WO1999066073A1 - PROCEDE DE CRIBLAGE D'AGENT DE POTENTIALISATION DU GENE Pax ET COMPOSITIONS MEDICINALES POUR LA POTENTIALISATION DU GENE Pax - Google Patents

PROCEDE DE CRIBLAGE D'AGENT DE POTENTIALISATION DU GENE Pax ET COMPOSITIONS MEDICINALES POUR LA POTENTIALISATION DU GENE Pax 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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English (en)
Japanese (ja)
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Yoshitaka Ueda
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Yamanouchi Pharmaceutical Co., Ltd.
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Priority to AU41666/99A priority Critical patent/AU4166699A/en
Publication of WO1999066073A1 publication Critical patent/WO1999066073A1/fr

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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

L'invention concerne une méthode de criblage permettant la sélection d'une substance potentialisant l'expression du gène Pax4 dans les cellules bêta pancréatiques, utile dans la protection des cellules bêta pancréatiques à l'état d'épuisement ou dans l'accélération de la fonction des cellules bêta pancréatiques. La substance obtenue par la méthode de criblage de l'invention et/ou l'activateur du récepteur de l'activine potentialisent l'expression du gène Pax4 et accélèrent l'expression du gène de l'insuline. Ainsi, les substances susmentionnées sont très utiles dans des compositions médicinales pour prévenir l'évolution d'une maladie due à l'épuisement des cellules bêta pancréatiques (diabète) et dans le traitement de cette maladie.
PCT/JP1999/003182 1998-06-16 1999-06-15 PROCEDE DE CRIBLAGE D'AGENT DE POTENTIALISATION DU GENE Pax ET COMPOSITIONS MEDICINALES POUR LA POTENTIALISATION DU GENE Pax WO1999066073A1 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998029566A2 (fr) * 1996-12-31 1998-07-09 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Nouveau procede pour tester l'etat de differenciation des cellules pancreatiques chez le mammifere

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998029566A2 (fr) * 1996-12-31 1998-07-09 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Nouveau procede pour tester l'etat de differenciation des cellules pancreatiques chez le mammifere

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
FUKUKAWA M, ET AL.: "SUI BETA SIBOU NO BUMKA", MODERN MEDICAL CARE, XX, XX, vol. 30, no. 10, 1 October 1998 (1998-10-01), XX, pages 2593 - 2598, XP002922084 *
INOUE H., ET AL.: "ISOLATION OF FULL-LENGTH CDNA OF MOUSE PAX4 GENE AND IDENTIFICATIONOF ITS HUMAN HOMOLOGUE.", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, ACADEMIC PRESS INC. ORLANDO, FL, US, vol. 243., 1 January 1998 (1998-01-01), US, pages 628 - 633., XP002922088, ISSN: 0006-291X, DOI: 10.1006/bbrc.1998.8144 *
KOJIMA ITARU: "DIFFERENTIATION OF PANCREATIC ENDOCRINE CELLS", TONYOBYO - JOURNAL OF THE JAPAN DIABETES SOCIETY, JAPAN DIABETES SOCIETY, TOKYO, JP, vol. 26, no. 03, 1 January 1998 (1998-01-01), JP, pages 97 - 103, XP002922083, ISSN: 0021-437X *
MATSUSHITA T., ET AL.: "MOLECULAR CLONING OF MOUSE PAIRED-BOX-CONTAINING GENE (PAX)-4 FROM AN ISLET SS CELL LINE AND DEDUCED SEQUENCE OF HUMAN PAX-4.", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, ACADEMIC PRESS INC. ORLANDO, FL, US, vol. 242., 1 January 1998 (1998-01-01), US, pages 176 - 180., XP002922087, ISSN: 0006-291X, DOI: 10.1006/bbrc.1997.7935 *
SOSA-PINEDA B., ET AL.: "THE PAX4 GENE IS ESSENTIAL FOR DIFFERENTIATION OF INSULIN-PRODUCINGSS CELLS IN THE MAMMALIAN PANCREAS.", NATURE, NATURE PUBLISHING GROUP, UNITED KINGDOM, vol. 386., 27 March 1997 (1997-03-27), United Kingdom, pages 399 - 402., XP002922086, ISSN: 0028-0836, DOI: 10.1038/386399a0 *
TAO T., ET AL.: "BRIEF GENETICS REPORT ISOLATION AND CHARACTERIZATION OF THE HUMAN PAX4 GENE.", DIABETES, AMERICAN DIABETES ASSOCIATION, US, vol. 47., 1 October 1998 (1998-10-01), US, pages 1650 - 1653., XP002922085, ISSN: 0012-1797, DOI: 10.2337/diabetes.47.10.1650 *
TOKUYAMA Y., ET AL.: "MOLECULAR CLONING OF RAT PAX4: IDENTIFICATION OF FOUR ISOFORMS IN RAT INSULINOMA CELLS.", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, ACADEMIC PRESS INC. ORLANDO, FL, US, vol. 248., 1 January 1998 (1998-01-01), US, pages 153 - 156., XP002922089, ISSN: 0006-291X, DOI: 10.1006/bbrc.1998.8925 *

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