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WO1999066073A1 - METHOD FOR SCREENING Pax4 GENE EXPRESSION POTENTIATOR AND MEDICINAL COMPOSITIONS FOR POTENTIATING Pax4 GENE EXPRESSION - Google Patents

METHOD FOR SCREENING Pax4 GENE EXPRESSION POTENTIATOR AND MEDICINAL COMPOSITIONS FOR POTENTIATING Pax4 GENE EXPRESSION Download PDF

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Publication number
WO1999066073A1
WO1999066073A1 PCT/JP1999/003182 JP9903182W WO9966073A1 WO 1999066073 A1 WO1999066073 A1 WO 1999066073A1 JP 9903182 W JP9903182 W JP 9903182W WO 9966073 A1 WO9966073 A1 WO 9966073A1
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Prior art keywords
cells
gene expression
pax4
activin
expression
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PCT/JP1999/003182
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French (fr)
Japanese (ja)
Inventor
Yoshitaka Ueda
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Yamanouchi Pharmaceutical Co., Ltd.
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Application filed by Yamanouchi Pharmaceutical Co., Ltd. filed Critical Yamanouchi Pharmaceutical Co., Ltd.
Priority to AU41666/99A priority Critical patent/AU4166699A/en
Publication of WO1999066073A1 publication Critical patent/WO1999066073A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/82—Translation products from oncogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention provides a method for screening a substance having an enhancing effect on Pax4 gene expression in rat j8 cells, which comprises a means for bringing a test substance into contact with the mouse / 3 cells and detecting a Pax4 gene expression site in the cells. And pharmaceuticals, in particular, the pancreas) 8.
  • Type 1 diabetes or non-insulin dependent diabetes mellitus accounts for 90% of diabetics and is an ever increasing disease in developed countries. Its characteristics are as follows (Diabetes Reviews (1997) 5, 177-269, J. Invest. Med (1996) 44, 413-428, Annu. Rev. Med. (1996) 47, 509-531, Eur. J. Clin. Invest. (1993) 23, 69-79).
  • insulin secretion in the kidney / 3 cells is normal, but some of the complex causes (genetic and environmental factors) are caused by the peripheral (muscle, fat) blood sugar utilization (uptake of glucose) by insulin. ), A condition called insulin resistance, which is inhibited by, and blood sugar levels rise.
  • IDDM insulin dependent diabetes mellitus
  • Drugs currently known as antidiabetic drugs are classified into two groups: drugs for controlling blood sugar and drugs for complications. Drugs for controlling blood sugar are further classified into the following five categories.
  • the first is an insulin preparation. Insulin shortage due to decreased secretion is directly replenished.
  • the second is an insulin secretagogue such as a sulfonylprea drug. This is characterized by blocking KATP channels, depolarizing the cell membrane of kidney j8 cells, and forcing Ca 2+ to flow into the cells, thereby forcibly secreting insulin regardless of blood glucose level ( Science (1995) 268, 423429).
  • Receptor ⁇ binds to a nuclear receptor called “receptor ⁇ ” and promotes adipocyte differentiation to take up blood sugar into fat and reduce blood sugar level (Diabetes (1998) 4L 507-514).
  • Fourth is an ⁇ -dalcosidase inhibitor. It has a saccharide structure and is characterized by inhibiting absorption of sugar from the intestine by inhibiting ⁇ -glucosidase in the small intestine.
  • PTF1 a transcription factor complex composed of p48 and E12. It has been clarified from experiments on knockout mice that p48 is essential for differentiation of excretory exocrine cells (Genes Dev. (1998) 12, 3752-3763).
  • Activin is a member of the transforming growth factor j8 (TGFp) superfamily. This family includes TGFp, BMP (bone morphogenetic protein), and MIS (Mullerian inhibiting substance). Activin has two subunits, 8 A and / 3 B, and the active form forms homodimer (/ 3A-i8 A,) 3 B-) 3 B) or heterodimer (j8A-i8 B) These are called activin A, activin B and activin AB, respectively.
  • TGFp transforming growth factor j8
  • FSH follicle stimulating hormone
  • the type I activin receptor phosphorylates a serine residue at the carboxyl terminus of a protein called Smad2 or Smad3, and these Smads form a heterodimer with Smad4 and translocate to the nucleus to target genes. It has been reported that various types of cells, such as Drosophila, Xenopus, and mammals, promote transcription of various species (Nature (1997) ⁇ Q, 465-471, Curr. Opin. Genet. (1997) 7, 467473).
  • An object of the present invention is to provide a method for screening a substance having Pax4 expression enhancing activity in mouse ⁇ cells, which comprises means for detecting the expression level of Pax4 gene in mouse 3 cells, and to effectively use the substance obtained by the screening method. It is intended to provide a pharmaceutical composition for enhancing expression of Pax4 gene in splanchnic i8 cells as a component, and a pharmaceutical composition for enhancing expression of Pax4 gene in splanchnic i8 cells, which comprises an activin receptor activator as an active ingredient.
  • the pharmaceutical composition for enhancing expression of Pax4 gene in the hepatic ⁇ -cell of the present invention alone or in combination with a drug for controlling blood sugar or a drug for complications, such as a sulfonylurea agent, an agent for improving dinsuline resistance, and an intestinal glucose absorption inhibitor. It can be expected to show an excellent diabetes treatment effect by using it.
  • the present invention aims to create a therapeutic drug based on such a new concept.
  • the inventor of the present invention believes that, under the state of the art, the presence of a substance having the Pax4 gene expression-enhancing activity in the xenal 0 cells may lead to the protection of the 0-cells from exhaustion or the enhancement of the function of the 0-cells. Focusing on the fact that it cannot be performed, the present inventors have found a screening method capable of measuring the expression level of Pax4 gene in three mature kidney cells, and completed the present invention. Furthermore, the present inventors screened the substance having the action of enhancing the expression level of the Pax4 gene using the screening method of the present invention. As a result, unexpectedly, an activin receptor activator that has never been reported in relation to the Pax4 gene has been unexpectedly reported.
  • the present invention is a.
  • a method for screening a substance having a Pax4 gene expression enhancing action which comprises a means for contacting a test substance with at least a mouse i8 cell and detecting the amount of Pax4 gene expression in the cell;
  • a pharmaceutical composition for enhancing the expression of Pax4 gene in cells (8) which contains an activin receptor activator as an active ingredient.
  • lean; three cells refers to mature three kidney cells after differentiation and regeneration, and is preferably a mammal-derived cell or an established cell. Specifically, RIN5 cells (Proc. Natl. Acad. Sci. USA (1977) 74, 628-630) and HIT cells (Proc. Natl. Acad. Sci. USA (1981)) used for cell studies 78, 4339-4342), MIN6 cells (Endocrinol. (1990) 127, 126-132), ⁇ TC cells (Endocrinol.
  • the ⁇ 8 intracellular Pax4 gene '' means that after formation of the spleen, exocrine and endocrine tissues are differentiated and have a paired-box that functions in the / 3 cell differentiation process in endocrine cell differentiation.
  • 1 shows a gene encoding the homeo transcription factor Pax4 or a partial fragment having the function.
  • test substance used in the "screening method for a substance having a Pax4 gene expression enhancing effect including a means for detecting a Pax4 gene expression level in a cell by contacting the test substance with at least 8 cells A novel protein (including an antibody or a partial fragment of an antibody), a peptide or a compound is indicated.
  • a compound registered in a chemical file, or a novel compound obtained by chemically modifying a substituent of a known compound by a conventional method Tetrahedron (1995) ⁇ 1, 8135). -8137
  • peptides or proteins obtained by conventional techniques, peptide engineering or phage display method ⁇ Mol. Bio (1991) 222, 301-310)
  • a random peptide group created by applying the method can be used as a test substance.
  • culture supernatants of microorganisms and natural components derived from plants and marine organisms can be used.
  • a test substance can be obtained by chemically modifying a substituent based on a known compound obtained by the screening method of the present invention, using a combinatorial chemistry technique or a phage display method.
  • the chemical modification of a substituent means a modification by a conventional method such as alkylation, amidation, esterification, oxidation, or reduction reaction.
  • peptide synthesis it can be synthesized using a liquid phase method such as a coupling method using a variety of protecting groups and coupling reagents, a carboxyl terminal activation method, and an amino terminal activation method.
  • a solid-phase method that can be produced and purified more easily [ ⁇ Am. Cem. Soc.
  • peptide synthesizer for example, 43 OA, a peptide synthesizer manufactured by Perchierma Applied Biosystems Co., Ltd. is available, and the peptide synthesis may be performed according to a standard operation program of this apparatus.
  • the genetic engineering technique is used, the gene encoding the amino acid of the target peptide or protein is transformed into E.
  • the gene is amplified, and the gene is transfected into animal cells (in this case, self- Any of a plasmid that is replicable and contains a transcription promoter region or a plasmid that can be incorporated into a chromosome of an animal cell) may be used to produce the protein.
  • animal cells in this case, self- Any of a plasmid that is replicable and contains a transcription promoter region or a plasmid that can be incorporated into a chromosome of an animal cell.
  • the substance also means the aforementioned natural component.
  • (Leather) (3) a substance having an effect of enhancing intracellular Pax4 gene expression '' can be obtained by the method obtained by the screening method of the present invention or other methods by stimulating or contacting the (liver) 8 cells
  • Any substance having the property of enhancing the expression of Pax4 gene in the gland 0 cells may be used, and specific examples include a protein (including an antibody or a partial fragment of an antibody), a peptide or a compound. These substances can be obtained in the same manner as the test substance.
  • it is a substance that activates an activin receptor, that is, an activin receptor activator.
  • the above-mentioned test substance or kidney 8A substance having an intracellular Pax4 gene expression enhancing action may form a salt with an acid or a base. It is an acid addition salt with an inorganic acid or an organic acid, or a salt with an inorganic base or an organic base, and a pharmaceutically acceptable salt is preferable.
  • these salts include mineral acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid and phosphoric acid, or formic acid, acetic acid, propionic acid, oxalic acid, and the like.
  • organic acid such as methanesulfonic acid or ethanesulfonic acid
  • acidic amino acid such as aspartic acid or glutamic acid Salt
  • sodium, potassium, magnesium examples include inorganic salts such as calcium and aluminum, organic base salts such as methylamine, ethylamine and ethanolamine, and salts with basic amino acids such as lysine and ordithine.
  • the term “pharmaceutical composition for enhancing Pax4 gene expression in mouse 0 cells” refers to a pharmaceutical composition administered to mammals, especially humans, and has the above-mentioned effect of enhancing Pax4 gene expression in mouse / 3 cells.
  • This is a pharmaceutical composition containing the following substances as active ingredients.
  • it is a pharmaceutical composition for enhancing Pax4 gene expression in porcine i8 cells, comprising an activin receptor activator as an active ingredient.
  • X-ray The pharmaceutical composition for the treatment or prevention of diabetes or diabetic complications based on ft-progression protection or function of ⁇ -cells from exhaustion.
  • “Protection of the three cells of the kidney from exhaustion or enhancement of the function of the cells” is as follows. During hyperinsulinemia or with the administration of existing diabetic drugs, protection from iS cells with excessive insulin synthesis or secretion of existing insulin, resulting in reduced or near-function of the relevant function Or it indicates functional recovery.
  • Each step used in the screening method of the present invention can be performed according to a known method (Maniatis, T. et al. (1982): “Molecular Cloning-A Laboratory Manua at Cold Spring Harbor Laboratory, NY). It is as follows.
  • RNA protection assay RPA
  • Guanidine thiosane 'hot' phenol method total RNA extraction method Anidinchi singer-guanidine-hydrochloric acid method.
  • mRNA may be purified from total RNA before performing the reverse transcription reaction.
  • Examples of the purification method include adsorption / elution using an oligo (dT) cellulose column and purification using oligo (dT) magnetic beads.
  • mRNA can be fractionated by sucrose density gradient centrifugation or the like.
  • a commercially available extracted mRNA may be used without extracting the mRNA.
  • first strand cDNA is synthesized by reverse transcriptase reaction of RNA in the presence of random primer or oligo (dT) primer, and a part of the target gene is sandwiched.
  • the first strand cDNA is subjected to PCR using two kinds of primers to amplify a target DNA fragment.
  • the DNA has been combined with a nucleic acid labeled with a fluorescent substrate called a TaqMAN probe, and combined with the PCR method, utilizing the 5 ' ⁇ 3' exonuclease activity of Taq DNA polymerase, the fluorescence that is hydrolyzed during the extension reaction Measures fluorescence from substrate
  • a method has been developed by PerkinElmer Applied Biosystems. Hereinafter, a method for formulating and administering a substance having an action of enhancing Pax4 gene expression in the pancreatic 0 cells of the present invention will be described in detail.
  • compositions containing one or more pharmaceutically acceptable salts of the substance of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient may be used in combination with carriers, excipients and other additives commonly used for pharmaceuticals. It is used to prepare tablets, powders, fine granules, granules, capsules, pills, solutions, injections, suppositories, ointments, patches, etc., and is administered orally or parenterally.
  • the clinical dose for the human of the present invention is appropriately determined in consideration of the patient's symptoms, weight, age, sex, and the like.
  • the substance having an effect of enhancing Pax4 gene expression in the three cells of the gut of the present invention may be used simultaneously or with a combination of other drugs such as a sulfonylurea agent dinsulin resistance improving agent, an ⁇ -dalcosidase inhibitor, a biguanide agent and the like. Can be used together.
  • Examples of the ⁇ sulfonyl ⁇ rea agent j include acetatehexamide, daliclazide, dalicloviramide, dalibenclamide, chlorpropamide, tolazamide or tolbutamide.
  • insulin sensitizer examples include troglitazone, pioglitazone, siglitazone, englitazone or rosiglitazone.
  • ⁇ ⁇ -darcosidase inhibitors include acarbose, voglibose, and miguri! And emigrites.
  • Examples of the “biguanide drug” include metformin and buformin.
  • the one or more active substances include at least one inert diluent, such as lactose, mannitol, glucose, hydroxypropylcellulose, microcrystalline cellulose, starch, polyvinylpyrrolidone. , Mixed with metasilicate or magnesium aluminate.
  • the composition may be formulated in accordance with the usual practice with additives other than inert diluents, such as lubricants such as magnesium stearate, disintegrants such as calcium fiber glycolate, stabilizers such as lactose, daltamine.
  • a solubilizing agent or solubilizing agent such as an acid or aspartic acid may be contained.
  • the tablets or pills may be coated with a gastric or enteric film such as sucrose, gelatin, hydroxypropylcellulose, or hydroxypropylmethylcellulose phthalate, if necessary.
  • Liquid compositions for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, elixirs and the like, and commonly used inert diluents, such as purified water. , Including ethyl alcohol.
  • the composition may contain, in addition to the inert diluent, solubilizing or solubilizing agents, wetting agents, auxiliary agents such as suspending agents, sweetening agents, flavoring agents, fragrances, and preservatives.
  • Injections for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • Diluents for aqueous solutions and suspensions include, for example, distilled water for injections and physiological saline.
  • examples of diluents for water-insoluble solutions and suspensions include, for example, propylene glycol, polyethylene glycol, vegetable oils such as crude oil, alcohols such as ethyl alcohol, and polysorbate 80 (trade name: Polyoxyl). Ethylene sorbitan higher fatty acid ester).
  • compositions may further comprise additives such as tonicity agents, preservatives, wetting agents, emulsifiers, dispersants, stabilizers (eg, lactose), and solubilizing or solubilizing agents. May be. These are sterilized by, for example, passage through a bacteria retaining filter, blending of a bactericide or irradiation. these Alternatively, a sterile solid composition can be prepared and dissolved in sterile water or a sterile injectable solvent before use.
  • additives such as tonicity agents, preservatives, wetting agents, emulsifiers, dispersants, stabilizers (eg, lactose), and solubilizing or solubilizing agents. May be. These are sterilized by, for example, passage through a bacteria retaining filter, blending of a bactericide or irradiation. these Alternatively, a sterile solid composition can be prepared and dissolved in sterile water or a sterile injectable solvent before use
  • solubilization treatment When the solubility of the compound of the present invention is low, a solubilization treatment may be performed.
  • solubilization treatment known methods applicable to pharmaceutical preparations, for example, surfactants (polyoxyethylene hydrogenated castor oils, polyxylene sorbitan higher fatty acid esters, polyxixylene oxypolyglycols, sucrose) Fatty acid esters, etc.), drugs and solubilizing agents such as polymers (water-soluble polymers such as hydroxypropylmethylcellulose (HPMC) and polyethylene glycol (PEG)), carboxymethylethylcellulose (CMEC) , Hydroxypropyl methylcellulose phthalate (HPMCP), methyl methacrylate-methacrylic acid copolymer
  • HPMC hydroxypropylmethylcellulose
  • PEG polyethylene glycol
  • CMEC carboxymethylethylcellulose
  • HPPMCP Hydroxypropyl methylcellulose phthalate
  • FIG. 1 Pax4 gene expression when 10 tM all-trans retinoic acid (atRA), LG100268 or 2 nM human activin A was allowed to act on ⁇ cells for 12, 24, and 48 hours (The expression level of Pax4 gene in untreated (None) is set to 100).
  • Fig. 2 is a diagram showing the expression level of Pax4 gene when activin A at each concentration was allowed to act on cells for 24 hours (Pax4 expression level in OpM is 100).
  • Fig. 3 shows the gene expression levels of insulin (Ins) and Pax4 when 2nM TGF / 31, activin A was allowed to act on cells for 24 hours. (Expression of each gene in untreated (None)) Set the amount to 100).
  • FIG. 4 Xenopus activin response element reporter gene and FAST-1 gene WT type WT cells or constitutive active type IB type activin receptor ALK4 (T206D) gene is stably expressed ⁇ cells ALK4 No. 6 is transfected into wild type NMT1 cells Fig. 4 shows the relative intracellular activin signal intensity measured by measuring the luciferase activity of each cell after 2 hours of the activin A (WT (Act)) was applied to 2 nM of the group. (Activin signal intensity in ⁇ cells of type (WT) is assumed to be 100).
  • FIG. 5 shows the expression level of Pax4 in ALK4 No. 6 ⁇ cells that stably express the activin receptor ALK4 (T206D) gene (Pax4 gene expression level in wild-type (WT) NIT1 cells was 100 and 100%).
  • BEST MODE FOR CARRYING OUT THE INVENTION-Examples will be described below and the contents will be described in detail, but the present invention is not limited to these examples.
  • Human activin A used in this example was obtained from Austral Biologicals (Cat. No. GF-530-3), and human transforming growth factor -beta 1 (TGF / 3 1) was purchased from SIGMA (Cat. No. T7039). Each amino acid sequence is shown in the sequence listing below. The one-letter and three-letter codes used in amino acid sequences are well known, but see the Biochemical Dictionary (2nd edition (1990), Tokyo Kagaku Dojin, p. 1468). ⁇ leen] Wild type ⁇ 1 cell, one of 8 cell lines, was purchased from ATCC (American Type Culture Collection) (Cat. No. CRL-2055) o
  • the expression plasmid of ALK4 (T206D) (GenBank Ac. No. Z22536) was kindly provided by Dr. Kohei Miyazono of the Biochemistry Department, Cancer Research Institute, Cancer Research Institute.
  • the ALK4 (T206D) expression plasmid was transfected into NIT1 cells, G418-resistant colonies were isolated and obtained and subcultured to isolate the ALK4 (T206D) stable expression cell clone ALK4 N0.6. .
  • the expression plasmid for the transcription factor FAST-1 (GenBank Ac. No. U70980, Nature (1996) 383, 691-696) from Xenopus was obtained from Harvard University
  • Total RNA was prepared from NIT1 cells using lsogen (Cat. No. 31 1-02501) of Futsubon Gene according to the instructions. ⁇ cells were cultured in Ham's F12 medium containing 10% fetal bovine serum. The prepared total RNA was then used for deoxyribonuclease.
  • RNA to cDNA was performed using ClonTech's Advantage TM RT-for-PCR Kit (Cat. No. K1402-2). After reverse transcription, it was stored at -20 ° C.
  • mice insulin Ins
  • Pax4 dalyseraldehyde 3-phosphate dehydrogenase
  • TFIIDT TATA-binding protein TFIIDT
  • Ins13-lns14 The genes for mouse insulin (Ins), Pax4, dalyseraldehyde 3-phosphate dehydrogenase (G3PDH), and TATA-binding protein TFIIDT (TBP) are primers Ins13-lns14, Pax401-Pax403, PCR amplification was performed using a combination of G3PDH F-G3PDH R and TF201-TF205. Each base sequence is described below.
  • Pax401 (SEQ ID NO: 6) GCCTGGGAGATCCAACACCA
  • Pax403 (SEQ ID NO: 7) GGGAAGAACTGGAGCCA
  • G3PDH F (SEQ ID NO: 8) AAAGTGGAGATTGTTGCCAT G3PDH R (SEQ ID NO: 9) TTGACTGTGCCGTTGAATT
  • TF201 (SEQ ID NO: 10) TCTTTAGTCCAATGATGCCTT TF205 (SEQ ID NO: 11) GGTTGCTGAGATGTTGATTG
  • the sequence of the TaqMAN probe labeled with the fluorescent substrate used for gene detection is as follows.
  • the measurement with the PRISM TM 7700 Sequence Detection System was performed according to the instruction manual for the system.
  • the PCR amplification conditions are as follows. Incubate at 50 ° C for 10 minutes, then at 95 for 10 minutes. Thereafter, 4045 cycles of a two-step PCR were performed at 95 ° C for 15 seconds, followed by 58 ° C for 1 minute.
  • the expression level of the target gene in each sample was corrected by the expression level of the G3PDH gene in FIGS. 1 to 3 and by the expression level of the TFIIDT gene in FIG. The corrected numerical value was calculated based on the following equation.
  • PGC-51) was diluted with water to lyse the cells.
  • a predetermined amount was mixed with a substrate of Luciferase, and the amount of luminescence was measured using Dynatech Pellet 3000.
  • the total amount of protein in each well was mixed with a Bioassay protein assay reagent (Cat. No. 500-0006) and colorimetric at 595 nm was measured.
  • the conversion of port degree was calculated from a calibration curve drawn with serum albumin as standard.
  • the activin signal strength was calculated by the following equation.
  • the secretion degree of activin was shaken between 0 and 2 nM to act on NIT1 for 24 hours, and the expression level of Pax4 gene was examined. It was found that the expression level increased in a concentration-dependent manner (Fig. 2).
  • TGF iS1 which belongs to the TGF8 superfamily as well as activin, was examined. Both are known to function as Smad2 or Smad3 as signal mediators. Both activin and TGF iS1 were allowed to act on NIT1 cells at 2 nM for 24 hours.
  • TGF iS1 reduced the gene expression of Ins, Pax4.
  • Pax4 gene expression enhancing action was specific to activin A (Fig. 3).
  • V Pax4 gene expression level in untreated or activin-treated wild-type ⁇ cells or N1 cells stably expressing constitutively active ALK4 (T206D)
  • Pax4 gene expression levels were measured in wild-type NIT1, a group treated with 2 nM human activin A for 24 hours and wild-type NIT1, and ALK4 (T206D) stably expressing 6 cells ALK4 No.6 ( ( Figure 5).
  • ALK4 N0.6 the Pax4 gene was expressed at a level equal to or higher than that of the 2 nM activin-treated group, and between the intracellular activin signal intensity shown in Fig. 4 and the Pax4 gene expression level in Fig. 5. Good response was seen.
  • the screening method of the present invention not only selects a substance having an action of enhancing Pax4 gene expression in the pancreas i8 cells, which is useful for protecting the pancreas) 8 cells from exhaustion or enhancing the function of the pancreas 8 cells. [Kidney] It is useful for diagnosis etc. by detecting the function or degree of exhaustion of 8 cells.
  • the substance obtained by the screening method of the present invention and / or the activin receptor activator is a disease caused by exhaustion of the [8] cells due to enhanced pax4 gene expression and increased insulin gene expression. It is extremely useful as a pharmaceutical composition for preventing and treating the progress of diabetes. INDUSTRIAL APPLICABILITY The present invention is useful for elucidating the mechanism of insulin expression in 8 cells of the kidney.

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Abstract

A screening method for selecting a substance having an effect of potentiating the expression of Pax4 gene in pancreatic β cells which is useful in protecting pancreatic β cells in an exhausted state or accelerating the function of pancreatic β cells. The substance obtained by the above screening method and/or activin receptor activator exhibit effects of potentiating the expression of Pax4 gene and accelerating the expression of insulin gene. Owing to these effects, the above substances are highly useful in medicinal compositions for preventing the progress of a disease caused by the exhaustion of pancreatic β cells (i.e., diabetes) and treating this disease.

Description

明 細 書  Specification
Pax4遺伝子発現増強物質のスクリ一二ング法並びに Pax4遺伝子発現増強用医薬組 成物 技術分野 Screening method of Pax4 gene expression enhancer and pharmaceutical composition for enhancing Pax4 gene expression
本発明は、 脖臓 /3細胞に被験物質を接触させ、 該細胞内の Pax4遺伝子発現置を検 出する手段を含む脖臓 j8細胞内 Pax4遺伝子発現増強作用を有する物質のスクリー二 ング方法、 及び医薬殊に脬臓) 8細胞内 Pax4遺伝子発現増強用医薬組成物に関する。 背景技術  The present invention provides a method for screening a substance having an enhancing effect on Pax4 gene expression in rat j8 cells, which comprises a means for bringing a test substance into contact with the mouse / 3 cells and detecting a Pax4 gene expression site in the cells. And pharmaceuticals, in particular, the pancreas) 8. A pharmaceutical composition for enhancing Pax4 gene expression in cells. Background art
1 1型糖尿病またはィンシユリン非依存性糖尿病（NIDDM; Non Insulin Dependent Diabetes Millitus)は糖尿病患者の 90%を占め、現在先進国で増加の一途をたどってい る疾患である。 その特徴は次の通リである (Diabetes Reviews (1997) 5, 177-269, J. Invest. Med (1996) 44, 413-428, Annu. Rev. Med. (1996) 47, 509-531， Eur. J. Clin. Invest. (1993) 23, 69-79参照）。 初期には脬臓 /3細胞におけるィンシユリン分泌は正 常に起きているが、 インシュリンによる末梢 （筋肉，脂肪） での血糖利用 （血糖取 リ込み） が何らかの複合的な原因 （遺伝的 ·環境的要因） により阻害されるインシ ュリン抵抗性と呼ばれる状態になり、 血糖値が上昇する。 それを代儍するために更 なるインシュリン分泌がなされ、 高インシュリン血症になる。 後期になるとそれま で脖臓 ]8細胞を酷使してィンシュリン分泌を行ってきた結果として脬臓 0細胞が疲 弊し、 インシュリン分泌量が減少し、 最終的にはほとんど分泌がなされなくなり、 I 型糖尿病またはィンシユリン依存性糖尿病（IDDM; Insulin Dependent Diabetes Millitus)へと移行していく。 現在糖尿病治療薬として知られている薬剤は、 血糖管理用薬剤と合併症用薬剤の 2つに分類され、 血糖管理用薬剤は更に主として次の 5つに分類される。 1 Type 1 diabetes or non-insulin dependent diabetes mellitus (NIDDM) accounts for 90% of diabetics and is an ever increasing disease in developed countries. Its characteristics are as follows (Diabetes Reviews (1997) 5, 177-269, J. Invest. Med (1996) 44, 413-428, Annu. Rev. Med. (1996) 47, 509-531, Eur. J. Clin. Invest. (1993) 23, 69-79). In the early stage, insulin secretion in the kidney / 3 cells is normal, but some of the complex causes (genetic and environmental factors) are caused by the peripheral (muscle, fat) blood sugar utilization (uptake of glucose) by insulin. ), A condition called insulin resistance, which is inhibited by, and blood sugar levels rise. In order to replace it, more insulin is secreted, resulting in hyperinsulinemia. In the late stage, until then, 8 cells were overworked to secrete insulin, resulting in exhaustion of 0 cells of the kidney, a decrease in insulin secretion, and ultimately little secretion. Transition to type diabetes or insulin dependent diabetes mellitus (IDDM; Insulin Dependent Diabetes Millitus). Drugs currently known as antidiabetic drugs are classified into two groups: drugs for controlling blood sugar and drugs for complications. Drugs for controlling blood sugar are further classified into the following five categories.
( 1 ) 第 1はインシュリン製剤である。 分泌置低下により不足したインシュリンを 直接補充することを特徴とする。  (1) The first is an insulin preparation. Insulin shortage due to decreased secretion is directly replenished.
( 2 )第 2はスルホニルゥレア剤などのィンシュリン分泌促進薬である。これは KATP チャネルをブロックし脖臓 j8細胞の細胞膜を脱分極させて Ca2+を細胞内に流入させ ることにより血糖値に関係なく強制的にインシュリンを分泌させることを特徴とす る (Science(1995) 268, 423429)。 (2) The second is an insulin secretagogue such as a sulfonylprea drug. This is characterized by blocking KATP channels, depolarizing the cell membrane of kidney j8 cells, and forcing Ca 2+ to flow into the cells, thereby forcibly secreting insulin regardless of blood glucose level ( Science (1995) 268, 423429).
( 3 ) 第 3はチアゾリジンジ才ン骨格を有するインシュリン抵抗性改善薬である。 これはまだ作用メカニズムが完全に理解されたわけではないが、 脂肪細胞の ΡΡΑί¾γ (3) Third is an insulin resistance improving drug having a thiazolidinediene skeleton. Although the mechanism of action has not yet been fully understood, 脂肪 γ in adipocytes
(ペル才キソー厶増殖剤応答性受容体 T ： Peroxisome Proliferator Activated (Peroxisome Proliferator Activated T)
Receptor γ) と呼ばれる核内受容体に結合し脂肪細胞の分化を促進することにより 血糖を脂肪に取り込み、 血糖値を低下させることを特徴とする (Diabetes(1998) 4L 507-514)。 Receptor γ) binds to a nuclear receptor called “receptor γ” and promotes adipocyte differentiation to take up blood sugar into fat and reduce blood sugar level (Diabetes (1998) 4L 507-514).
( 4 ) 第 4は αダルコシダーゼ阻害薬である。 これは糖類構造を有し、 小腸の αグ ルコシダーゼを阻害することにより腸からの糖の吸収を阻害することを特徴とする。  (4) Fourth is an α-dalcosidase inhibitor. It has a saccharide structure and is characterized by inhibiting absorption of sugar from the intestine by inhibiting α-glucosidase in the small intestine.
( 5 ) 第 5はビグアナイド薬である。 その作用機構はよくわかっていない。  (5) Fifth is biguanide drugs. Its mechanism of action is not well understood.
以上の 5つ以外にもいくつかの血糖管理薬剤はあるが、 これまでのところ糖尿病 における脖臓 β細胞の疲弊状態を保護又は機能亢進することによリインシュリン分 泌不全を治癒しょうとする治療薬は存在しない。  There are several other blood sugar management drugs in addition to the above five, but so far treatments that try to cure reinsulin hyposecretion by protecting or enhancing the exhaustion of skeletal β-cells in diabetes There is no drug.
薛臓 J8細胞の疲弊によるインシユリン分泌不全の治療を目的とする治療薬を開発 するためには脖臓、 特に) 8細胞の分化機構を解明する必要があるが、 その制御機構 は未だよくわかっていない。 現在までの知見をまとめると以下の通りである。  To develop a therapeutic drug for the treatment of insulin secretion deficiency caused by exhaustion of J8 cells, it is necessary to elucidate the mechanism of differentiation of the kidney, especially (8) cells, but the control mechanism is still well understood. Absent. The following is a summary of the findings to date.
まず vivoモデル動物系での) 3細胞再生現象はセロハンラップモデル (Tumor.Biol. (1993) 14, 184-200), 90%脖臓摘出モデル (Diabetes (1993) 42, 1715-1720), TGF α と gastrin共発現卜ランスジエニックモデル (丄 Clin. Invest. (1993) 92, 1349-1356), streptozotocin (5丁ヱ)投与モデル ^0(^1101.(1997)：1 ^ 1750-1762), alloxan部分灌 流モデソレ (Diabetes(1997) 4§, 1281-1290)などいくつかの系で見られている。 この中 には新生児でしか見られない現象もあれば adultでも観察されるものも含まれる。 近年の分子生物学の進展によりこのような脬臓 ]8細胞の新生 ·再生現象が如何な る機構によって起きているのかが徐々に解明されてきている （Diabetes (1998) Z, 1817-1823参照）。脖臓形成に一番重要な役割を果たしているのがホメ才ボックスを 持つ Pdx-1 (別名 IPF-1 , IDX-1. STF-1)と呼ばれる転写因子であることがノックァゥ卜 マウスにより証明されておリ（Nature (1994) 371, 606-609, Development (1996) 122. 983-995)、 また Pdx-1は若年性糖尿病 MODYの原因遺伝子のひとつ MODY4であるこ とが明らかとなっている （Nature Genet. (1997)：11， 138-139) 。 脬臓形成後、 外分 泌組織と内分泌組織が分化してくるわけであるが、 内分泌を司るランゲルハンス島 の形成に LIMホメ才ドメインを持つ lsl-1と呼ばれる転写因子が必要であることが、や はリノックァゥトマウスにより証明された (Nature(1997) 5, 257-260)。ランゲルハ ンス島が形成されるとその後、 そこから α , /3， δ , ΡΡの 4つの細胞が形成され、 それぞれグルカゴン、 インシュリン、 ソマトスタチン、 ΡΡ (脖臓ポリペプチド） を 特異的に産生するようになる。この過程で脖臓形成に必須であつた Pdx-1は )8細胞に 限局するようになリ、 インシユリン遺伝子の転写因子として機能するようになる。 そして β細胞の分化過程/成熟化に paired-boxを持つホメォ転写因子 Pax4が必須で あることが最近ノックァゥ卜マウスにより証明された (Nature(1997) 3 , 399-402, 387. 406-409, 国際公開特許 W098/29566)。 ここではゲノ厶上の Pax4 locusに βガ ラクトシダーゼを組み換え導入することにより、 胚発生期の成熟過程の膊臓または その結果としての新生児の脬臓における Pax4遺伝子発現をそれぞれ検出している。 しかし、 成熟し終えた 3細胞に低分子化合物、 蛋白質又は天然物等の物質を作用さ せて Pax4遺伝子発現の増強作用を調べ、 該作用物質を得ることは何等示唆はない。 First of all, the cell regeneration phenomenon in a cellophane wrap model (Tumor.Biol. (1993) 14, 184-200), 90% extirpated rat model (Diabetes (1993) 42, 1715-1720), transgenic model with co-expression of TGFα and gastrin (丄 Clin. Invest. (1993) 92, 1349-1356), streptozotocin (5-chome) administration model ^ 0 (^ 1101. (1997): 1 ^ 1750-1762), alloxan partial perfusion model (Diabetes (1997) 4§, 1281-1290) Seen in the system. Some of these phenomena are found only in newborn babies, while others are observed in adults. With the progress of molecular biology in recent years, it has been gradually elucidated by what kind of mechanism the regenerative and regenerative phenomena of this kind of [8] cells occur (see Diabetes (1998) Z, 1817-1823). ). The knockout mouse proved to be the transcription factor called Pdx-1 (also known as IPF-1 or IDX-1.STF-1) having the home box, which plays the most important role in kidney formation. (Nature (1994) 371, 606-609, Development (1996) 122. 983-995), and Pdx-1 has been shown to be one of the causative genes of juvenile diabetes MODY (MODY4). Nature Genet. (1997): 11, 138-139). After kidney formation, exocrine and endocrine tissues differentiate, and the formation of the islets of Langerhans, which is responsible for endocrine, requires a transcription factor called lsl-1, which has the LIM homeodomain. And were proved by linocquat mice (Nature (1997) 5, 257-260). After the islets of Langerhans are formed, they form α, / 3, δ, and 細胞 cells, which produce glucagon, insulin, somatostatin, and ΡΡ (intestinal polypeptide), respectively. Become. In this process, Pdx-1, which was essential for kidney formation, is now restricted to 8) cells and functions as a transcription factor for the inulin gene. Recently, it was proved by knockout mice that the home transcription factor Pax4 having a paired-box is essential for β cell differentiation / maturation (Nature (1997) 3, 399-402, 387.406-409, International Patent Publication W098 / 29566). Here, the expression of Pax4 gene in the arm during embryonic development or the resulting neonatal kidney is detected by recombinantly introducing β-galactosidase into Pax4 locus on the genome. However, there is no suggestion to obtain an active substance by examining the effect of increasing the expression of Pax4 gene by applying a substance such as a low molecular compound, a protein or a natural product to the three mature cells.
)8細胞が形成されるとィンシュリン遺伝子がどのように発現するかに焦点が当て られる。 関与している転写因子としては Pdx-1 ,E47， BETA2 (NeuroD) (Mol.  The focus is on how the insulin gene is expressed when 8 cells are formed. Pdx-1, E47, BETA2 (NeuroD) (Mol.
Endocrinol. (1994) 8, 1798-1806, Genes Dev. (1995) 9, 1009-1019), p300 (Mol. Cell. Biol. (1998) 18, 2957-2964)などが知られているが、 脖臓 ]8細胞株の MIN6, )8 -TC1で は Pdx-1の発現を抑制してもィンシュリン遺伝子発現に影響が現れないなど (丄 Clin. Invest. (1997) L 1840-1846), これらの転写因子群の関与は非常に複雑でぁリ、 未だ 十分な解明はなされていないのが実状である。 Endocrinol. (1994) 8, 1798-1806, Genes Dev. (1995) 9, 1009-1019), p300 (Mol.Cell.Biol. (1998) 18, 2957-2964), etc. In the case of MIN6,) 8-TC1 in 8 cell lines, suppression of Pdx-1 expression did not affect insulin gene expression (丄 Clin. Invest. (1997) L 1840-1846). The involvement of transcription factors is very complex and has not yet been fully elucidated.
以上は脬臓内分泌細胞についての知見であるが、 脖臓外分泌細胞についても近年 報告されている。 例えば PTF1と呼ばれる転写因子複合体は p48と E12から構成され ている。 p48が薛臓外分泌細胞の分化に必須であることがノックァゥ卜マウスの実験 から明らかになつた (Genes Dev. (1998) 12, 3752-3763) 。  The above is the knowledge of the endocrine secretory cells of the kidney. Recently, the report of exogenous secretory cells of the kidney has also been reported. For example, a transcription factor complex called PTF1 is composed of p48 and E12. It has been clarified from experiments on knockout mice that p48 is essential for differentiation of excretory exocrine cells (Genes Dev. (1998) 12, 3752-3763).
ところで脬臓外分泌腺房細胞株 AR42Jが HGFまたは betacellulin単独で、 もしくは それぞれがァクチビン （Activin) A共存下でインシュリン産生細胞へと変換するこ とが最近報告された (Endocrinol(1996) 137, 3969-3976， J. Clin. Invest. (1996) 97, 1647-1654)。 外分泌細胞から内分泌細胞への変換は今まで解明されてきた過程とは 異質なものであリ興味深い。 この現象は前述した alloxan部分灌流モデル  Recently, it was recently reported that the exogenous acinar acinar cell line AR42J converts HGF or betacellulin into insulin-producing cells alone or in the presence of activin (Activin) A (Endocrinol (1996) 137, 3969-). 3976, J. Clin. Invest. (1996) 97, 1647-1654). The conversion of exocrine cells to endocrine cells is foreign to the process that has been elucidated and is interesting. This phenomenon is based on the alloxan partial perfusion model described above.
(Diabetes(1997) 46, 1281 -1290)でも脬管細胞 (duct cell)で見られており、今後のメカ ニズ厶解明が待たれるが Pdx-1の発現が関与していることは示唆されている。 AR42J のインシュリン産生細胞への変換に関与したァクチビン Aは、 これまでに脬臓 /3細 胞において 1時間程度の短時間処理によリインシュリン分泌を促進するとの報告が なされているが (Endocrinol. (1993) 133, 624-630, Life Science (1993) 53, 1069- 1078)、 その機構は細胞内 Ca2+ »度を高めることによるインシュリン分泌であり (FEBS Lett. (1993) 329, 194-198, Mol. Cell. Endocrinol. (1995) 1 13, 83-87, J. Mol. Endocrinol. (1996) 16, 249-258)、 脖臓 i8細胞の分化あるいは機能維持に関与してい るとの報告ではない。一方インシュリン産生細胞 INS-1に各種成長因子を接触させた 結果、 ァクチビン Aがインシュリン含量増加作用を有するという結果が最近示され た (Endocrinology (1998) 139. 1494-1499) 。 (Diabetes (1997) 46, 1281-1290) also found in duct cells, suggesting that expression of Pdx-1 is involved, although further elucidation of the mechanism is awaited. ing. It has been reported that activin A, which is involved in the conversion of AR42J to insulin-producing cells, promotes the secretion of reinsulin by short-term treatment of about 1 hour in kidney / 3 cells (Endocrinol. (1993) 133, 624-630, Life Science (1993) 53, 1069-1078), and the mechanism is insulin secretion by increasing intracellular Ca 2+ » (FEBS Lett. (1993) 329, 194-198, Mol. Cell.Endocrinol. (1995) 1 13, 83-87, J. Mol. Endocrinol. (1996) 16, 249-258), It is not a report that it is involved in differentiation or function maintenance. On the other hand, as a result of contacting various growth factors with the insulin-producing cell INS-1, it was recently shown that activin A has an effect of increasing the insulin content (Endocrinology (1998) 139. 1494-1499).
ァクチビンは卜ランスフォーミング成長因子 j8 (TGFp)スーパーファミリ一に属 する蛋白質である。このファミリーには TGFpや BMP (bone morphogenetic protein)、 MIS (Mullerian inhibiting substance)などが含まれる。 ァクチビンには) 8 A、 /3 Bの 2 種類のサブユニットが存在し、 活性型はホモダイマー （/3A- i8 A， )3 B- )3 B) または ヘテロダイマー（j8A- i8 B)を形成したもので、 それぞれァクチビン A、 ァクチビン B およびァクチビン ABと呼ばれている。 発現は全身各所で見られ、 下垂体での卵胞刺 激ホルモン （FSH： follicle stimulating hormone)産生刺激、 血管内皮細胞の増殖、 赤 芽球細胞の分化、 Xenopusの中胚葉誘導など様々な活性を持っていることが知られ ている（蛋白質 '核酸 ·酵素（1998) 43, 49卜 501参照）。 ァクチビンはァクチビン受 容体に結合することにより活性が現れる。 シグナル伝達のカスケ一ドは次の通りで ある。 II型ァクチビン受容体はァクチビンの結合により活性化されて I型ァクチビン 受容体をリン酸化する。次に I型ァクチビン受容体が Smad2または Smad3と呼ばれる 蛋白質のカルボキシル末端のセリン残基をリン酸ィ匕し、これら Smadが Smad4とへテ 口ダイマーを形成して核へ移行し、 標的とする遺伝子の転写を促進することがショ ウジヨウバエ (Drosophila)、 アフリカッメガエル (Xenopus)、 哺乳類等、 種を問わず 様々な細胞において報告されている (Nature(1997) ^Q， 465-471 , Curr. Opin. Genet. (1997) 7, 467473参照）。  Activin is a member of the transforming growth factor j8 (TGFp) superfamily. This family includes TGFp, BMP (bone morphogenetic protein), and MIS (Mullerian inhibiting substance). Activin has two subunits, 8 A and / 3 B, and the active form forms homodimer (/ 3A-i8 A,) 3 B-) 3 B) or heterodimer (j8A-i8 B) These are called activin A, activin B and activin AB, respectively. Expression is found throughout the body and has various activities such as stimulation of follicle stimulating hormone (FSH) production in the pituitary gland, proliferation of vascular endothelial cells, differentiation of erythroid cells, and induction of mesoderm of Xenopus. (Protein 'Nucleic acid · Enzyme (1998) 43, 49, 501). Activin exhibits activity by binding to the activin receptor. The cascade of signal transmission is as follows. The type II activin receptor is activated by the binding of activin to phosphorylate the type I activin receptor. Next, the type I activin receptor phosphorylates a serine residue at the carboxyl terminus of a protein called Smad2 or Smad3, and these Smads form a heterodimer with Smad4 and translocate to the nucleus to target genes. It has been reported that various types of cells, such as Drosophila, Xenopus, and mammals, promote transcription of various species (Nature (1997) ^ Q, 465-471, Curr. Opin. Genet. (1997) 7, 467473).
このようにァクチビンとァクチビン受容体、 およびその下流のシグナル伝達機構 はかなり解明されてきているが、 Pax4との関係についての示唆や報告は全くない。 以上より、 薛臓 /3細胞内の Pax4遺伝子発現増強物質をスクリーニングする方法、 当該 Pax4遺伝子発現増強物質の存在、当該 Pax4遺伝子発現増強作用に基づく糖尿病 における脖臓) 8細胞の疲弊状態の保護ないし機能亢進させるというメカニズム、 及 び本メカニズムからなる糖尿病を治癒しょうとする治療薬へのアプローチは全く知 られていない。 発明の開示 Thus, although activin and the activin receptor, and the downstream signal transduction mechanism, have been elucidated considerably, there is no suggestion or report on the relationship with pax4. Based on the above, a method for screening for a Pax4 gene expression enhancer in the sarcolemma / 3 cells, the presence of the Pax4 gene expression enhancer, the pancreas in diabetes based on the Pax4 gene expression enhancer) There is no known mechanism of hyperfunction, and no approach to therapeutic drugs that cure diabetes that is composed of this mechanism. Disclosure of the invention
本発明の目的は、 脖臓 3細胞内 Pax4遺伝子発現量を検出する手段を含む脬臓 β細 胞内 Pax4発現増強作用を有する物質のスクリーニング方法、 当該スクリーニング法 によリ得られた物質を有効成分とする脖臓 i8細胞内 Pax4遺伝子発現増強用医薬組成 物、 並びにァクチビン受容体ァクチベータ一を有効成分とする脖臓 i8細胞内 Pax4遺 伝子発現増強用医薬組成物を提供するものである。 本発明脬臓 β細胞内 Pax4遺伝子 発現増強用医薬組成物を単独で、 またはスルホニルゥレア剤ゃィンシュリン抵抗性 改善薬、 腸管の糖吸収阻害薬などの血糖管理用薬剤や合併症用薬剤と併せて使用す ることによリ優れた糖尿病治療効果を示すことが期待できる。 本発明はこのような 新しい概念による治療薬の創製を目指している。  An object of the present invention is to provide a method for screening a substance having Pax4 expression enhancing activity in mouse β cells, which comprises means for detecting the expression level of Pax4 gene in mouse 3 cells, and to effectively use the substance obtained by the screening method. It is intended to provide a pharmaceutical composition for enhancing expression of Pax4 gene in splanchnic i8 cells as a component, and a pharmaceutical composition for enhancing expression of Pax4 gene in splanchnic i8 cells, which comprises an activin receptor activator as an active ingredient. The pharmaceutical composition for enhancing expression of Pax4 gene in the hepatic β-cell of the present invention alone or in combination with a drug for controlling blood sugar or a drug for complications, such as a sulfonylurea agent, an agent for improving dinsuline resistance, and an intestinal glucose absorption inhibitor. It can be expected to show an excellent diabetes treatment effect by using it. The present invention aims to create a therapeutic drug based on such a new concept.
本発明者はかかる技術水準下、 薛臓 0細胞内の Pax4遺伝子発現増強作用を有する 物質が存在すれば、 脬臓 0細胞の疲弊状態からの保護又は脖臓 β細胞の機能亢進に 結びつけられるかもしれないという点に着目し、 成熟脖臓 3細胞内の Pax4遺伝子発 現量を測定できるスクリーニング法を見出し本発明を完成した。 更に本発明スクリ 一ニング法を用いて当該 Pax4遺伝子発現量を増強させる作用を有する物質につき銳 意検討した結果、 予想外にもこれまで Pax4遺伝子との関連について全く報告されて いないァクチビン受容体ァクチベータ一が脖臓 β細胞内の Pax4遺伝子発現増強作用 を有することを、 更にまた当該 Pax4遺伝子発現量増強作用物質であるァクチビン受 容体ァクチべ一ターが脖臓; 8細胞におけるインシュリン遺伝子発現量も増強させる ことを見い出し本発明を完成した。 発明の実施の形態 The inventor of the present invention believes that, under the state of the art, the presence of a substance having the Pax4 gene expression-enhancing activity in the xenal 0 cells may lead to the protection of the 0-cells from exhaustion or the enhancement of the function of the 0-cells. Focusing on the fact that it cannot be performed, the present inventors have found a screening method capable of measuring the expression level of Pax4 gene in three mature kidney cells, and completed the present invention. Furthermore, the present inventors screened the substance having the action of enhancing the expression level of the Pax4 gene using the screening method of the present invention. As a result, unexpectedly, an activin receptor activator that has never been reported in relation to the Pax4 gene has been unexpectedly reported. One of which has the effect of enhancing the expression of Pax4 gene in rat β-cells. The present inventors have found that the receptor activator also enhances the expression level of the insulin gene in the kidney; 8 cells, and completed the present invention. Embodiment of the Invention
以下、 本発明について詳細に説明する。  Hereinafter, the present invention will be described in detail.
本発明は、 The present invention
( 1 ) 少なくとも脬臓 i8細胞に被験物質を接触させ、 該細胞内の Pax4遺伝子発 現量を検出する手段を含む Pax4遺伝子発現増強作用を有する物質のスクリーニング 法、  (1) a method for screening a substance having a Pax4 gene expression enhancing action, which comprises a means for contacting a test substance with at least a mouse i8 cell and detecting the amount of Pax4 gene expression in the cell;
( 2 ) ( 1 ) 記載のスクリーニング法より得られる物質を有効成分とする脖臓 )8細胞内 Pax4遺伝子発現増強用医薬組成物、 または、  (2) a kidney containing a substance obtained by the screening method according to (1) as an active ingredient) 8 a pharmaceutical composition for enhancing Pax4 gene expression in cells, or
( 3 ) ァクチビン受容体ァクチベータ一を有効成分とする薛臓) 8細胞内 Pax4遺 伝子発現増強用医薬組成物である'。  (3) A pharmaceutical composition for enhancing the expression of Pax4 gene in cells (8), which contains an activin receptor activator as an active ingredient.
さらに脖臓 )3細胞内 Pax4遺伝子発現増強用医薬組成物を製造するための前記（ 1 ) 記載のスクリーニング法により得られる物質の使用ゃ脖臓 0細胞内 Pax4遺伝子発現 増強用医薬組成物を製造するための前記 （3 ) 記載のァクチビン受容体ァクチべ一 ターの使用である。 本発明で使用される語句は、 以下の通りである。  (3) Use of the substance obtained by the screening method described in (1) above to produce a pharmaceutical composition for enhancing Pax4 gene expression in 3 cells; and 4) Preparation of a pharmaceutical composition for enhancing Pax4 gene expression in cells. The use of the activin receptor activator according to (3) for carrying out the above. The terms used in the present invention are as follows.
Γ脖臓; 3細胞」 とは、 分化 ·再生後の成熟した脖臓 3細胞を示し、 哺乳類由来の細 胞又は株化された細胞が好ましい。 具体的には、 細胞の研究に使用される RIN5細 胞（Proc. Natl. Acad. Sci. USA (1977) 74, 628-630)、 HIT細胞 (Proc. Natl. Acad. Sci. USA (1981) 78, 4339-4342) 、 MIN6細胞 (Endocrinol. (1990) 127, 126-132) 、 β TC細胞 （Endocrinol. (1990) 126, 2815-2822, Diabetes (1993) 42, 901-907) 、 NIT1 細胞（Diabetes (1991) 40, 842-849)、 INS-1細胞（Endocrinol. (1992) 130, 167-178)、 /3 HC細胞 (Mol. Cell. Biol.(1993) 13, 4223-4232) 等が樹立されている。 The term “lean; three cells” refers to mature three kidney cells after differentiation and regeneration, and is preferably a mammal-derived cell or an established cell. Specifically, RIN5 cells (Proc. Natl. Acad. Sci. USA (1977) 74, 628-630) and HIT cells (Proc. Natl. Acad. Sci. USA (1981)) used for cell studies 78, 4339-4342), MIN6 cells (Endocrinol. (1990) 127, 126-132), β TC cells (Endocrinol. (1990) 126, 2815-2822, Diabetes (1993) 42, 901-907), NIT1 Cells (Diabetes (1991) 40, 842-849), INS-1 cells (Endocrinol. (1992) 130, 167-178), / 3 HC cells (Mol. Cell. Biol. (1993) 13, 4223-4232) Etc. have been established.
Γ脖臓 )8細胞内 Pax4遺伝子」 とは、 前述の通り脬臓形成後、 外分泌組織と内分泌 組織が分化し、内分泌細胞分化における /3細胞の分化過程に機能している paired-box を持つホメォ転写因子 Pax4をコードする遺伝子または当該機能を有する部分断片を 示す。 As described above, the `` 8 intracellular Pax4 gene '' means that after formation of the spleen, exocrine and endocrine tissues are differentiated and have a paired-box that functions in the / 3 cell differentiation process in endocrine cell differentiation. 1 shows a gene encoding the homeo transcription factor Pax4 or a partial fragment having the function.
「少なくとも脖臓) 8細胞に被験物質を接触させ、 該細胞内の Pax4遺伝子発現量を 検出する手段を含む Pax4遺伝子発現増強作用を有する物質のスクリーニング法」 で 用いる被検物質とは、 公知又は新規のタンパク質 （抗体又は抗体の部分断片も包含 する） 、 ペプチド又は化合物を示す。 例えばケミカルファイルに登録されている化 合物や、 新規なものとしては公知化合物の置換基等を常法によリ化学修飾させたも の、 コンビナトリアル ·ケミストリー技術 (Tetrahedron (1995) ^1， 8135-8137)によ つて得られた化合物群、 常法のペプチド合成、 遺伝子工学的手法により得られるぺ プチド若しくはタンパク質やファージ ·ディスプレー法 (丄 Mol. Bioに（1991) 222, 301-310)などを応用して作成されたランダム ·ペプチド群を被検物質として用いる ことができる。 また微生物の培養上清や、 植物、 海洋生物由来の天然成分なども使 用できる。  The test substance used in the "screening method for a substance having a Pax4 gene expression enhancing effect including a means for detecting a Pax4 gene expression level in a cell by contacting the test substance with at least 8 cells" A novel protein (including an antibody or a partial fragment of an antibody), a peptide or a compound is indicated. For example, a compound registered in a chemical file, or a novel compound obtained by chemically modifying a substituent of a known compound by a conventional method (Tetrahedron (1995) ^ 1, 8135). -8137), peptides or proteins obtained by conventional techniques, peptide engineering or phage display method (丄 Mol. Bio (1991) 222, 301-310) A random peptide group created by applying the method can be used as a test substance. Also, culture supernatants of microorganisms and natural components derived from plants and marine organisms can be used.
更に本発明スクリ一二ング法で得られた公知化合物を基に置換基を化学的に修飾 したり、 コンビナ卜リアル ·ケミストリー技術やファージ ·ディスプレー法を用い ても被検物質は得られる。 置換基の化学的修飾とは、 常法のアルキル化、 アミド化、 エステル化、 酸化、 又は還元反応等による修飾を意味する。 ペプチド合成の場合は, 各種保護基、 カップリング試薬などを駆使して行うカップリング法、 カルボキシル 末端活性化法、 ァミノ末端活性化法などの液相法を用いて合成することが可能であ リ、 さらに簡便に生産及び精製することができる固相法 [丄 Am. C em. Soc. (1963) 85, 2185によって初めて紹介されその後改良が加えられている方法] を適用して合 成することもできる。 ペプチド合成機は例えばパーキエルマ一アプライド ·バイオ システム社のペプチド合成機、 4 3 O Aが市^されており、 此の装置の標準的運転 プログラムに従ってべプチド合成を行えばよい。遺伝子工学的手法を用いる場合は， 目的とするペプチド又はタンパク質のアミノ酸をコードする遺伝子を大腸菌等に形 質転換し， 遺伝子を増幅させその遺伝子を動物細胞に卜ランスフエク卜し （この場 合、 自己複製可能で転写プロモーター領域を含むプラスミドもしくは動物細胞の染 色体に組み込まれうるようなプラスミドの何れでもよい） 、 当該タンパク質を生産 させることができる。 また当該物質は前述の天然成分も意味する。 Furthermore, a test substance can be obtained by chemically modifying a substituent based on a known compound obtained by the screening method of the present invention, using a combinatorial chemistry technique or a phage display method. The chemical modification of a substituent means a modification by a conventional method such as alkylation, amidation, esterification, oxidation, or reduction reaction. In the case of peptide synthesis, it can be synthesized using a liquid phase method such as a coupling method using a variety of protecting groups and coupling reagents, a carboxyl terminal activation method, and an amino terminal activation method. A solid-phase method that can be produced and purified more easily [丄 Am. Cem. Soc. (1963) 85, 2185, which were first introduced and subsequently improved]. As the peptide synthesizer, for example, 43 OA, a peptide synthesizer manufactured by Perchierma Applied Biosystems Co., Ltd. is available, and the peptide synthesis may be performed according to a standard operation program of this apparatus. When the genetic engineering technique is used, the gene encoding the amino acid of the target peptide or protein is transformed into E. coli or the like, the gene is amplified, and the gene is transfected into animal cells (in this case, self- Any of a plasmid that is replicable and contains a transcription promoter region or a plasmid that can be incorporated into a chromosome of an animal cell) may be used to produce the protein. The substance also means the aforementioned natural component.
Γ脖臓) 3細胞内 Pax4遺伝子発現増強作用を有する物質」 とは、 脬臓) 8細胞を刺激又 は接触することにより、 本発明スクリーニング法により得られる方法またはそれ以 外の手法で得られる脬臓 0細胞内の Pax4遺伝子発現垦を増強させる性質を有するも のなら何れでもよく、具体的にはタンパク質（抗体又は抗体の部分断片も包含する）、 ぺプチド又は化合物を示す。 これらの物質は前記被検物質と同様な手法により入手 可能である。 好ましくはァクチビン受容体を活性化させる物質、 即ちァクチビン受 容体ァクチべ一ターである。  (Leather) (3) a substance having an effect of enhancing intracellular Pax4 gene expression '' can be obtained by the method obtained by the screening method of the present invention or other methods by stimulating or contacting the (liver) 8 cells Any substance having the property of enhancing the expression of Pax4 gene in the gland 0 cells may be used, and specific examples include a protein (including an antibody or a partial fragment of an antibody), a peptide or a compound. These substances can be obtained in the same manner as the test substance. Preferably, it is a substance that activates an activin receptor, that is, an activin receptor activator.
前述の被検物質又は脬臓) 8細胞内 Pax4遺伝子発現増強作用を有する物質は酸又は 塩基と塩を形成する可能性がある。 無機酸若しくは有機酸との酸付加塩， あるいは 無機塩基若しくは有機塩基との塩であり， 製薬学的に許容しうる塩が好ましい。 こ れらの塩としては， 具体的には塩酸， 臭化水素酸， ヨウ化水素酸， 硫酸， 硝酸若し くはリン酸等の鉱酸， 又は， ギ酸， 酢酸， プロピオン酸， シユウ酸， マロン酸， コ ハク酸， フマル酸， マレイン酸， 乳酸， リンゴ酸， 酒石酸， クェン酸， メタンスル ホン酸若しくはエタンスルホン酸等の有機酸， 又はァスパラギン酸若しくはグルタ ミン酸などの酸性アミノ酸との酸付加塩， ナトリウム， カリウム， マグネシウム， カルシウム， アルミニウムなど無機塩， メチルァミン， ェチルァミン， エタノール ァミンなどの有機塩基塩， リジン， オル二チンなどの塩基性アミノ酸との塩等を挙 げることができる。 (The above-mentioned test substance or kidney) 8A substance having an intracellular Pax4 gene expression enhancing action may form a salt with an acid or a base. It is an acid addition salt with an inorganic acid or an organic acid, or a salt with an inorganic base or an organic base, and a pharmaceutically acceptable salt is preferable. Specific examples of these salts include mineral acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid and phosphoric acid, or formic acid, acetic acid, propionic acid, oxalic acid, and the like. Acid addition with malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, organic acid such as methanesulfonic acid or ethanesulfonic acid, or acidic amino acid such as aspartic acid or glutamic acid Salt, sodium, potassium, magnesium, Examples include inorganic salts such as calcium and aluminum, organic base salts such as methylamine, ethylamine and ethanolamine, and salts with basic amino acids such as lysine and ordithine.
Γ脬臓 0細胞内 Pax4遺伝子発現増強用医薬組成物」 とは、 哺乳動物、 特にヒ卜に 投与される医薬組成物であリ、 前述の脬臓 /3細胞内 Pax4遺伝子発現増強作用を有す る物質を有効成分とする医薬組成物である。 好ましくはァクチビン受容体ァクチべ 一ターを有効成分とする脖臓 i8細胞内 Pax4遺伝子発現増強用医薬組成物である。 薛 臓 β細胞の疲弊からの保護又は当該細胞の機能： ft進に基づく糖尿病若しくは糖尿病 合併症治療又は予防用医薬組成物である。  The term “pharmaceutical composition for enhancing Pax4 gene expression in mouse 0 cells” refers to a pharmaceutical composition administered to mammals, especially humans, and has the above-mentioned effect of enhancing Pax4 gene expression in mouse / 3 cells. This is a pharmaceutical composition containing the following substances as active ingredients. Preferably, it is a pharmaceutical composition for enhancing Pax4 gene expression in porcine i8 cells, comprising an activin receptor activator as an active ingredient. X-ray The pharmaceutical composition for the treatment or prevention of diabetes or diabetic complications based on ft-progression protection or function of β-cells from exhaustion.
Γ脬臓 3細胞の疲弊からの保護又は当該細胞の機能亢進」 とは、 以下に示す通リ である。 高ィンシュリン血症状態時又は既存の糖尿病薬剤投与によリ脖隨 iS細胞に 過剰ィンシュリン合成または該細胞内既存のィンシュリン分泌を促した結果生じる、 当該機能の低下若しくは機能喪失に近い状態からの保護又は機能回復を示す。  “Protection of the three cells of the kidney from exhaustion or enhancement of the function of the cells” is as follows. During hyperinsulinemia or with the administration of existing diabetic drugs, protection from iS cells with excessive insulin synthesis or secretion of existing insulin, resulting in reduced or near-function of the relevant function Or it indicates functional recovery.
本発明スクリーニング法で使用される各工程は、 公知の方法 （Maniatis，T. et al. (1982)： "Molecular Cloning - A Laboratory Manuaに Cold Spring Harbor Laboratory, NY) 等に従って実施可能である。 例えば以下の通りである。  Each step used in the screening method of the present invention can be performed according to a known method (Maniatis, T. et al. (1982): "Molecular Cloning-A Laboratory Manua at Cold Spring Harbor Laboratory, NY). It is as follows.
まず被検物質を脬臓) 8細胞に接触させた後、 当該細胞から常法により全 RNAを抽 出する。これを（ 1 )ァガロースゲル電気泳動などによリサイズで分画後、当該 mRNA をノーザンブロッテイング法により検出したり、 （2 ) また当該 mRNAに相補的な RNAとハイブリダィズしたあとに RNaseにより消化を行い、 消化されない RNAを電 気泳動後に検出する RNA protection assay (RPA)法によリ当該 mRNAを検出するこ とができる。 （3 ) さらには当該 mRNAを逆転写酵素反応に供し目的の cDNAを得、 当該 cDNA発現量を常法によリ検出、 測定することも可能である。  First, a test substance is brought into contact with 8 cells, and then total RNA is extracted from the cells by a conventional method. This is fractionated by resizing such as (1) agarose gel electrophoresis, and then the mRNA is detected by Northern blotting. (2) The RNA is digested with RNase after being hybridized with RNA complementary to the mRNA. Alternatively, the mRNA can be detected by RNA protection assay (RPA), which detects undigested RNA after electrophoresis. (3) Furthermore, the mRNA can be subjected to a reverse transcriptase reaction to obtain a target cDNA, and the expression level of the cDNA can be detected and measured by a conventional method.
全 RNA抽出法としては、 グァニジンチオシァネー卜 'ホット 'フエノール法、 グ ァニジンチ才シァネ一卜—グァニジン ·塩酸法等が挙げられる。 Guanidine thiosane 'hot' phenol method, total RNA extraction method Anidinchi singer-guanidine-hydrochloric acid method.
上記 （1 ) の mRNAの検出においては3 2 P又は3 5 Sのラジオアイソトープでラベ ルした核酸と当該 mRNAを結合させてその放射活性を測定する。 上記 （2 ) の RPA 法においては、 当該 mRNAの部分断片に相補的な cRNAを3 2 P又は3 5 Sのラジオァ イソ卜ープでラベルして、電気泳動後にその放射活性を測定するのが普通であるが、 最近では Cytostar-Tというシンチレ一ティングプレート上で細胞を培養することに よリ、 全 RNAを抽出することなしに細胞内の当該 RNA量を RPA法を用いて直接測定 する方法がアマシャム社より開発されている。 上記 （3 ) の方法においては逆転写 反応を行う前に全 RNAから mRNAを精製してもよい。 精製方法としてはオリゴ (dT) セルロースカラムによる吸着■溶出やオリゴ (dT)磁気ビーズを用いた精製などが挙 げられる。 さらにショ糖密度勾配遠心法等によリ mRNAを分画することもできる。 また mRNAを抽出せずとも市販されている抽出済み mRNAを用いてもよい。 逆転写 反応による cDNAの合成法としては、 RNAをランダムプライマー又はオリゴ (dT)ブラ イマ一の存在下で逆転写酵素反応により第 1鎖 cDNAを合成し、目的の遺伝子の一部 の領域を挟んだ 2種類のプライマーを用いて当該第 1鎖 cDNAを PCRに供し目的と する DNA断片を増幅させる。 In the detection of the mRNA of (1) it is bound to 3 2 P or 3 5 S radioisotope in label nucleic acid and the mRNA of measuring its radioactivity. In RPA method (2), and labeled with 3 2 P or 3 5 S Rajioa iso Bok-loop of the complementary cRNA to a partial fragment of the mRNA, it is to measure its radioactivity after electrophoresis Usually, but recently, by culturing cells on a scintillating plate called Cytostar-T, a method for directly measuring the amount of RNA in cells using the RPA method without extracting total RNA Has been developed by Amersham. In the above method (3), mRNA may be purified from total RNA before performing the reverse transcription reaction. Examples of the purification method include adsorption / elution using an oligo (dT) cellulose column and purification using oligo (dT) magnetic beads. Furthermore, mRNA can be fractionated by sucrose density gradient centrifugation or the like. Alternatively, a commercially available extracted mRNA may be used without extracting the mRNA. As a method of synthesizing cDNA by reverse transcription reaction, first strand cDNA is synthesized by reverse transcriptase reaction of RNA in the presence of random primer or oligo (dT) primer, and a part of the target gene is sandwiched. The first strand cDNA is subjected to PCR using two kinds of primers to amplify a target DNA fragment.
次に DNAの検出法としては、 増幅させた DNAをァガロースゲル等で電気泳動して 分画後ェチヂゥ厶ブ口マイド染色して DNA量を半定量測定してもよいし、 また3 2 P のラジオアイソトープでラベルした核酸と当該 DNAを結合させてその放射活性を測 定するか、 又は蛍光基質ゃジゴキシゲニンなどのハプテンでラベルした抗体を用い て当該 DNAと結合させ、 化学発光 ·蛍光 ·発色などにより検出する方法が挙げられ る。 さらに最近では TaqMANプローブと呼ばれる蛍光基質でラベルした核酸と当該 DNAを結合させ、 PCR法と組み合わせて TaqDNAポリメラーゼの 5'→3'ェキソヌク レアーゼ活性を利用し、 伸長反応の際に加水分解される蛍光基質からの蛍光を測定 する方法がパーキンエルマ一アプライドバイオシステムズ社から開発されている。 以下に本発明脖臓 0細胞内 Pax4遺伝子発現増強作用を有する物質の製剤化法， 投 与方法を詳述する。 Next, as the detection of DNA, to the amount of DNA was electrophoresed to fractionation after Echidjiu厶Buguchi amide staining the amplified DNA in Agarosugeru like may be measured semi-quantitatively, also 3 2 P radio Either bind the isotope-labeled nucleic acid to the DNA and measure its radioactivity, or bind to the DNA using a hapten-labeled antibody such as the fluorescent substrate ゃ digoxigenin and bind to the DNA, and perform chemiluminescence, fluorescence, color development, etc. There is a method for detection. More recently, the DNA has been combined with a nucleic acid labeled with a fluorescent substrate called a TaqMAN probe, and combined with the PCR method, utilizing the 5 '→ 3' exonuclease activity of Taq DNA polymerase, the fluorescence that is hydrolyzed during the extension reaction Measures fluorescence from substrate A method has been developed by PerkinElmer Applied Biosystems. Hereinafter, a method for formulating and administering a substance having an action of enhancing Pax4 gene expression in the pancreatic 0 cells of the present invention will be described in detail.
本発明物質やその製薬学的に許容される塩の 1種又は 2種以上を有効成分として 含有する医薬組成物は， 通常用いられている製剤用の担体ゃ賦形剤， その他の添加 剤を用いて， 錠剤， 散剤， 細粒剤， 顆粒剤， カプセル剤， 丸剤， 液剤， 注射剤， 坐 剤， 軟膏， 貼付剤等に調製され， 経口的又は非経口的に投与される。  Pharmaceutical compositions containing one or more pharmaceutically acceptable salts of the substance of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient may be used in combination with carriers, excipients and other additives commonly used for pharmaceuticals. It is used to prepare tablets, powders, fine granules, granules, capsules, pills, solutions, injections, suppositories, ointments, patches, etc., and is administered orally or parenterally.
本発明のヒ卜に対する臨床投与量は適用される患者の症状， 体重， 年令や性別等 を考慮して適宜決定される。  The clinical dose for the human of the present invention is appropriately determined in consideration of the patient's symptoms, weight, age, sex, and the like.
通常成人 1 日当り経口で 0 . 1 ~ 5 0 0 mg， 非経口で 0 . 0 1〜1 0 0 mgであり， これを 1回あるいは数回に分けて投与する。 投与量は種々の条件で変動するので， 上記投与量範囲より少ない量で十分な場合もある。 なお、 本発明脖臓 3細胞内 Pax4 遺伝子発現増強作用を有する物質は、 他の薬物例えばスルホニルゥレア剤ゃィンシ ュリン抵抗性改善薬， αダルコシダーゼ阻害薬、 ビグアナイド薬等を組み合わせて 同時にあるいは時間をおいて併用することもできる。  It is usually 0.1 to 500 mg orally and 0.1 to 100 mg parenterally per adult per day, and is administered once or in several divided doses. Since the dosage varies under various conditions, an amount smaller than the above dosage range may be sufficient. The substance having an effect of enhancing Pax4 gene expression in the three cells of the gut of the present invention may be used simultaneously or with a combination of other drugs such as a sulfonylurea agent dinsulin resistance improving agent, an α-dalcosidase inhibitor, a biguanide agent and the like. Can be used together.
Γスルホニルゥレア剤 j としては、 ァセ卜へキサミド、 ダリクラジド、 ダリクロ ビラミド、 ダリベンクラミド、 クロルプロバミド、 トラザミドまたは卜ルブタミド 等が挙げられる。  Examples of the {sulfonyl} rea agent j include acetatehexamide, daliclazide, dalicloviramide, dalibenclamide, chlorpropamide, tolazamide or tolbutamide.
「インシュリン抵抗性改善薬」 としては卜ログリタゾン、 ピオグリタゾン、 シグ リタゾン、 ェングリタゾン又はロシグリタゾン等が挙げられる。  Examples of the “insulin sensitizer” include troglitazone, pioglitazone, siglitazone, englitazone or rosiglitazone.
Γ αダルコシダーゼ阻害薬」 としてはァカルボース、 ボグリボース、 ミグリ！ ^一 ル又はエミグリテ一卜等が挙げられる。  Γ α-darcosidase inhibitors include acarbose, voglibose, and miguri! And emigrites.
「ビグアナィド薬」 としてはメ卜ホルミン又はブホルミン等が挙げられる。  Examples of the “biguanide drug” include metformin and buformin.
本発明による経口投与のための固体組成物としては， 錠剤， 散剤， 顆粒剤等が用 いられる。 このような固体組成物においては， 一つ又はそれ以上の活性物質が， 少 なくとも一つの不活性な希釈剤， 例えば乳糖， マンニトール， ブドウ糖， ヒドロキ シプロピルセルロース， 微結晶セルロース， デンプン， ポリビニルピロリドン， メ タケイ酸又はアルミン酸マグネシウムと混合される。 組成物は， 常法に従って， 不 活性な希釈剤以外の添加剤， 例えばステアリン酸マグネシウムのような潤滑剤や織 維素グリコール酸カルシウムのような崩壊剤， ラクトースのような安定化剤， ダル タミン酸又はァスパラギン酸のような可溶化剤又は溶解補助剤を含有していてもよ い。 錠剤又は丸剤は必要によリショ糖， ゼラチン， ヒドロキシプロピルセルロース， ヒドロキシプロピルメチルセルロースフタレー卜などの胃溶性あるいは腸溶性物質 のフイルムで被膜してもよい。 As the solid composition for oral administration according to the present invention, tablets, powders, granules and the like are used. Can be. In such a solid composition, the one or more active substances include at least one inert diluent, such as lactose, mannitol, glucose, hydroxypropylcellulose, microcrystalline cellulose, starch, polyvinylpyrrolidone. , Mixed with metasilicate or magnesium aluminate. The composition may be formulated in accordance with the usual practice with additives other than inert diluents, such as lubricants such as magnesium stearate, disintegrants such as calcium fiber glycolate, stabilizers such as lactose, daltamine. A solubilizing agent or solubilizing agent such as an acid or aspartic acid may be contained. The tablets or pills may be coated with a gastric or enteric film such as sucrose, gelatin, hydroxypropylcellulose, or hydroxypropylmethylcellulose phthalate, if necessary.
経口投与のための液体組成物は， 薬剤的に許容される乳濁剤， 溶液剤， 懸濁剤， シロップ剤， エリキシル剤等を含み， 一般的に用いられる不活性な希釈剤， 例えば 精製水， エチルアルコールを含む。 この組成物は不活性な希釈剤以外に可溶化乃至 溶解補助剤， 湿潤剤， 懸濁剤のような補助剤， 甘味剤， 風味剤， 芳香剤， 防腐剤を 含有していてもよい。  Liquid compositions for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, elixirs and the like, and commonly used inert diluents, such as purified water. , Including ethyl alcohol. The composition may contain, in addition to the inert diluent, solubilizing or solubilizing agents, wetting agents, auxiliary agents such as suspending agents, sweetening agents, flavoring agents, fragrances, and preservatives.
非経口投与のための注射剤としては， 無菌の水性又は非水性の溶液剤， 懸濁剤， 乳濁剤を包含する。 水性の溶液剤， 懸濁剤の希釈剤としては， 例えば注射剤用蒸留 水及び生理食塩水が含まれる。 非水溶性の溶液剤， 懸濁剤の希釈剤としては， 例え ばプロピレングリコール， ポリエチレングリコール， 才リーブ油のような植物油， エチルアルコールのようなアルコール類， ポリソルベー卜 8 0 (商品名。 ポリオキ シエチレンソルビタン高級脂肪酸エステル） 等がある。 このような組成物は， さら に等張化剤， 防腐剤， 湿潤剤， 乳化剤， 分散剤， 安定化剤 （例えば， ラク卜ース） ， 可溶化乃至溶解補助剤のような添加剤を含んでもよい。 これらは例えばバクテリア 保留フィルターを通す通過， 殺菌剤の配合又は照射によって無菌化される。 これら は又無菌の固体組成物を製造し， 使用前に無菌水又は無菌の注射用溶媒に溶解して 使用することもできる。 Injections for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Diluents for aqueous solutions and suspensions include, for example, distilled water for injections and physiological saline. Examples of diluents for water-insoluble solutions and suspensions include, for example, propylene glycol, polyethylene glycol, vegetable oils such as crude oil, alcohols such as ethyl alcohol, and polysorbate 80 (trade name: Polyoxyl). Ethylene sorbitan higher fatty acid ester). Such compositions may further comprise additives such as tonicity agents, preservatives, wetting agents, emulsifiers, dispersants, stabilizers (eg, lactose), and solubilizing or solubilizing agents. May be. These are sterilized by, for example, passage through a bacteria retaining filter, blending of a bactericide or irradiation. these Alternatively, a sterile solid composition can be prepared and dissolved in sterile water or a sterile injectable solvent before use.
本発明化合物の溶解性が低い場合には， 可溶化処理を施してもよい。 可溶化処理 としては， 医薬製剤に適用できる公知の方法， 例えば界面活性剤 （ポリオキシェチ レン硬化ヒマシ油類， ポリ才キシエチレンソルビタン高級脂肪酸エステル類， ポリ 才キシェチレンポリオキシプロピレングリコール類， ショ糖脂肪酸エステル類等） を添加する方法， 薬物と可溶化剤例えば高分子 （ハイドロキシプロピルメチルセル ロース （H P M C ) , ポリエチレングリコール （P E G ) 等の水溶性高分子， カル ボキシメチルェチルセルロース （C M E C ) ， ハイドロキシプロピルメチルセル口 ースフタレート （H P M C P ) ， メタアクリル酸メチルーメタアクリル酸共重合体 When the solubility of the compound of the present invention is low, a solubilization treatment may be performed. As the solubilization treatment, known methods applicable to pharmaceutical preparations, for example, surfactants (polyoxyethylene hydrogenated castor oils, polyxylene sorbitan higher fatty acid esters, polyxixylene oxypolyglycols, sucrose) Fatty acid esters, etc.), drugs and solubilizing agents such as polymers (water-soluble polymers such as hydroxypropylmethylcellulose (HPMC) and polyethylene glycol (PEG)), carboxymethylethylcellulose (CMEC) , Hydroxypropyl methylcellulose phthalate (HPMCP), methyl methacrylate-methacrylic acid copolymer
(オイドラギッ卜し， S , 商品名；ローム ·アンド ·ハース社製） 等の腸溶性高分 子） との固体分散体を形成する方法が挙げられる。 図面の簡単な説明 (Eudragit, S, trade name; enteric polymer such as Rohm and Haas Co.) and the like to form a solid dispersion. BRIEF DESCRIPTION OF THE FIGURES
図 1 10 tMの全卜ランスレチノイン酸 （all-trans retinoic acid (atRA)) 、 LG100268または 2nMヒトァクチビン （human activin) Aを ΝΠΊ細胞に 12, 24, 48 時間作用させたときの Pax4の遺伝子発現量を示した図である （未処理 (None)におけ る Pax4遺伝子の発現量を 100とする） 。  Figure 1 Pax4 gene expression when 10 tM all-trans retinoic acid (atRA), LG100268 or 2 nM human activin A was allowed to act on ΝΠΊ cells for 12, 24, and 48 hours (The expression level of Pax4 gene in untreated (None) is set to 100).
図 2 各濃度のァクチビン Aを 24時間 ΝΠΊ細胞に作用させたときの Pax4の遺伝 子発現量を示した図である（OpMにおける Pax4発現量を 100とする） 。  Fig. 2 is a diagram showing the expression level of Pax4 gene when activin A at each concentration was allowed to act on cells for 24 hours (Pax4 expression level in OpM is 100).
図 3 2nMの TGF /3 1， ァクチビン Aを 24時間 ΝΠΊ細胞に作用させたときのイン シュリン（Ins) と Pax4の遺伝子発現量を示した図である （未処理 (None)における各 遺伝子の発現量を 100とする） 。  Fig. 3 shows the gene expression levels of insulin (Ins) and Pax4 when 2nM TGF / 31, activin A was allowed to act on cells for 24 hours. (Expression of each gene in untreated (None)) Set the amount to 100).
図 4 Xenopusのァクチビン応答配列レポーター遺伝子と FAST-1遺伝子を、 野生 型 (WT)の ΝΠΊ細胞または構成的活性型 （constitutive active type)の IB型ァクチビン 受容体 ALK4 (T206D)遺伝子を安定に発現する ΝΠΊ細胞 ALK4 No.6に遺伝子導入し、 野生型 NMT1細胞の内の一群には 2nMのヒ卜ァクチビン Aを 24時間作用させ (WT (Act))、各細胞のルシフェラーゼ活性を測定することにより、 相対的な細胞内ァクチ ビンシグナル強度を測定した図である （野生型 (WT)の ΝΠΊ細胞におけるァクチビン シグナル強度を 100とする） 。 Figure 4 Xenopus activin response element reporter gene and FAST-1 gene WT type WT cells or constitutive active type IB type activin receptor ALK4 (T206D) gene is stably expressed ΝΠΊ cells ALK4 No. 6 is transfected into wild type NMT1 cells Fig. 4 shows the relative intracellular activin signal intensity measured by measuring the luciferase activity of each cell after 2 hours of the activin A (WT (Act)) was applied to 2 nM of the group. (Activin signal intensity in ΝΠΊ cells of type (WT) is assumed to be 100).
図 5 野生型 (WT)の NIT1細胞、 野生型 ΝΠΊ細胞に 2nMのヒ卜ァクチビン Aを 24時 間作用させたとき (WT (Act)),および構成的活性型（constitutive active type)の IB型 ァクチビン受容体 ALK4 (T206D)遺伝子を安定に発現する ΝΠΊ細胞 ALK4 No.6にお いて Pax4の遺伝子発現量を示した図である （野生型 (WT)の NIT1細胞における Pax4 遺伝子発現量を 100とする） 。 発明を実施するための最良の形態 - 以下に実施例を掲記し内容を詳細に説明するが、 本発明はこれら実施例に限定さ れるものではない。  Fig. 5 When 2 nM human activin A was allowed to act on wild-type (WT) NIT1 cells and wild-type ΝΠΊ cells for 24 hours (WT (Act)), and constitutive active type IB FIG. 5 shows the expression level of Pax4 in ALK4 No. 6 ΝΠΊ cells that stably express the activin receptor ALK4 (T206D) gene (Pax4 gene expression level in wild-type (WT) NIT1 cells was 100 and 100%). Do). BEST MODE FOR CARRYING OUT THE INVENTION-Examples will be described below and the contents will be described in detail, but the present invention is not limited to these examples.
(材料）  (Material)
本実施例で使用されるヒト ァクチビン（human activin) Aは Austral Biologicals 社 (Cat. No. GF-530-3)より、 またヒト 卜ランスフォーミング成長因子 （human Transforming Growth Factor) -beta 1 (TGF /3 1 )は SIGMA社 (Cat. No.T7039)ょリ購 入した。 それぞれのアミノ酸配列は後述配列表に示した。 アミノ酸配列に使用され る 1文字表記及び 3文字表記は周知であるが、 生化学辞典 （第 2版 （1990) 、 東京 化学同人、 1468頁）参照のこと。脖臓 ]8細胞株の一つである野生型 ΝΓΓ1細胞は ATCC (American Type Culture Collection)から購入した (Cat. No. CRL-2055)o Human activin A used in this example was obtained from Austral Biologicals (Cat. No. GF-530-3), and human transforming growth factor -beta 1 (TGF / 3 1) was purchased from SIGMA (Cat. No. T7039). Each amino acid sequence is shown in the sequence listing below. The one-letter and three-letter codes used in amino acid sequences are well known, but see the Biochemical Dictionary (2nd edition (1990), Tokyo Kagaku Dojin, p. 1468).脖 leen] Wild type ΝΓΓ1 cell, one of 8 cell lines, was purchased from ATCC (American Type Culture Collection) (Cat. No. CRL-2055) o
206番目のスレオニン (T)をァスパラギン酸 (D)に置換することにより構成的活性型 受容体 (constitutive active type receptor； リガンドが結合していない状態においても 常に活性化された状態にあるタイプの受容体)にしたヒ卜 IB型ァクチビン受容体 Constitutively active form by replacing threonine (T) at position 206 with aspartic acid (D) Human IB-type activin receptor converted to a receptor (constitutive active type receptor; a type of receptor that is always activated even when no ligand is bound)
ALK4 (T206D) (GenBank Ac. No. Z22536)の発現プラスミドは （財） 癌研究会癌研 究所生化学部の宮園浩平先生より恵与された。 ALK4 (T206D)の発現プラスミドを NIT1細胞に卜ランスフエクシヨンし、 G418耐性コロニーを分離取得し継代すること により、 ALK4 (T206D)安定発現株の ΝΠΊ細胞クローン ALK4 N0.6を単離した。 ァフ リカツメガエル (Xenopus)由来の転写因子 FAST-1 (GenBank Ac. No. U70980、 Nature (1996) 383, 691-696)の発現プラスミドは Harvard大学 ·細胞生物学部の The expression plasmid of ALK4 (T206D) (GenBank Ac. No. Z22536) was kindly provided by Dr. Kohei Miyazono of the Biochemistry Department, Cancer Research Institute, Cancer Research Institute. The ALK4 (T206D) expression plasmid was transfected into NIT1 cells, G418-resistant colonies were isolated and obtained and subcultured to isolate the ALK4 (T206D) stable expression cell clone ALK4 N0.6. . The expression plasmid for the transcription factor FAST-1 (GenBank Ac. No. U70980, Nature (1996) 383, 691-696) from Xenopus was obtained from Harvard University
Whitman M.博士よリ恵与された。 ァクチビン応答配列 （EMBO J. (1995) 14, 5965- 5973、塩基配列は後述）の 5回繰り返しと TATAボックスをレポーターであるルシフ エラーゼをコードするプラスミド PGV-B2 (東洋インキ社、 Cat. No. 306-04831 ) の マルチクローニングサイ卜に挿入し、 ァクチビン応答配列レポータープラスミド PALUC5を作成した。 Thank you, Dr. Whitman M. An activin response element (EMBO J. (1995) 14, 5965-5973; base sequence is described below) 5 times and a plasmid PGV-B2 encoding a TATA box, luciferase, a reporter (Toyo Ink, Cat. No. 306-04831) to prepare an activin response element reporter plasmid PALUC5.
ヒ卜 ァクチビン A (配列番号 1 ： Swiss-Prot Ac. NO.P08476) Human Activin A (SEQ ID NO: 1: Swiss-Prot Ac. NO.P08476)
GLECDGKVN I CCKKQFFVSFKD I GWNDW 1 1 APSGYHANYCEGECPSH I AGTSGSSLSFHSTV I NHYR RGHSPF GLECDGKVN I CCKKQFFVSFKD I GWNDW 1 1 APSGYHANYCEGECPSH I AGTSGSSLSFHSTV I NHYR RGHSPF
ANLKSCCVPTKLRP S LYYDDGQN I I KKD I QNM I VEECGCS ANLKSCCVPTKLRP S LYYDDGQN I I KKD I QNM I VEECGCS
ヒ卜 TGF )8 1 (配列番号 2 ： Swiss-Prot Ac. NO.P01137)  Human TGF) 8 1 (SEQ ID NO: 2: Swiss-Prot Ac. NO.P01137)
ALDTNYCFSSTEKNCCVRQLY I DFRKDLGWKW I HEPKGYHANFCLGPCPY I WSLDTQYSKVLALYNQHNPGASA APCCVPQALEPLP I VYYVGRKPKVEQLSNM I VRSCKCS  ALDTNYCFSSTEKNCCVRQLY I DFRKDLGWKW I HEPKGYHANFCLGPCPY I WSLDTQYSKVLALYNQHNPGASA APCCVPQALEPLP I VYYVGRKPKVEQLSNM I VRSCKCS
アフリカッメガエル ァクチビン応答配列 （配列番号 3 ： GenBank Ac. No. U33914) African Megalactivin Response Element (SEQ ID NO: 3: GenBank Ac. No. U33914)
TTATCTGCTGCCCTAAAATGTGTATTCCATGGAAATGTCTGCCCTTCTCTCCG  TTATCTGCTGCCCTAAAATGTGTATTCCATGGAAATGTCTGCCCTTCTCTCCG
(遺伝子発現量の測定方法）  (Method of measuring gene expression level)
インシュリン、 又は Pax4等遺伝子の発現はパーキンエルマ一アプライドバイ才シス テムズ社の PRISM™ 7700 Sequence Detection Systemと Taq MAN™ PCR Core Reagent Kit (Cat. No. N808-0229)を用いて測定した。 原理を簡単に記すと PCR増幅 された DNA鎖を蛍光色素を用いてリアルタイムに定量的に検出するシステムである。Expression of insulin or Pax4 gene is determined by PerkinElmer The measurement was performed using Thames' PRISM ™ 7700 Sequence Detection System and Taq MAN ™ PCR Core Reagent Kit (Cat. No. N808-0229). The principle is simply a system that quantitatively detects PCR-amplified DNA strands in real time using fluorescent dyes.
( 1 ) 全 RNAの調製 (1) Preparation of total RNA
全 RNAは二ツボンジーン社の lsogen(Cat. No.31 1-02501)を用いて説明書に従い NIT1細胞から調製した。 ΝΠΊ細胞は 10%ゥシ胎児血清を含む Ham's F12培地を用い て培養した。 調製した全 RNAはその後デォキシリボヌクレアーゼ  Total RNA was prepared from NIT1 cells using lsogen (Cat. No. 31 1-02501) of Futsubon Gene according to the instructions. ΝΠΊ cells were cultured in Ham's F12 medium containing 10% fetal bovine serum. The prepared total RNA was then used for deoxyribonuclease.
[deoxyribonuclease (DNase) ；ニツボンジーン社 Cat. No. 313-01363) ]処理を行 い、 フエノール/クロ口ホルム処理、 エタノール沈殿して滅菌水に溶解し- 20°Cで保 存した。  [deoxyribonuclease (DNase); Nippon Gene Cat. No. 313-01363)] treatment, phenol / cloth form treatment, ethanol precipitation, dissolution in sterile water, and storage at -20 ° C.
( 2 ) cDNAの合成  (2) cDNA synthesis
RNAから cDNAへの逆転写は ClonTech社の Advantage™RT-for-PCR Kit (Cat. No. K1402-2)を用いて行った。 逆転写後、 -20°Cで保存した。  Reverse transcription from RNA to cDNA was performed using ClonTech's Advantage ™ RT-for-PCR Kit (Cat. No. K1402-2). After reverse transcription, it was stored at -20 ° C.
( 3 ) PCRプライマー （primer) ， TaqMANプローブ （probe) の作成  (3) Preparation of PCR primer (primer) and TaqMAN probe (probe)
マウスのインシュリン （Ins) 、 Pax4、 ダリセルアルデヒド 3—リン酸脱水素酵 素（Glyceraldehyde 3-phosphatedehydrogenase (G3PDH))、 TATA結合蛋白質 TFIIDT (TBP)の遺伝子はそれぞれプライマー Ins13-lns14, Pax401-Pax403, G3PDH F- G3PDH R, TF201-TF205の組み合わせによリ PCR増幅した。 それぞれの塩基配列を 以下に記す。  The genes for mouse insulin (Ins), Pax4, dalyseraldehyde 3-phosphate dehydrogenase (G3PDH), and TATA-binding protein TFIIDT (TBP) are primers Ins13-lns14, Pax401-Pax403, PCR amplification was performed using a combination of G3PDH F-G3PDH R and TF201-TF205. Each base sequence is described below.
I ns 1 3 (配列番号 4 ) ATGGCCCTGTGGATGCGCTTC  Ins 1 3 (SEQ ID NO: 4) ATGGCCCTGTGGATGCGCTTC
I ns l 4 (配列番号 5 ) TCAGTTGCAGTAGTTCTCCAG  Ins l 4 (SEQ ID NO: 5) TCAGTTGCAGTAGTTCTCCAG
Pax401 (配列番号 6 ) GCCTGGGAGATCCAACACCA  Pax401 (SEQ ID NO: 6) GCCTGGGAGATCCAACACCA
Pax403 (配列番号 7 ) GGGAAGAACTGGAGCCA  Pax403 (SEQ ID NO: 7) GGGAAGAACTGGAGCCA
G3PDH F (配列番号 8 ) AAAGTGGAGATTGTTGCCAT G3PDH R (配列番号 9 ) TTGACTGTGCCGTTGAATT G3PDH F (SEQ ID NO: 8) AAAGTGGAGATTGTTGCCAT G3PDH R (SEQ ID NO: 9) TTGACTGTGCCGTTGAATT
TF201 (配列番号 1 0 ) TCTTTAGTCCAATGATGCCTT TF205 (配列番号 1 1 ) GGTTGCTGAGATGTTGATTG  TF201 (SEQ ID NO: 10) TCTTTAGTCCAATGATGCCTT TF205 (SEQ ID NO: 11) GGTTGCTGAGATGTTGATTG
また遺伝子の検出に用いた蛍光基質ラベルされた TaqMAN probeの配列は次の通 リである。  The sequence of the TaqMAN probe labeled with the fluorescent substrate used for gene detection is as follows.
I ns (配列番号 1 2 ) 5' FAM-TCTACACACCCATGTCCCGCC-3' TAMRA Pax4 (配列番号 1 3 ) 5' FA -AGAGCTTGCACTGGACTCAACTCAGA-3' TA RA G3PDH (配列番号 1 4 ) 5' FAM-CATGTTCCAGTATGACTCCACTCACG-3' TAMRA TF I I DT (配列番号 1 5 ) 5 ' FAM-CCACAGCCTATTCAGAACACCAACAG-3 ' TAMRA この中で FAM (6—カルボキシフル才レシン (carboxyfluorescein)) , TAMRA (6-力 ルボキシテトラメチルロダミン (carboxytetramethylrhodamine))とはそれぞれフル ォレシン (Fluorescein)系、 ロダミン (Rhodamine)系の蛍光色素の名称である。  Ins (SEQ ID NO: 12) 5 'FAM-TCTACACACCCATGTCCCGCC-3' TAMRA Pax4 (SEQ ID NO: 13) 5 'FA-AGAGCTTGCACTGGACTCAACTCAGA-3' TA RA G3PDH (SEQ ID NO: 14) 5 'FAM-CATGTTCCAGTATGACTCCACTCACG-3' TAMRA TF II DT (SEQ ID NO: 15) 5 'FAM-CCACAGCCTATTCAGAACACCAACAG-3' TAMRA Wherein FAM (6-carboxyfluorescein), TAMRA (6-force carboxytetramethylrhodamine) and Are the names of fluorescein and rhodamine fluorescent dyes, respectively.
( 4 ) 遺伝子発現量の測定  (4) Measurement of gene expression level
PRISM™ 7700 Sequence Detection Systemによる測定は の系で説明書に従 つて行った。 PCR増幅条件は次の通りである。 50°Cで 10分間に続いて 95でで 10分間 ィンキュベー卜する。 その後 95°Cで 15秒間に続いて 58°Cで 1分間の 2ステップ PCR の工程を 4045サイクル行った。各試料における目的の遺伝子の発現量は図 1から図 3は G3PDH遺伝子の発現量で、 また図 5は TFIIDT遺伝子の発現量でそれぞれ補正し た。 補正後数値は下記式に基づいて算出された。  The measurement with the PRISM ™ 7700 Sequence Detection System was performed according to the instruction manual for the system. The PCR amplification conditions are as follows. Incubate at 50 ° C for 10 minutes, then at 95 for 10 minutes. Thereafter, 4045 cycles of a two-step PCR were performed at 95 ° C for 15 seconds, followed by 58 ° C for 1 minute. The expression level of the target gene in each sample was corrected by the expression level of the G3PDH gene in FIGS. 1 to 3 and by the expression level of the TFIIDT gene in FIG. The corrected numerical value was calculated based on the following equation.
式  Expression
[目的の遺伝子の発現量 （生データ） ] [Expression level of target gene (raw data)]
[補正発現量] = [Corrected expression level] =
[G3PDHまたは TFIIDT遺伝子の発現量（生データ）] ( 5 ) 相対ァクチビンシグナル強度の測定 いくつかの哺乳類の細胞においては、アフリカッメガエル (Xenopus)由来の転写因子 FAST-1存在下においてァクチビン応答配列レポーター系を用いることにより、 細胞 内のァクチビンシグナル強度を測定できることが示されている (Proc. Natl. Acad . Sci. USA (1998) 95, 2412-2416)0 そこで野生型またはァクチビン処理した ΝΠΊ細胞、 および ALK4 (T206D)の安定発現株の ΝΓΓ1細胞におけるァクチビンシグナル強度を 以下の方法により測定した。 2 4穴プレー卜にまいた各細胞に 1穴当たり pALUC5 (0.625 g)、FAST-1発現プラスミド （0·0625μ§)、リボフェクトァミン試薬（GibcoBRL 社 Cat. No. 18324-020) 6.25μΙと全量で 350μΙになるように残りの量の OPTI-MEM 培地（GibcoBRL社 Cat. No. 31985-070)を加え、遺伝子導入した。 12時間後に 10% ゥシ胎児血清を含む Ham's F12培地もしくはそれに 2nMのヒトァクチビン Aを含む 培地に交換した。 その 24時間後に培養細胞溶解剤 LC-β (東洋インキ社 Cat. No. PGC-51 ) を水で 5倍に希釈した液 150μΙを加え、 細胞を溶解した。 所定量をルシフ エラーゼの基質と混合し、 発光量を Dynatech社の Μレ 3000を用いて測定した。 得ら れた生データを補正するため各穴中の全蛋白質量を Biorad社プロテインアツセィ試 薬 （Cat. No. 500-0006) と混ぜて 595nmの比色を測定した。 港度の換算はスタンダ ードとしてゥシ血清アルブミンで検量線を書き、 そこから算出した。 ァクチビンシ グナル強度は下記の式によリ算出した。 [Expression level of G3PDH or TFIIDT gene (raw data)] (5) Measurement of relative activin signal intensity In some mammalian cells, the use of the activin response element reporter system in the presence of the transcription factor FAST-1 from Xenopus has shown that the intracellular activin signal intensity can be measured. (Proc. Natl. Acad. Sci. USA (1998) 95, 2412-2416) 0 Activin signal intensity in wild-type or activin-treated ΝΠΊ cells and in ALK4 (T206D) stably expressing ΝΓΓ1 cells Was measured by the following method. 2 Each cell in a 4-well plate was plated per well with pALUC5 (0.625 g), FAST-1 expression plasmid (0.0625 μ§), and Ribofectamine reagent (GibcoBRL Cat. No. 18324-020) 6.25 μΙ Then, the remaining amount of OPTI-MEM medium (GibcoBRL Cat. No. 31985-070) was added to bring the total volume to 350 μΙ, and the gene was introduced. Twelve hours later, the medium was replaced with Ham's F12 medium containing 10% fetal bovine serum or a medium containing 2 nM human activin A. Twenty-four hours later, 150 μΙ of a 5-fold diluted solution of a cell lysing agent LC-β (Cat. No. PGC-51) was diluted with water to lyse the cells. A predetermined amount was mixed with a substrate of Luciferase, and the amount of luminescence was measured using Dynatech Pellet 3000. To correct the obtained raw data, the total amount of protein in each well was mixed with a Bioassay protein assay reagent (Cat. No. 500-0006) and colorimetric at 595 nm was measured. The conversion of port degree was calculated from a calibration curve drawn with serum albumin as standard. The activin signal strength was calculated by the following equation.
式  Expression
[ルシフェラーゼの発光量（生データ） ] [補正ァクチビンシグナル強度] = [Luciferase luminescence (raw data)] [Corrected activin signal intensity] =
[検量線から算出した全蛋白質量 （換算データ） ]  [Total protein content calculated from calibration curve (converted data)]
(測定結果） (Measurement result)
(i) ァクチビン作用の絰時変化 核内受容体 RAR (レチノイン酸受容体： retinoic acid receptor)のリガンドである atRA (全卜ランスレチノイン酸： all-trans retinoic acid), RXR (レチノィド X受容体： retinoid X receptor)の合成リガンドである LG100268(Nature (1997) 386, 407410)を それぞれ 10μ Μ、または 2 ηΜヒ卜ァクチビン（human activin) Aを N I T1に 12, 24, 48 時間作用させた後に RNAを調製し、 cDNAに変換してインシュリンと Pax4の遺伝子 発現を測定した。 その結果、 核内受容体のリガンドは如何なる効果も示さなかった。 ァクチビンは顕著に Pax4遺伝子の発現を増加させ、 その作用は 12時間以内に起きて いること、 そして 48時間までは持続していることが判明した （図 1 ) 。 Pax4遺伝子 発現増強に追随する形でィンシュリンの遺伝子発現も増加していた。 (i) Temporal change of activin action Synthetic ligand for atRA (all-trans retinoic acid) and RXR (retinoid X receptor), which are ligands for nuclear receptor RAR (retinoic acid receptor) LG100268 (Nature (1997) 386, 407410) was reacted with 10μΜ or 2ηΜ human activin A, respectively, on NI T1 for 12, 24, 48 hours, and then RNA was prepared and converted to cDNA. Insulin and Pax4 gene expression were measured. As a result, the nuclear receptor ligand did not show any effect. Activin significantly increased Pax4 gene expression, and its effects were found to occur within 12 hours and persisted for up to 48 hours (Figure 1). Insulin gene expression was also increased following Pax4 gene expression enhancement.
(ii) ァクチビン作用の濃度依存性  (ii) Concentration dependence of activin action
ァクチビンの遘度を 0-2 nMの間で振つて 24時間 NIT1に作用させ Pax4の遺伝子発 現量を調べた。 濃度依存的に発現量が増加することが判明した （図 2 ) 。  The secretion degree of activin was shaken between 0 and 2 nM to act on NIT1 for 24 hours, and the expression level of Pax4 gene was examined. It was found that the expression level increased in a concentration-dependent manner (Fig. 2).
(iii) TGF iS 1とァクチビン Aの作用の比較  (iii) Comparison of the effects of TGF iS 1 and activin A
ァクチビンと同様に TGF 8スーパーファミリーに属する TGF iS 1の効果を検討し た。 これらは共にシグナル媒介因子として Smad2または Smad3が機能していること が知られている。 ァクチビン、 TGF iS 1共に 2 nMで 24時間 NIT1細胞に作用させた。  The effect of TGF iS1, which belongs to the TGF8 superfamily as well as activin, was examined. Both are known to function as Smad2 or Smad3 as signal mediators. Both activin and TGF iS1 were allowed to act on NIT1 cells at 2 nM for 24 hours.
TGF iS 1は lns,Pax4の遺伝子発現を低下させた。 つまリ Pax4遺伝子発現亢進作用は ァクチビン Aに特有であることが判明した （図 3 ) 。 TGF iS1 reduced the gene expression of Ins, Pax4. In other words, it was found that Pax4 gene expression enhancing action was specific to activin A (Fig. 3).
(iv)未処理またはァクチビン処理した野生型の NIT1細胞および構成的活性型の  (iv) Untreated or activin-treated wild-type NIT1 cells and constitutively active
ALK4 (T206D)を安定に発現する ΝΓΓ1細胞における細胞内ァクチビンシグナル強度 ァクチビン応答配列レポータープラスミ卜' (pALUC5)と FAST-1発現プラスミドを 野生型 (WT)の ΝΠΊ細胞または構成的活性型の旧型ァクチビン受容体 ALK4 (T206D) 遺伝子を安定に発現する ΝΠΊ細胞 ALK4 No.6に卜ランスフエクシヨンし、 野生型の うちの一群には 2nMのヒ卜ァクチビン Aを 24時間作用させたときの相対細胞内ァク チビンシグナル強度を測定した。 ALK4 Νο.6では 2nMのァクチビン A処理群と同等か それ以上のァクチビンシグナル強度が観察された （図 4 ) 。 Intracellular activin signal intensity in 細胞 1 cells that stably express ALK4 (T206D) ァ Activin response element reporter plasmid '(pALUC5) and FAST-1 expression plasmid were transferred to wild-type (WT) ま た は cells or constitutively active Stable expression of old-type activin receptor ALK4 (T206D) gene ΝΠΊ Transfected into ALK4 No.6 cell and exposed to 2nM human activin A for 24 hours Relative intracellular factor The tibin signal intensity was measured. In ALK4Ν.6, an activin signal intensity equivalent to or higher than that of the 2 nM activin A-treated group was observed (FIG. 4).
(V)未処理またはァクチビン処理した野生型の ΝΓΠ細胞または構成的活性型の ALK4 (T206D)を安定に発現する N 1細胞における Pax4遺伝子発現量  (V) Pax4 gene expression level in untreated or activin-treated wild-type ΝΓΠ cells or N1 cells stably expressing constitutively active ALK4 (T206D)
野生型の NIT1 , 野生型の NIT1に 2nMのヒ卜ァクチビン Aを 24時間処理した群、およ び ALK4 (T206D)を安定に発現する ΝΓΠ細胞 ALK4 No.6における Pax4遺伝子発現量 を測定した（図 5 )。 その結果、 ALK4 N0.6では 2nMァクチビン処理群と同等かそれ 以上の Pax4遺伝子が発現しており、 図 4で見られた細胞内ァクチビンシグナル強度 と図 5の Pax4遺伝子発現量の間に良い対応が見られた。  Pax4 gene expression levels were measured in wild-type NIT1, a group treated with 2 nM human activin A for 24 hours and wild-type NIT1, and ALK4 (T206D) stably expressing 6 cells ALK4 No.6 ( (Figure 5). As a result, in ALK4 N0.6, the Pax4 gene was expressed at a level equal to or higher than that of the 2 nM activin-treated group, and between the intracellular activin signal intensity shown in Fig. 4 and the Pax4 gene expression level in Fig. 5. Good response was seen.
上記の如くァクチビン受容体を活性化させた細胞内の Pax4遺伝子発現量が増加し ていることから、 ァクチビン受容体ァクチベータ一は、 Pax4遺伝子発現を増強させ ることが証明された。 産業上の利用可能性  As described above, since the amount of Pax4 gene expression in the cells in which the activin receptor was activated was increased, it was proved that the activin receptor activator-1 enhanced Pax4 gene expression. Industrial applicability
本発明スクリーニング法は、 脖臓 )8細胞の疲弊状態からの保護又は脖臓 ]8細胞の 機能亢進に有用な脬臓 i8細胞内 Pax4遺伝子発現増強作用を有する物質を選択するだ けでなく、 脖臓 ]8細胞の機能又は疲弊の程度を検出することにより診断等に有用で ある。  The screening method of the present invention not only selects a substance having an action of enhancing Pax4 gene expression in the pancreas i8 cells, which is useful for protecting the pancreas) 8 cells from exhaustion or enhancing the function of the pancreas 8 cells. [Kidney] It is useful for diagnosis etc. by detecting the function or degree of exhaustion of 8 cells.
さらに本発明スクリーニング法で得られる物質及び/又はァクチビン受容体ァク チベータ一は、 pax4遺伝子発現増強及びィンシュリン遺伝子発現冗進作用を示すた め脬臓 ]8細胞の疲弊に起因する疾患、 即ち、 糖尿病の進行予防及び治療用医薬組成 物として極めて有用である。 本発明は脬臓) 8細胞におけるインシュリン発現メカ二 ズ厶の解明に有用である。 Furthermore, the substance obtained by the screening method of the present invention and / or the activin receptor activator is a disease caused by exhaustion of the [8] cells due to enhanced pax4 gene expression and increased insulin gene expression. It is extremely useful as a pharmaceutical composition for preventing and treating the progress of diabetes. INDUSTRIAL APPLICABILITY The present invention is useful for elucidating the mechanism of insulin expression in 8 cells of the kidney.

Claims

請求の範囲 The scope of the claims
1 . 少なくとも睦臓 細胞に被験物質を接触させ、 該鈀胞内の Pax4遺伝子発現 量を検出する手段を含む Pax4遺伝子発現増強作用を有する物質のスクリーニング法。 1. A screening method for a substance having a Pax4 gene expression-enhancing action, which comprises at least a means for bringing a test substance into contact with murine cells and detecting the amount of Pax4 gene expression in the cell.
2 . 請求の範囲 1記載のスクリーニング法より得られる物質を有効成分とする 膝臓 細胞内 Pax4遺伝子発現増強用医薬組成物。  2. A pharmaceutical composition for enhancing Pax4 gene expression in knee cells, comprising a substance obtained by the screening method according to claim 1 as an active ingredient.
3 . ァクチビン受容体ァクチベータ一を有効成分とする膝臓 細胞内 Pax4遺伝子 発現増強用医薬組成物。  3. A pharmaceutical composition for enhancing Pax4 gene expression in knee cells, which comprises an activin receptor activator as an active ingredient.
PCT/JP1999/003182 1998-06-16 1999-06-15 METHOD FOR SCREENING Pax4 GENE EXPRESSION POTENTIATOR AND MEDICINAL COMPOSITIONS FOR POTENTIATING Pax4 GENE EXPRESSION WO1999066073A1 (en)

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