MXPA00008669A - Topical composition containing human epidermal growth factor - Google Patents

Abstract

The present invention relates to a topical composition containing human epidermal growth factor (hEGF). More specifically, the present invention relates to a topical composition prepared by using polyoxyethylene-polyoxypropylene copolymer (Generic name:Poloxamer) having a viscosity of 4-10 cps at 37°C, 60 rpm as the base for topical formulation to improve the wound healing ability of hEGF. The topical composition according to the present invention can sufficiently exhibit the wound healing effect of hEGF and therefore is useful for the treatment of cutaneous injuries including wounds, incisions, burns, etc.

Description

TOPICAL COMPOSITION CONTAINING HUMAN EPIDERMAL GROWTH FACTOR DESCRIPTION OF THE INVENTION The present invention relates to a topical composition containing human epidermal growth factor (hereinafter referred to as "hEGF"). More specifically, the present invention relates to a topical composition comprising a polyoxyethylene-polyoxypropylene copolymer (Generic name: Poloxamer) having a viscosity of 4-10 cps at 37 ° C, 60 rpm as the basis of the topical formulation of hEGF occurring naturally or recombinant human epidermal growth factor (hEGF). The topical composition of the present invention is useful for the treatment of skin lesions including wounds, incisions, burns, etc. HEGF is a polypeptide consisting of 53 amino acid residues with three disulfide bonds [see, Cohen, S.J. Biol. Chem., 237, 1555-1562, 1962; Savage, C. R., J. Biol. Chem. 248, 7669-7672, 1973]. It has been reported that hEGF plays a very important role in controlling the growth of epidermal and skin cells in mammals [see, Sporn, M. B. et al., Nature (London) 313, 745-747, 1985; Sporn M. B. et al., N. Engl. J. Med. 303, 878-880, 1980]. It is also involved in the process of wound healing [see, Buckley, A. et al., Proc.

Nati Acad. Sci. USA., 82, 7340-7344 1985] at a molecular level. Since then, several studies related to the recovery of skin wounds have been conducted with hEGF. Although HEGF shows a good activity to stimulate the differentiation of epidermal cells in vitro, the development of formulations containing hEGF for the treatment of topical wounds is very difficult due to the disadvantage that hEGF exhibits only a small effect in the treatment of wounds when applied chemically to wounds The reasons why hEGF does not show the desired effect sufficient to treat wounds in the human body are that hEGF as the protein is very unstable, particularly the presence of water, at room temperature, and it has less than one hour in half a life, which is much shorter in time of delay for the induction of DNA synthesis of the cells at the wound site, which is about 8-12 hours [ see, J. Surg, Res. 43.333.1987.] In addition, when hEGF is applied to the skin, hEGF loses its biological activity due to denaturation and decomposition of hEGF by e.g. Proteolytic enzymes present at the site of the wound. In order to provide the desired wound healing effect of hEGF, it is continually required to maintain the effective level of hEGF by applying the hEGF to the wound site at any time during the few initial days that are most important for wound healing [ see, J. Surg. Res., 43, 333, 1987, J. Lab. Clin. Med., 108, 103, 1986]. In this regard, studies have been conducted to develop the sustained release formulation that can continuously deliver the hEGF to the wound site. As one of the results of such studies, US Patent Specification No. 4,944,948 describes a gel formula of hEGF / liposome using neutral phospholipids, negatively charged phospholipids and cholesterol, which can continuously deliver the hEGF to the wound site; and EP Publication No. EP 0312208 discloses the aqueous formulation comprising a number of water soluble or pharmaceutically acceptable water soluble polymers as the base for consistently releasing hEGF. However, although the aforementioned prior art publications describe formulas that can continuously release the hEGF for 12 hours or more, they are not suitable for use in industry, since they have the disadvantage of a small wound healing effect in the site of the wound. Particularly, in EP Publication No. 0312208 when preparing the gel formula using polymers, as the base of continuous release of hEGF for 12 hours or more, at a high concentration, the hEGF is slowly released from the gel and continuously supplied to the wound site, and furthermore the. The gel formulation itself can provide the wound healing effect by forming the coating at the wound site to create the proper moisture condition at the wound site and prevent the invasion of pathogenic organisms within the wound site. However, since this gel formulation has a very high viscosity of 1,000-12,000,000 cps at room temperature, when applied to the wound site with rubbing, such rubbing can cause irritation at the wound site. It is very difficult to apply the formulation to the wound having a large injured area and suffering from severe pain, such as a burn. In addition, the gel formulation forms a coating at the wound site to physically inhibit the migration of epidermal cells by mitogenic activity of hEGF thereby retarding the effect of hEGE on wound healing. Therefore, the gel formula of the prior art publication suffers from the serious disadvantage that when clinically applied to the site of the wound, the wound healing effect of hEGF can not be sufficiently provided. As mentioned above, since topical formulations of the prior art of hEGF do not exhibit the desired sufficient wound healing effect at the wound site, it is highly desirable to develop the topical preparation which may sufficiently exhibit the wound healing effect. of hEGF in the human body. Thus, the inventors of the present have conducted numerous studies to develop the topical preparation which can exhibit a sufficient wound healing effect in the human body. As a result, topical preparations of hEGF have been identified using a polyoxyethylene-polyoxypropylene copolymer having a viscosity of 4-10 cps at 37 ° C, 60 rpm which may exhibit the desired good wound healing effect of hEGF, and thus complete the present invention. Thus, the present invention relates to a composition containing hEGF for topical use. More specifically, the present invention relates to a topical composition comprising the hEGF as the active ingredient and a polyoxythylene-polyoxypropylene copolymer having a viscosity of 4-10 cps at 37 ° C, 60 rpm as the base for the topical preparation. In the present specification, the degree of viscosity is indicated by the dimensions of dynes / sec. per cm. This dimension referred to herein, unless otherwise indicated, is in centipoise (cps) as measured using a Brookfield viscometer. All viscosity values are measured at 37 ° C, 60 rpm unless otherwise indicated. The polyoxythylene-polyoxypropylene copolymer which is used as the base in the present invention has a viscosity of 4-10 cps, preferably 4-6 cps, at 37 ° C, 60 rpm. When the viscosity of the polyoxyethylene-polyoxypropylene copolymer is less than 4 cps, the physical healing effect that is inherent in the polyoxyethylene-polyoxypropylene copolymer is decreased and the release rate of the hEGF is too rapid to reduce its healing effect. wound. On the other hand, the viscosity of the polyoxyethylene-polyoxypropylene copolymer greater than 10 cps is also undesirable, since the polyoxyethylene-polyoxypropylene copolymer forms an excessive coating at the wound site that inhibits the migration of the epithelial cells stimulated by the hEGF , with this decreasing the effect of wound healing. In the present invention, when formulating hEGF in a topical preparation for the treatment of wounds using the polyoxyethylene-polyoxypropylene copolymer having a viscosity of 4-10 cps, the coating formed of the polymer does not serve as a physical barrier during the wound healing process and exhibits the inherent physical healing effect and also induces a sufficient wound healing effect by means of hEGF. Therefore, the present topical formula can exhibit a good wound healing effect when applied to the wound site. The viscosity of the polyoxyethylene-polyoxypropylene copolymer is determined depending on the temperature and the molecular weight and the content of the polymer. Thus, to obtain the viscosity of the polyoxyethylene-polyoxypropylene copolymer of 4-10 cps at 37 ° C, 60 rpm it is preferable to use a polyoxyethylene-polyoxypropylene copolymer having a molecular weight of about 10,000 to 15,000 in a ratio of 10% based on weight to the total weight of the composition or use a polyoxyethylene-polyoxypropylene copolymer having a molecular weight of about 7,000 to 10,000 in a ratio of 7.5-15% by weight based on the total weight of the composition. More preferably, it is suitable to adjust the viscosity within the range of 4-6 cps using a polyoxyethylene-polyoxyprspylene copolymer having a molecular weight of about 10,000 to 15,000 in the proportion of 5-7.5% by weight based on the total weight of the composition or using a polyoxyethylene-polyoxypropylene copolymer having a molecular weight of about 7,000 to 10,000 in the proportion of 7.5-12.5% by weight based on the total weight of the composition. . In the topical composition according to the present invention, either the naturally occurring hEGF or the recombinant hEGF can be used as the active ingredient hEGF. The content of the hEGF in the composition is preferably 0.01-1,000 μg / ml, particularly 1-100 μg / ml. Since the topical composition of the present invention compounded as mentioned above is present in a liquid state at room temperature, the hEGF as the pharmacologically active ingredient can be uniformly dispersed in a polyoxyethylene-polyoxypropylene copolymer base. Therefore, contrary to the gel formulations of the prior art, when the composition of the present invention is applied to the wound site, it exhibits a good permeation of active ingredient within the wound site, and can evenly spread over the wound site. - site of the wound to increase contact of skin area with hEGF, with this providing a wound healing effect - excellent. In addition, the composition of the present invention also has the advantage that since it is in a liquid state having a low viscosity, it can be formulated into an aerosol preparation which can be applied to the wound site by simply spraying without rubbing, and therefore, does not cause a physical irritation to the wound. The composition according to the present invention can additionally contain pharmaceutically acceptable additives which can be conventionally used in preparing the topical preparation, for example, a stabilizer, excipient, etc., in addition to the active ingredient of hEGF and the polyoxyethylene-polyoxypropylene copolymer as the base . If required, the composition of the present invention can be stored in a lyophilized form and then dissolved in a suitable solvent when used. Therefore, the composition of the present invention can be stored stably for a long period of time. The present invention is explained more specifically by the following examples and experiments. However, it should be understood that the present invention is not limited to these examples in any way. Example 1 hEGF 5 mg Poloxamer 188 10 g methyl paraoxybenzoate 200 mg sodium hydrogen phosphate 340.23 mg sodium chloride 832.77 mg phosphoric acid q.s. purified water q.s. Total 100 ml The solution was prepared using the aforementioned components in the amounts given according to the conventional method. Specifically, a phosphate buffer was prepared from sodium hydrogen phosphate, sodium chloride and phosphoric acid. Poloxamer 188 was added to the resulting phosphate buffer and dispersed therein with stirring. Then, methyl paraoxybenzoate as preservative and hEGF as an active ingredient were added to it to obtain 100 ml of the topical solution according to the present invention. "Example 2 hEGF 5 mg Poloxamer 407 7.5 g methyl paraoxybenzoate 200 mg sodium hydrogen phosphate 340.23 mg sodium chloride 832.77 mg phosphoric acid cs purified water cs Total 100 ml The buffer of phosphate was prepared using phosphates of sodium hydrogen, sodium chloride and phosphoric acid in a given amount, Poloxamer 407 was added to the resulting phosphate buffer and dispersed therein with stirring, followed by methyl paraoxybenzoate as preservative and hEGF. as an active ingredient were added thereto to obtain 100 ml of the topical solution according to the present invention Experiment 1 Comparison of the wound healing effect of hEGF depending on the type of base in an animal wound model. rat torsos and a hair removal agent was spread (Neet Cream produced by II Dong Pharm.) to r Then, streptomycin (produced by Chong Kun Dang Corp.) was injected by intramuscular route in a dose of 0.5 g / kg to the rats. The rats were anesthetized by injecting sodium pentobarbital at a dose of 40 mg / kg via an intraperitoneal route. The wound was induced by removing epidermis and dermis by means of surgical scissors in an area having a diameter of 10 mm. Next, considering that the site of the wound is open, the size of the wound after one day of the wound induction was __ taken as the initial wound area. Depending on the types of bases used, the test animals were divided into a total of 11 groups-a control group without treatment, the experimental groups (Poloxamer + hEGF group, Polyvinylpyrrolidone + hEGF group, Dextran + hEGF group, Gelatin + hEGF group and Polymethacrylamine + hEGF group), and another control group to which only the base was applied (Poloxamer group, Polyvinylpyrrolidone group, Dextran group, Gelatin group and Polymethacrylamide group). After one day of wound induction, the wound site of each test animal was treated twice daily with the test drug in an amount of 0.5 ml each time. In the test groups, the preparation wherein the hEGF is contained in a proportion of 5 mg per 100 ml of the solution, as prepared according to the same procedure as in Example 2, except that the type and amount of base They were varied, it was used, the test drug. In the base control groups, the preparation was prepared according to the same procedure as in Example 2, except that the hEGF that was excluded was used. The wound was treated with the test drugs and the cutaneous wound area was measured every day. Next, the proportion of wound remaining for the days of treatment was obtained by calculating the proportion of the wound area measured according to the days of treatment compared to the initial measured area as measured after the wound induction. The proportion of wound remaining for the days of treatment and the days of treatment were analyzed by a linear regression to calculate 50% of the healing time (HT50) which is the days that exhibit 50% of proportion of remaining wound. The result obtained is described in the following Table 1. Table 1 Comparison of the days of wound treatment depending on the types of polymeric bases soluble in water (unit: days) Note: Poloxamer is the generic name of the polyoxyethylene-polyoxypropylene copolymer and is Poloxamer 407 having an average molecular weight of 10,000-15,000. As can be seen in "Table 1 above, when -Poloxamer 407 is used the polyoxyethylene-polyoxypropylene copolymer as the base in an amount of 5-10% by weight according to the present invention, shows a healing effect of excellent wounds with days of wound healing of 3.3-3.8 days., the use of the remaining water soluble polymer bases provides only a slight healing effect. Experiment 2 Wound healing effect of hEGF depending on the content of polyoxyethylene-polyoxypropylene copolymer in an animal wound model. To determine the wound healing effect of hEGF depending on the content of polyoxyethylene-polyoxypropylene copolymer that was identified as exhibiting the best wound healing effect in Experiment 1, the test preparation produced according to the procedure of Example 2 was used according to the same method as Experiment 1. The test animals were divided into untreated control group, group that has Poloxamer at 2.5, 5.0, 7.5, 10.0 and 12.5% by weight, and the groups containing Poloxamer in a respective content and hEGF as an active ingredient. In this way, the wound healing effect was determined depending on the content of the base.

Table 2 Comparison of wound healing days depending on the polyoxyethylene-polyoxypropylene copolymer (unit: days) As can be seen in Table 2 above, when the content of polyoxyethylene-polyoxypropylene copolymer is 5-10% by weight, it exhibits a good wound healing effect with days of wound healing of 3.1-3.8 days. Particularly, when the content of polyoxyethylene-polyoxypropylene copolymer is 5-7.5% by weight, it exhibits an excellent wound healing effect with days of wound healing of 3.1-3.3 days. • From the results obtained in Experiments 1 and 2 above, it can be identified that among several polymers that can be conventionally used as the base for the topical preparation, only the polyoxyethylene-polyoxypropylene copolymer in an amount of 5-10% in pesb, particularly 5-7.5% by weight can induce an excellent wound healing effect of hEGF.

Experiment 3 Viscosity of the polyoxyethylene-polyoxypropylene copolymer depending on molecular weight As can be seen in Experiments 1 and 2 above, the wound healing effect varied widely depending on the content of polyoxyethylene-polyoxypropylene copolymer. In addition, the viscosity of the polyoxyethylene-polyoxypropylene copolymers can be determined depending on their molecular weight and content, and temperature. Therefore, to identify the optimum content depending on the molecular weight of the polyoxyethylene-polyoxypropylene copolymers, the viscosity of the preparation containing Poloxamer 407 (average molecular weight: about 10,000-15,000), which shows the wound healing effect more excellent, in an amount of 5-10% eh weight and the content of Poloxamer 188 (average molecular weight: approximately 7,000-10,000) as low molecular weight polyoxyethylene-polyoxypropylene copolymers, which has the same viscosity as in the case of Poloxamer 407, were determined by measuring the viscosity in each case according to the following procedure. The viscosity was measured by means of a Brookfield Synchro-Lectric Viscometer (Model: RVF, U.S.) and the test preparations were prepared according to the following methods. Poloxamer was accurately taken depending on its content and slowly introduced into a laboratory beaker containing distilled water with agitation. Agitation continued until the Poloxamer completely dissolved. The resulting test solution was allowed to stand for 5 hours and then approximately 200 ml of the solution was taken, placed in an incubator at 25 ° C at 37 ° C before the experiment and then used. The viscometer was fitted vertically in the container containing the solution, it was rotated for 3 minutes at a speed of 60 rpm and then the numerical value was recorded in the graduation of the viscometer. The experiment was repeatedly conducted three times and the average value was obtained. Table 3 Viscosity of the polyoxyethylene-polyoxypropylene copolymer depending on its content (unit: cps) As can be seen in Table 3 above, when the content of Poloxamer 407 showing a good wound healing effect is 5-10% by weight, the viscosity was measured as 4-10 cps, and in case of preferred content of 5-7.5% by weight the viscosity was 4-6 cps. Therefore, as the basis for the topical preparation of hEGF for treating a wound, it is preferable to use the polyoxyethylene-polyoxypropylene copolymer having a viscosity of 4-10 cps, more preferably 4-6 cps, at 37 ° C. In addition, the content of a Poloxamer 188 of low molecular weight showing the same viscosity as in the case where content of Poloxamer 407 of high molecular weight is 5-10% by weight at 37 ° C was 7.5-15% by weight . Also the content of Poloxamer 188 showing the same viscosity as in the case where the most preferable content of Poloxamer 407 is 5-7.5% by weight at 37 ° C was 7.5-12.5% by weight. Thus, it can be identified that to obtain the preferable viscosity it is better to use a Poloxamer 188 of low molecular weight in an amount of 7.5-15% by weight and more preferably 7.5-12.5% by weight. From the results obtained in the above experiments, it can be seen that among several polymers that can be used as the base in a topical preparation to control the release rate of the active ingredient and physically protect the site of the wound, the polyoxyethylene copolymer -polyoxypropylene having a viscosity of 4-10 cps, particularly 4-6 cps, at 37 ° C, 60 rpm can be used according to the present invention to provide the topical preparation exhibiting a good wound healing effect.

Claims (7)

1. CLAIMS 1. A topical composition characterized in that it comprises an effective wound healing amount of human epidermal growth factor, and a polyoxyethylene-polyoxypropylene copolymer having a viscosity of 4-10 cps at 37 ° C, 60 rpm.
2. 2. The topical composition according to claim 1, characterized in that the human epidermal growth factor is contained in an amount of 0.01 to 1000 μg / ml. '
3. 3. The topical composition according to claim 1, characterized in that the viscosity of the polyoxyethylene-polyoxypropylene copolymer at 37 ° C, 60 rpm is 4-6 cps.
4. 4. The topical composition according to claim 1, characterized in that a polyoxyethylene-polyoxypropylene copolymer is a copolymer having a molecular weight of 10,000 to 15,000 is used in an amount of 5 to 10% by weight.
5. 5. The topical composition according to claim 1, characterized in that a polyoxyethylene-polyoxypropylene copolymer is a copolymer having a molecular weight of 10,000 to 15,000 is used in an amount of 5 to 7.5% by weight.
6. 6. The topical composition according to claim 1, characterized in that a polyoxyethylene-polyoxypropylene copolymer is a copolymer having a molecular weight of 7,000 to 10,000 is used in an amount of 7.5 to 15% by weight.
7. 7. The topical composition according to claim 1, characterized in that a polyoxyethylene-polyoxypropylene copolymer is a copolymer having a molecular weight of 7,000 to 10,000 is used in an amount of 7.5 to 12.5% by weight.