Patent Publication Number: US-2020297776-A1

Title: Preventive or therapeutic agent for muscle injury

Description:
This is a Division of application No. Ser. 16/073,986 filed Jul. 30, 2018, which in turn is a National Stage Application of PCT/JP2017/002973 filed Jan. 27, 2017, which claims the benefit of Japanese Patent Application No. 2016-016463 filed Jan. 29, 2016. The disclosure of the prior applications is hereby incorporated by reference herein in its entirety. 
    
    
     TECHNICAL FIELD 
     The present invention relates to a novel medical use of an extract from inflamed tissues inoculated with vaccinia virus and, more particularly, it relates to a preventive or therapeutic agent for muscle injury containing an extract from inflamed tissues inoculated with vaccinia virus as an active ingredient. 
     BACKGROUND ART 
     Muscle injury (i.e., injuries of muscle) is the injury of muscle fibers. Muscle injury is graded into three stages, i.e., stage I (slight injury of muscle fiber), stage II (partial rupture of muscle fiber) and stage III (complete rupture of muscle fiber), depending on the severity of the injury. 
     When muscle injury occurs, firstly vasodilatation and permeability enhancement are caused, and then a reaction of which the sign is enlargement or warmth, i.e., an inflammatory reaction, is induced. In an inflammatory reaction, inflammatory cells such as neutrophils or macrophages which are one type of leukocytes are infiltrated into an injury site to phagocytize necrotic muscle fibers and, at the same time, secrete cytokines that can proliferate muscle satellite cells or the like. The muscle satellite cells are activated rapidly in the injury site and are converted to myoblasts. The myoblasts are proliferated repeatedly, and are then fused to each other. In this manner, the myoblasts are differentiated into myotube cells. The myotube cells finally become mature into muscle fibers to finish the repair of the muscles (spontaneous remission). The period needed for the completion of the repair of muscles is normally about 3 weeks to 1 month in the case where the level of muscle injury is stage II. 
     As the method for treating muscle injury, a conservative treatment, a surgical treatment and the like can be mentioned. Among these methods, although a surgical treatment is usually employed when muscle injury is the complete rupture of muscles, a conservative therapy is generally employed. 
     However, in a conservative therapy which greatly relies on spontaneous remission, there is a risk such that a patient is forced to have a difficult life for a long period to some extent, as mentioned above. Therefore, a treatment method that can repair muscle injury earlier has been demanded. 
     The extract from inflamed tissues inoculated with vaccinia virus as an active ingredient in the preventive or therapeutic agent for muscle injury of the present invention (hereinafter referred to as “the medicament of the present invention”) is disclosed to have the following effects: an analgesic effect, sedative effect, anti-stress effect and anti-allergic effect (see Patent Document 1); an immunostimulating effect, anti-cancer effect and cirrhosis inhibitory effect (see Patent Document 2); a treatment effect against idiopathic thrombocytopenic purpura (see Patent Document 3); a treatment effect against postherpetic neuralgia, brain edema, dementia, spinocerebellar degeneration and the like (see Patent Document 4); a treatment effect against Raynaud syndrome, diabetic neuropathy, sequelae of subacute myelo-optico-neuropathy and the like (see Patent Document 5); a kallikrein production inhibitory effect and peripheral circulatory disorder improving effect (see Patent Document 6); a bone atrophy improving effect (see Patent Document 7); a nitric oxide production inhibitory effect effective for the treatment of sepsis and endotoxic shock (see Patent Document 8); a treatment effect against osteoporosis (see Patent Document 9); a treatment effect against AIDS based on a Nef action inhibitory effect and chemokine production inhibitory effect (see Patent Documents 10 and 11); a treatment effect against ischemic disorders such as cerebral infarction (see Patent Document 12); a treatment effect against fibromyalgia syndrome (see Patent Document 13); and a treatment effect against infections (see Patent Document 14); a prophylactic or alleviating effect for a peripheral nerve disorder induced by an anti-cancer agent (see Patent Document 15); a treatment effect against chronic prostatitis, interstitial cystitis an/or urination disorders (see Patent Document 16); an effect of promoting production of neurotrophic factor such as BDNF (see Patent Document 17); an effect of promoting the synthesis of collagen and proteoglycan in chondrocytes (see Patent Document 18) and the like. However, as far as we know, it is not known that the extract from inflamed tissues inoculated with vaccinia virus as an active ingredient in the medicament of the present invention is effective for prevention or therapy for muscle injury. 
     PRIOR ART DOCUMENTS 
     Patent Documents 
     
         
         Patent Document 1: Japanese Patent Laid-Open No. Sho-53-101515 
         Patent Document 2: Japanese Patent Laid-Open No. sho-55-87724 
         Patent Document 3: Japanese Patent Laid-Open No. Hei-1-265028 
         Patent Document 4: Japanese Patent Laid-Open No. Hei-1-319422 
         Patent Document 5: Japanese Patent Laid-Open No. Hei-2-28119 
         Patent Document 6: Japanese Patent Laid-Open No. Hei-7-97336 
         Patent Document 7: Japanese Patent Laid-Open No. Hei-8-291077 
         Patent Document 8: Japanese Patent Laid-Open No. Hei-10-194978 
         Patent Document 9: Japanese Patent Laid-Open No. Hei-11-80005 
         Patent Document 10: Japanese Patent Laid-Open No. Hei-11-139977 
         Patent Document 11: Japanese Patent Laid-Open No. 2000-336034 
         Patent Document 12: Japanese Patent Laid-Open No. 2000-16942 
         Patent Document 13: International Publication No. WO 2004/039383 
         Patent Document 14: Japanese Patent Laid-Open No. 2004-300146 
         Patent Document 15: International Publication No. WO2009/028605 
         Patent Document 16: International Publication No. WO2011/111770 
         Patent Document 17: International Publication No. WO2011/162317 
         Patent Document 18: International Publication No. WO2012/051173 
       
    
     SUMMARY OF THE INVENTION 
     Problem to be Solved by the Invention 
     The object of the present invention is to provide a medicament that is effective for the prevention or treatment of muscle injury and is highly safe. 
     Means for Solving the Problems 
     The present inventors have made extensive and intensive studies on a medicinal treatment for muscle injury for which an effective treatment method has been still demanded. As a result, it has been found that an extract from inflamed tissues inoculated with vaccinia virus has an excellent repair promotive effect on muscle injury. This finding leads to the accomplishment of the present invention. 
     According to the present invention, it becomes possible to greatly shorten the period required for the healing of muscle injury by repair. 
     Advantages of the Invention 
     An extract from inflamed tissues inoculated with vaccinia virus has an excellent pharmacological effect to promote the repair of muscle injury. 
     Thereby, the extract can decrease the extent of muscle injury when administered before the onset of the muscle injury, and can heal muscle injury in an earlier stage when administered after the onset of the muscle injury. 
     Therefore, a medicament of the present invention which contains the extract as an active ingredient is highly useful as a preventive or therapeutic agent for muscle injury. In particular, the medicament of the present invention has very little disadvantages such as side effects, and is therefore highly safe. 
     Mode for Carrying Out the Invention 
     The present invention relates to a preventive or therapeutic agent for muscle injury, which contains an extract from inflamed tissues inoculated with vaccinia virus as an active ingredient. The medicament of the present invention is useful for the prevention or treatment of muscle injury. 
     An extract from inflamed tissues inoculated with vaccinia virus has an effect to promote the repair of muscle injury. Therefore, the medicament of the present invention may be used as a muscle injury repair promoter. 
     The muscle in muscle injury to be prevented or treated by the medicament of the present invention is not particularly limited, as long as the muscle has a part in which muscle injury has been occurred or a part in which muscle injury may be occurred. Examples of the muscle include quadriceps femoris muscle, hamstrings, paraspinal muscle and gastrocnemius muscle. The cause of muscle injury is not particularly limited, and muscle injury associated with surgical invasion is also a target to be prevented or treated with the medicament of the present invention. 
     As for the extract from inflamed tissues inoculated with vaccinia virus, there are various reports on physiological active substances produced in the inflamed tissues inoculated with vaccinia virus, the method for extracting the substances from the diseased tissues, the pharmacological activities and the like as mentioned above (for example, Patent Documents 1 to 18). 
     Furthermore, a preparation of an extract from inflamed skins of rabbits inoculated with vaccinia virus is a commercially available pharmaceutical product which is available for the medicinal agent of the present invention. The preparation, as described in pages 3525 to 3527 of “Drugs in Japan, Ethical Drugs” (2016, edited and published by Japan Pharmaceutical Information Center), contains non-proteinous active substances extracted and separated from the inflamed skin tissues of rabbits inoculated with vaccinia virus. The preparation is known to be effective against low back pain, cervicobrachial syndrome, symptomatic neuralgia, periarthritis scapulohumeralis, osteoarthritis, itchiness accompanied with skin diseases (eczema, dermatitis, urticaria), allergic rhinitis, sequelae of subacute myelo-optico-neuropathy such as coldness, paresthesia and pain, postherpetic neuralgia and the like. The preparation is approved as an ethical drug in the form of hypodermic, intramuscular and intravenous injection products and of tablets and is commercially available. 
     Hereinafter, the process for producing the extract of inflamed tissues inoculated with vaccinia virus as an active ingredient in the medicinal agent of the present invention and the like will be described. 
     The extract from inflamed tissues inoculated with vaccinia virus used in the medicament of the present invention can be obtained by the following manner: inflamed tissues inflamed by the inoculation with vaccinia virus is crushed; an extraction solvent is added to remove the tissue fragments; then deproteinization is carried out; the deproteinized solution is adsorbed onto an adsorbent; and then the active ingredient is eluted. 
     As to basic extracting steps for the present extract, the following steps are used for example. 
     (A) Inflamed skin tissues of rabbits, mice or the like by the intradermal inoculation with vaccinia virus are collected, and the inflamed tissues are crushed. To the crushed tissues an extraction solvent such as water, phenol water, physiological saline or phenol-added glycerin water is added to conduct an extracting treatment for several days. Then, the mixture is filtrated or centrifuged to give a crude extract (filtrate or supernatant) wherefrom tissue fragments are removed. 
     (B) The crude extract obtained in (A) is adjusted to acidic pH, heated and then filtered or centrifuged to conduct a deproteinizing treatment. After that, the deproteinized solution is adjusted to basic pH, heated and then filtered or centrifuged to give a deproteinized filtrate or supernatant. 
     (C) The filtrate or the supernatant obtained in (B) is adjusted to acidic pH and adsorbed with an adsorbent such as activated carbon or kaolin. 
     (D) An extraction solvent such as water is added to the adsorbent obtained in (C), the mixture is adjusted to basic pH and the adsorbed component is eluted to give an extract from inflamed tissues inoculated with vaccinia virus. 
     The above is the basic steps and each of the steps will be more specifically illustrated as follows. 
     About (A): 
     As for animals in order to obtain the inflamed tissues by the inoculation of vaccinia virus, various animals that is infected with vaccinia virus such as rabbits, cows, horses, sheep, goats, monkeys, rats or mice can be used, and preferred inflamed tissues are inflamed skin tissues of rabbits. 
     These inflamed tissues are collected, and washed and disinfected using a phenol solution, etc. These inflamed tissues are crushed and an extraction solvent in 1-to 5-fold thereof by volume is added to make an emulsified suspension. Here, the term “crush” means to finely break down into minces using a mincing machine or the like. As to the extraction solvent, there may be used distilled water, physiological saline, weakly acidic to weakly basic buffer, etc. and stabilizer such as glycerin, bactericidal/antiseptic agent such as phenol, salts such as sodium chloride, potassium chloride or magnesium chloride, etc. may be appropriately added thereto. At that time, it is also possible that the cell tissue is destroyed by a treatment such as freezing/melting, ultrasonic wave, cell membrane dissolving enzyme or surfactant so as to make the extraction easier. The resulting suspension is allowed to stand for 5 to 12 days. During that period, the suspension may be heated at 30 to 45° C. with or without appropriate stirring. The resulting liquid is subjected to filtration, centrifugation, or the like to remove the tissue fragments whereupon a crude extract (filtrate or supernatant) is obtained. 
     About (B): 
     The crude extract obtained in (A) is subjected to filtration, centrifugation or the like to remove tissue fragments, and then deproteinized. The deproteinization-treatment may be carried out by a known method which has been usually conducted and a method such as heating treatment, treatment with a protein denaturant (such as acid, base, urea, guanidine or an organic solvent including acetone), isoelectric precipitation or salting-out may be applied. After that, a common method for the removal of insoluble matters such as filtration using filter paper (such as cellulose or nitrocellulose), glass filter, Celite or Seitz filter, ultrafiltration or centrifugation is conducted to give a filtrate or a supernatant wherefrom the separated insoluble protein is removed. 
     About (C) 
     The filtrate or supernatant obtained in (B) is adjusted to acidic or, preferably, to pH 3.5 to 5.5 with an acid such as hydrochloric acid, sulfuric acid or hydrobromic acid to conduct an operation of adsorbing with an adsorbent. Examples of the usable adsorbent include activated carbon and kaolin. An adsorbent is added to the extract followed by stirring or the extract is passed through a column filled with an adsorbent so that the active ingredient can be adsorbed with the adsorbent. When an adsorbent is added to the extract, the adsorbent with which the active ingredient is adsorbed can be obtained by means of filtration, centrifugation, etc. to remove the solution. 
     About (D) 
     For elution (desorption) of the active ingredient from the adsorbent obtained in (C), an elution solvent is added to said adsorbent, elution is conducted at room temperature or with suitable heating, or with stirring, and then the adsorbent is removed by a common method such as filtration or centrifugation. As to the extraction solvent used therefore, there may be used a basic solvent such as water, methanol, ethanol, isopropanol or the like adjusted to basic pH or an appropriate mixed solvent thereof and preferably, water adjusted to pH 9 to 12 may be used. Amount of the extracting solvent may be appropriately set. 
     The extract (eluate) obtained in this manner may be properly prepared in a suitable form as a raw material for a formulation or a pharmaceutical formulation. For example, the solution may be adjusted to have nearly neutral pH to be a raw material for a formulation, and may be adjusted to have a desired concentration by concentration or dilution. In addition, for a formulation for injection, sodium chloride may be added to prepare a solution isotonic to physiological saline. Furthermore, the solution may be concentrated to dryness or freeze-dried to prepare a solid form available for the raw material of tablets or the like. 
     Examples of an administration method to a patient include oral and other administrations such as subcutaneous, intramuscular and intravenous administrations. The dose can be suitably determined depending on the type of extract from inflamed tissues inoculated with vaccinia virus. The dose that is approved in the commercially available preparation according to the “Drugs in Japan, Ethical Drugs” (page 3525) is principally 16 NU per day by oral administration and 3.6 to 7.2 NU per day by injection. However, the dose may be appropriately increased or decreased depending on the type of disease, degree of seriousness, individual difference in the patients, method of administration, period of administration and the like (NU: Neurotropin unit. Neurotropin unit is defined by ED 50  value of analgesic effect measured by a modified Randall-Selitto method using SART-stressed mice that are chronic stressed animals showing a lowered pain threshold than normal animals. One NU indicates the activity of 1 mg of analgesic ingredients in Neurotropin preparations when the ED 50  value is 100 mg/kg of the preparation). 
     Hereinafter, examples of methods for producing an extract from inflamed tissues inoculated with vaccinia virus as well as pharmacological tests concerning novel pharmacological activity of the extract, that is, the repair promotive effect on muscle injury, are described. The present invention is not intended to be limited to the descriptions in Examples. 
    
    
     EXAMPLES 
     Example 1 
     Skins of healthy adult rabbits were inoculated with vaccinia virus. The inflamed skins were removed and crushed, and to the crushed skins, phenolated water was added. Then, the mixture was filtered under pressure, and the obtained filtrate was adjusted to pH 5 with hydrochloric acid, and then heated at 90 to 100° C. for 30 minutes. After deproteinization by filtration, the filtrate was adjusted to pH 9 with sodium hydroxide, further heated at 90 to 100° C. for 15 minutes, and then filtered. The filtrate was adjusted to about pH 4.5 with hydrochloric acid, and 2% activated carbon was added. The mixture was stirred for 2 hours and then centrifuged. To the collected activated carbon, water was added. The mixture was adjusted to pH 10 with sodium hydroxide, stirred at 60° C. for 1.5 hours, and then centrifuged and filtered to obtain a supernatant. To the collected activated carbon, water was added again. The mixture was adjusted to pH 11 with sodium hydroxide, stirred at 60° C. for 1.5 hours, and then centrifuged to obtain a supernatant. The two supernatants were combined and neutralized with hydrochloric acid to obtain an extract from inflamed skins of rabbits inoculated with vaccinia virus. 
     Example 2 
     Skins of healthy adult rabbits were inoculated with vaccinia virus to be infected. Subsequently, the inflamed skins were aseptically removed and chopped, and then phenol-added glycerin water was added. The mixture was ground with a homogenizer to be emulsified. Subsequently, the emulsion was filtered. The obtained filtrate was adjusted to weak acidity (pH 4.5 to 5.5) with hydrochloric acid, then heated at 100° C. and filtered. The filtrate was adjusted to weak alkalinity (pH 8.5 to 10.0) with sodium hydroxide, further heated at 100° C. and then filtered. The filtrate was adjusted to about pH 4.5 with hydrochloric acid, and about 1.5% activated carbon was added. The mixture was stirred for 1 to 5 hours and then filtered. To the activated carbon collected by the filtration, water was added. The mixture was adjusted to pH 9.4 to 10 with sodium hydroxide, stirred for 3 to 5 hours, and then filtered. The filtrate was neutralized with hydrochloric acid. 
     Example 3 
     Skins of healthy adult rabbits were inoculated with vaccinia virus to be activated. Then the activated skins were aseptically removed and chopped, and water was added. The mixture was ground with a homogenizer to be emulsified. Subsequently, the emulsion was filtered under pressure. The obtained filtrate was adjusted to pH 5.0 with hydrochloric acid, and then heated at 100° C. with flowing steam. After deproteinization by filtration, the filtrate was adjusted to pH 9.1 with sodium hydroxide, further heated at 100° C. and then filtered. The filtrate was adjusted to pH 4.1 with hydrochloric acid, and 2% activated carbon was added. The mixture was stirred for 2 hours and then filtered. To the filtrate, 5.5% activated carbon was further added, and the mixture was stirred for 2 hours and then filtered. To the activated carbon collected by the former filtration, water was added. The mixture was adjusted to pH 9.9 with sodium hydroxide, stirred at 60° C. for 1.5 hours, and then filtered. To the former activated carbon and the latter activated carbon, water was added. The mixture was adjusted to pH 10.9 with sodium hydroxide, stirred at 60° C. for 1.5 hours, and then filtered. The filtrates were combined and neutralized with hydrochloric acid. Then, the filtrate was desalted by electrodialysis using a membrane with a molecular weight of 100, and dried under reduced pressure. 
     Now there will be shown an example of the results of pharmacological test concerning the preventive and therapeutic action for muscle injury where the extract from inflamed tissues inoculated with vaccinia virus of the present invention (hereinafter, it will be sometimes referred to as “the present extract”) prepared in the above Example 1 was used as a test drug. 
     Pharmacological Test 
     Twenty-four 8-week-old male SD (Sprague-Dawley) rats were used. In each of the rats, a right side of the body relative to the spinous process that served as the center was defined as an injury side. Each of the rats was subjected to general anesthesia (0.3 to 0.5 mL of somnopentyl diluted into a double volume with physiological saline was administered intraperitoneally), and was then fixed to a testing bench by fastening rubber bands respectively around four legs thereof. An injury-scheduled site (right paraspinal muscle, the segment was a fourth lumbar vertebra level) was well shaved. A plastic-made cylindrical tip was arranged on the injury-scheduled site, and a heavy bob having a weight of 115 g was dropped from a height of 1 m (the heavy bob was free-fall dropped in the plastic-made cylinder) to cause the injury of paraspinal muscle [the drop mass method described in a reference document (Crisco J J, Joki P, Heinen G T, Connell M D, Panjabi M M. A muscle contusion injury model. Biomechanics, physiology, and histology. Am J Sports Med. 1994; 22(5)702-10.) was partly modified]. Immediately after the damaging, the rats were divided into a group in which physiological saline (5 μL) was subcutaneously injected to the injury site (an untreated group: n=12) and a group in which the extract of the invention (5 μL) was subcutaneously injected to the injury site (a treated group: n=12), and the evaluation was carried out on each of the groups. 
     The evaluation items were those which relate to an acute phase reaction and a tissue repair reaction in the staining of the injured paraspinal muscle with hematoxylin-eosin (HE) in each group (n=4) on day 1, day 3 and day 7 after the injury, and comparative examination on the evaluation items was carried out. 
     In each time course, each of the rats was subjected to general anesthesia (0.3 to 0.5 mL of somnopentyl diluted into a double volume with physiological saline was administered intraperitoneally), then paraspinal muscle was excised from the injury site, and then the excised piece was subjected to tissue fixation with a 4% paraformaldehyde (PFA) solution. The specimen that was subjected to the 4% PFA fixation was dehydrated with ethyl alcohol using Tissue-Tek [registered trademark] VIP (manufactured by Sakura Finetek Japan Co., Ltd.) to prepare a paraffin block, and the paraffin block was installed in a microtome SM2010R (manufactured by Leica microsystems, Wetzlar, Germany) to produce a paraffin slice slide having a thickness of about 4 μm. The HE staining was carried out in the following order: deparaffinization (three xylene vessels: 3 minutes and 30 seconds in total→four alcohol vessels: 5 minutes in total); washing with running water (3 minutes); washing with distilled water (1 minute); staining of nucleus (Mayer&#39;s hematoxylin solution (hematoxylin I: 20 seconds→hematoxylin II: 3 minutes)); washing with running water (3 minutes); development of color with phosphate buffered saline; washing with running water (1 minute); 50% alcohol (20 seconds); staining of a cytoplasm or the like (eosin: 30 seconds); dehydration (five 100% alcohol vessels: 4 minutes in total); clearing (four xylene vessels: 4 minutes in total); and mounting. Using the treated slide, the observation was carried out with respect to the above-mentioned evaluation items in the injury site using a microscope (BX51, manufactured by Olympus Corporation). 
     Results 
     As the method for evaluating the HE staining, the relative level (amount) of each of the observation items, i.e., the evidence of an acute phase reaction such as edema and bleeding, the proliferative evidence (initiation of tissue repair) such as neutrophil aggregation and fibroblasts appearance and the evidence (tissue delayed union) of fatty degeneration and muscle fiber atrophy, was rated in accordance with the following criteria and was scored: 0 point: the evidence was not observed; 1 point: the evidence was observed extremely slightly; 2 points: the evidence was observed moderately; and 3 points: the evidence was observed at a high degree. 
     The results of the average scores of paraspinal muscle HE staining evaluation on each group in each of time courses are shown in the following tables 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 On day 1 after the injury 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                 Fatty 
               
               
                   
                   
                   
                   
                   
                 degeneration 
               
               
                   
                   
                   
                 Neutrophil 
                 Fibroblast 
                 Muscle 
               
               
                   
                 Edema 
                 Bleeding 
                 aggregation 
                 appearance 
                 atrophy 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 Untreated 
                 2.25 ± 0.96 
                 2.50 ± 0.58 
                 0.25 ± 0.50 
                 0.50 ± 0.58 
                 0.25 ± 0.50 
               
               
                 group 
               
               
                 (average ± SD) 
               
               
                 Treated 
                 1.75 ± 0.50 
                 2.00 ± 0.00 
                 0.50 ± 0.58 
                 0.25 ± 0.50 
                 0.00 ± 0.00 
               
               
                 group 
               
               
                 (average ± SD) 
               
               
                   
               
            
           
         
       
     
     On day 1 after the injury, in the treated group, the scores of edema and bleeding were lower compared with those in the untreated group, and it was demonstrated that the occurrence of edema and bleeding in an acute phase was suppressed. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 On day 3 after the injury 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                 Fatty 
               
               
                   
                   
                   
                   
                   
                 degeneration 
               
               
                   
                   
                   
                 Neutrophil 
                 Fibroblast 
                 Muscle 
               
               
                   
                 Edema 
                 Bleeding 
                 aggregation 
                 appearance 
                 atrophy 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 Untreated 
                 2.00 ± 0.82 
                 2.00 ± 0.82 
                 1.75 ± 0.50 
                 1.75 ± 0.50 
                 0.50 ± 0.58 
               
               
                 group 
               
               
                 (average ± SD) 
               
               
                 Treated 
                 1.50 ± 0.58 
                  1.5 ± 1.00 
                 2.75 ± 0.50 
                  2.5 ± 0.58 
                 1.00 ± 0.00 
               
               
                 group 
               
               
                 (average ± SD) 
               
               
                   
               
            
           
         
       
     
     On day 3 after the injury, in the treated group, the scores of neutrophil aggregation and appearance of fibroblasts were higher compared with those in the untreated group, and it was demonstrated that the tissue repair process was initiated more surely. 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 On day 7 after the injury 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                 Fatty 
               
               
                   
                   
                   
                   
                   
                 degeneration 
               
               
                   
                   
                   
                 Neutrophil 
                 Fibroblast 
                 Muscle 
               
               
                   
                 Edema 
                 Bleeding 
                 aggregation 
                 appearance 
                 atrophy 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 Untreated 
                 1.50 ± 0.58 
                 2.00 ± 0.82 
                 2.75 ± 0.50 
                 2.75 ± 0.50 
                 2.75 ± 0.50 
               
               
                 group 
               
               
                 (average ± SD) 
               
               
                 Treated 
                 0.25 ± 0.50 
                 0.50 ± 0.58 
                 0.50 ± 0.58 
                 0.50 ± 0.58 
                 0.25 ± 0.50 
               
               
                 group 
               
               
                 (average ± SD) 
               
               
                   
               
            
           
         
       
     
     On day 7 after the injury, in the treated group, the scores of fatty degeneration and muscle atrophy were lower compared with those in the untreated group. Therefore, the tendency of a tissue delayed union was not observed, and it was demonstrated that a good tissue repair process (absorption of denatured cell tissues, repair of muscle fibers, and the like) proceeded. 
     From the results shown in Tables 1 to 3 above, in the treated group, the suppression of edema and bleeding on day 1 after the injury, the secured initiation of the tissue repair process on day 3 after the injury, and the progression of the good tissue repair process on day 7 after the injury were observed when compared with the untreated group. That is, it was demonstrated that the extract of the invention had a repair promotive effect on muscle injury. 
     INDUSTRIAL APPLICABILITY 
     As will be apparent from the above results of the pharmacological test, since it was confirmed that the medicament of the present invention had repair promotive effect on muscle injury, the medicament of the present invention has been shown to be useful as a preventing or therapeutic agent for muscle injury. A commercially available extract from inflamed skin inoculated with vaccinia virus has been used for many years and has been recognized to be a medicinal agent having very high safety. As such, the medicament of the present invention is effective as a preventing or therapeutic agent for muscle injury and is very highly useful with high safety and few side effects.