JPH0529333B2 - - Google Patents

Description

[Detailed description of the invention]

INDUSTRIAL APPLICATION FIELD The present invention relates to a wound treatment agent, and more particularly to a wound treatment agent characterized by containing ovomacroglobulin as an active ingredient. BACKGROUND TECHNOLOGY In the treatment of wounds, such as general trauma, anal fistulas, pressure ulcers, etc., and wounds resulting from major surgery that reach deep into the body, it is necessary to promote granulation formation in the injured area during the treatment process and to treat the epidermis. The promotion of formation is an important issue. Currently, compounds available for wound treatment include:
Retinoic acid, allantoin, asiaticoside, a plant component of the Umbelliferae family, and zinc are known. However, none of these drugs is capable of sufficiently promoting the above-mentioned granulation formation, etc. In addition, it is known that the use of steroidal and non-steroidal anti-inflammatory drugs during burns, etc., actually reduces resistance, and when administering these drugs, drugs that can further activate or strengthen resistance are used. It is necessary to use it in combination with Furthermore, it has recently been isolated from mammalian body fluids and has a molecular weight of about 5300 as an effective drug for wound treatment.
Polypeptide consisting of 52 amino acids
-38716) and compositions containing essential amino acids (Japanese Patent Application Laid-open No. 1980-80316).
In addition, eggs containing iodine at a high concentration of 300 μg or more are effective in promoting wound healing and preventing muscle diseases (Japanese Patent Application Laid-Open No. 116225/1983), and dried egg whites are blended as a base for skin cosmetics. Skin cosmetics (Special Publication No. 61-6801) have also been proposed;
Even these methods are still not sufficiently effective in treating wounds. Moreover, whole eggs such as egg whites are perishable even in the presence of preservatives, and preparations containing them have problems with storage stability, and egg whites do not dissolve unless alkaline, making them difficult to use as wound treatment agents. It also has drawbacks such as low affinity with the constituent raw materials of ointments, which are common formulations, and the formation of white precipitates containing proteins. Problems to be Solved by the Invention The purpose of the present invention is to eliminate the drawbacks of conventional drugs known to be used for this type of wound treatment, and to improve the effectiveness of epithelial cells and fibroblasts in the wound treatment stage. The object of the present invention is to provide a new wound treatment agent that has a proliferation promoting effect and exhibits an excellent wound treatment promoting effect. As a result of extensive research aimed at achieving the above objectives, the present inventors have discovered that ovomacroglobulin has an excellent wound healing promoting effect that meets the above objectives, and has now completed the present invention. Means for Solving the Problems That is, the present invention relates to a wound treatment agent characterized by containing ovomacroglobulin as an active ingredient. The wound treatment agent of the present invention includes, for example, abrasions, cuts, burns, scalds, frostbite, skin ulcers, dry skin, skin keratosis, cracks, scabs, dermatitis, athlete's foot sores, surgical wounds, and corneal wounds. In addition to the above, therapeutic agents for various wounds including anal fistulas, bedsores, etc., and agents for treating inflammation such as acne and sunburn are also included. Furthermore, the wound treatment agent of the present invention has excellent storage stability and safety. The drug of the present invention must contain ovomacroglobulin as its active ingredient. Here, ovomacroglobulin is known as a high molecular weight glycoprotein found in egg white, and its preparation method is already known [Feeney, RE].
et al., Comparative Biochemistry and Physiol.
54A, 281 (1976), Ikai et al., Journal of
Biochemistry (J.Biochem.), 92, 1679~
1682 (1982), 93, 121-127 (1983), and Nagase et al., Journal of Biological Chemistry (JBC), vol. 258, No. 12, 7481-7489
(1983) etc.]. Egg white as a raw material for preparing the above-mentioned ovomacroglobulin is not particularly limited, and any egg white from various animals can be used. In general, readily available egg whites from chicken, duck, quail, turkey, etc. are preferred. There are no particular restrictions on the method for preparing ovo macroglobulin from egg white, and various operations that utilize the physicochemical properties of ovo macroglobulin, such as protein precipitating agents, can be performed according to various methods commonly used for separating protein components. Treatment, molecular sieve chromatography (gel filtration), ion exchange chromatography, centrifugation, electrophoresis, dialysis, etc. can be performed alone or in combination. For example, a method can be exemplified in which egg white is mixed with a water-soluble solvent such as Tris-HCl buffer or polyethylene glycol is added thereto to remove insoluble proteins such as ovomucin, and then subjected to gel filtration. Ovomacroglobulin can be obtained as a glycoprotein of about 600,000 to 800,000. The wound treatment agent of the present invention can be put to practical use in the form of a general pharmaceutical preparation containing the obomacroglobulin obtained as described above as an active ingredient. As the pharmaceutical preparation form, various forms can be appropriately selected depending on the intended use of the obtained preparation. Examples include external preparations such as liquid liniments, lotions, aerosols, liniments, ointments, and poultices, as well as suppositories and injections. In preparing these various forms, various commonly used diluents, excipients, etc. can be used as appropriate. For example, when preparing an ointment for external use, conventional hydrophobic or hydrophilic base materials such as fat, fatty oil, lanolin, petrolatum, paraffin, wax, glycols, higher alcohols, glycerin, water, etc. are used. Can be used. Furthermore, various additives known to be commonly added, such as stabilizers, fragrances, colorants, etc., can also be added to the above-mentioned external preparations, if necessary. The amount of the active ingredient, that is, ovomacroglobulin, to be contained in the drug of the present invention is not particularly limited and is appropriately selected from a wide range.
It is blended in a range of 0.0001 to 30% by weight. The amount and method of application of the therapeutic agent of the present invention can be determined depending on the form of the preparation, the amount of active ingredient in the preparation, the age, sex and other conditions of the patient to whom it is applied, the degree of the wound, etc. For example, the wound treatment agent of the present invention in the form of an external preparation can be sprayed on the affected area once or multiple times a day in an amount sufficient to cover the entire affected area,
It can be applied by coating or the like. The same applies to other formulations. The thus obtained wound treatment agent of the present invention exhibits an excellent effect of promoting wound healing, as described above, and can exhibit outstanding effects in treating various wounds. EXAMPLES In order to explain the present invention in more detail, examples of preparation of ovomacroglobulin as an active ingredient, test examples of its effects, and various formulation examples of preparations of the present invention containing the same will be given below. Example 1 Preparation of ovomacroglobulin 20 kg of egg white was mixed with 10 kg of egg white containing an equal amount of 1% NaCl.
Suspended in mM Tris-HCl buffer (PH7.7),
This is added to polyethylene glycol (molecular weight = 8500,
(Manufactured by Tokyo Kasei Co., Ltd.) was added to a concentration of 2.5%,
The supernatant was collected by continuous centrifugation (10,000 rpm). The same polyethylene glycol as above was further added to the obtained supernatant until the concentration reached 10%, and the mixture was again subjected to continuous centrifugation (10,000 rpm) to collect the precipitate. This was dissolved in the above buffer and further centrifuged (10,000 rpm, 10 minutes) to collect the supernatant, which was applied to a column (252 x 900 mm) of Sepharose CL-6B (manufactured by Pharmacia). With buffer, flow rate
It was eluted at a rate of 3.6L/hour. For each elution section, trypsin inhibitory activity using casein as a substrate was measured using the method of Kitamoto et al. [T.
Kitamoto, M. Nakashima, and A. Ikai, J.
Biochem., 92 , 1679-1682 (1982)] and trypsin inhibitory activity fractions were collected. Next, the obtained active fraction was passed through a molecular sieve membrane.
While concentrating using a Pellicon cassette (manufactured by Millipore) equipped with a 100,000 membrane, the buffer was exchanged with 5 mM Tris-HCl buffer (PH7.7). The obtained sample was diluted with 10mM NaCl.
Equilibrated with mM Tris-HCl buffer (PH7.7)
It was applied to a DEAE Trisacryl M (Trisacryl M, manufactured by LKB) column (size 50 x 800 mm). After thoroughly washing the column with the same buffer, the column was washed with 675 ml of 10 mM Tris-HCl buffer (PH 7.7) containing 50 mM NaCl and 675 ml of 10 mM Tris-HCl buffer (PH 7.7) containing 150 mM NaCl for 2.5 hours each. Elution was performed over 5 hours. Under these conditions, the trypsin inhibitory activity fraction was eluted between 70 mM and 120 mM of sodium chloride concentration. The trypsin inhibitory activity fractions were collected and dialyzed against 1 mM phosphate buffer (PH7.4). After thorough dialysis, the dialyzed fluid was freeze-dried using a freeze dryer (manufactured by Labokone). As a result of the above, 5.9 to 7.1 g of a single purified ovomacroglobulin sample was obtained. For purified samples, with 4N methanesulfonic acid,
After hydrolysis at 110°C for 24 hours (in a vacuum sealed tube), it was analyzed using an amino acid analyzer (Model 835-50, Hitachi High Speed Amino Acid Analyzer, manufactured by Hitachi, Ltd.). The results are shown in Table 1 below.

[Table] Test Example 1 Test for Suppression of Vascular Permeability Due to Burns In this test, after causing a burn on the skin of a rat with an electric iron, a dye (Evans Blue) was administered to the rat, and the body fluid that oozed out from the burn area was measured. The wound treatment effect of the wound treatment agent of the present invention was investigated using the vascular permeability of the test animal as an index by quantifying the amount of dye leaked in the sample. The test was conducted using the following method. i.e. weight
Male Wistar rats weighing 200-250 g were divided into two groups of 5 rats each. After shaving the hair of the target area along the midline of the back of each group of rats with clippers, an electric iron (temperature scale: 100-110°C) was pressed in a circular shape with a diameter of 1 cm on one side for 20 seconds. I applied it to create a burn area. The other part was left as a non-burn area. Each rat in Group 1 received the above burns immediately after creation and 24 days later.
After an hour, 0.2 ml of physiological saline was intradermally injected into each of the burnt and non-burned areas to form a physiological saline administration group (control group). Each rat in the other group received a solution of ovomacroglobulin at a concentration of 10 mg/ml.
0.2 ml was similarly administered intradermally to form an ovomacroglobulin administration group (experimental group). 23.5 hours after each dose above, each group of rats received 0.5
% Evans blue dye solution 5 ml/Kg was administered intravenously. After 30 minutes, that is, 48 hours after creating the burn, each rat was exsanguinated to death, the skin of the burnt and non-burn areas was peeled off, and the attached fat was removed.
Cut into 1cm ^square pieces. This 1 cm ² square piece of skin was immersed in 1 ml of 1N KOH solution and left at 37° C. for 20 hours to dissolve. then
9 ml of a 0.6N-H ₃ PO ₄ :acetone (5:13) mixture was added, stirred with a mixer, centrifuged at 3000 rpm for 25 minutes, and the supernatant was collected. The absorbance of the obtained supernatant was measured using a spectrophotometer at a wavelength of 620 nm. From the measured absorbance, the amount of μg of Evans Blue leaked was determined using a standard curve prepared in advance. This value was expressed as the mean value ± standard error. The results are shown in Table 2 below.

[Table] From Table 2 above, (burn area) - (non-burn area)
The leakage value of Evans Blue was 25.0 ± 5.32 in the control group (physiological saline administration group), while it was 11.8 ± 3.55 in the experimental group (ovomacroglobulin administration group), It can be seen that the administration significantly suppresses vascular permeability. Test Example 2 Skin Inflammation Recovery Test Using Hair Removal Cream A total of 20 male BALB/c mice weighing 25 to 30 g were used in this test. The hair on the back of the test mouse was neatly trimmed with clippers, and 0.5 g of hair removal cream (Mavui Hair Remover, manufactured by Kanebo Co., Ltd.) was evenly applied to a 2.0 x 2.5 cm area. After leaving it for 30 minutes, the applied cream was wiped off with warm water to create a skin inflammation model. The above model mice were divided into 5 groups of 4 mice per group, and the mice in 4 groups were given the following ointment sample a total of 4 times each day on the day of skin inflammation creation, the next day, the 3rd day, and the 6th day. Experimental groups 1 to 4 were prepared by applying 0.3 g each. Experimental group 1: Application of Japanese Pharmacopoeia hydrophilic ointment (manufactured by Yoshida Pharmaceutical Co., Ltd.) Experimental group 2: Ovomacroglobulin 0.01 in the above ointment
Experimental group 3: Ovomacroglobulin added to the above ointment.
Application of ointment containing 0.005% Experimental group 4: Ovomacroglobulin added to the above ointment
Application of ointment containing 0.001% The remaining group served as a control group without any treatment. After creating each of the above experimental groups (applying ointment) (including the control group), the condition of the skin of the mice in each group was observed macroscopically and histologically every day. Histological observation was performed under a microscope after hematoxylin-eosin staining (HE staining). As a result, in the control group treated with hair removal cream on the first day, inflammation occurred in the epidermis and dermis, and inflammatory cells such as histiocytes and neutrophils gathered, and strong inflammation was observed in the epidermis and subsequent dermis. , materials that were strongly stained by eosin were widespread. Also, on the fourth day,
The histological images of the epidermis and subsequent dermis were replaced by images of inflammation, and the inflammation had spread to the entire hair follicle area. No difference was observed between experimental group 1 (hydrophilic ointment application group) and the control group. On the other hand, in experimental groups 2 to 4 (ovomacroglobulin-containing ointment application group), inflammation in the epidermis, dermis, and hair follicles was well suppressed compared to the control group and experimental group 1, and the dermal tissue The image was also found to be recovering well. In addition, in the group containing a low amount of ovomacroglobulin (experimental group 4), the above effect seemed to be somewhat weak. Furthermore, the results of histological observation on the 8th day of treatment with the hair removal cream showed that in experimental groups 2 to 4 of the present invention, the repair of the inflammatory surface was progressing. Test Example 3 Burn Recovery Test Five groups of BALB/c male mice each weighing 25 to 30 g were used in this test. The hair on the back of the test mouse was neatly trimmed with clippers, and a hair removal cream was applied and left for 5 minutes (to the extent that it did not cause inflammation), and then the applied cream was removed with warm water. Then 350-400℃ over a range of 1.5× ^2.5cm2
A burn was caused by applying an electric iron (T-27, paraffin cutting melting iron, manufactured by Takashima Shoten) for about 5 seconds. Starting from the day after the burn challenge, 0.2 g of each of the following test ointments was applied to the burn area every 3 days, and the test animals were observed for weight fluctuations, damage areas, and histological observations. Experimental group 1: Application of Japanese Pharmacopoeia hydrophilic ointment (manufactured by Yoshida Pharmaceutical Co., Ltd.) Experimental group 2: Ovomacroglobulin 0.01 in the above ointment
Experimental group 3: Ovomacroglobulin added to the above ointment.
Application of ointment containing 0.005% Experimental group 4: Ovomacroglobulin added to the above ointment
Application of ointment containing 0.001% The remaining group served as a control group without any treatment. The results of measuring changes in body weight in the above test are shown in FIG. The figure shows the weight change ratio of the test animals in each group for each day up to day 4, with the day when the electric iron burn was inflicted as "day 0" (the average weight before the burn injury is set as "1"), and the weight change relative to this is shown. The horizontal axis shows the number of days (days) and the vertical axis shows the weight change ratio. In addition, 1 in the figure indicates experimental group 4 (group applied with ointment containing 0.001% ovomacroglobulin;
2 represents experimental group 1 (group to which hydrophilic ointment without addition of ovomacroglobulin was applied) and 3 represents control group. The above body weight change well reflects the degree of burn injury. That is, after a burn injury, edema occurs due to increased vascular permeability due to local inflammation and a state of insensitivity and inability to excrete.Therefore, the edema increases depending on the size of the burn area, leading to an increase in body weight. From FIG. 1 showing the above body weight change results, in the ovomacroglobulin application group of the present invention (experimental group 4),
It is clearly seen that no weight gain was observed because edema was suppressed compared to the control group without application and the group to which hydrophilic ointment without ovomacroglobulin was applied (experimental group 1). From the macroscopic observation results in the above test, injury 6
Days later, compared to the control group and experimental group 1, very good recovery was observed in all experimental groups 2 to 4 of the present invention. Furthermore, histological observation using HE staining in the above test was carried out in the same manner as in Test Example 2. As a result, 4 days after the burn injury, in the control group and experimental group 1, the epidermis and dermis were all replaced by eosin-stained substances characteristic of burns, and the hair follicles remained in form. On the other hand, in the group to which the wound treatment agent of the present invention was applied (for example, experimental group 3), tissue repair progressed from the part of the dermis that was in contact with the subcutaneous tissue, and dermis-like tissue began to form, and hair root formation was progressing smoothly. It was confirmed that progress was made. From the above results, when the wound treatment agent of the present invention is applied, the effect of preventing edema after burn injury and the effect of restoring the injured area are confirmed macroscopically, and the effect of restoring skin and body hair (tissue formation) is also confirmed histologically. (promoting effect) was clearly observed. Test example 4 Skin abrasion wound recovery test A 25mm x 25mm incision was made on the back of a rabbit using a silver knife.
Five split-thickness skin defects with a depth of 1.2 mm are created.
At this time, in order to eliminate the influence of each wound healing mechanism, each defect wound is created at least 30 mm apart. Apply each test drug (ointment) below once a day to each defect wound created above, and after application, cover with gauze (25 mm x 25 mm size) to avoid contamination with the test drug. It is closed with a breathable nylon film (``Tegaderm'', manufactured by 3M), and then the entire area is closed with an elastic bandage. This treatment was continued throughout the observation period. <Test drugs> Experimental area 1: Hydrophilic petrolatum ointment (hydrophilic petrolatum base only) White beeswax 8g Stearyl alcohol 3g Cholesterol 3g White petrolatum 86g Propylparaben 0.0625g Experimental area 2: Hydrophilic petrolatum ointment containing 1% ovomacroglobulin 5% ovo Macroglobulin aqueous solution (containing 40mg methylparaben) 20g Hydrophilic petrolatum base 80g Experimental area 3: Hydrophilic petrolatum ointment containing 0.1% ovomacroglobulin 0.5% Ovomacroglobulin aqueous solution (containing 40mg methylparaben) 20g Hydrophilic petrolatum base 80g Experimental area 4: 0.01% Ovomacroglobulin-containing hydrophilic petrolatum ointment 0.05% ovomacroglobulin aqueous solution (containing 40mg methylparaben) 20g Hydrophilic petrolatum base 80g Wound healing was evaluated by the following items immediately after wound creation and on the 7th, 14th, and 21st days. was visually observed. (b) Epithelialization of the wound The healing process of the wound at each stage was photographed and observed and evaluated. (b) Histological observation The wound surface was biopsied at each stage, and the amount and morphology of collagen fibers in the granulation tissue and the formed epithelium were observed using HE-stained specimens. (c) Presence or absence of side effects The presence or absence of irritation to normal tissue (redness) and development of dermatitis was observed. In Experimental Groups 2 to 4, in which ovomacroglobulin-containing ointment was used for the above experimental skin abrasion wounds, the size of the wound on the 7th day of application was the same as that in the hydrophilic ointment application group (Experimental Group 1). It was slightly smaller in comparison, and histological findings also showed the formation of granulation tissue. Furthermore, on the 14th day after wound creation, the wound healing effect of ovomacroglobulin further progressed, and the size of the wound was clearly reduced compared to the hydrophilic ointment application group (experimental group 1). In the observation on the 14th day, the above-mentioned difference became clear histologically, and significant differences were observed in the formation of granulation tissue, the amount of collagen fibers, the proliferation of epithelial tissue, etc. On the 21st day of wound creation, a clear reduction in the wound healing period was observed in Experimental Groups 2 to 4 compared to Experimental Group 1. This was also evident histologically, with promotion of granulation formation, increase in the amount of collagen fibers, and promotion of epithelial cell proliferation observed. Each of the above effects is particularly
Group using hydrophilic petrolatum ointment containing 0.1% ovomacroglobulin (experimental group 3) and group using hydrophilic petrolatum ointment containing 0.01% ovomacroglobulin (experimental group 4)
It was noticeable in In addition, when each test drug used in Experimental Groups 2 to 4 above was applied to normal skin and the side effects of ovomacroglobulin were observed, no redness was observed on the normal skin, and no onset of dermatitis was observed. I couldn't see it at all. Test Example 5 Stretching test on corneal epithelial cells Domestic rabbits weighing 2.5 to 3.0 kg were used in this test. After the test rabbit was anesthetized in advance by intravenous administration of 30 mg/Kg of pentobarbital (manufactured by Pittman-Moore), the cornea was removed and a strip-shaped corneal piece measuring 2 x 4 mm was prepared. Add ovo macroglobulin to TC-199 culture solution.
The corneal pieces obtained above were dissolved in each culture solution at concentrations of 0.05 μg/ml, 0.5 μg/ml, and 5 μg/ml.
Cultured for hours. After culturing, the tissue pieces were soaked in 5% glacial acetic acid-95.
% ethanol and embedded in paraffin.
4μ sections were prepared. After staining with HE, this was observed under a microscope and the length of the stretched corneal epithelial cells was measured. The results are shown in Figure 2. In the figure, the horizontal axis represents the length (mμ) of corneal epithelial cells, and A on the vertical axis represents TC.
-199 culture solution only control group, B group containing ovomacroglobulin 0.05 μg/ml added to the above culture solution, C group
The same ovomacroglobulin 0.5 μg/ml added group,
D shows the group to which 5 μg/ml of the same ovomacroglobulin was added. From FIG. 2 above, it is clear that ovomacroglobulin can promote the spread of corneal epithelial cells in a dose-dependent manner. Examples of the preparation of the formulation of the present invention are shown below. In each example, parts indicate parts by weight. Prescription Example 1 Prescription for the wound treatment agent of the present invention Ovomacroglobulin 0.01g Preservative Appropriate amount Fragrance Appropriate amount Add distilled water to bring the total volume to 100ml. Add distilled water to the above ovomacroglobulin, preservative and fragrance to make a total volume of 100ml, then sterilize.
A wound treatment agent of the present invention in the form of a spray solution was prepared. Prescription Example 2 Prescription of the wound treatment agent of the present invention Ovomacroglobulin 0.05g Preservative Appropriate amount Fragrance Appropriate amount Add distilled water to bring the total volume to 100ml. In the same manner as in Formulation Example 1, a wound treatment agent of the present invention in the form of a spray solution having the above composition was prepared. Prescription example 3 Preparation of hydrophilic ointment formulated for the wound treatment agent of the present invention Ovomacroglobulin 0.5g White petrolatum 250g Stearyl alcohol 220g Propylene glycol 120g Sodium lauryl sulfate 15g Ethyl paraoxybenzoate or methyl paraoxybenzoate 0.25g Propyl paraoxybenzoate 0.15g Purified Water Appropriate amount Total amount 1000g The above ingredients were blended to prepare a wound treatment agent in the form of a hydrophilic ointment of the present invention containing ovomacroglobulin. Prescription Examples 4 to 7 Preparation of hydrophilic ointment formulated with the wound treatment agent of the present invention In Prescription Example 3, the amount of ovomacroglobulin was changed to 0.01 g, 0.05 g, 0.1 g, and 1 g, and the hydrophilic ointment of the present invention was prepared in the same manner. A wound treatment agent of the form was prepared.

[Brief explanation of the drawing]

FIG. 1 is a graph showing changes in body weight of test mice in a burn recovery test using burn-injured mice. FIG. 2 is a graph showing the influence of the drug of the present invention on the spread of corneal epithelial cells in Test Example 5.

[Claims]

Primary formula: (In the formula, R ₁ , R ₂ and R ₃ represent the same or different lower alkyl groups.) An iodinating agent for phenol or aniline or a derivative thereof, comprising a compound represented by the formula: 2 Phenol or aniline or their derivatives with the following formula: (In the formula, R ₁ , R ₂ and R ₃ represent the same or different lower alkyl groups.) A method for iodizing an aromatic compound, which comprises treating with a compound represented by the formula: