JP4022659B2 - Therapeutic and preventive agents for autoimmune diseases, inflammation and neurological diseases - Google Patents

Description

  The present invention relates to a therapeutic agent for autoimmune diseases, a therapeutic agent for inflammation, and a therapeutic agent for neurological diseases, comprising hyaluronan as an active ingredient. The present invention also relates to an autoimmune disease preventive agent, an inflammation preventive agent and a neurological disease preventive agent 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 fluids, or fermentation methods using bacteria of 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).

  Multiple sclerosis, on the other hand, usually develops between puberty and 40's and has symptoms such as staggering when walking, blurred vision, double vision, difficulty in urine, and pain and numbness. Epilepsy may occur when children or young people develop. One or more lesions that cause symptoms can be scattered in the cerebrum or spinal nerve. In addition, the lesions are not only spatially scattered, but also symptoms appear and disappear one after another. The pathology of multiple sclerosis involves the immune system and is regarded as an autoimmune disease / inflammation. It is also regarded as one of the neurological diseases because the spinal nerve is affected.

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.

  An object of the present invention is to provide a therapeutic agent for autoimmune diseases, a therapeutic agent for inflammation, and a therapeutic agent for neurological diseases. It is another object of the present invention to provide an autoimmune disease preventive agent, an inflammation preventive agent and a neurological disease preventive agent.

  The therapeutic agent for autoimmune diseases, the therapeutic agent for inflammation and the therapeutic agent for neurological diseases according to the present invention that have achieved the above-mentioned object contain hyaluronan as an active ingredient. Moreover, the autoimmune disease preventive agent, inflammation preventive agent, and neurological disease preventive agent according to the present invention contain hyaluronan as an active ingredient.

  Since the drug according to the present invention contains hyaluronan as an active ingredient, it has an advantage that it can be easily mass-produced at a relatively low cost. Hyaluronan is expected to be a therapeutic and prophylactic agent with very few side effects because it has little toxicity and antigenicity, and enhances the therapeutic action and disease prevention action inherent in the living body. Thus, according to the present invention, a novel drug effective for autoimmune diseases, inflammation and neurological diseases can be provided.

  Hereinafter, the present invention will be described in detail. In the following description, the therapeutic agent and prophylactic agent according to the present invention are collectively referred to as a drug.

  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 sugar and is basically composed of β-D-glucuronic acid and β-DN-acetylglucosamine, those elements are combined with one or more disaccharide units combined. May be used, and derivatives thereof such as those having a hydrolyzable protecting group such as an acyl group can also be used. The sugar may be an unsaturated sugar, and examples of the unsaturated sugar include non-reducing terminal sugars, usually those having unsaturated carbon atoms between positions 4 and 5 of glucuronic acid. 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 sodium hydroxide of about 1N 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 is selected from the group consisting of autoimmune diseases, inflammations and neurological diseases without adversely affecting the living body by administering an effective amount thereof to mammals including humans. At least one disease can be ameliorated. Autoimmune diseases, inflammation and neurological diseases can include multiple sclerosis. However, the autoimmune disease and inflammation are not limited to multiple sclerosis. For example, rheumatism, systemic lupus erythematosus, inflammatory enteritis, uveitis, nephritis, nephropathy, type I diabetes, atopic dermatitis, Sjogren Syndrome, insulin receptor abnormality, vasculitis, myasthenia gravis, polymyositis, asthma, Hashimoto's disease. Further, the neurological disease is not limited to multiple sclerosis, for example, neuritis, neuralgia, neuralgia, stroke, cerebral palsy, depression, senile dementia, Parkinson's disease, Alzheimer's disease, Recklinghausen disease, moyamoya Disease, Krabbe disease, acute disseminated encephalomyelitis, spinal radiculoneuropathy, acute disseminated encephalomyelitis, optic neuromyelitis, adrenoleukodystrophy, metachromatic leukodystrophy, amyotrophic lateral sclerosis, peripheral neuropathy (Peripheral nerve injury, Guillain-Barre syndrome, strangulation neuropathy, brachial plexus paralysis, diabetic neuropathy, etc.). That is, a drug containing hyaluronan as an active ingredient has a therapeutic effect and a preventive effect on the various autoimmune diseases, inflammations and neurological diseases described above.

  The agent of the present invention is administered hyaluronan or a salt thereof, as it is or as necessary, with a carrier, excipient, or other additive, orally or parenterally (intra-articular administration, intravenous, intramuscular, subcutaneous). As a pharmaceutical for administration (injection), enteral administration, transdermal administration, etc.), it can be formulated into any dosage form and administered to a patient by any administration method.

  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 described above or by adding a lubricant such as magnesium stearate or talc. The tablet or granule is coated with an enteric base such as hydroxypropyl methylcellulose phthalate or methyl methacrylate copolymer, or coated with ethyl cellulose, carnauba wax, hardened oil, etc. can do. The hard capsule can be obtained by filling the above powder or granule into a hard capsule. 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. The 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, add 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 intraspinal administration, intravenous administration and oral administration.

  (Dosage) The dosage is appropriately determined according to the target disease, the patient's 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 drug.

  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, HA4 was administered to experimental autoimmune myelitis (EAE), which is a multiple sclerosis model, and the effect was examined.

Multiple sclerosis model animals 4 weeks old Lewis rats were purchased and used after 5 weeks of age. According to the method of Shibaki et al. (Kenji Shibaki, Junichi Nomura, Ryozo Ohno, Kunio Shimazu: Suppression of experimental autoimmune encephalomyelitis with Ninjin Yoei-to. Neurotherapy 19 (2): 159-166,2002) , Guinea pig myelin basic protein (Guinea pig myelin basic protein (GPMBP) Sigma) 300μg / mouse was dissolved in PBS 50μl, equivalent to Freund Complete Adjuvant (FCA) manufactured by Difco) and Mycobacterium tuberculosis (MT, manufactured by Difco) The concentration was adjusted to 0.75 mg / ml, and 50 μl each was inoculated on the left and right hind footpads of the animals.

  In this example, a multiple sclerosis model animal immediately after inoculation and a multiple sclerosis model animal at the onset of multiple sclerosis were used.

Administration of test substance In this example, 1 mg / ml and 10 mg / ml HA4 were prepared. Specifically, HA4 is produced by the method of Tawada et al. (Tawada A, Masa T, Oonuki Y, Watanabe A, Matsuzaki Y, Asari A. Large-scale preparation, purification, and characterization of hyaluronan oligosaccharides from 4-mers to 52-mers. Glycobiology. 2002; 12 (7): 421-6.). Moreover, physiological saline (saline) was adjusted as a control.

  The catheter was placed in the medullary cavity of a multiple sclerosis model animal at two time points immediately after inoculation or when onset was confirmed by observation of neurological symptoms, and administered intradurally for a set administration period. For continuous administration, an osmotic pump (Model 2004, Alzet) was used. Table 1 shows the composition of the administration group.

Evaluation of EAE Neurological Symptoms Every day after inoculation, neurological symptoms were observed in two stages by two observers.
EAE grade
0: Asymptomatic 1: Decrease of tail tonus 2: Incomplete paralysis of hind limbs 3: Complete hind limb paralysis with occasional urinary incontinence 4: Paralysis of limbs

Result 1) Effect of HA4 intrathecal administration immediately after inoculation (cause) (prevention)
After the antigen inoculation, the neurologic signs of continuous administration of HA4 intrathecally were clearly mild compared with the EAE control group (FIG. 1). FIG. 1 is a graph in which the average value of the evaluation values of the above-mentioned EAE neurological symptoms is plotted on the vertical axis, and the number of days elapsed from day 0 of inoculation on the horizontal axis. In comparison with the neurological signs at the EAE extreme stage, the clinical score of the EAE control group on the 13th day after inoculation was 2.2 ± 0.41, compared with 0.2 ± 0.41 on the 13th day in the HA4 continuous administration group. A significantly low value (P <0.001) was shown, and in the HA4 continuous administration group, the number of cases was only 1/6. From the results of FIG. 1, it was demonstrated that a drug containing hyaluronan as an active ingredient is effective for the prevention of multiple sclerosis.

2) Effect of intrathecal administration of HA4 after onset (treatment)
After the onset, the neurologic signs of HA4 intrathecal continuous administration were clearly milder than those in the EAE control group (FIG. 2). FIG. 2 is a graph in which the average value of the evaluation values of the EAE neurological symptoms described above is plotted on the vertical axis, and the number of days elapsed from day 0 of inoculation on the horizontal axis. In comparison of the neurological signs at the EAE extreme stage, the clinical score of 2.2 ± 0.41 of the EAE control group on the 13th day after the inoculation was compared with 1.5 ± 1.0 on the 13th day in the HA4 continuous administration group. It showed a low value. In comparison of disease duration, the EAE control group 6.5 ± 0.55 days, the (GlcNAc-GlcA) ₂ continuous administration group showed a significant shortening (P <0.01), 4.3 ± 1.5 days. It was. From the results of FIG. 2, it was demonstrated that a drug containing hyaluronan as an active ingredient is effective for the treatment of multiple sclerosis.

3) Effect of HA4 single intrathecal administration after onset (treatment)
After onset, single signs of HA4 intrathecal administration showed clear mildness compared to the EAE control group (FIG. 3). FIG. 3 is a graph with the average value of the evaluation values of EAE neurological symptoms described above on the vertical axis and the number of days elapsed on day 0 of inoculation on the horizontal axis. In the comparison of neurological signs at the extreme EAE phase, the clinical score of the EAE control group on day 13 after inoculation was 2.2 ± 0.41, compared with 1.8 ± 0.5 days on day 13 in the HA4 continuous administration group. And showed a low value. In addition, in the comparison of the disease duration, a significant shortening P <0.001) was observed in the HA4 continuous administration group as compared with 6.5 ± 0.55 days in the EAE control group. From the results of FIG. 3, it was demonstrated that a drug containing hyaluronan as an active ingredient is effective for the treatment of multiple sclerosis.

It is the graph which made the average value of the evaluation value of the EAE neurological symptom observed about the multiple sclerosis model animal which administered HA4 from immediately after inoculation on the vertical axis, and set the elapsed days from inoculation on the 0th axis on the horizontal axis. It is the graph which made the average value of the evaluation value of the EAE neurological symptom observed about the multiple sclerosis model animal which administered HA4 for 11 days after onset on the vertical axis | shaft, and made the horizontal axis the elapsed days on the 0th day of inoculation. It is the graph which made the average value of the evaluation value of the EAE neurological symptom observed about the multiple sclerosis model animal which administered HA4 single time immediately after inoculation on the vertical axis, and made the horizontal axis the elapsed days which made inoculation the 0th day.

Claims (2)

  -D-glucuronic acid-β-1,3-D-N-acetylglucosamine-β-1,4- is a unit and multiple sclerosis containing hyaluronan, a tetrasaccharide containing two units, as an active ingredient Therapeutic or prophylactic agent.   -D-glucuronic acid-β-1,3-D-N-acetylglucosamine-β-1,4- is a unit and multiple sclerosis containing hyaluronan, a tetrasaccharide containing two units, as an active ingredient Therapeutic or preventive agent for neurological symptoms in

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