JP3148854B2 - New wound healing agent - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION The present invention relates to a wound healing agent comprising a novel peptide as effective components.

[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.

[0005] In view of the above, an object of the present invention is to provide a novel wound healing substance having high utility.

[0006]

SUMMARY OF THE INVENTION The present invention provides

Embedded image The above object is achieved by providing a wound healing substance (hereinafter, referred to as the wound healing substance of the present invention) comprising the novel peptide represented by the following (hereinafter, referred to as the peptide of the present invention) and a wound healing agent containing the peptide of the present invention as an active ingredient. To achieve.

[0007] The wound healing substance of the present invention is a strain producing the wound healing substance of the present invention belonging to the genus Streptomyces, for example, Streptomyces nobilis (Streptomyces nobilis).
ilis (hereinafter abbreviated as "S. nobilis")), and the obtained culture solution or a solid extract of the same solution or an extract extracted from the cultured cells with an organic solvent is subjected to various column chromatography. , By subjecting the column chromatography fraction containing the target substance to recrystallization treatment.

The actinomycetes S. producing the wound healing substance of the present invention.
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.

Actinomyces S. Cultivation of Nobilis is performed using a medium containing appropriate nutrients. In the case of liquid culture, the medium is preferably an aqueous solution containing one or more of sugars such as glucose, proteins such as peptone and malt extract, vitamins, nucleic acids, amino acids, and complex carbohydrates. Used. Typical culture media include starch and
Ammonium-based liquid medium (soluble starch, K ₂ HP
O ₄ and NH ₄ Cl). Liquid medium p
H is preferably 2 to 9, and the culture temperature is preferably 15 to 42 ° C. The preferred culture time of the liquid culture is 1 to 14 days. S. thus obtained. The peptide of the present invention is extracted from a culture solution of Nobilis or a dried product thereof or the cells itself using a solvent.

S. The culture solution of Nobilis or an extract of the dried product thereof or the cells itself may be treated with ammonium sulfate (ammonium sulfate), and the resulting precipitate may be subjected to solvent extraction. In the ammonium sulfate treatment, the ammonium sulfate added to the culture solution or the dried product thereof or the extract of the cells itself may be in a solid state or in a solution state. The amount of ammonium sulfate added is preferably in the range of 30 to 90% by weight of the saturation concentration. The processing time is not particularly limited, but is preferably in the range of 30 minutes to 5 hours. At the time of centrifugation for obtaining a precipitate, the centrifugal force is preferably 3,000 G to 20,000 G, and the centrifugation time is preferably 2 to 60 minutes.

The solvent used for the extraction is preferably an organic solvent. Representative examples thereof include esters such as ethyl acetate, alcohols such as methanol, ethanol and propanol, ethers such as ethyl ether and dioxane, acetone and methyl ethyl ketone. In addition to the ketones, dichloromethane, chloroform and the like,
The solvent that can be used is not limited to these. Further, a mixture of the above solvents can also be used. Particularly suitable solvents are ethyl acetate, dichloromethane, acetone and the like. The extraction time varies depending on the type of solvent, extraction temperature, etc.
Preferably it is in the range of 3 to 120 minutes. During the extraction, the liquid may be left standing or stirred. Preferably, the extraction operation is repeated a plurality of times for the same sample. The extraction temperature is not particularly limited.

Next, column chromatography for the solvent extract will be described.

As a packing material for column chromatography, ODS (octadecylsilanes such as octadecyldimethylchlorosilane) is preferable. The filling amount is not particularly limited, but it is preferable to fill the solvent extract in a weight ratio of 10 to 500 times the amount to be charged. When charging the solvent extract to the column, it is preferable to first adsorb it to the ODS. At this time, the amount of the ODS is not particularly limited. Preferably, the extract is adsorbed to the ODS with a weight ratio of 0.5 to 20 times the amount of the solvent extract to be adsorbed. And then charge the column. The elution solvent is not particularly limited, but preferably a solvent having a polarity between an aqueous solution containing a mixed solvent of methanol: acetonitrile = 1: 1 at 80% (v / v) and methanol is used.

Next, purification of the peptide of the present invention by recrystallization will be described.

The solvent used for recrystallization is not particularly limited as long as it dissolves the peptide of the present invention, but is preferably methanol, ethanol or the like. As a method of recrystallization, the column elution fraction containing the substance may be dissolved in a small amount of solvent under heating, and the resulting solution may be gradually cooled to recrystallize the substance, or may contain the substance. The column elution fraction to be dissolved may be dissolved in a solvent having high solubility of the substance, and a liquid having low solubility of the substance (for example, water) may be gradually added to recrystallize the substance.

In order to formulate the wound healing substance of the present invention, it is 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, which is generally used for preparing a drug according to a dosage form,
Flavoring agents, pH adjusters, solubilizers, suspending agents, buffers, stabilizers, preservatives, contaminants, surfactants, lubricants,
Excipients, antioxidants, dispersants, propellants, solubilizers, and solubilizers are exemplified. By selecting an appropriate solvent, the obtained peptide of the present invention can be used as it is as a liquid agent.

The unit dosage form of the wound healing agent formulated using the wound healing substance of the present invention may be, in addition to the above-mentioned liquid preparations, tablets, pills, drinking liquid preparations, limonase preparations, injections, powders, suspensions and the like. Preparations, emulsions, granules, extracts, fine granules,
Syrups, dips, decoctions, eye drops, troches, cataplasms, liniments, lotions, elixirs, eye ointments, plasters, capsules, suppositories, enemas, injections (solutions, suspensions, etc.) ), Patches, ointments, jellies, pastas, inhalants, creams, sprays, nasal drops, aerosols and the like.

The amount of the wound healing substance of the present invention to be contained in the wound healing agent is not particularly limited and may be appropriately selected from a wide range, but is preferably in the range of 10 ^-7 to 10% by weight in the wound healing agent. .

The wound-healing agent obtained from the wound-healing substance of the present invention 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 wound healing agent obtained from the wound healing substance of the present invention is appropriately selected depending on the purpose of use, symptoms and the like.
The range is about g / kg to 10 mg / kg. Of course, the above pharmaceutical composition may be administered once to four times a day.

[0021]

BEST MODE FOR CARRYING OUT THE INVENTION

Production Example of the Peptide of the Present Invention Example 1 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. This culture was centrifuged and the resulting supernatant 2
To 50 liters, 662 g of ammonium sulfate was added per liter of the supernatant, followed by stirring for 1 hour. Then, 20,000
Continuous centrifugation was performed at 0 G (flow rate: 360-500 liter / hour) to obtain about 1.2 kg of a precipitate.

To 1.2 kg of the precipitate obtained above, 3.6 liters of water was added, and the mixture was stirred at 80 ° C. for 30 minutes.
Next, an equal volume of ethyl acetate was added, and after shaking for 10 minutes, the whole was centrifuged at 5,000 G for 10 minutes. After separating the ethyl acetate phase, this operation was repeated several times for the aqueous phase to obtain a solvent extract.

Next, the solvent extract obtained as described above is
ODS (octadecyldimethylchlorosilane) carrier 25
g. Next, 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. This O
Purification was performed using a DS column under the following conditions. A) Methanol: acetonitrile: water = 7:
1.5 liters of 7: 6, b) methanol: acetonitrile: water = 1.2 liters of 8: 8: 4, c) 150 ml of methanol: acetonitrile: water = 9: 9: 2,
d) Methanol: acetonitrile: water = 19: 19: 2
Was flowed 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-80TM, 4.6 mm inner diameter
HPL using a 25.0 cm long Tosoh column
C (manufactured by Hitachi, Ltd., pump L-6000, L-6200, detector L-3000, column open 655A-52), at a detection wavelength of 210 nm, a column temperature of 40 ° C. and a flow rate of 1 ml / min, water was used as an eluent. : Acetonitrile: methanol = 6: 7: 7 (0 min) to 0: 1: 1 (3
(0 min). Of the above fractions, only those containing a peak having a retention time of 18 to 20 minutes were collected, made the same fraction (100 mg), and repeatedly recrystallized using methanol-water to obtain needle-like crystals (45 mg).

The structure of this substance was determined to be Formula [I] from various instrumental analysis data.

Example 2 Culture was performed in the same manner as in Example 1, and the obtained cells 215
2.15 liters of dichloromethane was added to g (wet weight), and the mixture was sonicated for 30 minutes, 8.6 liters 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
It was adsorbed on 28 g of ODS carrier. Then, ODS carrier 2
About 30 g of the above-mentioned extract-adsorbing carrier was charged onto the above-mentioned carrier in a 3.2 cm-diameter column packed with 75 g to prepare an ODS column. Purification was performed using the ODS column under the following conditions. 1.5 liters of a) methanol: acetonitrile: water = 7: 7: 6 as an elution solvent, b)
Methanol: acetonitrile: water = 8: 8: 4 = 1.2
Liter, c) methanol: acetonitrile: water = 9:
200 ml of 9: 2, d) methanol: acetonitrile: water = 1 liter of 19: 19: 2, e) 500 ml of methanol were flowed at a flow rate of 10.5 ml / min in this order. The fractionation is carried out every time the solvent composition is changed. In particular, the fraction eluted with methanol: acetonitrile: water = 19: 19: 2 is added little by little using a suitable fraction collector (2
(Every minute). For each eluted fraction, ODS-8
HPLC using a Tosoh column (0TM, inner diameter 4.6 mm x length 25.0 cm, pump L-6, manufactured by Hitachi, Ltd.)
000, L-6200, detector L-3000, column open 655A-52), detection wavelength 210n
m, column temperature 40 ° C., flow rate 1 ml / min, water: acetonitrile: methanol = 6: 7: 7 as eluent
The analysis was performed using (0 min) to 0: 1: 1 (30 min). Of the above fractions, only those containing a peak having a retention time of 18 to 20 minutes were collected and regarded as the same fraction (840).
mg) and methanol-water were repeatedly recrystallized to obtain 330 mg of the peptide of the present invention as needle crystals.

The structure of this substance was determined to be the formula [I] from various instrumental analysis data in the same manner as in Example 1.

Structural analysis data Instrument analysis data of the substances obtained in Examples 1 and 2 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 = 913.5079 (M + H-H2
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

[0030]

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

4. Compositional Formula From mass spectrometry and NMR data described below, it was found that the compositional formula of the novel peptide was C ₄₅ H ₇₀ N ₈ O ₁₃ .

5. NMR a) As a result of HMBC spectrum measurement of partial structure A, the carbonyl carbon of alanine (Ala) (173.6 ppm) and the CH3 of N-CH3-phenylalanine (Phe) (3.04 ppm)
Since the long-range correlation was observed, the partial structure of A was estimated.

[0034] b) partial structure B, ¹ H- ¹ H COSY for C, 2 with similar spin system from HOHAHA
Two components were estimated.

I) NH (4.38 ppm) CH ₂ (46.1, 2.89, 3.15 ppm) CH ₂ (21.3, 1.45, 1.63 ppm) CH ₂ (24.2, 1.93, 2.25 ppm) CH (51.7, 4.90 ppm) ii) NH (3.93 ppm) CH ₂ (47.9, 2.56, 3.30 ppm) CH ₂ (21.5, 1.55, 1.66 ppm) CH ₂ (24.4, 1.67, 2.58 ppm) CH (52.5, 5.18 ppm)

Since both ¹³ C shifts are in good agreement, it is considered that they have the same structure (residue). In addition, CH ₂
Since the protons are non-equivalent, it can be estimated to be a cyclic structure.

This shift is due to Monamycin.
This component was assumed to be a Pip (the left part of the partial structure B and the partial structure C) because it matches with that of Piperazic acid (Pip) contained in.

Α β γ δ Pip ¹⁾ 49.8 24.1 21.0 47.0 (CDCl ₃ solution) Actual value 52-53 24-25 21 46-48 (CDCl ₃ solution)

6.40 ppm of serine (Ser)
A long range was observed between the NH of the Pip and the carbonyl of 168.9 ppm of the Pip, whereby the partial structure B was deduced.

1) CHHassall, WAThomas, MCMosch
idis, J. Chem. Soc., Perkin Trans. I, 1977, 2371 (1977)

[0041] c) the partial structure D ¹ H- ¹ H spin coupling (CH _{3) 2} -CH-CH-
CH-NH- made moiety is introduced, a long-range ¹
This partial structure was confirmed by the H- ¹³ C bond, and 1
From the spin bond between 70.6 ppm of carbonyl and 4.88 ppm of CH, the structure of β-OH-leucine (Leu) was determined, and from 5.46 ppm of CH to β-OH-Le.
It was presumed that u was ester-linked at the β-position.

¹ H- ¹ H spin coupling, long range ¹
H- ^13C bonding and NOE (Nuclear Overhauser Effect)
As a result, the partial structure of the side chain (left) of the partial structure D was found.

From the long-range ¹ H- ¹³ C bond, β-
4.88 ppm CH and 8.34 p of OH-Leu
A long-range correlation was observed between pm of NH and 177 ppm of carbonyl in the side chain, and partial structure D was estimated.

D) Regarding the chemical structure of the novel peptide From the long-range ¹ H- ¹³ C bond, Ser.
5.46p of 2ppm carbonyl and β-OH-Leu
The long-range correlation was observed with CH of pm, so that the partial structures B and D were Ser and β-OH-Le.
It was found that an ester bond was formed between u. Also,
5.46 ppm CH of β-OH-Leu and 4.38 ppm NH of Pip (partial structure C), β-OH-Leu
8.34 ppm of NH and Pip (partial structure C)
NOE with 89 ppm CH ₂ and 4.90 ppm CH
Was observed, the partial structure C was changed to β-OH-Leu
Was found to be bonded to 170.6 ppm of a carbonyl group. The bonding method of the remaining partial structure A was one, and the chemical structure of the novel peptide was determined.

6.40 ppm of Ser, NH and N
N is also present between 3.04 ppm CH ₃ of —CH ₃ —Phe.
Observation of OE suggests that the structure is correct.

E) The NMR measurement results are as follows.

Table 1 and Table 2¹³C shift and carbon type
And join directly¹H, and Tables 3 to 5¹H shift
To spin-couple¹H,¹³C and long range
Join ¹³C and Tables 6 and 7 show the NOE measurement results.
And Tables 8 to 11¹³C and¹The H assignment is shown.

[0048]

[Table 1]

[0049]

[Table 2]

[0050]

[Table 3]

[0051]

[Table 4]

[0052]

[Table 5]

[0053]

[Table 6]

[0054]

[Table 7]

[0055]

[Table 8]

[0056]

[Table 9]

[0057]

[Table 10]

[0058]

[Table 11]

[0059]

Embedded image

[0060]

Embedded image

[0061]

Embedded image

[0062]

Embedded image

[0063]

Embedded image

[0064]

Embedded image

Preparation Example of Preparation Preparation Example 1 (Ointment) 1 mg of the peptide of the present invention 1 g of plastibase (manufactured by Taisho Pharmaceutical Co., Ltd.) 1 g 1 mg of the peptide of the present invention is finely ground using a mortar, and 1 g of plastibase is added. And mixed well to prepare an ointment.

Formulation Example 2 (Ointment) Peptide of the present invention 0.2 mg Methanol 12.5 μl Plastibase (Taisho Pharmaceutical Co., Ltd.) 1 g Methanol containing the peptide of the present invention obtained after dissolving 0.8 mg of the peptide of the present invention in 50 μl of methanol Was mixed well with 1 g of Plastibase in a mortar to prepare an ointment.

Formulation Example 3 (Solution) 0.5 mg of the peptide of the present invention 0.1 ml of DMSO 0.1 ml 5 wt% aqueous solution of gum arabic 1.9 ml 0.1 ml of DMSO was added to 0.5 mg of the peptide of the present invention to dissolve the former. 0.1 ml of the obtained DMSO solution containing the peptide of the present invention was added little by little to 1.9 ml of a 5% by weight gum arabic solution while stirring to prepare a uniformly suspended solution.

Formulation Example 4 (Liquid) 0.5 mg of the peptide of the present invention in 2 ml of physiological saline A small amount of physiological saline was added to 0.5 mg of the peptide of the present invention to form a uniform suspension using ultrasonic waves. A solution was prepared by adding saline to make the solution volume 2 ml.

Formulation Example 5 (Liquid) 25 mg of the peptide of the present invention was dissolved in 2.5 ml of ethanol after dissolving 25 mg of the peptide of the present invention in 2.5 ml of ethanol. 1 g of oil 60 was added. The obtained solution was added to 96.5 ml of physiological saline while stirring little by little to prepare a liquid preparation.

Formulation Example 6 (Solution) 10 mg of the peptide of the present invention, macrogol 400, 50 ml of physiological saline An appropriate amount of 10 mg of the peptide of the present invention was dissolved in 50 ml of macrogol, and physiological saline was added thereto to make 100 ml to prepare a liquid.

Pharmacological Test Test Example 1 Granulation Proliferation Effect The peptide of the present invention was prepared at a final concentration of 5 μg / ml,
50 μg / ml, 500 μg / ml and 5000 μg
/ Ml in methanol. 20 μl of each concentration of methanol solution (0.1 μg / peptide of the present invention)
The disc (-100 μg / disc) was impregnated twice into a paper disc (filter paper for antibiotics test manufactured by Advantech) having a diameter of 8 mm, and then methanol was removed by air drying.

As test animals, male Wistar rats weighing 120 to 200 g were used.

The rats were shaved under ether anesthesia and approximately 3 cm incisions were made with a scalpel along the midline of the back. The above-mentioned paper disc was implanted subcutaneously from the created cut portion, and the cut portion was sutured. One week later, the rats were euthanized under ether anesthesia, the paper disks were removed from the subcutaneous tissues, and the wet weight of the formed granulation was measured. As a control, methanol 2 containing no peptide of the present invention was used.
0 μl was soaked in a paper disk and then air-dried.

As a control drug, 25 μl of solcoseryl injection (manufactured by Taiho Pharmaceutical Co., Ltd.) (1 mg / solicoseryl / disc) impregnated in a paper disc was used.
Otherwise, the procedure was the same as above. The control used was a paper disc impregnated with 25 μl of a physiological saline solution.

This test was carried out using 5 rats each, and the wet weight of granulation was the average of the values obtained for these rats.

FIG. 1 shows the test results.

As can be seen from FIG.
In the group in which the disk containing g / disk or more was embedded, the wet weight of granulation was clearly increased as compared with the control group, and the wet weight of granulation was significantly increased as compared with the solcoseryl-administered group. Therefore, it is recognized that the peptide of the present invention has a cell proliferating action, and the action is more than 1 mg / disc of the peptide of the present invention over 1 mg / disk of solcoseryl.

Test Example 2 Effect on rat cut wound model The peptide of the present invention was used at final concentrations of 5 μg / ml and 20 μg / ml.
The suspension was suspended in a physiological saline solution to a concentration of 50 μg / ml and a test solution of the peptide of the present invention.

[0079] Wistar male rats weighing 250-300 g were used as test animals.

The rats were shaved under ether anesthesia, and a 3 cm incision was made with a scalpel in a direction perpendicular to the midline of the back.
The incised portion is sutured at three locations at equal intervals, and after 3 hours, 0.5 ml of the test solution (2.5 μg of the peptide of the present invention /
0 μg / wound and 25 μg / wound) were administered subcutaneously around the incision. A test solution prepared at the time of use for 3 days from the day after the cut wound.
5 ml (2.5 μg / wound, 10 μg / wound and 25 μg / wound) of the peptide of the present invention was subcutaneously administered once a day to the cut wound. Four days after the incision, the rat was euthanized under ether anesthesia, and after removing the thread, a strip-shaped skin section containing 2 cm of the central part of the incision was cut off. After removing the connective tissue of the skin section, the wound resistance (g / cm) of the cut was measured using a rheometer.

As a control, a physiological saline solution was used.

As a control drug, 0.5 ml of solcoseryl injection (solcoseryl 20 mg / creative) (manufactured by Taiho Pharmaceutical Co., Ltd.) was used.

This test was carried out using 15 rats each, and the wound resistance (g / cm) was the average of the values obtained for these rats.

FIG. 2 shows the test results.

As can be seen from FIG. 2, in the group to which the peptide of the present invention was administered, the wound tension was clearly increased as compared with the control group, and the wound tension was further increased as compared with the control drug, solcoseryl injection. . That is, it was confirmed that the peptide of the present invention exhibited a stronger wound healing promoting effect than solcoseryl injection.

Test Example 3 Action on Rat Skin Defect Model The peptide of the present invention and an ointment base (Plastibase manufactured by Taisho Pharmaceutical Co., Ltd.) were mixed to prepare an ointment containing 0.02% of the peptide of the present invention.

As test animals, male Wistar rats weighing 250 to 300 g were used.

Rats to which dexamethasone (1 mg / kg) was intraperitoneally administered for 2 days from the day before creation of the defect for 3 days were shaved under ether anesthesia, laid down and pulled, and punched (with an inner diameter of 16 mm).
mm) was used to create a skin defect at the midline on the back and a control site. Sterile filter paper (25 × 25 m) coated with 50 mg of ointment prepared at the time of use (containing 10 μg of the peptide of the present invention)
m) was affixed per skin defect site, covered with tegaderm (3M, dressing material) from above, and further covered and fixed with an adhesive stretchable bandage. Ointment was administered daily until healed once a day. The wound healing effect was determined by measuring the minor axis and major axis of the defect using calipers, determining the area of the defect (minor axis × major axis × 3.14 ÷ 4) by regarding the defect as an ellipse, and calculating the initial (0 Day) 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 (this is called an area ratio).

As a control, an ointment base (Plastibase manufactured by Taisho Pharmaceutical Co., Ltd.) was used, and the other points were the same as those described above.

As a control, Orsenone Ointment 5 was used.
0 mg (tocoretinate 125 μg / defect wound) (manufactured by Nippon Redary Co., Ltd.) was used.

This test was carried out using 7 rats each, and the area of the defect was determined by a total of 14 rats.
The average value of the areas of the missing parts at the three locations was taken. The change in the area ratio over time and the number of healing days in this test are shown in FIGS.
Shown in

[0092]

[Table 12]

As can be seen from FIGS. 3, 4 and Table 12,
In the group to which the peptide of the present invention was administered, a significant effect of reducing the area of the defective portion and the average number of healing days were significantly reduced as compared with the control group. Further, in the group to which the peptide of the present invention was administered, a remarkable effect of reducing the area of the defective part and a reduction in the average number of healing days were observed as compared with the control drug orsenone ointment. Thus, it was confirmed that the peptide of the present invention exhibited a remarkable effect of promoting wound healing.

Test Example 4 Action on Diabetic Mouse Skin Defect Model The peptide of the present invention was suspended in physiological saline to a final concentration of 50 μg / ml to prepare a test solution of the peptide of the present invention.

As test animals, female diabetic mice (db / db) weighing 30 to 45 g were used.

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. 20 μl of the test solution (1 μg of the peptide of the present invention) was applied to the wound surface (2c
m ² ), and covered with a polyurethane film material (manufactured by Johnson Corp., trade name: Bio-Cuisive) from above. Administration was performed once a day on days 0, 2, and 4, with the defect creation date as day 0. The wound healing effect was determined by measuring the minor axis and major axis of the defect using calipers, determining the area of the defect (minor axis × major axis × 3.14 ÷ 4) by regarding the defect as an ellipse, and calculating the initial (0 Day) 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 (this is called an area ratio).

As a control, only physiological saline was used, and the other points were the same as those described above.

As a control drug, basic fibroblast growth factor (b-FGF) (progen) was added at 10 μm.
g / ml was used by dissolving it in a physiological saline solution, and the other points were the same as those described above.

This test was carried out using five mice each, and the area of the defect was determined by taking the average value of the area of the defect obtained for these mice. FIG. 5 shows the time-dependent changes in the area ratio of this test, and Table 13 shows the area ratio on the 14th day of defect creation.

[0100]

[Table 13]

As can be seen from FIG. 5 and Table 13, in the group to which the peptide of the present invention was administered, the area ratio on the 14th day after the creation of the defect was reduced to about half as compared with the control group. Also,
In the group to which the peptide of the present invention was administered, the control drug b-FGF
The effect of reducing the area of the defective part was almost the same as that shown in FIG. Thus, it was confirmed that the peptide of the present invention exhibited a remarkable effect of promoting wound healing.

Test Example 5 Toxicity test The toxicity of the peptide of the present invention to rats was examined by the following method. First, the peptide of the present invention was suspended in a solution obtained by adding 5% by weight of dimethyl sulfoxide to a 5% by weight aqueous solution of gum arabic to prepare a test solution. Weight of test animal is 1
20-200 g Wistar male rats were used.

The above test solution was subcutaneously administered to rats to confirm the state of death. As a result, there was no death even with the administration of 20 mg / kg.

[0104]

According to the present invention, it is possible to provide a novel wound healing substance exhibiting a remarkable wound healing effect and a novel wound healing agent containing the same.

[Brief description of the drawings]

FIG. 1 is a graph showing the relationship between the concentration of the peptide of the present invention, a control and a control drug and the wet weight of granulation.

FIG. 2 is a graph showing the relationship between the concentration of the peptide of the present invention, a control drug, and a control drug and wound tension resistance.

FIG. 3 is a graph showing the relationship between the number of days from the date of defect creation and the area ratio for the peptide-containing ointment of the present invention and the control ointment.

FIG. 4 is a graph showing the relationship between the number of days from the defect creation date and the area ratio for the peptide-containing ointment of the present invention and the control ointment.

FIG. 5 is a graph showing the relationship between the number of days from the date of deletion and the area ratio for the peptide-containing agent of the present invention and the control.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification code FI C12R 1: 465) (72) Inventor Koji Inagaki 2-1 Hyakuyama, Shimamotocho, Mishima-gun, Osaka Sekisui Chemical Co., Ltd. (56 References JP-A-5-25053 (JP, A) JP-A-6-172194 (JP, A) JP-A-6-247863 (JP, A) JP-A-6-305975 (JP, A) 6-305974 (JP, A) Japanese Journal of Pharmacology (1994) Vol. 64, SUPPL 1, p. 95P, O-49 Japanese Journal of Pharmacology (1995) Vol. 67, SUPPL 1, p. 282P, P3-143 (58) Fields investigated (Int. Cl. ⁷ , DB name) A61K 38/00 A61P 43/00 107 C12P 21/00-21/02 CA (STN) REGISTRY (STN) BIOSIS (DIALOG) WPI (DIALOG)

Claims (1)

(57) [Claims] (1) A wound healing agent comprising a peptide represented by the formula (1) as an active ingredient .