Combined wound healing composition and wound healing method using the same

Abstract

Provided are a combined wound healing composition and a wound healing method using same, the composition comprising: a first active agent including NAD, a precursor thereof, a derivative thereof, or a combination thereof; and a second active agent composed of dECM.

Description

Composite wound treatment composition and wound treatment method using the same

[0001] The present invention relates to a composite wound treatment composition and a wound treatment method using the same.

[0002] Wound healing agents are applied to wounds to promote wound healing and prevent infection. Therefore, to be effective as wound healing agents, they must satisfy the following requirements: (1) maintaining a moist environment, (2) promoting regeneration, and (3) being convenient to use.

[0003] Wound healing agents include dressings, bandages, hydrogels, silver ion dressings, skin substitutes such as artificial skin, spray-type wound healing agents, collagen dressings, film dressings, and ointments.

[0004] Among these, ointments have the advantage of being able to act locally by being applied directly to the wounded area, of being able to provide or maintain a moist environment in the wounded area, of being able to take effect quickly by being applied directly to the wounded area, and of being able to be used while directly checking the condition of the wounded area.

[0005] Ointments currently available on the market include Madecassol containing Centella asiatica extract (Asiaticoside), Fucidin containing fusidic acid, Bepanthen containing dexpanthenol, DermaVate containing clobetasol propionate, and Atobarrier containing ceramide and panthenol.

[0006] However, while currently commercially available ointments may be effective for common abrasions and minor upper body abrasions, they have limitations for complex wounds such as severe burns, ulcers, post-surgical wounds, and diabetic foot ulcers, so a new ointment that can address these issues is required.

[0007] Furthermore, according to Korean Patent Publication Nos. 10-2008-0003886 and 10-2015-0003939, electrotherapy methods are disclosed that apply electric current to the wound to promote wound healing. Accordingly, demand for ointments suitable for use in such electrotherapy is also increasing.

[0008] One embodiment is to provide a composite wound healing composition that exhibits excellent efficacy even in complex wound healing.

[0009] Another embodiment is to provide a method for treating a wound using a composite wound treatment composition.

[0010] A composite wound healing composition according to one embodiment comprises a first activator comprising NAD, a precursor thereof, a derivative thereof, or a combination thereof, and a second activator comprising dECM.

[0011] The ratio of first activator: second activator can be from 1:0.3 to 1:500.

[0012] The composite wound healing composition may further comprise an electrolyte.

[0013] The electrolyte may be included in an amount of 0.1 to 0.5 wt% based on the total weight of the composite wound healing composition.

[0014] The composite wound healing composition may further comprise hyaluronic acid and a moisturizer.

[0015] The first active agent can be encapsulated in liposomes.

[0016] The first activator may be nicotinamide (NAM) or nicotinamide mononucleotide (NMN).

[0017] According to another embodiment, a wound treatment method comprises applying to a wound site a composite wound treatment composition comprising a first activator comprising NAD, a precursor thereof, a derivative thereof, or a combination thereof, and a second activator comprising dECM.

[0018] The ratio of first activator: second activator can be from 1:0.3 to 1:500.

[0019] The composite wound healing composition may further comprise an electrolyte.

[0020] The electrolyte may be included in an amount of 0.1 to 0.5 wt% based on the total weight of the composite wound treatment composition.

[0021] After placing an electrode patch on a wound site to which a composite wound treatment composition has been applied, electricity can be applied.

[0022] Electricity can be direct current.

[0023] Direct current can be a high voltage pulsed current.

[0024] 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.

[0025] The composite wound healing composition may further comprise hyaluronic acid and a moisturizer.

[0026] The first active agent can be encapsulated in liposomes.

[0027] The first activator may be nicotinamide (NAM) or nicotinamide mononucleotide (NMN).

[0028] According to one embodiment, a composite wound healing composition comprises a first activator that activates mitochondria in dermal cells surrounding a wound to enhance energy metabolism. Accordingly, autologous dermal cells surrounding the wound rapidly migrate to dECM, a second activator that provides a biomimetic microenvironment suitable for the growth and differentiation of autologous dermal cells. Furthermore, autologous dermal cells migrated into dECM, the second activator, rapidly grow in collaboration with the first activator present within dECM, thereby filling the tissues of the damaged wound area, thereby exhibiting a synergistic effect that accelerates wound healing. Furthermore, collagen production is promoted and epithelial regeneration is also actively activated.

[0029] A wound treatment method using a composite wound treatment composition according to another embodiment can increase the effect of wound treatment by applying the composite wound treatment composition and applying electrical stimulation as needed to promote the movement of fibroblasts in the dermal layer.

[0030] In addition, the efficiency of wound healing can be improved by periodically changing the polarity of the applied electrode to ensure uniform stimulation.

[0031] Figure 1 is a photograph measuring the change in wound size after applying each of the compositions disclosed in Table 1.

[0032] Figure 2 is a graph measuring changes in wound size after applying each of the compositions disclosed in Table 1.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] Figure 6 is a schematic diagram showing an experimental design for measuring the wound healing effect according to Example 2.

[0037] Figure 7 is a photograph measuring the change in wound size after applying each of the compositions disclosed in Table 2.

[0038] Figure 8 is a graph measuring changes in wound size after applying each of the compositions disclosed in Table 2.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] Figure 15 is a photograph measuring the change in wound size after applying each of the compositions disclosed in Table 3.

[0046] Figure 16 is a graph measuring changes in wound size after applying each of the compositions disclosed in Table 3.

[0047] Figure 17 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 3.

[0048] Figure 18 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 3.

[0049] Figure 19 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 3.

[0050] Figure 20 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 3.

[0051] Figure 21 is a photograph measuring the change in wound size after applying each of the compositions disclosed in Table 4.

[0052] Figure 22 is a graph measuring changes in wound size after applying each of the compositions disclosed in Table 4.

[0053] Figure 23 is a photograph of collagen stained using trichrome staining after tissue was removed on the last day of treatment after applying each of the compositions disclosed in Table 4.

[0054] Figure 24 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 4.

[0055] Figure 25 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 4.

[0056] Figure 26 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 4.

[0057] Figure 27 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 4.

[0058] Figure 28 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 4.

[0059] 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.

[0060] The following describes a composite wound treatment composition according to an embodiment.

[0061] Most of the wound treatment compositions currently on the market or for which patents have been applied are composed of a single main active ingredient, which presents limitations in treating complex wounds. Therefore, related research was conducted.

[0062] As a result, we were able to complete a composite wound treatment composition that can exhibit a synergistic effect of accelerating wound healing by combining a component that activates the mitochondria of dermal cells around the wound to accelerate the migration and growth of autologous dermal cells and a decellularized extracellular matrix (dECM) that provides a biomimetic microenvironment suitable for the growth and differentiation of autologous dermal cells.

[0063] A composite wound treatment composition according to one embodiment comprises a combination of a first active agent and a second active agent.

[0064] The first activator activates mitochondrial metabolism, accelerating the migration of dermal cells around the wound and promoting the growth of autologous dermal cells. The second activator provides a biomimetic microenvironment suitable for dermal cell growth and differentiation.

[0065] As the first activator, nicotinamide adenine dinucleotide (hereinafter, NAD), a precursor thereof, a derivative thereof, or a combination thereof may be used.

[0066] NAD, its precursors, or derivatives, are crucial cofactors for many redox reactions in living cells and are substrates for numerous enzymes. NAD production in cells declines rapidly with aging. Numerous studies have shown that increasing the concentration of NAD, its precursors, or derivatives in vivo is effective in promoting cellular activity and preventing aging.

[0067] Applying NAD, its precursors, or its derivatives to the wound site activates the mitochondria of dermal cells surrounding the wound, enhancing energy metabolism. Consequently, the migration of dermal cells around the wound is accelerated. Furthermore, it promotes collagen production and epithelial regeneration, thereby promoting the growth of autologous dermal cells.

[0068] The second 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.

[0069] Although dECM provides a biomimetic microenvironment to damaged tissue, it cannot sufficiently promote the migration and growth of autologous dermal cells, so its effect on wound healing when used alone is limited.

[0070] On the other hand, because it takes a long time for the damaged tissue of the wound to fill itself, there is a limit to the ability of autologous dermal cells to fill the tissue of the wound site even if NAD, its precursors, or its derivatives are supplemented.

[0071] Therefore, when the first activator and the second activator are supplied in combination, the second activator, dECM, fills the damaged tissue, and the first activator activates the mitochondrial metabolism of dermal cells, causing the autologous dermal cells to quickly move into the second activator, dECM, which is filling the damaged tissue. Then, the autologous dermal cells that have moved into the dECM grow rapidly as collagen production and epithelial regeneration are activated by the action of the first activator present in the dECM, completely filling the tissue of the damaged wound area, thereby exhibiting a synergistic effect that can accelerate wound healing.

[0072] To achieve this synergistic effect, the ratio of the first activator to the second activator may be 1:0.3 to 1:500.

[0073] In an embodiment, the first active agent may be present in an amount ranging from 0.1 to 5 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 1.5 to 50 wt%, any subrange thereof, or a single value, based on the total weight of the composition.

[0074] Precursors or derivatives of NAD may include nicotinic acid (NA), nicotinamide (NAM), nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), nicotinamide adenine dinucleotide plus hydrogen (NADH), nicotinamide adenine dinucleotide phosphate (NADP), nicotinic acid adenine dinucleotide phosphate (NAADP), nicotinamide adenine dinucleotide phosphate (NADPH), and the like.

[0075] Nicotinamide (NAM) or nicotinamide mononucleotide (NMN) may be preferred for cellular uptake. Larger molecules, such as NAD, its precursors, or its derivatives, may not readily undergo cellular uptake. Therefore, liposomes can be encapsulated to facilitate cellular uptake. Liposomes readily fuse with cells, allowing them to penetrate into surrounding cells. Once liposomes penetrate, substances within the liposomes (e.g., NAD) are released into the cytoplasm, maintaining a high intracellular concentration. This can activate the mitochondria of surrounding dermal cells and enhance energy metabolism.

[0076] Liposomes are formed when vesicle-forming lipids (e.g., phospholipids and their derivatives) are dispersed in an aqueous solvent (e.g., water). When dispersed in an aqueous solvent, the vesicle-forming lipids form closed vesicles called "liposomes," characterized by a lipid bilayer encapsulating an aqueous core.

[0077] Liposomes can be formed using vesicle-forming lipids comprising phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidic acid (PA), phosphatidylinositol (PI), sphingomyelin (SM), phosphatidylserine, phosphatidylglycerol, polyvinylpyrrolidone, polyvinylmethylether, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyloxazoline, polyhydroxypropylmethacrylamide, polymethacrylamide, polydimethylacrylamide, polyhydroxypropylmethacrylate, polyhydroxyethylacrylate, hydroxymethylcellulose, hydroxyethylcellulose, polyethylene glycol, polyaspartamide, lecithin, dipalmitoyl lecithin, distearoylphosphatidylcholine, or mixtures thereof.

[0078] 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, the cell membrane of a population of dermal tissue-derived cells cultured in vitro can be broken using a non-ionic detergent, Triton X-100, to remove intracellular components.

[0079] 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.

[0080] Potassium chloride, sodium chloride, etc. can be used as electrolytes.

[0081] 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 decreases, thereby lowering 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 should be understood that the content range may vary depending on the strength of the applied current.

[0082] 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.

[0083] The moisturizer may contain 0.5 to 2 wt% of glycerin.

[0084] As a viscosity modifier, carbomo may be included in an amount of 0.5 to 2 wt%.

[0085] The pH adjuster may include 0.1 to 0.5 wt of sodium bicarbonate.

[0086] Preservatives may be included in an amount of 0.1 to 0.2 wt for microbial inhibition.

[0087] When the composite wound treatment composition 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.

[0088] Specifically, first, a composite wound treatment composition according to an embodiment is applied to a 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 direct current, a high voltage pulsed current (HVPC). For example, the voltage 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.

[0089] 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.

[0090] 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.

[0091] 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.

[0092] [Example 1]

[0093] Preparation of a composition for wound treatment

[0094] Four different wound treatment composition treatment groups were set up under the conditions shown in Table 1 below.

[0095] Experimental group (wt%)By species / sex / age (at the time of experiment)By group (N)G1Negative Control (1wt% HA)C57BL / 6JF12M and above (aged animals)4G21wt% HA + 5wt% dECM5G31wt% HA + 5wt% NMN5G41wt% HA + 2.5wt% NMN + 2.5wt% dECM5

[0096] Evaluation of wound healing ability: Measurement of wound size changes. First, a 100㎟ wound was induced in the experimental mice, and then an ointment for each group was applied to the wound area. Specifically, each group was composed of G1 (normal group, composition containing 1 wt% HA), G2 (comparative group, dECM only), G3 (comparative group, NMN only), and G4 (experimental group, NMN + dECM). The change in wound size over 9 days was measured using a caliper. The results are depicted in the photograph in Fig. 1 and the graph in Fig. 2.

[0097] From the results of Figures 1 and 2, it can be confirmed that a superior wound healing effect is exhibited when NMN and dECM are mixed and treated compared to when NMN and dECM are treated alone.

[0098] Wound healing performance evaluation: measuring collagen production

[0099] 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.

[0100] From the results of Figures 3 and 4, it can be confirmed that a very high level of collagen is produced when NMN and dECM are mixed and treated compared to when NMN and dECM are treated alone.

[0101] Evaluation of wound healing capacity: Measurement of mRNA expression levels involved in collagen production

[0102] 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.

[0103] From the results in Fig. 5, it can be confirmed that the expression levels of Col1a1 and Col3a1 were the highest when NMN and dECM were mixed and treated compared to when NMN and dECM were treated alone.

[0104] [Example 2]

[0105] Five different wound treatment composition treatment groups were set up under the conditions shown in Table 2 below.

[0106] Experimental group (wt%)By speciesSexAge (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 + 5wt% NMN + 0.1wt% KCl5G41wt% HA + 2.5wt% NMN + 2.5wt% dECM + 0.1wt% KCl5G51wt% HA + 2.5wt% NR + 2.5wt% NMN + 0.1wt% KCl5

[0107] Evaluation of wound healing ability: Measurement of wound size changes First, a 100㎟ wound was induced in the experimental mice, and then an ointment for each group was applied to the wound area. Specifically, each group was composed of G1 (normal group, composition containing 1 wt% HA), G2 (comparative group, dECM only), G3 (comparative group, NMN only), G4 (experimental group, NMN + dECM), and G5 (comparative group, NR + NMN).

[0108] 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.

[0109] The changes in wound size at D3, D7, D10, and D13 were measured using calipers. The results are shown in the photograph in Fig. 7 and the graph in Fig. 8.

[0110] From the results of Figures 7 and 8, it can be confirmed that when NMN and dECM were mixed and electrical stimulation was applied, a superior wound healing effect was observed compared to when NMN and dECM were treated alone or when the combination of NR+NMN was used.

[0111] Wound healing performance evaluation: measuring collagen production

[0112] On the 13th day 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.

[0113] From the results of FIGS. 9 and 10, it can be confirmed that when NMN and dECM were mixed and electrical stimulation was applied, a very high level of collagen was produced compared to when NMN, dECM was treated alone or when the combination of NR and NMN was used. Comparing the results of FIGS. 4 and 10, it can be confirmed that when NMN + dECM was treated and electrical stimulation was added, the amount of collagen produced increased by more than 5 times compared to when no electrical stimulation was applied.

[0114] Wound healing performance evaluation: measurement of epithelial regeneration

[0115] On the 13th 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.

[0116] From the results of Figures 11 and 12, it can be confirmed that epithelial regeneration occurs at a very high rate when NMN and dECM are mixed and electrically stimulated compared to when NMN and dECM are treated alone or when the combination of NR+NMN is used.

[0117] Evaluation of wound healing capacity: Measurement of mRNA expression levels involved in collagen production

[0118] Tissue samples were sampled on the 13th day after wound healing, and PCR was performed to measure the expression levels of Col1a1 and Col3a1, which are involved in collagen production, and VEGF. The results are shown in Figure 13.

[0119] From the results of Figure 13, it can be confirmed that when NMN and dECM 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 NMN and dECM were treated alone.

[0120] Evaluation of wound healing ability: Measurement of the expression of inflammation-related substances

[0121] On the 13th day after wound healing, protein analysis (ELISA) was performed to measure TNF-α and IL-10. The results are shown in Figure 14.

[0122] From the results of Figure 14, it can be confirmed that when NMN and dECM were mixed and electrical stimulation was applied, the content of TNF-α, an inflammatory substance, decreased and the content of IL-10, which has the function of suppressing inflammation, increased compared to when NMN and dECM were treated alone.

[0123] [Example 3]

[0124] Five different wound treatment composition treatment groups were set up under the conditions shown in Table 3 below.

[0125] Experimental group (wt%)By speciesSexAge (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 + 5wt% Liposomal NAD + 0.1wt% KCl5G41wt% HA + 2.5wt% Liposomal NAD + 2.5wt% dECM + 0.1wt% KCl5G51wt% HA + 2.5wt% Liposomal NAD + 2.5wt% NMN + 0.1wt% KCl5

[0126] Evaluation of wound healing ability: Measurement of wound size change First, a 100㎟ wound was induced in the experimental mice, and then an ointment for each group was applied to the wound area. Specifically, each group was composed of G1 (normal group, composition containing 1 wt% HA), G2 (comparative group, dECM only), G3 (comparative group, NMN only), G4 (experimental group, Liposomal NAD + dECM), and G5 (comparative group, Liposomal NAD + NMN).

[0127] As in Experimental Example 2, 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 interpulse interval of 150 ms, and a pulse repetition count of 2100 x 2.

[0128] Changes in wound size were measured using calipers at D3, D7, D10, and D13. The results are shown in the photograph in Fig. 15 and the graph in Fig. 16.

[0129] From the results of Figures 15 and 16, it can be confirmed that a superior wound healing effect is exhibited when Liposomal NAD and dECM are mixed and electrically stimulated compared to when Liposomal NAD, dECM alone or a combination of Liposomal NAD and NMN.

[0130] Wound healing performance evaluation: measuring collagen production

[0131] On the 13th day after wound healing, tissue was excised and collagen was stained using trichrome staining. The results are shown in the photograph in Fig. 17 and the graph in Fig. 18.

[0132] From the results of Figs. 17 and 18, it can be confirmed that a very high level of collagen is produced when Liposomal NAD and dECM are mixed and electrically stimulated compared to when Liposomal NAD, dECM is treated alone or when the Liposomal NAD+NMN combination is used. Comparing the results of Figs. 18 and 4, it can be confirmed that when Liposomal NAD+dECM is treated and electrical stimulation is added, the amount of collagen produced increases by about 7 times compared to when only the NMN+dECM combination is used.

[0133] Wound healing performance evaluation: measurement of epithelial regeneration

[0134] On the 13th 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 19 and the graph in Figure 20.

[0135] From the results of Figures 19 and 20, it can be confirmed that epithelial regeneration occurs at a very high rate when Liposomal NAD and dECM are mixed and electrically stimulated compared to when Liposomal NAD, dECM alone or a combination of Liposomal NAD+NMN.

[0136] [Example 4]

[0137] Five different wound treatment composition treatment groups were set up under the conditions shown in Table 4 below.

[0138] Experimental group (wt%)By speciesSexAge(at the time of experiment)By group(N)G1Negative Control [1wt% HA]C57BL / 6JF12M and above(aged animals)4G21wt% HA + 5wt% dECM + 0.1wt% KCl4G31wt% HA + 5wt% NAM + 0.1wt% KCl4G41wt% HA + 2.5wt% dECM + 2.5wt% NAM + 0.1wt% KCl4G51wt% HA + 2.5wt% NMN + 2.5wt% NAM + 0.1wt% KCl4

[0139] Evaluation of wound healing ability: Measurement of wound size changes First, a 100㎟ wound was induced in the experimental mice, and then an ointment for each group was applied to the wound area. Specifically, each group was composed of G1 (normal group, composition containing 1 wt% HA), G2 (comparative group, dECM only), G3 (comparative group, NAN only), G4 (experimental group, NAM + dECM), and G5 (experimental group, NMN + NAM).

[0140] 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 interpulse interval of 150 ms, and a pulse repetition count of 2100 x 2 (see Figure 6).

[0141] Changes in wound size were measured using calipers at D3, D7, D10, and D13. The results are shown in the photograph in Fig. 21 and the graph in Fig. 22.

[0142] From the results of Figures 21 and 22, it can be confirmed that a superior wound healing effect is exhibited when NAM and dECM are mixed and electrical stimulation is applied compared to when NAM, dECM alone or the combination of NAM+NMN.

[0143] Wound healing performance evaluation: measuring collagen production

[0144] On the 13th day after wound healing, tissue was excised and collagen was stained using trichrome staining. The results are shown in the photograph in Fig. 23 and the graph in Fig. 24.

[0145] From the results of Figures 23 and 24, it can be confirmed that a very high level of collagen is produced when NAM and dECM are mixed and electrically stimulated compared to when NAM, dECM are treated alone or when the NAM+NMN combination is used.

[0146] Wound healing performance evaluation: measurement of epithelial regeneration

[0147] On the 13th 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 25 and the graph in Figure 26.

[0148] From the results of Figures 25 and 26, it can be confirmed that epithelial regeneration occurs at a very high rate when NAM and dECM are mixed and electrically stimulated compared to when NAM, dECM are treated alone or when NAM+NMN is combined.

[0149] Evaluation of wound healing capacity: Measurement of mRNA expression levels involved in collagen production

[0150] Tissue samples were sampled on the 13th day after wound healing, 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 27.

[0151] From the results of Figure 27, it can be confirmed that the expression levels of Col1a1, Col3a1, and VEGF were more than twice as high when NAM and dECM were mixed and electrically stimulated compared to when NAM, dECM were treated alone or when the NAM+NMN combination was used.

[0152] Evaluation of wound healing ability: Measurement of the expression of inflammation-related substances

[0153] On the 13th day after wound healing, protein analysis (ELISA) was performed to measure TNF-α and IL-10. The results are shown in Figure 28.

[0154] From the results of Figure 28, it can be confirmed that when NAM and dECM were mixed and electrical stimulation was applied compared to when NAM, dECM alone were treated or when NAM+NMN was combined, the content of TNF-α, an inflammatory substance, decreased and the content of IL-10, which has the function of suppressing inflammation, increased.

[0155] 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.

[0156] The present invention can be applied to a composite wound treatment medicine or a treatment method thereof.

Claims

1. A first activator comprising NAD, a precursor thereof, a derivative thereof or a combination thereof; and A composite wound healing composition comprising a second active agent comprising dECM.

2. In paragraph 1, A composite wound treatment composition in which the ratio of the first activator to the second activator is 1:0.3 to 1:

500.

3. In paragraph 1, A composite wound healing composition further comprising an electrolyte.

4. In paragraph 3, A composite wound treatment composition comprising the electrolyte in an amount of 0.1 to 0.5 wt% based on the total weight of the composite wound treatment composition.

5. In paragraph 1, A composite wound healing composition further comprising hyaluronic acid and a moisturizer.

6. In paragraph 1, A composite wound treatment composition wherein the first active agent is encapsulated in a liposome.

7. In paragraph 1, A composite wound treatment composition wherein the first active agent is nicotinamide (NAM) or nicotinamide mononucleotide (NMN).

8. A wound treatment method comprising the step of applying a composite wound treatment composition comprising a first activator comprising NAD, a precursor thereof, a derivative thereof, or a combination thereof, and a second activator comprising dECM to a wound site.

9. In paragraph 8, A wound treatment method wherein the ratio of the first activator to the second activator is 1:0.3 to 1:

500.

10. In paragraph 8, A wound treatment method wherein the above composite wound treatment composition further comprises an electrolyte.

11. In paragraph 10, A wound treatment method, wherein the electrolyte is included in an amount of 0.1 to 0.5 wt% based on the total weight of the composite wound treatment composition.

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

13. In paragraph 12, The above electric current is a wound treatment method using direct current.

14. In paragraph 13, The above direct current is a wound treatment method that is a high voltage pulse current.

15. In paragraph 13, 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.

16. In paragraph 8, A wound treatment method wherein the above composite wound treatment composition further comprises hyaluronic acid and a moisturizer.

17. In paragraph 8, A wound treatment method wherein the first active agent is encapsulated in a liposome.

18. In paragraph 8, A wound treatment method wherein the first active agent is nicotinamide (NAM) or nicotinamide mononucleotide (NMN).

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