JPH10338644A - Agent for curing wound - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject medicine exhibiting a strong wound cure- stimulating action by including a specific peptide and a peripheral circulation- improving agent. SOLUTION: This wound-curing agent contains 0.0001-10 wt.% of a peptide of the formula, and 0.01-99 wt.% of vitamin E, 0.01-40 wt.% of at least one kind of compound selected from d-camphor, dl-camphor, a heparin-like substance, ichthammol, cantharides and Guinea pepper tincture or 0.01-40 wt.% of at least one kind of compound selected from nicotinic acid, pharmacologically acceptable nicotinic acid esters, pharmacologically acceptable nicotinic acid amides and pharmacologically acceptable nicotinic salts. The peptide of the formula is obtained e.g. by culturing Streptomycin nobilis, extracting the culture solution, its dried product or cultured cells with an organic solvent, and subsequently purifying the extract by chromatography, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a therapeutic agent for wounds containing, as an active ingredient, a wound healing substance comprising a peptide represented by the following formula [I].

[0002]

BACKGROUND OF THE INVENTION Wounds are damages to surface tissue such as surgical incisions, gastrointestinal ulcers, burns, lacerations, or skin ulcers (such as pressure sores). As a method of treating a wound, it is a common practice to give emergency treatment to the injured part and then wait until the injured part is healed by the natural resilience of the living body. However, it takes a long time to recover, and the pain of the patient, including pain, is not uncommon. Therefore, it is desired to actively and directly promote healing without relying on natural healing. Wound healing generally depends on the formation of new connective tissue and epithelial tissue by cell proliferation, and it is considered that an agent that stimulates or promotes the differentiation and proliferation process of cells involved in wound healing is effective.

[0003] Hitherto, as an agent exhibiting the action of promoting wound healing, calf blood extract (solcoseryl) (applied pharmacology, 2
2, 565-579, 1981), lysozyme chloride and the like.

[0004]

However, the above-mentioned wound (damage to the surface tissue) is generally intractable, and the existing drugs often do not show a sufficient clinical effect. The reason is that the action of the existing drug itself is weak. Furthermore, the healing promotion in the wound healing process has several points, such as granulation and improvement of peripheral circulation, because existing drugs contribute only to a small part of them.

[0005] In view of the above, an object of the present invention is to provide a highly useful wound treatment agent.

[0006]

Means for Solving the Problems The present inventors have prepared the following formula [I]

Embedded image (Hereinafter referred to as a wound healing peptide)
Has a granulation effect at the point of promoting healing,
It is thought that a combination of this compound and a peripheral circulation improving agent will exert a strong wound healing promoting action, and as a result of earnest studies, the present invention has been completed.

[0007] That is, the agent for treating wounds of the present invention is characterized by containing the peptide represented by the formula [I] and an agent for improving peripheral circulation.

[0008] The wound healing peptide used in the present invention comprises:
The wound-healing peptide-producing strain belonging to the genus Streptomyces, for example, Streptomyces nobilis (Streptomyces nobilis, hereinafter abbreviated as "S. nobilis") is cultured, and the obtained culture solution or the dried solution thereof is dried. An extract obtained by extracting an organic solvent from a solid or cultured cells is subjected to various column chromatography, and the column chromatography fraction containing the target substance is recrystallized to obtain a solid.

An actinomycete S. aureus producing the above wound healing peptide. Nobilis is available from public preservation institutions, for example, bacteria such as the conserved bacterium of RIKEN (JCM4274), which is also stored as ATCC 19252 in the United States and CBS 198.65 in the Netherlands.

[0010] The method for obtaining the above-mentioned wound healing peptide can be obtained by the method described in WO 96/12732, which was previously filed by the present inventors in an international application.

The peripheral circulation improving agent used in the present invention includes, for example, vitamin E, d-camphor, dl-camphor, a heparin-like substance, itctamole, cantharis, capsicum tincture, nicotinic acid, a pharmacologically acceptable nicotine. Examples thereof include acid esters, pharmacologically acceptable amides of nicotinic acid, and pharmacologically acceptable salts of nicotinic acid. Those usable as pharmaceuticals are used.

The above-mentioned vitamin E means tocopherol (vitamin E) and its derivatives, and those listed in the Japanese Pharmacopoeia include, for example, tocopherol acetate, tocopherol succinate and the like. Other than the above, for example, α-tocopherol, β-
Tocopherol, γ-tocopherol, δ-tocopherol, tocopherol nicotinate, tocopherol phosphate, natural vitamin E, and the like, but are not limited thereto, and conjugates of tocopherol with monosaccharide, oligosaccharide or polysaccharide. And water-soluble vitamin E.

Examples of the nicotinic acid ester include, but are not limited to, nicotinic acid methyl ester, nicotinic acid ethyl ester and the like. Examples of the amide of nicotinic acid include, but are not particularly limited to, nicotinamide. Examples of the salt of nicotinic acid include salts with various bases. Specific examples include alkali metal salts (eg, lithium salt, sodium salt, potassium salt, etc.) and alkaline earth metal salts (eg, calcium salt, magnesium salt, etc.), but are not particularly limited. .

In order to formulate the wound healing peptide and the agent for improving peripheral circulation to obtain the agent for treating wounds of the present invention, these are usually in the form of a pharmaceutical composition together with a pharmaceutical carrier. As a carrier, a filler, a disintegrant, a bulking agent, a binder, a coloring agent, a flavoring agent, a pH adjusting agent, a solubilizing agent, a suspending agent, which are generally used for preparing a drug according to a dosage form, Buffers, stabilizers, preservatives, qualifiers, surfactants, lubricants, excipients,
Examples include antioxidants, dispersants, propellants, solubilizers, and solubilizers. Also, by selecting an appropriate solvent, the wound healing peptide and the peripheral circulation improving agent can be used as they are as a liquid preparation.

[0015] The above-mentioned carriers for preparations include, for example, sodium alginate, gelatin, corn starch, tragacanth gum, methyl cellulose, hydroxyethyl cellulose, carboxymethyl cellulose, dextrin, carboxymethyl starch, polyvinyl alcohol, sodium polyacrylate, methoxyethylene-maleic anhydride. Polymers, polymers such as polyvinyl ether and polyvinylpyrrolidone; oils and fats such as beeswax, olive oil, cocoa oil, sesame oil, soybean oil, camellia oil, peanut oil, beef oil, pork oil, lanolin; white petrolatum; paraffin;
Gelled hydrocarbons (for example, brand name Plastibase, manufactured by Bristol-Myers Squibb); higher fatty acids such as stearic acid; higher alcohols such as cetyl alcohol and stearyl alcohol; polyethylene glycol;

[0016] The unit dosage form of the wound healing agent formulated using the above-mentioned wound healing peptide and a peripheral circulation improving agent includes tablets, pills, drinking liquids,
Limonase agent, injection, powder, suspension, emulsion, granule,
Extracts, fine granules, syrups, dips, decoctions, eye drops,
Lozenges, poultices, liniments, lotions,
Elixirs, eye ointments, plasters, capsules, suppositories,
Examples include enemas, injections (solutions, suspensions, etc.), patches, ointments, jellies, pastas, inhalants, creams, sprays, nasal drops, aerosols and the like.

The amount of the wound healing peptide contained in the wound treatment agent is preferably 0.0001 to 10 in the wound treatment agent.
% By weight.

The amount of the peripheral circulation improving agent contained in the wound healing agent varies depending on its type.
Is preferably 0.01 to 99% by weight, more preferably 0.1 to 50% by weight, and in d-camphor 0.01 to 40% by weight.
% By weight, preferably 0.1 to 20% by weight, more preferably 0.01 to 40% by weight for nicotinic acid,
0.1-20% by weight is more preferred.

The wound healing agent obtained from the wound healing peptide and the peripheral circulation improving agent is administered by a method according to various forms upon use. For example, in the case of an external preparation, it is sprayed, affixed or applied directly to the required site such as skin or mucous membrane, and in the case of tablets, pills, drinking liquids, suspensions, emulsions, granules and capsules, It is administered intravenously, intramuscularly, intradermally, subcutaneously, intraarticularly or intraperitoneally in the case of injections, and rectally in the case of suppositories.

The dose of the therapeutic agent for wounds of the present invention is appropriately selected depending on the purpose of use, symptoms, etc., but is usually 10 pg / kg to 100 mg / day as a wound healing peptide per day.
kg, 1 ng / kg to 1 g /
It is in the range of about kg. Of course, the above pharmaceutical composition may be administered once to four times a day.

[0021]

BEST MODE FOR CARRYING OUT THE INVENTION

Example of Production of Wound Healing Peptide Actinomyces S. aureus obtained from RIKEN Nobilis (JCM
4274) was shake-cultured (pre-preculture) in 100 ml of starch / ammonium medium supplemented with 0.2% (w / v) of yeast extract, and then 3 liters of the same medium was inoculated with 60 ml of the pre-preculture culture solution. And cultured for 25 hours with shaking (seed culture). In addition, a starch / ammonium medium (100 ml of distilled water)
1 g of soluble starch and 0.02 g of dipotassium hydrogen phosphate
285 liters of the seed culture was inoculated into 285 liters (containing 5 g and 0.05 g of ammonium chloride) and cultured with shaking at about 30 ° C. for 8 days. To 215 g (wet weight) of the obtained cells, 2.15 l of dichloromethane was added, and the mixture was subjected to ultrasonic treatment for 30 minutes. Thereafter, 8.6 l of dichloromethane was further added, and the whole was stirred at room temperature for 1 hour. After filtering off the cells, the obtained filtrate was concentrated to dryness. After the dried product of the extract was dissolved in a small amount of hexane, the hexane phase was extracted three times with methanol. The hexane insolubles and the methanol extract were redissolved in methanol.

Next, the solvent extract obtained as described above is
ODS (octadecyldimethylchlorosilane) carrier 28
g. Then, about 30 g of the extract-adsorbing carrier was charged on the 3.2-cm-diameter column packed with 275 g of the ODS carrier to prepare an ODS column. Purification was performed using the ODS column under the following conditions. As an elution solvent, a) methanol: acetonitrile: water = 1.5 liter of 7: 7: 6, b) methanol: acetonitrile: water = 1.2 liter of 8: 8: 4, c) methanol: acetonitrile: water = 9 : 9: 2
200 ml, d) methanol: acetonitrile: water =
19: 19: 2 1 liter, e) 500 methanol
ml, in this order at a flow rate of 10.5 ml / min. The fractionation was carried out every time the solvent composition was changed. In particular, the fraction eluted with methanol: acetonitrile: water = 19: 19: 2 was fractionated little by little (by 2 minutes) using an appropriate fraction collector. For each eluted fraction, ODS-80T
M, HPLC using a Tosoh column having an inner diameter of 4.6 mm × length of 25.0 cm (Hitachi, Pump L-600)
0, L-6200, detector L-3000, column open 655A-52), under the conditions of a detection wavelength of 210 nm, a column temperature of 40 ° C., and a flow rate of 1 ml / min, water: acetonitrile: methanol = 6: 7: 7 (0
Min) to 0: 1: 1 (30 min). Of the above fractions, only those containing a peak with a retention time of 18 to 20 minutes were collected and treated as the same fraction (840 m
g), repeated recrystallization using methanol-water,
330 mg of a wound-healing peptide having needle-like crystals was obtained. The obtained wound healing peptide was used in the following preparation examples, examples and comparative examples.

The structure of this substance was confirmed to be the same as the substance described in WO96 / 12732 from various instrumental analysis data.

Structural analysis data Instrument analysis data of the substances obtained in the production examples are shown below.

1. MS ESI-MS: m / z = 913.6 (M + H-H
₂ O) ⁺ , 931.6 (M + H) ⁺ , 953.6 (M +
Na) ⁺ HRFAB-MS Found: m / z = 913.5507 (M + H-H
₂ O) ⁺ , m / z = 913, 953, 931 (913 is the main, 931 is very small) Calcd for: C ₄₅ H ₆₉ N ₈ O ₁₂ m / z = 913.55053

2. IR IR: 3,400 cm ^-1 : -OH, -NH, 2,90
0 cm ^-1: alkyl groups, 1,750cm ^-1: -C (=
O) -O-, 1,650 cm ^-1 : -C (= O) -NH-

3. Amino acid analysis D-serine, L-alanine and D-
N-methyl-phenylalanine was observed.

Preparation Example of Wound Healing Agent Preparation Example 1 Wound healing peptide 0.4 mg Gelated hydrocarbon (Plastibase, manufactured by Bristol-Myers Squibb) 9.99 g Tocopherol acetate (Wako Pure Chemical Industries) 10 mg Wound healing peptide 0 After finely pulverizing 0.4 mg in a mortar, the gelated hydrocarbon and tocopherol acetate in the above amounts were added, and these were mixed well in a mortar to prepare an ointment.

Formulation Example 2 Wound healing peptide 0.4 mg Gelated hydrocarbon (Plastibase, manufactured by Bristol-Myers Squibb) 9.99 g d-Camphor (Wako Pure Chemical Industries) 10 mg Wound healing peptide 0.4 mg in a mortar After finely pulverizing the mixture, the above amounts of gelled hydrocarbon and d-camphor were added, and these were mixed well in a mortar to prepare an ointment.

Formulation Example 3 Wound healing peptide 0.4 mg Gelated hydrocarbon (Plastibase, manufactured by Bristol-Myers Squibb) 9.99 g Nicotinic acid (Wako Pure Chemical Industries) 10 mg Wound healing peptide 0.4 mg in a mortar After finely pulverizing, the above amounts of the gelled hydrocarbon and nicotinic acid were added, and these were mixed well in a mortar to prepare an ointment.

Pharmacological Test Test Example Action on Diabetic Mouse Skin Defect Model Ointment containing wound healing peptide and tocopherol acetate was prepared according to Preparation Example 1 (Example 1), and ointment containing wound healing peptide and d-camphor was prepared Example 2 (Example 2), an ointment containing the wound healing peptide and nicotinic acid was prepared according to Formulation Example 3 (Example 3), and used as a test ointment. An ointment containing a wound healing peptide [wound healing peptide 0.4 mg, gelled hydrocarbon (plastibase) 10 g: Comparative Example 1], an ointment containing tocopherol acetate [tocopherol acetate 10 mg, gelled hydrocarbon (plastibase) 9] .99 g: Comparative Example 2],
Ointment containing d-camphor [d-camphor 10 mg,
9.99 g of gelled hydrocarbon (Plastibase): Comparative Example 3], ointment containing nicotinic acid [nicotinic acid 10 m
g, 9.99 g of gelled hydrocarbon (Plastibase): Comparative Example 4] and a gelled hydrocarbon base [10 g of gelled hydrocarbon (Plastibase): Comparative Example 5] were prepared in the same manner and used as a comparative example for the test. Was.

Female diabetic mice (db / db) weighing 30 to 45 g were used as test animals.

A mouse shaved 3 days before the defect was placed under the anesthesia with ether and having a diameter of 1
A 6 mm defect was made using ophthalmic scissors. 25 mg of test ointment (1 μg of wound healing peptide) was applied to the wound surface (2c
m ² ), and covered with a polyurethane film material (manufactured by Johnson Corp., trade name: Bioclusive) from above. Administration was performed once a day on days 0, 2, 4, and 7, with the defect creation day as day 0. The wound healing effect was measured using a caliper to measure the minor axis and major axis of the defect, and assuming the defect as an ellipse, the area of the defect (minor axis × major axis × 3.14 ÷ 4) was determined.
Evaluation was made by obtaining the ratio of the area of the defective part on the measurement day to the area of the defective part in the initial stage (day 0) (this is referred to as an area ratio).

As a control, the ointments of Comparative Examples 1 to 5 were used in place of the test ointment of Example 1, and the other points were the same as those described above.

This test was carried out using 5 mice each, and the area of the defective area was the average of the area of the defective areas obtained for these mice. Table 1 shows the area ratio on day 9 of the defect creation.

[0036]

[Table 1]

As can be seen from Table 1, in the group to which the test ointments of Examples 1 to 3 were administered, the area ratio on the 9th day of the defect creation was reduced to about half as compared with the ointment administration groups of Comparative Examples 2 to 5. In addition, the area ratio was smaller than that of the ointment administration group comprising the wound healing peptide and the gelled hydrocarbon of Comparative Example 1. Thus, it was confirmed that the wound treatment agent of the present invention exhibited a remarkable effect of promoting wound healing.

[0038]

According to the present invention, it is possible to provide a remedy for a wound which exhibits a remarkable wound healing effect.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI (C12P 21/04 C12R 1: 465) (72) Inventor Takashi Inagaki 2-1 Hyakuyama, Shimamoto-cho, Mishima-gun, Osaka Sekisui Chemical Co., Ltd. In company

Claims (4)

[Claims] 1. A compound represented by the following formula [I] A wound treatment agent comprising a peptide represented by the formula (I) and a peripheral circulation improving agent. 2. The method according to claim 1, wherein the content of the peptide represented by the formula [I] is 0.0001 to 10% by weight, and the content of vitamin E as a peripheral circulation improving agent is 0.01 to 99% by weight. Wound healing agent. (3) a content of the peptide represented by the formula (I) of 0.0001 to 10% by weight, and as a peripheral circulation improving agent, d-camphor, dl-camphor, a heparin-like substance,
Ikutamol, cantharis and at least one content selected from the group consisting of pepper tincture is 0.01
The agent for treating wounds according to claim 1, wherein the amount is from 40 to 40% by weight. 4. The content of the peptide represented by the formula [I] is 0.0001 to 10% by weight, and nicotinic acid, a pharmacologically acceptable ester of nicotinic acid, a pharmacologically acceptable ester as a peripheral circulation improving agent. The content of at least one selected from the group consisting of an acceptable amide of nicotinic acid and a pharmacologically acceptable salt of nicotinic acid is 0.01 to 40% by weight.
The wound treating agent according to claim 1, which is: