KR20210075051A - Composition for for treating wound or scar comprising hydrogel patches - Google Patents

Abstract

The present invention relates to a composition for treating wounds or scars comprising a hydrogel patch. According to the composition or hydrogel according to an aspect, it is possible to induce movement and proliferation of fibroblasts and keratinocytes to a wound site to induce early closure of the wound, and thus there is an effect that can be usefully used for the prevention, alleviation or treatment of skin wounds or scars by inducing structural reconstruction of a skin layer.

Description

Composition for treating wound or scar comprising hydrogel patches

It relates to a composition for treating wounds or scars comprising a hydrogel patch.

The skin acts as a barrier that protects the body from the outside. When a wound occurs on the skin, the wound site is filled with blood due to the natural healing action of the living body, and the decrease in platelet granulation and activation of the hageman factor begins, leading to the wound healing process. Blood clotting acts as a temporary defense, protecting exposed wound tissues and providing a basis for cells to migrate during the healing process.

Wound healing aims to minimize scarring and allow the wound to heal as early as possible. The mechanism involved in wound healing differs depending on the severity of the wound. It is divided into partial skin damage, which heals the wound due to proliferation of epithelial cells, and dermal layer skin damage, which requires the synthesis and decomposition of collagen and the complex action of macrophages. . When skin damage occurs, new blood vessels and collagen are formed through an inflammatory process, and then, as the wound area heals, new epithelial cells migrate and the wound is sutured.

As current wound healing agents, proteins such as epidermal growth factor (epidermal growth factor), cytokines, interleukins, transforming growth factor-β (transforming growth factor-beta) and madecasol (madecasol; asiaticoside) asiaticoside), etc. have been used, but in the case of protein, it is very expensive and unstable, so it is not widely used as a wound healing agent. In the case of madecassol, the effect is weak and it causes side effects such as contact dermatitis, so there is a risk of side effects There is a need for wound healing agents with low therapeutic efficacy and high safety.

In one aspect, fibrin and / or fibrinogen; laminin or laminin-derived peptides or proteins; And/or to provide a composition for the prevention, improvement or treatment of wounds or scars comprising hyaluronic acid or a salt thereof as an active ingredient, for example, a pharmaceutical composition, a composition for external application to the skin, or a cosmetic composition.

Another aspect is fibrin and/or fibrinogen; laminin or laminin-derived peptides or proteins; And / or a composition for the prevention, improvement or treatment of wounds or scars comprising a hydrogel or a hydrogel patch containing hyaluronic acid or a salt thereof as an active ingredient, for example, a pharmaceutical composition, a composition for external application to the skin, or a cosmetic to provide a composition.

In one aspect, fibrin and / or fibrinogen; laminin or laminin-derived peptides or proteins; And/or it provides a composition for the prevention, improvement or treatment of wounds or scars comprising hyaluronic acid or a salt thereof as an active ingredient, for example, a pharmaceutical composition, a composition for external application to the skin, or a cosmetic composition.

Another aspect is fibrin and/or fibrinogen; laminin or laminin-derived peptides or proteins; And / or a composition for the prevention, improvement or treatment of wounds or scars comprising a hydrogel or a hydrogel patch containing hyaluronic acid or a salt thereof as an active ingredient, for example, a pharmaceutical composition, a composition for external application to the skin, or a cosmetic A composition is provided.

Another aspect provides a method for preventing, ameliorating or treating a wound or scar comprising administering the hydrogel patch or composition to a subject.

Another aspect provides the use of the hydrogel, hydrogel patch or composition for use in the manufacture of a therapeutic agent for a disease.

The hydrogel patch or composition comprises fibrin and/or fibrinogen; laminin or laminin-derived peptides or proteins; and/or hyaluronic acid or a salt thereof. For example, the hydrogel patch or composition may include laminin or laminin-derived peptides or proteins; and hyaluronic acid or a salt thereof.

As used herein, the term “treat” may mean that a wound is healed in a shorter time compared to natural healing. The treatment may include amelioration and/or alleviation of a wound or scar. In addition, the treatment may include treatment of wounds and/or wound-related diseases. Said treatment may refer to healing and/or regeneration of damaged tissue resulting from a wound. The wound treatment may include the meaning of skin regeneration. In addition, the treatment may be to maintain the original composition of the damaged tissue. In addition, the treatment may be to promote healing and/or regeneration of the damaged tissue while minimizing complications and/or scarring of wound-related diseases.

As used herein, the term "pharmaceutically effective amount" means any amount of a composition used in the practice of the invention provided herein sufficient to alleviate, inhibit the progression or prevent a disease, disorder or condition, or one or more symptoms thereof. can

The terms “administering,” “applying,” “introducing,” and “implanting” are used interchangeably and refer to a method or route that results in at least partial localization to a desired site of a patch or composition according to one embodiment. may refer to the placement of a patch or composition according to one embodiment into a subject by

As used herein, the term “patch” may mean a means that has a predetermined shape and can be applied, attached, or contacted to a target site.

In one embodiment, the hydrogel patch or composition may have intermediate properties between a solid and a liquid. The hydrogel patch or composition may be amorphous, spherical, hemispherical, disc-shaped, or cylindrical. Also, for example, the hydrogel patch may have a diameter of 0.05 mm to 10 cm, 0.1 mm to 5 cm, 0.1 mm to 3 cm, or 0.2 mm to 1.5 cm, and may be provided in such a size or shape. . In addition, the hydrogel patch may be applied, implanted, attached, or contacted to a target site (eg, a tissue damaged site) to be deformed in shape according to the shape of the damaged site.

In another embodiment, the hydrogel patch or composition may be solid (including powder), semi-solid, or liquid. Also, for example, the hydrogel patch or composition may undergo a reversible phase transition (eg, according to temperature change) to a solid (including powder), semi-solid, or liquid state. Since the hydrogel patch may undergo a reversible phase transition depending on ambient conditions such as temperature conditions, the hydrogel patch according to one embodiment is produced and provided in a solid (including powder), or liquid state, before administration to the target site, Upon administration or after administration, it may be converted into a hydrogel patch and used. For example, the hydrogel patch is provided in a sol state containing fibrinogen, laminin, and hyaluronic acid, so that a user can convert fibrinogen to fibrin (e.g., thrombin) by using, A hydrogel patch according to one embodiment may be manufactured and used. Accordingly, the hydrogel patch according to one embodiment comprises fibrin and/or fibrinogen; laminin; and/or a composition comprising hyaluronic acid, for example, in the form of a prodrug of a solid (powder), liquid (sol), or semi-solid composition. A composition provided in the form of a prodrug may be prepared or modified to act as a hydrogel patch in vivo. In addition, the hydrogel patch according to an embodiment may further include thrombin, or thrombin may be provided together as a kit.

In another embodiment, the hydrogel patch or composition may be porous. Specifically, it may be one having porosity (micropores) on the surface of the hydrogel patch. Without being limited by a particular theory, the hydrogel patch according to one embodiment may have porosity, thereby enhancing the interaction between active materials.

In one embodiment, the hydrogel patch or composition may include fibrin and/or fibrinogen. The final pharmacological substance acting in vivo includes fibrin, but fibrinogen may be used instead of fibrin in the form of a prodrug. In addition, depending on the amount of a substance that converts fibrin into fibrinogen, the hydrogel patch or composition according to an embodiment may include some fibrinogen or thrombin. Accordingly, the present specification relates to fibrinogen; laminin; And/or a prodrug comprising hyaluronic acid may be additionally provided. The fibrin or fibrinogen is 0.5 to 25 mg/ml, 1 to 25 mg/ml, 1 to 20 mg/ml, 1 to 15 mg/ml, 0.5 to 8 mg/ml, 2 to 8 mg/ml, 3 to 12 It may be included in a concentration of mg/ml, 4 to 12 mg/ml, 8 to 12 mg/ml or 1 to 4 mg/ml.

The fibrinogen glycoprotein is a hexamer composed of soluble α, β, and γ subunits made in hepatocytes of the liver. Fibrinogen reacts with thrombin enzyme to undergo a phase change from soluble to insoluble fibrin polymer and fiber.

The thrombin enzyme is a serine protease and is an enzyme that transforms soluble fibrinogen into insoluble fibrin. The thrombin may serve to gel the sol-state hydrogel by changing fibrinogen to fibrin.

As used herein, the term "laminin" is an extracellular matrix protein constituting the basal lamina, and may refer to a heterotrimeric protein consisting of α, β, and γ subunits. Accordingly, the laminin may include not only the full-length laminin protein but also a laminin-derived peptide or protein. For example, the laminin is laminin-1, laminin-2, laminin-3, laminin-4, laminin-5A, laminin-5B, laminin-6, laminin-7, laminin-8, laminin-9, laminin-10 , laminin-11, laminin-12, laminin-14, or laminin-15. In addition, the laminin-derived peptide may be an α chain, a γ chain, or a β chain. The laminin is 1 to 100 μg/ml, 2 to 80 μg/ml, 2 to 40 μg/ml, 5 to 40 μg/ml, 15 to 25 μg/ml, 8 to 25 μg/ml, 15 to 35 μg/ml ml, 8 to 30 μg/ml, or 8 to 40 μg/ml.

The hyaluronic acid (Hyaluronic acid) glycosaminoglycan (glycosaminoglycan) is a glycoside bond with D-glucuronic acid and N-acetyl-D-glucosamine having changes in β-(1→4) and β-(1→3) It is a polysaccharide of disaccharide bonds composed of (glycosidic bonds) and has various molecular weights depending on the length of the disaccharide bond. In one embodiment, the molecular weight of hyaluronic acid may be 5,000 to 20,000,000 Da. More specifically, the molecular weight of hyaluronic acid may be 0.5 to 4.0 x 10 ⁶ Da, 1.0 to 2.0 x 10 ⁶ Da, or 1.5 to 1.8 x 10 ⁶ Da. The hyaluronic acid is 10 μg/ml to 10 mg/ml, 10 μg/ml to 5 mg/ml, 100 μg/ml to 3 mg/ml, 500 μg/ml to 5 mg/ml, 500 μg/ml to 8 mg/ml, 500 μg/ml to 4 mg/ml, 500 μg/ml to 2 mg/ml, 200 μg/ml to 2 mg/ml, 1 mg/ml to 8 mg/ml, or 1 mg/ml to 5 It may be included in a concentration of mg/ml.

In another embodiment, the hydrogel patch or composition may further include a cell growth factor. The cell growth factor is a nerve cell growth factor, a vascular endothelial growth factor, a fibroblast growth factor, a bone morphogenetic protein, an epidermal growth factor, a hepatocyte growth factor, a transforming growth factor or their It can be a combination. More specifically, the growth factors include placental growth factor, macrophage colony stimulating factor, granulocyte macrophage colony stimulating factor, neuropilin, fibroblast growth factor (FGF)-1, FGF-2 (bFGF), FGF-3, FGF -4, FGF-5, FGF-6, erythropoietin, BMP-2, BMP-4, BMP-7, TGF-beta, IGF-1, osteopontin, pleiotropin, activin, endo Celin-1 and combinations thereof. The nerve cell growth factor is BDNF (Brain-derived neurotropic factor), GDNF (Glial cell derived neurotropic factor), CNTF (Ciliary neurotropic factor), bFGF (basic fibroblast growth factor), cAMP (cyclic adenocyne monophosphate), NT (Neurotropin) , NT3 (Neurotropin-3), NT4 (Neurotropin-4), T3 (Triiodo-L-Thyronine), SHH (Sonic hedgehog), and PDGF (Platelet-derived growth factor) containing one or more selected from the group consisting of it could be The vascular endothelial growth factor may include vascular endothelial growth factor (VEGF)-A, VEGF-A, VEGF-B, VEGF-C, VEGF-D, or VEGF-E. The cell growth factor may have a different concentration included in the hydrogel patch or composition depending on the type of growth factor, but generally may be included at a concentration of 1 ng/ml to 1,000 ng/ml or 0.1 μM to 100 μM. The cell growth factor may serve to increase the tissue damage recovery effect of a hydrogel patch or composition comprising fibrin, laminin, and hyaluronic acid.

In other embodiments, the hydrogel patch or composition may or may not contain collagen. Without being limited by a particular theory, the collagen may or may not be substantially included as a component of the composition, but there may be cases in which it is more advantageous in one aspect than not being included.

In addition, the hydrogel patch or composition may be substantially free of cells. As used herein, "substantially does not comprise" means that collagen or cells are included or not included to such an extent that they do not affect the activity or pharmacological activity of the hydrogel patch or composition. The hydrogel patch or composition according to one embodiment may be distinguished from a cell therapy agent generally used for regeneration of damaged tissue by substantially not including cells.

In one embodiment, the hydrogel patch or composition comprises fibrin and/or fibrinogen; laminin or laminin-derived peptides; and hyaluronic acid or a salt thereof. In addition, one or more of the components mentioned herein may be additionally included and configured. For example, the hydrogel patch or composition may contain fibrin and/or fibrinogen; laminin or laminin-derived peptides; hyaluronic acid or a salt thereof; optionally thrombin; optionally collagen; and optionally cell growth factors.

The term “wound” refers to a disorder in which tissue is cut, torn, broken, burned, or traumatized, or causing such damage. Or it means damage to the human body resulting from a disease (injury). The wound may be an open wound or a closed wound with an open surface.

An example of the wound is the epidermis of the skin; dermis; epidermis and dermis; Alternatively, it may be a wound in which the epidermis, dermis and subcutaneous fat layer are damaged.

In addition, examples of the wound include cuts, incisions (eg, surgical incisions), abrasions, lacerations or lacerations, fractures, contusions, burns. , or amputations.

The wound may be selected from the group consisting of chronic wounds, acute wounds, surgical wounds, orthopedic wounds, traumatic wounds, combat wounds, and combinations thereof.

Also, the wound may be a wound caused by another disease. For example, the wound may be fibrosis, diabetes, diabetic ulcer, autoimmune skin disease, abrasions, lacerations, incisions, contusions, bruises, punctures, peels, burns, ulcers, pressure sores, or a combination thereof. .

The diabetes may be type I or type II diabetes. It is known that in patients with diabetes or its complications, for example, diabetic ulcer, wound healing is delayed due to various causes (Falanga V: Wound healing and its impairment in the diabetic foot. Lancet 366:1736-43 , 2005). The hydrogel patch or composition according to one embodiment promotes the migratory ability of epithelial cells, induces migration and proliferation of fibroblasts and keratinocytes to the wound site to induce early closure of the wound, or rebuilds the structure of the skin layer It can be effectively used for diabetic wounds or diabetic ulcers (diabetic skin ulcers or diabetic foot ulcers).

As used herein, "scar" may mean a fibrous tissue that replaces normal tissue destroyed by an injury or disease. Damage to the outer layers of the skin heals by rebuilding the tissue, in which case scarring may be minimal. However, when the thick layer of tissue beneath the skin is damaged, reconstruction becomes more complicated. The body accumulates collagen fibers (proteins that are produced naturally by the body), which usually result in marked scarring.

The scar may be a hypertrophic scar, a keloid scar, an atrophic scar, a stretch mark, or a combination thereof.

Hyperproliferative scars are raised scars that remain within the boundaries of the original lesion and can usually regress spontaneously after an initial injury. Hyperproliferative scars are hard, raised, red, itchy, tender, and constricted. These typically occur after burn injuries to the trunk and limbs. Clinically and histologically, hyperproliferative scars and keloid scars are very similar, but unlike keloids, hyperproliferative scars enlarge by pushing the scar border, whereas keloids infiltrate the surrounding tissue. Hyperproliferative scars mature and flatten over time. Hyperproliferative scars exhibit the same annular vitreous bundles of collagen as keloids and have more vessels and cells than normal scars.

Keloid scars are benign fibrous growths in the dermis that occur after skin trauma. It protrudes above the skin surface and extends beyond the boundaries of the original lesion. These scars are permanent and do not regress over time. Keloids are often cosmetically unsightly and can be painful.

Atrophic scars are flat and pressed under the surrounding skin. It is usually small and is often round with a sawtooth or inverted center. Atrophic scarring can be the result of surgery, trauma, acne and chickenpox.

Stretch marks occur when the fine lines of a surgical scar are gradually stretched and widened, usually within 3 weeks after surgery. This is typically a flat, faint, soft, asymptomatic scar often seen after knee or shoulder surgery.

The hydrogel patch or composition according to one embodiment may induce early closure of the wound by inducing movement and proliferation of fibroblasts and keratinocytes, which are cells constituting the skin, to the wound site. In addition, the hydrogel patch or composition according to an embodiment may inhibit scar formation by inducing structural reconstruction of the skin layer. In addition, the hydrogel patch or composition according to an embodiment may induce regeneration of blood vessels and hair follicles lost due to a wound to induce regeneration of the original structure of the skin at an early stage.

Accordingly, the composition can be usefully used for the treatment of wounds, prevention of scarring, improvement or treatment by having the above effects.

The dosage of the composition according to one embodiment is 0.01 mg to 10,000 mg, 0.1 mg to 1000 mg, 1 mg to 100 mg, 0.01 mg to 1000 mg, 0.01 mg to 100 mg, 0.01 mg to 10 mg, or 0.01 mg to 1 mg. However, the dosage may be prescribed in various ways depending on factors such as formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate and response sensitivity, and those skilled in the art The dosage may be appropriately adjusted in consideration of these factors. The number of administration may be one time or two or more within the range of clinically acceptable side effects, and may be administered to one or two or more sites for the administration site. For animals other than humans, the dose is the same as that of humans per kg, or the above dose is converted, for example, by the volume ratio (for example, the average value) of the target animal and the organ (heart, etc.) One dose can be administered. Possible routes of administration include oral, sublingual, parenteral (eg, subcutaneous, intramuscular, intraarterial, intraperitoneal, intrathecal, or intravenous), rectal, topical (including transdermal), inhalation, and injection, or implantable devices. or the insertion of a substance. Examples of the animal to be treated according to one embodiment include humans and other target mammals, specifically humans, monkeys, mice, rats, rabbits, sheep, cattle, dogs, horses, pigs, etc. Included.

The pharmaceutical composition according to one embodiment may include a pharmaceutically acceptable carrier and/or additive. For example, sterile water, physiological saline, conventional buffers (phosphoric acid, citric acid, other organic acids, etc.), stabilizers, salts, antioxidants (ascorbic acid, etc.), surfactants, suspending agents, isotonic agents, or preservatives, etc. can do. For topical administration, organic materials such as biopolymers, inorganic materials such as hydroxyapatite, specifically collagen matrix, polylactic acid polymers or copolymers, polyethylene glycol polymers or copolymers and chemical derivatives thereof, etc. may also be combined for topical administration. can

The pharmaceutical composition according to one embodiment, if necessary according to its administration method or formulation, a suspending agent, solubilizing agent, stabilizer, isotonic agent, preservative, adsorption inhibitor, surfactant, diluent, excipient, pH adjuster, analgesic agent, Buffers, reducing agents, antioxidants and the like may be included as appropriate. Pharmaceutically acceptable carriers and agents suitable for the present invention, including those exemplified above, are described in detail in Remington's Pharmaceutical Sciences, 19th ed., 1995. The pharmaceutical composition according to one embodiment is formulated in a unit dosage form by using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person of ordinary skill in the art to which the present invention pertains. It can be prepared as or by putting it in a multi-dose container. Herein, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or in the form of a powder, granules, tablets or capsules.

The composition may be formulated as an oral or parenteral dosage form. Oral dosage forms may be granules, powders, solutions, tablets, capsules, dry syrups, or a combination thereof. The parenteral dosage form may be an injection or an external preparation for skin.

The external preparation for skin may be a cream, gel, ointment, skin emulsifier, skin suspension, transdermally delivered patch, drug-containing bandage, lotion, or a combination thereof. The external preparation for skin is a component usually used in external preparations for skin such as cosmetics, pharmaceuticals, and quasi-drugs, for example, an aqueous component, an oily component, a powder component, alcohol, a moisturizer, a thickener, an ultraviolet absorber, a whitening agent, a preservative, an antioxidant, a surfactant , fragrance, colorant, various skin nutrients, or a combination thereof may be appropriately blended as needed. The external preparation for skin includes metal sequestering agents such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, and gluconic acid, caffeine, tannin, belapamil, licorice extract, glablidine, and kaline. Fruit hot water extract, various herbal medicines, tocopherol acetate, glitylitnic acid, tranexamic acid and derivatives or salts thereof, vitamin C, magnesium ascorbate phosphate, ascorbic acid glucoside, arbutin, kojic acid, glucose, fructose, Sugars, such as trehalose, etc. can be mix|blended suitably.

In addition, the composition may be a quasi-drug composition. The term "quasi-drug" refers to textiles, rubber products, or the like, which are used for the purpose of treating, alleviating, treating or preventing diseases of humans or animals, have weak action on the human body or do not directly act on the human body, and are not devices or machines. Products that fall under one of those used for sterilization, insecticide and similar purposes to prevent infection, and those used for the purpose of diagnosing, treating, alleviating, treating or preventing diseases of humans or animals , refers to items other than machines or devices, and items used for the purpose of pharmacologically affecting the structure and function of humans or animals, excluding those that are not instruments, machines, or devices, and may include external preparations for skin and personal care products. can When the hydrogel, hydrogel patch or composition is added to a quasi-drug composition for the purpose of preventing or improving wounds or scars, the hydrogel, hydrogel patch or composition may be added as it is or used together with other quasi-drug ingredients, It can be used appropriately depending on the phosphorus method. The mixing amount of the active ingredient may be appropriately determined depending on the purpose of use (prophylactic, health or therapeutic treatment). The quasi-drug composition is not particularly limited thereto, but includes personal care products, external preparations for skin, disinfection cleaner, shower foam, gargrin, wet tissues, detergent soap, hand wash, humidifier filler, mask, ointment or filter filler. The external preparation for skin is not particularly limited thereto, but is preferably prepared and used in the form of an ointment, lotion, spray, patch, cream, powder, suspension, gel or gel. The personal care product may be soap, wet tissue, tissue paper, shampoo, toothpaste, hair care product, air freshener gel or cleaning gel.

The composition may be a cosmetic composition for improving or alleviating wounds or scars. When the composition of the present invention is a cosmetic composition, it can be prepared in various forms, for example, emulsions, lotions, creams (oil-in-water, water-in-oil, multiphase), solutions, suspensions (anhydrous and water-based), anhydrous products (oils) and glycol-based), gels, masks, packs, powders, and the like. In addition, the cosmetic composition of the present specification may include an acceptable carrier in the cosmetic formulation. Here, "acceptable carrier in cosmetic preparations" refers to compounds or compositions that are already known and used that can be included in cosmetic preparations, or compounds or compositions to be developed in the future that have toxicity, instability, or irritation beyond what the human body can adapt when in contact with the skin. say nothing The carrier may be included in the cosmetic composition of the present specification in an amount of about 1% to about 99.99% by weight based on its total weight, preferably about 90% to about 99.99% by weight of the weight of the composition. Examples of the carrier include alcohol, oil, surfactant, fatty acid, silicone oil, humectant, humectant, viscosity modifier, emulsion, stabilizer, UV blocker, color developer, fragrance, and the like.

Another aspect is fibrinogen; laminin or laminin-derived peptides; And it provides a method for preparing a hydrogel patch by adding thrombin to a composition in a sol state comprising hyaluronic acid or a pharmaceutically acceptable salt thereof.

The method may further comprise the step of blending a cell growth factor to the composition in the sol state.

The method may also include the step of gelling by adding thrombin, followed by secondary gelation by adding thrombin again. The gelation may be performed at 10 to 40° C. for 5 minutes to 3 hours. The hydrogel patch may be manufactured in various shapes or sizes depending on the shape of the frame.

The method may also include cryopreserving or cryopreserving the hydrogel patch at 4 to -210°C in solution. The solution may include dimethyl sulfoxide (DMSO), but any solution that does not substantially change the chemical or physical properties of the hydrogel patch may be used. The hydrogel patch does not substantially change its shape or activity even when cryopreserved or cryopreserved.

The hydrogel patch, fibrin and/or fibrinogen, laminin, hyaluronic acid or cell growth factor is the same as described above.

Another aspect provides a method of cryopreserving or cryopreserving the hydrogel patch in solution (eg, DMSO) at 4 to -210°C.

According to the composition or hydrogel according to an aspect, it induces movement and proliferation of fibroblasts and keratin cells to the wound site to induce early closure of the wound, and induces structural reconstruction of the skin layer to prevent or improve skin wounds or scars Or there is an effect that can be usefully used for treatment.

1 is a hydrogel composition (fibrin, hyaluronic acid, and laminin) according to an embodiment and an untreated group (none) on the wound closure of human fibroblasts in vitro; is a view confirmed using an optical microscope; scale bars represent each 500 μm.
2 is a graph showing the quantification of the effects of the hydrogel according to an embodiment, each component alone, and the combination of the two components on wound repair in vitro of human fibroblasts; *p<0.05, **p<0.005.
3 is a photograph showing the wound and scar treatment effect of the hydrogel according to an embodiment.
Figure 4 is a graph showing the recovery rate of the wound according to the application of the hydrogel according to an embodiment.
Figure 5a is a photograph showing the skin structure regeneration effect of the hydrogel according to an embodiment; whole area; n = normal skin, w = wound area.
Figure 5b is a photograph showing the skin structure regeneration effect of the hydrogel according to an embodiment; enlargement of the hair follicle area; Arrow = hair follicles.

Hereinafter, the present invention will be described in detail by reference examples and examples. However, the following reference examples and examples are merely illustrative of the present invention, and should not be construed as limiting the present invention.

<Example> Preparation of hydrogel

1-1. Hydrogel formulation

Fibrinogen (Sigma, F8630) was dissolved in PBS at 20 mg/ml in a sol state, hyaluronic acid (Sigma, 53747) was dissolved in PBS at 5 mg/ml in a sol state, and thrombin 200 U/ml was dissolved in PBS. dissolved in the state. Thereafter, fibrinogen in a sol state, laminin (Thermofisher, 23017-015), and hyaluronic acid were mixed at 5 mg/ml, 20 μg/ml, and 1 mg/ml, respectively, to prepare a sol-state hydrogel.

1-2. Production of hydrogel patches

After making a circle of 0.3 to 0.8 mm on the sterilized parafilm and dispensing 6.7 U/ml of thrombin (Sigma, T4648) in the sol state, 15 μl of the hydrogel formulated in Example 1-1 was mixed with thrombin. Busy according to the circle frame. Thereafter, it was cured at 37° C. for 30 minutes to prepare a hydrogel patch through a sol-gel phase transition.

<Experimental Example 1> In vitro wound healing improvement effect confirmed

In order to confirm the in vitro wound healing effect of the hydrogel patch prepared in the above example, the effect of the hydrogel according to one embodiment on the proliferation and migration of seal fibroblasts compared to the comparative example material was confirmed.

Specifically, human fibroblasts (CRL-2097, ATCC) 5.0 x 10 to 100% confluency in 0.1% gelatin (sigma, G1890) coated 12 well culture dishes (corning, 07-200-82) ⁴ were cultured. Thereafter, identical wounds were made on the human fibroblasts with a 200 μl filter tip according to an in vitro scratch assay by a known method (Guan J et al. Nat Protoc, 2007). Next, the hydrogel (fibrin 5 mg/ml, laminin 20 μg/ml, and hyaluronic acid 1 mg/ml) prepared in the same manner as in the above example was applied and thrombin was added to form a hydrogel.

In addition, fibrin 15 mg/ml, laminin 60 μg/ml, and hyaluronic acid 3 mg/ml alone were treated, a combination of fibrin 5 mg/ml and laminin 20 μg/ml, laminin 20 μg/ml and hyaluronic acid A combination of 1 mg/ml and a combination of fibrin 5 mg/ml and hyaluronic acid 1 mg/ml were treated.

Thereafter, wound repair was taken using an optical microscope (Leica, DMI3000B) during incubation for 3 hours, 5 hours, 10 hours, 18 hours and 24 hours, and the results are shown in FIG. 1 . In addition, for the image observed with an optical microscope, the wound area was calculated using ImageJ (National Institutes of Health) to calculate the wound repair rate, and the results are shown in FIG. 2 . All statistical analyzes were performed using unpaired two-tailed Student's t-test, and significance was *P<0.05, or **P<0.005.

1 and 2, in the case of fibrin alone, hyaluronic acid alone, and the combination of fibrin and laminin, the recovery rate was about 50 to 60%, and it was confirmed that there was no significant wound healing effect compared to the control group. In contrast, in the case of laminin alone, fibrin and hyaluronic acid combination, and hyaluronic acid and laminin combination, the recovery rate was about 70 to about 80%. On the other hand, in the case of the combination of fibrin, hyaluronic acid, and laminin, it was confirmed that the wound repair rate increased by about 90%, which was about 2 times higher than that of a single material or a combination of two materials. . The above results indicate that the combination of hyaluronic acid and laminin has a remarkable synergistic effect on the proliferation and migration of human fibroblasts, and in particular, the combination of the three compositions causes a synergistic effect to increase the proliferation and migration of fibroblasts.

In addition, the wound healing effect was confirmed by additionally producing hydrogel patches of various concentrations.

Specifically, sol-state fibrinogen, laminin, and hyaluronic acid of 3 mg/ml, 10 μg/ml, and 5 mg/ml, respectively (Formulation 1), each of 10 mg/ml, 30 μg/ml, and 500 μg/ml Formulation (Formulation 2), Formulation of 10 mg/ml, 30 µg/ml, and 5 mg/ml, respectively (Formulation 3), and Formulation of 3 mg/ml, 10 µg/ml, and 500 µg/ml, respectively (Formulation 4) was prepared, and the wound healing effect was confirmed after 24 hours in the same manner as above. The results are shown in Table 1 below.

control formulation 1 formulation 2 formulation 3 formulation 4 Wound closure 62.03 86.58 88.92 85.66 89.54

<Experimental Example 2> In vivo wound treatment and scar improvement effect confirmed

In order to confirm the wound healing and scar improvement effects of the hydrogel prepared in the above example, it was evaluated whether the skin structure was regenerated in an animal model.

Specifically, C57BL/6 mice (Hyochang Science, Korea) were anesthetized by inhalation with isofluorine, and then their backs were shaved and hair removed with a hair removal cream. Thereafter, the hair removal site was sterilized with povidone and 70% ethanol. Next, cut 4 places on the epithelium of the mouse's back with a 5 mm biopsy punch, and then sterilize it with sodium hypochlorite and 70% ethanol on a 15 mm round silicone sheet with a 6 mm internal hole (0.5-mm- thick) was attached to the wound area with an instant adhesive and then sutured to the skin. After applying about 30 ul of the prepared hydrogel to the wound site, Tegaderm (3M, 2-3/8 inches X 2-3/4 inches) is attached to the wound area and wrapped with a compression bandage. gave. In the control group, Tegaderm was attached to the wound site without any treatment and wrapped with a compression bandage.

Thereafter, the wound site was photographed every day for 14 days, and the results are shown in FIG. 3 .

In addition, the upper and lower and left and right sizes of the wound site were measured with a caliper every day for 14 days, and the average value was converted to the 0 wound day to measure the recovery rate, and the results are shown in FIG. 4 and Table 2 below.

number of experimental animals recovery rate number of doses recovery period administration group untreated group administration group untreated group 15 15 94% 76% 1 time 10-14 days

In addition, for histological analysis of the skin regeneration of the wound site, the skin tissue of the wound site was extracted 14 days after application of the hydrogel of the above example. Thereafter, the extracted skin tissue is stored in 4% paraformaldehyde for one day, treated with 50%, 70%, 90%, 100% ethanol, and xylene sequentially, and then preserved in paraffin for one day to obtain a paraffin block. produced. After cutting the paraffin block into 10 μm, H&E staining was performed, and the results are shown in FIG. 5 .

3 is a photograph showing the wound and scar treatment effect of the hydrogel according to an embodiment.

4 is a graph showing the recovery rate of the wound according to the application of the hydrogel according to an embodiment.

5 is a photograph showing the skin structure regeneration effect of the hydrogel according to an embodiment; (a) the entire site; (b) enlargement of the hair follicle area; Arrow = hair follicles, n = normal skin, w = wound area.

3 and 4, it can be seen that the hydrogel according to one embodiment induces movement and proliferation of fibroblasts and keratin cells, which are cells constituting the skin, to the wound site, thereby inducing early closure of the wound. there was.

In addition, as shown in FIG. 5 , it was found that the hydrogel according to one embodiment could inhibit scar formation by inducing structural reconstruction of the skin layer. In addition, it was found that the hydrogel according to one embodiment induces regeneration of blood vessels and hair follicles lost due to wounds, thereby inducing regeneration of the original structure of the skin at an early stage.

In addition, in order to further confirm the effect of treating diabetic wounds, the effect of treating wounds in diabetic mice was confirmed. Specifically, after acclimatization to male db/db 　 diabetic mice until the blood sugar became 250 mg/dL or more, the wound healing effect in vivo was confirmed in the same manner as above. The control group and the experimental group each used 5 animals, and the upper and lower and left and right sizes of the wound site were measured with a caliper every day for 14 days, and the average value was converted to the 0th day of the wound to measure the recovery rate. The results are shown in Table 3 below.

control Example hydrogel recovery rate 62% 90%

In general, despite many advances in wound healing and management, in diabetes, wound healing is delayed because all the carefully prepared sequences of the cellular and biochemical processes of wound healing are hampered (Brown, 1992; Shukla, 1998). ; Mustoe, 2004). In addition, healing of diabetic skin ulcers is delayed because of impaired blood vessel formation, insufficient blood flow, increased inflammation, decreased proliferation of fibroblasts, and decreased re-epithelialization by keratinocytes.

As shown in Table 3 above, the hydrogel patch according to one embodiment was found to exhibit a significant wound healing effect even in a diabetic animal model. These results mean that the hydrogel patch according to one embodiment can be usefully used in the treatment of diabetic wounds, such as diabetic skin ulcers and diabetic foot ulcers.

Claims (16)

fibrin or fibrin and fibrinogen;
laminin; and
A pharmaceutical composition for the prevention or treatment of skin wounds or scars comprising hyaluronic acid or a salt thereof as an active ingredient. The pharmaceutical composition of claim 1, wherein the composition is a hydrogel or a hydrogel patch. The pharmaceutical according to claim 1, comprising fibrin or fibrinogen at a concentration of 0.5 to 25 mg/ml, laminin at a concentration of 1 to 100 μg/ml, or hyaluronic acid at a concentration of 10 μg/ml to 10 mg/ml. enemy composition. The pharmaceutical composition according to claim 1, further comprising a cell growth factor The method according to claim 4, wherein the cell growth factor is nerve cell growth factor, vascular endothelial growth factor, fibroblast growth factor, bone morphogenetic protein, epidermal growth factor (epidermal growth factor), hepatocyte growth factor, trait A transforming growth factor or a combination thereof, a pharmaceutical composition. The pharmaceutical composition of claim 2, wherein the hydrogel or hydrogel patch does not contain cells. The pharmaceutical composition of claim 2, wherein the hydrogel or hydrogel patch has porosity on the surface. The pharmaceutical composition of claim 2, wherein the hydrogel or hydrogel patch undergoes a reversible phase transition to a solid, semi-solid, or liquid state depending on temperature. The method according to claim 2, wherein the hydrogel or hydrogel patch is applied to the wound site, the shape is modified according to the shape of the wound site, the pharmaceutical composition. The pharmaceutical composition of claim 1 , wherein the wound is a chronic wound, an acute wound, a surgical wound, an orthopedic wound, a traumatic wound, a combat wound, or a combination thereof. The method according to claim 1, wherein the wound is fibrosis, diabetes, diabetic ulcer, autoimmune skin disease, abrasions, lacerations, incisions, contusions, bruises, punctures, peels, burns, ulcers, pressure sores, or a combination thereof. pharmaceutical composition. The pharmaceutical composition of claim 1, wherein the scar is a hypertrophic scar, a keloid scar, atrophic scar, a stretch mark, or a combination thereof. fibrin or fibrin and fibrinogen;
laminin; and
A composition for external application for skin for preventing or improving wounds or scars, comprising hyaluronic acid or a salt thereof as an active ingredient. The composition for external application for skin according to claim 13, wherein the composition is a hydrogel or a hydrogel patch. fibrin or fibrin and fibrinogen;
laminin; and
A cosmetic composition for preventing or improving wounds or scars comprising hyaluronic acid or a salt thereof as an active ingredient. The cosmetic composition of claim 15, wherein the composition is a hydrogel or a hydrogel patch.

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