol, 2,4 —Esters with dinitrophenol, cyanomethyl alcohol, paranitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyphthalimide, HOB t)].
  • alcohol eg, pentachlorophenol, 2,4,5-trichloromouth phenol, 2,4 —Esters with dinitrophenol, cyanomethyl alcohol, paranitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyphthalimide, HOB t
  • the activated amino group of the raw material for example, a corresponding phosphoramide is used. .
  • Methods for removing (eliminating) protecting groups include, for example, catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd-black or Pd-carbon, or hydrogen fluoride anhydride, methanesulfonic acid, trifluoromethane, or the like.
  • the elimination reaction by the above acid treatment is generally performed at a temperature of about 120 ° C. to 40 ° C.
  • anisol for example, anisol, phenol, thioanisole, methacrylol, paracresol, dimethylsulfur
  • a cation scavenger such as ido, 1,4-butanedithiol or 1,2-ethanedithiol.
  • the 2,4 ⁇ dinitrophenyl group used as an imidazole protecting group of histidine is removed by thiophenol treatment, and the formyl group used as an indole protecting group of tributofan is 1,2-ethanedithiol, 1,4 —In addition to deprotection by acid treatment in the presence of butanedithiol, etc., it is also removed by alkali treatment with dilute sodium hydroxide solution or dilute ammonia.
  • Protection of functional groups that should not participate in the reaction of The elimination of the protecting group, the activation of the functional group involved in the reaction, and the like can be appropriately selected from known groups or known means.
  • the receptor or the partial peptide thereof of the present invention for example, first, after protecting the carboxy-terminal amino acid at the amino group side with a peptide ( After extending the chain to the desired length, only the polypeptide except the N-terminal a-amino group protecting group of the peptide chain was removed, and only the C-terminal carboxyl group protecting group was removed. A polypeptide is produced, and both polypeptides are condensed in a mixed solvent as described above. Details of the condensation reaction are the same as described above. After purifying the protected polypeptide obtained by the condensation, all the protecting groups are removed by the above-mentioned method, and a desired crude polypeptide can be obtained. The crude polypeptide is purified by using various known purification means, and the main fraction is lyophilized to obtain a desired polypeptide, receptor or an amide of a partial peptide thereof.
  • the receptor or its partial peptide of the present invention for example, after condensing an ⁇ -hydroxyl group of a carboxyl-terminal amino acid with a desired alcohol to form an amino acid ester, in the same manner as the amide of a peptide, a receptor or a partial peptide thereof, an ester of a desired polypeptide, a receptor or a partial peptide thereof can be obtained.
  • the polypeptide, receptor or partial peptide thereof of the present invention can be produced by a known peptide synthesis method, or, for a partial peptide of the receptor, by cleaving the receptor with an appropriate peptide. can do.
  • a method for synthesizing a peptide for example, either a solid phase synthesis method or a liquid phase synthesis method may be used. That is, the partial peptide or amino acid which can constitute the polypeptide, receptor or its partial peptide of the present invention is condensed with the remaining portion, and when the product has a protecting group, the protecting group is removed.
  • Known methods for condensation and elimination of protecting groups include, for example, the methods described in the following (a) to (e).
  • polypeptide, receptor or partial peptide thereof of the present invention can be purified and isolated by combining crystals and the like.
  • the polypeptide, receptor or its partial peptide obtained by the above method is a free form, it can be converted to an appropriate salt by a known method or a method analogous thereto, and conversely, it can be obtained as a salt. In such a case, it can be converted to a free form or another salt by a known method or a method analogous thereto.
  • the DNA encoding the polypeptide of the present invention, the receptor or the partial peptide thereof may be any DNA containing the above-described nucleotide sequence encoding the polypeptide of the present invention, the receptor or the partial peptide thereof. There may be. In addition, any of genomic DNA, genomic DNA library, cDNA derived from the above-mentioned cells and tissues, cDNA library derived from the above-described cells and tissues, and synthetic DNA may be used.
  • the vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. Alternatively, it can also be directly amplified by Reverse Transcriptase Polymerase Chain Reaction (hereinafter abbreviated as RT-PCR) using a preparation of a total RNA or mRNA fraction from the cells and tissues described above.
  • RT-PCR Reverse Transcriptase Polymerase Chain Reaction
  • DNA encoding the polypeptide of the present invention examples include (a) SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: : 17, SEQ ID NO: 18, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 59, 'SEQ ID NO: 60, Sequence No .: 61, SEQ ID NO: 62, SEQ ID NO: No .: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 86 Or a DNA containing the base sequence represented by SEQ ID NO: 88,
  • SEQ ID NO: 3 SEQ ID NO: 4, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, Sequence SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 86 or SEQ ID NO: 88 Or (ii) hybridized with the nucleotide sequence represented by SEQ ID NO: 5, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 28 or SEQ ID NO:
  • DNA containing a nucleotide sequence having a homology of about 70% or more, preferably about 80% or more, more preferably about 90% or more, and even more preferably about 95% or more with the nucleotide sequence represented by 34 is used.
  • Hybridization is performed by a known method or a method based thereon, for example,
  • High stringency conditions refer to, for example, sodium concentration of about 19 to 4: 0 mM, preferably about 19 to 20 mM, temperature of about 50 to 70, and preferably about 60 to 65 ° C. .
  • the case where the sodium concentration is about 19 mM and the temperature is about 65 ° C is most preferable.
  • the DNA encoding the polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 includes a DNA containing the nucleotide sequence represented by SEQ ID NO: 3, and a DNA containing the nucleotide sequence represented by SEQ ID NO: 88 DNA etc. are used,
  • the DNA encoding the polypeptide containing the amino acid sequence represented by SEQ ID NO: 7 includes a DN containing the base sequence represented by SEQ ID NO: 13. A and so on.
  • DNA encoding the polypeptide containing the amino acid sequence represented by SEQ ID NO: 10 DNA containing the base sequence represented by SEQ ID NO: 16 or the like is used,
  • DNA encoding the polypeptide containing the amino acid sequence represented by SEQ ID NO: 11 is, for example, DNA containing the base sequence represented by SEQ ID NO: 17;
  • the DNA encoding the polypeptide containing the amino acid sequence represented by SEQ ID NO: 12 is a DNA containing the base sequence represented by SEQ ID NO: 18.
  • DNA encoding the polypeptide containing the amino acid sequence represented by SEQ ID NO: 25 includes DNA containing the base sequence represented by SEQ ID NO: 27, and the like.
  • DNA encoding the polypeptide containing the amino acid sequence represented by SEQ ID NO: 30 includes DNA containing the base sequence represented by SEQ ID NO: 32, and the like.
  • the DNA encoding the polypeptide containing the amino acid sequence represented by SEQ ID NO: 31 contains the base sequence represented by SEQ ID NO: 33.
  • the DNA encoding the polypeptide containing the amino acid sequence represented by SEQ ID NO: 41 is a DNA encoding the polypeptide comprising the base sequence represented by SEQ ID NO: 59.
  • the DNA encoding the polypeptide containing the amino acid sequence represented by SEQ ID NO: 46 is a DNA containing the nucleotide sequence represented by SEQ ID NO: 64.
  • DNA encoding the polypeptide containing the amino acid sequence represented by SEQ ID NO: 47 DNA containing the base sequence represented by SEQ ID NO: 65, or the like is used.
  • DNA encoding the polypeptide containing the amino acid sequence represented by SEQ ID NO: 48 a DNA containing the base sequence represented by SEQ ID NO: 26 or the like is used,
  • DNA encoding the polypeptide containing the amino acid sequence represented by SEQ ID NO: 50 includes DNA containing the base sequence represented by SEQ ID NO: 3 ′, and the like.
  • DNA encoding the polypeptide containing the amino 9-noic acid sequence represented by SEQ ID NO: 51 DNA containing the base sequence represented by SEQ ID NO: 3 or the like is used,
  • DNA containing the amino acid sequence represented by SEQ ID NO: 54: ⁇ -dose is used as DNA, such as DNA having the base sequence represented by SEQ ID NO: 68,
  • DNA encoding the polypeptide containing the amino acid sequence represented by SEQ ID NO: 55 includes, for example, DNA containing the base sequence represented by SEQ ID NO: 69,
  • DNA As the DNA encoding the polypeptide containing the amino acid sequence represented by SEQ ID NO: 21, DNA such as' DNA containing the base sequence represented by SEQ ID NO: 70 is used,
  • DNA encoding the polypeptide containing the amino acid sequence represented by SEQ ID NO: 56 contains the nucleotide sequence represented by SEQ ID NO: 66 DNA etc. are used, ⁇
  • DNA encoding the polypeptide containing the amino acid sequence represented by SEQ ID NO: 71 includes DNA having the base sequence represented by SEQ ID NO: 72, and the like.
  • DNA encoding the polypeptide containing the amino acid sequence represented by SEQ ID NO: 85 DNA containing the base sequence represented by SEQ ID NO: 86, DNA and the like are used.
  • Examples of the DNA encoding the receptor of the present invention include: (1) DNA containing the nucleotide sequence represented by SEQ ID NO: 74, or the nucleotide sequence represented by SEQ ID NO: 74 and under conditions of high stringency. DNA encoding a protein having a nucleotide sequence that hybridizes with: and having substantially the same activity as the protein containing the amino acid represented by SEQ ID NO: 73; (2) SEQ ID NO: 76 Or a protein having a base sequence that hybridizes under high stringent conditions to a DNA containing the base sequence represented by SEQ ID NO: 76 or a protein containing the amino acid represented by SEQ ID NO: 75 (3) DNA containing the nucleotide sequence represented by SEQ ID NO: 78, or DNA encoding the protein having the same activity, Gent DNA encoding a protein having a nucleotide sequence that hybridizes under the conditions and having substantially the same activity as the amino acid-containing protein represented by SEQ ID NO: 77; (4) SEQ ID NO
  • SEQ ID NO: 81 A DNA encoding a protein having substantially the same activity as a protein containing an amino acid represented by (6), (6) a DNA containing a base sequence represented by SEQ ID NO: 84, or a SEQ ID NO: 84 A nucleotide sequence that hybridizes under high stringent conditions with a nucleotide sequence represented by the following: and encodes a protein having substantially the same activity as the protein containing the amino acid represented by SEQ ID NO: 83 Any material such as A may be used.
  • DNAs that can hybridize with the nucleotide sequence represented by SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78 or SEQ ID NO: 80 under high stringent conditions include, for example, SEQ ID NO: 74 and SEQ ID NO: 76, respectively. About 70% or more, preferably about 80% or more, more preferably about 90% or more, and still more preferably about 95% or more with the nucleotide sequence represented by SEQ ID NO: 78 or SEQ ID NO: 80. DNA having a base sequence having the same is used.
  • Examples of the DNA that can hybridize with the base sequence represented by SEQ ID NO: 82 under high stringency conditions include, for example, the base sequence represented by SEQ ID NO: 82 and 82% or more, preferably about 85% or more, more preferably
  • DNA containing a nucleotide sequence having a homology of about 90% or more, more preferably about 95% or more is used.
  • Examples of the DNA that can hybridize with the base sequence represented by SEQ ID NO: 84 under high stringency conditions include, for example, 92% or more, preferably about 95% or more, more preferably the base sequence represented by SEQ ID NO: 84.
  • DNA containing a base sequence having about 98% or more homology is used.
  • Hybridization can be performed by a known method or a method analogous thereto, for example, Molecular Cloning 2nd (J. Sarabrook et al., Cold Spring Harbor Lab.
  • High stringency conditions include, for example, a sodium concentration of about 19 to 40 mM, preferably about 19 to 2 OmM, and a temperature of about 50 to 70 ° C, preferably about
  • the DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 73 includes a DNA containing the base sequence represented by SEQ ID NO: 74, and an amino acid represented by SEQ ID NO: 75.
  • Examples of the DNA encoding the protein containing the sequence include DNA containing the base sequence represented by SEQ ID NO: 76, and DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 77 include: DNA containing the nucleotide sequence represented by SEQ ID NO: 78; DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 79; DNA containing the nucleotide sequence represented by SEQ ID NO: 80;
  • SEQ ID NO: DNA containing the base sequence represented by 84 is used
  • the DNA encoding the partial peptide of the receptor of the present invention may be any as long as it contains the nucleotide sequence encoding the partial peptide of the receptor of the present invention described above. Further, it may be any of genomic DNA, genomic DNA library, the above-described cell / tissue-derived cDNA, the above-described cell / tissue-derived cDNA library, and synthetic DNA.
  • Examples of the DNA encoding the partial peptide of the receptor of the present invention include: (1) a DNA having a partial nucleotide sequence of a DNA having the nucleotide sequence represented by SEQ ID NO: 74, or a nucleotide represented by SEQ ID NO: 74 Has a nucleotide sequence that hybridizes with the sequence under eight stringent conditions, and has a partial nucleotide sequence of a DNA encoding a protein having substantially the same activity as the protein containing the amino acid represented by SEQ ID NO: 73 DNA, (2) DNA having a partial nucleotide sequence of DNA having the nucleotide sequence represented by SEQ ID NO: 76, or SEQ ID NO:
  • DNAs capable of hybridizing with the base sequence represented by SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82 or SEQ ID NO: 84 have the same significance as described above.
  • the DNA encoding the polypeptide, receptor or partial peptide thereof of the present invention may be labeled by a known method, and specifically, isotope-labeled DNA, fluorescently-labeled DNA (for example, Fluorescent labeling with fluorescein), biotinylated or enzyme-labeled ones.
  • isotope-labeled DNA for example, Fluorescent labeling with fluorescein
  • biotinylated for example, Biotinylated or enzyme-labeled ones.
  • a polypeptide of the present invention labeled with isotop is used.
  • the polypeptide, the receptor or its partial peptide of the present invention (hereinafter, in the description of the cloning and expression of DNA encoding these polypeptides, etc., when these polypeptides, etc. are simply abbreviated as the polypeptides of the present invention.
  • a DNA is amplified by a known PCR method using a synthetic DNA primer having a partial nucleotide sequence of the polypeptide of the present invention, or an appropriate method is used.
  • the DNA incorporated into the vector can be selected by hybridization with a DNA fragment encoding a part or the entire region of the polypeptide of the present invention or a DNA fragment labeled with a synthetic DNA. The method of hybridization is described, for example, in Molecul ar Cloning 2nd (J.
  • Conversion of the DNA base sequence can be performed using a known kit, for example, Mutan TM -super Express Km (Takara Shuzo Co., Ltd.), Mutan TM -K (Takara Shuzo Co., Ltd.), etc., using the OM-LA PCR method and the Gapped method. It can be carried out according to a known method such as the duplex method and the Kunkel method, or a method analogous thereto.
  • the DNA encoding the cloned polypeptide can be used as it is depending on the purpose, or it can be used after digesting with a restriction enzyme or adding a linker, if desired.
  • the DNA has ATG as a translation initiation codon at its 5, terminal end. It may also have TAA, TGA or TAG as a translation stop codon on the 3 'end side. These translation initiation codon and translation termination codon can be added using an appropriate synthetic DNA adapter.
  • the expression vector of the polypeptide of the present invention can be prepared, for example, by (a) cutting out a DNA fragment of interest from DNA encoding the polypeptide of the present invention, and (mouth) placing the DNA fragment in an appropriate expression vector. Can be produced by ligating downstream of this promoter.
  • the vector examples include a plasmid derived from Escherichia coli (eg, pBR322, pBR325, pUC12, pUC13), a plasmid derived from Bacillus subtilis (eg, pUBll0, TP5, pC194), a plasmid derived from yeast (eg, SH19, pSH15), bacteriophages such as ⁇ phage, animal viruses such as retrovirus, vaccinia virus, baculovirus, etc., pAl-11, pXT1, Rc / CMV, pRc / RSV And pc DNA I / Neo. , 1
  • Escherichia coli eg, pBR322, pBR325, pUC12, pUC13
  • Bacillus subtilis eg, pUBll0, TP5, pC194
  • yeast eg, SH19, pSH15
  • bacteriophages such as
  • the promoter used in the present invention may be any promoter as long as it is suitable for the host used for expression of the gene.
  • examples include the SRa promoter, SV40 promoter, HIV / LTR promoter, CMV promoter, and HSV-TK promoter.
  • CMV cytomegalovirus
  • SRCK promoter cytomegalovirus promoter
  • the host is Escherichia, trp promoter, lac promoter, recA promoter, ⁇ PL promoter, lpp promoter, T7 promoter, etc., and if the host is Bacillus, SP ⁇ 1 promoter
  • yeast such as SPO2 promoter, penP promoter overnight, PH05 promoter, PGK promoter, GAP promoter, ADH. Promoter and the like are preferable.
  • a polyhedrin promoter, a P10 promoter and the like are preferable.
  • expression vectors include enhancers and splicing if desired.
  • a signal containing a signal, a polyA addition signal, a selection marker, an SV40 replication origin (hereinafter sometimes abbreviated as SV40 ori '), or the like can be used.
  • selectable markers include dihydrofolate reductase (hereinafter sometimes abbreviated as dhfr) gene (methotrexate (MTX) resistance), ampicillin resistance gene (hereinafter sometimes abbreviated as Amp 37 ), neomycin Syn-resistance gene (hereinafter sometimes abbreviated as Neor., G418 resistance) and the like.
  • dh fr gene when used as a selection marker using Chinese hamster cells deficient in the dh fr gene, the target gene can be selected using a thymidine-free medium.
  • a signal sequence suitable for the host is added to the N-terminal side of the polypeptide of the present invention.
  • the PhoA * signal sequence and the 0-A signal sequence are used.
  • the amylase-signal sequence and subtilisin-signal sequence are used.
  • the host is yeast, MFcK signal sequence, SUC2 signal sequence, etc.
  • the host is an animal cell, insulin signal sequence, ⁇ - ⁇ f interferin signal sequence, antibody molecule, signal sequence, etc. Are available respectively.
  • a transformant can be produced using the thus constructed vector containing the DNA encoding the polypeptide of the present invention.
  • Escherichia bacteria for example, Bacillus bacteria, yeast, insect cells, insects, animal cells, and the like are used.
  • Escherichia bacteria include, for example, Escherichia coli
  • Bacillus bacteria include, for example, Bacillus subtilis (Bacillus subtilis) MI 114 [Gene, 24, 255 (1983)], 207-21 [Journal of Biochemistry, Vol. 95] , 8.7 (1 984)].
  • yeast examples include Saccharomyces cerevisiae AH 22, AH 22 R ⁇ , NA87-11A, DKD-5D, 20B-12 and Schizosaccharomyces pombe N CYC 1913. , NCYC 2036, Pichia astoris KM 71 and the like.
  • Insect cells include, for example, 'If the virus is Ac NPV, cell lines derived from night larvae of moth larvae (Spodoptera frugiperda cells; S f cells), MG1 cells derived from the midgut of Trichoplusia ni, and eggs derived from eggs of Trichoplusia ni High Five TM cells,
  • Cells derived from Mamestra brassicae or cells derived from Estigmena acrea are used.
  • a silkworm-derived cell line (Bombyx mori cell; BmN cell) is used.
  • Sf cells include Sf9 cells (ATCC CRL1711) and Sf21 cells (Vaughn, JL et al., In Vivo, 13, 213-217, (1977)). Are used.
  • insects for example, silkworm larvae and the like are used [Maemura, Nature, Vol. 315, 592 (1985)].
  • animal cells examples include monkey cell COS-7, Vero, Chinese hamster cell CHO (hereinafter abbreviated as CHO cell), dh ⁇ r gene-deficient Chinese hamster cell CHO (hereinafter, CHO (dh fr—) cell Abbreviations), mouse L cells, mouse AtT-'20, mouse myeloma cells, rat GH3, human FL cells, and the like. '
  • Insect cells or insects can be transformed, for example, according to the method described in Bio / Technology, 6, 47-55 (1988). ,
  • a liquid medium is suitable as a medium for cultivation, and a carbon source necessary for the growth of the transformant is contained therein.
  • Carbon sources include, for example, glucose, dextrin, soluble starch, sucrose, etc.
  • Nitrogen sources include, for example, ammonium salts, nitrates, corn chips.
  • the inorganic or organic substance such as a liquid and the inorganic substance include calcium chloride, sodium dihydrogen phosphate, and magnesium chloride.
  • yeast extract, vitamins, growth promoting factors and the like may be added.
  • the pH of the medium is preferably about 5-8.
  • Examples of a medium for culturing Escherichia bacteria include, for example, M9 medium containing glucose and casamino acids (Miller, Journal of Journal of Experiments in Molecular Genetics). Molecular Genetics), 431-433, Cold Spring Harbor Laboratory, New York 1972].
  • a drug such as 3i3-indolylacrylic acid can be added.
  • the cultivation is usually performed at about 15 to 43 ° C for about 3 to 24 hours, and if necessary, aeration and stirring may be added.
  • the cultivation is usually carried out at about 30 to 40 ° C for about 6 to 24 hours, and if necessary, aeration and stirring can be applied.
  • the medium used is Grace s Insect Medium (Grace, T.,
  • the pH of the medium is preferably adjusted to about 6.2 to 6.4. Culture is usually performed at about 27 ° C for about 3 to 5 days, with aeration and agitation as necessary. ,
  • the medium When culturing a transformant in which the host is an animal cell, the medium may be, for example, a MEM medium containing about 5 to 20% fetal bovine serum [Science, 122, 501 (1952)], a DMEM medium [Virology, 8, 396 (1959)], RPMI 1640 medium [Journal of the American Medical Association 'Yon (The Journal of the American Medical Association) 199, 519 ( 1967) 3, 199 medium [Proceding of the Society for the Biological Medicine, 73, 1 (1950)]. pH is about 6-8 It is preferred that Cultivation is usually carried out at about 30 ° C to 40 ° C for about 15 to 60 hours, and aeration and stirring are added as necessary.
  • polypeptide of the present invention can be produced in the cells, in the cell membrane, or outside the cells of the transformant.
  • polypeptide of the present invention can be separated and purified from the culture by, for example, the following method. '
  • the cells or cells are collected by a known method, suspended in an appropriate buffer, and subjected to ultrasonication, lysozyme and / or lysozyme. After the cells or cells are destroyed by freeze-thawing or the like, a method of obtaining a crude extract of the polypeptide by centrifugation or filtration is appropriately used.
  • the buffer may contain a protein denaturant such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100 TM .
  • Purification of the polypeptide contained in the thus obtained culture supernatant or extract can be carried out by appropriately combining known separation and purification methods.
  • known separation and purification methods include methods using solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis.
  • a method utilizing a difference in hydrophobicity such as isoelectric focusing
  • a method utilizing an isoelectric point difference such as isoelectric focusing, and the like are used.
  • the polypeptide thus obtained when obtained in a free form, it can be converted to a salt by a known method or a method analogous thereto, and conversely, when the polypeptide is obtained as a salt, a known method or analogous method Depending on the method, it can be converted into a free form or another salt.
  • the polypeptide produced by the recombinant can be arbitrarily modified or treated with a suitable protein-modifying enzyme before or after purification. It can also be partially removed.
  • a suitable protein-modifying enzyme for example, trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like are used.
  • an antibody against the polypeptide or the receptor of the present invention may be a polyclonal antibody, a monoclonal antibody if it can recognize the polypeptide or the receptor of the present invention. Any of antibodies may be used.
  • An antibody against the polypeptide or the receptor of the present invention can be produced by using the polypeptide or the receptor of the present invention as an antigen according to a known method for producing an antibody or an antiserum.
  • the polypeptide or receptor of the present invention is administered to a warm-blooded animal itself or together with a carrier or diluent at a site where antibody production is possible by administration.
  • Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered to enhance the antibody-producing ability upon administration. Administration is usually performed once every 2 to 6 weeks, for a total of about 2 to 10 times.
  • Examples of the warm-blooded animal to be used include monkeys, rabbits, rabbits, dogs, guinea pigs, mice, rats, sheep, goats, and chickens, and mice and ruds are preferably used.
  • a warm-blooded animal immunized with the antigen for example, an individual with an antibody titer from a mouse is selected, and spleen or lymph nodes are collected 2 to 5 days after the final immunization.
  • a monoclonal antibody-producing hybridoma can be prepared by fusing the antibody-producing cells contained with myeloma cells of the same or different species.
  • the measurement of the antibody titer in the antiserum can be performed, for example, by reacting a labeled polypeptide described below with the antiserum, and then measuring the activity of the labeling agent bound to the antibody.
  • the fusion operation can be performed according to a known method, for example, the method of Keller and Milstein [Nature, 256, 495 (1975)].
  • the fusion promoter include polyethylene daricol (PEG) and Sendai virus, and PEG is preferably used.
  • PEG polyethylene daricol
  • myeloma cells include myeloma cells of warm-blooded animals such as NS-1, P3U1, SP 2/0, and AP-1, but P3U1 is preferably used.
  • the preferred ratio between the number of antibody-producing cells (spleen cells) and the number of myeloma cells used is about 1: 1 to 20: 1, and PEG (preferably PEG 1000 to PEG 6000) is used at a concentration of about 10 to 80%.
  • Cell fusion can be carried out efficiently by adding the mixture and incubating at 20 to 40 ° C, preferably 30 to 37 ° C, for 1 to 10 minutes.
  • hybridomas can be cultured on a solid phase (eg, microplate) onto which a polypeptide (protein) antigen is directly or adsorbed together with a carrier. Then, an anti-immunoglobulin antibody labeled with a radioactive substance or an enzyme (if the cell used for cell fusion is a mouse, a mouse immunoglobulin antibody is used) or protein A, is added.
  • a solid phase eg, microplate
  • an anti-immunoglobulin antibody labeled with a radioactive substance or an enzyme if the cell used for cell fusion is a mouse, a mouse immunoglobulin antibody is used
  • protein A protein A
  • a method for detecting a monoclonal antibody bound to a solid phase adding a hybridoma culture supernatant to a solid phase to which an anti-immunoglobulin antibody or protein A is adsorbed, adding a polypeptide labeled with a radioactive substance, an enzyme, or the like, A method for detecting a monoclonal antibody bound to a solid phase is exemplified.
  • the selection of the monoclonal antibody can be performed according to a known method or a method analogous thereto. Usually, it can be performed in a medium for animal cells supplemented with HAT (hypoxanthine, aminopterin, thymidine).
  • any medium can be used as long as it can grow a hybridoma.
  • RPMI 1640 medium containing 1-20%, preferably 10-20% fetal bovine serum
  • GIT medium containing 1-10% fetal bovine serum (Wako Pure Chemical Industries, Ltd.)
  • the culture temperature is usually 20-40 ° C, preferably about 37 ° C.
  • the culturing time is usually between 5 days and 3 days, preferably between 1 week and 2 weeks. Culture can be usually performed under 5% carbon dioxide gas.
  • the antibody titer of the hybridoma culture supernatant can be measured in the same manner as the measurement of the antibody titer in the antiserum described above.
  • Monoclonal antibodies can be separated and purified by known methods, for example, immunoglobulin separation and purification methods (eg, salting out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis method, ion exchanger (eg, DEAE)). Adsorption / desorption method, ultracentrifugation method, gel filtration method, antigen-binding solid phase or specific purification method in which only the antibody is collected using an active adsorbent such as protein A or protein G and the bond is dissociated to obtain the antibody). You can do it.
  • immunoglobulin separation and purification methods eg, salting out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis method, ion exchanger (eg, DEAE)
  • the polyclonal antibody of the present invention can be produced according to a known method or a method analogous thereto.
  • an immunogen polypeptide antigen itself or a complex thereof with a carrier protein is formed, and immunization is performed on a warm-blooded animal in the same manner as in the above-described method for producing a monoclonal antibody.
  • the antibody can be produced by collecting an antibody-containing substance for the peptide and separating and purifying the antibody.
  • the type of the carrier protein and the mixing ratio of the carrier and the hapten depend on the hapten immunized by cross-linking with the carrier. As long as ⁇ can be efficiently formed, any kind may be cross-linked at any ratio.For example, serum serum albumin, thyroglobulin, hemocyanin, etc. are used in a weight ratio of about 0.1 to 1 for hapten. A method of coupling at a rate of 1 to 20, preferably about 1 to 5 is used. In addition, various condensing agents can be used for force coupling between the hapten and the carrier.
  • daltaraldehyde carbodiimide, a maleimide active ester, an active ester reagent containing a thiol group or a dithioviridyl group, or the like is used.
  • the condensation product is administered to a warm-blooded animal at a site where antibody production is possible or together with a carrier or diluent.
  • Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance antibody production ability upon administration.
  • the administration is usually performed once every about 2 to 6 weeks, for a total of about 3 to 10 times.
  • the polyclonal antibody can be collected from the blood, ascites, etc., preferably from the blood, of the warm-blooded animal immunized by the above method.
  • the measurement of the polyclonal antibody titer in the antiserum is based on the measurement of the antibody titer in the antiserum described above. Can be measured in the same manner as described above. Separation and purification of the polyclonal antibody can be carried out according to the same immunoglobulin separation and purification method as the above-described separation and purification of the monoclonal antibody.
  • Bases complementary to or substantially complementary to DNA encoding the polypeptide, receptor or partial peptide thereof of the present invention may be abbreviated as the DNA of the present invention.
  • An antisense polynucleotide preferably DNA having a sequence (hereinafter sometimes abbreviated as antisense DNA) has a base sequence complementary to or substantially complementary to the DNA of the present invention.
  • any antisense DNA may be used as long as it has an action of suppressing the expression of the DNA.
  • the nucleotide sequence substantially complementary to the DNA of the present invention refers to, for example, the entire nucleotide sequence or a partial nucleotide sequence of the nucleotide sequence complementary to the DNA of the present invention (that is, the complementary strand of the DNA of the present invention). Nucleotide sequences having about 70% or more, preferably about 80% or more, more preferably about 90% or more, and most preferably about 95% or more homology.
  • an antisense DNA having a homology of preferably about 80% or more, more preferably about 90% or more, and most preferably about 95% or more is suitable. These antisenses can be produced using a known DNA synthesizer or the like.
  • the antisense DNA of the present invention may contain altered or modified sugars, bases, or bonds, and may be provided in a special form such as ribosome or microsphere, applied by gene therapy, or added. Can be given in a written form.
  • polycations such as polylysine, which acts to neutralize the charge of the phosphate skeleton
  • lipids which increase the interaction with the cell membrane or increase the uptake of nucleic acids (
  • hydrophobic substances such as phospholipid and cholesterol
  • Preferred lipids for addition include cholesterol and its derivatives (eg, cholesteryl chromate formate, cholic acid, etc.).
  • nucleic acids Can be attached to the 3 'end or 5' end of the protein, and can be attached via a base, sugar, or intramolecular nucleoside bond.
  • Other groups include cap groups specifically arranged at the 3 'end or 5' end of a nucleic acid for preventing degradation by nucleases such as exonuclease and RNase.
  • capping groups include, but are not limited to, hydroxyl-protecting groups known in the art, including glycols such as polyethylene glycol and tetraethylene glycol.
  • the antisense DNA inhibitory activity 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 peptide or receptor of the present invention. be able to.
  • the following are (a) the polypeptide of the present invention, (b) the receptor of the present invention (hereinafter also including a partial peptide thereof), (c) the DNA of the present invention, (d) the antibody of the present invention, and (e) The use of the antisense DNA and the like of the present invention will be described.
  • the polypeptide of the present invention has a cell stimulating activity on an expression cell such as the receptor of the present invention (eg, GPR8, GPR7, rat TGR26, mouse TGR26, ⁇ sher GPR8, Gsher GPR7), and the like. Is an endogenous ligand.
  • the receptor of the present invention eg, GPR8, GPR7, rat TGR26, mouse TGR26, ⁇ sher GPR8, Gsher GPR7, and the like. Is an endogenous ligand.
  • the polypeptide of the present invention or the DNA of the present invention is abnormal or defective, or when the receptor of the present invention or the DNA encoding the receptor is abnormal or defective.
  • urinary storage disorders eg, frequent urination, urinary incontinence (eg, urge incontinence, stress urinary incontinence, functional urinary incontinence, etc.)
  • polyuria eg, diabetes insipidus (eg, pituitary urine) Dyspnea, renal diabetes insipidus, etc.), hypernatremia, metabolic allolysis, hypokalemia, Gushing syndrome and the likelihood is high.
  • the polypeptide of the present invention and the DNA of the present invention can be used, for example, as an antidiuretic, for example, for urinary storage disorders [eg, frequent urination, urinary incontinence (eg, urge incontinence, bariatric urine) Incontinence, functional urinary incontinence, etc.), polyuria, diabetes insipidus (eg, pituitary diabetes insipidus, renal diabetes insipidus, etc.), hypernatremia, metabolic alkalosis, low potassium. It can be used as a preventive 'treatment for muemia, Cushing's syndrome, etc.
  • urinary storage disorders eg, frequent urination, urinary incontinence (eg, urge incontinence, bariatric urine) Incontinence, functional urinary incontinence, etc.
  • polyuria eg, diabetes insipidus (eg, pituitary diabetes insipidus, renal diabetes insipidus, etc.
  • urinary incontinence eg, urge incontinence, stress urinary incontinence, functional urinary incontinence, etc.
  • polyuria e.g, diabetes insipidus (eg, pituitary insipidus, renal insipidus, etc.)
  • Hypernatremia e.g., metabolic alkalosis, hypokalemia, Cushing syndrome and other prophylactic treatment.
  • the polypeptide of the present invention and the DNA of the present invention can be obtained, for example, by administering the DNA of the present invention to a patient when the polypeptide of the present invention is reduced or deleted in vivo.
  • the DNA of the present invention By expressing the polypeptide of the present invention in vivo, (oral) the DNA of the present invention is inserted into cells, and after expressing the polypeptide of the present invention, the cells are transplanted into a patient.
  • C By administering the polypeptide of the present invention to the patient, the role of the polypeptide of the present invention in the patient can be sufficiently or normally exerted.
  • the DNA of the present invention is used as the above-mentioned prophylactic or therapeutic agent
  • the DNA is used alone or in an appropriate vector such as a retrovirus vector, an adenovirus vector, an adenovirus-associated virus vector, or the like. After that, it can be administered to humans or warm-blooded animals according to conventional means.
  • the DN N of the present invention can be administered as it is or as a formulation with a physiologically acceptable carrier such as an adjuvant for promoting uptake, and administered by a catheter such as a gene gun or a hydrogel catheter. '
  • polypeptide of the present invention When used as the above prophylactic / therapeutic agent, it is purified to at least 90%, preferably at least 95%, more preferably at least 98%, and even more preferably at least 99%. It is preferred to use
  • the polypeptide of the present invention can be used, for example, as a sugar-coated tablet, capsule, elixir, microcapsule or the like, if necessary, orally, or water or other pharmaceutically acceptable liquid. It can be used parenterally (preferably subcutaneous administration) in the form of an injection such as a sterile solution or suspension.
  • the polypeptides of the present invention can be combined with physiologically acceptable carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, and the like, in the form of unit dosages generally required for accepted pharmaceutical practice. It can be manufactured by mixing. The amount of active ingredient in these preparations is such that a suitable dosage in the specified range can be obtained. You. .
  • Additives that can be incorporated into tablets, capsules, etc. include, for example, binders such as gelatin, corn starch, tragacanth, gum arabic, excipients such as crystalline cellulose, Swelling agents such as gelatin, alginic acid and the like, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, and flavoring agents such as peppermint, cocoa oil or cherry.
  • the unit dosage form is a capsule
  • the above-mentioned ingredients of the evening may further contain a liquid carrier such as oil and fat.
  • Sterile compositions for injection can be formulated according to standard pharmaceutical practice, such as dissolving or suspending the active substance in a vehicle such as water for injection, or naturally occurring vegetable oils such as sesame oil or coconut oil. .
  • aqueous liquids for injection include physiological saline, isotonic solutions containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium salt, etc.) and the like.
  • Solubilizers for example, alcohols (eg, ethanol, etc.), polyalcohols (eg, propylene glycol, polyethylene glycol, etc.), nonionic surfactants (eg, Polysorbate 80 TM, HCO-50, etc.) ) May be used together.
  • the oily liquid include sesame oil and soybean oil, and may be used in combination with benzyl benzoate, benzyl alcohol and the like as a solubilizing agent.
  • buffers for example, phosphate buffer, sodium acetate buffer, etc.
  • soothing agents for example, benzalkonium chloride ,: pro-protein hydrochloride, etc.
  • stabilizers for example, human serum albumin, polyethylene glycol: Alcohol, etc.
  • preservatives eg, benzyl alcohol, phenol, etc.
  • antioxidants antioxidants and the like.
  • the prepared injection solution is usually filled in a suitable ampoule.
  • the vector into which the DNA of the present invention is inserted is also formulated in the same manner as described above, and is usually used parenterally.
  • the preparations obtained in this way are safe and have low toxicity, and are therefore useful, for example, in humans or warm-blooded animals (eg, rats, mice, guinea pigs, egrets, birds, higgs, bushes, dogs, dogs, Cats, dogs, monkeys, etc.).
  • the dose of the polypeptide of the present invention may vary depending on the target disease, the subject of administration, the administration route, and the like.For example, when the polypeptide of the present invention is administered subcutaneously for the purpose of treating diabetes insipidus, it is generally In adults (as 60 kg), about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg of the polypeptide is administered per day. In the case of other animals, the dose can be administered in terms of 60 kg.
  • the polypeptide of the present invention has a function as a ligand of the receptor of the present invention
  • the polypeptide of the present invention or the present invention Compounds or salts thereof that promote the function of the receptor are useful, for example, as antidiuretics, etc., and have impaired urinary storage [eg, urinary frequency, urinary incontinence (eg, urge incontinence, stress urinary incontinence, functional Urinary incontinence, etc.), polyuria, diabetes insipidus (eg, pituitary diabetes insipidus, renal diabetes insipidus, etc.), hypernatremia, metabolic alkalosis, hypokalemia, Cushing syndrome, etc.
  • urinary storage eg, urinary frequency, urinary incontinence (eg, urge incontinence, stress urinary incontinence, functional Urinary incontinence, etc.)
  • polyuria eg, diabetes insipidus (eg, pituitary diabetes insipid
  • incontinence eg, urge incontinence, stress urinary incontinence, functional urinary incontinence, etc.
  • polyuria e.g, diabetes insipidus (eg, pituitary insipidus, renal insipidus, etc.)
  • hypernatremia e.g, metabolic alkalosis, hypokalemia, Cushing syndrome. It is a therapeutic agent.
  • the polypeptide of the present invention or a compound or a salt thereof that inhibits the function of the receptor of the present invention is useful as, for example, a diuretic, and produces renal edema, urinary excretion disorder (eg, , Dysuria, urinary pain, urinary tract obstruction, etc.), hyponatremia, antidiuretic hormone inadequate syndrome (SIADH), hypertension, etc. can be used as preventive and therapeutic agents.
  • the screening can be performed by using the polypeptide of the present invention or constructing a recombinant expression system of the polypeptide of the present invention, and using a receptor binding assay system using the expression system.
  • Compounds that alter the binding between the polypeptide and its receptor a compound that promotes or inhibits the activity of the polypeptide of the present invention, a compound that promotes or inhibits the activity of the receptor of the present invention
  • a compound that promotes or inhibits the activity of the receptor of the present invention eg, peptide, protein, etc.
  • Such compounds and through the receptor of the present invention cell stimulating activity (e.g., Arakidon acid release, acetylcholine release, intracellular C a 2 + release, intracellular AM P generated Z suppression, intracellular c GM P Production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, activation of c-fos, decrease in pH, activity to promote GTPTS binding activity, etc. (ie, the polypeptide of the present invention) Receptor agonist) and a compound having no cell stimulating activity (ie, a receptor antagonist of the polypeptide of the present invention).
  • "Altering the binding to the polypeptide of the present invention” includes both cases of inhibiting the binding to the polypeptide of the present invention and promoting the binding to the polypeptide of the present invention. Things.
  • a method for screening a compound or a salt thereof that promotes or inhibits the activity of the polypeptide of the present invention or the receptor of the present invention characterized by using the polypeptide and the receptor of the present invention or the receptor of the present invention.
  • a method for screening for a compound that alters the binding property of the polypeptide of the present invention to the receptor of the present invention a compound that promotes or inhibits the activity of the polypeptide of the present invention or a salt thereof, characterized in that: No.
  • a labeled polypeptide of the present invention When a labeled polypeptide of the present invention is brought into contact with a cell having the receptor of the present invention or a membrane fraction of the cell, a labeled polypeptide of the present invention and a test compound are compared with each other. Measuring and comparing the amount of the labeled polypeptide of the present invention bound to the cell or the membrane fraction when the cell is contacted with the cell containing the receptor of the present invention or the membrane fraction of the cell. A method for screening a compound (a compound that promotes or inhibits the activity of the polypeptide of the present invention) or a salt thereof, which alters the binding between the polypeptide of the present invention and the receptor of the present invention,
  • a compound activating the receptor of the present invention for example, the polypeptide of the present invention
  • a compound activating the receptor of the present invention When a test compound is brought into contact with a cell containing the receptor of the present invention, cell stimulating activity via the receptor of the present invention (for example, araki'donic acid release, acetylcholine release, intracellular Ca 2+ Release, Intracellular CAMP production Z suppression, Intracellular cGMP production, Inositol phosphate production, Cell membrane potential fluctuation, Intracellular protein phosphorylation, c-fos activation, pH decrease, GTP r S
  • a compound that alters the binding property between the polypeptide of the present invention and the receptor of the present invention which is characterized by measuring and comparing the activity of promoting or suppressing the binding activity and the like (the polypeptide of the present invention) Promotes or inhibits the activity of Compounds) or screening method of
  • a compound that activates the receptor of the present invention for example, And the like, when a transformant containing DNA encoding the receptor of the present invention is contacted with the receptor of the present invention expressed on the cell membrane by culturing the transformant.
  • the activating compound and the test compound are brought into contact with the receptor of the present invention expressed on the cell membrane by culturing a transformant containing the DNA encoding the receptor of the present invention
  • Cell stimulating activity mediated by the receptor of the invention e.g., arachidonic acid release, acetylcholine release, intracellular Ca 2+ release, inhibition of intracellular cAMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular Protein phosphorylation, activation of c: fos, decrease in pH, activity to promote or inhibit GTPaS binding activity, etc.
  • Peptide and the present invention Compounds that alter the binding property between receptor or screening method of a compound represented by the formula (compound promoting or inhibiting the activity of the polypeptide of the present invention), and the like.
  • SEQ ID NO were respectively-labeled with [125 1]: 1, SEQ ID NO: 2, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 36, SEQ ID NO : 37, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO .: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO :
  • the receptor of the present invention used in the screening method of the present invention may be any receptor that recognizes the polypeptide of the present invention as a ligand. And the like are preferred. However, since it is particularly difficult to obtain human-derived organs, the receptor of the present invention, etc., which is expressed in large amounts using recombinants, is suitable for screening. You.
  • the preparation method described later when cells containing the receptor of the present invention or the cell membrane fraction are used, the preparation method described later may be followed.
  • the cells When cells containing the receptor of the present invention are used, the cells may be immobilized with dartartaldehyde, formalin, or the like.
  • the immobilization method can be performed according to a known method. ⁇
  • the cell containing the receptor of the present invention refers to a host cell expressing the receptor of the present invention.
  • the host cell include the aforementioned Escherichia coli, Bacillus subtilis, yeast, insect cells, animal cells, and the like.
  • Can be The host cell that has expressed the receptor of the present invention includes the same method as the above-described method for producing a transformant that has been transformed with the expression vector containing the polypeptide of the present invention.
  • the membrane fraction refers to a fraction containing a large amount of cell membrane obtained by a known method after cell disruption.
  • Cells can be disrupted by crushing cells with a Potter-Elvehj em-type homogenizer, such as a Warinda blender or Polytron.
  • centrifugal fractionation methods such as differential centrifugation and density gradient centrifugation are mainly used.
  • the cell lysate is centrifuged at a low speed (500-3000 ⁇ 111) for a short time (typically about 1-10 minutes), and the supernatant is further centrifuged at a higher speed (15000-30000 rpm) for 30 minutes to 2 hours.
  • the resulting precipitate is used as the membrane fraction.
  • the membrane fraction is rich in the expressed receptor of the present invention and membrane components such as cell-derived phospholipids and membrane proteins.
  • the amount of the receptor of the present invention in the cell or membrane fraction containing the receptor of the present invention is preferably 10 3 to 10 8 molecules per cell, and more preferably 10 5 to 10 7 molecules per cell. It is suitable.
  • the receptor fraction of the present invention is preferably a natural receptor fraction of the present invention or a recombinant receptor fraction of the present invention having an activity equivalent thereto.
  • equivalent activity refers to equivalent ligand binding activity and the like.
  • the labeled polypeptide for example [3 ⁇ 4], [125 1], and the like can be used [14 C], C 35 S] labeled polypeptide or the like. Of these, a polypeptide labeled with [ 125 1] is preferable.
  • a cell containing the receptor of the present invention or a membrane fraction of the cell is used. Is prepared in a buffer suitable for screening to prepare a receptor preparation.
  • the buffer may be any buffer, such as a phosphate buffer having a pH of 4 to 10 (preferably pH 6 to 8) or a buffer such as a tris-monohydrochloride buffer, which does not inhibit the binding between the ligand and the receptor.
  • a surfactant such as CHAPS, Tween-80 TM (Kao-Atlas), digitonin, and dexcholate can be added to the buffer.
  • a protease inhibitor such as PMS F, leptin, E-64 (manufactured by Peptide Research Institute), or pepstatin is added to suppress the degradation of the receptor of the present invention or the polypeptide of the present invention by a protease.
  • One NSB obtained by subtracting the non-specific binding amount (NSB) from the count when there is no antagonist (B.) is defined as 100%, the specific binding amount (B—NSB) ) Is, for example, 50% or less. It is selected as auxiliary material Can be
  • the methods (d) to (e) for screening for a compound that changes the binding property of the polypeptide of the present invention to the receptor of the present invention to carry out the fine ⁇ intense activity mediated by the receptor of the present invention (e.g., Arakidon acid release, acetylcholine release, intracellular C a 2 + release, intracellular c AM P generated Z suppression, intracellular c GM P generated Inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, activation of c-fos, decrease in pH, activity to promote or inhibit GTPaS binding activity, etc.) It can be measured using a commercially available measurement kit.
  • cells containing the receptor of the present invention are cultured in a multiwell plate or the like. Before conducting screening, replace the cells with fresh medium or an appropriate buffer that is not toxic to cells, add test compounds, etc., incubate for a certain period of time, and then extract cells or collect supernatant.
  • the products produced are quantified according to the respective methods.
  • a substance for example, arachidonic acid
  • an inhibitor for the degrading enzyme may be added to perform the assay.
  • activities such as inhibition of cAMP production can be detected as production inhibiting effects on cells whose basic production has been increased with forskolin or the like.
  • cells expressing an appropriate receptor of the present invention are required.
  • the above-described cell lines expressing the receptor of the present invention and the like are desirable.
  • Test compounds include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and the like.
  • a screening kit for a compound that changes the binding property of the polypeptide of the present invention to the receptor of the present invention comprises the receptor of the present invention or the salt thereof.
  • Examples of the screening kit of the present invention include the following.
  • CHO cells expressing the receptor of the present invention were subcultured on a 12-well plate at 5 ⁇ 10 5 cells / well and cultured for 2 days in 37, 5% CO 2 , 95% air.
  • a solution of the polypeptide of the present invention labeled with [ ⁇ ], [I], [ 14C ], [], etc. in a suitable solvent or buffer solution is stored at 4 ° C or -20, and stored at the time of use. Dilute to 1 M with assay buffer.
  • polypeptide of the present invention is dissolved in P'BS containing 0.1% Pserum serum albumin (manufactured by Sigma) so as to be ImM, and stored at ⁇ 20 ° C.
  • a compound or a salt thereof obtained by using the above-described screening method or screening kit is a compound that alters (inhibits or promotes the binding) the binding between the polypeptide of the present invention and the receptor of the present invention (the present invention).
  • a compound which promotes or inhibits the activity of the polypeptide of the present invention specifically, a compound having a cell stimulating activity via the receptor of the present invention or a salt thereof (a so-called receptor agonist of the present invention), or The compound does not have the stimulating activity (so-called receptor antagonist of the present invention).
  • the compound include peptides, proteins, non-peptidic compounds, synthetic compounds, and fermentation products. These compounds may be novel compounds or known compounds.
  • the specific method of evaluating whether the receptor agonist of the present invention is an antagonist or an antagonist may be according to the following (i) or (ii).
  • the binding assay shown in the screening methods (a) to (c) is performed to change the binding between the polypeptide of the present invention and the receptor of the present invention (particularly, the binding is inhibited).
  • the compound having no activity or a salt thereof is the receptor antagonist of the present invention.
  • test compound is brought into contact with a cell containing the receptor of the present invention, and the cell stimulating activity via the receptor of the present invention is measured.
  • the compound having a cell stimulating activity or a salt thereof is the receptor agonist of the present invention.
  • a compound that activates the receptor of the present invention for example, the polypeptide of the present invention or the receptor agonist of the present invention
  • the cell stimulating activity mediated by the receptor of the present invention when a compound activating the receptor of the present invention and a test compound are brought into contact with cells containing the receptor of the present invention is measured and compared.
  • the compound capable of reducing the cell stimulating activity of the compound that activates the receptor of the present invention or a salt thereof is the receptor agonist of the present invention.
  • the receptor agonist of the present invention has the same activity as the physiological activity of the polypeptide of the present invention on the receptor of the present invention, it can be used as an antidiuretic like the polypeptide of the present invention.
  • urinary storage disorders eg, frequent urination, urinary incontinence (eg, urge incontinence, stress urinary incontinence, functional urinary incontinence, etc.) etc.
  • polyuria eg, diabetes insipidus ( Eg, pituitary diabetes insipidus, renal diabetes insipidus, etc.), hypernatremia, metabolic alkalosis, hypokalemia, Cushing syndrome, etc. It can be used as a preventive and therapeutic agent.
  • urinary incontinence eg, urge incontinence, stress urinary incontinence, functional urinary incontinence, etc.
  • polyuria e.g., a preterm evolution, a preterm evolution, a preterm evolution, a preterm evolution, a preterm evolution, a preterm evolution, etc.
  • diabetes insipidus e.g, pituitary insipidus, renal insipidus, etc.
  • hypernatremia eg, metabolic alkalosis, hypokalemia, Cushing's syndrome, etc.
  • the receptor antagonist of the present invention can suppress the physiological activity of the polypeptide of the present invention with respect to the receptor of the present invention, it is useful as a diuretic, and is safe and low toxic.
  • Edema urinary discharge disorder (eg, bladder contraction disorder, urethral passage disorder, difficulty urinating, urinary pain, urinary obstruction, etc.), hyponatremia, antidiuretic hormone secretion inadequate syndrome (SIADH), prevention of hypertension, etc.
  • SIADH antidiuretic hormone secretion inadequate syndrome
  • ⁇ Can be used as a therapeutic agent.
  • Compounds or salts thereof obtained using the screening method or screening kit of the present invention include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, and animals.
  • salt of the compound those similar to the aforementioned salts of the polypeptide of the present invention can be used. Used.
  • a compound obtained by using the screening method or the screening kit of the present invention is used as the above-mentioned prophylactic / therapeutic agent, it can be carried out in a conventional manner.
  • tablets, capsules, elixirs, microcapsules, sterile solutions, suspensions, and the like can be prepared in the same manner as the above-mentioned drug containing the polypeptide of the present invention.
  • the preparations obtained in this way are safe and low toxic and can be used, for example, in humans or in warm-blooded animals (eg, mice, rats, puppies, higgs, bushus, puppies, pumas, birds, cats, dogs). , Monkeys, chimpanzees, etc.).
  • the dose of the compound or a salt thereof varies depending on its action, target disease, subject to be administered, route of administration, and the like.For example, it promotes the activity of the polypeptide of the present invention for the treatment of diabetes insipidus.
  • the compound is administered subcutaneously, in general, for adults (per 60 kg body weight), about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1 to 50 mg of the compound is used per day. Administer 0-20 mg. In the case of other animals, the dose can be administered in terms of 60 kg.
  • a compound that inhibits the activity of the polypeptide of the present invention when administered subcutaneously for the purpose of treating renal edema, generally, in an adult (per 60 kg body weight), the compound is used in an amount of about 0.
  • the antibody of the present invention can specifically recognize the polypeptide or receptor of the present invention, it can be used for quantification of the polypeptide or receptor of the present invention in a test wave, particularly for quantification by sandwich immunoassay. Can be used.
  • the present invention is a.
  • one antibody is an antibody that recognizes the N-terminal of the polypeptide or the receptor of the present invention, and the other antibody is a C-terminal of the polypeptide or the receptor of the present invention. It is desirable that the antibody be reactive.
  • the polypeptide or the receptor of the present invention can be quantified using a monoclonal antibody against the polypeptide or the receptor of the present invention, and can also be detected by tissue staining or the like.
  • the antibody molecule itself may be used, or F (ab ') 2 , Fab', or Fab fraction of the antibody molecule may be used.
  • the method for quantifying the polypeptide or the receptor of the present invention using the antibody of the present invention is not particularly limited, and may be an antibody, an antigen or an antibody corresponding to the amount of an antigen (for example, the amount of a polypeptide) in a test solution. Any method that detects the amount of one antigen complex by chemical or physical means and calculates it from a standard curve prepared using a standard solution containing a known amount of antigen can be used. Is also good. For example, nephelometry, a competition method, an immunometric method and a sandwich method are preferably used, but the sandwich method described later is particularly preferable in terms of sensitivity and specificity.
  • a labeling agent used in a measurement method using a labeling substance for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance and the like are used.
  • the radioisotope e.g., [125 1], [131 1], [3 ⁇ 4], and the like are used [14 c].
  • the enzyme those which are stable and have a large specific activity are preferable.
  • 3-galactosidase, / 3-dalcosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used.
  • fluorescent substance for example, fluorescamine, fluorescein isothiosinate and the like are used.
  • luminescent substances include luminol, luminol derivatives, luciferin, lucigenin, etc. You can.
  • a biotin-avidin system can be used for binding the antibody or antigen to the labeling agent.
  • the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose; synthetic resins such as polystyrene, polyacrylamide, and silicon; and glass.
  • the test solution is reacted with the insoluble monoclonal antibody of the present invention (primary reaction), and further reacted with another labeled monoclonal antibody of the present invention (secondary reaction).
  • primary reaction the insoluble monoclonal antibody of the present invention
  • secondary reaction another labeled monoclonal antibody of the present invention
  • the primary reaction and the secondary reaction may be performed in the reverse order, may be performed simultaneously, or may be performed at staggered times.
  • the labeling agent and the method of insolubilization can be in accordance with those described above.
  • the antibody used for the solid phase antibody or the labeling antibody does not necessarily need to be one kind, and a mixture of two or more kinds of antibodies is used for the purpose of improving measurement sensitivity and the like. May be used.
  • the monoclonal antibody of the present invention used in the primary reaction and the secondary reaction an antibody having a different site to which the polypeptide of the present invention binds is preferably used. That is, the antibody used in the primary reaction and the secondary reaction is, for example, when the antibody used in the secondary reaction recognizes the C-terminal of the polypeptide of the present invention, the antibody used in the primary reaction is Preferably, an antibody that recognizes other than the C-terminal, for example, the N-terminal, is used.
  • the monoclonal antibody of the present invention can be used in a measurement system other than the sandwich method, for example, a competition method, an immunometric method, or a nephrometry method.
  • the antigen in the test solution and the labeled antigen are allowed to react competitively with the antibody, and then the unreacted labeled antigen (F) and the labeled antigen (B) bound to the antibody are separated.
  • BZF separation The amount of labeling of either B or F is measured, and the amount of antigen in the test wave is quantified.
  • B / F separation was performed using polyethylene as a soluble antibody.
  • Liquid phase method using a glycol, a second body against the above-mentioned body, and using an immobilized antibody as the first antibody, or using a soluble first antibody as the second antibody And an immobilization method using an antibody.
  • the antigen in the test solution and the immobilized antigen are subjected to a competitive reaction with a certain amount of the labeled antibody, and then the solid phase and the liquid phase are separated. Then, the unreacted labeled antibody is bound to the solid phase, and the solid phase and the liquid phase are separated. Next, the amount of the label in either phase is measured to determine the amount of the antigen in the test wave.
  • nephrometry the amount of insoluble sediment generated as a result of an antigen-antibody reaction in a gel or in a solution is measured. Even when the amount of antigen in the test solution is small and only a small amount of sediment is obtained, laser nephrometry utilizing laser scattering is preferably used.
  • the measurement system for the polypeptide of the present invention may be constructed by adding ordinary technical considerations of those skilled in the art to ordinary conditions and operation methods in each method. For details of these general technical means, reference can be made to reviews, written documents, and the like.
  • the polypeptide or receptor of the present invention can be quantified with high sensitivity by using the antibody of the present invention.
  • a decrease in the concentration of the polypeptide or the receptor of the present invention is detected by quantifying the concentration of the polypeptide or the receptor of the present invention using the antibody of the present invention, for example, a urinary storage disorder (eg, frequent urination) , Urinary incontinence (eg, urge incontinence, stress urinary incontinence, functional urinary incontinence, etc.), etc., polyuria, diabetes insipidus (eg, pituitary insipidus, renal insipidus, etc.) It can be diagnosed as a disease such as hypernatremia, metabolic alkalosis, hypokalemia, Cushing's syndrome, or is likely to be affected in the future.
  • a urinary storage disorder eg, frequent urination
  • Urinary incontinence eg, urge incontinence, stress urinary incontinence, functional urinary incontinence, etc.
  • polyuria eg, diabetes insipidus (eg,
  • renal edema e.g., renal contraction disorder, urethral passage disorder, dysuria, dysuria, urinary tract
  • Obesity e.g., hyponatremia, antidiuretic hormone inadequate syndrome (SIADH), hypertension, etc.
  • SIADH antidiuretic hormone inadequate syndrome
  • the antibody of the present invention can be used for detecting the polypeptide or the receptor of the present invention present in a subject such as a body fluid or a tissue. Further, preparation of an antibody column used for purifying the polypeptide or the receptor of the present invention, detection of the polypeptide or the receptor of the present invention in each fraction at the time of purification, and detection of the polypeptide or the receptor in the test cells For analysis of the behavior of the polypeptide or the receptor of the present invention.
  • the DNA of the present invention can be used as a probe, for example, in humans or warm-blooded animals (eg, rats, mice, guinea pigs, egrets, birds, higgies, pigs, pigs, dogs, cats, dogs, Abnormalities (genetic abnormalities) of the DNA or the mRNA encoding the polypeptide of the present invention in monkeys, etc.), for example, damage, mutation or reduced expression of the DNA or mRNA. Or a gene diagnostic agent for increasing or overexpressing the DNA or mRNA. It is useful.
  • the above-described genetic diagnosis using the DNA of the present invention can be performed, for example, by the known Northern hybridization or the PCR-SSCP method (Genomics, Vol. 5, pp. 874-879).
  • the expression of the polypeptide or receptor gene of the present invention when the expression of the polypeptide or receptor gene of the present invention is detected to be decreased by Northern hybridization, it may be, for example, a urinary storage disorder [eg, frequent urination, urinary incontinence (eg, urge incontinence) , Stress urinary incontinence, functional urinary incontinence, etc.), polyuria, diabetes insipidus (eg, pituitary diabetes insipidus, renal insipidus, etc.), hypernatremia, metabolic alkalosis, low It can be diagnosed that the disease is likely to be a disease such as kalemia or Cushing's syndrome, or is likely to be affected in the future.
  • a urinary storage disorder eg, frequent urination, urinary incontinence (eg, urge incontinence) , Stress urinary incontinence, functional urinary incontinence, etc.
  • polyuria eg, diabetes insipidus (e
  • the polypeptide or receptor gene of the present invention when overexpression of the polypeptide or receptor gene of the present invention is detected by Northern hybridization, for example, renal edema, urinary excretion disorder (eg, bladder contraction disorder, transurethral passage) Disorders, difficulty urinating, urinary pain, urinary tract obstruction, etc.), hyponatremia, anti-diuretic hormone secretion syndrome (SIADH), hypertension, etc. It can be diagnosed. (5) Pharmaceuticals containing antisense DNA
  • the antisense DNA of the present invention can be used as a diuretic and the like, for example, renal edema, urinary discharge disorder (eg, bladder contraction disorder, urethral passage disorder, dysuria, dysuria, urinary tract obstruction, etc. ), Hyponatremia, antidiuretic hormone secretion inadequate syndrome (SIADH), hypertension and other prophylactic and therapeutic agents can be used.
  • urinary discharge disorder eg, bladder contraction disorder, urethral passage disorder, dysuria, dysuria, urinary tract obstruction, etc.
  • Hyponatremia eg, antidiuretic hormone secretion inadequate syndrome (SIADH), hypertension and other prophylactic and therapeutic agents
  • SIADH antidiuretic hormone secretion inadequate syndrome
  • the antisense DNA when used, the antisense DNA may be used alone or after being inserted into an appropriate vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector, or the like, and then used in a conventional manner.
  • an appropriate vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector, or the like
  • the antisense DNA may be used as it is or together with a physiologically acceptable carrier such as an adjuvant to promote uptake. It can be formulated and administered via a catheter such as a gene gun or hydrogel catheter.
  • the antisense DNA can also be used as a diagnostic oligonucleotide probe for examining the presence and expression of the DNA of the present invention in tissues and cells.
  • the antibody of the present invention having the action of neutralizing the polypeptide of the present invention can be used, for example, as a diuretic.
  • a diuretic for example, renal edema, urinary excretion disorder (eg, bladder contraction disorder, urethral passage disorder, urination) It is useful as a prophylactic / therapeutic agent for hyponatremia, antidiuretic hormone inadequate syndrome (SIADH), hypertension, etc.
  • SIADH antidiuretic hormone inadequate syndrome
  • the agent for the above-mentioned diseases containing the antibody of the present invention can be used as it is as a liquid preparation or as a pharmaceutical composition in an appropriate dosage form, in humans or mammals (eg, rat, puppies, sheep, pigs, puppies, cats, cats). Can be administered orally or parenterally.
  • the dosage varies depending on the administration subject, target disease, symptoms, administration route, and the like.For example, when used for the treatment of renal edema in adults, the antibody of the present invention is usually administered in a single dose.
  • the antibodies of the present invention can be administered by themselves or as a suitable pharmaceutical composition.
  • the pharmaceutical composition used for the above administration contains the above or a salt thereof and a pharmacologically acceptable carrier, diluent or excipient.
  • Such compositions are provided in dosage forms suitable for oral or parenteral administration.
  • compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (soft capsules and the like). Including), syrup, emulsion, suspension Agents and the like.
  • Such a composition is produced by a known method and contains a carrier, diluent or excipient commonly used in the pharmaceutical field. For example, lactose, starch, sucrose, magnesium stearate and the like are used as carriers and excipients for tablets.
  • injections for example, injections, suppositories, etc. are used.
  • Injections are in the form of intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, drip injections, etc. Is included.
  • Such injections are prepared according to known methods, for example, by dissolving, suspending or emulsifying the antibody or a salt thereof in a sterile aqueous or oily solution usually used for injections.
  • aqueous solution for injection for example, physiological saline, isotonic solution containing glucose and other adjuvants, and the like, suitable solubilizing agents, for example, alcohol (eg, ethanol), polyalcohol (eg, Propylene glycol, polyethylene glycol), nonionic surfactants [eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduc t of hydrogenated castor oil)], etc.
  • suitable solubilizing agents for example, alcohol (eg, ethanol), polyalcohol (eg, Propylene glycol, polyethylene glycol), nonionic surfactants [eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduc t of hydrogenated castor oil)], etc.
  • oily liquid for example, sesame oil, soybean oil and the like are used, and benzyl benzoate, benzyl alcohol and the like may be used in combination as a solubilizing agent.
  • the above-mentioned oral or parenteral pharmaceutical composition is conveniently prepared in the form of a dosage unit so as to conform to the dose of the active ingredient.
  • the dosage unit include tablets, pills, capsules, injections (ampoules), suppositories, etc., and usually 5 to 500 mg, especially 5 to 100 mg for each dosage unit.
  • other dosage forms preferably contain 10 to 250 mg of the above antibody.
  • compositions may contain another active ingredient as long as the composition does not cause an undesirable interaction with the above-mentioned antibody.
  • DNA encoding an exogenous polypeptide or receptor of the present invention (hereinafter referred to as A non-human mammal having the exogenous DNA of the present invention or its mutant DNA (sometimes abbreviated as the foreign mutant DNA of the present invention) is also used for screening diuretics and the like.
  • Non-human mammals having the exogenous DNA of the present invention or the mutant DNA thereof include unfertilized eggs, fertilized eggs, germ cells including spermatozoa and their progenitor cells, and the like.
  • the calcium phosphate method, the electric pulse method, and the ribofection method It can be produced by transferring the desired DNA by a coagulation method, microinjection method, particle gun method, DEAE-dextran method or the like.
  • the exogenous DNA of the present invention can be transferred to somatic cells, organs of living organisms, tissue cells, and the like, and used for cell culture, tissue culture, and the like. Can be fused with the above-mentioned germinal cells by a known cell fusion method to produce the DNA transgenic animal of the present invention.
  • mice for example, porcupines, pigs, higgins, goats, magpies, dogs, cats, guinea pigs, hamsters, mice, rats and the like are used.
  • rodents that have relatively short ontogeny and biological cycles in terms of the creation of disease animal model systems and are easy to breed, especially mice (for example, pure strains such as C57BLZ6 and DBA2 strains, , BSCSFi strain, BDF 1 strain, BeDSFi strain, BALBZc strain, ICR strain, etc.) or rat (for example, Wistar, SD etc.) is preferable.
  • “Mammals” in a recombinant vector that can be expressed in mammals include humans and the like in addition to the above-mentioned non-human mammals.
  • the exogenous DNA of the present invention is not the DNA of the present invention originally possessed by a non-human mammal, but refers to the DNA of the present invention once isolated and extracted from a mammal.
  • Examples of the mutant DNA of the present invention include those having a mutation (for example, mutation) in the base sequence of the original DNA of the present invention, specifically, addition or deletion of bases, or other bases.
  • a DNA having substitution with a DNA is used, and an abnormal DNA is also included.
  • the abnormal DNA means a DNA that expresses an abnormal polypeptide or receptor of the present invention, and for example, expresses a polypeptide that suppresses the function of the normal polypeptide or receptor of the present invention. DNA or the like is used.
  • the exogenous DNA of the present invention may be derived from a mammal that is the same or different from the animal of interest.
  • the human DNA of the present invention when transferred, it is derived from various mammals (eg, egrets, dogs, cats, guinea pigs, hamsters, rats, mice, etc.) having the DNA of the present invention having high homology thereto.
  • a DNA construct eg, a vector, etc.
  • a DNA construct comprising the human DNA of the present invention downstream of various promoters capable of expressing the DNA of the present invention into a fertilized egg of a target mammal, for example, a mouse fertilized egg.
  • a DNA-transferred mammal that highly expresses the DNA of the present invention can be created.
  • expression vectors for the polypeptide or receptor of the present invention include plasmids derived from Escherichia coli, plasmids derived from Bacillus subtilis, plasmids derived from yeast, bacteriophages such as phage ⁇ , retroviruses such as Moroni monoleukemia virus, and xinia viruses. Alternatively, animal viruses such as baculovirus are used. Among them, a plasmid derived from Escherichia coli, a plasmid derived from Bacillus subtilis or a plasmid derived from yeast are preferably used.
  • Examples of the promoter that regulates DNA expression include: (a) DNA promoter derived from virus (eg, Simian virus, cytomegalovirus, Moroni monoleukemia virus, JC virus, breast cancer virus, poliovirus, etc.). Motors, (b) Promoters from various mammals (humans, egrets, dogs, cats, guinea pigs, hamsters, rats, mice, etc.), such as albumin, insulin II, perobrakin II, elastase, erythropoietin, Endothelin, muscle creatine kinase, glial fibrillary acidic protein, dalyuthion S-transferase, platelet-derived growth factor j3, keratin K1,10, W 200
  • Collagen type I and type II cyclic AMP-dependent protein kinase / 31 subunit, dystrophin, tartrate-resistant alkaline phosphatase, atrial natriuretic factor, endothelial receptor thymic synkinase (generally abbreviated as Tie.2) ), Sodium potassium adenosine 3-phosphatase (Na, K-ATPase), neurofilament light chain, meta-oral thionein I and II, A, meta-oral proteinase 1 tissue inhibitor, MHC class II (H—2L) ), H-ras, renin, dopamine j8-hydroxylase, thyroid peroxidase (TPO), polypeptide chain elongation factor 1 (EF-1), ⁇ -actin, a and / 3 myosin heavy chain, myosin Light chains 1 and 2, myelin basic protein, thyroglobulin, Thy-1, immunoglobulin, heavy chain variable region (VNP
  • the vector preferably has a sequence that terminates transcription of the messenger RNA of interest in the DNA-transferred mammal (generally referred to as "Yuichi Mineta-1").
  • a sequence of each DNA can be used, and preferably, SV40 terminator of simian virus or the like is used.
  • the splicing signal of each DNA, the enhancer region, a part of the intron of eukaryotic DNA, etc. are placed 5 'upstream of the promoter region, between the promoter region and the translation region, in order to further express the target exogenous DNA.
  • it can be connected to the 3 'downstream of the translation area depending on the purpose.
  • the normal translation region of the polypeptide or receptor of the present invention may be a liver, kidney, thyroid cell, or the like derived from humans or various mammals (eg, egrets, dogs, cats, guinea pigs, hamsters, rats, mice, etc.). All or part of genomic DNA from fibroblast-derived DNA and various commercially available genomic DNA libraries, or by known methods from liver, kidney, thyroid cells, or fibroblast-derived RNA The prepared complementary DNA can be obtained as a raw material.
  • the exogenous abnormal DNA can produce a translation region obtained by mutating the translation region of a normal polypeptide obtained from the above cells or tissues by point mutagenesis.
  • the translation region can be prepared as a DNA construct that can be expressed in a transposed animal by a conventional DNA engineering technique in which it is ligated downstream of the above-mentioned promoter and, if desired, upstream of the transcription termination site.
  • Transfer of the exogenous DNA of the present invention at the fertilized egg cell stage is ensured to be present in all germ cells and somatic cells of the target mammal.
  • the presence of the exogenous DNA of the present invention in the germ cells of the produced animal after DNA transfer means that the progeny of the produced animal retains the exogenous DNA of the present invention in all of its germ cells and somatic cells Means to do.
  • the progeny of such an animal that inherits the exogenous DNA of the present invention has the exogenous DNA of the present invention in all of its germ cells and somatic cells.
  • the non-human mammal to which the exogenous normal DNA of the present invention has been transferred is confirmed to stably retain the exogenous DNA by mating, and is subcultured as an animal having the DNA in a normal breeding environment. You can do it.
  • Transfer of the exogenous DNA of the present invention at the fertilized egg cell stage is ensured to be present in excess in all germinal and somatic cells of the target mammal.
  • Excessive presence of the exogenous DNA of the present invention in the germinal cells of the animal after transfer of DNA indicates that the offspring of the animal in which the foreign DNA of the present invention is present in excess of the foreign DNA of the present invention in all of its germinal and somatic cells. Means to have. Progeny of this type of animal that has inherited the exogenous DNA of the present invention have an excess of the exogenous DNA of the present invention in all of its germinal and somatic cells.
  • the non-human mammal having the normal DNA of the present invention expresses the normal DNA of the present invention at a high level, and eventually promotes the function of endogenous normal DNA to thereby finally produce the polypeptide of the present invention.
  • Hyperfunction may develop, and its pathological model It can be used as a thing. For example, using the normal DNA-transferred animal of the present invention, elucidation of the hyperfunction of the polypeptide of the present invention and the pathological mechanism of diseases associated with the polypeptide of the present invention, and examination of treatment methods for these diseases. It is possible to do so.
  • the non-human mammal having the foreign abnormal DNA of the present invention should be subcultured in a normal breeding environment as an animal having the DNA after confirming that the foreign DNA is stably retained by the crossing. Can be done. Furthermore, the desired foreign DNA can be incorporated into the above-mentioned plasmid and used as a raw material.
  • a DNA construct with a promoter can be prepared by ordinary DNA engineering techniques. The transfer of the abnormal DNA of the present invention at the fertilized egg cell stage is ensured to be present in all germ cells and somatic cells of the target mammal.
  • the presence of the abnormal DNA of the present invention in the germinal cells of the animal produced after transfer of the DNA means that the offspring of the animal produced have the abnormal DNA of the present invention in all of the germinal and somatic cells.
  • the progeny of this type of animal that has inherited the exogenous DNA of the present invention has the abnormal DNA of the present invention in all of its germinal and somatic cells.
  • the non-human mammal having the abnormal DNA of the present invention expresses the abnormal DNA of the present invention at a high level, and finally inhibits the function of the endogenous normal DNA to thereby finally produce the polypeptide of the present invention. In some cases, it becomes functionally inactive refractory, and can be used as a model animal for the disease. For example, it is possible to elucidate the pathological mechanism of a functionally inactive refractory state of the polypeptide of the present invention and to examine a method for treating this disease using the abnormal DNA transgenic animal of the present invention.
  • the abnormal DNA-highly expressing animal of the present invention can be used for the abnormal polypeptide of the present invention in the function-inactive refractory disease of the polypeptide of the present invention.
  • the mammal into which the foreign abnormal DNA of the present invention has been transferred has an increased symptom of the released polypeptide of the present invention
  • the therapeutic agent for the polypeptide of the present invention or a functionally inactive refractory disease thereof is used. It can also be used for screening tests.
  • other possible uses of the above two types of DNA transgenic animals of the present invention include, for example,
  • the DNA-transferred animal of the present invention it is possible to examine the clinical symptoms of a disease associated with the polypeptide or the receptor of the present invention, including the inactive refractory type of the polypeptide of the present invention, etc.
  • a more detailed pathological finding in each organ of the disease model related to the polypeptide or receptor of the present invention can be obtained, a new treatment method can be developed, and a secondary disease caused by the disease can be studied. And contribute to treatment.
  • the present invention ⁇ ) It is possible to take out each organ from a DNA-transferred animal, cut it into small pieces, and then use a protease such as trypsin to obtain free DNA-transferred cells, culture them, or systematize the cultured cells. It is possible. Furthermore, the specification of the polypeptide or receptor-producing cells of the present invention, the relationship with apoptosis, differentiation or proliferation, or the signal transduction mechanism therein, and their abnormalities are examined. Thus, it becomes an effective research material for elucidating the polypeptide or receptor of the present invention and its action.
  • a protease such as trypsin
  • a therapeutic agent for a disease associated with the polypeptide or the receptor of the present invention including the inactive refractory type of the polypeptide or the receptor of the present invention, is developed. Therefore, it is possible to provide an effective and rapid screening method for the therapeutic agent for the disease by using the above-mentioned test method and quantitative method. Further, using the DNA transgenic animal of the present invention or the exogenous DNA expression vector of the present invention, it is possible to examine and develop a method for treating a DNA associated with the polypeptide of the present invention.
  • the non-human mammalian embryonic stem cells in which the DNA of the present invention has been inactivated and the non-human mammal deficient in the expression of the DNA of the present invention are also used for screening an antidiuretic agent.
  • the non-human mammalian embryonic stem cells in which the DNA of the present invention has been inactivated refers to whether the DNA of the present invention possessed by the non-human mammal is artificially mutated to suppress the DNA expression ability.
  • the DNA substantially does not have the ability to express the polypeptide of the present invention (hereinafter, the present invention Knockout DNA) (referred to as ES cells) of a non-human mammal embryo.
  • the non-human mammal the same one as described above is used.
  • the method of artificially mutating the DNA of the present invention can be performed, for example, by deleting a part or all of the DNA sequence and inserting or substituting another DNA by a genetic engineering technique.
  • the knockout DNA of the present invention may be produced by, for example, shifting the reading frame of a codon or disrupting the function of a promoter or exon by these mutations.
  • non-human mammalian embryonic stem cells in which the DNA of the present invention has been inactivated include, for example, The DNA of the present invention possessed by a non-human mammal And its exon portion is represented by the neomycin resistance gene, the drug resistance gene represented by the hygromycin resistance gene, or lac Z (3-galactosidase-ze gene) and cat (chloramphenico-l-acetyltransferase gene)
  • the exon function may be disrupted by inserting a repo overnight gene, or a DNA sequence that terminates gene transcription (for example, a polyA addition signal) may be inserted into the introns between the exons.
  • a DNA chain (hereinafter abbreviated as targeting vector 1) having a DNA sequence constructed so as to disrupt the gene by disabling the synthesis of a novel messenger RNA, for example, by homologous recombination.
  • the resulting ES cells are transferred to or near the DNA of the present invention.
  • PCR using as primers the DNA sequence of the neighboring region other than the DNA sequence of the present invention used for Southern hybridization analysis or targeting vector preparation and targeting vector preparation It can be obtained by selecting the knockout ES cells of the present invention by analysis according to the method.
  • the original ES cells for inactivating the DNA of the present invention by the homologous recombination method or the like for example, those already established as described above may be used, and the method of the known Evans and Kaufma may be used. It may be newly established according to. For example, in the case of mouse ES cells, currently, 129 ES cells are generally used, but since the immunological background is not clear, it is an alternative pure immunological and genetic background.
  • BDFi mice C57BLZ6 and DBA / 2 BDI ⁇ mice can be used satisfactorily because they have a high number of eggs collected and their eggs are robust, and they have C57BLZ6 mice as their background.
  • the ES cells obtained as described above can be advantageously used when a pathological model mouse is produced, because the genetic background can be replaced by C57BLZ6 mice by backcrossing with C57BLZ6 mice.
  • blastocysts 3.5 days after fertilization are generally used, but it is also effective to collect 8-cell stage embryos and culture them to blastocysts. A large number of early embryos can be obtained efficiently.
  • male ES cells are generally more convenient for producing a germ line chimera. It is also desirable to discriminate between males and females as soon as possible in order to reduce the complexity of culturing.
  • An example of a method for determining the sex of ES cells is a method of amplifying and detecting a gene in the sex-determining region on the Y chromosome by PCR.
  • this method conventionally, for example G-banding method, requires about 10 6 cells for karyotype analysis, than suffices ES cell number of about 1 colony (about 50),
  • the primary selection of ES cells in the early stage of culture can be performed by discriminating between male and female, and the early stage of culture can be greatly reduced by enabling early selection of male cells.
  • Embryonic stem cell lines obtained in this way usually have very good proliferative potential, but must be carefully subcultured because they tend to lose their ontogenetic potential.
  • a suitable feeder cell such as STO fibroblast
  • a carbon dioxide incubator preferably 5% carbon dioxide
  • trypsin ZEDTA solution usually 0.001 to 0.5% trypsin Z0.
  • a single cell is obtained by treatment with 5 mM EDTA (preferably about 0.1% trypsin ZlmM EDTA), and the cells are seeded on a freshly prepared feeder cell.
  • Such subculture is usually performed every 1 to 3 days. At this time, it is desirable to observe the cells, and if morphologically abnormal cells are found, discard the cultured cells.
  • ES cells can be cultured in a monolayer up to high density, or in suspension culture until cell clumps are formed, under appropriate conditions, for example, parietal muscle, visceral muscle, myocardium, etc. [MJ Evans and MH Kaufman, Nature 292, 154, 1981; GR Martin Proc. Natl. Acad. Sci. USA 78, 7634; 1981; TC Doetschman et al., Journal of Embryology and Experimental Morphology, Vol. 87, p. 27, 1985], DNA of the present invention obtained by differentiating the ES cell of the present invention. Expression-deficient cells are useful in in vitro cell biology studies of the polypeptides of the invention or the receptors of the invention.
  • the non-human mammal deficient in DNA expression of the present invention can be distinguished from a normal animal by measuring the mRNA amount of the animal using a known method and indirectly comparing the expression level. . .
  • non-human mammal those similar to the above can be used.
  • the non-human mammal deficient in expression of the DNA of the present invention can be obtained, for example, by introducing the evening-getting vector prepared as described above into a mouse embryonic stem cell or a mouse egg cell, and introducing the DNA of the evening-getting vector of the present invention. Knocking out the DNA of the present invention by homologous recombination of the inactivated DNA sequence with the DNA of the present invention on the chromosome of mouse embryonic stem cells or mouse egg cells by gene homologous recombination Can be.
  • the cells in which the DNA of the present invention has been knocked out can be obtained by combining the DNA sequence on the DNA of the present invention or a DNA sequence on a nearby DNA with a DNA sequence on or near the DNA, and a DNA sequence on a vector.
  • the DNA can be determined by PCR analysis using the DNA sequence of a mouse-derived neighboring region other than the DNA sequence of the present invention as a primer.
  • a cell line in which the DNA of the present invention has been inactivated is cloned by homologous gene recombination, and the cell is cloned at an appropriate time, for example, at the 8-cell stage.
  • the chimeric embryo is injected into a non-human mammal embryo or blastocyst, and the resulting chimeric embryo is transplanted into the uterus of the pseudo-pregnant non-human mammal.
  • the produced animal is a chimeric animal composed of both a cell having a normal DNA locus of the present invention and a cell having an artificially mutated DNA locus of the present invention.
  • the individual obtained in this manner is usually an individual lacking heterologous expression of the polypeptide of the present invention, and mated with individuals lacking heterologous expression of the polypeptide of the present invention or the receptor of the present invention.
  • An individual deficient in homoexpression of the polypeptide of the present invention or the receptor of the present invention can be obtained from the offspring.
  • a transgenic non-human mammal having a chromosome into which a gettering vector has been introduced can be obtained by injecting a DNA solution into the nucleus of an egg by a microinjection method. It can be obtained by selecting a gene having a mutation in the DNA locus of the present invention by gene homologous recombination as compared with a transgenic non-human mammal.
  • the germline can be obtained and maintained according to a conventional method. That is, by crossing male and female animals having the inactivated DNA, a homozygous animal having the inactivated DNA on both homologous chromosomes can be obtained. The obtained homozygous animal can be efficiently obtained by rearing the mother animal in such a manner that one normal individual and a plurality of homozygous animals are obtained. By crossing male and female heterozygous animals, homozygous and heterozygous animals having the inactivated DNA are bred and subcultured.
  • the non-human mammalian embryonic stem cells in which the DNA of the present invention has been inactivated are extremely useful for producing the non-human mammal deficient in expression of the DNA of the present invention.
  • the non-human mammal deficient in DNA expression of the present invention lacks various biological activities that can be induced by the polypeptide of the present invention or the receptor of the present invention. It can be used as a model for diseases caused by inactivation of the biological activity of the receptor of the present invention, and is useful for investigating the causes of these diseases and studying treatment methods.
  • the non-human mammal deficient in DNA expression of the present invention can be used for screening for a compound having a therapeutic / preventive effect against diseases caused by DNA deficiency or damage of the present invention.
  • the present invention comprises administering a test compound to a non-human mammal deficient in expression of the DNA of the present invention, and observing and measuring changes in the animal.
  • a method for screening a compound or a salt thereof having a therapeutic or preventive effect on a disease caused by the disease is provided.
  • Examples of the non-human mammal deficient in DNA expression of the present invention used in the screening method include those described above.
  • Test compounds include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and plasma.These compounds are novel compounds. Or a known compound.
  • a non-human mammal deficient in expression of the DNA of the present invention is treated with a test compound and compared with a non-treated control animal, and the change in each organ, tissue, symptom of disease or the like of the animal is used as an index.
  • the therapeutic and prophylactic effects of the test compound can be tested.
  • Methods for treating a test animal with a test compound include, for example, oral administration and intravenous injection, which are appropriately selected according to the symptoms of the test animal, properties of the test compound, and the like.
  • the dose of the test compound can be appropriately selected according to the administration method, properties of the test compound, and the like.
  • a non-human mammal deficient in expression of the DNA of the present invention is subjected to a drinking water treatment, and a test compound is administered before or after the drinking water treatment, and the urine output of the animal is changed. Is measured over time.
  • the urine output of the test animal decreases by about 10% or more, preferably about 30% or more, more preferably about 50% or more.
  • Therapeutic and prophylactic effects of compounds on the above diseases Can be selected as the compound having
  • the compound obtained by using the screening method is a compound selected from the test compounds described above, and has a therapeutic / preventive effect against a disease caused by deficiency or damage of the polypeptide of the present invention. It can be used as a safe and low toxic preventive and therapeutic agent for the disease. Furthermore, compounds derived from the compounds obtained by the above screening can be used in the same manner.
  • the compound obtained by the screening method may form a salt.
  • the salt of the compound include physiologically acceptable acids (eg, inorganic acids, organic acids, etc.) and bases (eg, And the like, and salts with alkali metals and the like are used, and especially preferred are physiologically acceptable acid addition salts.
  • salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.) and organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid) Succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, etc.).
  • inorganic acids eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.
  • organic acids eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid
  • Succinic acid tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, etc.
  • a medicament containing the compound or a salt thereof obtained by the screening method can be produced in the same manner as the aforementioned medicament containing the polypeptide of the present invention.
  • the preparations obtained in this way are safe and of low toxicity and can be used, for example, in humans or mammals (eg, rats, mice, guinea pigs, egrets, sheep, pigs, pigs, dogs, cats, dogs). , Monkeys, etc.).
  • the dose of the compound or a salt thereof varies depending on the target disease, the administration subject, the administration route, and the like.
  • the compound when administered subcutaneously, it is generally used in an adult patient (assuming a body weight of 60 kg). About 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg of the compound is administered per day.
  • the single dose of the compound when administered parenterally, the single dose of the compound varies depending on the administration subject, target disease, and the like.
  • the compound of the present invention that promotes promoter-one activity on DNA may be in the form of an injection.
  • the compound when administered to an adult (with a body weight of 60 kg) anorexia nervosa patient, the compound is administered in an amount of about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 30 mg per day. It is convenient to administer about 0.1 to about 10 mg by intravenous injection. In the case of other animals, the dose can be administered in terms of weight per 60 kg.
  • the present invention provides a non-human mammal deficient in expression of the DNA of the present invention, which comprises administering a test compound and detecting the expression of a repo overnight gene. To provide a method for screening a compound or a salt thereof that promotes or inhibits cell growth.
  • the non-human mammal deficient in expression of the DNA of the present invention includes, among the aforementioned non-human mammals deficient in the expression of the DNA of the present invention, the DNA of the present invention obtained by introducing a repo overnight gene. Those inactivated and capable of expressing the repo overnight gene under the control of the promoter for the DNA of the present invention are used.
  • test compound examples include the same compounds as described above.
  • reporter gene the same as those described above are used, / 3-galactosidase gene (1 a c Z), soluble alkaline phosphatase gene or luciferase gene and the like.
  • the polypeptide of the present invention when a part of the DNA region encoding the polypeptide of the present invention is replaced with a 3-galactosidase gene (1acZ) derived from Escherichia coli, the polypeptide of the present invention is originally expressed. In tissues, j3-galactosidase is expressed instead of the polypeptide of the invention.
  • a reagent that is a substrate for 3-galactosidase such as 5-bromo-4-monocloth-3-indolyl / 3-galactopyranoside (X-gal) is more convenient.
  • the expression state of the polypeptide of the present invention in an animal body can be easily observed.
  • the polypeptide-deficient mouse of the present invention or a tissue section thereof is treated with daltaraldehyde.
  • PBS phosphate-buffered saline
  • X-gal staining solution containing X-gal at room temperature or around 37 ° C for about 30 minutes to 1 hour.
  • the ⁇ -galactosidase reaction can be stopped by washing the sample with an ImM EDT AZPBS solution, and the color can be observed.
  • mRNA encoding 1 ac ⁇ ⁇ may be detected according to a conventional method.
  • the compound or a salt thereof obtained by the above-mentioned screening method is a compound selected from the above-mentioned test compounds, and is a compound that promotes or inhibits the promoter activity for DNA of the present invention.
  • the compound obtained by the screening method may form a salt.
  • the salt of the compound include physiologically acceptable acids (eg, inorganic acids) and bases (eg, organic acids). And the like, and a physiologically acceptable acid addition salt is particularly preferable.
  • such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.) and organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid) , Succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, etc.).
  • inorganic acids eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.
  • organic acids eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid
  • Succinic acid tart
  • the compound or a salt thereof that promotes the promoter activity for the DNA of the present invention can promote the expression of the polypeptide or the receptor of the present invention and promote the function of the polypeptide or the receptor. It is useful as an antidiuretic, etc., for example, urinary storage disorders [eg, frequent urination, urinary incontinence (eg, urge incontinence, stress urinary incontinence, functional urinary incontinence, etc.)], polyuria, diabetes insipidus ( Eg, pituitary diabetes insipidus, renal diabetes insipidus, etc.), hypernatremia, metabolic alkalosis, hypokalemia,
  • urinary storage disorders eg, frequent urination, urinary incontinence (eg, urge incontinence, stress urinary incontinence, functional urinary incontinence, etc.)
  • polyuria eg, diabetes insipidus ( Eg, pituitary diabetes insipidus, renal diabetes ins
  • It can be used as an agent for preventing and treating Cushing's syndrome.
  • the compound of the present invention or a salt thereof that inhibits the promoter activity on DNA can inhibit the expression of the polypeptide or receptor of the present invention and inhibit the function of the polypeptide or receptor.
  • a diuretic for example, for example, renal edema, urinary drainage disorder (eg, bladder contraction disorder, urethral passage disorder, difficulty urinating, urinary pain, urinary tract obstruction, etc.), hyponatremia, antidiuretic hormone It can be used as a preventive and remedy for secretory improper syndrome (SIADH), hypertension, etc.
  • SIADH secretory improper syndrome
  • a compound derived from the compound obtained by the above screening can be used in the same manner.
  • a drug containing the compound or a salt thereof obtained by the screening method can be produced in the same manner as the above-mentioned drug containing the polypeptide of the present invention or a salt thereof.
  • the preparations obtained in this way are safe and of low toxicity and can be used, for example, in humans or mammals (eg, rats, mice, guinea pigs, egrets, sheep, pigs, pigs, dogs, cats, dogs). , Monkeys, etc.).
  • the dose of the compound or a salt thereof varies depending on the target disease, the administration subject, the administration route, and the like.
  • the compound of the present invention which promotes the promoter activity against DNA is administered orally
  • the single dose of the compound varies depending on the administration subject, target disease and the like.
  • the compound for promoting the promoter activity for DNA of the present invention is usually in the form of an injection.
  • the compound When administered to an adult patient (assuming 60 kg), the compound may be administered intravenously at a dose of about O.Ql to about 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day. It is convenient to administer by injection. For other animals, the equivalent amount per 60 kg can be administered.
  • the compound of the present invention that inhibits the promoter activity for DNA is orally administered, generally, in an adult patient (with a body weight of 60 kg), the compound is used in an amount of about 0.1 to 100 mg per day, preferably About 1.0-50 mg, more preferably about
  • the single dose of the compound may vary depending on the administration subject, target disease, etc.
  • the compound of the present invention which inhibits the promoter activity on DNA against DNA is usually in the form of an injection.
  • the compound of the present invention which inhibits the promoter activity on DNA against DNA is usually in the form of an injection.
  • about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg of the compound per day is injected intravenously. It is convenient to administer the drug by. In the case of other animals, it is possible to administer the equivalent amount per 60 kg. Include.
  • the non-human mammal deficient in expression of the DNA of the present invention is extremely useful for screening a compound or a salt thereof that promotes or inhibits the activity of the promoter for the DNA of the present invention. It can greatly contribute to the investigation of the causes of various diseases caused by insufficient DNA expression or the development of therapeutic drugs.
  • genes encoding various proteins are ligated downstream thereof and injected into an egg cell of an animal to produce a so-called transgenic animal (gene transfer). Animal), it is possible to specifically synthesize the polypeptide and examine its effects in the living body. Furthermore, by binding an appropriate repo overnight gene to the above promoter portion and establishing a cell line in which this is expressed, the polypeptide of the present invention has an action of specifically promoting or suppressing the in vivo production ability of the polypeptide itself. It can be used as a search system for low molecular weight compounds.
  • bases, amino acids, and the like are indicated by abbreviations based on the abbreviations by IUPAC-IUB Communication on Biochemical Nomenclature or commonly used abbreviations in the art, and examples thereof are described below.
  • amino acids can have optical isomers, the L-form is indicated unless otherwise specified.
  • Y Thymine (T) or cytosine (C)
  • M Adenine (A) or cytosine (C) K guanine (G) or thymine (T)
  • B cytosine (C), guanine (G) or thymine (T) D adenine (A), guanine (G) or thymine (T) V adenine (A), guanine (G) or cytosine (C) N adenine ( A), guanine (G), cytosine (C) or thymine (T) or unknown or other base
  • BSA serum CHAPS 3— [(3-colamidopropyl) dimethyl ammonium]
  • H is or H histidine
  • FIG. 2 shows the amino acid sequence of a part of the human GPR8 ligand peptide precursor.
  • FIG. 2 shows the amino acid sequence of a synthetic human GPR8 ligand (1-29).
  • Fig. 3 shows the amino acid sequence of a synthetic human GPR8 ligand (1-27).
  • Fig. 3 shows the amino acid sequence of a synthetic human GPR8 ligand (1-26).
  • 1 shows the amino acid sequence of a synthetic human GPR8 ligand (1-25).
  • FIG. 2 shows the amino acid sequence of a human GPR8 ligand peptide precursor.
  • FIG. 2 shows the amino acid sequence of a GPR8 ligand peptide.
  • FIG. 2 shows the amino acid sequence of a GPR8 ligand peptide.
  • FIG. 2 shows the amino acid sequence of a GPR8 ligand peptide.
  • FIG. 2 shows the sequence of the cDNA encoding the rat GPR8 ligand peptide precursor.
  • FIG. 4 shows the amino acid sequence of rat GPR8 ligand peptide precursor.
  • FIG. 2 shows the amino acid sequence of rat GPR8 ligand peptide.
  • FIG. 2 shows the amino acid sequence of rat GPR8 ligand peptide.
  • FIG. 2 shows the amino acid sequence of a mouse GPR8 ligand peptide precursor.
  • FIG. 2 shows the amino acid sequence of a mouse GPR8 ligand peptide.
  • FIG. 2 shows the amino acid sequence of a mouse GPR8 ligand peptide.
  • FIG. 2 shows the amino acid sequence of an oxidized synthetic human GPR8 ligand (1-23).
  • 1 shows the amino acid sequence of a synthetic human GPR8 ligand (1-18).
  • 1 shows the amino acid sequence of a synthetic human GPR8 ligand (1-17).
  • 1 shows the amino acid sequence of a synthetic human GPR8 ligand (1-16).
  • FIG. 2 shows the amino acid sequence of a synthetic Fmoc-modified human GPR8 ligand (1-23).
  • FIG. 2 shows the amino acid sequence of a synthetic human GPR8 ligand (2-23).
  • Fig. 3 shows the amino acid sequence of a synthetic human GPR8 ligand (4-23).
  • [SEQ ID NO: 56] Shows the amino acid sequence of synthetic [N_Acetyrant Tyr 2 ] -human GPR8 ligand (2-23).
  • 1 shows the nucleotide sequence of cDNA encoding rat TGR26.
  • 1 shows the nucleotide sequence encoding human GPR7.
  • ⁇ Shows the nucleotide sequence of the 5 'upstream of cDNA encoding GPR8.
  • ⁇ Shows the nucleotide sequence of the 5 'upstream of cDNA encoding GPR7.
  • ⁇ Egret Shows the nucleotide sequence of the 5 'upstream of cDNA encoding GPR8 ligand peptide precursor protein.
  • ⁇ Egret Shows the nucleotide sequence of the 3 'downstream of cDNA encoding GPR8 ligand peptide precursor protein.
  • ⁇ Shows the nucleotide sequence containing the cDNA coding for the heron GPR8 ligand peptide precursor protein.
  • ⁇ Shows the nucleotide sequence containing the cDNA coding for the heron GPR8 ligand peptide precursor protein.
  • Escherichia coli DH5 ⁇ / ⁇ rab-rabbit GPR7 obtained in Example 3 described below has been obtained from September 25, 2003 at 1-1-1, Higashi 1-chome, Tsukuba-shi, Ibaraki Prefecture. Deposited at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology under the accession number FERM BP-8496.
  • Escherichia coli DH5 ⁇ / ⁇ -rbbit prepro-NPW obtained in Example 4 described later has been located at 1-1 1-1 Higashi, Tsukuba City, Ibaraki Prefecture since September 25, 2003.
  • the present invention will be described in more detail with reference to Reference Examples and Examples, but these examples do not limit the scope of the present invention.
  • the protected peptide resin was dried. Deprotection of the resulting protected peptide and separation of the peptide from the resin carrier were performed by TFA treatment. The obtained crude peptide was extracted with 0.1% TFA water and freeze-dried to obtain a powder solid. Subsequently, the crude peptide was subjected to preparative purification by reversed-phase high-performance chromatography (Shimadzu Corporation, preparative instrument: model LC8A) using acetonitrile-0.1% TFATK system (15-35%, 80 min). Thus, 35 mg of the purified peptide to be obtained was obtained.
  • the amino acid analysis value of the hydrolyzate obtained by hydrolyzing the purified product with 4N methanesulfonic acid containing 0.2% 3- (2-aminoethyl) indole at 110 ° C for 22 hours is as follows. Value) as follows.
  • the column used was 0DS-80 ⁇ (4.6 thigh ⁇ 15 cm) (Tosoh, one company), 10% acetonitrile / 0.1% TFA as eluent A, and 60% acetonitrile / 0.1% TFA as eluate B.
  • the daradient elution method of -0 (2 min), 0-27 (5 min), 27-32 (40 min)% B / A + B was performed.
  • the flow rate was 1 mL / min
  • the column temperature was 40 ° C
  • the detection was 215 nm.
  • DPhe 2, 125 I-Tyr 10] human GPR8 ligand (1-20) was eluted around 25 minutes.
  • Urethane ethyl carbamate
  • a ureteral catheter was inserted into both ureters, and arterial blood pressure, heart rate and urine volume were measured using Polygraph 363.
  • Human GPR8 ligand (1-23) (SEQ ID NO: 1) (sometimes described as hNPW23) prepared in the same manner as described in WO 01/98494 is a physiological saline (Otsuka Pharmaceutical) And doses were set at 12.5, 25, 50 and 100 mnol / kg. Physiological saline was administered to the control group, and the treatment was performed in 5 cases in each group. The urine volume before and after administration of hNPW23 was measured for 4 minutes, and the F-test was performed using the ratio (%) of the change in urine volume before and after administration.
  • hNPW23 had no effect on blood pressure or heart rate at any dose.
  • TotalTotal RNA of whole egret brain was purchased from UNITECH. After preparing the Poly (A) + RNA fraction using the mRNA purification kit (Amersham Biosciences), manually prepare 1.0 g of poly (A) + RNA from the egret whole brain using PowerScript Reverse Transcriptase (Clontech). After reverse transcription was performed according to the above, a type II, egret whole brain double-stranded cDNA for 5, -RACE was prepared using a Marathon cDNA Amplification kit (Clontech).
  • the primer sets used for the RACE PCR reaction were Adapter Primer 1 and Primer 1 (SEQ ID NO: 89) attached to the kit for the first PCR reaction, and Nested Adapter Primer 2 and Primer 2 ( SEQ ID NO: 90) was used.
  • the reaction was performed in the form of ⁇ with a volume of 50 ⁇ 1 for 2 ng of mRNA of reverse transcribed cDNA.
  • the composition of the reaction solution was primer concentration 0.2 M, dNTP mixture 0.2 mM, LA-Taq (Takara Shuzo) 1/100 volume, and 2 times concentrated GC Buffer I 1/2 volume.
  • the cycle for amplification is that both PCRs are kept at 94 ° C for 120 seconds, 94 ° C ⁇ 30 seconds, 60 ° C (increases by 0.5 ° C for each additional cycle), 15 cycles of 72 ° C ⁇ 4 minutes, 94 ° C ⁇ 30 seconds, 68 ° C ⁇ 4 After repeating the cycle for 15 minutes for 15 minutes, the mixture was kept at 72 ° C for 10 minutes.
  • the reaction mixture was electrophoresed on a 1.2% agarose gel, and a band near 900 bp, which was visible when stained with ethidium umide, was extracted with a DNA Extraction Kit (Qiagen) and the plasmid vector "PGEM" was extracted using a DM Ligation Kit (Takara Shuzo). After subcloning into -T Easy Vector (Promega), the cells were introduced into E. coli DH5 (T0Y0B0) Plasmid DNA was purified from the resulting transformant using the QIAGEN Plasmid Mini Kit (Qiagen).
  • the reaction for nucleotide sequence determination was performed using the BigDye Terminator Cycle Sequence Ready Reaction Kit (available from Kinery Reema Co., Ltd.) The sequence was read using a fluorescent automatic sequencer, and the 5 'upstream of the cDNA encoding Egret GPR8 A base sequence represented by SEQ ID NO: 91, which is a side sequence, was obtained.
  • TotalTotal RNA of whole egret brain was purchased from UNITECH. Poly (A) + RNA fractions were prepared using mRNA purification kit (Amersham Biosciences), and then were prepared from Egret whole brain poly (A) + RNA 1.0 / ig using PowerScript Reverse Transcriptase (Clontech). After reverse transcription was performed according to the manual, a type 2 rabbit egret whole brain double-stranded cDNA for 3'-RACE was prepared using a Marathon cDNA Amplification kit (Clontech).
  • the primer set used for the RACE PCR reaction was Adapter Primer 1 and Primer 1 (SEQ ID NO: 92) attached to the kit for the first PCR reaction, and Nested Adapter Primer 2 and Primer 2 for the second PCR reaction. (SEQ ID NO: 93) was used.
  • the reaction was carried out in the form of ⁇ with a volume of 501, corresponding to 2 ng of mRNA of the reverse-transcribed cDNA.
  • the composition of the reaction solution was a primer concentration of 0.2 M, a dNTP mixed solution of 0.2 mM, LA-Taq (Takara Shuzo) 1/100 vol hidden, and a 2-fold concentrated GC Buffer I 1/2 volume.
  • the cycle for amplification is that both PCRs are kept at 94 ° C for 120 seconds,
  • Plasmid DNA was purified from the resulting transformant using QIAGEN Plasmid Mini Kit (Qiagen). The reaction for base sequence determination was performed using BigDye Terminator Cycle Sequence Ready Reaction Kit (Perkin Elmer). Decoding was performed using a fluorescent automatic sequencer to obtain a base sequence represented by SEQ ID NO: 94, which is the 3 'downstream sequence of cDNA encoding the magpie GPR8.
  • TotalTotal RM of egret whole brain was purchased from UNITECH. After preparing the Poly (A) + RNA fraction using the mRNA purification kit (Amersham Biosciences), use manual PowerScript Reverse Transcriptase (Clontech) from 1.0 g of dried whole brain poly (A) + RNA. After reverse transcription was performed according to the above, a double heron brain double stranded cDNA was prepared using a Marathon cDNA Amplification kit (Clontech).
  • a PCR reaction was performed using the whole brain double-stranded cDNA as type II.
  • the reaction was carried out using 100 liters of cDNA corresponding to 1 ng of mRNA for type III.
  • the composition of the reaction solution was a primer concentration of 0.5 zM, a dT mixed solution of 0.2 mM, LA-Taq (Takara Shuzo) 1/100 volume, and a 2-fold concentrated GC Buffer I 1/2 volume.
  • the cycle for amplification was as follows: after incubating at 94 ° C for 120 seconds, a cycle of 94 ° C for 30 seconds and 68 ° C for 2 minutes was repeated 40 times, and then incubated at 72 ° C for 10 minutes.
  • the resulting reaction solution was purified using the QIAquick PCR purification Kit (Qiagen), subcloned into a plasmid vector — pGEM-T Easy Vector (Promega) using the DNA Ligation Kit (Takara Shuzo), and then transformed into E. coli DH5a ( T0Y0B0). Plasmid DNA was purified from the resulting transformant using QIAGEN Plasmid Mini Kit (Qiagen).
  • the reaction for base sequence determination was performed using a BigDye Terminator Cycle Sequence Ready Reaction Kit (No., Kin-Elema). Decoding was performed using a fluorescent automatic sequencer to obtain a nucleotide sequence represented by SEQ ID NO: 97. This sequence contained the nucleotide sequence encoding the entire amino acid sequence of the Great Egret GPR8 (SEQ ID NO: 81).
  • the plasmid After digesting the DNA with the restriction enzymes Sal I and Spe I (Takara Shuzo), the reaction solution was electrophoresed on a 1.2% agarose gel, and the band near 1000 bp, which was visible when stained with ethidium amide, was extracted using the DNA Extraction Kit ( (Qiagen).
  • TotalTotal RNA of whole egret brain was purchased from UNITECH. After preparing a Poly (A) + RNA fraction using mRNA purification kit (Amersham Biosciences), PowerScript Reverse Transcriptase (Clontech) was prepared from 10% whole brain poly (A) 1 "RNA 1.Og. After reverse transcription was performed according to the manual according to the manual, using a Marathon cDNA Amplification kit (Clontech), a type II, egosa whole brain double-stranded cDNA for RACE was prepared using the RACE PCR reaction.
  • Adapter Primer 1 and primer 1 SEQ ID NO: 98
  • Nested Adapter Primer 2 and Primer 2 SEQ ID NO: 99
  • the reaction was performed using 2 ng of reverse transcribed cDNA in the amount of 50 ⁇ 1 for the type II mRNA at a primer concentration of 0.2 / M, dNTP mixture 0.2 mM, LA- Taq (Takara Shuzo) 1/100 volume, 2x concentrated GC Buffer I 1/2 volume.
  • the cycle for, after both of the PCR was also kept at 94 to 120 seconds,
  • TotalTotal RNA of whole egret brain was purchased from UNITECH. After preparing the Poly (A) + RNA fraction using the mRNA purification kit (Amersham Biosciences), PowerScript Reverse Transcriptase (Clontech) was used from Escherichia coli whole brain poly (A) + RNA 1. O ⁇ g. After reverse transcription was performed according to the manual using the manual, Marathon cDNA Amplification kit (Clontech) was used to prepare a type II Egat whole brain double-stranded cDNA for 3, -RACE.
  • the primer set used for the RACE PCR reaction was: Adapter Primer 1 and primer 1 (SEQ ID NO: 10 1) attached to the kit in the first PCR reaction, and Nested Adapter Primer 2 and primer 1 in the kit for the second PCR reaction. (SEQ ID NO: 102) was used.
  • the reaction was carried out in the form of type II in a volume of 50 with 2 ng of mRNA of the reverse transcribed cDNA.
  • the composition of the reaction solution was a primer concentration of 0.2 M, a dMT mixed solution of 0.2 mM, LA-Taq (Takara Shuzo) 1/100 volume, and GC buffer I 1/2 volume concentrated twice.
  • the cycles for amplification were as follows: Both PCRs were kept at 94 ° C for 120 seconds, then 94 ° C for 30 seconds, 72T: 5 cycles of 4 minutes, 94 ° C for 30 seconds, 70 ° C The cycle was repeated 5 times at 4 minutes, 94 ° C for 30 seconds, 68 DC for 4 minutes 20 times, and then incubated at 72 ° C for 10 minutes.
  • the reaction mixture was electrophoresed on a 1.2% agarose gel, and the band at around 1000 bp, which was visible when stained with ethidium bromide, was extracted with a DNA Extraction Kit (Qiagen) and the plasmid was extracted using a DNA Ligation Kit (Takara Shuzo). After subcloning into the vector pGEM-T Easy Vector (Promega), E. coli DH5
  • Plasmid DNA was purified from the resulting transformant using a QIAGEN Plasmid Mini Kit (Qiagen). The reaction for base sequence determination was carried out using a BigDye Terminator Cycle Sequence Ready Reaction Kit (NO. Decoding was performed using a fluorescent automatic sequencer to obtain a base sequence represented by SEQ ID NO: 103, which is a 3 'downstream sequence of cDNA encoding Egret GPR7. (3) Cloning of full length cDNA encoding Egret GPR7
  • TotalTotal RNA of whole egret brain was purchased from UNITECH. After preparing Poly (A) + RNA fraction using mRNA purification kit (Amersham Biosciences), using PowerScript Reverse Transcriptase (Clontech) from 1.0 xg of egos whole brain poly (A) + RNA, After reverse transcription was performed according to the manual, a rabbit egret whole brain double-stranded cMA was prepared using Marathon cDNA Amplification kit (Clontech).
  • a PCR reaction was performed using whole brain double-stranded cDNA as type II.
  • the reaction was carried out using a cDNA equivalent to 1 ng of mRNA as a type II in a volume of 1001.
  • the composition of the reaction solution was primer concentration 0.5 iM, dNTP mixture 0.2 mM, LA-Taq (Takara Shuzo) 1/100 volume, and 2 times concentrated GC Buffer I 1/2 volume.
  • the cycle for amplification was as follows: after incubating at 94 ° C for 120 seconds, a cycle of 94 ° C for 30 seconds and 68 ° C for 2 minutes was repeated 40 times, and then incubated at 72 ° C for 10 minutes.
  • the resulting reaction solution was purified using the QIAquick PCR purification Kit (Qiagen), subcloned into the plasmid vector pGEM-T Easy Vector (Promega) using the DNA Ligation Kit (Takara Shuzo), and then E. coli DH5Q! T0Y0B0). Plasmid DNA was purified from the resulting transformant using QIAGEN Plasmid Mini Kit (Qiagen).
  • the base sequence was determined using the BigDye Terminator Cycle Sequence Ready Reaction Kit (Zo-Kin Elma). Decoding was performed using a fluorescent automatic sequencer to obtain a nucleotide sequence represented by SEQ ID NO: 106. This sequence contained the nucleotide sequence encoding the entire amino acid sequence of the Great Egret GPR7 (SEQ ID NO: 83).
  • the plasmid DNA was digested with the restriction enzymes Sal I and Spe I (Takara Shuzo), and the reaction solution was electrophoresed on a 1.2% agarose gel, and the 1000 bp region observed when stained with ethidium bromide was observed. Bands were collected using DNA Extraction Kit (Qiagen).
  • TotalTotal RNA of the egret whole brain was purchased from UNI TECH. After preparing the Poly (A) + RNA fraction using the mRNA purification kit (Amersham Biosciences), PowerScript Reverse Transcriptase (Clontech) was used from Escherichia coli whole brain poly (A) + RNA 1. O ⁇ g. After reverse transcription was carried out according to the manual using the manual, Marathon cDNA Amplification kit (Clontech) was used to prepare a type II, egos whole brain double-stranded cDNA for 5-RACE.
  • the primer set used for the RACE PCR reaction was Adapter Primer 1 and Primer 1 (SEQ ID NO: 10 7) attached to the kit in the first PCR reaction, and Nested Adapter Primer 2 and Kit 1 in the kit for the second PCR reaction. 2 (SEQ ID NO: 108) was used.
  • the reaction was performed using a 2 ⁇ g equivalent of mRNA of the reverse transcribed cDNA as a type II solution at a volume of 50 A.
  • the composition of the reaction solution was a primer concentration of 0.2 ⁇ iM, a dNTP mixed solution of 0.2 mM, LA-Taq (Takara Shuzo) 1/100 volume, and a 2-fold concentrated GC Buffer II 1/2 volume.
  • the amplification cycle was as follows: After incubating both PCRs at 94 ° C for 120 seconds, 5 cycles of 94 ° C for 30 seconds, 72 ° C for 4 minutes, 94 ° C for 30 seconds, and 70 ° C ⁇ 4 cycles of 5 minutes, 94t ⁇ 30 seconds, 68 ° C ⁇ After repeating the cycle of 4 minutes 20 times, it was kept at 72 ° C for 10 minutes.
  • the reaction mixture was electrophoresed on a 1.5% agarose gel, and the band around 350 bp, which was visible when stained with ethidium umide, was extracted with a DNA Extraction Kit (Qiagen) and the plasmid vector pGEM-T was used with the DNA Ligation Kit (Takara Shuzo). After subcloning into Easy Vector (Promega), E. coli DH5a
  • Plasmid DNA was purified from the resulting transformant using QIAGEN Plasmid Mini Kit (Qiagen). The reaction for base sequence determination was performed using BigDye Terminator Cycle Sequence Ready Reaction Kit (Kinrylema). Decoding was performed using a fluorescent automatic sequencer to obtain a base sequence represented by SEQ ID NO: 109, which is a 5 'upstream sequence of cDNA encoding a persimmon GPR8 ligand peptide precursor protein.
  • TotalTotal RNA of the egret whole brain was purchased from Ring I TECH.
  • Poly (A) + RNA fractions were prepared using mRNA purification kit (Amersham Biosciences), and 1.0 g of egos whole brain poly (A) + RNA was purified using PowerScript Reverse Transcriptase (Clontech). After reverse transcription was performed according to the manual, a type II, egret whole brain double-stranded cDNA for 3, -RACE was prepared using a Marathon cDNA Amplification kit (Clontech).
  • the primer set used in the RACE PCR reaction was Adapter Primer 1 and degenerate primer 1 (SEQ ID NO: 1 10) attached to the kit in the first PCR reaction, and Nested Adapter Primer attached to the kit in the second PCR reaction. 2 and degenerate primer 1 (SEQ ID NO: 1 1 1) were used.
  • the reaction was carried out using the equivalent of 2 ng of the mRNA of the reversely transcribed cDNA as a ⁇ in a liquid volume of 501.
  • the composition of the reaction solution was a primer concentration of 0.2 iM, a dNTP mixture of 0.2, LA-Taq (Takara Shuzo) 1/100 vol, and a 2 ⁇ concentrated GC Buffer I 1/2 volume.
  • the cycles for amplification are as follows: After incubating both PCRs at 94 ° C for 120 seconds, 5 cycles of 94 ° C for 30 seconds, 72 ° C for 4 minutes, 94 ° C for 30 seconds, 70 ° C ⁇ 5 cycles of 4 minutes, 94 ° C ⁇ 30 seconds, 68 ° C ⁇ After repeating the cycle of 4 minutes 20 times, it was kept at 72 ° C for 10 minutes.
  • the reaction mixture was electrophoresed on a 1.2% agarose gel, and the band near 600 bp, which was visible when stained with ethidium bromide, was
  • Extraction was performed using Extraction Kit (Qiagen), subcloned into a plasmid vector pGEM-T Easy Vector (Promega) using DNA Ligation Kit (Takara Shuzo), and then introduced into E. coli DH5Q! (T0Y0B0). Plasmid DNA was purified from the resulting transformant using QIAGEN Plasmid Mini Kit (Qiagen). The seventh round of nucleotide sequence determination was performed using the BigDye Terminator Cycle Sequence Ready Reaction Kit (PerkinElmer). Decoding was performed using a fluorescent automatic sequencer to obtain a base sequence represented by SEQ ID NO: 112, which is a 3, downstream sequence of a cDNA encoding a ⁇ sagi GPR8 ligand peptide precursor protein.
  • TotalTotal RNA of whole egret brain was purchased from UNITECH. After preparing Po (A) + RNA fraction using mRNA purification kit (Amersham Biosciences), After reverse transcription was performed according to the manual using PowerScript Reverse Transcriptase (Clontech) from poly (A) + RNA 1.0 ig of Giant Whole Brain, and then using the Marathon cDNA Amplification kit (Clontech), ⁇ Sagi whole brain double-stranded cDNA was prepared.
  • Primer 1 SEQ ID NO: 113 prepared based on the 5'-side sequence of the gene encoding the heron GPR8 ligand peptide precursor protein and the 3'-side of the gene encoding the ⁇ sagi GPR8 ligand peptide precursor protein Using a primer II (SEQ ID NO: 114) prepared on the basis of the sequence, a PCR reaction was performed on the type II of double-stranded cDNA of the egret whole brain.
  • the reaction was performed using 200 liters of cDNA corresponding to 1 ng of mRNA in the form of type II.
  • the composition of the reaction solution was a primer concentration of 0.5 M, a dNTP mixed solution of 0.2 mM, LA-Taq (Takara Shuzo) 1/100 volume, and a 2-fold concentrated GC Buffer II 1/2 volume. Cycles for amplification were kept at 94 ° C for 120 seconds, followed by 5 cycles of 9430 seconds, 72 minutes, 5 cycles of 94:30 seconds, 70 ° C, 1 minute, A cycle of 94 ° C for 30 seconds and 68 minutes for 1 minute was repeated 35 times, followed by incubation at 72 for 7 minutes.
  • the resulting reaction solution was purified using the QIAquick PCR purification Kit (Qiagen), subcloned into the plasmid vector pGEM-T Easy Vector (Promega) using the DNA Ligation Kit (Takara Shuzo), and then E. coli DH5a (T0Y0B0). Plasmid DNA was purified from the resulting transformant using QIAGEN Plasmid Mini Kit (Qiagen).
  • the reaction for base sequence determination was performed using BigDye Terminator Cycle Sequence Ready Reaction Kit (Perkin Elmer). Decoding was performed using a fluorescent automatic sequencer to obtain a nucleotide sequence represented by SEQ ID NO: 115.
  • the amino acid sequence of this hypothetical rabbit heron GPR8 ligand peptide precursor protein is shown in SEQ ID NO: 116. ⁇ A normal bioactive peptide is cut out from the expected amino acid sequence of the heron GPR8 ligand peptide to the amino acid sequence, similar to the human, porcine, rat or mouse homolog precursor protein of the GPR8 ligand peptide. There were two Arg_Arg sequences (Ann. NY Acad. Sci. 839, 9-24, 1998). From these facts, it was deduced that the amino acid sequence of the egret homolog of the GPR8 ligand peptide was either or both of SEQ ID NOs: 85 and 87.
  • amino acid sequence of the 23-residue heron GPR8 ligand peptide of SEQ ID NO: 87 is identical to the amino acid sequence of the 23-residue human GPR8 ligand peptide (SEQ ID NO: 1).
  • telomere sequence 50 ng was transformed into a type II primer 1 (SEQ ID NO: 1) prepared based on the 5'-side sequence of the heron GPR8 ligand peptide precursor protein gene.
  • PCR was carried out with the use of primer 1 (SEQ ID NO: 118) prepared based on the 3′-side sequence of the 117 ′) and egret GPR8 ligand peptide precursor protein gene.
  • the composition of the reaction solution was a primer concentration of 0.5 M dNTP mixed solution 0.2 mM LA-Taq (Takara Shuzo) 1/100 vol 2 times concentrated GC Buffer II 1/2 volume.
  • the cycle for amplification was as follows: after incubating at 94 ° C for 120 seconds, a cycle of 94 ° C for 30 seconds and 68 ° C for 1 minute was repeated 15 times, and then incubated at 72 ° C for 7 minutes.
  • the obtained reaction solution was purified using QIAquick PCR purification Kit (Qiagen), and then digested with restriction enzymes Sal I and Spe I (Takara Shuzo). This DNA fragment was subcloned into the SalI site and SpeI site of the animal cell expression vector pAKKO using the DNA Ligation Kit (Takara Shuzo), and then introduced into Escherichia coli DH5Q! (T0Y0B0).
  • Plasmid DNA was purified from the resulting transformant using QIAGEN 'Plasmid Mini Kit (Qiagen). The reaction for base sequence determination was performed using BigDye Terminator Cycle Sequence Ready Reaction Kit (PerkinElmer). Decoding was performed using a fluorescent automatic sequencer to obtain a nucleotide sequence represented by SEQ ID NO: 119. Escherichia coli DH5 (T0Y0B0) was transformed with this plasmid to obtain Escherichia coli DH5 ⁇ / ⁇ -rabbit prepro_NPW. Industrial applicability
  • the polypeptide or receptor of the present invention and the DNA of the present invention are useful for screening antidiuretics or diuretics.
  • urinary storage disorders eg, frequent urination, urinary incontinence (eg, urge incontinence, stress urinary incontinence, functional urinary incontinence, etc.)
  • polyuria eg, diabetes insipidus (eg, pituitary incontinence) ⁇
  • urinary excretion disorder eg, fl parapleural contraction
  • urethral passage obstruction dysuria, urination pain, urinary tract obstruction, etc.
  • hyponatremia antidiuretic hormone secretion inadequate syndrome
  • the polypeptide or receptor of the present invention, the DNA of the present invention, the compound promoting the activity of the polypeptide or the receptor of the present invention, or a salt thereof is useful as a low-toxicity and safe antidiuretic, and has an urinary storage disorder ( Eg, urinary frequency, urinary incontinence (eg, urge incontinence, stress urinary incontinence, functional urinary incontinence, etc.), etc., polyuria, diabetes insipidus (eg, pituitary diabetes insipidus, renal urine) It is useful as a preventive and therapeutic agent for hypernatremia, metabolic alkalosis, hypokalemia, Cushing syndrome, etc.
  • urinary storage disorder Eg, urinary frequency, urinary incontinence (eg, urge incontinence, stress urinary incontinence, functional urinary incontinence, etc.), etc., polyuria, diabetes insipidus (eg, pituitary diabetes insipidus, renal urine) It is
  • the antibody of the present invention, the antisense DNA of the present invention, the polypeptide of the present invention, or a compound that inhibits the activity of the receptor or a salt thereof is useful as a low-toxicity and safe diuretic, and has renal edema and urinary dysfunction.
  • a compound that inhibits the activity of the receptor or a salt thereof is useful as a low-toxicity and safe diuretic, and has renal edema and urinary dysfunction.
  • SIADH antidiuretic hormone secretion syndrome

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Organic Chemistry (AREA)
  • Diabetes (AREA)
  • Epidemiology (AREA)
  • Hematology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Endocrinology (AREA)
  • Cardiology (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Urology & Nephrology (AREA)
  • Molecular Biology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

L'invention concerne des polypeptides (par exemple, un ligand GPR8) et des composés stimulant l'activité de ces polypeptides ou des récepteurs de ces polypeptides (par exemple, GPR8, GPR7 et TGR26) ou des sels des ces composés, qui sont utiles comme antidiurétiques présentant d'excellentes caractéristiques. Les polypeptides selon l'invention (par exemple, un ligand GPR8) et leurs récepteurs (par exemple, GPR8, GPR7 et TGR26) sont utiles dans le criblage d'un antidiurétique présentant d'excellentes caractéristiques et d'un diurétique.
PCT/JP2003/014102 2002-11-06 2003-11-05 Antidiuretiques Ceased WO2004041301A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2003277553A AU2003277553A1 (en) 2002-11-06 2003-11-05 Antidiuretics

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2002-322715 2002-11-06
JP2002322715 2002-11-06

Publications (1)

Publication Number Publication Date
WO2004041301A1 true WO2004041301A1 (fr) 2004-05-21

Family

ID=32310396

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2003/014102 Ceased WO2004041301A1 (fr) 2002-11-06 2003-11-05 Antidiuretiques

Country Status (2)

Country Link
AU (1) AU2003277553A1 (fr)
WO (1) WO2004041301A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7763716B2 (en) 2003-05-28 2010-07-27 Takeda Pharmaceutical Company Limited Antibody against NPW

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001098494A1 (fr) * 2000-06-21 2001-12-27 Takeda Chemical Industries, Ltd Ligand de gpr8 et son adn
WO2002044368A1 (fr) * 2000-11-30 2002-06-06 Takeda Chemical Industries, Ltd. Nouvelles proteines du recepteur couple a la proteine g et leurs adn

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001098494A1 (fr) * 2000-06-21 2001-12-27 Takeda Chemical Industries, Ltd Ligand de gpr8 et son adn
WO2002044368A1 (fr) * 2000-11-30 2002-06-06 Takeda Chemical Industries, Ltd. Nouvelles proteines du recepteur couple a la proteine g et leurs adn

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7763716B2 (en) 2003-05-28 2010-07-27 Takeda Pharmaceutical Company Limited Antibody against NPW

Also Published As

Publication number Publication date
AU2003277553A1 (en) 2004-06-07

Similar Documents

Publication Publication Date Title
JP2002112772A (ja) 新規ポリペプチドおよびそのｄｎａ
JP4841054B2 (ja) 新規インスリン／ｉｇｆ／リラキシンファミリーポリペプチドおよびそのｄｎａ
EP1239039A1 (fr) Nouveau polypeptide et son adn
US7235531B2 (en) Tachykinin-like polypeptides and use thereof
WO2001002564A1 (fr) Nouveau polypeptide et adn de ce polypeptide
WO2004041301A1 (fr) Antidiuretiques
WO2002066050A1 (fr) Inhibiteurs de caspase 3
CA2372696A1 (fr) Nouvelle proteine et son adn
JP4090092B2 (ja) 新規Ｆａｓリガンド様タンパク質およびそのＤＮＡ
WO2001048203A1 (fr) Nouvelle proteine et adn associe
WO2002103018A1 (fr) Nouveau polypeptide et son utilisation
WO2004041850A1 (fr) Nouveaux ligands fprl1 et utilisations de ceux-ci
JP4320151B2 (ja) 新規ポリペプチドおよびその用途
JP2004166699A (ja) 抗利尿剤
JP4676608B2 (ja) 新規タヒキニン様ポリペプチドおよびその用途
JP4320355B2 (ja) 新規Ｆａｓリガンド様タンパク質およびそのＤＮＡ
JP4959880B2 (ja) Ｇｐｒ８に対するリガンドおよびそのｄｎａ
JP2002335973A (ja) 新規タンパク質およびそのｄｎａ
JP2001299362A (ja) 新規ポリペプチドおよびそのｄｎａ
JP2001299364A (ja) 新規タンパク質およびそのｄｎａ
WO2004080485A1 (fr) Substance preventive/therapeutique pour les maladies de l'appareil digestif superieur
JP2001299363A (ja) 新規タンパク質およびそのｄｎａ
JP2001069994A (ja) 新規ポリペプチドおよびそのｄｎａ
JP2002365287A (ja) 糖尿病治療薬のスクリーニング方法
JP2000053700A (ja) 新規タンパク質およびそのｄｎａ

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): BW GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP

WWW Wipo information: withdrawn in national office

Country of ref document: JP