JP5414366B2 - Pain treatment - Google Patents

Description

  The present invention relates to pain, particularly trauma, infection of foreign organisms including bacteria, invasion or parasitism, breakdown of autoimmunity such as rheumatism, pain or hyperalgesia caused by arthritis or cancer, or various nerve injuries or shingles The present invention relates to a pain therapeutic agent that exhibits an excellent therapeutic effect on abnormal pain caused by the pain, a method for treating pain using such a therapeutic agent, and the like. More specifically, the present invention relates to a pain therapeutic agent containing sialidase as an active ingredient, a pain treatment method using the therapeutic agent, and the like.

  When a living tissue is damaged, the damage is recognized by feeling it as pain. Excessive pain due to inflammation or the like not only causes pain to the patient, but has little biological significance. Therefore, it is desirable to quickly relieve this pain (pain). However, the pain-sensing mechanism is complex and various mechanisms have been reported, but there are many unexplained parts (for example, Okuse K Int J Biochem Cell Biol. 39 (3): 490-6 2007, Ueda H. Pharmacol Ther. 109 (1-2) 57-77 2006, Scholz J, and Woolf CJ. Nat Neurosci. 5 1062-7 2002).

  Currently, various analgesics and narcotics are used for pain suppression, but there are also pains for which these drugs do not exhibit pain suppression effects. As analgesics that have been used in conventional pharmacotherapy, central opioid receptor agonists represented by morphine, nonsteroidal anti-inflammatory drugs (NSAIDs) represented by indomethacin, and the like are known. However, these analgesics have problems in terms of usefulness, safety and addictiveness.

  It is known that the pain biology mechanism is caused by the production of pain mediators such as bradykinin and ATP in the damaged site, and the peripheral nerve detects this and excites it. (Scholz J, and Woolf CJ. Nat Neurosci. 5 1062-7 2002). However, as for the mediator, bradykinin and ATP have been found (Scholz J, and Woolf CJ. Nat Neurosci. 5 1062-7 2002), but the whole picture is still unclear. For this reason, research aimed at the discovery of new mediators has been vigorously conducted, and the development of new analgesics targeting each mediator discovered through such research has been promoted (for example, about glutamic acid). Renbaeck K, Beirith A, et.al. Brain Res. 924 (2) 219-28 2002, Jin YH et. Al., J Pharmacol Sci. 109 (2) 233-41. et. al., Brain Res Mol Brain Res. X 75 (2), 350-4.2000, etc.).

  Gangliosides are a group of glycosphingolipids with sialic acid, and all endogenous glycosphingolipids are biosynthesized from ceramide through a series of enzymatic reactions. In gangliosides, many variations such as higher gangliosides having a plurality of sialic acids in the molecule have been found. Gangliosides are glycolipids present on the cell surface and are abundant in the central nervous system, especially in vivo, and thus research focused on nerve function has been conducted. However, the function of gangliosides in the initiation and transmission of pain, one of the major roles of peripheral nerves, has not been studied.

Okuse K Int J Biochem Cell Biol. 39 (3): 490-6 2007 Ueda H. Pharmacol Ther. 109 (1-2) 57-77 2006, Scholz J, and Woolf CJ. Nat Neurosci. 5 1062-7 2002 Scholz J, and Woolf CJ. Nat Neurosci. 5 1062-7 2002 Beirith A, et.al.Brain Res. 924 (2) 219-28 2002 Jin YH et. Al., J Pharmacol Sci. 109 (2) 233-41. 2009 Renbaeck K et.al., Brain Res Mol Brain Res.X 75 (2), 350-4.2000

  Under such circumstances, it has been desired to provide a novel pain therapeutic agent that exhibits a superior therapeutic effect.

  The present inventors have conducted research based on an original idea to solve the above problems, and found that high-grade gangliosides are involved in the generation of pain in the periphery. As a result of further research based on this finding, the present inventors have found that sialic acid degrading enzyme (sialidase) converts higher ganglioside into a substance not involved in pain and completed the present invention.

That is, the present invention provides the following therapeutic agents for pain, pharmaceutical compositions for treating pain, methods for treating pain, and the like.
(1) A therapeutic agent for pain containing sialidase as an active ingredient.
(2) The sialidase is derived from Arthrobacter ureafaciens sialidase, Clostridium perfringens sialidase, Vibrio cholerae sialidase, Streptococcus pneumoniae sialidase, Clostridium perfringens sialidase, Micromonospora viridifaciens sialidase, Influenzad virus Sella sialidase The pain therapeutic agent according to (1) above, which is an enzyme having an α-sialidase activity selected from the group consisting of typhimurium-derived sialidase, Newcastle disease virus-derived sialidase, Streptococcus sp.-derived sialidase, and vertebrate-derived sialidase.
(3) treating pain selected from the group consisting of pain or hyperalgesia caused by trauma, infection of different organisms, invasion or parasitism, failure of autoimmunity or cancer, and abnormal pain caused by nerve damage or shingles Therefore, the pain therapeutic agent according to (1) or (2) above.
(4) A pharmaceutical composition for treating pain comprising sialidase and a pharmaceutically acceptable carrier.
(5) Use of sialidase for pain treatment.
(6) A method for treating pain by administering an effective amount of sialidase to a mammal.
(7) Use of sialidase or a pharmaceutically acceptable salt thereof for producing a therapeutic agent for pain.

  The pain therapeutic agent of the present invention is, for example, pain caused by inflammation caused by trauma, infection and invasion of foreign organisms including bacteria, parasitism, breakdown of autoimmunity such as rheumatism, and hyperalgesia. It is also effective for treating pain that presents symptoms such as abnormal pain caused by various nerve injuries.

FIG. 1 shows a schematic diagram in which higher gangliosides including ganglioside GT1b are decomposed into ganglioside GM1 by sialidase. FIG. 2 shows the results of measuring the time of licking, which is a pain-related behavior, by administering ganglioside GT1b and the control (GM1) to the sole of the right hind limb of mice. Fig. 3 (a) shows the foot licking behavior when inducing pain by administering GT1b or PBS to the sole of the right hind limb of mice and administering 0.05% formalin to the same site after 20, 40 and 100 minutes. The result of measuring the time to cause licking) is shown. FIG. 3 (b) shows the results of measuring the time for licking behavior by administering 0.05% formalin in the same manner 40 minutes after each dose of GT1b or GM1 ganglioside. FIG. 4 shows the results of measuring the time for licking behavior after administration of sialidase and administration of 2% formalin.

  The present inventors have found that higher gangliosides are involved in the development of pain in the periphery, and specifically, higher ganglioside GT1b clearly increases the time to cause foot licking in mice. Based on this knowledge, high ganglioside GT1b, a causative agent that enhances pain, is administered to the affected area by administering sialic acid degrading enzyme (sialidase), which degrades ganglioside GM1, which has less sialic acid and has no pain-sustaining effect, to the affected area. Has been found to reduce the pain of the second phase, especially during formalin administration. This second phase pain is caused by a variety of factors, and is a particularly inflammatory model and one of the models enhanced in neuropathic pain. Various types of higher gangliosides are known. However, there have been no reports on the relationship between these higher gangliosides and pain in pain models. Surprisingly, the present inventors have found for the first time that these higher gangliosides act as pain-inducing substances and that these higher gangliosides have a therapeutic effect on pain by decomposing them. The present invention has been completed based on such findings.

Hereinafter, the present invention will be described in detail.
The present invention provides a pain therapeutic agent containing sialidase as an active ingredient.
In the present specification, “sialidase” refers to a glycolytic enzyme that desorbs sialic acid from a glycolipid or glycoprotein sugar chain by hydrolysis. “Α-sialidase” refers to an enzyme that eliminates sialic acid that is α-coordinated ketoside-bonded to the sugar chain terminal. Sialidase is an enzyme that catalyzes the initial reaction of glycolysis. The present invention has found that higher gangliosides such as GT1b are causative agents of pain. In the present invention, sialidase that decomposes such higher gangliosides into substances that do not induce pain is used as an active ingredient. More specifically, the active ingredient used in the present invention preferably has an action of decomposing or metabolizing higher gangliosides such as GT1b into gangliosides of monosialic acid.

  Ganglioside is a general term for glycosphingolipids having sialic acid, and at least 80 gangliosides are known. It is universally present in various organs and tissues in the body, especially in the cranial nervous system. In the present specification, “higher ganglioside” means a ganglioside containing two or more sialic acids in the molecule. Gangliosides are indicated by common abbreviations. Specifically, G is ganglioside, M, D, T, Q ... are the number of sialic acids, and Arabic numerals are the order of mobility in thin-layer chromatography. And sialic acid binding positions a, b, c,..., Α, β,. In the present specification, examples of the higher ganglioside include GQ1b, GT1b, GT1a, GD1b, GD1a, and the like. In the present invention, the higher ganglioside is preferably GT1b. In the specification, examples of the monosialic acid ganglioside include GM1, GM2, GM3, and GM4. In the present invention, the ganglioside monosialic acid is preferably GM1.

  Examples of the sialidase used in the present invention include Acinetobacter baumannii, Actinomyces johnsonii, Actinomyces naeslundii, Actinomyces oris, Actinomyces viscosus, Akkermansia muciniphila, Arthrobacter aurescens, Arthrobacter eroacter chlorophenolicus, Arcanobacterium b Beutenbergia cavernae, Bifidobacterium longum, Capnocytophaga canimorsus, Clostridium perfringens, Clostridium septicum, Clostridium tertium, Corynebacterium diphtheriae, Corynebacterium glutamicum, Erysipelothrix rhusiopathiae, Haemophilus parasuis, Mannheimia haemolytica, Micromonospora viridifaciens, Mycoplasma alligatoris, Mycoplasma gallisepticum, Mycoplasma synoviae, Parabacteroides distasonis, Pasteurella multocida , Porphyromonas gingivalis, Propionibacterium acnes, Salmonella typhimurium, Pseudoalteromonas haloplanktis, Rhodopirellula baltica, Saccharopolyspora erythraea, Shewanella p ealeana, Solibacter usitatus, Streptococcus agalactiae, Streptococcus equi, Streptomyces avermitilis, Streptococcus mitis, Streptococcus pneumoniae, Streptococcus sp., Streptomyces coelicolor, Streptomyces griseus, Tannerella forsythia, Treponema dencho , Bos taurus, Ciona intestinalis, Cricetulus griseus, Callithrix jacchus, Danio rerio, Homo sapiens, Mus musculus, Pongo abelii, Rattus norvegicus, Takifugu rubripes, Xenopus laevis, Xenopus Sialidase with amino acid sequence similar to NEU1, NEU2, NEU3, NEU4 derived from vertebrates such as rats, Arabidopsis thaliana, Cryptococcus neoformans, Macrobdella decora, Medicago truncatula, Oryza sativa, Penicillium chrysogenum, Penicillium chrysogenum, Philodinabros Trypanosoma carassii, Trypanosoma con Golense, Trypanosoma cruzi, Trypanosoma evansi, Trypanosoma rangeli, Zea mays, etc. Canine parainfluenza virus, Emiliania huxleyi virus, Equine influenza virus, Fer-de-lance virus, Frog adenovirus, Goose paramyxovirus, Hendra virus, HPIV-1, Human parainfluenza virus, Influenza A virus, Influenza B virus, Mapuera virus, Menangle virus, Mossman virus, Mumps virus, Murayama virus, Newcastle disease virus, Nipah virus, Nipah virus, Pigeon paramyxovirus, Porcine rubulavirus, Porcine rubulavirus, Sendai virus, Simian virus, Swine parainfluenza virus, Tioman virus, Tupaia paramyxovirus, Turkey adenovirus, Yucaipa virus, etc. Examples include virus-derived sialidase.

  Preferably, as the sialidase used in the present invention, Neuraminidase isozyme L, M1, M2, and S (nacalai: product number 24229-61, etc.) that are Arthrobacter ureafaciens-derived sialidase, Clostridium perfringens-derived sialidase (EC 3.2.1. 18) (ROCHE: product number 1585886, etc.), Vibrio cholerae-derived sialidase (EC 3.2.1.18) (ROCHE: product number 1080725, etc.), Streptococcus pneumoniae-derived sialidase (EC 3.2.1.18) (Sigma; N7271 Clostridium perfringens sialidase (EC 3.2.1.18) (Sigma; N2876 etc.), Micromonospora viridifaciens sialidase (EC 3.2.1.18) (R & D systems; 5084-NM-010 etc.), Influenza A Virus H1N1-derived sialidase (EC 3.2.1.18) (R & D systems; 4858-NM-005), Macrobdella decora-derived sialidase (EC 3.2.1.18) (Calbiochem; 480706, etc.), Salmonella typhimurium-derived sialidase (EC 3.2.1.18) (Prozyme, Inc .; GKX-5018, etc.), Newcastle disease virus-derived sialidase (EC 3.2. 1.18) (Prozyme, Inc .; GKX-5017 etc.), Streptococcus sp.-derived sialidase (EC 3.2.1.18) (TOYOBO; NRH-301 etc.), other bacteria, viruses, algae, plants, fungi, Examples include sialidase derived from invertebrates, or sialidase Neu3 derived from vertebrates such as human, mouse, and rat (the above EC means Enzyme Commission Number). The function of sialidase is known and described in, for example, Schauer R. Glycoconj J. 17 (7-9), 485-499.2000.

  The term “treatment” as used herein generally means ameliorating the symptoms of humans and non-human mammals. The term “improvement” means, for example, a case where the degree of the disease is reduced or aggravated as compared with the case where the therapeutic agent of the present invention is not administered, and also includes the meaning of prevention. Furthermore, the term “pharmaceutical composition” means a composition containing an active ingredient (sialidase etc.) useful in the present invention and an additive such as a carrier used in the preparation of a medicine.

  In the present specification, the term “containing sialidase as an active ingredient” means a pharmaceutically acceptable form of sialidase (for example, a salt, ester, amide, hydrated or solvated form thereof, racemic mixture, optical In a purely pure form etc.).

  Therefore, the sialidase as an active ingredient used in the present invention may be a free form or a pharmaceutically acceptable salt. Such “salts” include acid salts and base salts. Examples of the acid salt include hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acidic phosphate, acetate, lactate, citrate, Acid citrate, tartrate, bitartrate, succinate, maleate, fumarate, gluconate, saccharide, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate , P-toluenesulfonate, 1,1′-methylene-bis- (2-hydroxy-3-naphthoic acid) salt, and the like. Examples of the base salt include alkali metal salts such as sodium salts and potassium salts, alkaline earth metal salts such as calcium salts and magnesium salts, water-soluble amine addition salts such as ammonium salts and N-methylglucamine salts, and lower alkanols. Mention may be made of ammonium salts, salts derived from other bases of pharmaceutically acceptable organic amines.

  The therapeutic agent and pharmaceutical composition of the present invention are effective for treating various pains. In the present invention, as pain, for example, headache (for example, migraine, myotonic headache, cluster headache, fever and other symptomatic headache), orofacial pain (for example, toothache, glossodynia, temporomandibular disorder, Glossopharyngeal neuralgia, trigeminal neuralgia, eye pain, glaucoma, ear pain, etc., cervical shoulder pain (eg, cervical disc herniation, degenerative cervical spondylosis, cervical shoulder arm syndrome, peri-shoulderitis (fifty shoulder), cervical spinal canal stenosis , Chest exit syndrome, brachial plexus withdrawal injury, shoulder-hand syndrome, traumatic cervical syndrome (whipping), chest pain (angina, acute pericarditis, esophagitis, pulmonary embolism, etc.), abdominal pain (eg, Acute abdomen, cholelithiasis, acute pancreatitis, pain due to organ lesions such as urolithiasis), low back pain (eg osteoporosis, lumbar disc herniation, degenerative lumbar spondylosis, lumbar spinal canal stenosis, lumbar sequestration, Lumbar spondylolisthesis, facet joints, etc.), knee pain, musculoskeletal pain [For example, muscle pain (eg, myofascial pain syndrome (MPS), fibromyalgia syndrome (FMS), etc.), joint pain (eg, arthritis, rheumatoid arthritis (RA), gout, etc.), spinal pain , Bone pain, etc.], pain due to blood flow disorders (eg, obstructive arteriosclerosis (ASO), Buerger's disease (TAO), etc.), pain due to trauma, infection with foreign organisms such as bacteria, pain due to invasion or infestation, Neuropathic pain [eg, neuralgia (eg, nerve damage, trigeminal neuralgia, intercostal neuralgia, sensory dysfunctional femoral neuralgia, inguinal neuralgia, saphenous neuralgia, median neuralgia, ulnar neuralgia, sciatica, nerve root pain, etc.), herpes zoster Pain (eg, acute herpes zoster pain, postherpetic pain (chronic phase), etc.), diabetic pain (eg, diabetic neuropathy, large fiber neuropathy, small fiber neuropathy, proximal muscle dominant neuropathy) -Acute mononeuropathy, paralysis due to compression, etc., strangulation neuropathy (eg, thoracic outlet syndrome, suprascapular nerve strangulation disorder, scapulodorsal nerve strangulation disorder, quadrilateral gap syndrome, circular gyrus syndrome) , Anterior interosseous nerve syndrome, elbow canal syndrome, delayed ulnar nerve palsy, posterior interosseous nerve syndrome, carpal tunnel syndrome, ulnar neural tube syndrome, Wartenberg's disease, Blowler's thumb, sensory dysfunction femoral pain, piriformis Syndrome, Hunter tube syndrome, common peroneal nerve strangulation disorder, tarsal tunnel syndrome, anterior tarsal tunnel syndrome, Morton disease, cervical spinal canal stenosis, lumbar spinal canal stenosis, extensive spinal canal stenosis), low back pain related neuropathy, Brachial plexus withdrawal injury, reflex sympathetic dystrophy (complex regional pain syndrome type 1), reflex sympathetic atrophy, causalgia (burning pain, complex local pain syndrome type 2), painful neuropathy, Pain and phantom after spinal cord injury (Eg, phantom limb pain, phantom tooth pain, etc.), afferent block pain, iatrogenic neuropathy, sympathetic nerve-dependent pain, retrograde C-fiber excitability syndrome (ABC syndrome [Angry Backffiring C-nociceptor syndrome]), pain due to cancer HIV-related neuropathic pain, stone-induced pain (for example, pain due to urinary calculi (eg, kidney stones, ureteral stones, bladder stones, urethral stones, etc.), pain due to gallbladder stones, pain due to vagina stones, etc.) Postoperative pain, chronic headache, oral and facial pain (eg, toothache, glossodynia, temporomandibular disorders, trigeminal neuralgia), atypical facial pain (eg, atypical facial pain after extraction, etc.), shoulder joint Peripheritis, osteoarthritis, arthritis, pain associated with rheumatism, back pain, multiple sclerosis, pain induced by medication, pain induced by radiation therapy, sufficient effects of narcotic analgesics No pain etc.] .

  In the present invention, in particular, from trauma, infection of foreign organisms such as bacteria, invasion or parasitic, pain of autoimmunity such as rheumatism or pain or hyperalgesia caused by cancer, abnormal pain caused by various nerve damage, etc. It is effective in the treatment of pain selected from the group consisting of

  There are no particular restrictions on the dosage form of the therapeutic agent for pain of the present invention, and it can be administered orally or parenterally. The active ingredient of the pain-treating agent of the present invention may be blended alone or in combination. However, a pharmaceutically acceptable carrier or formulation additive may be blended with the active ingredient and provided in the form of a formulation. it can. In this case, the active ingredient of the present invention can be contained, for example, in the preparation in an amount of 0.1 to 99.9% by weight.

  Examples of pharmaceutically acceptable carriers or additives include excipients, disintegrants, disintegration aids, binders, lubricants, coating agents, dyes, diluents, solubilizers, solubilizers, isotonic agents. Agents, pH adjusters, stabilizers and the like can be used.

  Examples of preparations suitable for oral administration include powders, tablets, capsules, fine granules, granules, liquids or syrups. For oral administration, various excipients such as microcrystalline cellulose, sodium citrate, calcium carbonate, dipotassium phosphate, glycine are added to starch, preferably corn, potato or tapioca starch, and alginic acid and certain species. It can be used with various disintegrants such as silicate double salts and granulating binders such as polyvinylpyrrolidone, sucrose, gelatin, gum arabic. In addition, lubricants such as magnesium stearate, sodium lauryl sulfate, and talc are often very effective for tablet formation. The same kind of solid composition can also be used by filling gelatin capsules. Suitable substances in this connection include lactose or lactose as well as high molecular weight polyethylene glycols. When an aqueous suspension and / or elixir is desired for oral administration, the active ingredient is used in combination with various sweeteners or flavors, colorants or dyes, and if necessary, an emulsifier and / or suspending agent is also used. And can be used with water, ethanol, propylene glycol, glycerin, and the like, and diluents that combine them.

  Examples of preparations suitable for parenteral administration include injections and suppositories. In the case of parenteral administration, a solution in which the active ingredient of the present invention is dissolved in either sesame oil or peanut oil or dissolved in an aqueous propylene glycol solution can be used. The aqueous solution should be appropriately buffered as necessary (preferably pH 8 or more), and the liquid diluent must first be made isotonic. Such aqueous solutions are suitable for intravenous injection and oily solutions are suitable for intra-articular, intramuscular and subcutaneous injection. All these solutions can be prepared aseptically by standard pharmaceutical techniques well known to those skilled in the art. Furthermore, the active ingredient of the present invention can also be administered locally such as on the skin. In this case, topical administration in the form of creams, jellies, pastes, ointments is desirable according to standard pharmaceutical practice.

  Subjects of the above formulation include human and non-human animals. Examples thereof include animals generally used in pharmaceutical tests such as monkeys, chimpanzees, dogs, cats, guinea pigs, rats, mice, pigs, sheep, horses and the like. Preferred are monkeys, chimpanzees, rats and mice, and more preferred are mice and rats.

  The dose of the pain treatment agent of the present invention is not particularly limited, and an appropriate dose is selected according to various conditions such as the type of pain, the age and symptoms of the patient, the administration route, the purpose of treatment, the presence or absence of a concomitant drug, etc. Is possible. The dosage of the therapeutic agent for pain of the present invention is, for example, about 0.01 to 2500 mg, preferably 0.1 to 900 mg per day for an adult (for example, body weight 60 kg). The dosage when administered as an injection is, for example, about 10 to 500 mg, preferably 18 to 180 mg per day for an adult (for example, 60 kg body weight). These daily doses may be administered in two to four divided doses.

EXAMPLES Hereinafter, although this invention is demonstrated more concretely based on an Example, this invention is not limited to these Examples at all.
(Experimental materials used and general experimental methods)
(1) Model animal As an experimental animal, ICR mice (Japan Clare, male, 5 weeks old) were purchased and used for the experiment one week later. After moving to the experimental container, it was acclimated for 1 hour and administered ganglioside and the like.
(2) Preparation of administered substance For the administered substance, (Wako Pure Chemicals, 075-03911) was used as ganglioside GT1b. Ganglioside GM1 (Wako Pure Chemicals, 079-03931). As sialidase, sialidase derived from Arthrobacter ureafaciens (nacalai: product number 24229-61 etc.) was used.
(3) Administration method The test substance dissolved in PBS was administered by subcutaneous injection into the right hind foot sole of the mouse. In order to induce pain stimulation, 0.05% or 2% formalin was administered subcutaneously to the right hind limb of mice.
(4) Evaluation of pain In order to evaluate that the administered substance has an effect on pain, the foot licking action (licking) which is a pain related action was visually evaluated, and the time was measured. As a measuring method, for example, Ferreira J et. Al. Pain. 117 (1-2), 171-81. 2005, Sakurada C et. Al. Neurochem Int. 38 (5), 417-23. 2001, Piovezan AP et. Al. Eur J Pharmacol. 351 (1), 15-22. The specific procedure is as follows.
1: Move mouse to observation container and place for 1 hour.
2: The reagent is injected subcutaneously into the right hind foot sole of the mouse and returned to the container.
3: Immediately after that, within a predetermined time, the time for licking the foot (right hind limb) injected with the reagent is visually measured with a stopwatch.
4: Describe the licking time for each reagent.
In order to observe the effect of enhancing pain (0.05% formalin administration), various gangliosides or PBS were administered to the sole of the right hind leg of the mouse, and after a predetermined time, 0.05% formalin was administered to the same site, and licking was caused for 5 minutes. The time was measured. In order to observe the analgesic effect, 30% after sialidase was administered to the sole of the right hind limb of the mouse, 2% formalin was administered to the same site. As the two phases, the time during which licking was triggered was measured.

Example 1: Pain test with ganglioside As to whether ganglioside is involved in the development of pain in the periphery, it is considered that mice take when pain is felt by injecting a pain-causing substance subcutaneously into the hind limbs ( licking) was measured. In other words, the longer the licking time, the stronger the pain.

  PBS or ganglioside (dissolved in PBS) 40 μl is administered to the sole of the right hind limb of an ICR mouse placed in a 20 cm diameter transparent cylinder for 1 hour, and then for 20 minutes, licking the foot, which is a pain-related behavior (licking Pain was evaluated by measuring the time at which) occurred.

  As a result of the experiment, it was found that the licking time of ganglioside GT1b was clearly increased compared to administration of buffer alone (FIG. 2). From the above, it was shown that GT1b may be a pain-causing substance. On the other hand, GM1 had no significant effect.

Example 2: Examination of pain enhancement effect Similarly, PBS or ganglioside is administered to the sole of the right hind limb of ICR mice, 0.05% formalin is administered 40 minutes later, and pain caused by low concentration formalin is enhanced by administration of ganglioside. I examined.

  When GT1b was administered to the sole of the right hind limb of the mouse and 0.05% formalin was administered to the same site 20 minutes, 40 minutes, and 100 minutes later, the time to cause licking, which is a pain-related behavior, compared to PBS It increased (Fig. 3 (a)). Further, each amount of GT1b or GM1 ganglioside was administered, and after 40 minutes, 0.05% formalin was similarly administered. GT1b showed a significant pain enhancement effect, but GM1 did not show a significant effect (FIG. 3 (b)).

  Therefore, ganglioside GT1b also enhanced pain caused by low concentrations of formalin (FIG. 3). This result also indicated that GT1b could be a pain-causing substance. In addition, ganglioside GM1, which has less sialic acid than GT1b, did not show any effect of enhancing pain.

Example 3: Examination of analgesic effect by sialidase As shown in FIG. 1, gangliosides such as GT1b contain a plurality of sialic acids, and thus can be decomposed to GM1 by removing sialic acids by sialidase treatment. Note that sialic acid such as GM1 is accumulated because it is difficult to cleave in a three-dimensional structure. Therefore, sialidase was administered to mice for the purpose of degrading endogenous GT1b, which was presumed to be a pain-causing substance, into GM1, and the pain-suppressing effect of sialidase was examined.

  0.9% NaCl + 50 mM acetate buffer (pH 5.2) or sialidase 8 mU (about 0.13 μg, nacalai: product number 24229-61) (dissolved in 0.9% NaCl in 50 mM acetate buffer pH 5.2) After sufficient time (after 30 minutes) to remove sialic acid, 2% formalin, a painful stimulus, was administered. With this painful stimulus, the mouse feels direct pain due to formalin, and once the pain has subsided, a second phase of pain that seems to be due to inflammation occurs. Licking was measured with 0-5 minutes immediately after formalin administration as the first phase and 10-20 minutes as the second phase. As a result, the administration of sialidase particularly relieved this second-phase pain (FIG. 4). Therefore, it was demonstrated that inflammatory pain can be suppressed by degrading endogenous higher ganglioside with an enzyme.

  As described above, the pain treatment agent containing the sialidase of the present invention as an active ingredient can be effectively used for pain treatment.

Claims (3)

A therapeutic agent for local administration of peripheral pain involving GT1b, comprising sialidase having α-sialidase activity as an active ingredient , wherein the peripheral pain involving GT1b is caused by trauma, infection, invasion or parasitism of foreign organisms Selected from the group consisting of: failure of autoimmunity, pain or hyperalgesia caused by arthritis or cancer, and abnormal pain caused by nerve damage or shingles, wherein the local administration is the location of peripheral pain associated with GT1b A therapeutic agent which is intraarticular injection, intramuscular injection, or subcutaneous injection .   The sialidase is derived from Arthrobacter ureafaciens sialidase, Clostridium perfringens sialidase, Vibrio cholerae sialidase, Streptococcus pneumoniae Streptococcus pneumonia, Micromonospora viridifaciens sialidase, Influenza A Virus H1N1 sialidase, Macrobdella decora sialidase, Salmonella typhimurium sialidase, Newcastle disease Group consisting of sialidase derived from virus (Newcastle disease virus), sialidase derived from Streptococcus sp., And vertebrate sialidase Ri is selected, the therapeutic agent according to claim 1. The therapeutic agent according to claim 1 or 2 , further comprising a pharmaceutically acceptable carrier.