JP2007153766A - Expression enhancing agent of map kinase kinase kinase gene - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an expression enhancing agent of MAPKKK gene. <P>SOLUTION: This medicine contains hyaluronan as an effective ingredient. It is favorable that the hyaluronan is a tetrasaccharide (HA4) containing two units of -D-glucuronic acid-β-1,3-D-N-acetylglucosamine-β-1,4-. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a MAP kinase kinase kinase gene expression enhancer comprising hyaluronan as an active ingredient.

  Hyaluronan is a long-chain polysaccharide composed of disaccharide repeating units of D-glucuronic acid and N-acetyl-D-glucosamine, while oligosaccharides are also known. Hyaluronan is produced by extraction from biological tissues such as chicken crowns, corpses, skin, joint fluid, or fermentation methods using bacteria belonging to the genus Streptococcus, and has no toxicological or immunological effects. For example, treatment of arthritis by intraarticular injection of hyaluronan is well known. In the following description, the tetrasaccharide hyaluronan is referred to as HA4.

HA4 has been reported to have therapeutic / suppressive effects on organ preservation, liver damage, and gastric ulcers (see Patent Document 1). Moreover, it is known that HA4 has a stress protein expression enhancing action and a cell death suppressing action (see Non-Patent Document 1). Furthermore, it has also been reported that hyaluronic acid oligosaccharides have various physiological activities (see Non-Patent Document 2).

  On the other hand, MAP kinase kinase kinase (hereinafter referred to as MAPKKK) has an activity to phosphorylate MAP kinase kinase (hereinafter referred to as MAPKK) in MAP kinase signal transduction. Among MAPKKKs, MAPKKK13 is involved in the Stress responsive MAP kinase (hereinafter referred to as SAPK) pathway and is a kinase also referred to as leucine zipper-bearing kinase (hereinafter referred to as LZK). The SAPK signaling pathway is known to control stress adaptation, apoptosis, cell proliferation, canceration, differentiation, immune response, etc. by transmitting extracellular stimuli to the nucleus and regulating gene expression (non-) (See Patent Documents 3 to 5).

  LZK is a protein kinase belonging to the Mixed-lineage kinase (MLK) family (characterized by a serine / threonine kinase and tyrosine kinase hybrid catalytic region and two adjacent leucine zipper structures). LZK has serine / threonine kinase activity and is a MAPKKK that activates MKK7, which is a MAPKK, and specifically activates the JNK / SPAK pathway. In this pathway, it binds to the scaffold protein JIP-1 (see Non-Patent Documents 3 to 5). LZK is strongly expressed in the pancreatic islets of Langerhans and the brain (see Non-Patent Documents 3 to 5).

  However, the knowledge that HA4 is involved in MAP kinase signaling or has some influence has not been known.

WO2002 / 004471 Xu H, Ito T, Tawada A, Maeda H, Yamanokuchi H, Isahara K, Yoshida K, Uchiyama Y, Asari A. Effect of hyaluronan oligosaccharides on the expression of heat shock protein 72. J Biol Chem. 2002 10; 277 (19 ): 17308-14. Asari A. Novel Functions of Hyaluronan Oligosaccharides. In Science of Hyaluronan Today. Editors: Vincent C. Hascall. Masaki Yanagishita. Glycoforum, (http://www.glycoforum.gr.jp/science/hyaluronan/HA12/HA12J.html) . 2005. Masaki M, Ikeda A, Shiraki E, Oka S, Kawasaki T. Mixed lineage kinase LZK and antioxidant protein-1 activate NF-kappaB synergistically. Eur J Biochem. 2003 Jan; 270 (1): 76-83. Ikeda A, Sakuma H, Masaki M, Kozutsumi Y, Oka S, Inazawa J, Kawasaki T. Genomic organization and fine-mapping of the human leucine zipper-bearing kinase (LZK) gene.J Biochem Mol Biol Biophys. 2002 Apr; 6 (2): 113-7. Ikeda A, Hasegawa K, Masaki M, Moriguchi T, Nishida E, Kozutsumi Y, Oka S, Kawasaki T. Mixed lineage kinase LZK forms a functional signaling complex with JIP-1, a scaffold protein of the c-Jun NH (2) -terminal kinase pathway. J Biochem (Tokyo). 2001 Dec; 130 (6): 773-81.

  Accordingly, an object of the present invention is to provide a novel expression enhancer for MAPKKK gene.

  The MAPKKK gene expression enhancer according to the present invention that has achieved the above-mentioned object contains hyaluronan as an active ingredient. That is, it has been found that hyaluronan has a novel function of enhancing the expression of the MAPKKK gene, and the present invention has been completed.

  That is, the MAPKKK gene expression enhancer according to the present invention contains hyaluronan as an active ingredient. Here, the hyaluronan is preferably a tetrasaccharide containing 1 unit of -D-glucuronic acid-β-1,3-DN-acetylglucosamine-β-1,4-. In addition, as the MAPKKK gene, expression of the MAPKKK13 gene involved in the SAPK signaling pathway can be enhanced.

  Since the MAPKKK gene expression enhancer according to the present invention contains hyaluronan as an active ingredient, it has the advantage of being easily mass-produced at a relatively low cost. Hyaluronan is expected to be an inhibitor with very few side effects because it has almost no toxicity and antigenicity, and enhances the therapeutic action and disease prevention action inherent in the living body. Therefore, according to the present invention, it is possible to provide a novel drug effective for the treatment of diseases caused by suppression of MAPKKK gene expression.

  Hereinafter, the present invention will be described in detail. The MAPKKK gene expression enhancer according to the present invention is an agent having a function of enhancing the expression of various MAPKKK genes. The expression of the MAPKKK gene is enhanced when the expression level of the gene in untreated animal cells is compared with the expression level of the gene in animal cells treated with this drug. It means that. The gene expression level can be measured using a DNA chip in which a large number of probe DNAs are immobilized on a substrate.

  In the following description, the MAPKKK gene expression enhancer according to the present invention is simply referred to as a drug. That is, the drug according to the present invention can be used for the treatment of diseases caused by suppression of MAPKKK gene expression. The hyaluronan contained in the drug according to the present invention basically includes at least one disaccharide unit in which the 1-position of β-D-glucuronic acid and the 3-position of β-D-N-acetylglucosamine are bonded. As long as it is more than a sugar and is composed of β-D-glucuronic acid and β-DN-acetylglucosamine, those elements are bound to one or more disaccharide units bound together. Sugars may be used, and derivatives thereof such as those having a hydrolyzable protecting group such as an acyl group may also be used. The sugar may be an unsaturated sugar, and examples of the unsaturated sugar include non-reducing terminal sugars, usually those in which the 4- and 5-position carbons of glucuronic acid are unsaturated. As the hyaluronan used in the present invention, specifically, those extracted from natural products such as animals, those obtained by culturing microorganisms, those synthesized chemically or enzymatically, etc. should be used. Can do. For example, it can be obtained by known extraction and purification methods from biological tissues such as chicken crown, corpse, skin, and joint fluid. It can also be produced by a fermentation method using Streptococcus bacteria or the like.

  In the present invention, hyaluronan oligosaccharides are also included in hyaluronan, and use from low molecular weight hyaluronan such as the above-mentioned disaccharide consisting of one disaccharide unit and its derivatives to high molecular weight hyaluronan having a weight average molecular weight of about 4 million. be able to. Preferably, hyaluronan having a weight average molecular weight of about 380 to 900,000, which is excellent in terms of permeability in tissues, and the like, more preferably about 2 to 20 sugars.

  Specifically, hyaluronan having a low molecular weight can be obtained by known methods such as enzymatic decomposition, alkaline decomposition, heat treatment, and ultrasonic treatment (Biochem., 33 (1994) p6503-6507). It is preferably produced by a method of reducing the molecular weight, a method of chemically or enzymatically synthesizing (Glycoconjugate J., (1993) p435-439, WO93 / 20827). For example, hyaluronan degrading enzymes (hyaluronidase (derived from testicles), hyaluronidase (derived from Streptomyces), hyaluronidase SD, etc.), chondroitinase AC, chondroitinase ACII, chondroitinase ACIII, chondroitinase ABC, etc. A method to produce hyaluronan oligosaccharides by causing an enzyme that degrades hyaluronan to produce hyaluronan oligosaccharides (see Shinsei Chemistry Laboratory "Carbohydrate II-Proteoglycans and Glycosaminoglycans" p244-248, published in 1991, Tokyo Chemical Dojin) Can be mentioned.

  As an alkali decomposition method, for example, a base such as about 1N sodium hydroxide is added to a hyaluronan solution, heated for several hours to lower the molecular weight, and then neutralized by adding an acid such as hydrochloric acid. And a method for obtaining a low molecular weight hyaluronan. Hyaluronan used in the present invention includes a salt form, and a pharmaceutically acceptable salt thereof can be used as necessary in the preparation. For example, alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as calcium salt and magnesium salt, amine salts such as tri (n-butyl) amine salt, triethylamine salt, pyridine salt and amino acid salt Can do.

  The drug of the present invention can be used without particular limitation, such as hyaluronan having a certain molecular weight alone or a combination of hyaluronan having various molecular weights. The drug of the present invention comprises hyaluronan as an active ingredient, and administration of the effective amount to mammals including humans can enhance the expression of the MAPKKK gene without adversely affecting the living body. That is, the drug of the present invention can be used for the treatment of diseases caused by suppression of MAPKKK gene expression without adverse effects on the living body. In particular, the drug of the present invention can enhance the expression of the MAPKKK13 gene among the MAPKKK genes.

  The MAPKKK13 gene is LZK involved in the SAPK signaling pathway. The SAPK signaling pathway controls stress adaptation, apoptosis, cell proliferation, canceration, differentiation, immune response, etc. by transmitting extracellular stimuli to the nucleus. Therefore, if the expression of the MAPKKK13 gene can be enhanced by the agent of the present invention, particularly neuropathy (multiple sclerosis, Alzheimer's disease, Parkinson's disease, Guillain-Barre syndrome, stroke, spinal cord injury, uveitis, glaucoma, etc.) , Various injuries (brain contusion, spinal cord injury, wound wound), myocardial infarction, angina pectoris, myocarditis, autoimmune diseases (rheumatic, SLE, inflammatory bowel disease, multiple sclerosis, etc.), cancer, various allergies It is expected to improve symptoms such as disease, hepatitis, cirrhosis, pancreatitis, pneumonia, pulmonary fibrosis, asthma, various ulcers, nephritis, kidney prize, diabetes, and the like.

  The drug of the present invention is for oral or parenteral administration of hyaluronan or a salt thereof, as it is or if necessary, together with a carrier, excipient, or other additives for the purpose of treating the disease as described above or improving symptoms. Can be formulated into any dosage form as a pharmaceutical for manual administration (intra-articular administration, intravenous, intramuscular, subcutaneous, intra-tissue administration (injection), enteral administration, transdermal administration, etc.) And is administered to the patient by any method of administration. In particular, the drug according to the present invention is preferably an injection or an oral preparation.

  Examples of oral preparations include solid preparations such as powders, granules, capsules and tablets; liquid preparations such as syrups, elixirs and emulsions. The powder can be obtained by mixing with excipients such as lactose, starch, crystalline cellulose, calcium lactate, calcium hydrogen phosphate, magnesium aluminate metasilicate, and silicic anhydride. In addition to the above excipients, the granule is optionally combined with a binder such as sucrose, hydroxypropylcellulose, polyvinylpyrrolidone, a binder such as carboxymethylcellulose, carboxymethylcellulose calcium, carboxymethylrose, carboxymethylcellulose calcium, etc. Further, a disintegrant may be added and granulated by wet or dry method. Tablets can be obtained by compressing the powder or granules as they are or by adding a lubricant such as magnesium stearate or talc. The tablet or granule is coated with an enteric base such as hydroxypropylmethylcellulose phthalate or methyl methacrylate copolymer, or coated with ethylcellulose, carnauba wax, hardened oil, etc. can do. A hard capsule can be obtained by filling a hard capsule with the above powder or granule. Soft capsules can be obtained by mixing hyaluronan or a salt thereof with glycerin, polyethylene glycol, sesame oil, olive oil or the like and coating it with a gelatin film. A syrup can be obtained by dissolving a sweetener such as sucrose, sorbitol, and glycerin and hyaluronan or a salt thereof in water. In addition to sweeteners and water, essential oils, ethanol and the like can be added to make elixirs, or gum arabic, tragacanth, polysorbate 80, sodium carboxymethylcellulose and the like can be added to make emulsions or suspensions. Moreover, a corrigent, a coloring agent, a preservative, etc. can be added to these liquid preparations as needed.

  Examples of parenteral preparations include injections, rectal administration agents, pessaries, external preparations for skin, inhalants, aerosols, eye drops and the like. Injections include hyaluronan or salts thereof, pH regulators such as hydrochloric acid, sodium hydroxide, lactic acid, sodium lactate, sodium monohydrogen phosphate, sodium dihydrogen phosphate; isotonic agents such as sodium chloride and glucose; and It can be obtained by adding distilled water for injection, sterilizing and filtering and then filling ampoules. Further, mannitol, dextrin, cyclodextrin, gelatin and the like can be added and lyophilized in a vacuum to obtain an injection that can be dissolved at the time of use. Moreover, after adding an emulsifier, such as lecithin, polysorbate 80, polyoxyethylene hydrogenated castor oil, to hyaluronan or its salt, it can also be set as the emulsion for injection emulsified in water.

  For rectal administration, after adding a suppository base such as mono-, di- or triglyceride of cacao fatty acid or polyethylene glycol to hyaluronan or a salt thereof, dissolve by heating, and pour it into a mold to cool, Alternatively, hyaluronan or a salt thereof can be mixed with polyethylene glycol, soybean oil or the like and then coated with a gelatin film. The external preparation for skin can be obtained by adding white petrolatum, beeswax, liquid paraffin, polyethylene glycol or the like to hyaluronan or a salt thereof, and heating and kneading as necessary. The tape agent can be obtained by kneading hyaluronan or a salt thereof with a pressure-sensitive adhesive such as rosin or an alkyl acrylate polymer and spreading it on a nonwoven fabric or the like. The inhalant can be obtained by, for example, dissolving or dispersing hyaluronan or a salt thereof in a propellant such as a pharmaceutically acceptable inert gas and filling the pressure-resistant container.

(Administration method) The administration method of the drug comprising the hyaluronan of the present invention as an active ingredient is not particularly limited, and examples thereof include intravenous administration, oral administration and intra-articular administration.
(Dosage) The dosage is appropriately determined according to the target disease, patient age, health condition, body weight, etc. Generally, 0.05 to 50 mg / kg is divided once or more once a day. To administer.
(Toxicity) The hyaluronan used in the present invention showed little or no cytotoxicity at doses showing biological activity as a medicine.

  Hereinafter, although the chemical | medical agent which concerns on this invention is demonstrated in detail using an Example, the technical scope of this invention is not limited to a following example.

[Example 1]
In this example, the MAPKKK gene expression enhancer by the drug according to the present invention was evaluated using a DNA chip that can monitor gene expression enhancement / expression suppression.

Experimental Method First, K562 was cultured in RPMI medium divided into 1 and 2 groups. Both groups had culture conditions of incubation at 42 ° C. for 20 minutes and then at 37 ° C. for 30 minutes. Note that HA4 (10 ng / ml) was added to the two groups of media.

Next, after culture, the medium was removed by centrifugation at 1000 rpm. The obtained cells were stored in a -60 ° C deep freezer. Next, RNA was extracted from the preserved cells according to a conventional method. Using the extracted RNA, gene expression was analyzed using a DNA chip on which an oligonucleotide that specifically hybridizes to the MAPKKK13 gene was immobilized. Gene expression analysis using a DNA chip was outsourced to the DNA chip laboratory. In detail, the product name: AceGene Human Oligo Chip 30K 1 Chip Version made by DNA Chip Laboratory was used.
result
As a result of the expression analysis of the MAPKKK13 gene using a DNA chip, the ratio between the expression intensity in group 1 and the expression intensity in group 2 ([expression intensity of group 2] / [expression intensity of group 1]) was calculated. The results are shown in Table 1. The expression intensity ratio for the MAPKKK13 gene was 11.43.

  An image of the DNA chip used in this example is shown in FIG.

Discussion In this example, expression of MAPKKK gene was enhanced by treatment with HA4. In this example, since cells other than K562 are not contained, the influence of other types of cells is excluded. Further, in this example, after completion of heat shock at 42 ° C. for 20 minutes, at the time of incubation at 37 ° C. for 30 minutes, although there is new mRNA expression, it is considered that new protein expression hardly occurs. For example, for Hsp72, it can be said that there is no change at the protein level even though mRNA expression is enhanced. Therefore, it can be said that the mRNA expression enhancement of the MAPKKK gene shown in this Example is a direct influence by HA4 and not a secondary phenomenon mediated by other factors. From the above results, it was demonstrated that HA4 functions as an expression enhancer of MAPKKK gene.

It is the photograph which imaged the DNA chip used in the Example.

Claims (4)

  MAP kinase kinase kinase gene expression enhancer comprising hyaluronan as an active ingredient.   The hyaluronan is a tetrasaccharide containing two units of -D-glucuronic acid-β-1,3-DN-acetylglucosamine-β-1,4-. 1. The expression enhancer for MAP kinase kinase kinase gene according to 1.   The MAP kinase kinase kinase gene expression enhancer according to claim 1, wherein the MAP kinase kinase kinase gene is a MAP kinase kinase kinase 13 gene.   The expression enhancer for a MAP kinase kinase kinase gene according to claim 1, which is a kinase involved in the Stress responsive MAP kinase (SAPK) pathway and also called leucine zipper-bearing kinase (LZK).

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