JPH08225452A - Ameliorative agent to abnormal perception - Google Patents

Abstract

PURPOSE: To provide an ameliorative agent having excellent bloodstream- ameliorative action and useful for amelioration of abnormal perception such as Raynaud's syndrome or cryesthesia, numbness or pain according to bloodstream disturbance by including a specific physiologically active substance extracted from infected tissue as an active component. CONSTITUTION: This ameliorative agent contains a physiologically active substance extracted from infected tissue and has following properties as an active component. Properties: amorphous hygroscopic powder of light-yellowish brown color; solubility: soluble to water, methanol and ethanol; absorption of ultraviolet part: UV max at 255-275nm; ninhydrin reaction: positive; phosphorus qualitative analysis: positive; pentose qualitative analysis: positive; chloride qualitative analysis: positive; contains nucleic acid bases; protein detecting reaction: negative. The physiologically active substance may be obtained by aseptically triturating infected tissue infected to virus, extracting with physiological saline water, etc., removing the tissue piece, removing protein, making it adsorbed to an adsorbent such as activated charcoal and eluting the adsorbed component. As the virus, virus belonging to family Poxviridae is preferable.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an agent for improving paresthesia containing a physiologically active substance extracted from infected tissue as an active ingredient.

[0002]

2. Description of the Related Art Organic blood vessel lesions such as arteriosclerosis, cold, physical and mental stress, and various triggers such as drugs cause local blood flow disorders to cause ischemia. Symptoms such as functional disorder, cold sensation, numbness, pain, and hypoesthesia appear. If this state continues for a long time, the local tissues will atrophy, degenerate, and eventually become necrotic. In an aging society, many patients on a daily basis, especially in the elderly, have numbness,
I suffer from symptoms such as pain and dysfunction. Therefore, there is a demand for the development of a safe drug that improves the blood flow locally in the pathological condition, has a function of repairing the function of tissues that have decreased, and has no side effects.

[0003]

DISCLOSURE OF THE INVENTION The present inventors have conducted a search and research on physiologically active substances extracted from infected animal tissues (hereinafter simply referred to as infected tissues) in which various viruses were inoculated into animal or cultured tissues to cause inflammation. As a result, it was found that the substance of the present invention has an excellent effect of improving blood flow in the pathological local region, and is useful as a drug for improving paresthesia such as cold sensation, numbness, pain, and paresthesia, and completed the present invention. did. An object of the present invention is to provide a paresthesia improving agent containing a physiologically active substance extracted from infected tissue as an active ingredient.

[0004]

The agent for improving paresthesia of the present invention is to grind infected tissue, add an extraction solvent to remove a tissue piece, and then deproteinize it to adsorb it to an adsorbent. Then, it contains a physiologically active substance obtained by eluting the adsorbed component as an active ingredient.

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION As the virus used in the present invention, viruses having a respiratory action, preferably orthopox virus such as vaccinia virus, cowpox virus, variola virus, ectromelia virus, sarpox virus, orf Parapoxvirus such as virus, paravaccinia virus, bovine papillary stomatitis virus, sheep pox virus, goat pox virus,
Goat pox virus such as tuberculosis virus, chicken pox virus, avian pox virus such as hare fibroma virus, rabbit myxoma virus, rabbit pox virus such as rabbit fibroma virus, swinepox virus, Yava monkey tumor virus, Tara Viruses belonging to the family Poxviridae such as poxvirus can be mentioned.

As animals for obtaining infected tissues, various mammals and birds such as rabbits, sheep, goats, pigs, cows, horses, monkeys, hamsters, guinea pigs, rats, mice and chickens can be used. It can be selected according to the virus type and purpose.

As the culture tissue, culture cells capable of propagating the type of poxvirus used can be used, and examples thereof include rabbit, sheep, goat, pig, cow, horse, monkey, hamster, guinea pig, rat, Mouse and their fetus kidney, skin, lung, testicle, liver, muscle,
Examples thereof include adrenal gland, thyroid gland, brain, nerve cells, cultured cells of various tissues such as blood cells, tumor cell cultures, human-derived cultured tissues such as Hela cells, and chorioallantoic membrane.

[0008] These infected tissues are aseptically collected and ground, and 1 to 5 times the amount of the extraction solvent is added to obtain an emulsified suspension. As the extraction solvent, distilled water, physiological saline, weakly acidic to weakly basic buffer, etc. can be used. Stabilizers such as glycerin, bactericidal / preservative agents such as phenol, sodium chloride, potassium chloride, chloride You may add inorganic salts, such as magnesium, suitably. At this time, the cell tissue can be destroyed by treatment with freeze-thaw, ultrasonic waves, cell membrane lysing enzyme or a surfactant to facilitate extraction.

The milky extract thus obtained is filtered or centrifuged to remove the tissue pieces, and then deproteinized. Deproteinization is
It can be carried out by known methods, heating, ultrasonic waves, protein denaturing agents such as acids, bases, urea, guanidine, organic solvents,
A method such as treatment with a surfactant or the like, isoelectric precipitation, salting out, etc. can be applied. Next, the precipitated insoluble protein is removed by filtration using filter paper (cellulose, nitrocellulose, etc.), glass filter, Celite, Zeitz filter plate, etc., ultrafiltration, gel filtration, ion exchange resin, centrifugation and the like.

The extract containing the active ingredient thus obtained is
An acid such as hydrochloric acid, sulfuric acid or hydrobromic acid is used to adjust the acidity, preferably pH 3.5 to 5.5, and the adsorption operation to the adsorbent is performed. Examples of adsorbents that can be used include activated carbon, kaolin, ion exchange resins, etc., and the active ingredient can be added by adding the adsorbent to the extract and stirring or by passing through a column packed with the adsorbent. Can be adsorbed.

In order to elute the substance of the present invention from the adsorbed components, an elution solvent is added to the adsorbent, and the adsorbent is removed by a usual method such as filtration at room temperature or by appropriately heating or stirring. Can be achieved. The elution solvent used is
It is possible to use water, methanol, ethanol, isopropanol or the like or a suitable mixed solution thereof, or a basic solvent, preferably the above-mentioned solvent adjusted to pH 9 to 12.

The eluate thus obtained is preferably adjusted to a pH of around 6.5 to 8.5 near neutrality and then evaporated to dryness or freeze-dried under reduced pressure to obtain the desired physiological activity. The substance can be obtained.

The active ingredient of the present invention extracted and purified by the above operation has the following physicochemical properties. Property: Light yellow brown amorphous hygroscopic powder Solubility: Soluble in water, methanol, ethanol Ultraviolet absorption: UVmax 255-275nm Ninhydrin reaction: Positive Phosphorus qualitative analysis: Positive Take 2 mg of the substance of the present invention, 1 ml of perchloric acid Is added and heated until the liquid becomes colorless, and 3 ml of dilute sulfuric acid, 0.4 g of amidol hydrochloride and 8 g of sodium bisulfite are added to 100 ml of water.
2 ml of added solution and 1 part of ammonium molybdate
When 2 ml of a solution prepared by adding 30 ml of water to g and adding the mixture and allowing to stand, the liquid exhibits a blue color. Pentose Qualitative analysis: Positive Take 5 mg of the substance of the present invention and dissolve it by adding water to make 10 ml. To 1 ml of this solution, add 0.2 g of orcin and 0.135 g of ferric ammonium sulfate and dissolve 5 ml of ethanol, and dissolve this solution in 83 ml of hydrochloric acid. In addition to 100, add water
When adding 3 ml of liquid and heating in a boiling water bath,
The liquid has a green color. Chloride Qualitative Analysis: Positive An aqueous solution of the substance of the present invention precipitates with the silver nitrate reagent. Contains nucleobases. Various protein detection reactions against the substance of the present invention are negative.

The following are examples of the method for producing the substance of the present invention. However, these do not limit the scope of the present invention.

【Example】

Example 1. After infecting healthy mature rabbit skin with vaccinia virus and infecting it, aseptically exfoliate the varicella-like skin, shred it, add phenol-glycerin water, and grind with a homogenizer to give a milky form. . Then, this was centrifugally filtered, and the obtained filtrate was weakly acidified with hydrochloric acid (about pH 4.5 to 5.
After adjusting to 5), it was heat-treated at 100 ° C. under flowing steam and filtered. The filtrate is further filtered using a Zeitz filter plate,
Weakly alkaline with sodium hydroxide (about pH 8.5 to 1)
0.0), heat treatment at 100 ° C., and filtration. The filtrate was adjusted to pH 4.5 with hydrochloric acid, added with activated carbon 1.5%, stirred for 1 to 5 hours, and then filtered. Water was added to the activated carbon collected by filtration, the pH was adjusted to 9.4 to 10 with sodium hydroxide, the mixture was stirred for 3 to 5 hours, and then filtered. The filtrate was neutralized with hydrochloric acid to a pH of about 7 and concentrated to dryness under reduced pressure to obtain the substance of the present invention.

Example 2. Methanol was added to the adsorbed activated carbon obtained in the same manner as in Example 1, and the mixture was stirred for 1 hour and then filtered. It was dried under reduced pressure to obtain the active ingredient.

[0016]

Next, an example of the results of toxicity test and clinical test of the agent for improving paresthesia of the present invention will be shown. (1) Toxicity test A male or female mouse and a male or female rat were administered with a physiologically active substance, which is an active ingredient of the drug of the present invention, by oral, subcutaneous, intraperitoneal, or intravenous routes to conduct an acute toxicity test. As a result, the LD ₅₀ of the substance of the present invention was 5,000 mg / kg or more regardless of the animal species and sex regardless of the route of administration.
In addition, although various safety tests such as subacute toxicity and reproductive tests were conducted, no abnormalities were found in each organ, and in the reproductive tests, there was no evidence of reproductive ability of the mother, fetus, newborn, and offspring. Did not affect.

(2) Blood Flow Improving Action 50 to 400 mg / kg of the substance of the present invention was orally administered to male Wistar rats (body weight: about 200 g). One hour later, 0.1 ml of 0.2% carrageenin per site was subcutaneously administered to the right hind leg, and the skin temperature of the legs and feet was measured by thermography until 48 hours later. As a result, it was found that the substance of the present invention dose-dependently improved the decrease in skin temperature of the legs and limbs that occurred 45 minutes to 3 hours after carrageenin administration, and had an excellent blood flow improving action.

(3) Clinical test For the improvement of the above symptoms by administering the drug of the present invention to patients with Raynaud's syndrome, diabetic neuropathy, SMON sequelae, etc. suffering from symptoms such as numbness, pain, cold limbs, and paresthesia. Examined. For example, in the case of injection, the substance of the present invention was intravenously administered at 3 to 12 mg per day for 1 to 2 weeks. In the overall improvement degree, a clear significant improvement effect was obtained compared with placebo for symptoms such as numbness, pain, cold limbs, and paresthesia. For example, 70% was moderate improvement or higher, 9 There was a test example in which the effect of mild improvement or more was obtained. In the above clinical trial, not only serious side effects but also insomnia,
Almost no mild side effects such as sweating, dry mouth, digestive disorders were observed.

[0019]

EFFECTS OF THE INVENTION As is clear from the above test results, the substance of the present invention promotes improvement of blood flow in various tissues that have entered an ischemic state due to blood flow disorder and normalizes the tissue function without imposing a burden on the heart. It has an excellent effect of Therefore, vibration disease, white fistula, arteriosclerosis obliterans, thromboangiopathy obliterans, progressive muscular atrophy, cerebrovascular disorders, periarteritis nodosa, systemic erythematosus, rheumatoid arthritis, aortitis syndrome. Raynaud's syndrome and diabetic neuropathy, SMON sequelae, ischemic neuropathy, frostbite, frostbite, tinnitus, deafness, various diseases associated with blood flow disorders such as central retinal artery occlusion, numbness,
The agent for improving paresthesia of the present invention is very useful as a therapeutic agent for treating or alleviating concomitant symptoms of the above diseases such as pain, hypoesthesia, dysfunction, atrophy and necrosis.

The agent for improving paresthesia of the present invention has an action of working only in a pathological condition in which blood flow is impaired and functionally deteriorated to restore tissues to a normal state, has low toxicity, and can be orally administered without side effects. It can be safely used for a long time, and is particularly advantageous for treating chronic diseases.

The paresthesia improving agent containing the substance of the present invention as an active ingredient can be in the form of tablets, capsules, powders, granules, powders, solutions, injections, suppositories and the like. In the prescription, the substance of the present invention may be used alone, or may be used as a compounding agent with other pharmaceutically active ingredients.

For oral administration preparations, crystalline cellulose, cellulose derivatives, gum arabic, corn starch, gelatin, etc. as they are or together with suitable additives such as lactose, mannitol, corn starch, potato starch and other conventional excipients are used. Binders, corn starch, potato starch, disintegrating agents such as carboxymethyl cellulose calcium, talc, lubricants such as magnesium stearate, and other fillers, wetting agents, buffers, preservatives, flavors, etc. , Powder, granules or capsules.

As the injection, an aqueous solvent such as distilled water for injection, physiological saline, 5 to 20% glucose injection, or a solution of a non-aqueous solvent such as vegetable oil, synthetic fatty acid glyceride, higher fatty acid ester, propylene glycol, etc., It can be a suspension or an emulsion, and if necessary, a solubilizing agent,
Additives that are normally used, such as an isotonicity agent, a suspending agent, an emulsifier, a stabilizer, and a preservative, may be appropriately added. Alternatively, it can be used as a freeze-dried preparation by putting it in a vial bottle or the like and appropriately dissolving it in the above solvent at the time of use. Further, the substance of the present invention is
It can be mixed with various bases such as an emulsion base or a water-soluble base to prepare a suppository, or can be formulated into other inhalants, aerosols and the like.

Although the desirable dose of the drug of the present invention varies depending on the administration subject, dosage form, administration method, administration period, etc., in order to obtain a desired effect, generally, the dose of the active ingredient is 1 to 1 per day for an adult. Oral administration can be 100 mg, preferably 4 to 40 mg. Parenteral administration (eg injection)
In this case, the daily dose is preferably a dose level which is 3 to 1/10 of the above dose. The prescription examples of the paresthesia improving agent of the present invention are shown below.

[0025]

[0026]

[0027]

Claims (3)

[Reference number] PC-247 [Claims] 1. An agent for improving paresthesia, which comprises, as an active ingredient, a physiologically active substance having the following physicochemical properties extracted from infected tissue. Property: Light yellow brown amorphous hygroscopic powder Solubility: Soluble in water, methanol, ethanol Ultraviolet absorption: UVmax 255-275nm Ninhydrin reaction: positive Phosphorus qualitative analysis: positive Pentose qualitative analysis: positive Chloride qualitative analysis: positive Contains nucleobases Protein detection reaction: Negative 2. The paresthesia improving agent according to claim 1, which is a therapeutic agent for Raynaud's syndrome. 3. The paresthesia improving agent according to claim 1, which is a therapeutic agent for SMON sequelae.