JP2539669B2 - Diabetic neuropathy treatment - Google Patents

Description

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

(Prior Art) Organic blood vessel lesions such as arteriosclerosis, cold, physical and mental stress, and various triggers such as drugs cause local blood flow disorders, resulting in ischemic tissues and organs. 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 the aging society, many patients are suffering from symptoms such as numbness, pain, and dysfunction on a daily basis, especially in elderly people. 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.

The present inventor 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 an animal or cultured tissues to cause inflammation, and as a result, the substance of the present invention was found to be The present invention has been completed by discovering that it has an excellent local blood flow improving action.

(Problems to be Solved by the Invention) An object of the present invention is to provide a therapeutic agent for diabetic neuropathy containing a physiologically active substance extracted from infected tissue as an active ingredient.

(Means for Solving Problems) The therapeutic agent of the present invention is to grind infected tissue, add an extraction solvent to remove a tissue piece, perform deproteinization, and adsorb the adsorbent, It contains a physiologically active substance obtained by eluting the adsorbed component as an active ingredient.

As the virus used in the present invention, viruses having a respiratory action, preferably, vaccinia virus, cowpox virus, pox virus, ectromelia virus, orthopox virus such as sarpox virus, orf virus, paravaccinia virus, Parapoxvirus such as 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 fiber Examples include viruses belonging to the poxviridae family such as rabbit poxviruses such as tumor virus, swinepox virus, Yava monkey tumor virus, and Tara poxvirus.

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. And can be selected according to the purpose.

In addition, as the cultured tissue, it is possible to use cultured cells in which the type of poxvirus used can proliferate, for example, rabbit, sheep, goat, pig, cow, horse, monkey,
Humans such as hamsters, guinea pigs, rats, mice and their fetal kidneys, skin, lungs, testicles, liver, muscle, adrenal gland, thyroid, brain, nerve cells, blood cells, tumor cell cultures, Hela cells, etc. The derived culture tissue, chorioallantoic membrane and the like are included.

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, a weakly acidic to weakly basic buffer and the like can be used, a stabilizer such as glycerin, a bactericidal / preservative such as phenol, sodium chloride,
Inorganic salts such as potassium chloride and magnesium chloride may be appropriately added. 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 resulting milky extract is filtered or centrifuged to remove tissue fragments, and then subjected to deproteinization. Deproteinization can be carried out by a known method, such as heating, ultrasonication, treatment with a protein denaturing agent such as an acid, a base, urea, guanidine, an organic solvent, a surfactant, isoelectric point precipitation, salting out and the like. Can be applied. Then, 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 acidified with an acid such as hydrochloric acid, sulfuric acid, or hydrobromic acid, preferably at pH 3.5.
Adjust to 5.5 to perform the adsorption operation to the adsorbent. 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.

The elution of the substance of the present invention from the adsorbed component can be achieved by adding an elution solvent to the adsorbent, eluting it at room temperature or by appropriately heating or stirring, and removing the adsorbent by a usual method such as filtration. . As the elution solvent used, water, methanol, ethanol, isopropanol or the like or a suitable mixed solution thereof, or a basic solvent, preferably pH 9 to 12
The solvent adjusted to the above can be used.

The eluate thus obtained preferably has a pH of 6.5.
The target physiologically active substance can be obtained by adjusting the pH to about 8.5 to around neutrality and then evaporating to dryness or freeze-drying under reduced pressure.

The active ingredient of the present invention extracted and purified by the above operation has the following physicochemical properties.

Property: Light yellowish brown amorphous hygroscopic powder Solubility: Soluble in water, methanol, ethanol Ultraviolet absorption: UVmax255-275nm Ninhydrin reaction: Positive Phosphorus qualitative analysis: Positive Take 2 mg of the substance of the present invention and take 1 ml of perchloric acid. In addition, the solution was heated until it became colorless, and 100 ml of water was added to 3 ml of dilute sulfuric acid, 0.4 g of amidol hydrochloride and 8 g of sodium bisulfite, and dissolved.
When 2 ml of a solution prepared by adding 30 ml of water to 1 ml of ammonium molybdate and adding 2 ml is left to stand, the liquid turns blue.

Pentose Qualitative analysis: Positive Take 5 mg of the substance of the present invention, dissolve in water to make 10 ml,
0.2 g of orcin and ferric ammonium sulfate in 1 ml of this solution
To 0.135 g, add 5 ml of ethanol and dissolve.
In addition to 3 ml, water is added to make 100 ml, and when 3 ml is added and heated in a boiling water bath, the liquid turns green.

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 is an example of a 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 the skin of a healthy mature rabbit with a vaccinia virus to infect it, the varicella-exposed skin was aseptically exfoliated and then finely chopped, followed by addition of phenol-added glycerin water, It was ground with a homogenizer to give a milky form. Then, this was subjected to centrifugal filtration, the obtained filtrate was adjusted to be weakly acidic (about pH 4.5 to 5.5) with hydrochloric acid, and then heat-treated at 100 ° C. under flowing steam and filtered. After filtering the filtrate with a Zeitz filter plate, make it weakly alkaline (about pH 8.5 to 10.0) with sodium hydroxide, and then add 100 ° C.
The mixture was heat treated with and filtered. The filtrate is adjusted to pH 4.5 with hydrochloric acid,
Activated carbon (1.5%) was added, and the mixture was stirred for 1 to 5 hours and then filtered. Water was added to the activated carbon collected by filtration, and the pH was adjusted to 9 with sodium hydroxide.
The mixture was adjusted to 4 to 10, stirred for 3 to 5 hours, and then filtered. The filtrate was neutralized with hydrochloric acid to a pH value of around 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.

(Operation) Next, an example of the results of the toxicity test and clinical test of the therapeutic agent of the present invention will be shown.

(1) Toxicity test A male and female mouse and a male and female rat were administered with a physiologically active substance, which is an active ingredient of the therapeutic agent of the present invention, by oral, subcutaneous, intraperitoneal, or intravenous route, and an acute toxicity test was conducted.

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 effect Wistar male rats (body weight: about 200 g) were orally administered with 50 to 400 mg / kg of the substance of the present invention. 0.1m per site after 1 hour
0.2% carrageenin (l) was subcutaneously administered to the right hind leg, and the skin temperature of the paw was measured by thermography until 48 hours later.

As a result, it was confirmed that the substance of the present invention dose-dependently improved the lowering of the skin temperature of the legs and limbs that occurred 45 minutes to 3 hours after the administration of carrageenin, and had an excellent blood flow improving action.

(3) Clinical study The therapeutic agent of the present invention was administered to patients with Raynaud's syndrome, diabetic neuropathy, SMON sequelae, etc., who suffer from symptoms such as numbness, pain, cold limbs, paresthesia, etc. . The substance of the present invention is, for example, in the case of injection, 3 times a day.
˜12 mg was administered intravenously for 1 day 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 test, not only serious side effects but also mild side effects such as insomnia, sweating, dry mouth, and gastrointestinal tract abnormalities were hardly observed.

(Effect) As is clear from the above test results, the substance of the present invention is excellent in promoting blood flow improvement in various tissues that have fallen into an ischemic state due to blood flow disorder and normalizing tissue function without giving a burden to the heart and the like. Has the effect.

Therefore, vibration disease, white fistula, obstructive arteriosclerosis, obstructive thromboangiopathy, progressive muscular atrophy, cerebrovascular disorder, periarteritis nodosa, systemic erythematosus, rheumatoid arthritis, aortitis syndrome. Raynaud's syndrome, diabetic neuropathy, SMON sequelae, ischemic neuropathy, frostbite, frostbite, tinnitus, deafness, various diseases associated with blood flow disorders such as central retinal artery occlusion, coldness, numbness, pain, hypoesthesia ,Dysfunction,
The therapeutic agent of the present invention is very useful as a therapeutic agent for treating or alleviating concomitant symptoms of the above diseases such as atrophy and necrosis.

INDUSTRIAL APPLICABILITY The therapeutic agent of the present invention has an action of working only in a pathological condition in which blood flow disorder is impaired and functionally restored to restore the normal condition, and is orally administrable with low toxicity and no side effect, and therefore safe and long-term. It can be used and is particularly advantageous for treating chronic diseases.

(Example) The biological function activator 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 orally administered preparations, as it is or with suitable additives, for example, conventional excipients such as lactose, mannitol, corn starch, potato starch, etc., and binders such as crystalline cellulose, cellulose derivatives, gum arabic, corn starch, gelatin etc. , Corn starch,
Tablets, powders, granules or capsules by appropriately combining disintegrating agents such as potato starch, carboxymethyl cellulose calcium, lubricants such as talc and magnesium stearate, and other fillers, wetting agents, buffers, preservatives, and flavors. It can be an agent.

As injections, distilled water for injection, physiological saline, 5 to
Aqueous solution such as 20% glucose injection solution, or a solution, suspension or emulsion of non-aqueous solvent such as vegetable oil, synthetic fatty acid glyceride, higher fatty acid ester, propylene glycol, and the like, a solubilizing agent as necessary, 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 mixed with various bases such as an emulsion base or a water-soluble base to form a suppository, or other inhalants,
It can be formulated into an aerosol and the like.

The desired dose of the therapeutic agent of the present invention is the administration subject, dosage form,
Although it varies depending on the administration method, administration period and the like, in order to obtain a desired effect, generally, 1 to 100 mg, preferably 4 to 40 mg, of the active ingredient per day can be orally administered to an adult.

In the case of parenteral administration (eg, injection), the daily dose is preferably a dose level which is 3 to 1/10 of the above dose.

 The prescription examples of the therapeutic agent of the present invention are shown below.

Prescription example 1. (tablet) composition per tablet (mg) Substance of the present invention 4 Lactose 106 Crystalline cellulose 40 Carboxymethyl cellulose calcium 20 Magnesium stearate 10 Total 180 mg Prescription example 2. (capsule) composition Per capsule (mg ) Inventive substance 10 Lactose 200 tal 40 Total 250 mg Prescription example 3. (Injection) Component Ingredient per ampoule (mg) Inventive substance 1 Sodium chloride Suitable amount Distilled water for injection Suitable amount Total 1 ml

Claims (1)

(57) [Claims] 1. A therapeutic agent for diabetic neuropathy containing as an active ingredient a physiologically active substance having the following physicochemical properties extracted from infected tissue. Property: Light yellowish brown amorphous hygroscopic powder Solubility: Soluble in water, methanol, ethanol Ultraviolet absorption: UVmax255-275nm Ninhydrin reaction: Positive Phosphorus Qualitative analysis: Positive Pentose Qualitative analysis: Positive Chloride Qualitative analysis: Positive Nucleic acid Contains bases Protein detection reaction: Negative