Crosslinked hyaluronic acid gels with good diffusivity and stability and uses thereof

Abstract

The invention provides cross-linked hyaluronic acid with good diffusivity and stability and application of the cross-linked hyaluronic acid.

Description

Technical Field

[0001] Disclosed is a cross-linked hyaluronic acid gel with good diffusibility and stability and its uses. Background Art

[0002] Hyaluronic acid is a natural component of the dermis. Hyaluronic acid plays an important role in skin hydration and elasticity. As the skin ages, the quantity and quality of hyaluronic acid in the skin decline. These changes cause skin dryness and wrinkle formation.

[0003] Hyaluronic acid can be used for hydration, wrinkles or wound healing. However, hyaluronic acid is degraded in vivo by factors such as enzymes, temperature and free radicals. Accordingly, the lifespan of hyaluronic acid in vivo is limited.

[0004] Therefore, when using hyaluronic acid for hydration, a hyaluronic acid gel that can be maintained in vivo for a long time, has good diffusibility, and does not form lumps is needed. Summary of the Invention

[0005] Technical Problem

[0006] On the one hand, provided is a cross-linked hyaluronic acid gel that has good diffusibility and stability when injected into the body, wherein its elastic modulus (G') is from 0.01 Pa to 20 Pa, its viscous modulus (G”) is from 0.1 Pa to 20 Pa, and its elasticity percentage is from 10% to 40%. The average particle size of the cross-linked hyaluronic acid gel can be from 35 μm to 100 μm. On the other hand, provided is a composition for filling tissues or hydrating the skin, which comprises the cross-linked hyaluronic acid gel. On the other hand, provided is a method for filling an individual's tissues or hydrating an individual's skin, which comprises the step of administering to the individual a composition for filling tissues or hydrating the skin that comprises the cross-linked hyaluronic acid gel.

[0007] Solution to the Problem

[0008] On the one hand, provided is a cross-linked hyaluronic acid gel that has good diffusibility and stability when injected into the body, wherein its elastic modulus (G') is from 0.01 Pa to 20 Pa, its viscous modulus (G”) is from 0.1 Pa to 20 Pa, and its elasticity percentage is from 10% to 40%. The average particle size of the cross-linked hyaluronic acid gel can be from 35 μm to 100 μm.

[0009] In this specification, the term "cross-linked hyaluronic acid gel" refers to a composition containing injectable cross-linked hyaluronic acid. The composition may be an injectable dermal filler composition. The term "gel" as used in this specification generally refers to a substance having a fluidity intermediate between a liquid and a solid at room temperature. Additionally, the term "gel" refers to a substance that can absorb water (i.e., a hydrogel). The composition generally may include a nonpyrogenic isotonic buffer, for example, a physiologically acceptable carrier fluid such as buffered saline.

[0010] The gel may include 1.5 wt% to 3.0 wt% of cross-linked hyaluronic acid based on the weight of the gel composition.

[0011] The term "injectable" means that the composition is suitable for injection into the skin or other tissues to deliver the composition to a desired target site.

[0012] The "injectable" composition can be dispensed through a syringe under normal conditions and normal pressure.

[0013] In this specification, the term "hyaluronic acid" refers to hyaluronan, hyaluronate, or a pharmaceutically acceptable salt thereof having the chemical formula of Formula 1.

[0014]

[0015] In Formula 1, n is the number of repeating units. Hyaluronic acid from all sources, including bacterial and algal sources, is useful.

[0016] The term "cross-linked" as mentioned in this specification refers to two or more hyaluronic acid polymer chains covalently linked by a cross-linking agent. This cross-linking can be distinguished by intermolecular or intramolecular dehydration in a single polymer or two or more chains that causes the formation of lactones, anhydrides, or esters. Intramolecular cross-linking may also be included in the compositions mentioned in this specification.

[0017] The term "cross-linking agent" includes at least two reactive functional groups that form covalent bonds between two or more molecules. The cross-linking agent can be homobifunctional or heterobifunctional. The cross-linking agent used in this specification may include functional groups complementary to the functional groups of hyaluronic acid, thereby allowing the cross-linking reaction to proceed.

[0018] The term "elasticity%" is defined as "(storage modulus (G') / (storage modulus (G') + loss modulus (G''))) × 100". Whether the loss modulus of a substance is high or low, its elasticity% can be similar. Elasticity% represents the direction of the hardness or softness of the cross-linked hyaluronic acid gel. The lower the elasticity%, the softer the cross-linked hyaluronic acid gel and the better its spreadability.

[0019] The phrase "spreadability" and its grammatical variations refer to the degree to which a substance initially spreads into the surrounding tissue after injection, without agglomeration, swelling, lifting, or caking at the injection site. Additionally, "good spreadability" and its grammatical variations may refer to a volume percentage (%) of 100% or less relative to the initial volume of the injection site at the initial stage of injection. For example, the volume ratio of the substance of the present invention between day 0 and day 3, between day 0 and day 2, or between day 0 and day 1 after injection (day 0) may be 100% to 90%, 100% to 80%, 100% to 60%, 100% to 50%, 100% to 30%, 100% to 20%, 100% to 10%, 100% to 0%, 90% to 80%, 90% to 60%, 90% to 50%, 90% to 30%, 90% to 20%, 90% to 10%, 90% to 0%, 80% to 60%, 80% to 50%, 80% to 30%, 80% to 20%, 80% to 10%, or 80% to 0% relative to the volume at the time of injection. The volume ratio is calculated by the following formula.

[0020] Volume ratio (%) = Volume after injection / Initial injection volume × 100

[0021] The volume ratio measurement site is within a range with a radius of 0.5 cm to 1 cm centered on the central injection site.

[0022] The term "Degradation 50" refers to the time required for the elastic modulus (G') of hyaluronic acid to decrease to 50% of its initially measured value as hyaluronic acid degrades after being treated with degrading enzymes such as hyaluronidase in vitro. It is known that the longer the degradation time 50, the higher the resistance to degradation, and it can remain stable in the body for a long time. The high resistance to degradation of hyaluronic acid means that the hyaluronic acid has high stability in the body.

[0023] The elastic modulus (G') of the crosslinked hyaluronic acid gel may be 0.01 Pa to 20 Pa, for example, 0.01 Pa to 15 Pa, 0.01 Pa to 10 Pa, 0.01 Pa to 5 Pa, 0.01 Pa to 4.0 Pa, 0.01 Pa to 3.5 Pa, 0.01 Pa to 3.0 Pa, 0.01 Pa to 2.5 Pa, 0.01 Pa to 2.0 Pa, 0.01 Pa to 1.5 Pa, 0.01 Pa to 1.0 Pa, 0.1 Pa to 20 Pa, 0.1 Pa to 15 Pa, 0.1 Pa to 10 Pa, 0.1 Pa to 5 Pa, 0.1 Pa to 4 Pa, 0.1 Pa to 3.5 Pa, 0.1 Pa to 3.0 Pa, 0.1 Pa to 2.5 Pa, 0.1 Pa to 2.0 Pa, 0.1 Pa to 1.5 Pa, 0.1 Pa to 1.0 Pa, 0.05 Pa to 5 Pa, 0.05 Pa to 4.0 Pa, or 0.105 Pa to 1.035 Pa.

[0024] The viscous modulus (G") of the crosslinked hyaluronic acid gel may be 0.1 Pa to 20 Pa, for example, 0.1 Pa to 15 Pa, 0.1 Pa to 10 Pa, 0.1 Pa to 5 Pa, 0.1 Pa to 4 Pa, 0.1 Pa to 3.5 Pa, 0.1 Pa to 3.0 Pa, 0.1 Pa to 2.5 Pa, 0.1 Pa to 2.0 Pa, 0.1 Pa to 1.5 Pa, 0.1 Pa to 1.0 Pa, 0.5 Pa to 1.0 Pa, 0.6 Pa to 1.0 Pa, or 0.7 Pa to 1.0 Pa.

[0025] The elastic ratio of the crosslinked hyaluronic acid gel may be 10% to 40%, for example, 10% to 30%, 20% to 30%, 20% to 27%, 20% to 25%, 21% to 25%, or 22% to 24%.

[0026] The average particle size of the cross-linked hyaluronic acid gel can be from 35 μm to 100 μm, such as from 35 μm to 90 μm, from 35 μm to 80 μm, from 35 μm to 70 μm, from 35 μm to 60 μm, from 35 μm to 65 μm, from 35 μm to 50 μm, from 35 μm to 45 μm, from 40 μm to 65 μm, from 40 μm to 60 μm, from 40 μm to 55 μm, from 40 μm to 50 μm, from 45 μm to 50 μm, from 40 μm to 45 μm, or from 41 μm to 44 μm.

[0027] The degradation time of the cross-linked hyaluronic acid gel can be from 90 minutes to 300 minutes, for example, from 90 minutes to 290 minutes, from 90 minutes to 280 minutes, from 90 minutes to 270 minutes, from 90 minutes to 260 minutes, from 90 minutes to 250 minutes, from 90 minutes to 245 minutes, from 100 minutes to 250 minutes, from 100 minutes to 245 minutes, from 120 minutes to 350 minutes, from 130 minutes to 350 minutes, from 140 minutes to 300 minutes, from 150 minutes to 300 minutes, from 200 minutes to 300 minutes, from 200 minutes to 290 minutes, from 200 minutes to 280 minutes, from 200 minutes to 270 minutes, from 200 minutes to 260 minutes, from 200 minutes to 145 minutes, from 210 minutes to 300 minutes, from 210 minutes to 290 minutes, from 210 minutes to 280 minutes, from 210 minutes to 270 minutes, from 210 minutes to 260 minutes, from 220 minutes to 300 minutes, from 230 minutes to 260 minutes, from 235 minutes to 260 minutes, from 235 minutes to 250 minutes, or from 240 minutes to 260 。

[0028] The cross-linked hyaluronic acid gel can be cross-linked by a cross-linking agent having a bifunctional epoxy group. The cross-linking agent having a bifunctional epoxy group can be 1,4-butanediol diglycidyl ether (BDDE), ethylene glycol diglycidyl ether (EGDGE), 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), biscarbodiimide (BCDI), diglycerol polyglycidyl ether, divinylsulfone (DVS), polyethylene glycol diglycidyl ether (PEGDE), polypropylene glycol diglycidyl ether (PPGDE), polytetramethylene glycol diglycidyl ether, polyglycerol polyglycidyl ether, glycerol diglycidyl ether, triethylene glycol diglycidyl ether, trimethylolpropane triglycidyl ether, ethylene glycol diglycidyl ether, neopentyl glycol diglycidyl ether, 1,6-hexanediol diglycidyl ether or a combination thereof.

[0029] The cross-linked hyaluronic acid gel may be in the form of a gel in a solution such as an aqueous solution. In this case, the term hyaluronic acid is used interchangeably with "hyaluronic acid gel".

[0030] In the cross-linked hyaluronic acid gel, the hyaluronic acid before cross-linking may be from any source. The hyaluronic acid before cross-linking may be from, for example, a non-animal source. The hyaluronic acid before cross-linking may be from bacteria. The bacteria may be from the genus Streptococcus. The Streptococcus bacteria may be Streptococcus equi, S. pyogenes, or S. zooepidemicus. The hyaluronic acid before cross-linking may also be commercially available. The intrinsic viscosity of the hyaluronic acid before cross-linking may be 1.0 m 3 / kg to 4.0 m 3 / kg.

[0031] The cross-linked hyaluronic acid gel may be manufactured by a method including the following steps: incubating a reaction mixture including a cross-linking agent and hyaluronic acid to carry out a cross-linking reaction. The incubating may be carried out while heating. The heating may be heating to 30°C to 60°C, 30°C to 50°C, or 35°C to 45°C. The incubating may be carried out for 10 hours to 40 hours, 10 hours to 30 hours, 10 hours to 25 hours, or 15 hours to 25 hours. The cross-linking agent may have a polyfunctional group. The cross-linking agent may have a bifunctional epoxy group, as described above.

[0032] The cross-linked hyaluronic acid gel may have a cross-linking degree of 0.5% to 7%. The cross-linking degree may be, for example, 0.6% to 7%, 0.7% to 7%, 0.8% to 7%, 0.9% to 7%, 1.0% to 7%, 0.5% to 5%, 0.5% to 3.0%, 0.5% to 2.0%, 1.0% to 5%, 1.0% to 3.0%, 1.0% to 2.5%, or 1.0% to 2.0%.

[0033] In this specification, the term "cross-linking degree (%, cross-link rate)" represents the ratio of the cross-linking agent covalently bonded in every 100 hyaluronic acid disaccharide repeating units. The disaccharide repeating unit is composed of D-glucuronic acid and N-acetylglucosamine. The cross-linking degree may be confirmed by a known method. For example, it may be confirmed by ion exchange chromatography (IEC) or NMR. Ion exchange chromatography is a method for separation and analysis by utilizing the difference in the affinity of sample ions for the stationary phase through reversible ion exchange between the stationary phase and the mobile phase. In the case of using IEC, the formula for the cross-linking degree is as follows.

[0034] Degree of crosslinking (%) = ∑(peak area × number of hyaluronic acid disaccharide repeating units × proportion of crosslinked disaccharide repeating units) × 100 / ∑(peak area × number of hyaluronic acid disaccharide repeating units)

[0035] The crosslinked hyaluronic acid gel can be more stable in vivo than natural hyaluronic acid or some of the known crosslinked hyaluronic acid gels available. The crosslinked hyaluronic acid gel can be a hyaluronic acid gel that is more stable than, for example, Belotero soft lidocaine (Merz Aesthetics). The crosslinked hyaluronic acid gel can be more stable in vivo than administering a composition comprising natural hyaluronic acid or, for example, Belotero soft lidocaine.

[0036] The cross-linked hyaluronic acid gel can stably exist in the body for 28 days or longer, 29 days or longer, 30 days or longer, 31 days or longer, 32 days or longer, 33 days or longer, 34 days or longer, 35 days or longer, 36 days or longer, 37 days or longer, 38 days or longer, 39 days or longer, 40 days or longer, 41 days or longer, 42 days or longer, 43 days or longer, 44 days or longer, 45 days or longer, 46 days or longer, 47 days or longer, 48 days or longer, 49 days or longer, 50 days or longer, 51 days or longer, 52 days or longer, 53 days or longer, 54 days or longer, 55 days or longer, 56 days or longer, 57 days or longer, 58 days or longer, 59 days or longer, 60 days or longer, 61 days or longer, 62 days or longer, 63 days or longer, 64 days or longer, 65 days or longer, 66 days or longer, 67 days or longer, 68 days or longer, 69 days or longer, 70 days or longer, 71 days or longer, 72 days or longer, 73 days or longer, 74 days or longer, 75 days or longer, 76 days or longer, 77 days or longer, 78 days or longer, 79 days or longer, 80 days or longer, 81 days or longer, 82 days or longer, 83 days or longer, 84 days or longer, 85 days or longer, 86 days or longer, 87 days or longer, 88 days or longer, 89 days or longer, 90 days or longer, 91 days or longer, 92 days or longer, 93 days or longer, 94 days or longer, 95 days or longer, 96 days or longer, 97 days or longer, 98 days or longer, 99 days or longer, 100 days or longer, 101 days or longer, 102 days or longer, 103 days or longer, 104 days or longer, 105 days or longer, 106 days or longer, 107 days or longer, 108 days or longer, 109 days or longer, 110 days or longer, 111 days or longer, 112 days or longer, 120 days or longer, 130 days or longer, 140 days or longer, 150 days or longer, 160 days or longer, 170 days or longer, 180 days or longer, from 28 days to 84 days, from 29 days to 84 days, from 30 days to 84 days, from 35 days to 84 days, from 40 days to 84 days, from 45 days to 84 days, from 50 days to 84 days, from 54 days to 84 days, from 55 days to 84 days, from 60 days to 84 days, from 28 days to 112 days, from 29 days to 112 days, from 30 days to 112 days, from 35 days to 112 days, from 40 days to 112 days, from 45 days to 112 days, from 50 days to 112 days, from 54 days to 112 days, from 55 days to 112 days, from 60 days to 112 days, from 70 days to 112 days, from 80 days to 112 days, from 90 days to 112 days, from 100 days to 112 days, from 105 days to 112 days, from 28 days to 180 days, from 56 days to 180 days, from 56 days to 180 days, or from 112 days to 180 days.

[0037] In this specification, the term "lump" phenomenon refers to an uneven elevation on the skin surface at or around the injection site caused by the injected composition, forming an unnatural appearance compared to the surrounding area. Generally, the lower the viscoelastic value or the lower the elastic modulus, the better it diffuses into the surrounding area within the tissue, thus tending to reduce the lump phenomenon.

[0038] On the other hand, a composition for filling tissues or hydrating the skin is provided, which comprises the cross-linked hyaluronic acid gel. In the composition, the cross-linked hyaluronic acid gel may have the physical properties described for the cross-linked hyaluronic acid gel, such as elastic modulus, viscous modulus, elastic modulus, average particle size, degree of cross-linking, and degradation time 50, etc.

[0039] In the composition, the concentration of the cross-linked hyaluronic acid gel may be 10 mg / ml or higher, 15 mg / ml or higher, 18 mg / ml or higher, or 19 mg / ml or higher. For example, the concentration of the cross-linked hyaluronic acid gel may be 10 mg / ml to 30 mg / ml, 12 mg / ml to 28 mg / ml, 14 mg / ml to 26 mg / ml, 15 mg / ml to 25 mg / ml, 16 mg / ml to 24 mg / ml, 18 mg / ml to 22 mg / ml, or about 20 mg / ml.

[0040] The composition can be used for tissue repair or hydration in an individual.

[0041] In the composition, the individual may be a mammal. The mammal may be a human, a dog, a cat, a cow, a pig, a mouse, or a sheep.

[0042] The composition may not additionally include non-cross-linked hyaluronic acid. "Additionally including" non-cross-linked hyaluronic acid means further artificially adding non-cross-linked hyaluronic acid to the cross-linked hyaluronic acid gel. Additionally, "not additionally including non-cross-linked hyaluronic acid" means not further artificially adding non-cross-linked hyaluronic acid to the cross-linked hyaluronic acid gel.

[0043] The composition may further include an anesthetic. The anesthetic may be a local anesthetic. The local anesthetic may be ambucaine, amolanone, amylocaine, benoxinate, benzocaine, betoxycaine, biphenamine, bupivacaine, butacaine, butamben, butanilicaine, butethamine, butoxycaine, carticaine, chloroprocaine, cocaethylene, cocaine, cyclomethycaine, dibucaine, dimethysoquin, dimethocaine, diperodon, dycyclomine, ecgonidine, ecgonine, ethyl chloride, etidocaine, beta-eucaine, euprocin, fenalcomine, formocaine, hexylcaine, hydroxytetracaine, isobutyl paminobenzoate, leucinocaine mesylate, levoxadrol, lidocaine, mepivacaine, meprylcaine, metabutoxycaine, methylchloride), myrtecaine, naepaine, octacaine, orthocaine, oxethazaine, parethoxycaine, phenacaine, phenol, piperocaine, piridocaine, polidocanol, pramoxine, prilocaine, procaine, propanocaine, proparacaine, propipocaine, propoxycaine, pseudococaine, pyrrocaine, ropivacaine, salicyl alcohol, tetracaine, tolycaine, trimecaine, zolamine, or one or more of their salts, but not limited thereto.

[0044] The composition may further comprise a pharmacologically active substance or a substance having a biological function. The substance may be a functional polymer substance. The functional polymer substance may be a natural polymer or a synthetic polymer. The pharmacologically active substance or the substance having a biological function may be one or more substances selected from the group consisting of, for example, collagen, chitosan, alginate, gelatin, polynucleotide, polydeoxyribonucleotide, fibroin, elastin, tropoelastin, carrageenan, γ-polyglutamic acid, sulfated hyaluronic acid, chondroitin sulfate, fucoidan, heparin, heparan sulfate, pentosan polysulfate, pullulan, and keratin.

[0045] The collagen may be one or more substances selected from the group consisting of bovine-derived, porcine-derived, fish-derived, human recombinant-derived, human recombinant collagen peptides, telopeptide-free collagen, human recombinant collagen α1 chain, human recombinant collagen peptide derivatives, RGD sequence-enhanced human recombinant collagen peptides, and RGD sequence-enhanced human recombinant collagen peptide α1 chain.

[0046] The collagen may be selected from the group consisting of type I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XVII, XVIII, XIX, XX, XXI, XXII, XXIII, XXIV, XXV, XXVI, XXVII, and XXVIII collagen.

[0047] The pharmacologically active substance or biologically functional substance may be one or more substances selected from the group consisting of polyethylene glycol, polylactic acid, polyglycolic acid, poly lactic - co - glycolic acid, polycaprolactone, poloxamer, polyphosphazene, and poly(p - dioxanone).

[0048] The composition may be filled in a syringe.

[0049] The composition may be used for one or more uses selected from the group consisting of skin moisturization, facial plastic surgery, wrinkle improvement, facial contour surgery, breast augmentation, treatment of urinary incontinence, treatment of arthritis, and drug delivery.

[0050] In the composition, when measuring the composition filled in a 1 - ml syringe at a speed of 12 mm / min using a needle of size 22 to 31 gauge (G) and 10 mm to 13 mm, the injection force may be 50 N or less. In the composition, when measuring the composition filled in a 1 - ml syringe at a speed of 12 mm / min using a 27 - gauge (G) and 13 - mm needle, the injection force may be 50 N or less.

[0051] The composition may further comprise a pharmaceutically acceptable carrier, excipient, and diluent. The carrier may include, for example, water or a buffer solution. The buffer solution may cause the pH of the solution to hardly change with the addition of the components of the composition. The composition may be an aqueous liquid composition. The composition may be an aqueous buffer composition. The pH of the aqueous buffer composition may be within the physiological pH range, for example, between about 6.0 and 8.0, such as between about 6.0 and about 7.5, between about 6.5 and about 7.5, or between about 6.8 and about 7.2. The pH may be adjusted by adding a suitable acid or base, such as HCl, Na 2c O3 or NaOH. In a specific embodiment, the aqueous buffer composition may include phosphate buffered saline (PBS). In another specific embodiment, the aqueous buffer composition may include tris(hydroxymethyl)aminomethane (Tris). In some specific embodiments, additional solutes, such as sodium chloride, calcium chloride, and potassium chloride, may be added to adjust the osmolarity and ionic concentration.

[0052] The composition can be obtained by suspending the cross-linked hyaluronic acid gel in an aqueous solvent. The aqueous solvent can be a buffer such as PBS, water, or saline. The composition can be sterilized.

[0053] The composition can be included in a container. The container can be a syringe. The composition can be pre-filled into the syringe before use. The composition can be administered by the pre-filled syringe.

[0054] In addition, the composition can be used for administration into the skin to hydrate the tissue. In this specification, "hydration" can be increasing or maintaining the moisture in the tissue. The increasing or maintaining includes supplying moisture to tissues such as the skin by injecting hyaluronic acid and cross-linked hyaluronic acid gel which originally have a high water content, or absorbing moisture from the surrounding tissues of the injection site by highly hydrophilic hyaluronic acid, thereby supplementing the moisture at the injection site. Therefore, the hydration includes supplying and retaining moisture to the tissue. In some specific embodiments, the hydration can be used interchangeably with "moisturizing". The composition can be used for administration into the skin for hydration. The composition can be used for administration into wrinkle-free skin for hydration.

[0055] On the other hand, a device including the composition is provided. The device can be a pre-filled syringe. The device can be sterilized.

[0056] On the other hand, a kit including the pre-filled syringe is provided. The kit can include instructions, and the instructions include information for administering the composition.

[0057] On the other hand, there is provided a method for filling individual tissues or hydrating the skin of an individual, which comprises the step of administering a therapeutically effective amount of the composition to the individual. The method can be used to enhance, repair or strengthen individual tissues, or fill body cavities, or hydrate the skin. From this perspective, enhancing, repairing or strengthening individual tissues or filling body cavities can be a phenomenon of skin enhancement caused by the injected composition or a secondary phenomenon that occurs with skin hydration. The components forming the composition can be evenly diffused from the injection site to the surrounding sites without caking. In the method, "composition" and "individual" are as described above. The administration can be, for example, intradermal administration to the skin or intra-articular administration. In the method, the administration can be carried out by a syringe, such as a pre-filled syringe, to the skin, such as intradermally. In the method, the administration can be 0.1 ml to 50 ml, 0.5 ml to 30 ml, 0.5 ml to 20 ml, 0.5 ml to 15 ml, or 0.8 ml to 12 ml per administration. In the method, the administration can be once every 3 months or longer, once every 4 months or longer, once every 5 months or longer, once every 6 months or longer, once every 12 months or longer, or once every 18 months or longer.For example, the administration may be once or more than once every 28 days or longer, 29 days or longer, 30 days or longer, 31 days or longer, 32 days or longer, 33 days or longer, 34 days or longer, 35 days or longer, 36 days or longer, 37 days or longer, 38 days or longer, 39 days or longer, 40 days or longer, 41 days or longer, 42 days or longer, 43 days or longer, 44 days or longer, 45 days or longer, 46 days or longer, 47 days or longer, 48 days or longer, 49 days or longer, 50 days or longer, 51 days or longer, 52 days or longer, 53 days or longer, 54 days or longer, 55 days or longer, 56 days or longer, 57 days or longer, 58 days or longer, 59 days or longer, 60 days or longer, 61 days or longer, 62 days or longer, 63 days or longer, 64 days or longer, 65 days or longer, 66 days or longer, 67 days or longer, 68 days or longer, 69 days or longer, 70 days or longer, 71 days or longer, 72 days or longer, 73 days or longer, 74 days or longer, 75 days or longer, 76 days or longer, 77 days or longer, 78 days or longer, 79 days or longer, 80 days or longer, 81 days or longer, 82 days or longer, 83 days or longer, 84 days or longer, 85 days or longer, 86 days or longer, 87 days or longer, 88 days or longer, 89 days or longer, 90 days or longer, 91 days or longer, 92 days or longer, 93 days or longer, 94 days or longer, 95 days or longer, 96 days or longer, 97 days or longer, 98 days or longer, 99 days or longer, 100 days or longer, 101 days or longer, 102 days or longer, 103 days or longer, 104 days or longer, 105 days or longer, 106 days or longer, 107 days or longer, 108 days or longer, 109 days or longer, 110 days or longer, 111 days or longer, 112 days or longer, 180 days or longer, from 28 days to 84 days, from 29 days to 84 days, from 30 days to 84 days, from 35 days to 84 days, from 40 days to 84 days, from 45 days to 84 days, from 50 days to 84 days, from 54 days to 84 days, from 55 days to 84 days, from 60 days to 84 days, from 28 days to 112 days, from 29 days to 112 days, from 30 days to 112 days, from 35 days to 112 days, from 40 days to 112 days, from 45 days to 112 days, from 50 days to 112 days, from 54 days to 112 days, from 55 days to 112 days, from 60 days to 112 days, from 70 days to 112 days, from 80 days to 112 days, from 90 days to 112 days, from 100 days to 112 days, from 105 days to 112 days, from 28 days to 180 days, from 56 days to 180 days, from 56 days to 180 days, or from 112 days to 180 days. The administration may be local administration to areas in need of tissue filling or skin hydration. The administration may be applied to the skin, face, chest, joints, or a combination thereof.

[0058] Advantageous effects of the present disclosure

[0059] The crosslinked hyaluronic acid gel requested according to one aspect has good diffusibility and good stability when injected into the body without lumping, so it can be maintained for a long time.

[0060] A composition for filling or skin moisturizing comprising a crosslinked hyaluronic acid gel according to another aspect can be used for filling or skin moisturizing.

[0061] By a method of filling an individual tissue according to another aspect, an individual tissue can be effectively filled.

[0062] By a method of moisturizing an individual's skin according to another aspect, an individual's skin can be effectively moisturized and the skin moisturizing environment can be improved. Description of the Drawings

[0063] Figure 1 It is a figure showing the result of measuring the volume of the injection site after injecting a crosslinked hyaluronic acid composition into mice or the like.

[0064] Figure 2a 、 Figure 2b and Figure 2c They are photos showing the volume change in the injection site observed with the naked eye after injecting a crosslinked hyaluronic acid gel into mice.

[0065] Figure 3a and Figure 3b They are photos of cross-sections of the central and side parts of the injection site stained on the 28th day after injecting a crosslinked hyaluronic acid gel into mice.

[0066] Figure 4a and Figure 4b They are photos of cross-sections of the central and side parts of the injection site stained on the 56th day after injecting a crosslinked hyaluronic acid gel into mice.

[0067] Figure 5a and Figure 5b They are photos of cross-sections of the central and side parts of the injection site stained on the 84th day after injecting a crosslinked hyaluronic acid gel into mice.

[0068] Figure 6a and Figure 6b They are photos of cross-sections of the central and side parts of the injection site stained on the 112th day after injecting a crosslinked hyaluronic acid gel into mice.

[0069] Figure 7a and Figure 7b They are photos of cross-sections of the central and side parts of the injection site stained on the 180th day after injecting a crosslinked hyaluronic acid gel into mice.

[0070] Best Mode

[0071] Hereinafter, the present invention will be described in more detail by way of examples. However, these examples are only for exemplarily describing the present invention, and the scope of the present invention is not limited to these examples.

[0072] Materials and Methods

[0073] In the following examples, the following materials and methods were used.

[0074] 1. Preparation of Control Group and Crosslinked Hyaluronic Acid Gel

[0075] (1) Crosslinked Hyaluronic Acid Gel of Control Group

[0076] As the control group, the following commercially available crosslinked hyaluronic acid gel products were used, namely Belotero soft with lidocaine (Merz Aesthetics), Juvederm Volite (Allergan), and Neuramis Light Lidocaine (Medy-Tox). Belotero soft with lidocaine (hereinafter referred to as "BSL") has the property of being able to spread well from the injection site to the surrounding tissues in the body without the phenomenon of lifting or lump formation. The G', G", and elastic modulus of BSL are significantly low, so it can spread well from the injection site to the surrounding tissues without swelling, lifting, or lump formation at the initial injection site, but its duration is short.

[0077] Juvederm Volite (hereinafter referred to as "JV") and Neuramis Light Lidocaine (hereinafter referred to as "NLL") are products commonly used for correcting fine wrinkles, which are products that cause swelling and lifting at the injection site in the initial stage of injection and tend to increase the volume of the injection site. It is known that the volumes of both of these products increase after injection.

[0078] (2) Preparation of Crosslinked Hyaluronic Acid Gel of Test Group

[0079] First, dissolve NaOH in water to prepare a 0.25N NaOH solution. Mix 7.5 g of sodium hyaluronate (IV 2.0 to 3.0) into the prepared 0.25N NaOH solution to make the concentration reach 15.0% (w / w), and then stir to fully dissolve it. Here, IV represents intrinsic viscosity. Add 0.189 g (5 mol% relative to 1 mole of hyaluronic acid monomer) of butanediol diglycidyl ether (BDDE) (Sigma - Aldrich) to the solution, and further stir to fully mix it. Then, take it out and carry out a cross - linking reaction at 40 °C for 18 hours to prepare a cross - linked hyaluronic acid gel.

[0080] Next, use an aqueous NaCl solution and an aqueous PBS (pH 7.0) solution as dialysis solutions to dialyze the obtained cross - linked hyaluronic acid gel to remove the unreacted cross - linker. After dialysis is completed, considering the loss rate of the initially added sodium hyaluronate weight, content correction is carried out using 1×PBS until the concentration of the finally removed sodium hyaluronate reaches 20 mg / mL. At this time, content correction is carried out on lidocaine hydrochloride hydrate so that it reaches 3 mg / mL. The obtained cross - linked hyaluronic acid gel includes a 20 mg / mL cross - linked hyaluronic acid gel in PBS and 3 mg / mL lidocaine hydrochloride hydrate. The obtained cross - linked hyaluronic acid gel is crushed as follows to be used as samples for the following test groups 1 to 2.

[0081] Put the prepared cross - linked hyaluronic acid gel into the container of a blender (Retsch GM - 200) and crush it at 4000 rpm for 3 minutes. As a result, a composition containing 20 mg / mL of the crushed cross - linked hyaluronic acid gel and 3 mg / mL of lidocaine was prepared, and it was called test group 1 or TA1.

[0082] According to the same method as the manufacturing method of test group 1, put the prepared cross - linked hyaluronic acid gel into the container of a blender (Retsch GM - 200) and crush it at 4000 rpm for 10 minutes. As a result, a composition containing 20 mg / mL of the crushed cross - linked hyaluronic acid gel and 3 mg / mL of lidocaine was prepared. This is a composition containing the crushed cross - linked hyaluronic acid gel and lidocaine, which has a smaller particle size compared to test group 1, and is hereinafter called test group 2 or TA2.

[0083] Then, fill 1 ml of the test groups 1 (TA1) and 2 (TA2) of the composition containing the cross - linked hyaluronic acid gel and lidocaine into glass syringes respectively, and then carry out high - temperature steam sterilization.

[0084] The average particle sizes of the cross-linked hyaluronic acid gels in the compositions of Test Group 1 and Test Group 2 were 111.2 μm and 42.3 μm, respectively. The particle size measurement was carried out according to the following method.

[0085] In addition, the cross-linked hyaluronic acid gels of Test Group 3 and Test Group 4 were prepared as follows.

[0086] NaOH was dissolved in water to prepare a 0.25N NaOH solution. 391 g of sodium hyaluronate (IV 2.0 to 3.0) was mixed into the prepared 0.25N NaOH solution to make the concentration reach 15.0% (w / w), and then stirred to dissolve thoroughly. Here, IV represents the intrinsic viscosity. 9.8 g (5 mol% relative to 1 mole of hyaluronic acid monomer) of butanediol diglycidyl ether (Sigma-Aldrich) was added to the solution, and further stirred to mix well, and then taken out for an 18-hour cross-linking reaction at 40 °C to produce a cross-linked hyaluronic acid gel.

[0087] Next, the obtained cross-linked hyaluronic acid gel was put into a dialysis membrane and sealed, and then dialyzed using an aqueous NaCl solution and an aqueous PBS (pH 7.0) solution as dialysis solutions to remove the unreacted cross-linking agent. After dialysis was completed, considering the loss rate of the initially added sodium hyaluronate weight, content correction was carried out using 1×PBS until the concentration of the finally removed sodium hyaluronate reached 20 mg / mL. At this time, content correction of lidocaine hydrochloride hydrate was carried out to make it reach 3 mg / mL. The obtained cross-linked hyaluronic acid gel included 20 mg / mL cross-linked hyaluronic acid gel and 3 mg / mL lidocaine hydrochloride hydrate in PBS. The obtained cross-linked hyaluronic acid gel was pulverized with a pulverizer (MP-50, IKA) at 6,500 rpm for 60 minutes and 90 minutes to obtain Test Group 3 and Test Group 4, respectively.

[0088] Then, Test Group 3 and Test Group 4 of the compositions containing the cross-linked hyaluronic acid gel and lidocaine were filled into glass syringes at 1 ml each, and then sterilized by high-temperature steam.

[0089] The average particle sizes of the cross-linked hyaluronic acid gels in the compositions of Test Group 3 and Test Group 4 were 61.3 μm and 46.4 μm, respectively. The particle size measurement was carried out according to the following method after sterilization.

[0090] 2. Method

[0091] (2.1) Measurement method of elastic modulus (G') and viscous modulus (G'')

[0092] Analysis conditions of the DHR-2 rheometer (TA instruments company)

[0093] (1) Frequency: 0.1 Hz

[0094] (2) Temperature: 25 °C

[0095] (4) Strain: 1.5%

[0096] (5) Measuring geometry: 40 mm plate

[0097] (6) Measuring gap: 1.0 mm

[0098] (2.2) Method for measuring elastic modulus

[0099] Using the values measured by the method for measuring viscosity modulus and elastic modulus in 2.1, calculate the elastic modulus (%) according to the following formula.

[0100] Elastic modulus (elasticity %) = (Elastic modulus (G') / (Elastic modulus (G') + Viscosity modulus (G''))) × 100

[0101] (2.3) Method for measuring degradation time

[0102] Use the rheometer in 2.1 to measure the degradation time according to the following method.

[0103] (1) Hyaluronidase (from Streptomyces ≥ 300 units, Sigma - Aldrich, CAS No. 37259 - 53 - 3), final test solution concentration: 3.27 unit / ml)

[0104] (2) Frequency: 0.1 Hz

[0105] (3) Temperature: 37 °C

[0106] (4) Strain: 1.5%

[0107] (5) Measuring geometry: 40 mm plate

[0108] (6) Measuring gap: 1.0 mm

[0109] (7) Degradation time confirmation: Calculate the value closest to 50% of the initial measured value of the elastic modulus as the degradation time 50 value

[0110] (2.4) Method for measuring particle size

[0111] Use the Particle Size Analyzer S3500 of Microtrac company to analyze the particle size measurement, and the conditions are as follows.

[0112] (1) Sample preparation: Add 900 μl of purified water to 100 μl of gel to disperse the particles.

[0113] (2) Refractive Index: Hyaluronic acid gel (HA Gel): 1.37, water: 1.333

[0114] (3) Flow rate: 70%

[0115] (4) Measured value: Percentile 50%

[0116] (5) Use the average value of three measurements as the measured value of the sample.

[0117] Example 1: Confirm the properties of cross-linked hyaluronic acid gel

[0118] The physical properties of the cross-linked hyaluronic acid gels of the control group and the test group were confirmed, and the degree of diffusion, the presence or absence of caking, and the degree of non-degradation and retention over time, i.e., stability, were confirmed after injecting the substances of the control group and the test group into mice.

[0119] 1. Physical properties of cross-linked hyaluronic acid gel

[0120] Table 1 shows the physical properties of the cross-linked hyaluronic acid gels of the control group and the test group.

[0121]

Table 1

[0122]

[0123] In Table 1, Control Groups 1, 2, and 3 refer to Belotero Soft with Lidocaine (Merz Aesthetics) (BSL), New Fill Light Lidocaine (Medytox) (NLL), and Juvederm Volift (Allergan) (JV), respectively. 2. Confirm the in vivo diffusibility of cross-linked hyaluronic acid gel

[0124] Next, the cross-linked hyaluronic acid gels of the control group and the test group were injected into mice, and the volume changes at the injection sites were observed to determine the degree of caking and diffusion.

[0125] The hairless mice used were 6-week-old female mice weighing 20±3 g. They were placed in the breeding room and acclimated for 1 week, and then administered at the administration site with a 0.1 mL syringe in the 7th week. The observation period for the cross-linked hyaluronic acid gel was 6 months in total. Measurements were taken immediately after administration (day 0), 3 days, 7 days, and 14 days, and the height of the injection site, the maximum length, and the volume of the injection were measured at 1, 2, 3, 4, and 6 months after administration using Primos lite (PRIMOS 45X 33mm, Canfield Scientific Inc, New Jersey, USA). Each substance was injected once at each site, and a total of 17 animals were injected.

[0126] Figure 1 The figure shows the results of measuring the volume of the injection site after injecting the cross-linked hyaluronic acid gel composition into the back of the mouse. As Figure 1 shown, in Test Group 2 (TA2) and Control Group 1 (BSL), starting from the first measurement day after injection, which is the 3rd day, when the volume of the initially injected content was 100%, there was no additional volume increase. In other words, the initially injected volume of the cross-linked hyaluronic acid gel in Test Group 2 (TA2) and Control Group 1 (BSL) decreased by 100% and remained at 0% until the end of the 6-month observation period. This indicates good diffusion, and there was no swelling, lifting, or lumping in the initial injection site.

[0127] Figure 2a 、 Figure 2b and Figure 2c are photos showing the volume changes in the injection site observed with the naked eye after injecting the cross-linked hyaluronic acid gel into the mouse. Figure 2a 、 Figure 2b and Figure 2c were observed at 0, 3, and 7 days after injection respectively. As Figure 2a shown, at day 0 after injection, swelling occurred at the injection site in all individuals. As Figure 2b shown, at day 3 after injection, the volume of the injection site in Test Group 1 and 2 and Control Group 1 decreased significantly. In contrast, the volume of the injection site in Control Group 2 and 3 remained swollen, and uneven lumps were observed. The volume of the injection site was Control Group 2 and Control Group 3 >>> Test Group 1 > Test Group 2 and Control Group 1.

[0128] As Figure 2c shown, at day 7 after injection, there was no swelling or uneven lumps at the injection site in Test Group 2 and Control Group 1. However, the injection sites in Experimental Example 1, Control Group 2, and 3 remained swollen, and uneven lumps were observed. In the figure, green represents the floor mat.

[0129] Based on such macroscopic observations, the cross-linked hyaluronic acid gel of Test Group 2 had the same appearance as that of Control Group 1. On about the 3rd day, no swelling or lumping was observed, thus confirming its good diffusion into the surrounding tissues.

[0130] 3. Confirm the in vivo stability of the cross-linked hyaluronic acid gel

[0131] Next, the cross-linked hyaluronic acid gels of the control group and the test groups were injected into mice, and the remaining degree at the injection site over time was confirmed to confirm the anti-degradability, i.e., stability, of the substance. The administration site, dosage form, and method were the same as those described in Part 2.

[0132] At the designated dates after administration, cross-sections with an area of approximately 1×1 cm were obtained from the median plane of the administration site and the sagittal site 4 mm away from it, and were embedded in paraffin. By confirming both the central and sagittal sites of the administration site, the degree of diffusion of the cross-linked hyaluronic acid gel from the injection site into the surrounding tissues was confirmed. The tissue sections embedded in paraffin were stained with Alcian blue.

[0133] Figure 3a and Figure 3b are photographs of the cross-sections of the central and sagittal sites of the injection site stained on the 28th day after injecting the cross-linked hyaluronic acid gel into mice. Figure 3a and Figure 3b are respectively for Test Group 1, Test Group 2, and Control Group 2, as well as Control Group 3 and Control Group 1.

[0134] Figure 4a and Figure 4b are photographs of the cross-sections of the central and sagittal sites of the injection site stained on the 56th day after injecting the cross-linked hyaluronic acid gel into mice. Figure 4a and Figure 4b are respectively for Test Group 1, Test Group 2, and Control Group 2, as well as Control Group 3 and Control Group 1.

[0135] Figure 5a and Figure 5b are photographs of the cross-sections of the central and sagittal sites of the injection site stained on the 84th day after injecting the cross-linked hyaluronic acid gel into mice. Figure 5a and Figure 5b are respectively for Test Group 1, Test Group 2, and Control Group 2, as well as Control Group 3 and Control Group 1.

[0136] Figure 6a and Figure 6b are photographs of the cross-sections of the central and sagittal sites of the injection site stained on the 112th day after injecting the cross-linked hyaluronic acid gel into mice. Figure 6a andFigure 6b For experimental group 1, experimental group 2, and control group 2, as well as control group 3 and control group 1, respectively.

[0137] Figure 7a and 7b are photos of cross-sections of the central and lateral parts of the injection site stained on the 180th day after injecting cross-linked hyaluronic acid gel into mice. Figure 7a and Figure 7b For experimental group 1, experimental group 2, and control group 2, as well as control 3 and control group 1, respectively.

[0138] At Figure 3a and Figure 3b , Figure 4a and Figure 4b , Figure 5a and Figure 5b , Figure 6a and Figure 6b , as well as Figure 7a and Figure 7b In, blue represents cross-linked hyaluronic acid gel, and red represents dermis. As shown in the figure above, until the 84th day after injection, the cross-linked hyaluronic acid gel in experimental group 1, control group 2, and control group 3 hardly spread to the side and accumulated at the injection site. The degree of accumulation was control group 3 > control group 2 > experimental group 1. In addition, in experimental group 1, control group 2, and control group 3, lumps were observed on the surface of the dermis.

[0139] In experimental group 2 and control group 1, it spread to the side roughly evenly without accumulating at the injection site, i.e., the central surface, and no or almost no lumps on the surface of the dermis were observed. However, on the 28th day, there was a difference in the staining intensity between experimental group 2 and control group 1, and experimental group 2 was stained deeper than control group 1, thus confirming that more cross-linked hyaluronic acid gel substances remained. Control group 1 gradually degraded from the 28th day and there was no residual filling substance from the 56th day. This means that control group 1, i.e., BSL, showed excellent dispersibility but had low anti-degradability compared to the gel of the experimental group.

[0140] According to Tables 2, 3, 4, 5, and 6, as well as Figure 3a , Figure 3b , Figure 4a , Figure 4b , Figure 5a , Figure 5b , Figure 6a , Figure 6b , Figure 7a , as well as Figure 7b The results shown indicate the residual degree of cross-linked hyaluronic acid gel on the 28th day, 56th day, 84th day, 112th day, and 180th day after injection.

[0141]

Table 2

[0142]

[0143]

[0144]

Table 3

[0145]

[0146]

Table 4

[0147]

[0148]

Table 5

[0149]

[0150]

Table 6

[0151]

[0152] In Tables 2, 3, 4, 5, and 6, O indicates that cross-linked hyaluronic acid gel was observed as a whole under the skin, △ indicates that cross-linked hyaluronic acid gel was observed partially under the skin, and X indicates that cross-linked hyaluronic acid gel was not observed.

Claims

1. A cross-linked hyaluronic acid gel having good diffusibility and stability when injected into the body, wherein, its elastic modulus (G') is from 0.01 Pa to 20 Pa, its viscous modulus (G") is from 0.1 Pa to 20 Pa, and its elastic ratio (%) is from 10% to 40%.

2. The crosslinked hyaluronic acid gel according to claim 1, wherein The diffusibility is the volume ratio calculated by the following formula from 0 day to 3 days after injection, and the volume ratio is from 0% to 100%: Volume ratio (%) = volume after injection / volume at initial injection × 100.

3. The crosslinked hyaluronic acid gel according to claim 1, wherein, Its degradation time 50 is from 90 minutes to 100 minutes.

4. The crosslinked hyaluronic acid gel according to claim 1, wherein Its average particle size is from 35 μm to 100 μm.

5. The crosslinked hyaluronic acid gel according to claim 1, wherein, It is cross-linked by a cross-linking agent having a bifunctional epoxy group.

6. The cross-linked hyaluronic acid gel according to claim 4, wherein the cross-linking agent having a bifunctional epoxy group is selected from one or more of the group consisting of 1,4-butanediol diglycidyl ether, polyethylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, polytetramethylene glycol diglycidyl ether, polyglycerol polyglycidyl ether, glycerol diglycidyl ether, triethylene glycol diglycidyl ether, trimethylolpropane triglycidyl ether, ethylene glycol diglycidyl ether, neopentyl glycol diglycidyl ether, and 1,6-hexanediol diglycidyl ether.

7. The crosslinked hyaluronic acid gel according to claim 1, wherein Its cross-linking degree is from 0.5% to 7%.

8. A composition for filling or moisturizing, which comprises the cross-linked hyaluronic acid gel according to any one of claims 1 to 6.

9. The composition according to claim 7, wherein the concentration of the cross-linked hyaluronic acid gel is from 10 mg / mL to 30 mg / mL.

10. The composition according to claim 7, which does not additionally include non-cross-linked hyaluronic acid.

11. The composition according to claim 7, which further includes a local anesthetic.

12. The composition according to claim 7, which is filled in a syringe.

13. The composition according to claim 7, which is used for one or more uses selected from the group consisting of skin moisturizing, facial plastic surgery, wrinkle improvement, facial contour surgery, breast plastic surgery, breast augmentation surgery, genital enlargement, urinary incontinence treatment, arthritis treatment, and drug delivery.

14. The composition according to claim 7, which is used for administration in the skin to moisturize within the tissue.

15. The composition according to claim 1, which further includes a pharmacologically active substance or a substance having a biological function.

16. The composition according to claim 15, wherein the pharmacologically active substance or the substance having a biological function is selected from one or more of the group consisting of anesthetic, collagen, chitosan, alginate, gelatin, polynucleotide, polydeoxyribonucleotide, fibroin, elastin, tropoelastin, carrageenan, γ-polyglutamic acid, sulfated hyaluronic acid, chondroitin sulfate, fucoidan, heparin, heparan sulfate, polysulfated pentosan, pullulan, keratin, polyethylene glycol, polylactic acid, polyglycolic acid, poly(lactic-co-glycolic acid) copolymer, polycaprolactone, poloxamer, polyphosphazene, and poly(p-dioxanone).

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