HK1036221B - Topical composition containing human epidermal growth factor - Google Patents
Topical composition containing human epidermal growth factor Download PDFInfo
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- HK1036221B HK1036221B HK01106872.9A HK01106872A HK1036221B HK 1036221 B HK1036221 B HK 1036221B HK 01106872 A HK01106872 A HK 01106872A HK 1036221 B HK1036221 B HK 1036221B
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- hegf
- polyoxyethylene
- wound
- polyoxypropylene copolymer
- viscosity
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Description
Technical Field
The present invention relates to a topical composition containing human epidermal growth factor (hereinafter referred to as "hEGF"). More particularly, the present invention relates to a topical composition comprising a polyoxyethylene-polyoxypropylene copolymer (trade name: Poloxamer) having a viscosity of 4-10cps at 37℃ and 60rpm as a base for a topical formulation of naturally occurring hEGF or recombinant human epidermal growth factor (rhEGF). The topical compositions of the present invention are useful for treating skin lesions, including wounds, incisions, burns, and the like.
Background
hEGF is a polypeptide consisting of 53 amino acid residues with 3 disulfide bonds [ see: cohen, s., "journal of biochemistry" 237: 1555 and 1562, 1962; savage, c.r., journal of biochemistry 248: 7669 and 7672, 1973. hEGF has been reported to play a very important role in controlling epidermal and skin growth in mammals [ see: sporn, m.b. et al, nature (london) 313: 745 and 747, 1985; sporn m.b. et al, new england journal of medicine 303: 878-880, 1980). It is also related to the healing process of wounds at the molecular level [ see: buckley, a. et al, "proceedings of the american academy of sciences", 82: 7340 7344, 1985). Since then, a number of studies have been conducted on the healing of skin wounds with hEGF.
Although hEGF exhibits good activity for stimulating epidermal cell differentiation in vitro, it is very difficult to develop an hEGF-containing preparation for treating a surface wound because hEGF shows only a little effect for treating a wound when it is clinically applied to a wound. The reason that hEGF does not exhibit the desired adequate wound treatment in vivo is that hEGF is very unstable as a protein at room temperature, particularly in the presence of water, with a half-life of less than 1 hour, much shorter than the lag time required to induce cellular DNA synthesis at the wound site, which is about 8-12 hours (see: journal of surgical research 43: 333, 1987 ]. Furthermore, when hEGF is applied to the skin, hEGF loses its biological activity due to the denaturing and degrading effects of proteases present at the wound site.
To provide the desired wound healing effects of hEGF, effective levels of hEGF need to be continuously maintained by applying hEGF to the wound site over the first few days which are most important for wound healing (see: journal of surgical research 43: 333, 1987, journal of experimental and clinical medicine 108: 103, 1986). In this regard, research has been conducted in an attempt to develop sustained release formulations that continuously release hEGF to the wound site.
As a result of one of these studies, U.S. patent specification No. 4,944,948 discloses an hEGF/liposome gel formulation using neutral phospholipids, negatively charged phospholipids and cholesterol, which is capable of continuously delivering hEGF to a wound site; EP publication No. EP 0312208 discloses aqueous formulations comprising various water-soluble or water-swellable polymers pharmaceutically acceptable as a base to stably release hEGF.
However, although the above prior publications disclose preparations that can release hEGF for 12 hours or more continuously, they are not suitable for industrial use because they have a disadvantage in that there is only a little wound healing effect at the wound site.
In EP publication 0312208 in particular, a gel formulation is prepared which can release hEGF for 12 hours or more continuously by using a polymer as a matrix, from which hEGF is slowly released and continuously released to a wound site at a high concentration, and which itself can provide a wound healing effect by forming a coating on the wound site to form a suitable moist condition at the wound site and prevent pathogenic organisms from invading the wound site. However, since the viscosity of the gel formulation at room temperature is very high, up to 1000-12,000,000cps, when it is applied to a wound site by friction, the friction may cause irritation at the wound site. It is difficult to apply the preparation to wounds having large-area injuries and suffering from severe pain, such as burn wounds. In addition, the gel formulation forms an over-coating at the wound site, physically inhibiting the migration of epidermal cells through the mitogenic activity of hEGF, thereby retarding the effect of hEGF on wound healing. Therefore, the gel formulation disclosed in the prior publication has serious drawbacks, namely: when clinically applied to a wound site, the wound healing effect of hEGF is not sufficiently achieved.
As described above, since the topical preparations of hEGF of the prior art do not exhibit the desired sufficient wound healing effect at the wound site, it is highly desirable to develop a topical preparation that can sufficiently exhibit the wound healing effect of hEGF in the living body.
Accordingly, the present inventors have conducted extensive studies to develop a topical preparation that can exhibit sufficient wound healing effects in a living body. As a result, we have determined that a topical formulation of hEGF using a polyoxyethylene-polyoxypropylene copolymer having a viscosity of 4-10cps at 37 ℃ and 60rpm shows a desired good wound healing effect of hEGF, thereby completing the present invention.
Disclosure of the invention
Accordingly, the present invention relates to a composition comprising hEGF for topical use.
More particularly, the present invention relates to a topical composition comprising hEGF as an active ingredient and a polyoxyethylene-polyoxypropylene copolymer having a viscosity of 4-10cps at 37 ℃, 60rpm as a base for a topical formulation.
In the present specification, the viscosity is expressed by the dimension "dyne/sec/cm2"indicates. Unless otherwise indicated, the dimensions referred to herein are measured in centipoise (cps) using a Brookfield viscometer. All viscosity values were measured at 37 ℃ and 60rpm, unless otherwise stated.
Best Mode for Carrying Out The Invention
The polyoxyethylene-polyoxypropylene copolymer used as a base in the present invention has a viscosity of 4-10cps, preferably 4-6cps at 37 ℃ and 60 rpm. When the viscosity of the polyoxyethylene-polyoxypropylene copolymer is less than 4cps, the inherent physical healing effect of the polyoxyethylene-polyoxypropylene copolymer is reduced, and the release rate of hEGF is too fast, thereby reducing its wound healing effect. On the other hand, a polyoxyethylene-polyoxypropylene copolymer having a viscosity of more than 10cps is also undesirable because the polyoxyethylene-polyoxypropylene copolymer forms an excessive coating on the wound site, inhibiting migration of epidermal cells stimulated by hEGF, thereby reducing the wound healing effect.
In the present invention, hEGF is formulated into a topical preparation for treating a wound by using a polyoxyethylene-polyoxypropylene copolymer having a viscosity of 4-10cps, and a coating layer formed from the polymer does not act as a physical barrier during a wound healing process and can exhibit an inherent physical healing effect and can further induce a sufficient wound healing effect of hEGF. Therefore, when the topical preparation of the present invention is applied to a wound site, a good wound healing effect can be exhibited.
The viscosity of the polyoxyethylene-polyoxypropylene copolymer is determined according to the temperature and the molecular weight and content of the polymer. Therefore, in order to obtain a polyoxyethylene-polyoxypropylene copolymer having a viscosity of 4-10cps at 37 ℃ and 60rpm, it is preferable to use 5-10 wt% of the polyoxyethylene-polyoxypropylene copolymer having a molecular weight of about 10,000-15,000 based on the total weight of the composition, or to use 7.5-15 wt% of the polyoxyethylene-polyoxypropylene copolymer having a molecular weight of about 7,000-10,000 based on the total weight of the composition.
More preferably, the viscosity is adjusted within the range of 4 to 6cps by using 5 to 7.5 wt% of a polyoxyethylene-polyoxypropylene copolymer having a molecular weight of about 10,000-15,000 based on the total weight of the composition or using 7.5 to 12.5 wt% of a polyoxyethylene-polyoxypropylene copolymer having a molecular weight of about 7,000-10,000 based on the total weight of the composition.
In the topical composition of the present invention, natural or recombinant hEGF may be used as the active ingredient hEGF. The content of hEGF in the composition is preferably 0.01-1,000. mu.g/ml, in particular 1-100. mu.g/ml.
Since the topical composition of the present invention having the above-mentioned composition exists in a liquid state at room temperature, hEGF, which is a pharmacologically active ingredient, can be uniformly dispersed in the polyoxyethylene-polyoxypropylene copolymer matrix. Therefore, in contrast to the gel preparation of the prior art, when the composition of the present invention is applied to a wound site, it shows a good effect of active ingredient penetration into the wound site, and it can be uniformly spread at the wound site to increase the contact area of skin with hEGF, thereby providing an excellent wound healing effect. In addition, since the composition of the present invention is in a liquid state having a low viscosity, it has an advantage that: it can be formulated as a spray which can be applied to the wound site by simple spraying rather than rubbing, and thus does not cause physical irritation to the wound.
The composition of the present invention may contain pharmaceutically acceptable additives, such as stabilizers, excipients, etc., which are generally used in the preparation of topical preparations, in addition to the active ingredient hEGF and the polyoxyethylene-polyoxypropylene copolymer as a base.
If desired, the compositions of the present invention may be stored in lyophilized form and then dissolved in a suitable solvent at the time of use. Therefore, the composition of the present invention can be stably stored for a long period of time.
The present invention is more specifically explained by the following examples and experiments. However, it should be understood that: the invention is not in any way restricted to these examples.
Example 1
hEGF 5mg
Poloxamer (Poloxamer) 18810 g
200mg of methyl paraben
Disodium hydrogen phosphate 340.23mg
Sodium chloride 832.77mg
Proper amount of phosphoric acid
Proper amount of purified water
The total volume is 100ml
The solutions were prepared according to conventional methods using the components and amounts given above. Specifically, a phosphate buffer was prepared using disodium hydrogen phosphate, sodium chloride and phosphoric acid. Poloxamer188 was added to the resulting phosphate buffer and dispersed therein by stirring. Methyl paraben as preservative and hEGF as active ingredient were then added to this to give 100ml of topical solution according to the invention.
Example 2
hEGF 5mg
Poloxamer 407 7.5g
200mg of methyl paraben
Disodium hydrogen phosphate 340.23mg
Sodium chloride 832.77mg
Proper amount of phosphoric acid
Proper amount of purified water
The total volume is 100ml
Phosphate buffer was prepared using the given amounts of disodium hydrogen phosphate, sodium chloride and phosphoric acid. Poloxamer407 was added to the resulting phosphate buffer and dispersed therein by stirring. Methyl paraben as preservative and hEGF as active ingredient were then added to this to give 100ml of topical solution according to the invention.
Experiment 1
Contrasting the wound healing effects of hEGF on matrix species in animal wound models
The hair on the back of the rat was shaved and a depilatory (Neet Cream produced by II Dong pharm) was applied thereto to remove the hair. Then, the rats were injected with streptomycin (produced by Chong Kun Dang Corp.) at a dose of 0.5g/kg by the intramuscular route. Rats were anesthetized by intraperitoneal injection of 40mg/kg dose of sodium pentobarbital. Wounds were caused by removing the epidermis and dermis over an area of 10mm in diameter with surgical scissors. Then, considering that the wound site is open, the size of the wound 1 day after the wound is caused is considered as the initial wound area.
The test animals were divided into a total of 11 groups according to the kind of matrix used: untreated controls, experimental groups (Poloxamer + hEGF group, polyvinylpyrrolidone + hEGF group, dextran + hEGF group, gelatin + hEGF group and polymethacrylamide + hEGF group) and another control group to which only the matrix was applied (Poloxamer group, polyvinylpyrrolidone group, dextran group, gelatin group and polymethacrylamide group). 1 day after the initiation of wound induction, the wound site of each test animal was treated with 0.5ml of the test drug twice a day. In the experimental group, preparations containing 5mg of hEGF per 100ml of solution were used as test drugs, and they were prepared in the same manner as in example 2, except that the kind and amount of the substrate used were changed during the preparation. In the vehicle control group, a preparation prepared according to the same method as example 2, but excluding hEGF, was used. Daily, wounds were treated with test drugs and the area of the skin wound site was measured. The remaining rate of the wound after the treatment of several days was then obtained by calculating the ratio of the area of the wound measured in terms of the number of days of treatment to the initial wound area measured after the wound was induced. Analysis by linear regression of the wound residual rate after several days of treatment and the number of days of treatment to calculate 50% Healing Time (HT)50) I.e. the number of days showing a 50% remaining rate of the wound. The results obtained are shown in table 1 below.
TABLE 1 based on water-soluble polymer baseComparison of wound treatment days of quality type (Unit: day)
| Experimental group | Content (wt%) | |||
| 5 | 10 | 15 | 20 | |
| Poloxamer*，hEGF | 3.3 | 3.8 | 4.8 | 5.3 |
| Polyvinylpyrrolidone, hEGF | 4.9 | 5.2 | 5.1 | 5.6 |
| Dextran, hEGF | 4.8 | 5.1 | 5.5 | 5.6 |
| Gelatin, hEGF | 5.4 | 5.2 | 5.3 | 5.8 |
| Polymethacrylamide, hEGF | 5.1 | 5.1 | 5.4 | 5.7 |
| Control group | 7.3 | |||
| Poloxamer* | 4.8 | 5.2 | 5.8 | 6.3 |
| Polyvinylpyrrolidone | 5.5 | 5.8 | 5.8 | 6.2 |
| Glucan | 5.4 | 5.7 | 6.2 | 6.2 |
| Gelatin | 5.8 | 6.1 | 6.3 | 6.5 |
| Polymethacrylamide | 5.9 | 6.1 | 6.4 | 6.2 |
Note: poloxamer is the generic name for polyoxyethylene-polyoxypropylene copolymers, Poloxamer407 has an average molecular weight of 10,000-15,000.
As can be seen from Table 1 above, Poloxamer407, as a polyoxyethylene-polyoxypropylene copolymer, showed very good wound healing effect when used as a matrix in the present invention in an amount of 5-10% by weight, with wound healing days of 3.3-3.8 days. In contrast, the use of the remaining water-soluble polymer matrix provides only a slight wound healing effect.
Experiment 2
Wound healing effect of hEGF based on polyoxyethylene-polyoxypropylene copolymer content in animal wound model
In order to determine the hEGF wound healing effect based on the content of the polyoxyethylene-polyoxypropylene copolymer, which has been confirmed to show the optimal wound healing effect in experiment 1, the test formulation produced according to the method of example 2 was used in the same manner as experiment 1. The test animals were divided into untreated control groups, groups containing 2.5, 5.0, 7.5, 10.0 and 12.5 wt% Poloxamer, and groups containing different amounts of Poloxamer and hEGF as an active ingredient. The wound healing effect based on matrix content was thus determined.
TABLE 2 comparison of the number of wound healing days based on the polyoxyethylene-polyoxypropylene copolymer content (unit: day)
| Experimental group | Content (wt%) | ||||
| 2.5 | 5.0 | 7.5 | 10.0 | 12.5 | |
| Poloxamer407，hEGF | 5.8 | 3.3 | 3.1 | 3.8 | 4.8 |
| Poloxamer407 | 6.4 | 4.7 | 4.8 | 5.3 | 5.6 |
| Control group | 7.2 | ||||
As can be seen from Table 2 above, when the polyoxyethylene-polyoxypropylene copolymer is contained in an amount of 5-10% by weight, it shows good wound healing effects for 3.1-3.8 days. In particular, when the polyoxyethylene-polyoxypropylene copolymer is contained in an amount of 5-7.5% by weight, it shows very good wound healing effects for 3.1-3.3 days.
From the results obtained in the above experiments 1 and 2, it was confirmed that only 5 to 10 wt%, particularly 5 to 7.5 wt% of the polyoxyethylene-polyoxypropylene copolymer among various polymers conventionally used as a base for topical preparations induces superior wound healing of hEGF.
Experiment 3
Viscosity of polyoxyethylene-polyoxypropylene copolymer based on molecular weight
It can be seen from the above experiments 1 and 2 that the wound healing effects are greatly different depending on the content of the polyoxyethylene-polyoxypropylene copolymer. Further, the viscosity of the polyoxyethylene-polyoxypropylene copolymer can be determined according to its molecular weight and content, and temperature.
Therefore, in order to determine the optimum content based on the molecular weight of the polyoxyethylene-polyoxypropylene copolymer, the viscosity of a formulation containing Poloxamer407 (having an average molecular weight of about 10,000-10,000) in an amount of 5-10% by weight, which exhibited the optimum wound healing effect, and the content of Poloxamer188 (having an average molecular weight of about 7,000-10,000) as a low-molecular-weight polyoxyethylene-polyoxypropylene copolymer in a formulation having the same viscosity as that when Poloxamer407 was used, were determined by measuring the viscosity in each case according to the following procedures.
The viscosity was measured using a Brookfield Synchro-left viscometer (model: RVF, USA), and the test formulations were prepared as follows. Poloxamer was weighed precisely according to its content and added slowly to a beaker containing distilled water with stirring. Stirring was continued until the Poloxamer was completely dissolved. The resulting test solution was allowed to stand for 5 hours, and then about 200ml of the solution was taken out before the experiment into an incubator at 25 ℃ to 37 ℃ and then used. The viscometer was mounted vertically in a vessel containing the solution, rotated at 60rpm for 3 minutes, after which the value on the viscometer scale was recorded. The experiment was repeated 3 times and the average was obtained.
TABLE 3 viscosity (unit: cps) of polyoxyethylene-polyoxypropylene copolymer based on its content
| Temperature of | Content (wt%) | ||||||
| 5 | 7.5 | 10 | 12.5 | 15 | 20 | ||
| Poloxamer407 | 25℃ | 4.5 | 6 | 11 | |||
| 37℃ | 4 | 6 | 10.6 | ||||
| Poloxamer188 | 25℃ | 4.2 | 5 | 7 | 8.6 | 10 | 16 |
| 37℃ | 3.5 | 4.1 | 5 | 6.5 | 8 | 12.5 | |
As can be seen from Table 3 above, the viscosity was measured to be 4-10cps when the Poloxamer407 content, which showed good wound healing, was 5-10 wt%, and 4-6cps with the preferred 5-7.5 wt% content. Accordingly, as a base for the hEGF topical preparation for treating wounds, it is preferable to use a polyoxyethylene-polyoxypropylene copolymer having a viscosity of 4-10cps, more preferably 4-6cps at 37 ℃.
Further, when the content of the high molecular weight Poloxamer407 is 5 to 10 wt%, the content of the low molecular weight Poloxamer188 showing the same viscosity at 37 ℃ is 7.5 to 15 wt%. While the amount of Poloxamer188, which exhibits the same viscosity at 37 deg.C, is 7.5-12.5 wt% when Poloxamer407 is more preferably 5-7.5 wt%. It has therefore been determined that in order to obtain the preferred viscosity, it is advantageous to use the low molecular weight Poloxamer188 in an amount of 7.5 to 15% by weight, more preferably 7.5 to 12.5% by weight.
As can be seen from the results obtained from the above experiments, among various polymers that can be used as a base for a topical preparation to control the release rate of an active ingredient and to physically protect a wound site, a polyoxyethylene-polyoxypropylene copolymer having a viscosity of 4-10cps, particularly 4-6cps at 37 ℃ and 60rpm can be used according to the present invention to provide a topical preparation exhibiting good wound healing effects.
Claims (7)
1. A topical composition comprising an effective wound healing amount of human epidermal growth factor and a polyoxyethylene-polyoxypropylene copolymer having a viscosity of 4-10cps at 37 ℃ and 60 rpm.
2. The topical composition according to claim 1, wherein the human epidermal growth factor is present in an amount of 1-100 μ g/ml.
3. The topical composition according to claim 1, wherein the polyoxyethylene-polyoxypropylene copolymer has a viscosity of 4-6cps at 37 ℃ at 60 rpm.
4. The topical composition according to claim 1, wherein the copolymer having a molecular weight of 10,000-15,000 as the polyoxyethylene-polyoxypropylene copolymer is used in an amount of 5-10% by weight.
5. The topical composition according to claim 4, wherein the copolymer having a molecular weight of 10,000-15,000 as polyoxyethylene-polyoxypropylene copolymer is used in an amount of 5-7.5% by weight.
6. The topical composition according to claim 1, wherein the amount of the copolymer having a molecular weight of 7,000-10,000 as the polyoxyethylene-polyoxypropylene copolymer is from 7.5 to 15% by weight.
7. The topical composition according to claim 6, wherein the amount of the copolymer having a molecular weight of 7,000-10,000 as the polyoxyethylene-polyoxypropylene copolymer is from 7.5 to 12.5% by weight.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/KR1998/000043 WO1999044631A1 (en) | 1998-03-07 | 1998-03-07 | Topical composition containing human epidermal growth factor |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| HK1036221A1 HK1036221A1 (en) | 2001-12-28 |
| HK1036221B true HK1036221B (en) | 2005-06-03 |
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