Injectable composition for skin texture improvement and / or skin moisturization, comprising gamma-polyglutamic acid

Abstract

The present invention relates to an injectable composition for regenerating tissue and / or promoting collagen production, comprising non-crosslinked gamma-polyglutamic acid (γ-PGA) or a pharmaceutically acceptable salt thereof. The injectable composition according to one embodiment of the present invention exhibits excellent spreadability without a lifting phenomenon or a lump phenomenon at an initial injection site when injected into the skin or subcutaneous tissue, and provides effects of promoting collagen production and regenerating tissue in skin tissue.

Description

Injectable composition for improving skin texture and / or moisturizing the skin containing gamma-polyglutamic acid

[0001] The present invention relates to an injectable composition based on non-crosslinked gamma-polyglutamic acid (abbreviated as 'γ-PGA'), and more specifically, to an injectable composition for promoting tissue regeneration and / or collagen production in the skin or subcutaneous tissue.

[0002] γ-PGA is a substance known to possess fibroblast proliferation, collagen production, and moisturizing effects. Due to these characteristics, it is applied in the medical field (wound healing), the cosmetics field (topical cosmetics such as skin toners and lotions), and the functional materials field. However, most skin moisturizing compositions containing γ-PGA relate to cosmetics (Korean Patent No. 1096393; Korean Patent No. 2091587, etc.) and appear in the form of topical application formulations, while technology regarding injectable formulations injected into the skin or subcutaneous tissue is limited. Although prior art regarding γ-PGA injectable fillers exists, it is intended for skin volume enhancement. Furthermore, in the case of such compositions intended for skin volume enhancement, γ-PGA hydrated hydrogels cannot achieve the volume enhancement objective due to their low viscoelasticity; therefore, prior art discloses the use of γ-PGA crosslinked (using radiation, etc.) (Korean Patent No. 2017741, etc.).

[0003] However, there is no prior art regarding an injectable composition that induces microscopic regeneration of skin or subcutaneous tissue, with non-crosslinked γ-PGA as the main component, while having the primary purpose of promoting tissue regeneration or collagen production.

[0004] Meanwhile, most compositions currently used for improving skin texture or skin hydration are primarily hyaluronic acid hydrogel-based. When these hydrogel compositions are injected into the skin, the surface at the injection site or the surrounding area may become uneven and raised, resulting in an unnatural appearance, known as a "lump." The duration of this lump phenomenon can vary from a few days to a week depending on the commercially available product. Therefore, there is a need to develop an injectable composition that exhibits natural diffusion upon injection while simultaneously demonstrating efficacy such as tissue regeneration and collagen production promotion.

[0005] The present invention aims to provide a non-crosslinked γ-PGA-based injectable composition that provides the effect of promoting tissue regeneration or collagen production in the skin or subcutaneous tissue as described above, while preventing the lump phenomenon that may occur after injection. However, this objective is exemplary and does not limit the scope of the present invention.

[0006] According to one aspect of the present invention, an injectable composition for promoting tissue regeneration and / or collagen production in the skin or subcutaneous tissue of a subject is provided, comprising non-crosslinked γ-PGA or a pharmaceutically acceptable salt thereof.

[0007] According to another aspect of the present invention, a medical device for promoting tissue regeneration and / or collagen production is provided, which is filled with the injectable composition.

[0008] According to another aspect of the present invention, uncrosslinked γ-PGA or a pharmaceutically acceptable salt thereof is provided for use in promoting tissue regeneration and / or collagen production of a subject.

[0009] According to another aspect of the present invention, a use is provided for preparing an injectable agent for tissue regeneration and / or collagen production promotion of non-crosslinked γ-PGA or a pharmaceutically acceptable salt thereof.

[0010] According to another aspect of the present invention, a method for promoting tissue regeneration and / or collagen production is provided, comprising the step of administering a pharmaceutically effective amount of uncrosslinked γ-PGA or a pharmaceutically acceptable salt thereof to the skin or subcutaneous tissue of a subject.

[0011] The injectable composition of the present invention, as described above, can improve skin condition by exhibiting effects of increasing collagen production and promoting tissue regeneration in the skin or subcutaneous tissue without causing lumps. Accordingly, the injectable composition of the present invention can be used in a procedure for skin cosmetic purposes as a medical device manufactured in a state filled into a syringe. Of course, the scope of the present invention is not limited by these effects.

[0012] Figure 1 is a graph showing the experimental results confirming the cytotoxicity of an injectable composition according to one embodiment of the present invention.

[0013] FIG. 2 is a schematic diagram showing an animal experiment schedule for the in vivo effects and tissue analysis of an injectable composition according to one embodiment of the present invention.

[0014] FIG. 3 is a series of photographs of the skin of an experimental animal after administering an injectable composition according to one embodiment of the present invention once to the animal and after a certain period of time has elapsed.

[0015] FIG. 4 is a series of photographs of the skin of an experimental animal after administering an injectable composition according to one embodiment of the present invention twice and after a certain period of time has elapsed.

[0016] FIG. 5 is a series of photographs of the skin of an experimental animal after administering an injectable composition according to one embodiment of the present invention three times and after a certain period of time has elapsed.

[0017] FIG. 6 is a series of microscopic photographs showing the results of collagen analysis performed on tissue specimens of skin tissue obtained from experimental animals that were sacrificed 28 days after administering an injectable composition according to one embodiment of the present invention to experimental animals 1 to 3 times.

[0018] Definition of Terms

[0019] The term "gamma-polyglutamic acid (γ-PGA)" used in this document refers to a polymer composed of multiple linked glutamic acids, a substance produced by bacteria. Unlike typical proteins, it is a polymer in which amine groups are linked by peptide bonds with gamma-carboxyl groups rather than with alpha-carboxyl groups. γ-PGA is known to provide excellent moisturizing effects on both the inner and outer skin, as well as contribute to maintaining the skin barrier. These characteristics can improve the skin microenvironment and provide favorable conditions for tissue regeneration processes. γ-PGA can absorb water molecules up to approximately 5,000 times its own molecular weight and is known to stabilize skin conditions by producing Natural Moisturizing Factors (NMFs) within the skin, form a foundation for collagen production and tissue repair, and effectively stabilize the skin's pH value to inhibit the activity of pathogenic microorganisms. γ-PGA is typically a polymer of L-glutamic acid, but polymers of D-glutamic acid or polymers in the form of a mixture of D- / L-glutamic acid may also be used, provided there is no immunogenicity or other side effects.

[0020] As used in this document, the term “pharmaceuticalally acceptable salt” means a salt prepared using a compound according to one aspect and a relatively non-toxic acid or base. When the compound contains relatively acidic functional groups, a base addition salt may be obtained by contacting a sufficient amount of base with the neutral form of the compound in a pure solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include salts of sodium, potassium, calcium, ammonium, organic amines, or magnesium, or similar salts. When the compound contains relatively basic functional groups, an acid addition salt may be obtained by contacting a sufficient amount of acid with the neutral form of the compound in a pure solution or a suitable inert solvent. Pharmaceutically acceptable acid addition salts include salts of inorganic acids such as hydrochloric acid, hydrobromide, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrogen sulfate, hydroiodic acid, or phosphoric acid, and salts of organic acids such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, souveric acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-tolylsulfonic acid, citric acid, tartaric acid, and methanesulfonic acid, and further include salts of amino acids (e.g., arginine) and salts of organic acids such as glucuronic acid.

[0021] The above pharmaceutically acceptable salts can be synthesized by conventional chemical methods from parent compounds containing an acidic or basic portion. Generally, such salts are prepared by reacting the free acid or base form of these compounds with a stoichiometrically appropriate amount of base or acid in water, an organic solvent, or a mixture of the two. Generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred.

[0022] The term "lump phenomenon" as used in this document refers to a phenomenon in which the skin at the injection site or the surrounding area swells for a certain period as the hydrogel within the composition swells when an injectable composition is injected into the skin; if this persists for 3 days or more, it is defined as a lump phenomenon. Although the lump phenomenon does not pose any particular health problems, it has a negative aesthetic effect because it becomes more noticeable in appearance when the composition is injected at multiple locations at regular intervals.

[0023] As used in this document, the term “storage modulus (G')” refers to a physical property representing a material’s ability to store deformation energy when deformed by external stress, defined as the ratio of stress to strain. The storage modulus (G') reflects the elastic properties of a material; the higher the G' value, the more elastic and solid-like the material's characteristics.

[0024] The term “loss modulus (G”)” used in this document represents the degree of energy dissipation during deformation caused by external forces and reflects the amount of energy lost during one cycle of deformation motion. The higher the G” value, the more viscous and liquid-like the material becomes.

[0025] The term “tangent delta (tan δ)” as used in this document is defined as follows:

[0026] tan δ = G" / G'

[0027] When tan δ > 1, viscous properties are dominant over elastic properties, exhibiting liquid-like behavior, and when tan δ < 1, elastic properties are dominant, exhibiting solid-like behavior. Therefore, tan δ can be used as an indicator to assess the fluidity, spreadability, and tissue diffusion characteristics of an injectable composition.

[0028] Detailed description of the invention

[0029] According to one aspect of the present invention, an injectable composition for promoting tissue regeneration and / or collagen production in the skin or subcutaneous tissue of a subject is provided, comprising non-crosslinked γ-PGA or a pharmaceutically acceptable salt thereof.

[0030] The above γ-PGA may have the structure of the following structural formula I:

[0031] (Structural Formula I)

[0032] (In the above structural formula, n is an integer from 10 to 20000).

[0033] In the above injectable composition, the γ-PGA may be included in an amount of 10 to 100 mg / mL, more preferably 15 to 80 mg / mL, 16 to 70 mg / mL, 17 to 60 mg / mL, 18 to 55 mg / mL, or 19 to 52 mg / mL, and most preferably 20 to 50 mg / mL.

[0034] In the above injectable composition, the molecular weight of the γ-PGA may be 10 to 3000 kDa, and more preferably 100 to 2500 kDa, 200 to 2400 kDa, 300 to 2300 kDa, 400 to 2200 kDa, or 500 to 2000 kDa.

[0035] The above-mentioned injectable composition may further include a local anesthetic, and the local anesthetic is ambucaine, amolanone, amylocaine, benoxinate, benzocaine, betoxycaine, biphenamine, bupivacaine, butacaine, butamben, butanilicaine, butethamine, butoxycaine, carticaine, chloroprocaine, cocaethylene, cocaine, cyclomethycaine, dibucaine, dimethysoquin, dimethocaine, Diperodon, dicyclonine, ecgonidine, ecgonine, ethyl chloride, etidocaine, beta-eucaine, euprocin, fenalcomine, formocaine, hexylcaine, hydroxytetracaine, isobutyl p-aminobenzoate, leucinocaine mesylate, levoxadrol, lidocaine, mepivacaine, meprylcaine, metabutoxycaine, methyl chloride, Myrtecaine, naepaine, octacaine, orthocaine,It may be selected from the group consisting of oxethazaine, parethoxycaine, phenacaine, phenol, piperocaine, pyridocaine, polidocanol, pramoxine, prilocaine, procaine, propanocaine, proparacaine, propipocaine, proroxycaine, pseudococaine, pyrrocaine, ropivacaine, salicyl alcohol, tetracaine, tolycaine, trimecaine, zolamine, and pharmaceutically acceptable salts thereof. It is preferably lidocaine, and more preferably lidocaine-HCl, but is not limited thereto.

[0036] In addition to the above, the injectable composition may further include additives such as buffers, antioxidants, bacteriostatic agents, diluents, dispersants, surfactants, binders, lubricants, polysaccharides, collagen, and peptides.

[0037] The non-crosslinked γ-PGA-based injectable composition to be provided in the present invention is a composition that causes minimal foreign body sensation when injected into the skin, is easy to inject, and exhibits tissue regeneration and / or collagen production-promoting effects without lifting or lumping at the injection site during the initial injection stage.

[0038] The above composition may have an elastic modulus (G') in the range of 0.0001 to 5 Pa, preferably in the range of 0.00013 to 3.5 Pa, more preferably in the range of 0.00016 to 2 Pa, but is not limited thereto.

[0039] The above composition may have a viscosity coefficient (G) in the range of 0.05 to 50 Pa, preferably in the range of 0.052 to 40 Pa, more preferably in the range of 0.055 to 35 Pa, but is not limited thereto.

[0040] The above composition may have a tangent delta (tan δ) of 10 or more, preferably 13.5 or more, more preferably 15 or more, but is not limited thereto.

[0041] An injectable composition according to one embodiment of the present invention may be injected into an animal, e.g., a mouse, at a dose of 50 to 500 mg / kg once, more preferably at a dose of 70 to 450 mg / kg, 80 to 400 mg / kg, 90 to 350 mg / kg, or 100 to 320 mg / kg, and most preferably at a dose of 100 to 300 mg / kg.

[0042] In addition, the above dosage is a single dose and may be administered once a day, or repeated at intervals of 2 to 4 days rather than once a day. For example, it is possible to administer it on the 1st day, take a rest period of two days, and then administer it again on the 4th day.

[0043] An injectable composition according to one embodiment of the present invention may be administered to humans at a predicted dose of 1 to 30 mg / kg. More preferably, a composition according to one embodiment of the present invention may be administered to humans at a dose of 1 to 25 mg / kg, 1 to 24 mg / kg, 1 to 23 mg / kg, 1 to 22 mg / kg, 1.25 to 22 mg / kg, 1.5 to 22 mg / kg, or 1.75 to 21.5 mg / kg, and most preferably at a dose of 1.78 to 21.36 mg / kg.

[0044] When administered to humans, the above-described dosage may also be applied as a single dose, administered once a day, or divided into multiple doses. In addition, it may be administered repeatedly at intervals of 2 to 4 days rather than once a day. For example, it is possible to administer it on the 1st day, take a rest period of two days, and then administer it again on the 4th day.

[0045] The prediction of the human dosage from the dosage measured in animals can be performed using the following formula:

[0046] Human Equivalent Dose = Animal Dose (mg / kg) X (Animal Body Weight (kg) / Human Body Weight (kg))^(1-0.67) [Equation 1]

[0047] In addition, the injectable composition according to one embodiment of the present invention may be administered simultaneously or sequentially to multiple locations on the target skin in a grid pattern with a constant spacing, for example, 2 to 10 mm, and in this case, the total dosage may be equivalent to the dosage.

[0048] According to another aspect of the present invention, a medical device for promoting tissue regeneration and / or collagen production is provided, wherein a single dose of the injectable composition is filled inside a syringe.

[0049] According to another aspect of the present invention, uncrosslinked γ-PGA or a pharmaceutically acceptable salt thereof is provided for use in promoting tissue regeneration and / or collagen production of a subject.

[0050] In the above-mentioned non-crosslinked γ-PGA or a pharmaceutically acceptable salt thereof, the γ-PGA may have the structure of the following structural formula I:

[0051] (Structural Formula I)

[0052] (In the above structural formula, n is an integer from 10 to 20000).

[0053] In the above-mentioned non-crosslinked γ-PGA or pharmaceutically acceptable salt thereof, the γ-PGA may be formulated into an injectable formulation, in which case it may be contained in the formulation at 10 to 100 mg / mL, more preferably 15 to 80 mg / mL, 16 to 70 mg / mL, 17 to 60 mg / mL, 18 to 55 mg / mL, or 19 to 52 mg / mL, and most preferably at 20 to 50 mg / mL.

[0054] In addition, the molecular weight of the γ-PGA may be 10 to 3000 kDa, and more preferably 100 to 2500 kDa, 200 to 2400 kDa, 300 to 2300 kDa, 400 to 2200 kDa, or 500 to 2000 kDa.

[0055] A non-crosslinked γ-PGA or a pharmaceutically acceptable salt thereof according to one embodiment of the present invention may be administered to animals, e.g., mice, at a single dose of 50 to 500 mg / kg, more preferably at a dose of 70 to 450 mg / kg, 80 to 400 mg / kg, 90 to 350 mg / kg, or 100 to 320 mg / kg, and most preferably at a dose of 100 to 300 mg / kg.

[0056] In addition, the above dosage is a single dose and may be administered once a day or divided into multiple doses. Furthermore, it may be administered repeatedly at intervals of 2 to 4 days instead of once a day. For example, it is possible to administer it on the 1st day, take a rest period of two days, and then administer it again on the 4th day.

[0057] Non-crosslinked γ-PGA or a pharmaceutically acceptable salt thereof according to one embodiment of the present invention may be administered to humans at a predicted dose of 1 to 30 mg / kg once. More preferably, a composition according to one embodiment of the present invention may be administered to humans at a dose of 1 to 25 mg / kg, 1 to 24 mg / kg, 1 to 23 mg / kg, 1 to 22 mg / kg, 1.25 to 22 mg / kg, 1.5 to 22 mg / kg, or 1.75 to 21.5 mg / kg, and most preferably at a dose of 1.78 to 21.36 mg / kg.

[0058] When administered to humans, the non-crosslinked γ-PGA of the present invention or a pharmaceutically acceptable salt thereof may be administered once a day as a single dose as described above, or the single dose may be divided into multiple doses. In addition, it may be administered repeatedly at intervals of 2 to 4 days rather than once a day. For example, it is possible to administer it on the 1st day, take a rest period of two days, and then administer it again on the 4th day.

[0059] The prediction of the human dosage from the dosage measured in animals can be performed using the following formula:

[0060] Human Equivalent Dose = Animal Dose (mg / kg) X (Animal Body Weight (kg) / Human Body Weight (kg))^(1-0.67) [Equation 1]

[0061] In addition, the non-crosslinked γ-PGA or a pharmaceutically acceptable salt thereof according to one embodiment of the present invention may be administered to multiple target skin locations simultaneously or sequentially in a grid pattern with a constant spacing, for example, 2 to 10 mm, and in this case, the total dosage may be equivalent to the above dosage.

[0062] According to another aspect of the present invention, a use is provided for preparing an injectable agent for tissue regeneration and / or collagen production promotion of non-crosslinked γ-PGA or a pharmaceutically acceptable salt thereof.

[0063] For the above use, the γ-PGA may have the structure of the following structural formula I:

[0064] (Structural Formula I)

[0065] (In the above structural formula, n is an integer from 10 to 20000).

[0066] For the above use, the γ-PGA may be included in the injectable at 10 to 100 mg / mL, more preferably 15 to 80 mg / mL, 16 to 70 mg / mL, 17 to 60 mg / mL, 18 to 55 mg / mL, or 19 to 52 mg / mL, and most preferably at 20 to 50 mg / mL.

[0067] For the above use, the molecular weight of the γ-PGA may be 10 to 3000 kDa, and more preferably 100 to 2500 kDa, 200 to 2400 kDa, 300 to 2300 kDa, 400 to 2200 kDa, or 500 to 2000 kDa.

[0068] In this case, the above-mentioned injectable is synonymous with the injectable formulation, and the description of the additives for its composition is as described above.

[0069] According to another aspect of the present invention, a method for promoting tissue regeneration and / or collagen production is provided, comprising the step of administering a pharmaceutically effective amount of uncrosslinked γ-PGA or a pharmaceutically acceptable salt thereof to the skin or subcutaneous tissue of a subject.

[0070] In the above method, the γ-PGA may have the structure of the following structural formula I:

[0071] (Structural Formula I)

[0072] (In the above structural formula, n is an integer from 10 to 20000).

[0073] In the above method, the γ-PGA may be formulated into an injectable form and may be included in the formulation in an amount of 10 to 100 mg / mL, more preferably 15 to 80 mg / mL, 16 to 70 mg / mL, 17 to 60 mg / mL, 18 to 55 mg / mL, or 19 to 52 mg / mL, and most preferably 20 to 50 mg / mL.

[0074] At this time, the description of the additive for the composition of the above-mentioned injectable formulation is as described above.

[0075] In the above method, the molecular weight of the γ-PGA may be 10 to 3000 kDa, and more preferably 100 to 2500 kDa, 200 to 2400 kDa, 300 to 2300 kDa, 400 to 2200 kDa, or 500 to 2000 kDa.

[0076] In the above method, a local anesthetic may be administered together with or separately from the γ-PGA, wherein the local anesthetic is ambucaine, amolanone, amylocaine, benoxinate, benzocaine, betoxycaine, biphenamine, bupivacaine, butacaine, butamben, butanilicaine, butethamine, butoxycaine, carticaine, chloroprocaine, cocaethylene, cocaine, cyclomethycaine, dibucaine, dimethysoquin, dimethocaine, Diperodon, dicyclonine, ecgonidine, ecgonine, ethyl chloride, etidocaine, beta-eucaine, euprocin, fenalcomine, formocaine, hexylcaine, hydroxytetracaine, isobutyl p-aminobenzoate, leucinocaine mesylate, levoxadrol, lidocaine, mepivacaine, meprylcaine, metabutoxycaine, methyl chloride, Myrtecaine, naepaine, octacaine, orthocaine,It may be selected from the group consisting of oxethazaine, parethoxycaine, phenacaine, phenol, piperocaine, pyridocaine, polidocanol, pramoxine, prilocaine, procaine, propanocaine, proparacaine, propipocaine, proroxycaine, pseudococaine, pyrrocaine, ropivacaine, salicyl alcohol, tetracaine, tolycaine, trimecaine, zolamine, and pharmaceutically acceptable salts thereof. It is preferably lidocaine, and more preferably lidocaine-HCl, but is not limited thereto.

[0077] In a method according to one embodiment of the present invention, the non-crosslinked γ-PGA or a pharmaceutically acceptable salt thereof may be injected into an animal, e.g., a mouse, at a dose of 50 to 500 mg / kg once, more preferably at a dose of 60 to 450 mg / kg, 70 to 4000 mg / kg, 80 to 350 mg / kg, or 90 to 320 mg / kg, and most preferably at a dose of 100 to 300 mg / kg.

[0078] In addition, the above-mentioned non-crosslinked γ-PGA or pharmaceutically acceptable salt thereof may be administered as a single dose once a day, or divided into multiple doses. Furthermore, it may be administered repeatedly at intervals of 2 to 4 days rather than once a day. For example, it is possible to administer it on the 1st day, take a rest period of two days, and then administer it again on the 4th day.

[0079] In a method according to one embodiment of the present invention, the non-crosslinked γ-PGA or a pharmaceutically acceptable salt thereof may be administered to a human at a predicted dose of 1 to 30 mg / kg once. More preferably, the use according to one embodiment of the present invention may be administered to a human at a dose of 1 to 25 mg / kg, 1 to 24 mg / kg, 1 to 23 mg / kg, 1 to 22 mg / kg, 1.25 to 22 mg / kg, 1.5 to 22 mg / kg, or 1.75 to 21.5 mg / kg, and most preferably at a dose of 1.78 to 21.36 mg / kg.

[0080] When administered to humans, the above-described non-crosslinked γ-PGA or its pharmaceutically acceptable salt may be administered once a day as a single dose, or the single dose may be divided into multiple doses. In addition, it may be administered repeatedly at intervals of 2 to 4 days rather than once a day. For example, it is possible to administer it on the 1st day, take a rest period of two days, and then administer it again on the 4th day.

[0081] The prediction of the human dosage from the dosage measured in animals can be performed using the following formula:

[0082] Human Equivalent Dose = Animal Dose (mg / kg) X (Animal Body Weight (kg) / Human Body Weight (kg))^(1-0.67) [Equation 1]

[0083] In addition, the above-mentioned non-crosslinked γ-PGA or pharmaceutically acceptable salt thereof may be administered to the target skin simultaneously or sequentially in multiple locations in a grid pattern with a fixed spacing, for example, 2 to 10 mm, and in this case, the total dosage may be equivalent to the above dosage.

[0084] The present invention will be explained in more detail below through examples. However, these examples are intended to illustrate the invention and the scope of the invention is not limited to these examples.

[0085] Example: Control group and preparation of a γ-PGA composition according to one embodiment of the present invention

[0086] The inventors prepared solutions with concentrations of 60 mg / mL, 40 mg / mL, and 20 mg / mL, respectively, by simply dissolving non-crosslinked γ-PGA (molecular weights 1,920 kDa and 588 kDa) in the solvent PBS. The compositions of Examples 1 to 6 were prepared by filter-sterilizing the dissolved γ-PGA solutions using a 0.22 μm syringe filter, filling them into glass syringes to a volume of 1 mL, and sealing them with a suction cup (Table 1). As a control group, only cells treated with no reagents (i.e., DMEM + 2% FBS medium alone) were used.

[0087] Composition Ratio of a Composition According to One Embodiment of the Present Invention Example Substance Molecular Weight (kDa) Concentration (mg / mL) Treatment Concentration (μg / mL) 1γ-PGA 1,9 20 60 300 24 0 200 32 01 00 45 88 60 300 54 0 200 62 01 00 Control Group No Treatment (DMEM + 2% FBS) N / A--

[0088] Experimental Example 1: Cytotoxicity Test

[0089] The inventors evaluated the cytotoxicity test using a cell viability test to determine whether the composition prepared in the above examples exhibited cytotoxicity. The cell viability test was performed to evaluate the safety of the composition of the present invention as a method for evaluating the cytotoxicity of a treated substance.

[0090] To this end, the samples of each example and control were treated with γ-PGA solution diluted in medium to treatment concentrations of 20, 40, and 60 mg / mL, considering the substance concentration range suitable for cytotoxicity evaluation, and treated with the cells.

[0091] The cytotoxicity test method was performed according to the following procedure.

[0092] 1) Thawing and culture of HDF cell lines

[0093] After thawing the HDF (human dermal fibroblast) stock vial (passage 12) by placing it in a constant temperature water bath to half-thaw it, 1 mL of pre-warmed 10% FBS DMEM medium was added to the vial and mixed. After thawing completely, the vial was transferred to a 15 mL centrifuge tube, filled with 10% FBS DMEM medium up to the 10 mL mark, and centrifuged at 1500 rpm for 3 minutes. The supernatant was removed and the vial was resuspended in 4 mL of fresh DMEM medium. Then, 11 mL of 10% FBS DMEM medium was placed in a T-75 flask, and 1 mL of the cell suspension was added to the T-75 flask and cultured for 72 hours under conditions of 37 ℃ and 5% CO2 to obtain the required cell quantity for the test.

[0094] 2) Cytotoxicity test using HDF cell line

[0095] To use HDF cell lines in the experiment, the culture medium in the T-75 flask was removed, and cells were detached by treatment with 0.25% trypsin-EDTA. The cells were collected in a 15 ml centrifuge tube and centrifuged at 1500 rpm for 3 minutes. After removing the supernatant and resuspending the cells in fresh medium, 10 μL of 10% FBS DMEM medium and 10 μL of Trypan blue were mixed into the cell suspension. The cell count was performed using a cell counter, and the total required number of cells was calculated as 2.5 × 10⁶. 4 Under the cell / well condition, 600 μL of 10% FBS DMEM medium resuspended with cells was seeded into a total of 44 wells of two 24-well plates.

[0096] After 24 hours, the samples were diluted to the treatment concentrations in 2% FBS DMEM medium using a syringe as shown in Table 1. For the control group, 2% FBS DMEM medium was injected into the syringe to maintain identical conditions. Then, the drugs from each example, diluted in 2% FBS DMEM medium, were added to the original 10% FBS DMEM medium in the same volume, and the samples were incubated for 48 hours under conditions of 37°C and 5% CO2. To obtain samples for ELISA analysis, 300 μL of medium was transferred into Eppendorf tubes. 30 μL of CCK-8 reaction reagent was loaded into each well and gently mixed. After a 2-hour reaction, the OD (Optical Density) at 450 nm was measured using a SpectraMax i3.

[0097] 3) Evaluation

[0098] Cell viability (%) compared to control group

[0099] Cell viability was calculated using the absorbance value of CCK-8, and the calculation was performed by the following Equation 2:

[0100] Cell viability = (A_Sample-A_Blank) / (A_Control-A_Blank)×100 [Equation 2]

[0101] In the above mathematical formula 2, A_Control represents the absorbance value of a well containing only culture medium, A_Sample represents the absorbance value of a well treated with drugs, and A_Blank represents the absorbance value of a well containing only drugs and culture medium without cells.

[0102] As confirmed in Fig. 1, the results of the cytotoxicity test showed that the compositions of all examples did not exhibit cytotoxicity compared to the control group. These results indicate that no toxicity is induced in cells within the molecular weight and concentration ranges of γ-PGA used in the compositions prepared in Examples 1 to 6, which ultimately indicates that the composition according to one embodiment of the present invention is a safe substance (Fig. 1).

[0103] Experimental Example 2: Measurement of Viscoelastic Properties (G', G", tan δ)

[0104] While cytotoxicity evaluations were conducted at concentrations (20, 40, 60 mg / mL) intended for verifying cell responsiveness, it is necessary to verify the injectable formulation characteristics of non-crosslinked γ-PGA compositions—particularly viscoelastic properties (G', G", tan δ) directly related to diffusivity, injectability, and the occurrence of lumps—over a wider concentration range. Accordingly, to verify the viscoelastic properties of γ-PGA-based compositions over a wider concentration range, the inventors prepared solutions with concentrations of 50 mg / mL, 100 mg / mL, and 250 mg / mL, respectively, by simply dissolving non-crosslinked γ-PGA (molecular weights 1,800 kDa and 588 kDa) in PBS, a solvent. These concentrations are experimental settings intended to comprehensively verify the rheological properties of the injectable formulation compositions and are compositions for physical property analysis performed independently of the cytotoxicity test concentrations.

[0105] The compositions of Examples 7 to 12 were prepared by filter-sterilizing the dissolved γ-PGA solution using a 0.22 μm syringe filter, filling it into a glass syringe to a volume of 1 mL, and sealing it with a suction device (Table 2).

[0106] Composition ratio of an injectable formulation according to one embodiment of the present invention Example substance molecular weight (kDa) Concentration (mg / mL) 7γ-PGA 1,800 508 100 9250 10588 5011 100 12250

[0107] For the compositions of Examples 7 to 12, the elastic modulus (G', Storage modulus), viscosity modulus (G", Loss modulus), and tangent delta (tan δ) were measured. Since rheological properties serve as indicators of the diffusivity and spreadability exhibited by the composition within the skin or subcutaneous tissue after injection, they are utilized as important physical property values ​​to explain the characteristics of the compositions of the present invention.

[0108] Viscoelasticity measurements were performed using a DHR-2 rheometer (TA Instruments), and the analysis conditions are as shown in Table 3 below.

[0109] DHR-2 Rheometer Analysis Conditions Item Condition Frequency: 0.1 Hz Temperature: 25 ℃ Strain: 0.2% Measurement Geometry: 40 mm plate Measurement Gap: 1.0 mm

[0110] The tangent delta (tan δ) was calculated according to the following formula (Equation 3).

[0111] tan δ = G" / G'(Equation 3)

[0112] The higher this value, the more the composition exhibits liquid-like behavior, which is associated with diffusivity within the skin and spreading characteristics immediately after injection.

[0113] Table 4 below shows the G', G", and tan δ values ​​when high molecular weight (approx. 1,800 kDa) and low molecular weight (approx. 588 kDa) γ-PGA were formulated at different concentrations (50, 100, 250 mg / mL).

[0114] Viscoelastic properties according to concentration and molecular weight of γ-PGA composition Example Molecular Weight (kDa) Concentration (mg / mL) G'(Pa) G"Pa) tan δ(=G" / G') 7 1,800 500.00 7 32500.28 1724 40.06 04 81000.02 796 600.85 214 830.47 99 2501.96 6803 4.70 4117.6 44 910 588500.000 186 7080.05 746 33307.77 4111000.00 489 2360.55 1923 112.81 3122500.03 0917 51.48 994 48.1909

[0115] For both high molecular weight and low molecular weight γ-PGA, the tan δ values ​​ranged from tens to hundreds at all concentrations, indicating that the composition possesses dominant flow characteristics. These flow characteristics allow the composition to rapidly diffuse around the injection site when injected into the skin or subcutaneously, which can contribute to suppressing swelling or lump formation immediately after the initial injection.

[0116] In particular, the characteristics of the composition, in which the G' value exists in a low elastic range of 0.0001 to 1.97 Pa and the G" value exhibits very dominant characteristics, suggest that the composition of the present invention exhibits viscosity-dominated fluidity.

[0117] These rheological properties exhibit rheological properties that are not problematic for injectable formulations, and the effect of minimizing lump phenomena is achieved through the property of spreading uniformly within the tissue immediately after injection into the skin, thus possessing technical compatibility with the present invention.

[0118] Experimental Example 3: Measurement of In Vivo Volume Change

[0119] Next, the inventors injected the compositions of Examples 1 to 6 and the control group prepared as described above into mice subcutaneously using a syringe and observed volume changes at the injection site, and in particular, visually observed the lump phenomenon on the skin of the mice (Table 5 and Figs. 3 to 5).

[0120] Example of an animal experiment using a composition according to one embodiment of the present invention Number of substance administration Molecular weight (kDa) Concentration (mg / mL) Number of individuals 1γ-PGA 11,920 60 3233 421 40 3233 431 20 3233 441 58 860 3233 451 40 3233 461 20 3233 4 Control group No treatment (DMEM + 2% FBS)---10

[0121] The mice used were 6-week-old female hairless mice with a body weight of 20 ± 3 g. After being brought into the breeding facility, they were used for animal experiments after a 2-week quarantine and acclimatization period.

[0122] For the control group and test substance administration, anesthesia was performed on each mouse by intraperitoneal injection of a mixed anesthetic of ketamine (100 mg / kg) and rumpun (10 mg / kg). After anesthesia, 0.1 mL of the test substance was administered subcutaneously to a specific location on the dorsal side of the mouse.

[0123] Injection of the test substance was performed at 0.1 mL per site for each test substance at 3-day intervals, divided into a 1-dose group, a 2-dose group, and a 3-dose group (Fig. 2).

[0124] More specifically, a visual evaluation was performed after administering the test substance to 3 animals in the single-dose group, 3 animals in the double-dose group, and 4 animals in the triple-dose group of the compositions and control groups of each example (Examples 1 to 6: 3 animals in the single-dose group, 3 animals in the double-dose group, 4 animals in the triple-dose group, total 10 animals x 6 test groups, total 60 animals; control group: 10 animals).

[0125] To confirm the volume change of the substance injected into the body, the volume change of the injection site according to the number of injections was visually evaluated using a digital camera (IXUS 8x, CANNON) at intervals of 0, 1, 3, and 6 days after administration of the substance (immediately after administration).

[0126] Visual evaluation was performed using the following procedure:

[0127] Ten mice were placed at 0 (immediately after administration), 1, 3, and 6 days after a single injection, depending on the number of injections, and the injection site of the test substance was photographed using a digital camera (Examples 1 to 6 and control group, Fig. 3).

[0128] Seven mice that received two injections were placed at day 0 (immediately after administration) and the injection site of the test substance was photographed using a digital camera (Fig. 4).

[0129] Four mice that had been administered three times were placed at 0 days (immediately after administration) and 1 day after three administrations according to the number of injections, and the site of administration of the test substance was photographed using a digital camera (Fig. 5).

[0130] A test substance injected into the body exhibits a tendency to maintain volume at the injection site for a certain period, and if the phenomenon of maintaining volume persists for 3 days or more, it is defined as a lump phenomenon. The better the characteristic of the injected substance spreading to surrounding tissues, the shorter the duration of the lump phenomenon tends to be.

[0131] Figure 3 shows representative results of subjects in which the volume of the injection site was photographed after injecting the test substances of Examples 1 to 6 once into mice, etc. As shown in Figure 3, in all Examples 1 to 6, the volume of the initially injected substance was maintained until the 6th day without being observed with the naked eye, starting from the 1st day after the injection of the test substance. This means that the test substances of Examples 1 to 6 did not cause swelling, lifting, or lumping at the initial injection site.

[0132] Figure 4 is a representative result of subjects in which the injection site was photographed after an additional injection (total of 2 injections) 3 days after the initial injection of the test substances of Examples 1 to 6. As shown in Figure 4, for all subjects of the test substances of Examples 1 to 6, the volume of the initially injected test substance was not visually observed from 3 days after the injection.

[0133] Figure 5 shows representative results of subjects in which the injection site was photographed after an additional injection (a total of three injections) at the time three days after the second injection of the test substances of Examples 1 to 6. As shown in Figure 5, for all subjects of the test substances of Examples 1 to 6, the volume of the injected substance was not visually observed starting from the time one day after injection. This means that the test substances of Examples 1 to 6 have good tissue spreadability and exhibit the same spreadability even when administered repeatedly.

[0134] Experimental Example 4: Histological Analysis

[0135] Collagen in the skin is a substance that plays an important role in maintaining skin elasticity, so an increase in the amount of collagen leads to increased skin elasticity.

[0136] To confirm these effects, the effects according to the number of administrations, molecular weight, and concentration of the composition according to one embodiment of the present invention were investigated. In particular, histological analysis was performed to confirm the effect of the composition of the present invention on the formation and distribution of collagen fibers within the skin or subcutaneous tissue. The effect of the administered substance was indirectly confirmed by determining the degree of collagen production using Masson's Trichrome tissue staining method, which allows for the identification of collagen.

[0137] The administration site, formulation, and method involved sacrificing the subjects of the animal experiment performed in Experimental Example 2 28 days after the last administration date, extracting skin tissue, and embedding it in paraffin to create tissue specimens. Masson's Trichrome staining method, which allows for the selective staining of three dyes including muscle (red), collagen fibers (blue), red blood cells (red), and nuclei (black), was used.

[0138] FIG. 6 is a tissue staining photograph taken at 100x magnification using a microscope of a cross-section of a stained tissue specimen after administering the test substances of Examples 1 to 6 to mice once, twice, and three times according to the administration plan of FIG. 2, followed by tissue excision at the injection site and tissue staining. Tissue excision for the 1-dose group was 28 days after the first administration date. Tissue excision for the 2-dose group was 31 days after the first administration date. Tissue excision for the 3-dose group was 34 days after the first administration date.

[0139] In Figure 6 above, the control group was the untreated group, and tissue staining was performed to confirm the patterns of cell nuclei, epidermal fibers, muscle fibers, and collagen fibers observed in general tissues and to compare them with the test substance administration groups of Examples 1 to 6.

[0140] In general tissues, through Masson's Trichrome staining, cell nuclei distributed within the tissue appear black, epidermal fibers and muscle fibers appear pink, and collagen fibers appear blue. In Figure 6 above, in the test substance administration groups of Examples 1 to 6, cell nuclei, epidermal fibers, muscle fibers, and collagen fibers observed in general tissues were stained and observed in the same way as in the control group.

[0141] Collagen fibers were identified in all test groups upon a single administration, and a staining pattern was observed in which collagen fibers were more densely distributed within the subcutaneous tissue in the groups administered the compositions of Examples 1 to 6 compared to the control group.

[0142] Even when administered twice, the collagen fibers in the test substance administration groups of Examples 1 to 6 were observed to be more densely distributed compared to the control group, and the amount of collagen fibers was confirmed to be similar to that of the single administration group.

[0143] Even after three administrations, the test substance administration groups of Examples 1 to 6 showed a more dense distribution of collagen fibers compared to the control group, and the thickness of the subcutaneous tissue also showed increased subcutaneous tissue thickness compared to the control group.

[0144] As confirmed in Figure 6, in the test substance administration groups of Examples 1 to 6, a large amount of collagen fibers was observed compared to the control group regardless of molecular weight, concentration, and number of administrations, and the result of increased thickness of the subcutaneous tissue was confirmed.

[0145] These results indicate that when the composition according to one embodiment of the present invention is administered to the skin, skin regeneration and skin elasticity increase equivalent to or greater than that of the control group.

[0146] Although the present invention has been described with reference to the embodiments described above, this is merely illustrative, and those skilled in the art will understand that various modifications and equivalent alternative embodiments are possible therefrom. Accordingly, the true technical scope of protection of the present invention should be determined by the technical spirit of the appended claims.

[0147] An injectable composition according to one embodiment of the present invention is manufactured as a pharmaceutical or quasi-pharmaceutical product for promoting tissue regeneration and / or collagen production in the skin or subcutaneous tissue of a subject, and can be efficiently used for purposes such as tissue regeneration of the skin or skin tissue of a subject.

Claims

1. An injectable composition comprising non-crosslinked γ-PGA or a pharmaceutically acceptable salt thereof, for promoting tissue regeneration and / or collagen production in the skin or subcutaneous tissue of a subject.

2. In Paragraph 1, A composition characterized by not causing a lump at the injection site.

3. In Paragraph 1, A composition having an elastic modulus (G′) in the range of 0.0001 to 5 Pa.

4. In Paragraph 1, A composition having a viscosity coefficient (G″) in the range of 0.05 to 50 Pa.

5. In Paragraph 1, The above γ-PGA is a composition having the structure of the following structural formula 1: (Structural Formula 1) (In the above structural formula, n is an integer from 10 to 20,000).

6. In Paragraph 1, A composition comprising 10 to 100 mg / mL of the above γ-PGA.

7. In Paragraph 1, A composition in which the molecular weight of the above γ-PGA is 10 to 3,000 kDa.

8. In Paragraph 1, The above subject is a mouse, and the composition is administered at a dose of 50 to 500 mg / kg as a single injection.

9. In Paragraph 1, The above subject is a human, and the above composition is administered at a dose of 1 to 30 mg / kg as a single injection.

10. In Paragraph 1, A composition further comprising a local anesthetic.

11. In Paragraph 7, The above-mentioned local anesthetic is lidocaine, a composition.

12. A medical device having an internally filled composition for tissue regeneration and / or collagen production promotion, filled with the injectable composition of claim 1.

13. Non-crosslinked γ-PGA or a pharmaceutically acceptable salt thereof for use in promoting skin tissue regeneration and / or collagen production in a subject.

14. Use of non-crosslinked γ-PGA or pharmaceutically acceptable salts thereof for preparing injectables for promoting skin tissue regeneration and / or collagen production.

15. A method for promoting tissue regeneration and / or collagen production, comprising the step of administering a pharmaceutically effective amount of uncrosslinked γ-PGA or a pharmaceutically acceptable salt thereof to the skin or subcutaneous tissue of a subject.

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