JPH0782171A - Film preparation containing fibroblast growth factor - Google Patents

Abstract

PURPOSE:To provide a preparation containing a fibroblast growth factor (FGF), capable of stably holding the FGF and applicable to the wound without causing the problems inherent in conventional preparations. CONSTITUTION:This film preparation is produced by compounding a fibroblast growth factor (FGF) with a water-soluble cellulose lower alkyl ether and optionally a polyhydric alcohol and/or a surfactant.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a film preparation containing stable fibroblast growth factor (hereinafter, also referred to as "FGF"). More specifically, the present invention provides a stable composition of FGF with a water-soluble cellulose lower alkyl ether, and if necessary, a plasticizer selected from polyhydric alcohols and surfactants, a buffering agent, a bactericide, etc. An excellent film formulation.

[0002]

FGF was first detected in bovine pituitary gland in 1974, and by the mid-1980s, two types, namely, acidic fibroblast growth factor (isoelectric point (pI) 5-6) , "AFGF"), and basic fibroblast growth factor having a pI of 9 to 10 (hereinafter, "bFG").
F ") was purified. Currently, these aFGF
And bFGF, INT2 protein, HST
1 protein, FGF5, HST2 protein / FGF
6 and 7 growth factors of KGF are said to belong to the FGF family. Among them, aFGF and bFGF exist in various human organs and have a strong proliferative action on fibroblasts and vascular endothelial cells. As its clinical use, treatment of diabetic skin ulcer and varicose leg ulcer, facilitation of cornea, skin and bone graft, shortening of treatment period for cutting wounds, fractures and tendons are expected. Is also considered to be applicable to degenerative diseases of the cranial nerves such as Alzheimer's disease and myocardial infarction.

[0003]

[Problems to be Solved by the Invention] However, FGF
Is extremely unstable physicochemically and is inactivated immediately by acids, alkalis, heat, etc., so that it is not easy to develop it as a drug even though it is an excellent physiologically active substance. Therefore, as an attempt to stabilize the formulation containing FGF,
Method for preparing an aqueous pharmaceutical composition containing a sufficient amount of a water-soluble polysaccharide for stabilizing a growth factor (JP-A-63-1523)
4), a method of adding glycosaminoglycan to an aqueous solution of FGF mutein (JP-A-2-40399), a method of adding sulfated glucan to FGF mutein or an aqueous solution thereof (JP-A-2-138223), and water-insoluble in FGF protein Of hydroxypropyl cellulose (JP-A-4-128239), a method of combining FGF and a salt of sucrose octasulfate (SOS) or forming a complex (JP-A-4-330017), FGF and dextran sulfate. Formulation in combination with (International patent application
WO 9003797) and the like are disclosed. However, all of them are limited to stabilization of FGF in an aqueous composition and a powder composition.

In general, FG having a strong wound healing promoting action on intractable skin ulcers such as burns, pressure ulcers and diabetic ulcers.
It is believed that administration of F is effective. The above-mentioned conventional stabilized FGF aqueous composition and powder composition may be prepared into an external preparation such as a liquid, an ointment, a cream and a gel by a usual method and directly administered to the ulcer site. However, in the case of a liquid agent, it may adhere to the wound because it is impregnated with gauze or the like and may damage the neoepithelium during gauze replacement.In the case of ointment, cream or gel, the wound must be applied by hand. However, it is not preferable to handle a small amount of highly active substance such as FGF. Furthermore, the injury site is naturally considered to be painful, and physical irritation during application may be excessive irritation for patients with low pain thresholds.
Also, a hydrogel formulation containing FGF (sheet form)
(International Patent Application WO 9003810) has also been proposed, but due to lack of adhesiveness to the uneven wound surface, exudate may be stored between the wound surface and the preparation surface, and there is a risk of purulence due to various bacteria. Further, unlike the film preparation, since it contains a large amount of water, there remains a problem regarding the stability of FGF.

The present inventors have diligently studied a method of overcoming the drawbacks of conventional preparations and stabilizing FGF in order to use FGF having an action of promoting wound healing in the treatment of intractable skin ulcers as described above. As a result, a water-soluble polymer film preparation is a preparation which can be used by dissolving FGF into a deep ulcer by a simple method of cutting it according to the size of the affected area and placing it on the wound site. I found a thing. In addition, since the water-soluble polymer film formulation is thin and flexible, there is little physicochemical irritation, and because it follows, it adheres even to uneven wounds, and it is necessary to administer a small amount of active substance accurately and effectively. It is easy to manage the wound because it can be washed out with a liquid and can be easily washed away. In addition, they have found that FGF, which is extremely unstable in an aqueous solution, is stable in a film preparation having a low water content without using a specific stabilizer, and thus completed the present invention.

[0006]

[Means for Solving the Problems] That is, the present invention comprises a water-soluble cellulose lower alkyl ether compounded in FGF, and if necessary, a plasticizer selected from a polyhydric alcohol and a surfactant, a buffering agent, and a sterilizer. Provided is a stable FGF film preparation characterized by containing an agent and the like. Examples of the FGF used in the present invention include a recombinant FGF that can be mass-produced by expressing a human FGF gene cloned by a gene manipulation technique in a microorganism or an animal cell. The compounding amount is 5 μg to 10 mg / (10 × 10) in the film preparation.
cm ² . Examples of the water-soluble cellulose lower alkyl ether used in the present invention include hydroxypropyl cellulose, hydroxypropylmethyl cellulose, methyl cellulose and the like, and hydroxypropyl cellulose is particularly preferable. The viscosity range of these water-soluble cellulose lower alkyl ethers is preferably 5 to 50,000 cps (2% aqueous solution, 20 ° C., B-type viscometer). The amount of the water-soluble cellulose lower alkyl ether is 50 to 1,500,000 parts by weight, more preferably 100 to 250,000 parts by weight, and most preferably 300 to 2,000 parts by weight, based on 1 part by weight of FGF. It is a range.

The film preparation of the present invention contains glycerin, propylene glycol, and glycerin for the purpose of improving the flexibility of the preparation.
Polyethylene glycol, 1,3-butanediol, 1,
3-butylene glycol, polyhydric alcohol such as sorbitol, polyoxyethylene sorbitan monooleate,
A plasticizer selected from surfactants such as polyoxyethylene hydrogenated castor oil, polyoxyethylene lauryl ether, and polyethylene glycol monostearate can be added. The amount added is 0.3 to 80 of the total formulation weight.
The range of wt% is preferable, and 5 to 60 wt% is more preferable. If the amount added exceeds 80% by weight, the shape of the film preparation cannot be maintained, which is not preferable.

The film preparation of the present invention may further contain a buffering agent and a bactericidal agent, if necessary. Examples of the buffer used in the film preparation of the present invention include phosphate buffer, citrate buffer, borate buffer, acetate buffer and the like. Its pH is 4.5-7.5, preferably pH 5.0-6.5. Examples of the bactericide used in the film preparation of the present invention include benzalkonium chloride, chlorhexidine gluconate, and benzethonium chloride. The compounding amount thereof is 0.0005 to 0.1% by weight, preferably 0.001 to 0.1% by weight. The thickness of the film preparation of the present invention is 5 to 500 μm, preferably 10 to 100 μm. Preparation thickness is 5 μm
The following cases are not preferable because the strength of the film becomes weak, breakage easily occurs, and handling is difficult. Conversely 500
If it exceeds μm, it is not preferable because it lacks flexibility and the adhesion to the wound becomes poor.

As a general method for producing the film preparation of the present invention, a plasticizer and a bactericidal agent are first added to water or a buffer solution, if necessary, and then water-soluble cellulose lower alkyl ether is gradually added and dispersed and dissolved. After that, a buffer solution containing FGF is gradually added and stirred to obtain a uniform solution.
After degassing this solution, it is spread and dried to obtain an FGF-containing film preparation. In this case, if the drying temperature is too high, FG
Since the F may be deactivated, the drying temperature is 30 to 50.
A temperature as low as possible at about 0 ° C. is preferable.

[0010]

EXAMPLES Next, the present invention will be described in more detail by way of examples, but the present invention is not limited to these. Example 1 Hydroxypropylcellulose (HPC-M) ¹⁾ (6.0 g) was gradually added to water (92.33 g), and stirring was continued until the particles were completely dispersed and dissolved. While stirring this solution, citrate buffer solution (1.67 ml) containing 5 mg / ml human bFGF was gradually added, and the stirring was continued until it became uniform. After degassing, spread and dry to prepare a film preparation.

Example 2 Hydroxypropylmethylcellulose 2910 (HPMC 2) was added while heating water (88.5 g) to 70 ° C. or higher.
910) ²⁾ (9.0 g) is gradually added. After forming a uniform hot water slurry, stir while cooling from the outside,
Citrate buffer (1.67 ml) containing mg / ml human bFGF is added slowly and stirring is continued until uniform. After degassing, spread and dry to prepare a film preparation.

Example 3 Methylcellulose (MC) ³⁾ (9.0 g) was gradually added while heating water (88.5 g) to 70 ° C. or higher. After forming a uniform hot water slurry, stir while cooling from the outside, gradually add citrate buffer (1.67 ml) containing 5 mg / ml human bFGF, and continue stirring until uniform. . After degassing, spread and dry to prepare a film preparation.

Example 4 Film formulations A to D are prepared according to the ingredients shown in Table 1 below. That is, water or buffer (78.33 g)
To the plasticizer (1.0 g), and in the case of the film preparation D, a bactericidal agent (1.0 mg) was further added, and hydroxypropyl cellulose (HPC-L) (19.0 g) was gradually added to completely remove particles. Continue stirring until dispersed and dissolved.
While stirring this solution, citrate buffer solution (1.67 ml) containing 5 mg / ml human bFGF was gradually added, and the stirring was continued until it became uniform. After degassing, the film is spread and dried to prepare film preparations A to D.

[0014]

[Table 1] 1) HPC-M (manufactured by Nippon Soda Co., Ltd.) / Viscosity 150-
400 centipoise / hydroxypropyl group 52.4-
77.5% 2) TC-5S (manufactured by Shin-Etsu Chemical Co., Ltd.) / Viscosity 15.0 centipoise / methoxy group 28.0 to 30.0% / hydroxypropyl group 7.0 to 12.0% 3) Metrose SM-15 (Shin-Etsu Chemical Co., Ltd.) / viscosity 1
3.0-18.0 centipoise / Methoxyl group 27.5
31.5% 4) HPC-L (manufactured by Nippon Soda Co., Ltd.) / viscosity 6.0-
10.0 centipoise / hydroxypropyl group 52.4-
77.5%

Test Example 1 The stability of FGF in the FGF-containing film preparation of the present invention was examined. (Preparation of Test Sample) The film preparation used in this experiment was manufactured as follows. Hydroxypropyl cellulose (HPC) (6.20 g) was gradually added to water (93.35 g), and stirring was continued until the particles were completely dispersed and dissolved. While stirring this solution, 5 mg / ml human bFG
Citrate buffer containing F (3.8 ml) is added slowly and stirring is continued until uniform. After degassing, the film is spread and dried to prepare a film preparation, which is used as a sample. (Method) Human bFGF-containing isotonic phosphate buffer (pH 7.
4) and the human bFGF-containing film preparation were stored in a thermostatic chamber at 15 ° C. After that, bFGF was determined by the HPLC method.
The residual rate was measured and the results are shown in Table 2.

(Result)

[Table 2] From the results shown in Table 2, the film preparation of the present invention is bFGF
It can be seen that it holds stable.

Test Example 2 [Effect on Wound Treatment] The effect of the FGF-containing film preparation of the present invention on wound treatment was examined using a model of full-thickness skin cut of a genetically diabetic mouse. (Preparation of Test Sample) The film preparation used in this experiment was manufactured as follows. Hydroxypropyl cellulose (HPC) (6.20 g) was gradually added to water (93.35 g), and stirring was continued until the particles were completely dispersed and dissolved. While stirring this solution, 5 mg / ml human bFG
Citrate buffer containing F (3.8 ml) is added slowly and stirring is continued until uniform. After degassing, the product is spread and dried to prepare a film preparation, which will be referred to as Sample 3. A sample prepared in the same manner without adding human bFGF is designated as Sample 1. As a control, a physiological saline solution (20 μl / site) was used, and human bFGF was prepared with physiological saline to a predetermined concentration to prepare a sample 2. Samples 1 and 3 are finely chopped (1.4
(cm × 1.4 cm) was used. Table 3 shows the composition of the test group
Shown in.

[0018]

[Table 3]

(Test method) On the day before the test, the back of the mouse was carefully shaved using an electric clipper and an electric shaver, and under ether anesthesia, the back skin was wiped with ethanol for disinfection, and then curved surgical scissors were used. A circular full-thickness skin excision wound (2 cm ² ) was created in the midline of the back. After the skin excision, each sample was dropped or attached once a day for 5 days (total 5 times), and the wound surface was covered with BIOCLUSIVE ^R (Johnson & Johnson Medical). After tracing the wound surface on a plastic sheet at intervals of 2 to 4 days, the wound area was measured by an Oscon digitizer, and the ratio to the area immediately after excision was calculated to obtain the wound healing effect (Fig. 1). Furthermore, the degree of angiogenesis / granulation on the wound surface was visually scored (0: no effect, 1: mild, 3: moderate, 4: strength), and the daily change was examined (Figs. 2 and 3). ).

(Statistical analysis) The average value and standard deviation of the area variation rate of each sample group were calculated, and in the case of Hartley's test, one-way ANOVA was used for equal variance, and Kruskal-for non-uniform variance. A Kruskal-Wallis H test was performed. As a result, when a significant difference was observed at 5%, the difference between the control group and the sample 2 administration group, and the difference between the sample 1 administration group and the sample 3 administration group were subjected to multiple comparison. In addition, the Kruskal-Wallis H test was used to compare the macroscopic scores of angiogenesis and granulation on the wound surface.

(Results) In the results shown in FIG. 1, the wound areas of the control group and the sample 1-administered group gradually decreased, and the wound areas 18 days after excision were 25.5% and 2 on average, respectively.
It was 3.4%. On the other hand, the wound area of the sample 2 and sample 3 administration groups decreased rapidly and significantly from the 4th day of administration as compared to the control and sample 1 administration groups, and the wound area was 1
Average wound area on day 8 was 0.4% and 2.6%, respectively
Showed a clear wound healing promoting action. No statistical difference was observed in the change in the wound area between Sample 2 and Sample 3. From the results of FIGS. 2 and 3, it was considered that angiogenesis and granulation in Samples 2 and 3 were strongly recognized earlier than in Control and Sample 1 and contributed to the promotion of wound healing. In addition, no difference was observed between the two groups, Sample 2 and Sample 3, in the degree of angiogenesis and granulation. From the above results, it is understood that the human bFGF-containing film preparation of the present invention is effective as a wound healing agent.

[0022]

INDUSTRIAL APPLICABILITY The present invention has a medicinal effect by incorporating a medicinal component, FGF, a water-soluble cellulose lower alkyl ether, and if necessary, a specific plasticizer, a buffer, a bactericide, etc. into a film preparation. Provided is a formulation which can hold FGF as a component in a physicochemical stable state and can be directly applied to a skin ulcer site. The FGF-containing film preparation of the present invention, when applied to a skin ulcer site as a wound healing agent, can administer the main ingredient by a simple method of simply placing it on the wound site, and since the preparation is thin and flexible, it can be applied to the affected area. It has the advantage that there is little irritation and that the patient does not feel pain during administration. Furthermore, since the FGF-containing film preparation of the present invention has conformability, it can adhere even to an uneven wound, and a minute amount of the active substance (FGF) can be accurately administered to the affected area. Furthermore, since the preparation can be easily washed off with a liquid, it has a feature as an excellent therapeutic agent for skin ulcer that the wound can be easily managed.

[Brief description of drawings]

FIG. 1 Physiological saline (control), human bFGF
Rate of change in wound area in mouse skin full-thickness resection model when non-containing film preparation (Sample 1), human bFGF-containing physiological saline solution (Sample 2) and human bFGF-containing film preparation of the present invention (Sample 3) were applied. A graph showing the change over time in.

FIG. 2 Physiological saline (control), human bFGF
Angiogenesis of a wound site in a mouse skin full-thickness resection model when a film preparation containing no film (Sample 1), a physiological saline solution containing human bFGF (Sample 2) and a film preparation containing human bFGF of the present invention (Sample 3) were applied. A graph showing the daily change in the degree of.

FIG. 3: Physiological saline (control), human bFGF
Granulation at the wound site in the mouse full-thickness resection model when the film preparation containing no film (Sample 1), the physiological saline solution containing human bFGF (Sample 2) and the film preparation containing human bFGF of the present invention (Sample 3) were applied. A graph showing the daily change in the degree of.

─────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] November 2, 1993

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0019

[Correction method] Change

[Correction content]

(Test method) On the day before the test, the back of the mouse was carefully shaved using an electric clipper and an electric shaver, and under ether anesthesia, the back skin was wiped with ethanol for disinfection, and then curved surgical scissors were used. A circular full-thickness skin excision wound (2 cm ² ) was created in the midline of the back. After the skin excision, each sample was dropped or attached once a day for 5 days (total 5 times), and the wound surface was covered with BIOCLUSIVE ^R (Johnson & Johnson Medical). After tracing the wound surface on a plastic sheet at intervals of 2 to 4 days, the wound area was measured by a trace type graphic analysis system, and the ratio to the area immediately after excision was calculated to obtain the wound healing effect (FIG. 1). Furthermore, the degree of angiogenesis / granulation on the wound surface was visually scored (0: no effect, 1: mild, 3: moderate, 4 ::
Intensity) and changes over time were examined (FIGS. 2 and 3).

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical indication location A61K 47/38 N (72) Inventor Kazuhiro Fukunaga 301 Gengensuke, Fujieda City, Shizuoka Kaken Pharmaceutical Co., Ltd. (72) Inventor, Kimitaka Ishimura, 301 Gensuke, Fujieda-shi, Shizuoka, Central Research Institute, Kaken Pharmaceutical Co., Ltd. (72) Inventor, Makoto Okumura, 14 Minamikawara-cho, Shinnomiya, Yamashina-ku, Kyoto, Japan (72) ) Inventor Toshiaki Okuda 14 Minamigawara-cho, Shinnomiya, Yamashina-ku, Kyoto City Kyoto Research Institute, Central Research Institute

Claims (4)

[Claims] 1. A film preparation comprising a fibroblast growth factor (FGF) and water-soluble cellulose lower alkyl ether. 2. The water-soluble cellulose lower alkyl ether is one kind or two or more kinds selected from hydroxypropyl cellulose, hydroxypropylmethyl cellulose and methyl cellulose, and the blending amount thereof is 50 to 1,500 relative to 1 part by weight of FGF. The film preparation according to claim 1, which is in the range of 000 parts by weight. 3. The film preparation according to claim 1, further comprising a polyhydric alcohol and / or a surfactant. 4. The film preparation according to claim 3, wherein the content of the polyhydric alcohol and / or the surfactant is in the range of 0.3 to 80% by weight based on the total weight of the preparation.