Title of Invention

SUSTAINED RELEASE FILM FORMULATION FOR HEALING WOUND COMPRISING EPIDERMAL GROWTH FACTOR

Abstract

ABSTRACT The present invention relates to a sustained release film formulation for healing wound comprising epidermal growth factor, chitosan, viscosity modifiers, plasticizers, and stabilizers. Specifically, the present invention relates to a sustained release film formulation for healing wound comprising epidermal growth factor as an effective ingredient and chitosan as a main base, and additionally comprising one or more antioxidants selected from the group consisting of EDTA and vitamin C; one or more viscosity modifiers selected from the group consisting of hydroxypropylmethylcellulose, gellan gum and pullulan; and one or more plasticizers selected from the group consisting of glycerin, propylene glycol, polyethylene glycol, polyvinyl alcohol and polyvinylpyrrolidone thereto. When the present film is attached to wound site, it absorbs exudation from wound site, and so is changed to hydrogel to keep wound site humid, which is good for wound-healing. And, the present film has superior attachment property to human body, and antifungal activity. Thus, the present film has more merits than simple ointment/cream formulation.

Full Text

SUSTAINED RELEASE FILM FORMULATION FOR HEALING WOUND COMPRISING EPIDERMAL GROWTH FACTOR TECHNICAL FIELD The present invention relates to a sustained release film formulation for healing wound comprising epidermal growth factor; chitosan; one or more viscosity modifiers selected from the group consisting of hydroxypropylmethylcellulose, gellan gum and pullulan; one or more piasticizers selected from the group consisting of glycerin, propylene glycol, polyethylene glycol, polyvinyl alcohol and polyvinylpyrrolidone; and one or more antioxidants selected from the group consisting of EDTA and vitamin C. BACKGROUND ART Epidermal growth factor is polypeptide whose molecular weight is 6045, having three disulfide bonds and fifty-three (53) amino acids known as urogastrone. The factor is known to have such activities as mitosis promotion and cell growth promotion of various cells including epidermal cell and mesenchymal cell, inhibition of gastric acid secretion, etc., and so be effective for treating skin or cornea wound, or gastric ulcer (Carpenter, Experimental Cell Research, 164:1-10, 1986). However, epidermal growth factor is too unstable at room temperature, particularly, in the presence of water. And, the latent period of 8 to 12 hrs is required in order to induce cells to synthesize DMA at wound site. However, the latent period of epidermal growth factor is about 1 hr or less, which is too short to achieve desired effect. Also, the epidermal growth factor is difficult to apply to external skin wound since the factor is denaturalized, decomposed, condensed and precipitated by proteolytic enzyme existing at wound site when applied to skin, and so loses biological activity (Manning et al.. Pharmaceutical Res.,: 903-917, 1989). To overcome biological instability among the above drawbacks of epidermal growth factor, and sufficiently maintain therapeutic effect, it was reported that an effective concentration of epidermal growth factor should be consistently maintained at wound site (Franklin et al., J. Lab. Clin. Med., 108:103-108, 1986). Thus, many studies have been made to develop a sustained release formulation which can consistently provide epidermal growth factor into wound site. USP No. 4,944,948 discloses epidermal growth factor/liposome gel formulation which consistently provides epidermal growth factor to wound site by using neutral phospholipids, negatively charged phospholipids, and cholesterol. In particular, this invention provides a method for consistently releasing epidermal growth factor for 12 hrs or more, but the method has limits in attaining storage stability, and so is difficult to apply in the industry. Therefore, to show a sufficient therapeutic effect, epidermal growth factor as drug should retain consistent drug-releasing property, with maintaining biological activity and physicochemical stability related to constant purity and content, under long term storage. Also, EP No. 267015 discloses a film prepared by using epidermal growth factor with hydrophiiic cellulose polymer, particularly, with hydroxypropylmethylcellulose used in the present invention. However, the film has a problem that the effect of epidermal growth factor is not significant enough to heal wound. DITAILED DESCRIPTION OF THE INVENTION TECHNICAL SUBJECT The object of the present invention is to provide a sustained release film formulation for healing wound comprising epidermal growth factor which can consistently release main ingredient for 1 week, with maintaining the biological activity and physicochemical stability of epidermal growth factor, by using chitosan having antibacterial activity as main base for maximizing would-healing effect of epidermal growth factor; optionally hydroxypropylmethyicellulose, gellan gum, pullulan, etc. as film base for consistently releasing main ingredient with maintaining stability of epidermal growth factor; antioxidant such as EDTA, vitamin C, etc. as stabilizing agent; and glycerin, propylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, polyethylene glycol, etc. as plasticizer. When the film according to the present invention is applied to wound site, the dried film absorbs exudation to be changed to hydrogel, and so can be easily attached to living body. Also, the film can maximize the therapeutic effect of epidermal growth factor, and can keep the effective therapeutic concentration only by one application a day since the main base, chitosan, itself has antibacterial activity. BRIEF DESCRIPTION OF THE DRAWINGS Figs. 1 to 3 are graphs showing in vitro release rate of the film for healing wound comprising epidermal growth factor and chitosan according to the present invention, for one day. Fig. 4 is a graph showing production rates of impurities of the film for healing wound comprising epidermal growth factor and chitosan according to the present invention, under the cold-storage condition for a year. Figs. 5 and 6 each are photographs of wound site before and after treatment of the control group, and the film for healing wound comprising epidermal growth factor and chitosan according to the present invention. MODE FOR CARRYING OUT THE INVENTION The present invention relates to a sustained release film formulation for healing wound comprising epidermal growth factor; chitosan; one or more viscosity modifiers selected from the group consisting of hydroxypropylmethylcellulose, gellan gum and pullulan; one or more plasticizers selected from the group consisting of glycerin, propylene glycol, polyethylene glycol, polyvinyl alcohol and polyvinylpyrrolidone; and one or more antioxidants selected from the group consisting of EDTA and vitamin C And, the present invention relates to a film formulation characterized in * additionally comprising form type of dressing or hydrocolloidal type of dressing to the above sustained release film formulation. The epidermal growth factor which is an active ingredient of the sustained release film formulation of the present invention can be applied to either natural protein or recombination protein. The epidermal growth factor in the present film formulation is used in an amount of 0.1 to 10,000µg/g, more preferably 1 to 5,000µg/g which is a pharmacologically effective concentration. If the content of epidermal growth factor is less than 0.1µg/g, the film formulation is not pharmacologically effective. If the content is more than 10,00Oµg/g, the film formulation may be toxic. Here, the pH of the film formulation is preferably adjusted to 5 to 7, which is the range to maintain the stability of epidermal growth factor. Chitosan which is the main base of the present invention is preferably a polymer having 10 to 150 kDa of molecular weight. If the molecular weight is smaller than lOkDa, chitosan cannot keep the viscosity as hydrogel. If the molecular weight is more than 150kDa, the viscosity of polymer solution is so high that the preparation is very difficult. Also, chitosan in the film formulation is preferably used in an amount of 1.0 to 10.0 w/w% based on the total weight. If the amount of chitosan is out of said range, the viscosity is so low that the preparation is difficult. Antioxidant used for stabilizing epidermal growth factor is preferably one or more selected from the group consisting of EDTA and vitamin C. In the present invention, antioxidant is preferably used in an amount of 0.01 to 10 w/w% based on the total weight, more preferably, 0.05 to 5 w/w%. If the amount is out of said range, it is difficult to achieve the stability of epidermal growth factor. Also, preferably, the present film formulation may additionally comprise one or more buffers selected from the group consisting of sodium hydrogen phosphate, sodium hydrogen carbonate, and sodium citrate; or one or more amino acids selected from the group consisting of L-histidine, L-lysine and L-arginine for stabilizing the formulation. In the present invention, viscosity modifiers may be preferably one or more selected from the group consisting of hydroxypropylmethylcellulose, gellan gum and puUulan. Here, hydroxypropylmethylcellulose may be classified into low-viscosity hydroxypropylmethylcellulose and high-viscosity hydroxypropylmethylcellulose according to viscosity. The low-viscosity hydroxypropylmethylcellulose functions as a film auxiliary base, and the high-viscosity hydroxypropylmethylcellulose plays a role to control sustained release. Therefore, it is preferable to use low-viscosity hydroxypropylmethylcellulose of 10 to 100 cps and high-viscosity hydroxypropylmethylcellulose of 1,000 to 100,000 cps in mixture. The viscosity modifier is preferably used in an amount of 0.01 to 20.0 w/w%, more preferably, 0.01 to 5 ) w/w%. If the amount is out of said range, the viscosity of base before drying is affected enough to cause a problem in the preparation. The sustained release film formulation of the present invention additionally comprises one or more plasticizers selected from the group consisting of glycerin, polypropylene glycol, polyvinylpyrrolidone and polyethylene glycol. The amount of plasticizer is preferably 1 to 40 w/w%, more preferably, 1 to 20 w/w%. If the amount of plasticizer is less than 1 w/w%, the film would get loose from the coating paper. If the amount is more than 40 w/w%, the drying time and total weight are increased to facilitate gelation, which may cause a problem during the process. To the sustained release film formulation of the present invention, water is added to make the total volume 100 w/w%. Form type of dressing or hydrocolloidal type of dressing known in the art may be added to the film formulation according to the present invention. Also, to keep the film shape and prevent external pollution, a dressing additionally comprising silver or alginate may be added to form type of dressing or hydrocolloidal type of dressing. The sustained release film formulation for healing wound according to the present invention may be prepared by the following methods. Chitosan, a viscosity modifier, and a plasticizer are put into a suitable container. Then, distilled water is added thereto, and stirred to be completely homogenized. Antioxidant as a stabilizer, and epidermal growth factor are added thereto, and homogeneously mixed to prepare a composition for preparing the present film. Here, the viscosity of the composition is preferably 1,000 to 150,000 cps, more preferably, 4,000 to 120,000 cps. If the viscosity is out of said range, the preparation itself would be difficult because the casting is difficult. The composition as prepared above is kept in a refrigerator of 4-10 for about a day, and then is cast onto coated paper, and dried to prepare the sustained release film formulation for healing wound. The thickness of the film prepared above is preferably 0.001 to 1 mm which is the range easy for the preparation and treatment, more preferably, 0.01 to 0.5 mm. The content of water in the film, which is an important factor to affect the stability of epidermal growth factor and the preparation condition of film, is preferably 1 to 30 %, more preferably, 10 to 25 %. Hereinafter, the present invention is specifically described by examples. However, the examples are provided to explain the present invention, and so the scope of the present invention is not limited by them in any manner. The sustained release film formulation of the present invention comprising epidermal growth factor, chitosan, viscosity modifier, plasticizer and stabilizer was prepared by the following process: Chitosan, hydroxypropylmethylcellulose (HPMC), one or more viscosity modifiers selected from the group consisting of gellan gum and pullulan, and one or more plasticizers selected from the group consisting of glycerin, polypropylene glycol, polyvinylpyrrolidone (PVP), and polyethylene glycol (PEG) were put into a suitable container. Then, distilled water was added thereto, and stirred to be completely homogenized. One or more antioxidants selected from the group consisting of EDTA and vitamin C as stabilizer, and recombinant epidermal growth factor, i.e., lyophilized epidermal growth factor formulation (Daewoong Co.), were added thereto and homogeneously mixed. Then, the mixture was kept in a refrigerator of 4-10 for about a day, and then was cast onto coated paper, and ventilated or lyophilized to prepare the sustained release film formulation for healing wound according to the present invention. To analyze the physicochemical property of the sustained release film formulation according to the present invention by ingredients, films (DWFl to DWF6) comprising epidermal growth factor were prepared by using the ingredients described in the following Table 1 according to the same method as Example I. Only, the thickness of films was 1.0mm when the film is cast, and the method of dryness was ventilation at 40 for 1 hr. The purity of epidermal growtii factor in DWFl to DWF6 prepared above was determined by reverse phase HPLC. And, the property and condition, thickness, and moisture content of the film prepared above were measured, and the results were shown in the following Table 2. As shown in the above Table 2, it was confirmed that the films prepared by mixing other polymers such as HPMC, glycerine, PEG, and PVP (DWF2-DWF6) have more superior property and condition than DWFl film prepared by chitosan only. Only, when gelatin was used as polymer (DWF4), the epidermal growth factor was degraded to result in decreasing the purity of epidermal growth factor during the process of increasing the temperature at the time of preparing the film base. In particular, when hydroxypropylmethylcellulose (HPMC) was used as a viscosity modifier, the epidermal growth factor was most superior in the property and condition, and purity. viscosity and thickness of film> To confirm the release property of epidermal growth factor according to the viscosity of film and the contents of main ingredients, the films (DWF7 to DWF14) were prepared by using the ingredients described in the following Table 3 according to the same methods as Example 1 and Example 2. DWF13 and DWF14 films were prepared in different casting thicknesses, and the viscosity and thickness of each film were measured and shown in the following Table 3. To analyze each release amount of epidermal growth factor from DWF7 to DWF12 films prepared above, the release test was conducted. The results were shown in the following Table 4 and Figs. 1 and 2. The release test was conducted by using Franz diffusion apparatus. The release medium was filled with phosphoric acid buffer (pH 7.4, 0.02% Tween 20), kept at 32 ± 0.5 , and set to directly contact to buffer without membrane. Thus prepared films were cut to the test size for application. The mixtures of 300µl each were taken from the films at the designated times of 1, 3, 6, 9, 15, 18 and 24 hrs. Then, the release amounts of EGF were determined by HPLC, and the determined values were shown as release % to initial value. As shown in Table 4 and Fig. 1, it was confirmed that the higher the viscosity of ■) film is, the more the release of epidermal growth factor is delayed. Also, as shown in Fig. 2, it was confirmed that the release amount of epidermal growth factor from DWF12 (l.Omg) containing the smallest amounts of main ingredients were less than those from DWFli (5.Omg), whereby it was concluded that the smaller the amounts of main ingredients are, the more the release is delayed. This confirmed that the release rate of epidermal growth factor is controlled by the viscosity and amount, and so the epidermal growth factor shows linear release for 24 hrs. Also, the release amounts of epidermal growth factor from DWF13 and DWF14 were measured, and shown in the following Table 5 and Fig. 3. As shown in Table 5, it was confirmed that the thicker the film is, the more the release rate of epidermal growth factor is delayed. In sum, it was confirmed that the release rate of epidermal growth factor is controlled by the viscosity and thickness of film. formulation of the present invention according to the addition of stabilizer, the content and activity of epidermal growth factor by time were measured from the films comprising epidermal growth factor, chitosan, viscosity modifier and plasticizer for each case that stabilizer is not added (DWF 15); only antioxidant is added (DWF 17 and 18); only amino acid is added (DWF 16); antioxidant and amino acid are added (DWF 19); and antioxidant and buffer are added (DWF 20). For this experiment, DWF15 to DWF20 films were prepared by using the ingredients described in the following Table 6 according to the same methods as Example 1 and Example 2. DWF15 to DWF20 films prepared above were sealed airtight by aluminum packing paper, and then stored at room temperature (25 ) for 3 months, and at cold temperature (2-8 ) for 1 year. The content and activity of epidermal growth factor in each film were measured by time, and shown in the following Table 7 and Fig. 4. As shown in the above Table 7, it was confirmed that the production rate of impurities of other films than DWF15 which does not comprise stabilizer was very low. Also, the stabilities of DWF16 film comprising amino acid, and DWFI7 and DWF18 films comprising antioxidant were a little better. DWFl 9 film comprising both amino acid and antioxidant, or DWF20 film comprising both antioxidant and buffer showed the most superior stability. To confirm the pharmacological effect of epidermal growth factor in the sustained release film formulation of the present invention, the comparison test was conducted by using the epidermal growth factor film prepared with hydroxypropyicelluiose as control group, and DWF20 film described in Table 6 of Example 4 prepared by the same method as Example 1 as test group. The conditions of the comparison test were shown in the following Table 8. The photograph of wound site after 1 day from the wound was shown in Fig. 5, and the photographs of wound site after 4 days from treating the control and test groups were shown in Fig. 6. As shown in the figures, it was confirmed that the rat's wound treated with the present film comprising epidermal growth factor, chitosan, viscosity modifier, plasticizer, antioxidant and buffer was healed much faster than that treated with the control. The above result was from that EGF was consistently released from the present film, and an effective concentration at wound site was maintained, by treating the present sustained film once, compared with treating the control twice. Also, the treatment effect became better by increasing the antifungal activity through adding chitosan. INDUSTRIAL APPLICABILITY The present invention can maximize wound-healing effect by providing a sustained release film formulation comprising chitosan, viscosity modifier, plasticizer, and stabilizer, in addition to epidermal growth factor as effective ingredient. And, the present sustained film formulation can maintain an effective concentration by once-a-day attachment to wound site unlike ointment/cream formulation or gel formulation which should be applied twice or three times a day. Thus, the present film is not required to be removed from wound site to lower the exposure of wound site, and provide patients and medical workers more convenience. Also, the present film absorbs exudation, and so is changed to hydrogel to keep wound site humid, and make a favorable condition for wound-healing. And, the present film has superior attachment property to human body, antifungal activity, and stability under cold-storage condition for one or more years. Thus, the present film has more advantages than simple ointment/cream formulation or gel formulation. WHAT IS CLAIMED IS 1. A sustained release film formulation for healing wound comprising epidermal growth factor; chitosan; one or more viscosity modifiers selected from the group consisting of hydroxypropylmethylceilulose, gellan gum and puliulan; one or more plasticizers selected from the group consisting of glycerin, propylene glycol, polyethylene glycol, polyvinyl alcohol and polyvinylpyrrolidone; and one or more antioxidants selected from the group consisting of EDTA and vitamin C. 2. The film formulation according to claim 1, additionally comprising one or more buffers selected from the group consisting of sodium hydrogen phosphate, sodium hydrogen carbonate and sodium citrate. 3. The film formulation according to claim 1, additionally comprising one or more amino acids selected from the group consisting of L-histidine, L-lysine and L-arginine. 4. The film formulation according to claim 1, wherein the molecular weight of chitosan is 10 kDa to 150 kDa. 5. The film formulation according to claim 1 or 4, wherein the content of chitosan is 1 to 10 w/w% based on the total weight. 6. The film formulation according to claim 1, wherein the content of epidermal growth factor is O.I to 10.000 g/g based on the total weight. 7. The film formulation according to claim 1, wherein the hydroxypropylmethylcellulose is one or more selected from those whose viscosity is 5 to 100,000 cps. 8. The film formulation according to claim I or 7, wherein the hydroxypropylmethylcellulose comprises low-viscosity hydroxypropylmethylcellulose of 10 to 100 cps and high-viscosity hydroxypropylmethylcellulose of 1,000 to 100,000 cps. 9. The film formulation according to claim 1 or 7, wherein the content of viscosity modifier is 0.01 to 20 w/w % based on the total weight. 10. The film formulation according to claim 1, wherein the content of plasticizer is 1 to 40 w/w% based on the total weight. 11. The film formulation according to claim 1, wherein the content of antioxidant is 0.01 to 10 w/w% based on the total weight. 12. The film formulation according to claim 1, wherein the viscosity of film formulation is 1,000 to 150,000 cps. 13. The film formulation according to claim 1, wherein the thickness of film is 0.001 to 1 mm. 14. The film formulation according to any one of claims 1 to 3, additionally comprising form type of dressing or hydrocolloidal type of dressing. 15. The film formulation according to claim 14, further adding silver or alginate to form type of dressing or hydrocolloidal type of dressing.

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2353-CHENP-2008 AMENDED CLAIMS 27-02-2013.pdf

2353-CHENP-2008 AMENDED PAGES OF SPECIFICATION 27-02-2013.pdf

2353-CHENP-2008 EXAMINATION REPORT REPLY RECEIVED 27-02-2013.pdf

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2353-CHENP-2008 OTHERS 27-02-2013.pdf

2353-CHENP-2008 CORRESPONDENCE OTHERS 23-04-2012.pdf

2353-chenp-2008 abstract.pdf

2353-chenp-2008 claims.pdf

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2353-chenp-2008 description(complete).pdf

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