JPH10259134A - Wound healing promoter - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject medicine having high wound healing effect by embedding leukocyte or bone marrow cell into a matrix or attaching leukocyte or bone marrow cell to the matrix. SOLUTION: This agent is obtained by separating leukocyte or bone marrow cell from blood, mixing the separated leukocyte or bone marrow cell with a matrix (preferably biodegradable collagen, chitin, fibrin, etc.) in solution state, gelatinizing the mixture, as necessary, culturing the resultant gel in a solution containing cytokine (e.g. γ-interferon), a cell growth factor or a stimulating agent (e.g. a cyclic peptide of the formula and obtained by culturing Streptomyces nobilis, preferably in an amount of 10<-16> to 10<-2> based on culture medium). The agent has activated leukocyte or bone marrow cell. Strong wound healing promoting effect is exhibited by bringing the agent into contact with the wound part.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a wound healing promoter.

[0002]

2. Description of the Related Art Conventionally, in the treatment of skin ulcers and pressure sores, the formation of granulation and the regeneration of the epidermis are expected by the natural healing power of patients while removing necrotic tissue and suppressing the growth of bacteria with antibacterial agents. The main method was to do it. In other words, in many cases, an ointment containing an antibiotic is used, and a method of waiting for the formation of granulation and regeneration of the epidermis by the natural healing power of the patient while suppressing the growth of bacteria and the like has been adopted.

[0003] However, in recent years, along with the progress of basic research on wound healing, drugs and treatments that actively promote granulation and epidermis formation have been desired.
Substances that promote granulation and epidermal regeneration have come to be applied clinically. Recently, various cell growth factors and the like have been developed as wound healing agents. For example, in patients with various diseases such as diabetes and elderly people, there is a problem that ulcers and pressure sores tend to occur and are difficult to heal. In particular, in diseases such as diabetes, the production of various cell growth factors required in the wound is suppressed, and as a treatment method, administration of purified cell growth factors or genetically modified cell growth factors can be considered. ing. For example, genetically modified basic fibroblast growth factor and the like have been developed for clinical application.

[0004] Neutrophils, monocytes and macrophages are essential cells for granulation tissue formation during wound healing, and infiltration of these cells into the wound site is an important process for wound healing. Therefore, transplantation of bone marrow cells or the like to a wound site has been considered in order to promote wound healing more quickly. For example, the formation of capillary-rich granulation tissue by autologous bone marrow transplantation into the subcutaneous tissue of adult dogs has been reported (Marat, Doulet et a).
l. , Artificial Organ Vol. 25, p486-489, 1
996).

[0005] On the other hand, matrices such as collagen, chitin, chitosan, and fibrin have an effect of promoting healing in wounds, and are used as wound coatings and adhesives. For example, collagen plays an important role as a cell substrate as an extracellular matrix, and is used as a substrate for various cells in vitro. Various wound dressings such as collagen membranes have been developed for the treatment of shallow burns in which epidermal cells remain in burns and the like. Further, in the case of a deep burn in which epidermal cells are completely lost or a wound with complete loss of skin, autogenous skin transplantation or the like is required, and development of artificial skin that survives as it is to form skin has been desired. Therefore, as artificial skin with a structure equivalent to living skin,
Hybrid artificial skin in which fibroblasts and epidermal basal cells are cultured on collagen gel has been developed (E. Bel).
l, P. Ehrlich, D .; J. Butle, T .;
Nakatsuji: Living Tissue F
ormed in vitro and Accept
ed as Skin-Equivalent Tis
sue of Full Thickness. Sci
ence Vol. 211. p1052, 1981,
I. V. Yannas, J .; F. Burke, D .; P.
Orgill, E .; M. Skrabut: Wound
Tissue Can Utilize a Poly
meric Template to Synthel
size a Functional Extension
no of Skin. Science Vol. 21
5. 174, 1982). Thus, skin cells are used for artificial skin because they can be easily collected from a small amount of skin pieces of the patient themselves, are relatively easy to culture, and can proliferate to the required number of cells.

[0006] As described above, in the treatment of skin ulcers and pressure sores, along with the progress of basic research on wound healing, drugs and treatments that actively promote granulation and epidermis formation have been desired. Substances that promote granulation and epidermal regeneration have come to be applied clinically. Hybrid artificial skin in which fibroblasts and epidermal basal cells are cultured on collagen gel is also used as artificial skin.

[0007] However, since cytokines and cell growth factors form a complex network, administration of a single cytokine to a living body in a high volume results in increased side effects. In addition, leukocytes and bone marrow are major cytokine-producing cells, but even if they are transplanted locally, they may not stay at the target diseased area but spread, causing a decrease in effects and increased side effects. is there. Administration of collagen or the like as an extracellular matrix does not exert a sufficient healing promoting action when the wound healing power of a living body such as an elderly person is deteriorated. Even with artificial skin, it is effective after leukocyte infiltration and granulation, which are early stages of wound healing, but does not exert a strong effect on promoting healing from the early stage of wounding. Thus, in the treatment of wounds, a therapeutic agent that promotes wound healing more strongly is desired.

[0008]

SUMMARY OF THE INVENTION The present invention has been made to solve the above problems, and an object of the present invention is to provide a wound healing promoting agent having a higher wound healing effect at a wound site or an ulcer site. .

[0009]

Means for Solving the Problems In order to solve the above-mentioned problems, the present inventors have conducted intensive studies and as a result, implanted their own leukocytes and bone marrow cells in a matrix such as a collagen gel and implanted them into a wound. It has been found that by doing this, a strong wound healing promoting effect is exhibited. In addition, they found that a strong wound healing promoting effect was exhibited by implanting these cells, activating them with cytokines, cell growth factors, stimulants, etc., and transplanting them into a wound, thereby completing the present invention.

That is, the agent for promoting wound healing of the present invention is characterized in that leukocytes or bone marrow cells are embedded or adhered to a matrix.

The matrix is not particularly limited as long as it is capable of embedding or attaching leukocytes or bone marrow cells and is not dermatologically toxic. Polysaccharides such as cellulose, chitin, chitosan, agarose, and calcium alginate; proteins such as collagen, gelatin, albumin, and fibrin; and synthetic organic polymers such as polymethyl methacrylate, polystyrene, polyethylene, and polylactic acid. Silicone resin, polytetrafluoroethylene resin and the like can be mentioned.

The matrix is preferably a biodegradable one, for example, collagen, chitin, chitosan, fibrin and the like. When the matrix is a matrix having biodegradability, the wound healing promoting agent of the present invention is used in contact with the wound site, and after the wound healing has progressed, it is not necessary to peel off or wash off the wound. is there.

When the matrix itself has a wound healing promoting effect, for example, collagen, chitin, chitosan, fibrin, etc., the healing promoting effect is less than that of leukocytes or bone marrow cells. It is particularly preferable because it is synergistically exhibited.

The matrix has biodegradability,
It has a wound healing promoting effect by itself and is easy to obtain,
Collagen gel is particularly preferred because it is easy to use.

The above-mentioned leukocytes or bone marrow cells refer to leukocytes or bone marrow cells of humans or animals, and are preferably those of humans who are to treat wounds. As leukocytes,
For example, macrophages, neutrophils, eosinophils, basophils and the like can be mentioned, and a leukocyte mixture may be used without separating them.

In the agent for promoting wound healing of the present invention, the number of leukocytes or bone marrow cells embedded or adhered to the matrix can be freely increased or decreased according to the required number of cells.

The method for obtaining a wound healing promoter in which leukocytes or bone marrow cells are embedded or adhered to a matrix is not particularly limited, and examples thereof include the following methods. First,
Separate leukocytes or bone marrow cells from blood. This is mixed with the matrix in a solution state to gel the mixture. After the mixture is gelled, it may be cultured as necessary.

When separating white blood cells or bone marrow cells from the above blood, in order to separate the own peripheral white blood cells or bone marrow cells, it is possible to sufficiently collect and separate them by a technique of blood transfusion or bone marrow transplantation. Culture is relatively easy, and it is possible to grow to the required number of cells.

The agent for promoting wound healing of the present invention is used in contact with a wound site. The above-mentioned contact may be brought into contact with the wound site by being inserted into the wound site in addition to being brought into contact with the wound site with a bandage, tape, bandage, or the like.

The wound healing promoter according to claim 4 of the present invention, wherein the leukocytes or bone marrow cells are activated with a cytokine, a cell growth factor or a stimulant. It is a healing promoter.

[0021] The wound healing promoting agent according to claim 4 is obtained by culturing a matrix having leukocytes or bone marrow cells embedded or attached thereto in a solution containing cytokines, cell growth factors or stimulants. Can be. Examples of the cytokine include interferon γ. As the stimulant, for example, the following formula [I]

Embedded image (The wound healing promoting agent according to claim 5 is limited to this cyclic peptide as a stimulant)
And lectins. Alternatively, it can also be obtained by activating the separated leukocytes or bone marrow cells by culturing them in a solution containing the above-mentioned cytokine, cell growth factor or stimulant, and then embedding or attaching the cells to a matrix.

The cyclic peptide represented by the above formula (I) is
The peptide-producing strain belonging to the genus Streptomyces, for example, Streptomyces nobilis (Streptomyces)
ycesnobilis (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. cerevisiae producing the cyclic peptide Nobilis is available from public preservation institutions, such as the RIKEN archival strain (JCM4274), which is ATCC 19252 in the United States and CCC in the Netherlands.
(Preserved also as BS198.65).

The method for obtaining the cyclic peptide is, for example, as follows:
It can be obtained by the method described in WO 96/12732 filed by the present inventors in an international application earlier.

The wound healing promoting agent according to claim 5 can be obtained by culturing a matrix in which leukocytes or bone marrow cells are embedded or adhered, in a solution containing the cyclic peptide. Alternatively, it can also be obtained by activating the separated leukocytes or bone marrow cells by culturing them in a solution containing the cyclic peptide, and then embedding or attaching the cells to a matrix. In the culture, the amount of the cyclic peptide used is not particularly limited and is widely used.
It is in the range of ^-16 to 10 ^-2 % by weight.

The method of using the wound healing promoter according to claim 4 or 5 is the same as that of the wound healing promoter according to claim 1 described above.

The shape of the wound healing promoter according to claims 1 to 5 of the present invention is not particularly limited, and can be adapted to the shape of the wound site. For example, pellets, membranes, microcapsules and the like can be used. Various shapes can be freely obtained by molding.

In the wound healing promoter of the present invention, fibroblasts and epidermal basal cells used in conventional artificial skin are mixed together with leukocytes or bone marrow cells and embedded in a matrix. May be attached.

[0029]

When the wound healing promoter of the present invention is brought into contact with a wound site, various cytokines and cell growth factors are produced from leukocytes or bone marrow cells at the wound site, thereby promoting wound healing. .

Further, the agent for promoting wound healing of the present invention can satisfactorily stay at the wound site because leukocytes or bone marrow cells are embedded or adhered to the matrix.

Further, the wound healing promoter of the present invention can assist infiltration of wounds such as leukocytes by itself even in patients with various diseases such as diabetes and elderly people. Also promotes wound healing.

In particular, the wound healing promoter according to claim 4 is
Since leukocytes or bone marrow cells are artificially activated,
In particular, it has a high effect of promoting wound healing, and is also effective in treating wounds of patients with various diseases such as diabetes and the elderly.

In particular, in the wound healing promoting agent according to the fifth aspect, since the above-mentioned cyclic peptide is used as a stimulant to artificially activate leukocytes or bone marrow cells, the effect of promoting wound healing is particularly high. .

[0034]

BEST MODE FOR CARRYING OUT THE INVENTION

Reference Example 1 Actinomyces S. aureus obtained from RIKEN Nobilis (JCM4274) was added to yeast extract 0.2% (w /
v) 500 containing 50 ml of added starch / ammonium medium
5 ml Sakaguchi Srasco, 26 ° C, 150 rpm, 1
The cells were shake-cultured (pre-preculture) for 20 hours. Subsequently, the same medium 12
Inoculate 240 ml of the pre-preculture culture solution into a 20-liter jar fermenter containing 20 liters, at 26 ° C. and 410 rpm.
m, and aeration (pre-culture) for 24 hours at an aeration rate of 4 L / min. In addition, a starch / ammonium medium (100 ml of distilled water)
1 g of soluble starch and 0.02 g of dipotassium hydrogen phosphate
A 200-liter jar fermenter containing 140 liters (containing 5 g and 0.05 g of ammonium chloride) was inoculated with 12 liters of the precultured bacterial solution and seed-cultured at 26 ° C. for 24 hours.

Further, 140 liters of a seed culture solution was inoculated into a 2,000 liter tank containing 1400 liters of a starch / ammonium medium, and cultured at 26 ° C., 140 rpm, aeration rate 700 liters / min, and pH 7.5 for 7 days.

After completion of the culture, the cells were separated by filtration.
Of 6.34 kg (wet weight) of the cells thus obtained, 3 liters of dichloromethane was added to 1 kg (wet weight) of the cells, and the mixture was stirred at room temperature for 15 hours. A liquid was obtained. The same operation was repeated three times for the cells. After concentrating the obtained bacterial cell extract, the silica gel carrier 12
It was adsorbed to 0 g. This adsorbed silica gel carrier was purified by a silica gel column.

7. Diameter filled with 800 g of silica gel carrier
About 120 g of the above-mentioned extract adsorption carrier was charged into a 0 cm column to prepare a silica gel column. This silica gel column was purified under the following conditions. As the elution solvent, 4 liters of a) hexane: ethyl acetate = 4: 6, 3.5 liters of b) ethyl acetate, and 2 liters of c) methanol were flowed in this order at a flow rate of 500 ml / hour. The fractionation was carried out every time the solvent composition was changed. In particular, the fraction eluted with ethyl acetate was fractionated by 500 ml (therefore, the number of eluted fractions for ethyl acetate was 7 in total).

For each of the above eluted fractions,
HPLC using a Tosoh column (DS-80TM, inner diameter 4.6 mm × length 25.0 cm, manufactured by Hitachi, Ltd., pump L-6000, L-6200, detector L-3000,
The detection wavelength is 2 by the column oven 655A-52).
Purity was confirmed under the conditions of 10 nm, a column temperature of 40 ° C., and a flow rate of 1 ml / min, using water: acetonitrile = 3: 7 as an eluent.

In the above purity confirmation by HPLC,
The elution fractions of the silica gel column confirmed that the peak area eluted at a retention time of 12 to 15 minutes occupies 80% or more of the total elution peak area were combined to obtain the same fraction. This fraction was concentrated and dried, and then recrystallized repeatedly using a methanol-dichloromethane system to obtain columnar crystals.
5 g were obtained.

The structure of this substance was the same as the substance described in WO 96/12732 from various instrumental analysis data, and was determined to be the formula [I].

Structural analysis data Instrument analysis data of the substance obtained above is 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−H ₂ O) ⁺ , m / z = 913, 953, 931 (913 is main, 931 is very small) Calcd for: C ₄₅ H ₆₉ N ₈ O ₁₂ m / z = 913.5053

[0043]

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

(Example 1) A 3% by weight thioglycolate medium was administered to the abdominal cavity of a 7-week-old Wister rat, and after 4 days, peritoneal exuded macrophages were collected. 5x these cells
The suspension was suspended in a calf skin-derived acid-soluble type I collagen solution (CELLGEN, manufactured by Koken Co., Ltd.) at a concentration of 10 ⁶ cells / ml. This was mixed and stirred under ice-cooling (final concentration of collagen: 0.25% by weight), and 100 µl of the mixture was poured into a 96-well microplate (manufactured by Iwaki Glass Co., Ltd.). Was prepared as a wound healing promoter. This was cultured in an RPMI1640 medium containing 10% by weight of rat serum for 2 days and subjected to the following test.

Example 2 A disk-shaped collagen gel embedded with macrophages was prepared in the same manner as in Example 1. This is called interferon gamma (RECOMBINANT RA).
T IFN-γ: manufactured by GENZYME) 500 Unit
/ Ml solution for 2 days and used as a wound healing promoter and subjected to the following tests.

Example 3 Blood was collected from a 7-week-old Wister rat by heart blood sampling, and leukocytes were collected by centrifugation using a specific gravity solution. The cells were suspended in a calf skin-derived acid-soluble type I collagen solution (CELLGEN, manufactured by Koken Co., Ltd.) at 5 × 10 ⁶ cells / ml.
This was mixed and stirred under ice cooling (collagen final concentration: 0.1%).
100 μl was injected into a 96-well microplate (manufactured by Iwaki Glass Co., Ltd.), and heated at 37 ° C. for 30 minutes to prepare a disk-shaped leukocyte-embedded collagen gel, which was used as a wound healing promoter. RP containing 10% by weight rat serum
The cells were cultured in MI1640 medium for 2 days and subjected to the following tests.

(Example 4) Bone marrow cells were collected from the femur of a 7-week-old Wister rat. 5 × 10
^The cells were suspended in calf skin-derived acid-soluble type I collagen solution (CELLGEN, manufactured by Koken Co., Ltd.) at a concentration of ⁶ cells / ml. This was mixed and stirred under ice cooling (final collagen concentration:
0.25% by weight), 100 μl was injected into a 96-well microplate (manufactured by Iwaki Glass Co., Ltd.), and heated at 37 ° C. for 30 minutes to produce a disc-shaped bone marrow cell-embedded collagen gel, which was used as a wound healing promoter. And This was cultured in an RPMI1640 medium containing 10% by weight of rat serum for 2 days and subjected to the following test.

Example 5 A disk-shaped macrophage-embedded collagen gel was prepared in the same manner as in Example 1. This contains the cyclic peptide obtained in Reference Example 1 at a concentration of 10 ⁻⁶ wt%, and RPMI 1640 containing 10 wt% rat serum.
The cells were cultured in a medium for 2 days and used as a wound healing promoter and subjected to the following tests.

Comparative Example 1 An acid-soluble type I collagen solution (CELLGEN, manufactured by Koken Co., Ltd.) derived from calf skin was stirred under ice-cooling (collagen concentration: 0.25% by weight), and a 96-well microplate (Iwaki) After injection of 100 μl of the mixture into glass (manufactured by Glass Co., Ltd.), the mixture was heated at 37 ° C. for 30 minutes to produce a disc-shaped collagen gel, which was RPM containing 10 wt% rat serum.
The cells were cultured in the I1640 medium for 2 days and subjected to the following tests.

(Comparative Example 2) A 3% by weight thioglycolate medium was administered to the abdominal cavity of a 7-week-old Wister rat, and after 4 days, peritoneal exuded macrophages were collected. 10 cells
A cell suspension was prepared at 5 × 10 ⁶ per ml in RPMI 1640 medium containing rat serum by weight, and 100 μl of this macrophage suspension was subjected to the following test.

Comparative Example 3 Blood was collected from a 7-week-old Wister rat by cardiac blood sampling, and leukocytes were collected by centrifugation using a specific gravity solution. The cells were placed at 5 × / ml in RPMI 1640 medium containing 10% by weight rat serum.
A cell suspension of 10 ⁶ was prepared, and this leukocyte suspension 1
00 μl was subjected to the following test.

Comparative Example 4 Bone marrow cells were collected from the femur of a 7-week-old Wister rat. A cell suspension was prepared at 5 × 10 ⁶ cells / ml in RPMI1640 medium containing 10% by weight of rat serum, and 100 μl of the bone marrow cell suspension was subjected to the following test.

(Test) The wound healing promoters of Examples 1, 2, 3, 4, and 5 and the samples of Comparative Examples 1, 2, 3, and 4 were subjected to the following tests, one by one. .

Seven-week-old male Wister rats were purchased from SLC Japan, preliminarily reared for one month, and weighed 2 weeks.
Twenty-five rats of 25-300 g were subjected to the experiment per test.

The back skin of the rat, which had been shaved using a clipper and a shaver, was incised (about 3 cm) along the midline under ether anesthesia. A control sample was inserted on the side. In addition, as a control sample, only the collagen gel was used for the samples of Examples 1 to 5 and Comparative Example 1, and 100 μl of the RPMI1640 medium containing 10% by weight rat serum was used for the samples of Comparative Examples 2 to 4.

After the insertion of the sample, the incised portion was sutured at three places at equal intervals with a metal suturing device (Natsume Seisakusho). After inserting the sample, the granulation tissue formed on the seventh day was taken out and weighed. The ratio of the weight of the granulation tissue of the sample to the weight of the granulation tissue of the control was defined as the growth granulation tissue ratio (%), and the average value of the four rats subjected to the test is shown in Table 1.

[0058]

[Table 1]

Table 1 shows that the agent for promoting wound healing of the present invention has a higher wound healing effect than the transfer of collagen gel, leukocytes, bone marrow cells, and the like.

[0060]

The composition of the wound healing promoter according to claim 1 is as described above, and is used in a wound site or an ulcer site.
A wound healing promoter having a higher wound treatment effect can be provided.

The composition of the agent for promoting wound healing according to claim 2 comprises:
As described above, in the wound site or ulcer site, while having a higher wound treatment effect, the matrix is biodegradable, so after the wound healing progresses, it is not necessary to peel off or wash away, so the wound healing Useful.

The composition of the agent for promoting wound healing according to claim 3 comprises:
As described above, since the matrix is a collagen gel, it has biodegradability, itself has a wound healing promoting effect, and is easily available and easy to use, so it has a higher wound treatment effect In addition, it is easy to use and manufacture.

The composition of the agent for promoting wound healing according to claim 4 comprises:
As described above, since leukocytes or bone marrow cells are activated with cytokines, cell growth factors or stimulants,
It is possible to provide a wound healing promoter having a particularly high wound treatment effect at a wound site or an ulcer site.

The composition of the agent for promoting wound healing according to claim 5 comprises:
As described above, since the cyclic peptide represented by the chemical formula [I] is used as a stimulant to artificially activate leukocytes or bone marrow cells, a particularly high wound treatment effect is obtained at a wound site or an ulcer site. Can be provided.

Claims (5)

[Claims] 1. An agent for promoting wound healing, wherein leukocytes or bone marrow cells are embedded or adhered to a matrix. 2. The wound healing promoter according to claim 1, wherein the matrix is a biodegradable matrix. 3. The agent for promoting wound healing according to claim 1, wherein the matrix is a collagen gel. 4. The method according to claim 1, wherein the leukocyte or bone marrow cell is a cytokine,
A cell growth factor or a stimulant that is activated.
4. The agent for promoting wound healing according to any one of 3. 5. The stimulant according to the following formula [I]: The wound healing promoter according to claim 4, which is a cyclic peptide represented by the formula: