US20110059508A1 - Improving agent for dysfunction due to neuropathy and rho kinase activation inhibitor - Google Patents

Improving agent for dysfunction due to neuropathy and rho kinase activation inhibitor Download PDF

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US20110059508A1
US20110059508A1 US12/667,178 US66717808A US2011059508A1 US 20110059508 A1 US20110059508 A1 US 20110059508A1 US 66717808 A US66717808 A US 66717808A US 2011059508 A1 US2011059508 A1 US 2011059508A1
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improving agent
keratanase
dysfunction
keratan sulfate
nerve damage
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Kenji Kadomatsu
Yukihiro Matsuyama
Akiomi Tanaka
Sawako Takeshita
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Nagoya University NUC
Seikagaku Corp
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Nagoya University NUC
Seikagaku Corp
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/47—Hydrolases (3) acting on glycosyl compounds (3.2), e.g. cellulases, lactases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A61P21/02—Muscle relaxants, e.g. for tetanus or cramps
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/04—Centrally acting analgesics, e.g. opioids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01096—Mannosyl-glycoprotein endo-beta-N-acetylglucosaminidase (3.2.1.96)

Definitions

  • the present invention relates to an improving agent for dysfunction due to nerve damage and to an Rho kinase activation inhibitor.
  • the environment surrounding the central nervous system is equipped with a regeneration inhibiting function via plurality of regeneration suppressing factors for neuronal axons for preventing the disordered formation of synapse and this function disturbs the regeneration of neuronal network.
  • Chondroitin sulfate proteoglycan has been known as one of the regeneration suppressing factors for neuronal axon and has been reported to inhibit the axonal regeneration in the injured area (refer, for example, to Non-Patent Literature 1).
  • Patent Literature 1 the fact that keratan sulfate plays an important role in the formation of glial scar inhibiting the axonal regeneration is clarified by an experiment in brain-injured model using mice where N-acetylglucosamine 6-O-sulfotransferase 1 (GlcNAc6ST-1) gene which is necessary for biosynthesis of keratan sulfate in the brain is knocked out.
  • GlcNAc6ST-1 N-acetylglucosamine 6-O-sulfotransferase 1
  • neuropathic pain is a pain caused by disorder of the central nerve or the peripheral nerve and examples thereof include spontaneous pain, hyperalgesic reaction where the threshold for invasive stimulation lowers and mechanical allodynia where non-invasive mechanical stimulation and tactile stimulation which usually do not induce the pain are erroneously recognized as sharp pain.
  • diseases expressing neuropathic pain include cerebral disorder, multiple sclerosis and spinal cord injury as central ones and diabetes mellitus and herpes zoster as peripheral ones.
  • neuropathic pains allodynia is characterized in that intractable and burning pain and piercing pain continue for a long period of time without intermittence and is also a cause of the reduction in the effect of rehabilitation due to the pain.
  • drug therapy for neuropathic pain and there has been a demand for the development of drugs which satisfy both pharmaceutical effect and safety.
  • the development of the drug has been unable to make satisfactory progress.
  • One of the reasons therefor is that it is thought that the mechanism of pathogenesis is not single but many of them are entangled in a complicated manner.
  • Non-Patent Literature 2 It has been also being aware of that spinal microglia of dorsal horn is activated by nerve injury and that stimulation of P2X4 receptor which is strongly expressed therein causes neuropathic pain, and it has been proposed of the participation of Rho kinase signal transduction system as one of the routes of cascade of the activation as such (Non-Patent Literature 2).
  • Patent Literature 1 JP-A-2006-290842
  • Non-Patent Literature 1 Niederost, B. P., Zimmermann, D. R., Schwab, M. E. & Bandtlow, C. E. Bovine CNS myelin contains neurite growth-inhibitory activity associated with chondroitin sulfate proteoglycans. J. Neurosci. 19, 8979-8989 (1999).
  • Non-Patent Literature 2 Nissan, S. et al., 2001. Extracellular ATP or ADP Induce Chemotaxis of Cultured Microglia through Gi/o-Coupled P2Y Receptors. J. Neurosci. 21(6), 1975-1982.
  • an object of the present invention is to provide a substance which is able to be an active ingredient for the improvement of dysfunction caused by nerve damage.
  • an endo-O—N-acetylglucosaminidase type enzyme which is one of the keratan sulfate degrading enzymes and hydrolyzes an N-acetylglucosaminide bond in a keratan sulfate backbone improves dysfunction due to nerve damage such as motor neuron dysfunction or sensory neuron dysfunction (such as neuropathic pain represented by a pain caused by allodynia and hyperalgesic reaction) and that such an action is mediated by the suppression of an Rho kinase activation by keratan sulfate.
  • an endo- ⁇ -N-acetylglucosaminidase type enzyme which hydrolyzes an N-acetylglucosaminide bond in a keratan sulfate backbone (hereinafter, it will be sometimes referred to as “an endo- ⁇ -N-acetylglucosaminidase type keratan sulfate degrading enzyme”) is continuously administered to the injured area of an individual immediately after nerve damage, the spinal cord injury happens for example, so as to remove a keratan sulfate backbone of keratan sulfate proteoglycan, that releases the continuous abnormal activation of nerve cells and non-nerve cells such as glia cell caused by proteoglycan and results in the release of an abnormal axonal guidance function by proteoglycan and also in the therapeutic effect for neuropathic pain.
  • the present inventors have for the first time found that, when an endo- ⁇ -N-acetylglucosaminidase type keratan sulfate degrading enzyme is continuously administered to the injured area in the intrathecal cavity of model rats for spinal cord injury, improvement of motor neuron function and sensory neuron function is promoted and that, particularly due to the improvement of the latter, neuropathic pain, particularly allodynia, is able to be improved.
  • an improving agent for dysfunction due to nerve damage of the present invention achieved by the above-mentioned finding is characterized in that, as mentioned in claim 1 , it comprises an endo- ⁇ -N-acetylglucosaminidase type enzyme which hydrolyzes an N-acetylglucosaminide bond in a keratan sulfate backbone as an active ingredient.
  • the improving agent mentioned in claim 2 is characterized in that, in the improving agent mentioned in claim 1 , the nerve damage is that arising from spinal cord injury.
  • the improving agent mentioned in claim 3 is characterized in that, in the improving agent mentioned in claim 1 , the nerve damage is amyotrophic lateral sclerosis.
  • the improving agent mentioned in claim 4 is characterized in that, in the improving agent mentioned in any of claims 1 to 3 , the dysfunction due to nerve damage is motor neuron dysfunction.
  • the improving agent mentioned in claim 5 is characterized in that, in the improving agent mentioned in any of claims 1 to 3 , the dysfunction due to nerve damage is sensory neuron dysfunction.
  • the improving agent mentioned in claim 6 is characterized in that, in the improving agent mentioned in claim 5 , the sensory neuron dysfunction is neuropathic pain.
  • the improving agent mentioned in claim 7 is characterized in that, in the improving agent mentioned in claim 6 , the neuropathic pain is a pain caused by allodynia or hyperalgesic reaction.
  • an improving agent for neuropathic pain of the present invention is also characterized in that, it comprises an endo- ⁇ -N-acetylglucosaminidase type enzyme which hydrolyzes an N-acetylglucosaminide bond in a keratan sulfate backbone as an active ingredient.
  • an Rho kinase activation inhibitor of the present invention (hereinafter, it will be sometimes referred to as an inhibitor of the present invention) is characterized in that, it comprises an endo- ⁇ -N-acetylglucosaminidase type enzyme which hydrolyzes an N-acetylglucosaminide bond in a keratan sulfate backbone as an active ingredient.
  • an agent for treating nerve damage of the present invention comprising an endo- ⁇ -N-acetylglucosaminidase type enzyme which hydrolyzes an N-acetylglucosaminide bond in a keratan sulfate backbone as an active ingredient for a continuous administration to a neuropathic site at the dose of 0.3 milliunit (mU) to 15000 mU per day to an adult human.
  • an endo- ⁇ -N-acetylglucosaminidase type keratan sulfate degrading enzyme is provided as an active ingredient for an improving agent for dysfunction of motor neuron and of sensory neuron due to nerve damage such as spinal cord injury or, to be more specific, for an improving agent for neuropathic pain for example.
  • an agent for administering to nerve damage for a continuous administration to a neuropathic site which comprises a specific amount of an endo- ⁇ -N-acetylglucosaminidase type keratan sulfate degrading enzyme.
  • FIG. 1 Experimental Result of Example 1, No. 1: This is a graph showing the dose response of the therapeutic effect in an evaluation of the recovery of hind paw motor function (BBB Test) of Bc keratanase II.
  • FIG. 2 (the same as above): This is a graph showing the dose response of the therapeutic effect of Ks36 keratanase II.
  • FIG. 3 (the same as above): This is a graph showing the comparison in the therapeutic effects by Bc keratanase II and Ks36 keratanase II when the administered concentration is 0.05 U/200 ⁇ l.
  • FIG. 4 (the same as above): This is a graph showing the comparison in the therapeutic effects by Bc keratanase II and Ks36 keratanase II when the administered concentration is 0.000025 U/200 ⁇ l.
  • FIG. 5 (the same as above): This is a graph showing the comparison in the therapeutic effects by Bc keratanase II, inactivated Bc keratanase II and Ps keratanase when the administered concentration is 0.05 U/200 ⁇ l.
  • FIG. 6 Experimental Result of Example 1, No. 2: This is a graph showing the comparison in the therapeutic effects by Bc keratanase II and Ks36 keratanase II when the administered concentration is 0.05 U/200 ⁇ L in an evaluation of the recovery of hind paw motor function (Grid Test).
  • FIG. 7 (the same as above): This is a graph showing the comparison in the therapeutic effects by Bc keratanase II and Ks36 keratanase II when the administered concentration is 0.000025 U/200 ⁇ L.
  • FIG. 8 Experimental Result of Example 1, No. 3: This is a graph showing the comparison in the therapeutic effects by Bc keratanase II and Ks36 keratanase II when the administered concentration is 0.05 U/200 ⁇ L in an evaluation of the recovery of hind paw motor function (Foot Print Test).
  • FIG. 9 Experimental Result of Example 1, No. 4: This is a graph showing the comparison in the therapeutic/improving effects by Bc keratanase II and Ks36 keratanase II when the administered concentration is 0.05 U/200 ⁇ L in an evaluation of a therapeutic/improving effect for thermal allodynia (Tail Flick Test).
  • FIG. 10 Experimental Result of Example 1, No. 5: This is a graph showing the comparison in the therapeutic/improving effects by Bc keratanase II and Ks36 keratanase II when the administered concentration is 0.05 U/200 ⁇ L in an evaluation of the therapeutic/improving effect for mechanical allodynia (Touch Test).
  • FIG. 11 Experiment of Example 2, No. 1: This is a graph showing a releasing effect of Ks36 keratanase II to a neurite outgrowth-inhibiting action by keratan sulfate.
  • FIG. 12 Experiment of Example 2, No. 2: These are pictures under a fluorescence microscope showing a releasing effect of Y27632 (Rho kinase inhibitor, Sigma) to a neurite outgrowth-inhibiting action by keratan sulfate.
  • FIG. 13 (the same as above): This is a graph showing an Rho kinase activation suppressing action of Ks36 keratanase II.
  • FIG. 14 This is a result of western blotting showing the expression of keratan sulfate in spinal cord of ALS model mice in Example 3.
  • FIG. 15 are pictures under a fluorescence microscope showing the expression of keratan sulfate in microglia.
  • An improving agent of the present invention is characterized in that the agent comprises an endo- ⁇ -N-acetylglucosaminidase type enzyme which hydrolyzes an N-acetylglucosaminide bond in a keratan sulfate backbone (an endo- ⁇ -N-acetylglucosaminidase type keratan sulfate degrading enzyme) as an active ingredient.
  • an endo- ⁇ -N-acetylglucosaminidase type enzyme which hydrolyzes an N-acetylglucosaminide bond in a keratan sulfate backbone (an endo- ⁇ -N-acetylglucosaminidase type keratan sulfate degrading enzyme) as an active ingredient.
  • the endo- ⁇ -N-acetylglucosaminidase type enzyme which hydrolyzes an N-acetylglucosaminide bond in a keratan sulfate backbone is a keratan sulfate degrading enzyme, also known as keratanase II, and the enzyme per se is a known substance (hereinafter, the endo- ⁇ -N-acetylglucosaminidase type enzyme which hydrolyzes an N-acetylglucosaminide bond in a keratan sulfate backbone will be abbreviated as keratanase II).
  • Keratanase II is an enzyme derived from microbes and has been known to be produced by Ks36 strain of bacteria belonging to genus Bacillus (Accession Number for the microbe: FERM P-10204) (refer, if necessary, to Japanese Patent No. 2726274; hereinafter, keratanase II produced by Ks36 strain will be abbreviated as Ks36 keratanase II).
  • Ks36 keratanase II A heat resistant keratanase II having an excellent stability against heat where the optimum reaction temperature is 50 to 60° C.
  • Bc keratanase II Bacillus circulans KsT202 strain (Accession Number for the microbe: FERM BP-5285) (hereinafter, the heat resistant keratanase II will be abbreviated as Bc keratanase II).
  • Bc keratanase II is characterized in that it degrades keratan sulfate whereupon sulfated keratan sulfate disaccharide and keratan sulfate tetrasaccharide are mainly produced (refer, if necessary, to Japanese Patent No. 3734504, U.S. Pat. No. 5,840,546, European Patent No. 0798376 B1, etc.).
  • Bc keratanase II may be a genetically recombinant type heat resistant keratanase II which is produced, for example, by transferring the heat resistant keratanase II gene cloned from Bacillus circulans KsT202 strain to a host such as Escherichia coli (refer, if necessary, to JP-A-2004-24189 and Gen Bank: Accession No.
  • rBC keratanase II genetically recombinant type heat resistant keratanase II will be abbreviated as rBC keratanase II).
  • the total amino acid sequence of rBC keratanase II described in the above-mentioned JP-A-2004-24189 is attached hereto as SEQ ID No. 1.
  • Ks36 keratanase II and Bc keratanase II are the endo- ⁇ -N-acetylglucosaminidase type enzymes, they are different in terms of reactivity to high concentrated keratan polysulfate (keratan sulfate having a high degree of sulfation), optimum pH, pH stability, optimum temperature, temperature stability, influence of inhibition by drugs, etc. and, therefore, they are essentially different enzymes (refer, if necessary, to Japanese Patent No. 3734504, U.S. Pat. No. 5,840,546, European Patent No. 0798376 B1, etc.).
  • those two kinds of enzymes are advantageously listed wherein Bc keratanase II is preferred and rBC keratanse II is more preferred.
  • keratanase II used in the present invention is not limited to the above-mentioned exemplified enzymes so far as it is an endo-O—N-acetylglucosaminidase type enzyme which hydrolyzes an N-acetylglucosaminide bond in a keratan sulfate backbone and, further, there is no limitation for the origin and the enzymatic properties thereof.
  • Rho kinase II improves motor neuron dysfunction and sensory neuron dysfunction caused by various nerve damages such as nerve damage arising from spinal cord injury or amyotrophic lateral sclerosis, and the action as such is mediated by the suppression of an Rho kinase activation by keratan sulfate.
  • Rho kinase is a small GTPase (guanosine triphosphate phosphatase) existing in cytoplasm and having an activity of hydrolyzing GTP (guanosine triphosphate) and is an important enzyme for phosphorylation which conducts various controls for cell shape and locomotion by controlling an actin cytoskeletal system or tubulin.
  • Rho This enzyme was found in 1990s and is activated by several intracellular factors such as Rho.
  • Rho kinase signal transduction system When an Rho kinase signal transduction system is activated, an actin cytoskeletal system is inactivated and, as a result, an axonal regeneration is inhibited and decay of growth cone is induced.
  • Keratanase II such as Ks36 keratanase II or Bc keratanase II which is an active ingredient for an improving agent of the present invention or an inhibitor of the present invention (hereinafter, both agents will be sometimes referred to as the drug of the present invention) is made into a parenteral preparation by a common method same as in the case of common parenteral enzyme preparations, parenterally administered to mammals (such as humans, non-human primates, rats, mice, rabbits, cattles, horses, pigs, dogs or cats) by an administration route corresponding to object disease and used for the treatment or the prevention of the aimed disease of the animal.
  • mammals such as humans, non-human primates, rats, mice, rabbits, cattles, horses, pigs, dogs or cats
  • parenteral preparation examples include liquid preparation (such as solution preparation, suspension preparation, eye drop, nose drop or locally infusion agent to brain, intrathecal cavity, skin, etc.), solid preparation (such as freeze-dried preparation, powder preparation, granule preparation, microcapsule, liposome or liposphere) and semisolid preparation (such as ointment).
  • liquid preparation such as solution preparation, suspension preparation, eye drop, nose drop or locally infusion agent to brain, intrathecal cavity, skin, etc.
  • solid preparation such as freeze-dried preparation, powder preparation, granule preparation, microcapsule, liposome or liposphere
  • semisolid preparation such as ointment
  • the drug of the present invention is manufactured as liquid preparation for example, it is able to be manufactured by dissolving or dispersing keratanase II of a pharmaceutically acceptable grade into a solution to which water for injection, pharmaceutically acceptable additives or carriers such as isotonizing agent (e.g., sodium chloride, potassium chloride, glycerin, mannitol, sorbitol, boric acid, borax, glucose or propylene glycol), buffer agent (e.g., phosphate buffer, acetate buffer, borate buffer, carbonate buffer, citrate buffer, Tris buffer, glutamate buffer or c-aminocaproate buffer), preservative (e.g., p-hydroxybenzoate, chlorobutanol, benzyl alcohol, benzalkonium chloride, sodium dehydroacetate, sodium edetate, boric acid or borax), nonionic surfactant (e.g., polyoxyethylene sorbitan fatty acid ester, polyoxyethylene hydrogenated cast
  • liquid preparation is dried by a drying method (such as freeze-drying) which does not affect the pharmacological function of keratanase II such as activity whereupon solid preparation which is a type being dissolved before use is prepared.
  • a drying method such as freeze-drying
  • keratanase II of a pharmaceutically acceptable grade is dissolved or suspended in physiological saline, water for injection, isotonic liquid, oily liquid or the like to prepare liquid preparation.
  • the drug of the present invention prepared as such is administered by an administering method depending upon object disease, degree of symptom, subject to be administered, etc. whereby dysfunction caused by nerve damage is able to be improved, treated or prevented.
  • An example thereof is a method where it is locally administered to the site suffering from nerve damage or surroundings thereof and such a method is preferred.
  • Dose and administering period of the drug of the present invention is to be appropriately determined by medical specialists such as a medical doctor taking the conditions such as object disease, animal species, age or body weight to be administered, degree of symptom or health condition of the subject into consideration and there is no particular limitation provided that the dose and the period are effective for suppressing an activation of Rho kinase in diseased site or for degrading a keratan sulfate backbone of keratan sulfate proteoglycan in glial scar.
  • Daily dose for intrathecal administration of an improving agent of the present invention to a rat (body weight: 0.3 kg; cerebrospinal fluid amount: 0.3 mL) of a spinal cord-injured model is about 1.5 microunit ( ⁇ U) (0.000025 U/200 ⁇ L) to 30 milliunit (mU), preferably about 0.3 mU (0.005 U/200 ⁇ L) to 10 mU, and most preferably about 3.0 mU (0.05 U/200 ⁇ L) in terms of enzyme unit of keratanase II.
  • “U/200 ⁇ L” in the parentheses means a concentration and an enzyme liquid preparation having each concentration is administered in a dose of 12 ⁇ L per day.
  • the dose to an adult human is as follows.
  • the daily dose for intrathecal administration in terms of a conversion based on body weight is about 0.3 mU to 6000 mU, preferably about 60 mU to 2000 mU, and most preferably about 600 mU.
  • the daily dose for intrathecal administration in terms of a conversion based on cerebrospinal fluid amount is about 0.75 mU to 15000 mU, preferably about 150 mU to 5000 mU, and most preferably about 1500 mU.
  • the daily dose for intrathecal administration is about 0.3 mU to 15000 mU, preferably about 60 mU to 5000 mU, and most preferably about 600 mU to 1500 mU.
  • the administering period is to be decided by medical specialists such as a medical doctor depending upon the symptoms taking expressed amount of keratan sulfate proteoglycan in the injured area and possibility of adverse event, etc. into consideration and a yardstick thereof in the case of a continuous administration is up to about eight weeks, preferably up to about four weeks, and more preferably up to about two weeks after the injury. Time for starting the administration is preferred to be immediately after the injury or three days to about one week after the injury.
  • the above-mentioned daily dose for intrathecal administration is that about 0.3 mU, 15000 mU, 60 mU, 5000 mU, 600 mU and 1500 mU correspond to not more than about 0.15 microgram ( ⁇ g), not more than about 7500 ⁇ g, not more than about 30 ⁇ g, not more than about 2500 ⁇ g, not more than about 300 ⁇ g and not more than about 750 ⁇ g, respectively in terms of the weight of enzyme protein.
  • One unit (1 U) of enzyme amount is defined as enzyme amount producing reducing terminal corresponding to 1 ⁇ mol of galactose per minute when a reaction is conducted at 37° C. for 10 minutes using keratan polysulfate derived from cartilage of shark as a substrate (refer, if necessary, to JP-A-2004-24189).
  • the enzyme amount or the concentration per unit preparation of the drug of the present invention is set depending upon the above-mentioned dose and the preparation is not always necessary to make the enzyme amount or the concentration upon the administration but, for example, dilution may be conducted immediately before the administration or upon the administration to an effective and safe concentration by using diluent. Accordingly, the present invention also provides a kit in which such diluent is combined with the above-mentioned drug of the present invention.
  • keratanase II which is an active ingredient is in a pharmaceutically acceptable grade and that impurities such as endotoxin, nucleic acid or protease derived from the microbes wherefrom the present enzyme is produced are to be removed as much as possible.
  • Amounts of endotoxin, nucleic acid and protease are to be not more than the detecting limit by the conventional analytic method.
  • endotoxin its amount is preferred to be not more than 5.0 pg/100 U when measured by Toxicolor (registered trade mark) System of Seikagaku Corporation.
  • nucleic acid it is preferred to be not more than the detecting limit when measured by Threshold Method (DNA measuring device: Threshold (manufactured by Molecular Devices)).
  • Threshold Method DNA measuring device: Threshold (manufactured by Molecular Devices)
  • protease its amount is preferred to be not more than 0.1% to the total protein when measured by using FITC-casein as a substrate.
  • the improving agent of the present invention is used for improvement, treatment or prevention of dysfunction caused by acute disorder, subacute disorder or chronic disorder of central nerve system (CNS) including spinal cord and brain or peripheral nerve system.
  • CNS central nerve system
  • object disease for the improving agent of the present invention include nerve injury, neural degenerative disorder or neural dysfunction including contusion of central nerve, traumatic brain injury, other brain injury, apoplexy, amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), brachial plexus injury, ambliophia, spinal cord injury, Alzheimer's disease, Parkinsonism, although it is not limited thereto.
  • Spinal cord injury includes disease such as crush of neuron and traumatic injury caused by traffic accident, accidental fall, contusion, gunshot wound and other injury.
  • motor neuron dysfunction such as motor function disorder of the four limbs and the trunk caused by dysfunction, disorder and paralysis of motor neuron
  • sensory neuron dysfunction such as neuropathic pain represented by a pain caused by allodynia and hyperalgesic reaction (such as hypertarachia, dysesthesia, numbness of the four limbs, facial nerve paralysis, etc. caused by dysfunction, disorder and paralysis of sensory neuron) of the object to be treated (patient).
  • the drug of the present invention is used for the treatment together with other drug which has been known to have an improving effect for nerve damage such as spinal cord injury.
  • An example of such a drug is that which comprises chondroitinase having an action of degrading chondroitin sulfate proteoglycan as an active ingredient.
  • Chondroitinase is a lyase which degrades chondroitin sulfate into an unsaturated disaccharide in an eliminating manner. Examples of the enzyme which is available at present will be listed below.
  • Chondroitinase ABC [EC 4.2.2.20 or EC 4.2.2.4] is an enzyme which cleaves an N-acetylhexosaminide bond of glycosaminoglycan containing chondroitin sulfate in an elimination reaction manner whereupon an unsaturated disaccharide having a ⁇ 4-hexuronic acid residue in a non-reducing terminal is mainly produced.
  • chondroitin sulfate A derived from cartilage of mammals (containing abundant chondroitin-4-sulfate), chondroitin sulfate C derived from cartilage of shark (containing abundant chondroitin-6-sulfate) and chondroitin sulfate B (dermatan sulfate) derived from skin of mammals while weakly catalyzing the degradation of hyaluronic acid, and it also degrades chondroitin sulfate A, dermatan sulfate and chondroitin sulfate C in a chondroitin sulfate side chain of chondroitin sulfate proteoglycan (Yamagata, T.
  • This enzyme is commercially available as a reagent for the research in, for example, removal of mucopolysaccharide from animal tissue or identification of mucopolysaccharide in the tissue, as an enzyme product produced by bacteria such as Proteus vulgaris from Seikagaku Biobusiness Corporation with a code number 100330 (Chondroitinase ABC ( Proteus vulgaris )) and code number 100332 (Chondroitinase ABC Protease Free ( Proteus vulgaris )).
  • an enzyme which is purified in higher purity being a single protein containing no endotoxin, nucleic acid, contaminating proteins, etc.
  • an enzyme which is purified in higher purity being a single protein containing no endotoxin, nucleic acid, contaminating proteins, etc.
  • the chondroitinase ABC having high purity and high specific activity described in Japanese Patent No. 3980657, U.S. Pat. No. 6,184,023, U.S. Pat. No. 5,773,277, and U.S. Pat. No. 5,763,205, etc. is most preferred.
  • the above chondroitinase ABC is the same enzymes as the following enzymes having other names.
  • amino acid sequence that which is described in the database for amino acid sequences (UniProtKB/Swiss-Prot: Entry name CABCPROVU, Primary accession number P59807, Protein name Chondroitin ABC endolyase 1 [Precursor]) may be exemplified. Although this amino acid sequence also comprises 1021 amino acid residues, it is different in two amino acids from the amino acid sequence described in U.S. Pat. No. 5,578,480 (No. 694: Q ⁇ E, No. 738: D ⁇ N).
  • amino acid sequence described in SEQ ID NO: 1 of US 2006/0233782 A1 is of a mature protein whereby it comprises 997 amino acid residues
  • it is different in four amino acids from amino acids of 25 to 1021 in the amino acid sequence described in U.S. Pat. No. 5,578,480 (No. 154: A ⁇ T, No. 295: I ⁇ T, No. 694: Q ⁇ E, No. 738: D ⁇ N).
  • an enzyme is that where at least four amino acids (such as Nos. 154, 295, 694 and 738) are substituted with other amino acids based on the amino acid sequence described in U.S. Pat. No. 5,578,480, that is still able to be regarded as an enzyme having the identical chondroitinase ABC activity.
  • the drug where the improving agent of the present invention is made into a pharmaceutical preparation is used for the treatment together with other drug which has been known to have an improving effect for neuropathic pain.
  • the other drug include NSAIDs, opioid and adjuvant analgesics as exemplified below.
  • opioid analgesic include morphine, fentanyl, oxycodone, codeine phosphate, pethidine, buprenorphine, tramadol, pentazocine and butorphanol.
  • adjuvant analgesics include a tricyclic type (such as amitriptyline or nortriptyline), a tetracyclic type (such as Tetramide), SSRI (such as paroxetine or fluvoxamine and SNRI (milnacipran) which are antidepressants;
  • examples of anticonvulsant include carbamazepine, lamotrigine, zonisamide, valproic acid and clonazepam;
  • examples of antispasmodic include baclofen;
  • examples of antiarrhythmic include lidocaine and mexiletine;
  • examples of NMDA receptor antagonist include ketamine, dextromethorphan, amantadine and ifenprodil; and examples of a preparation containing an extract from the inflamed skin of rabbit inoculated with vaccinia virus include Neurotropin.
  • Calcium channel blocker (bonding to ⁇ 2 ⁇ subunit) which has been receiving public attention recently such as gabapentin (anticonvulsant) and pregabalin may be also listed. It is also possible to use together with a drug having an antiinflammatory action such as steroidal agent (e.g., methylprednisolone sodium succinate, dexamethasone and betamethasone) or fasudil which is an Rho kinase inhibitor.
  • steroidal agent e.g., methylprednisolone sodium succinate, dexamethasone and betamethasone
  • fasudil which is an Rho kinase inhibitor.
  • the tube and the pump were sutured to the interspinal ligament and the muscle. Afterward, the muscles and skin were closed in layers. After the operation, all animals were orally given antibiotics for two weeks.
  • the bladder was compressed by manual abdominal pressure once a daily until bladder function restored.
  • the test sample was continuously administered intrathecally for 14 days since the stage of immediately after the injury (administering rate: 12 ⁇ l/24 hours; total dose: 168 ⁇ L/14 days).
  • the test sample was the following five types.
  • Bc keratanase II (a heat resistant keratanase II produced by the bacteria of the species Bacillus circulans KsT202 strain described in Japanese Patent No. 3734504) (0.000005 U/200 ⁇ L, 0.000025 U/200 ⁇ L and 0.05 U/200 ⁇ L)
  • Ks36 keratanase II Keratanase II produced by Ks36 strain of bacteria belonging to genus Bacillus described in Japanese Patent No. 2726274 (0.000025 U/200 ⁇ L, 0.0005 U/200 ⁇ L, and 0.05 U/200 ⁇ L)
  • Bc keratanase II an enzyme prepared by inactivation of the enzymatic activity of Bc keratanase II (0.05 U/200 ⁇ L) at 100° C. (for 10 minutes)
  • Ps keratanase (although it is an enzyme which degrades keratan sulfate, it is an endo-O-galactosidase type enzyme derived from Pseudomonas sp strain which hydrolyzes a ⁇ -galactoside bond of non-sulfated galactose of keratan sulfate; hereinafter, it will be referred to as Ps keratanase; refer, if necessary, to the reference document by Kiyoshi Nakazawa and Sakaru Suzuki, J. Biol. Chem., 250:(3) 912-917 (1975), Purification of Keratan sulfate-endogalactosidase and Its Action on Keratan Sulfate of Different Origin) (0.05 U/200
  • BBB Basso-Beattie-Bresnahan
  • the % grip values in the groups to which Bc keratanase II and Ks36 keratanase II were administered were nearly the same when the administered concentration was 0.05 U/200 ⁇ L and any of them was higher than the % grip value in the group to which physiological saline was administered.
  • each of right and left pressure stimulations of the filament by which the rat felt the pain and moved the hind paws was measured and expressed in terms of their average value (unit; gram(s)).
  • the result where each of Bc keratanase II and Ks36 keratanase II was administered in the administered concentration of 0.05 U/200 ⁇ L is shown in FIG. 10 .
  • the pressure stimulation of the filament showed a high value from one week to two weeks after the injury as compared with the stage before the injury showing an insensitivity reaction to the stimulation to the hind paws.
  • the pressure stimulation of the filament showed low values as compared with the stage before the injury and, after six weeks from the injury, a sensory hypersensitivity symptom where the response was resulted even by a slight stimulation to the hind paws was noted.
  • HBSS calcium and magnesium free Hanks buffer
  • DNase deoxyribonuclease
  • the test sample was the following three types.
  • FIG. 11 The result is shown in FIG. 11 .
  • an elongation of neurite lengths was inhibited about 70% in the presence of PG containing keratan sulfate, while an inhibiting action for elongation of the neurite lengths induced by PG was released to an extent of nearly 100% in the co-presence of Ks36 keratanase II. From the above result, it has now become clear that keratanase II has a releasing effect for an inhibiting action for the elongation of neurite lengths.
  • Rho kinase participation of Rho kinase was investigated in order to investigate the action mechanism of keratanase II in the above Experiment 1.
  • the neurons were seeded onto an 8-well chamber to which a test sample was applied by the same manner as in the method of Experiment 1. Twenty-four hours after starting the incubation of the neurons, 15 ⁇ M of Y27632 (Rho kinase inhibitor, Sigma) was added to the neurons followed by incubating for 24 hours more. After that, the chamber was fixed with 4% of paraformaldehyde and the neurons were identified by an immunohistochemical means with Neuronal Class III ⁇ tubulin (TUJ1) antibody (COVANCE). The neurite lengths were measured of from 80 to 90 neurons per condition, an average value thereof was calculated and shown by a graph.
  • the test sample was the following four types.
  • FIG. 12 shows the pictures under a fluorescent microscope showing the degree of an elongation of the neurite lengths in (a) the case where Y27632 was not added to the test sample (2) and (b) the case where it was added thereto.
  • FIG. 13 shows the results of measurement of the neurite lengths for each test sample where Y27632 was not added and was added.
  • an inhibiting action for an elongation of neurite lengths induced by keratan sulfate was released by the addition of Y27632. From such results, it was noted that keratan sulfate induces the inhibition of the elongation of the neurite lengths via an Rho kinase activation.
  • SOD1-G93A mice which are ALS model mice were purchased from Jackson Laboratory. Spinal cords were excised from two ALS model mice and, after extraction of protein, Western blotting was carried out. Amount of keratan sulfate was detected by anti-keratan sulfate antibody 5D4 (Seikagaku Corporation). As a result, in ALS model mice, increase of the expression of keratan sulfate was detected as compared with the wild type mice (WT) ( FIG. 14 ).
  • ALS has been known that gliosis occurs therein and, from the above result, it was noted that keratan sulfate is not expressed in astrocyte which is a main body of gliosis but is expressed specifically in microglia which has been said to be closely participated in the onset of disease. Such a phenomenon is able to be said to be an expression mode which is specific to ALS when the fact that, in spinal cord injury and brain stab injury, keratan sulfate is expressed not only in microglia but also in oligodendrocyte is taken into consideration.
  • rBc keratanase II (genetically recombinant type heat resistant keratanase II) was dissolved in physiological saline to prepare a solution preparation where the concentration was 0.000025 U/mL to 0.05 U/200 ⁇ L.
  • the present invention has an industrial applicability in such a respect that an endo-D-N-acetylglucosaminide type enzyme which hydrolyzes an N-acetylglucosaminide bond in a keratan sulfate backbone is able to be provided as an active ingredient for an improving agent for dysfunction of motor neuron and of sensory neuron due to nerve damage such as spinal cord injury or, to be more specific, for an improving agent for neuropathic pain for example.

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