JPH0543453A - Local sustained-release preparation for accelerating wound curing - Google Patents

Abstract

PURPOSE:To provide a local preparation capable of locally administering a physiologically active substance (e.g. growth hormone or insulin-like growth gene-1) having a wound cure-promoting action at a wound site and capable of continuously releasing the physiologically active substance. CONSTITUTION:A carrier containing collagen as an essential component is mixed with the physiologically active substance and subsequently processed into a filmy or powdery article to produce the local preparation. The preparation exhibits a significantly rapid wound cure-promoting effect in comparison with the single employment of the physiologically active substance.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a pharmaceutical preparation for promoting the treatment of wounds, more specifically, a preparation containing a drug in a sustained release carrier containing collagen as an essential component, The present invention relates to a sustained-release preparation for local use for promoting wound healing, which locally and continuously releases an effective amount of a drug by sticking or applying.

[0002]

2. Description of the Related Art Since various growth factors, cell growth factors, hormones and the like have an action of promoting cell migration, proliferation and differentiation, these physiologically active substances are used for treatment of wounds by systemic administration such as injection. Things have already been done. However, the application of these physiologically active substances by systemic administration has a serious drawback that a high dose is required to maintain an effective concentration of the drug at the wound site, and thus there is a high risk of side effects. .. From this situation,
Recently, attempts have been made to develop topical formulations for applying these physiologically active substances only to wounds. For example, the preparations described in JP-A-63-54328, JP-A-63-96135, and JP-A-2-112 are wounds comprising a pharmaceutically acceptable gel carrier, growth factors, hormones and the like. Although healing compositions, gels are usually not suitable for long-term retention of the entrapped drug at the wound topical site, and these formulations did not achieve sustained release of the drug over the day. That is, since the gel is in the middle of the solid state and the liquid state and constantly exchanges the solvent and solute with the surrounding environment, the release rate of the included drug is relatively faster than that of the solid carrier. Become. Furthermore, gels and ointments are generally easily washed out by exudate when applied to the wound surface, requiring repeated administration. In addition, JP-A-6
The composition described in JP-A-4-22254 comprises a microcapsule layer containing a cell growth factor incorporated in a coacervate structure composed of denatured collagen and mucopolysaccharide, and a sheet-like collagen film layer supporting the microcapsule layer. However, it is necessary to heat the wound dressing to 37 ° C. or higher, substantially 60 ° C. or so in order to cause a coacervation phenomenon when the preparation is prepared. It is practically not applicable to heat-labile proteinaceous drugs such as PDGF (plasma plate-derived growth factor). JP-A-1-158963 discloses a collagen matrix composition containing a cell growth factor, which is used as a medium for artificial skin and cell growth culture. No mention is made of the wound healing promoting effect of the composition.

On the other hand, a long-term sustained-release preparation which is biodegradable and can be embedded in a living body has an international application 900900.
And JP-A-60-126217, the purpose of which is to cause a systemic effect by implanting the drug in the body and releasing the drug for a long period of time, and for healing the wound locally. It is not what

[0004]

As described above, a preparation which has satisfactory results in terms of pharmaceutics and therapeutics has not yet been reported, and a topical preparation effective for wound healing has been desired.

[0005]

Under such circumstances, the present inventors can treat a wound by applying it to the wound site and releasing the drug consisting of the aforementioned physiologically active substance for a long period of time. As a result of repeated intensive studies to develop a preparation capable of promoting the following, a carrier consisting only of collagen, or collagen and albumin, proteins such as gelatin, sugars such as chitin, polylactic acid, polyglycolic acid,
And a carrier composed of a carrier component selected from the group consisting of one kind or a mixture of two or more kinds selected from the group consisting of synthetic polymers such as copolymers thereof, and the aforementioned physiologically active substance having a wound healing promoting activity. It has been found that the mixed composition gives a satisfactory therapeutic effect, and has completed the present invention. That is, the present invention provides a topical sustained-release preparation for promoting wound healing, which is obtained by mixing a carrier containing collagen as an essential component with a physiologically active substance having an activity of promoting wound healing.

The carrier component used in the preparation according to the present invention is defined as a substance having biocompatibility or biodegradability and capable of stabilizing or controlling the release of a physiologically active substance as an active ingredient. However, in addition to the above-mentioned specific examples, these include carrier components used in conventional sustained-release pharmaceutical preparations for oral or implant. Examples of the method of using these carrier components include a method of uniformly mixing with collagen according to the purpose. In this case, the mixing ratio is preferably 0 to 50% by weight, more preferably 0 to 30% by weight based on collagen. %. Further, it is also possible to form a layer separately from collagen using these carrier components to control the release of the active ingredient.

Physiologically active substances having a wound healing promoting activity include GH (growth hormone), IGF (insulin-like growth factor) -1, FGF (fibroblast growth factor), PDG.
Examples thereof include, but are not limited to, F, TGF (transforming growth factor) -α, β, NGF (nerve growth factor), insulin, and EGF. The compounding ratio of the physiologically active substance to the carrier can be up to about 80% by weight, but it is preferably about 30% by weight or less. Incidentally, I
There have been attempts to utilize cell growth factors and hormones such as GF-1, PDGF and FGF for wound treatment (Japanese Patent Publication No. 64-500199, Japanese Patent Publication No. 2-231429).
JP-A-63-54328, JP-A-63-54328
No. 96135, Japanese Patent Application Laid-Open No. 2-112, and Japanese Patent Application Laid-Open No. 64-22254) and GH have not been reported to try to use them locally. Therefore, the present invention will be described in more detail below by taking GH as an example.

GH has a skeletal elongation activity during growth.
Since then, it has been clinically applied to the treatment of pituitary dwarfism.
However, in addition to its growth-promoting effect, it also contains proteins, sugars, lipids and electrolysis.
Hormo plays an important role in regulating substance metabolism such as quality
Are becoming more and more popular. like this
One of the diverse actions of GH is the tissue repair action.
Healing by systemic administration of GH in a wound healing model system using
There are a number of studies that have shown a promoting effect, including: Kowal
euski, K., Young, S.,; Acta Endocrinol.,5
9, 53-66 (1968); Criglencza, W. et al., Act.
a Med. Pol.,24, 1-4 (1983); Barbul, A .;
Et al., Am. J. Clin. Nutr.,37, 786-794
(1983); Pessa, M .; E. Et al, Surgical Forum,
36, 6-8 (1986); Hollandar, D .; M. Et al, S
urgical Forum,35, 612-614 (1984); J.
orgensen, p. H. Endocrinol.,121, 1637
-1641 (1987); Jorgensen, P .; H. Et Acta
Chir. Scand.,154, 623-626 (1988)

However, as described above, GH
If a high dose of GH is administered in anticipation of the effect on the local area of the wound, a systemic effect will appear, which may lead to exacerbation of diabetes in acromegaly and especially in diabetic patients.

The healing-promoting action of GH is mainly mediated by the systemic administration of IGF-1 in the liver. Recently, it has been reported that GH has local as well as systemic effects. That is, the following literature suggests that GH directly acts on cartilage and fibroblasts to promote cell proliferation, and that its action is mediated by IGF-1 production stimulation from these target cells. :
Nilsson, A., J. Isgaard, A. Lindahl, A. Dalsto
m, A. Skottner, and O. Isaksson, Science 2
33 , 571-574 (1986); Cook, J. et al. J., H
aynes, KM. And Werther, G .; A .: J. Clin.
Invest., 81 , 206-212 (1988). But,
These results were only confirmed at the cellular level, and the effect of GH when locally administered to the wound site has not been clarified yet.

Waggo has reported an attempt to administer an ointment mixed with GH locally as a method for treating refractory ulcer of the heel of diabetic patients (Waggo, H .: Lancet, 27 , 14).
85 (1987)). However, in this case, the patient died during the treatment, and its effect of promoting wound healing is not clear.

[0012] The present inventors prepared a formulation that releases an effective amount of GH continuously and locally in the wound by the method described in detail below, and when administered to the wound locally, it has a significant effect of promoting wound healing. It was confirmed. That is, when the GH aqueous solution or the freeze-dried powder is administered alone to the wound site,
Since GH rapidly disappears from the wound local area, a wound healing promoting effect was not observed, but a sustained release preparation containing the same amount of GH was prepared using the carrier defined in the present specification to prepare a wound site. It was found that the formulation had a wound healing promoting action when administered to.

Collagen, which is an essential component of the carrier, is excellent in biocompatibility and has almost no immunological rejection reaction. Therefore, it has been widely used as a surgical thread or a wound dressing material from a medical viewpoint. In the present invention, atelocollagen may be used, in which the antigenicity of collagen is reduced by enzymatic treatment (for example, treatment with pepsin) for the purpose of further improving safety. Regarding the wound healing promoting effect of collagen, collagen and collagen degradation products show a chemotactic effect on fibroblasts, and some of the applied collagen is reused for fibril formation. Backed up. As described above, when GH was added to collagen having appropriate properties as a wound dressing to prepare a sustained-release preparation, and the preparation was locally administered to the wound, active promotion of wound healing was observed, which was not observed in the case of collagen alone. I was able to bring out the effect.

The method for producing the topical sustained release preparation for promoting wound healing of the present invention will be described in detail below. That is, a physiologically active substance as an active ingredient is added to a solution containing a carrier component having collagen as an essential component, and the mixture is stirred, and this mixture is optionally air-dried or freeze-dried. At this time, pharmaceutically acceptable excipients, stabilizers, preservatives, soothing agents, solubilizers, proteolytic enzyme inhibitors, etc., and additives for controlling moldability and sustained release are required. Can be added according to For example, glycerin can be added to impart softness to the formulation. The carrier having collagen as an essential component can be produced without heating operation or use of an organic solvent, and inactivation of the drug and denaturation of collagen can be avoided. For the purpose of avoiding drug burst, it is preferable to use non-fibrillar collagen. The solid composition thus obtained is used as it is or after being appropriately processed according to the purpose. For example, it may be pulverized with an ordinary pulverizer so as to have an appropriate particle size and processed into a powder, or a plurality of film-shaped compositions having different compositions may be layered and compressed to be processed into one film. it can.

The pharmaceutical preparation prepared by the above process has the ability to continuously release the drug contained therein, and has the sustainability required for wound healing, that is, preferably 1 day or more, more preferably 3 days or more. Sex can be achieved. The sustained-release preparation of the present invention can be easily applied and applied to a wound site, and since it is biodegradable, it spontaneously decomposes and disappears with healing, and therefore does not need to be peeled off after healing. Therefore, the pain of the patient due to the peeling can be avoided. The topical sustained-release preparation for promoting wound healing of the present invention is for treating burns, skin-scratching wounds, incisions, surgical wounds, wounds at the joint site of skin transplantation, chronic skin ulcers such as pressure ulcers, and diabetic ulcers. Effective as a formulation. As for the form of the preparation, an appropriate dosage form such as a film form or a powder form can be selected according to the therapeutic purpose. Further, a film having a laminated structure composed of two or more layers having the same or different compositions can be used. Also,
When using this formulation, a commonly used coating material,
For example, it can be covered with a polyurethane film or silicone. Further, it goes without saying that these sheets can be used as the back layer of the present preparation.

Hereinafter, the present invention will be described in more detail with reference to examples, but these examples do not limit the present invention in any sense.

Example 1 Preparation of GH-containing collagen film Example 1-1 Human growth hormone (hGH) aqueous solution 10 mg / ml (1N-HC
(Adjusted to pH3 with l) 10.52 ml was added to 12.28 g of 2% atelocollagen aqueous solution, and after stirring, 1N-HCl was added.
The pH was adjusted to 3.3 to 3.5. This mixed solution is 20.8 for each plastic petri dish having a diameter of 6 cm.
By injecting 0 g and air-drying, 30% (w / w) GH /
A collagen film was obtained. Example 1-2 hGH aqueous solution 10 mg / ml (adjusted to pH 3 with 1N-HCl), glycerin aqueous solution 10 mg / ml (1N-HCl pH)
(Adjusted to 3) 18.58 ml of each was added to 12.40 g of a 2% atelocollagen aqueous solution, and after stirring, the pH was adjusted with 1N-HCl.
Adjusted to 3.3-3.5. By injecting 47.59 g of this mixed solution per plastic dish having a diameter of 9 cm and air-drying, 30% (w / w) GH / 30% (w
/ W) Glycerin / collagen film was obtained. Example 1-3 hGH aqueous solution 10 mg / ml (adjusted to pH3 with 1N-HCl)
4.30 ml of 2% atelocollagen aqueous solution 40.73 g
In addition, the mixture was stirred and the pH was adjusted to 3.3 to 3.
Adjusted to 5. 12.62 g of this mixed solution was injected per plastic dish having a diameter of 6 cm and freeze-dried to obtain 5% (w / w) GH / collagen sponge. Meanwhile, 11.42 g of 2% atelocollagen aqueous solution
Similarly, was injected into one plastic petri dish having a diameter of 6 cm and freeze-dried to obtain a collagen sponge. 5% (w / w) G between two collagen sponges
By sandwiching the H / collagen sponge and applying a pressure of 400 kgf / cm ² for 80 minutes, a three-layer structure preparation was obtained.

Examples 1-4 hGH aqueous solution 10 mg / ml (adjusted to pH 3 with 1N-HCl)
17.41 ml of 2% atelocollagen aqueous solution 165.3
After adding 8 g and stirring, pH is 3.3- with 1N-HCl.
Adjusted to 3.5. By injecting 16.80 g of this mixed solution per plastic dish having a diameter of 6 cm and air-drying, a 5% (w / w) GH / collagen film was obtained. By adding 5.71 g of a 2% atelocollagen aqueous solution thereto and freeze-drying, a two-layer preparation of a GH-containing film layer-sponge layer was obtained. Example 1-5 2 ml of a polylactic acid-ethyl acetate solution (100 mg / ml) was dried with hot air to prepare a circular film having a diameter of 1.5 cm. A polylactic acid-GH-containing collagen two-layer structure was produced by pressing a circular 5% (w / w) GH-containing collagen film having a diameter of 1.5 cm, which was prepared in the same manner as in Example 1-1. A formulation was obtained. Example 1-6 Polyglycolic acid powder 400 mg, 2.5 cm in diameter 2.5 cm prepared in the same manner as in Example 1 and further prepared in the same manner as in Example 1-1. w / w) A GH-containing collagen film is placed on the GH-containing collagen film, and 400 mgf / cm ² of pressure is applied for 15 minutes while 400 mg of polyglycolic acid powder is uniformly placed on the GH-containing collagen film. A three-layer structure preparation was obtained.

Example 1-7 hGH 19.39 mg, human serum albumin (HSA) 1
6.23 mg of 2% atelocollagen aqueous solution 30.59 g
In addition, after stirring, the pH was adjusted to 3.5 with 1N HCl. This mixed solution was once freeze-dried and water was added to prepare a gel having a collagen concentration of 10%. By injecting 5.28 g of this gel per plastic petri dish having a diameter of 9 cm and air-drying, 3% (W / W) GH / 2.
A 5% (W / W) HSA / collagen film was obtained. Example 1-8 hGH 19.96 mg, human serum albumin (HSA) 1
99.29 mg of 2% atelocollagen aqueous solution 22.31
After adding to g and stirring, the pH was adjusted to 3.5 with 1N HCl. This mixed solution was once freeze-dried and water was added to prepare a gel having a collagen concentration of 10%. By injecting 3.77 g of this gel into a plastic dish having a diameter of 9 cm and air-drying, 3% (W / W) GH / 30 was obtained.
% (W / W) HSA / collagen film was obtained.

Experimental Example 1 In Vitro GH Release Experiment Examples 1-1, 1-3, 1- were placed in a plastic test tube.
GH / collagen film 1 cm x 1 cm produced by the same method as 4, 1-7, 1-8 was put in each (Example 1-4
As a back sheet, a silicone sheet was attached to the GH-containing film layer side), 1.5 ml of a phosphate buffer (containing 0.01% sodium azide) was added, and the mixture was allowed to stand at 37 ° C. The amount of GH in the external liquid was quantified by liquid chromatography. The results are shown in FIGS. 1, 2, 3, and 4, respectively.

Experimental Example 2 Change in blood concentration of GH in vivo Genetically diabetic mice (female C57BL / KsJ db / db mice) were anesthetized with ether, their backs were hair-removed, and 1.5 cm square was obtained using ophthalmic scissors. Full-thickness wounds were prepared. 10 mg of the film prepared in Examples 1-1 and 1-2 (corresponding to 3 mg of hGH) was administered to the wound surface and then covered with a polyurethane film Op-Site ^R. 4,8,12,16,2 after administration
After 4, 48 and 72 hours, blood was collected from the heart under ether anesthesia and serum was separated. GH concentration in serum was measured by two-site immunoradiometry. Results are shown in FIG. As a comparative control, FIG. 5 shows changes in blood concentration when 3 mg of hGH was administered alone. When hGH was administered alone, the blood GH concentration reached a peak (80 μg / ml) 2 hours after administration and then decreased with a half-life of 3 hours.
When a collagen film containing% GH / 30% glycerin was administered, the blood GH concentration reached the peak (11,14 μg / ml) 8 hours after the administration, and the half-life was 1 thereafter.
It decreased in each of 5 and 12 hours.

Experimental Example 3 Transition of GH Remaining Amount in Vivo The GH / collagen film used in Experimental Example 2 was collected from the wound site after blood collection, and 0.5% HSA (human serum albumin) aqueous solution (1N-HCl The pH was adjusted to pH 3) and the amount of GH remaining in the film was quantified by liquid chromatography. The results are shown in Fig. 7. GH residual amount decreases with time, but after 72 hours, 30% GH /
About 50% of the collagen film remained, and about 30% GH / 30% glycerin / collagen film remained about 20% GH.

Experimental Example 4 Wound Healing Promoting Effect A genetically diabetic mouse (female C57BL / KsJ db / db mouse) was anesthetized with ether and the back part was hair-removed. A 1.5 cm square full-thickness wound was obtained using ophthalmic scissors. Was produced. Immediately after the preparation of the skin defect and on the 6th day, 3 mg of hGH, 7 mg of collagen or 10 mg of the film prepared in Example 1 (hGH
(Equivalent to 3 mg) was administered, and the polyurethane film Op-S
Covered with it ^R. The outline of the defect wound surface was transferred to a polyvinyl film under anesthesia with time, the wound surface was recorded, and the wound area was measured with a planimeter. The healing process is the healing rate (%)
Displayed in. The results are shown in Table 1.

[Table 1] Healing promotion effect by hGH collagen film Time healing rate (%) Mean value ± SD (n = 10) (day) hGH collagen hGH collagen untreated 14 14.4 ± 5.9 13.4 ± 5.9 21 41.0 ± 20.5 33.8 ± 17.1 14 22.0 ^a) ± 14.5 8.9 ± 6.0 13.3 ± 3.4 21 45.0 ^a) ' ^b) ± 34.9 17.2 ± 5.3 19.0 ± 4.6 Healing rate (%) = (1-wound area / initial wound area) x 100 b) P <0. 05 vs. no treatment a) P <0.05 vs. Collagen Student's t-test By GH alone, no healing promoting effect was observed, but the wound area was statistically significantly reduced by the administration of GH / collagen film.

[Brief description of drawings]

FIG. 1 is a graph showing the elution behavior of GH from a 30% (W / W) GH / collagen preparation according to the present invention to a phosphate buffer (containing 0.01% sodium azide).

FIG. 2 is a graph showing a GH elution behavior from a three-layer structure film containing 5% (W / W) GH / collagen sponge according to the present invention to a phosphate buffer solution (containing 0.01% sodium azide).

FIG. 3 GH from a bilayer film formulation according to the present invention to a phosphate buffer (containing 0.01% sodium azide)
The graph which shows elution behavior.

FIG. 4 3% (W / W) GH with HSA added according to the present invention
/ Collagen film preparation from phosphate buffer (0.01
The graph which shows the elution behavior of GH to (% sodium azide content).

FIG. 5 is a graph showing changes in blood concentration when 3 mg of GH was administered alone to the wound surface of diabetic mice.

FIG. 6 is a graph showing changes in blood concentration when the preparation according to the present invention (containing 3 mg of GH) was administered to the wound surface of diabetic mice.

FIG. 7 is a graph showing the results of time-dependent measurement of the amount of GH remaining in the preparation after the preparation according to the present invention (containing 3 mg of GH) was administered to the wound surface of diabetic mice.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification number Office reference number FI Technical indication location A61K 37/26 8314-4C 37/36 ADA 8314-4C 45/00 8415-4C 47/34 C 7329 −4C 47/36 C 7329−4C 47/42 B 7329−4C C 7329−4C (72) Inventor Keiji Fujioka 1-3-45 Kuragakiuchi, Ibaraki City, Osaka Prefecture Sumitomo Pharmaceuticals Co., Ltd. (72) Inventor Akihiko Sano Sumitomo Pharmaceutical Co., Ltd. 1-345, Kuragakinai, Ibaraki City, Osaka Prefecture (72) Inventor Miho Maeda 1-34-45, Kuragakiuchi, Ibaraki City, Osaka Prefecture (72) Inventor, Kajiwara ▲ Tadako Osaka Sumitomo Pharmaceutical Co., Ltd. 1-345, Kuragakiuchi, Ibaraki City, Japan

Claims (9)

[Claims] 1. A sustained-release preparation for local use for promoting wound healing, which is obtained by mixing a carrier having collagen as an essential component with a physiologically active substance having an activity of promoting wound healing. 2. The carrier consists only of collagen.
The preparation according to. 3. The preparation according to claim 1, wherein the carrier is composed of a carrier component selected from the group consisting of collagen, protein, sugar, and synthetic polymer. 4. The carrier is composed of a carrier component consisting of collagen and albumin, gelatin, chitin, polylactic acid, polyglycolic acid and a mixture of one or more selected from copolymer polymers thereof. The formulation according to 3. 5. The physiologically active substance is growth hormone, insulin-like growth factor-1, fibroblast growth factor, plasma plate-derived growth factor, transforming growth factor-α, β, insulin,
The preparation according to claim 1, which is selected from the group consisting of nerve growth factor and epidermal growth factor. 6. The preparation according to claim 5, wherein the physiologically active substance is growth hormone. 7. The preparation according to claim 1, which is in the form of a film. 8. The preparation according to claim 1, which is in the form of powder. 9. The preparation according to claim 1, which has a laminated structure consisting of two or more layers having the same or different structures and compositions.