Diabetic chronic wound healing composition and wound healing method using same

Abstract

Provided are: a diabetic chronic wound healing composition comprising a first active agent consisting of dECM, and a second active agent comprising metformin, a precursor thereof, or a derivative thereof; and a wound healing method using same.

Description

Composition for treating diabetic chronic wounds and method for treating wounds using the same

[0001] The present invention relates to a composition for treating diabetic chronic wounds and a wound treatment method using the same.

[0002] According to research results published in The Lancet by the Institute for Health Metrics and Evaluation at the University of Washington in the United States, the number of diabetes patients is increasing, and a warning is being issued that one in ten people worldwide may suffer from diabetes in 30 years.

[0003] The first complications to appear in diabetic patients are those farthest from the heart, the feet. Foot ulcers are known to develop in 15-20% of diabetic patients. These ulcers are caused by a lack of oxygen supply due to poor blood circulation in the microvasculature.

[0004] Chronic wounds caused by diabetic foot ulcers (hereafter referred to as diabetic chronic wounds) are dehiscent wounds that do not heal well on their own. They are very wide and deep, often accompanied by significant tissue loss. Therefore, while conventional wound treatments may promote wound healing, they often do not heal quickly enough, resulting in delayed wound closure. Furthermore, in diabetic patients, hyperglycemia impedes the wound healing process, increasing the apoptosis index and inhibiting collagen synthesis. Therefore, ointments containing ingredients that possess anti-apoptotic effects and promote collagen synthesis are needed. However, existing products have limitations. Therefore, there is a need for the development of therapeutic compositions suitable for diabetic chronic wounds and similar wounds.

[0005] Furthermore, Korean Patent Publication Nos. 10-2008-0003886 and 10-2015-0003939 disclose electrotherapy methods that promote wound healing by applying electric current to the wound site during the treatment of chronic diabetic wounds. Accordingly, demand for ointments suitable for use in such electrotherapy is also increasing.

[0006] One embodiment is to provide a composition for treating diabetic chronic wounds that exhibits excellent effects in treating diabetic chronic wounds in which the wound area is very wide and deep and a lot of tissue is lost.

[0007] Another embodiment is to provide a method for treating chronic wounds using a composition for treating diabetic chronic wounds.

[0008] A composition for treating diabetic chronic wounds according to one embodiment comprises a first active agent comprising dECM and a second active agent comprising metformin or a derivative thereof.

[0009] The ratio of the first activator: the second activator can be from 1:0.002 to 1:1.35.

[0010] The composition for treating diabetic chronic wounds may further comprise an electrolyte.

[0011] The electrolyte may be included in an amount of 0.1 to 0.5 wt% based on the total weight of the composition for treating diabetic chronic wounds.

[0012] The composition for treating diabetic chronic wounds may further comprise hyaluronic acid and a moisturizer.

[0013] According to another embodiment, a method for treating a diabetic chronic wound comprises applying a composition for treating a diabetic chronic wound, the composition comprising a first active agent comprising dECM and a second active agent comprising metformin or a derivative thereof, to a site of a diabetic chronic wound.

[0014] The ratio of the first activator: the second activator can be from 1:0.002 to 1:1.35.

[0015] The composition for treating diabetic chronic wounds may further comprise an electrolyte.

[0016] The electrolyte may be included in an amount of 0.1 to 0.5 wt% based on the total weight of the composition for treating diabetic chronic wounds.

[0017] After placing an electrode patch on a wound site to which a composition for treating diabetic chronic wounds has been applied, electricity can be applied.

[0018] Electricity can be direct current.

[0019] Direct current can be a high voltage pulsed current.

[0020] When applying electricity, the polarity of the electrodes can be changed so that current flows alternately from the first electrode to the second electrode or from the second electrode to the first electrode.

[0021] The composition for treating diabetic chronic wounds may further comprise hyaluronic acid and a moisturizer.

[0022] According to one embodiment, a composition for treating diabetic chronic wounds comprises a first activator, dECM, which fills the tissue of a damaged wound site where a large amount of tissue has been lost, and a second activator, metformin, which is distributed within dECM, which increases collagen synthesis and enhances anti-apoptotic action to promote tissue cell growth. Therefore, the synergistic effect of the first activator and the first activator effectively and widely and deeply fills the tissue of a wound site where a large amount of tissue has been lost, thereby exhibiting a synergistic effect that can accelerate wound healing.

[0023] According to another embodiment, a wound treatment method using a composition for treating diabetic chronic wounds comprises applying the composition for treating diabetic chronic wounds and, if necessary, applying electrical stimulation to promote the migration of fibroblasts in the dermal layer, thereby enhancing the effectiveness of wound healing. Furthermore, by periodically varying the polarity of the applied electrode, a uniform stimulus can be applied, thereby enhancing the effectiveness of wound healing.

[0024] Figure 1 is a photograph measuring the change in wound size after applying each of the compositions disclosed in Table 1.

[0025] Figure 2 is a graph measuring changes in wound size after applying each of the compositions disclosed in Table 1.

[0026] Figure 3 is a photograph of collagen stained using trichromatic staining after tissue was removed on the last day of treatment after applying each of the compositions disclosed in Table 1.

[0027] Figure 4 is a graph showing the collagen content measured using trichromatic staining after tissue was removed on the last day of treatment after applying each of the compositions disclosed in Table 1.

[0028] Figure 5 is a graph showing the expression levels of Col1a1 and Col3a1, which are involved in collagen production, measured by PCR after tissue was removed on the last day of treatment after applying each of the compositions disclosed in Table 1.

[0029] Figure 6 is a schematic diagram showing an experimental design for measuring the wound healing effect according to Example 2.

[0030] Figure 7 is a photograph measuring the change in wound size after applying each of the compositions disclosed in Table 2.

[0031] Figure 8 is a graph measuring changes in wound size after applying each of the compositions disclosed in Table 2.

[0032] Figure 9 is a photograph of collagen stained using trichromatic staining after tissue was removed on the last day of treatment after applying each of the compositions disclosed in Table 2.

[0033] Figure 10 is a graph showing the content of collagen measured using trichromatic staining after tissue was removed on the last day of treatment after applying each of the compositions disclosed in Table 2.

[0034] Figure 11 is a photograph showing epithelial regeneration measured using hematoxylin & eosin staining after tissue was removed on the last day of treatment after applying each of the compositions disclosed in Table 2.

[0035] Figure 12 is a graph showing the measurement of epithelial regeneration using hematoxylin & eosin staining after tissue was removed on the last day of treatment after applying each of the compositions disclosed in Table 2.

[0036] Figure 13 is a graph showing the expression levels of Col1a1 and Col3a1, which are involved in collagen production, and VEGF, which are measured by performing PCR after tissue was extracted on the last day of treatment after applying each of the compositions disclosed in Table 2.

[0037] Figure 14 is a graph showing the measurement of TNF-α and IL-10 by performing protein analysis (ELISA) on tissues extracted on the last day of treatment after applying each of the compositions disclosed in Table 2.

[0038] Hereinafter, embodiments of the present invention will be described in detail so that those skilled in the art can easily implement the invention. However, the present invention may be implemented in various different forms and is not limited to the embodiments described herein.

[0039] The following describes a composite wound treatment composition according to an embodiment.

[0040] Most of the diabetic chronic wound treatment compositions currently on the market or patented are composed of a single main active ingredient, which presents limitations in treating deep and wide wounds with significant tissue loss. This study was conducted with this in mind.

[0041] As a result, a composition suitable for the treatment of diabetic chronic wounds was completed that can exhibit a synergistic effect of accelerating wound healing by combining decellularized extra cellular matrix (dECM), which provides a biomimetic microenvironment suitable for the growth and differentiation of autologous dermal cells and can fill the damaged wound area where a lot of tissue has been lost, with metformin, which can reverse the side effects caused by diabetes, promote collagen production, exhibit anti-apoptotic effects, and increase cell proliferation of autologous dermal cells.

[0042] A composite wound treatment composition according to one embodiment comprises a combination of a first active agent and a second active agent.

[0043] The first activator is a component capable of filling wide and deep wounds and providing a biomimetic microenvironment suitable for dermal cell growth and differentiation. The second activator is a component capable of stimulating collagen synthesis, increasing cell proliferation, and reducing cell death in patients suffering from diabetes-related side effects such as impaired collagen synthesis and cell proliferation and increased apoptosis.

[0044] The first active agent includes decellularized extracellular matrix (dECM). dECM refers to the extracellular matrix remaining after the nucleus, cell membrane, and nucleic acids have been removed from cells or tissues. Decellularized extracellular matrix removes only the nucleus and cell membrane from a cell population, allowing the entire extracellular matrix component to be utilized, providing a more natural, biomimetic microenvironment for cell growth and differentiation.

[0045] Although dECM provides a biomimetic microenvironment to damaged tissue, it cannot sufficiently promote the growth of autologous dermal cells, so its effect on wound healing when used alone is limited.

[0046] Here, a second activator is mixed with the first activator to reverse the side effects of diabetes and enable sufficient growth of autologous dermal cells.

[0047] The second active agent comprises metformin or a derivative thereof.

[0048] Metformin is a biguanide compound represented by the following chemical formula 1.

[0049]

[0050] Originally known as an oral antidiabetic drug, it exhibits a wound healing effect when applied topically, and is particularly effective in wound healing in diabetic patients.

[0051] Derivatives of metformin can be represented by the chemical formula 2 below.

[0052]

[0053] In the above formula, R1 and R2 independently represent a hydrogen atom, a C1∼C7 alkyl group, a cycloalkyl group, a heterocycle, a C2∼C7 alkenyl group, an aryl group, an aralkyl group, an aryloxyalkyl group or a heteroaryl group, or R1 and R2 taken together represent a C2∼C7 alkylene group containing one or more heteroatoms,

[0054] R3 represents a primary, secondary or tertiary amine.

[0055] The term "C1∼C7 alkyl group" refers to a linear or branched substituted or unsubstituted C1∼C7 alkyl group and isomers thereof, such as, for example, a methyl group, an ethyl group, a propyl group, an isopropyl group or a butyl group.

[0056] The term "cycloalkyl group" refers to a cycloalkyl group containing 3 to 7 carbon atoms, such as, for example, a cyclohexyl group.

[0057] The term "heterocycle" refers to a ring containing 3 to 7 atoms, at least one of which is a heteroatom, such as a nitrogen atom, an oxygen atom, or a sulfur atom, and the remainder are carbon atoms.

[0058] The term "C2∼C7 alkenyl group" refers to a linear or branched substituted or unsubstituted C2∼C7 alkenyl group such as a vinyl group or an allyl group.

[0059] The term "aryl group" refers to a hydrocarbon aromatic group including one or more substituents, for example, a C1∼C7 alkyl group as described above, a C2∼C7 alkenyl group as described above, or a phenyl group including a halogen.

[0060] The term "aralkyl group" means an aryl group as described above linked by an alkyl group as described above. Advantageously, when the alkyl group represents a CH2 group and the aryl group represents a phenyl group, the phenyl group is substituted as described above, and when the alkyl group does not represent CH2, the aryl group is as described above, and advantageously a phenyl group.

[0061] The term "aryloxyalkyl group" refers to an aryl group as described above in which an alkyl moiety is linked via an oxyalkyl group as described above.

[0062] The term "heteroaryl group" refers to a hydrocarbon aromatic group that contains one or more heteroatoms, such as, for example, sulfur, nitrogen, or oxygen atoms, and may have one or more substituents, such as, for example, the above-mentioned C1∼C7 alkyl group, the above-mentioned C2∼C7 alkenyl group, or halogen. Examples of heteroaryl groups include a furyl group, an isoxazolyl group, a pyridyl group, and a pyrimidyl group.

[0063] The term "C1∼C7 alkylene group" refers to a C2∼C7 alkenyl group, such as, for example, an ethylene group, a trimethylene group, a tetramethylene group, or a pentamethylene group.

[0064] Preferably, R1 and R2 may independently be a hydrogen atom, a C1∼C7 alkyl group, a cycloalkyl group, a heterocycle, a C2∼C7 alkenyl group, an aryloxyalkyl group, or a heteroaryl group. R3 may be a secondary amine of the following chemical formula 3.

[0065]

[0066] Treating diabetic chronic wounds with metformin or its derivatives activates mitochondria in dermal cells surrounding the wound, increasing energy metabolism. Consequently, dermal cell migration around the wound accelerates. It also promotes collagen production and stimulates epithelial regeneration.

[0067] Therefore, when the first and second activators are combined and supplied, the first activator, dECM, fills the wound tissue and provides a biomimetic microenvironment. The second activator, metformin, stimulates collagen synthesis, increases cell proliferation, and reduces the cell death index through its anti-apoptotic effect. Therefore, the collaboration of the first and second activators produces a synergistic effect that accelerates wound healing by rapidly growing autologous dermal cells and filling the damaged wound tissue.

[0068] To achieve this synergistic effect, the ratio of the first activator to the second activator may be 1:0.002 to 1:1.35.

[0069] In an embodiment, the first active agent may be present in an amount ranging from 1.5 to 50 wt%, any subrange thereof, or a single value, based on the total weight of the composition. The second active agent may be present in an amount ranging from 0.1 to 2 wt%, any subrange thereof, or a single value, based on the total weight of the composition.

[0070] The dECM may be obtained from cells cultured in vitro. The dECM may be more preferably an extracellular matrix obtained from a cell population cultured in vitro than an extracellular matrix derived from tissue formed within an individual. The decellularized extracellular matrix, when obtained from cells cultured in vitro, can solve the supply problem, which is a disadvantage of tissue-derived extracellular matrix, and has a low possibility of immune rejection because autologous cells can be used. The cells for obtaining the extracellular matrix may be, for example, one or more selected from the group consisting of dermal tissue-derived cells, fibroblasts, chondrocytes, osteoblasts, vascular endothelial cells, myocytes, smooth muscle cells, hepatocytes, neural cells, cardiomyocytes, intervertebral disc cells, and mesenchymal stem cells. The decellularization method for obtaining the decellularized extracellular matrix may be performed by a known method or an appropriate modification thereof. In one specific example, a population of dermal tissue-derived cells cultured in vitro can be treated with Triton X-100, a non-ionic detergent, to break the cell membrane and remove intracellular components.

[0071] Meanwhile, in the embodiment, the composition may further comprise an electrolyte. If the composition further comprises an electrolyte, it may be more suitable for use with a microcurrent-based wound treatment device (e.g., a patch) disclosed in prior literature.

[0072] Electrolytes that can be used include potassium chloride and sodium chloride.

[0073] The electrolyte may be present in an amount ranging from 0.1 to 0.5 wt%, any sub-range thereof, or a single value, based on the total weight of the composition. If the content of the electrolyte is less than 0.1 wt%, the conductivity may decrease, which may lower the efficiency of the wound treatment device. If the content of the electrolyte exceeds 0.5 wt%, the high conductivity may cause side effects or discomfort at the site of application, so it may be preferable to use it within the mentioned range. However, it is obvious that the content range may vary depending on the strength of the applied current.

[0074] In an embodiment, the composition may further comprise a residual amount of hyaluronic acid, a moisturizer, etc. In addition, it may comprise other ingredients that are incorporated into a typical pharmaceutical composition. For example, it may further comprise a maintenance component, a moisturizer, a viscosity modifier, an emollient, a surfactant, an organic or inorganic pigment, an organic powder, an ultraviolet absorber, a preservative, a bactericide, an antioxidant, a plant extract, a pH adjuster, an alcohol, a pigment, a fragrance, a blood circulation promoter, a cooling agent, an antiperspirant, purified water, etc.

[0075] The moisturizer may contain 0.5 to 2 wt% of glycerin.

[0076] As a viscosity modifier, carbomo may be included in an amount of 0.5 to 2 wt%.

[0077] The pH adjuster may include 0.1 to 0.5 wt of sodium bicarbonate.

[0078] Preservatives may be included in an amount of 0.1 to 0.2 wt for microbial inhibition.

[0079] When the composition for treating diabetic chronic wounds according to the embodiment comprises an electrolyte, it can be used together with a wound treatment device using high voltage pulse current electrical stimulation including an electrode patch disclosed in KR10-2023-0167069 filed by the present applicant.

[0080] Specifically, first, a composition for treating diabetic chronic wounds according to an embodiment is applied to the wound site. Next, an electrode patch is placed on the wound site, and then electricity is applied. The electricity applied at this time may be a high voltage pulsed current (HVPC) as a direct current. For example, the voltage magnitude may be 100 to 500 V, the duration of one pulse (A) may be 1 μs to 100 μs, the interval between pulses (B) may be 100 ms to 10 sec, the duration of one pulse with one polarity (C) may be 2.5 min to 30 min, the time for changing electrodes (D) may be 200 μs to 1 ms, the duration of a pulse bundle, that is, the time for performing one procedure may be 5 min to 60 min, and the interval between pulse bundles may be 1 h to 12 h.

[0081] High-voltage current has excellent skin penetration ability, and electrical stimulation by HVPC can be effective in wound treatment due to the galvanotaxix phenomenon in which fibroblasts in the dermal layer move toward the cathode in proportion to the voltage of the applied current.

[0082] By changing the polarity of the electrodes, the current can flow alternately from one direction (positive direction) to the opposite direction (negative direction) (from the first electrode to the second electrode) and from the opposite direction to one direction (from the second electrode to the first electrode), thereby achieving a uniform stimulation. In the case of alternating current or ultrasound, since the energy vibrates, it only generates heat energy by causing vibrations in cells or intercellular substances in the skin tissue, making it difficult to stimulate cells or infectious agents such as bacteria in a consistent direction. However, direct current can stimulate cells or infectious agents in a consistent direction, and by changing the direction of the electrodes, it can also stimulate in the opposite direction. This polarity change can make wound treatment more effective.

[0083] The following examples illustrate embodiments of the present invention in more detail. However, the following examples are provided for illustrative purposes only and do not limit the scope of the present invention.

[0084] [Example 1]

[0085] Preparation of a composition for treating diabetic chronic wounds

[0086] Four different wound treatment composition treatment groups were set up under the conditions shown in Table 1 below.

[0087] Experimental group (wt%)By speciesBy age(at the time of experiment)By group(N)G1Negative Control (1wt% HA)C57BL / 6JF8W4G21wt% HA + 5wt% dECM5G31wt% HA + 5wt% Metformin5G41wt% HA + 3wt% dECM + 2wt% Metformin5

[0088] Evaluation of wound healing ability: Measurement of wound size changes First, diabetic chronic wounds were formed by inducing a 100㎟ wound in diabetic rats, and then ointments for each group were applied to the wound area. Specifically, each group was composed of G1 (normal group, composition containing 1 wt% HA), G2 (dECM only, comparison group), G3 (Metformin only, comparison group), and G4 (dECM + Metformin, experimental group). The change in wound size over 9 days was measured using a caliper. The results are shown in the photograph in Fig. 1 and the graph in Fig. 2.

[0089] From the results of Figures 1 and 2, it can be confirmed that a superior wound healing effect is exhibited when dECM and metformin are mixed and treated compared to when dECM and metformin are treated alone.

[0090] Wound healing performance evaluation: measuring collagen production

[0091] On the 9th day after wound healing, tissue was excised and collagen was stained using trichrome staining. The results are shown in the photograph in Fig. 3 and the graph in Fig. 4.

[0092] From the results of Figures 3 and 4, it can be confirmed that collagen is produced at a very high rate when dECM and metformin are mixed and treated compared to when dECM and metformin are treated alone.

[0093] Evaluation of wound healing capacity: Measurement of mRNA expression levels involved in collagen production

[0094] Tissue samples were sampled on the 9th day after wound healing, and PCR was performed to measure the expression levels of Col1a1 and Col3a1, mRNAs involved in collagen production. The results are shown in Figure 5.

[0095] From the results of Figure 5, it can be confirmed that the expression levels of Col1a1 and Col3a1 were the highest when dECM and metformin were mixed and treated compared to when dECM and metformin were treated alone.

[0096] [Example 2]

[0097] Four different wound treatment composition treatment groups were set up under the conditions shown in Table 2 below.

[0098] Experimental group (wt%)By species / sex / weekly age (at the time of experiment)By group (N)G1Negative Control (1wt% HA + 0.1wt% KCl)C57BL / 6JF12M and above (aged animals)4G21wt% HA + 5wt% dECM + 0.1wt% KCl5G31wt% HA + 2wt% Metformin + 0.1wt% KCl5G41wt% HA + 3wt% dECM + 2wt% Metformin + 0.1wt% KCl5

[0099] Evaluation of wound healing ability: Measurement of wound size change First, a 100㎟ sized wound was induced in diabetic rats to form a diabetic chronic wound, and then an ointment for each group was applied to the wound site. Specifically, each group was composed of G1 (normal group, composition containing 1 wt% HA), G2 (dECM only, comparison group), G3 (Metformin only, comparison group), and G4 (dECM + Metformin, experimental group).

[0100] Unlike Experimental Example 1, as illustrated in Fig. 6, the therapeutic composition was applied in the morning and afternoon, respectively, and electrical stimulation was applied for 10 minutes each. The electrical stimulation was performed with a voltage of 100 V, a pulse duration of 100 μs, an interval between pulses of 150 ms, and a pulse repetition count of 2100 x 2.

[0101] Changes in wound size at D1, D2, D3, and D6 were measured using calipers. The results are shown in the photograph in Fig. 7 and the graph in Fig. 8.

[0102] From the results of Figures 7 and 8, it can be confirmed that a superior wound healing effect is exhibited when dECM and metformin are mixed and electrical stimulation is applied compared to when dECM and metformin are treated alone.

[0103] Wound healing performance evaluation: measuring collagen production

[0104] Six days after wound healing, tissue was excised and collagen was stained using trichrome staining. The results are shown in the photograph in Fig. 9 and the graph in Fig. 10.

[0105] From the results in FIGS. 9 and 10, it can be confirmed that when dECM and metformin are mixed and electrical stimulation is applied, a very high level of collagen is produced compared to when dECM and metformin are treated alone. Comparing the results in FIGS. 4 and 10, it can be confirmed that when dECM and metformin are mixed and treated and electrical stimulation is added, collagen production increases by more than 8 times compared to when no electrical stimulation is applied.

[0106] Wound healing performance evaluation: measurement of epithelial regeneration

[0107] On the sixth day after wound healing, tissue was excised and epithelial regeneration was measured using hematoxylin & eosin (H&E) staining. The results are depicted in the photograph in Figure 11 and the graph in Figure 12.

[0108] From the results of Figures 11 and 12, it can be confirmed that epithelial regeneration occurs at a very high rate when dECM and metformin are mixed and electrical stimulation is applied, compared to when dECM and metformin are treated alone.

[0109] Evaluation of wound healing capacity: Measurement of mRNA expression levels involved in collagen production

[0110] Six days after wound healing, tissue samples were sampled and PCR was performed to measure the expression levels of Col1a1 and Col3a1, which are involved in collagen production, as well as the expression levels of VEGF. The results are shown in Figure 13.

[0111] From the results of Figure 13, it can be confirmed that when dECM and metformin were mixed and electrical stimulation was applied, the expression levels of Col1a1, Col3a1, and VEGF were more than two times, or even three times, higher than when dECM and metformin were treated alone.

[0112] Evaluation of wound healing ability: Measurement of the expression of inflammation-related substances

[0113] Six days after wound healing, protein analysis (ELISA) was performed to measure TNF-α and IL-10. The results are shown in Figure 14.

[0114] From the results of Figure 14, it can be confirmed that when dECM and metformin are mixed and electrical stimulation is applied, the content of TNF-α, an inflammatory substance, decreases and the content of IL-10, which has the function of suppressing inflammation, increases compared to when dECM and metformin are treated alone.

[0115] Although the preferred embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and improvements made by those skilled in the art using the basic concept of the present invention defined in the following claims also fall within the scope of the present invention.

[0116]

[0117] The present invention can be used in medicines and treatment devices for treating diabetic chronic wounds.

Claims

1. A first activator comprising dECM; and A composition for treating diabetic chronic wounds comprising a second active agent comprising metformin or a derivative thereof.

2. In paragraph 1, A composition for treating diabetic chronic wounds, wherein the ratio of the first activator to the second activator is 1:0.002 to 1:1.

35.

3. In paragraph 1, A composition for treating diabetic chronic wounds further comprising an electrolyte.

4. In paragraph 3, A composition for treating diabetic chronic wounds, wherein the electrolyte is included in an amount of 0.1 to 0.5 wt% based on the total weight of the composition for treating diabetic chronic wounds.

5. In paragraph 1, A composition for treating diabetic chronic wounds further comprising hyaluronic acid and a moisturizer.

6. A wound treatment method comprising the step of applying a composition for treating diabetic chronic wounds, comprising a first active agent consisting of dECM and a second active agent comprising metformin or a derivative thereof, to a diabetic chronic wound site.

7. In paragraph 6, A wound treatment method wherein the ratio of the first activator to the second activator is 1:0.002 to 1:1.

35.

8. In paragraph 6, A wound treatment method wherein the composition for treating diabetic chronic wounds further comprises an electrolyte.

9. In paragraph 8, A wound treatment method comprising the electrolyte in an amount of 0.1 to 0.5 wt% based on the total weight of the composition for treating diabetic chronic wounds.

10. In paragraph 8, A wound treatment method comprising placing an electrode patch on a wound site to which the above diabetic chronic wound treatment composition has been applied and then applying electricity.

11. In paragraph 10, The above electric current is a wound treatment method using direct current.

12. In paragraph 11, The above direct current is a wound treatment method that is a high voltage pulse current.

13. In paragraph 11, A wound treatment method in which the polarity of the electrodes is changed when applying the above electricity so that the current flows alternately from the first electrode to the second electrode or from the second electrode to the first electrode.

14. In paragraph 6, A wound treatment method wherein the composition for treating diabetic chronic wounds further comprises hyaluronic acid and a moisturizer.

Images