Filler composition for tissue repair comprising double cross-linked hyaluronic acid as active ingredient using cross-linking agent having excellent biocompatibility

Abstract

The present invention relates to: a method for preparing crosslinked hyaluronic acid, comprising the steps of: (a) crosslinking hyaluronic acid and ornithine; and (b) additionally crosslinking the hyaluronic acid crosslinked with ornithine in step (a) with sodium trimetaphosphate (STMP); a crosslinked hyaluronic acid comprising hyaluronic acid, ornithine, and STMP; and a composition for tissue repair, a filler composition, and a composition for in vivo administration, comprising the crosslinked hyaluronic acid.

Description

A filler composition for tissue repair containing hyaluronic acid as an active ingredient based on double cross-linking using a cross-linking agent with excellent biocompatibility

[0001] The present invention relates to a method for producing cross-linked hyaluronic acid, comprising the steps of (a) cross-linking hyaluronic acid and ornithine; and (b) further cross-linking the hyaluronic acid cross-linked with ornithine in (a) with sodium trimetaphosphate (STMP); cross-linked hyaluronic acid produced by the method; hyaluronic acid cross-linked with ornithine and STMP; a composition for tissue repair, a filler composition, and a composition for bioadministration comprising the cross-linked hyaluronic acid; a composition for producing a cross-linked hyaluronic acid, a biomaterial for tissue repair, a filler, and a bioadministration agent, comprising ornithine, STMP, and hyaluronic acid; a composite composition comprising at least one of the units represented by Chemical Formulas 3 to 7; and a composition for tissue repair, a filler composition, and a composition for bioadministration comprising the same.

[0002]

[0003] Tissue repair biomaterials are being used to restore tissue volume in an injectable form, and the use of fillers is increasing, particularly for wrinkles and other facial contour changes. For example, due to increasing global demand, the global dermal filler market is projected to grow from approximately $5 billion in 2021 to approximately $31 billion in 2031, with a compound annual growth rate of 8% between 2022 and 2031. Hyaluronic acid fillers, a key component of biodegradable fillers due to their high stability and low side effects, account for 90% of the total market.

[0004]

[0005] Generally, when manufacturing hyaluronic acid fillers, a cross-linking agent is used to ensure that hyaluronic acid is maintained in the body for a long time without being decomposed (KR 10-2051467 B1). However, DVS (Divinyl Sulfone) and BDDE (1,4-Butanediol diglycidyl ether), which are widely used as existing hyaluronic acid cross-linking agents, are biotoxic (Korea Chemical Safety Corporation Material Safety Data Sheet System), and thus the development of fillers with excellent stability is minimal.

[0006]

[0007] One object of the present invention is to provide a method for producing cross-linked hyaluronic acid, comprising the steps of (a) cross-linking hyaluronic acid and ornithine; and (b) further cross-linking the hyaluronic acid cross-linked with ornithine in (a) with STMP (Sodium Trimetaphosphate).

[0008] Another object of the present invention is to provide cross-linked hyaluronic acid manufactured by the manufacturing method of the present invention.

[0009] Another object of the present invention is to provide hyaluronic acid cross-linked with ornithine and STMP.

[0010] Another object of the present invention is to provide a tissue repair composition, a filler composition, and a composition for bioadministration comprising the cross-linked hyaluronic acid of the present invention.

[0011] Another object of the present invention is to provide a composition for producing cross-linked hyaluronic acid comprising ornithine, STMP, and hyaluronic acid, a composition for producing a biomaterial for tissue repair, and a composition for producing a filler.

[0012] Another object of the present invention is to provide a composite composition comprising at least one of the units of chemical formulae 3 to 7.

[0013]

[0014] The present invention can be used stably as a composition for bioadministration, a composition for tissue repair, and a filler composition by utilizing the composition having excellent biocompatibility and physical properties.

[0015]

[0016] Figure 1 is a diagram showing the results of biocompatibility evaluation of various crosslinking agents.

[0017] Figure 2 is a diagram showing elasticity results according to ornithine concentration.

[0018] Figure 3 is a diagram showing the shape of a single STMP cross-linked formulation.

[0019] Figure 4 shows the results of confirming the elasticity effect according to the cross-linking order of ornithine and STMP.

[0020] Figure 5 is a diagram showing the effect of ornithine concentration on elasticity in ornithine and STMP double crosslinking.

[0021] Figure 6 is a diagram showing the effect of STMP concentration on elasticity in ornithine and STMP double crosslinking.

[0022] Figure 7 is a diagram showing the effect of molecular weight on elasticity of hyaluronic acid in ornithine and STMP double crosslinking.

[0023]

[0024] Hereinafter, the present invention will be described in detail. Meanwhile, the description and embodiments of one aspect disclosed in the present invention may also be applied to the descriptions and embodiments of other aspects regarding common elements. Furthermore, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. Furthermore, the documents described in the present invention may be incorporated herein by reference. Furthermore, the scope of the present invention should not be considered limited by the specific descriptions set forth below.

[0025] One aspect of the present invention provides a method for producing cross-linked hyaluronic acid, comprising: (a) a step of cross-linking hyaluronic acid and ornithine; and (b) a step of cross-linking hyaluronic acid cross-linked with ornithine in (a) with STMP (Sodium Trimetaphosphate).

[0026] The step (a) above may include a step of mixing hyaluronic acid and ornithine, and the step (b) may include a step of further mixing hyaluronic acid cross-linked with ornithine with STMP.

[0027] As an example of implementation, the manufacturing method may include, but is not limited to, at least one of a step of dissolving hyaluronic acid and ornithine in a buffer prior to step (a); and a step of adding a cross-linking agent.

[0028] As an example of an embodiment, the manufacturing method may further include, but is not limited to, one or more of a step of adding a basic solution between steps (a) and (b) to break down ester bonds between hyaluronic acids; a paste step; and a step of adding STMP.

[0029] As an example of implementation, the manufacturing method may further include, but is not limited to, one or more of a step of neutralizing by adding an acidic solution after step (b); a dialysis step; a grinding step; a final concentration adjustment step; a mixing step; and a sterilization step.

[0030] In one embodiment of the above-described embodiment, the cross-linking agent may be an activator and / or a coupling reagent, and the activator may include at least one selected from the group consisting of NHS (N-Hydroxysuccinimide), HOBt (1-hydroxybenzotriazole), HOOBt (3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine), HOAt (1-hydroxy-7- azabenzotriazole), Sul-NHS (Sulfo-N-hydroxysulfosuccinimide), TBTU [O-(1H-benzotriazole-1-yl)- N,N,N',N'-tetramethyluronium tetrafluoroborate], and NMM (4-methylmorpholine), but is not limited thereto.

[0031] In one embodiment of the above-described embodiment, the coupling reagent may include at least one selected from the group consisting of EDC (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide), CMC [1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluenesulfonate], DCC (dicyclohexylcarbodiimide), DIC (diisopropylcarbodiimide), and CDMT (2-chloro-dimethoxy-1,3,5-triazine).

[0032] The above cross-linking agent may include two or more, and they may be added simultaneously, sequentially, or in reverse order. For example, in the above manufacturing method, EDC may be added after NHS, but is not limited thereto.

[0033] Meanwhile, the order of the above steps may be changed.

[0034] Additionally, the above steps may be performed more than once, and the number of times may be adjusted by a person skilled in the art depending on the size or shape of the desired cross-linked hyaluronic acid.

[0035] As an example of one implementation, the crosslinking time in steps (a) and / or (b) of the present invention may be 30 minutes or more. Specifically, 30 minutes to 36 hours, 30 minutes to 24 hours, 30 minutes to 12 hours, 30 minutes to 10 hours, 30 minutes to 8 hours, 30 minutes to 7.5 hours, 30 minutes to 7 hours, 30 minutes to 6.5 hours, 30 minutes to 6 hours, 30 minutes to 5.5 hours, 30 minutes to 5 hours, 30 minutes to 4 hours, 30 minutes to 3 hours, 30 minutes to 2 hours, 1 to 36 hours, 1 to 24 hours, 1 to 12 hours, 1 to 10 hours, 1 to 8 hours, 1 to 7.5 hours, 1 to 7 hours, 1 to 6.5 hours, 1 to 6 hours, 1 to 5.5 hours, 1 to 5 hours, 1 to 4 hours, 1 to 3 hours, 1 to 2 Hours, 3 to 48 hours, 3 to 36 hours, 3 to 24 hours, 3 to 12 hours, 3 to 10 hours, 3 to 8 hours, 3 to 7.5 hours, 3 to 7 hours, 3 to 6.5 hours, 3 to 6 hours, 3 to 5.5 hours, 3 to 5 hours, 3 to 4 hours, 6.5 to 48 hours, 6.5 to 36 hours, 6.5 to 24 hours, 6.5 to 12 hours, 6.5 to 10 hours, 6.5 to 8 hours, 6.5 to 7.5 hours, 6.5 to 7 hours, 8 to 48 hours, 8 to 36 hours, 8 to 24 hours, 8 to 12 hours, 8 to 10 hours, 10 to 48 hours, 10 to 36 hours, 10 to It may be, but is not limited to, 24 hours, 10 to 12 hours, 12 to 48 hours, 12 to 36 hours, 12 to 24 hours, 24 to 48 hours, or 24 to 36 hours.

[0036] Other conditions of the above steps (e.g., reaction temperature) may range from 100 to 200°C, 100 to 150°C, or 121°C, but are not limited thereto.

[0037]

[0038] The present invention is characterized in that it has developed cross-linked hyaluronic acid with excellent safety, biocompatibility, elasticity, etc. by double-cross-linking hyaluronic acid using not only ornithine and STMP but also both of them, and can be used for tissue repair, filler, etc.

[0039]

[0040] Another aspect of the present invention provides a cross-linked hyaluronic acid produced by the production method of the present invention. Specifically, the cross-linked hyaluronic acid is provided, comprising hyaluronic acid, ornithine, and STMP.

[0041] The above manufacturing method is as described in other aspects.

[0042] In the present invention, the term “cross-linked hyaluronic acid” means two or more hyaluronic acid molecules that are cross-linked, and may be used interchangeably with “cross-linked hyaluronic acid.”

[0043] The cross-linked hyaluronic acid of the present invention may be double-cross-linked with ornithine and STMP, and may be a hydrogel.

[0044] In the present invention, the term "ornithine" refers to one of the amino acids existing in the body, and may include L-ornithine and D-ornithine. The ornithine may include a physiologically acceptable salt, and specifically, may be in the form of an inorganic salt such as hydrochloride or phosphate, or an organic salt such as citrate, malate, α-ketoglutarate, or aspartate, and may have the following chemical formula 1, but is not limited thereto. In addition, the ornithine may be obtained by a chemical synthesis method, a fermentation production method, or a commercially available product.

[0045]

[0046] [Chemical Formula 1]

[0047]

[0048] The STMP (Sodium Trimetaphosphate) of the present invention may have the following chemical formula 2, but is not limited thereto.

[0049]

[0050] [Chemical Formula 2]

[0051]

[0052] In one embodiment, the hyaluronic acid of the present invention has a molecular weight of 10 kDa to 10,000 kDa, specifically 10 kDa to 5,000 kDa, 10 kDa to 4,500 kDa, 10 kDa to 4,000 kDa, 10 kDa to 3,500 kDa, 10 kDa to 3,000 kDa, 10 kDa to 2,500 kDa, 10 kDa to 2,000 kDa, 10 kDa to 1,500 kDa, 10 kDa to 1,000 kDa, 10 kDa to 900 kDa, 10 kDa to 800 kDa, 10 kDa to 700 kDa, 10 kDa to 600 kDa, 10 kDa to 500 kDa, 10 kDa to 450 kDa, 10 kDa to 400 kDa, 10 kDa to 350 kDa, 10 kDa to 300 kDa, 10 kDa to 250 kDa, 10 kDa to 200 kDa, 10 kDa to 180 kDa, 10 kDa to 150 kDa, 10 kDa to 100 kDa, 10 kDa to 50 kDa, 100 kDa to 5,000 kDa, 100 kDa to 4,500 kDa, 100 kDa to 4,000 kDa, 100 kDa to 3,500 kDa, 100 kDa to 3,000 kDa, 100 kDa to 2,500 kDa, 100 kDa to 2,000 kDa, 100 kDa to 1,500 kDa, 100 kDa to 1,000 kDa, 100 kDa to 900 kDa, 100 kDa to 800 kDa, 100 kDa to 700 kDa, 100 kDa to 600 kDa, 100 kDa to 500 kDa, 100 kDa to 450 kDa, 100 kDa to 400 kDa, 100 kDa to 350 kDa, 100 kDa to 300 kDa, 100 kDa to 250 kDa, 100 kDa to 200 kDa, 100 kDa to 180 kDa, 100 kDa to 170 kDa, 100 kDa to 160 kDa,100 kDa to 150 kDa, 200 kDa to 10,000 kDa, 200 kDa to 5,000 kDa, 200 kDa to 4,500 kDa, 200 kDa to 4,000 kDa, 200 kDa to 3,500 kDa, 200 kDa to 3,000 kDa, 200 kDa to 2,500 kDa, 200 kDa to 2,000 kDa, 200 kDa to 1,500 kDa, 200 kDa to 1,000 kDa, 200 kDa to 900 kDa, 200 kDa to 800 kDa, 200 kDa to 700 kDa, 200 kDa to 600 kDa, 400 kDa to 10,000 kDa, 400 kDa to 5,000 kDa, 400 kDa to 4,500 kDa, 400 kDa to 4,000 kDa, 400 kDa to 3,500 kDa, 400 kDa to 3,000 kDa, 400 kDa to 2,500 kDa, 400 kDa to 2,000 kDa, 400 kDa to 1,500 kDa, 400 kDa to 1,000 kDa, 600 kDa to 10,000 kDa, 600 kDa to 5,000 kDa, 600 kDa to 4,500 kDa, 600 kDa to 4,000 kDa, 600 kDa to 3,500 kDa, 600 kDa to 3,000 kDa, 600 kDa to 2,500 kDa, 600 kDa to 2,000 kDa, 600 kDa to 1,500 kDa, 600 kDa to 1,000 kDa, 800 kDa to 10,000 kDa, 800 kDa to 5,000 kDa, 800 kDa to 4,500 kDa, 800 kDa to 4,000 kDa, 800 kDa to 3,500 kDa, 800 kDa to 3,000 kDa, 800 kDa to 2,500 kDa, 800 kDa to 2,000 kDa, 800 kDa to 1,500 kDa, 1,000 kDa to 10,000 kDa, 1,000 kDa to 5,000 kDa, 1,000 kDa to 4,500 kDa, 1,000 kDa to 4,000 kDa, 1,000 kDa to 3,500 kDa, 1,000 kDa to 3,000 kDa, 1,000 kDa to 2,500 kDa, 1,000 kDa to 2,000 kDa, 1,000 kDa to 1,500 kDa, 1,500 kDa to 10,000 kDa, 1,500 kDa to 5,000 kDa, 1,500 kDa to 4,500 kDa, 1,500 kDa to 4,000 kDa, The hyaluronic acid of the present invention may have an average molecular weight of 1,500 kDa to 3,500 kDa, 1,500 kDa to 3,000 kDa, 1,500 kDa to 2,500 kDa, or 1,500 kDa to 2,000 kDa, but is not limited thereto. In addition, the hyaluronic acid of the present invention may be classified into ultra-low molecular weight hyaluronic acid having 10 kDa to 200 kDa, low molecular weight hyaluronic acid having 200 kDa to 600 kDa, medium molecular weight hyaluronic acid having 600 kDa to 1,000 kDa, and high molecular weight hyaluronic acid having 800 kDa to 1,500 kDa, but is not limited thereto.

[0053] In one embodiment, the initial dosage of hyaluronic acid of the present invention, hyaluronic acid included in the composition for preparation, and / or hyaluronic acid in the final formulation is 1 mg / mL to 100 mg / mL, specifically 1 mg / mL to 90 mg / mL, 1 mg / mL to 80 mg / mL, 1 mg / mL to 70 mg / mL, 1 mg / mL to 60 mg / mL, 1 mg / mL to 55 mg / mL, 1 mg / mL to 50 mg / mL, 1 mg / mL to 45 mg / mL, 1 mg / mL to 40 mg / mL, 1 mg / mL to 35 mg / mL, 1 mg / mL to 30 mg / mL, 1 mg / mL to 25 mg / mL, 1 mg / mL to 20 mg / mL, 1 mg / mL to 15 mg / mL, 1 mg / mL to 10 mg / mL, 10 mg / mL to 100 mg / mL, 10 mg / mL to 90 mg / mL, 10 mg / mL to 80 mg / mL, 10 mg / mL to 70 mg / mL, 10 mg / mL to 60 mg / mL, 10 mg / mL to 55 mg / mL, 10 mg / mL to 50 mg / mL, 10 mg / mL to 45 mg / mL, 10 mg / mL to 40 mg / mL, 10 mg / mL to 35 mg / mL, 10 mg / mL to 30 mg / mL, 10 mg / mL to 25 mg / mL, 10 mg / mL to 20 mg / mL, 10 mg / mL to 15 mg / mL, 15 mg / mL to 100 mg / mL, 15 mg / mL to 90 mg / mL, 15 mg / mL to 80 mg / mL, 15 mg / mL to 70 mg / mL, 15 mg / mL to 60 mg / mL, 15 mg / mL to 55 mg / mL, 15 mg / mL to 50 mg / mL, 15 mg / mL to 45 mg / mL, 15 mg / mL to 40 mg / mL, 15 mg / mL to 35 mg / mL, 15 mg / mL to 30 mg / mL, 15 mg / mL to 25 mg / mL, 15 mg / mL to 20 mg / mL,20 mg / mL to 100 mg / mL, 20 mg / mL to 90 mg / mL, 20 mg / mL to 80 mg / mL, 20 mg / mL to 70 mg / mL, 20 mg / mL to 60 mg / mL, 20 mg / mL to 55 mg / mL, 20 mg / mL to 50 mg / mL, 20 mg / mL to 45 mg / mL, 20 mg / mL to 40 mg / mL, 20 mg / mL to 35 mg / mL, 20 mg / mL to 30 mg / mL, 20 mg / mL to 25 mg / mL, 25 mg / mL to 100 mg / mL, 25 mg / mL to 90 mg / mL, 25 mg / mL to 80 mg / mL, 25 mg / mL to 70 mg / mL, 25 mg / mL to 60 mg / mL, 25 mg / mL to 55 mg / mL, 25 mg / mL to 50 mg / mL, 25 mg / mL to 45 mg / mL, 25 mg / mL to 40 mg / mL, 25 mg / mL to 35 mg / mL, 25 mg / mL to 30 mg / mL, 30 mg / mL to 100 mg / mL, 30 mg / mL to 90 mg / mL, 30 mg / mL to 80 mg / mL, 30 mg / mL to 70 mg / mL, 30 mg / mL to 60 mg / mL, 30 mg / mL to 55 mg / mL, 30 mg / mL to 50 mg / mL, 30 mg / mL to 45 mg / mL, 30 mg / mL to 40 mg / mL, 30 mg / mL to 35 mg / mL, 50 mg / mL to 100 mg / mL, 50 mg / mL to It may have a concentration of, but is not limited to, 90 mg / mL, 50 mg / mL to 80 mg / mL, 50 mg / mL to 70 mg / mL, 50 mg / mL to 60 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, or 50 mg / mL.

[0054] As a specific example, the composition of the present invention may have a variety of elasticity ranges that can be applied to various parts of the body, including ultra-low molecular weight, low molecular weight, medium molecular weight, and high molecular weight hyaluronic acid, depending on the molecular weight of the hyaluronic acid.

[0055]

[0056] As an example of implementation, the ornithine of the present invention may have a concentration of 0.5 to 25 wt% relative to the initial amount of hyaluronic acid or hyaluronic acid included in the composition for preparation. Specifically, 0.5 wt% to 25 wt%, 0.5 wt% to 21 wt%, 0.5 wt% to 15 wt%, 0.5 wt% to 10.5 wt%, 0.5 wt% to 10 wt%, 0.5 wt% to 8.7 wt%, 0.5 wt% to 5 wt%, 0.5 wt% to 4.5 wt%, 0.5 wt% to 4 wt%, 0.5 wt% to 3.5 wt%, 0.5 wt% to 3 wt%, 0.1 wt% to 2.5 wt%, 0.5 wt% to 2 wt%, 0.5 wt% to 1.7 wt%, 0.1 wt% to 1.5 wt%, 0.1 wt% to 1 wt%, 0.7 wt% to 25 wt%, 0.7 wt% to 21 wt%, 0.7 % to 15 wt%, 0.7 wt% to 10.5 wt%, 0.7 wt% to 10 wt%, 0.7 wt% to 8.7 wt%, 0.7 wt% to 5 wt%, 0.7 wt% to 3.5 wt%, 0.7 wt% to 1.7 wt%, 0.7 wt% to 1.5 wt%, 1.7 wt% to 25 wt%, 1.7 wt% to 21 wt%, 1.7 wt% to 15 wt%, 1.7 wt% to 10.5 wt%, 1.7 wt% to 10 wt%, 1.7 wt% to 8.7 wt%, 1.7 wt% to 5 wt%, 1.7 wt% to 3.5 wt%, 1.7 wt% to 1.5 wt%, 1 wt% to 25 wt%, 1 wt% to 21 % by weight, 1 wt% to 20 wt%, 1 wt% to 15 wt%, 1 wt% to 10.5 wt%, 1 wt% to 10 wt%, 1 wt% to 8.7 wt%, 1 wt% to 5 wt%, 1 wt% to 4.5 wt%, 1 wt% to 4 wt%, 1 wt% to 3.5 wt%, 1 wt% to 3 wt%, 1 wt% to 2.5 wt%, 1 wt% to 2 wt%, 1 wt% to 1.7 wt%, 1 wt% to 1.5 wt%, 2 wt% to 25 wt%, 2 wt% to 21 wt%, 2 wt% to 20 wt%, 2 wt% to 15 wt%, 2 wt% to 10.5 wt%, 2 wt% to 10 wt%, 2 wt% to 8.7 wt%, 2 wt% to 5 wt%, 2 wt% to 4.5 wt%, 2 wt% to 4 wt%, 2 wt% to 3.5 wt%, 2 wt% to 3 wt%, 2 wt% to 2.5 wt%, 3.5 wt% to 25 wt%, 3.5 wt% to 21 wt%, 3.5 wt% to 15 wt%, 3.5 wt% to 10.7 wt%, 3.5 It may have a concentration of, but is not limited to, 3.5 wt% to 10 wt%, 3.5 wt% to 8.7 wt%, 3.5 wt% to 5 wt%, 3.5 wt% to 4.5 wt%, 3.5 wt% to 4 wt%, 5 wt% to 25 wt%, 5 wt% to 20 wt%, 5 wt% to 15 wt%, or 5 wt% to 10 wt%.

[0057] As an example of implementation, the STMP of the present invention may have a concentration of 1 to 130 wt% relative to the initial amount of hyaluronic acid or the hyaluronic acid included in the composition for preparation. Specifically, 1 wt% to 150 wt%, 1 wt% to 130 wt%, 1 wt% to 127 wt%, 1 wt% to 100 wt%, 1 wt% to 63.7 wt%, 1 wt% to 50 wt%, 1 wt% to 40 wt%, 1 wt% to 38 wt%, 1 wt% to 35 wt%, 1 wt% to 30 wt%, 1 wt% to 25 wt%, 1 wt% to 20 wt%, 1 wt% to 12.7 wt%, 1 wt% to 10 wt%, 1 wt% to 5 wt%, 1 wt% to 2.5 wt%, 1 wt% to 1.25 wt%, 1.25 wt% to 150 wt%, 1.25 wt% to 130 wt%, 1.25 wt% to 127 % by weight, 1.25 wt% to 100 wt%, 1.25 wt% to 63.7 wt%, 1.25 wt% to 50 wt%, 1.25 wt% to 40 wt%, 1.25 wt% to 38 wt%, 1.25 wt% to 35 wt%, 1.25 wt% to 30 wt%, 1.25 wt% to 25 wt%, 1.25 wt% to 20 wt%, 1.25 wt% to 12.7 wt%, 1.25 wt% to 10 wt%, 1.25 wt% to 5 wt%, 1.25 wt% to 2.5 wt%, 2.5 wt% to 150 wt%, 2.5 wt% to 130 wt%, 2.5 wt% to 127 wt%, 2.5 wt% to 100 wt%, 2.5 wt% to 63.7 wt%, 2.5 wt% to 50 wt%, 2.5 wt% to 40 wt%, 2.5 wt% to 38 wt%, 2.5 wt% to 35 wt%, 2.5 wt% to 30 wt%, 2.5 wt% to 25 wt%, 2.5 wt% to 20 wt%, 2.5 wt% to 12.7 wt%, 2.5 wt% to 10 wt%, 2.5 wt% to 5 wt%, 5 wt% to 150 wt%, 5 wt% to 130 wt%, 5 wt% to 127 wt%, 5 wt% to 100 wt%, 5 wt% to 63.7 wt%, 5 wt% to 50 wt%, 5 wt% to 40 wt%, 5 wt% to 38 wt%, 5 wt% to 35 wt%, 5 wt% to 30 wt%, 5 wt% to 25 wt%, 5 wt% to 20 wt%, 5 wt% to 12.7 wt%, 5 wt% to 10 wt%, 10 wt% to 150 wt%, 10 wt% to 130 wt%, 10 wt% to 127 wt%, 10 wt% to 100 wt%, 10 wt% to 63.7 wt%, 10 wt% to 50 wt%, 10 wt% to 40 wt%, 10 wt% to 38 wt%, 10 wt% to 35 wt%, 10 wt% to 30 wt%, 10 wt% to 25 wt%, 10 wt% to 20 wt%, 12.7 wt% to 150 wt%, 12.7 wt% to 130 wt%, 12.7 wt% to 127 wt%, 12.7 wt% to 100 wt%, 12.7 wt% to 63.7 wt%, 12.7 wt% to 50 wt%, 12.7 wt% to 40 wt%, 12.7 wt% to 38 wt%, 12.7 wt% to 35 wt%, 12.7 wt% to 30 wt%, 12.7 wt% to 25 wt%, 12.7 wt% to 20 wt%, 25 wt% to 150 wt%, 25 wt% to 130 wt%, 25 wt% to 127 wt%, 25 wt% to 100 wt%, 25 wt% to 63.It may have a concentration of, but is not limited to, 7 wt%, 25 wt% to 50 wt%, 25 wt% to 40 wt%, 25 wt% to 38 wt%, 25 wt% to 35 wt%, 25 wt% to 30 wt%, 38 wt% to 150 wt%, 38 wt% to 130 wt%, 38 wt% to 127 wt%, 38 wt% to 100 wt%, 38 wt% to 63.7 wt%, 38 wt% to 50 wt%, 38 wt% to 45 wt%, 50 wt% to 150 wt%, 50 wt% to 130 wt%, 50 wt% to 127 wt%, 50 wt% to 100 wt%, or 50 wt% to 63.7 wt%.

[0058] The molecular weight of the above hyaluronic acid and the concentration of hyaluronic acid, STMP, and ornithine may refer to the molecular weight and concentration of the components included in the initial input amount or the composition for manufacturing in the manufacturing method of the present invention.

[0059] In one embodiment, the cross-linked hyaluronic acid may be one in which the concentrations of hyaluronic acid, ornithine, and STMP can be adjusted depending on the desired elasticity.

[0060] It may be important to control the elasticity of the cross-linked hyaluronic acid of the present invention so that the desired filler can be appropriately injected into the desired tissue. Specifically, the cross-linked hyaluronic acid may exhibit an elastic modulus of about 100 Pa to about 1,000 Pa, specifically about 100 Pa to about 800 Pa, so that it can be used in various areas (tissues).

[0061] In one embodiment, in order for the cross-linked hyaluronic acid to have the desired elasticity, ornithine may be included in an amount of 0.5 wt% to 25 wt% relative to the amount of hyaluronic acid added, and STMP may be included in an amount of 1 wt% to 130 wt% relative to the amount of hyaluronic acid added. Specifically, ornithine may be included in an amount of 0.7 wt% to 21 wt%, and STMP may be included in an amount of 1.25 wt% to 127 wt% relative to the amount of hyaluronic acid added.

[0062] In one embodiment, in order for the cross-linked hyaluronic acid to have the desired elasticity, ornithine may be included in an amount of 0.7 wt% to 21 wt% relative to the amount of hyaluronic acid added, and STMP may be included in an amount of 38 wt% relative to the amount of hyaluronic acid added.

[0063] For example, when ornithine is included in an amount of 0.7 wt% to 21 wt% relative to the amount of hyaluronic acid input, and STMP is included in an amount of 38 wt% relative to the amount of hyaluronic acid input, the cross-linked hyaluronic acid of the present invention may have an elasticity range of about 100 Pa or more, specifically about 100 Pa to about 1,000 Pa; when ornithine is included in an amount of 1.7 wt% to 10.5 wt% relative to the amount of hyaluronic acid input, and STMP is included in an amount of 38 wt% relative to the amount of hyaluronic acid input, the cross-linked hyaluronic acid of the present invention may have an elasticity range of about 300 Pa or more, specifically about 300 Pa to about 1,000 Pa; when ornithine is included in an amount of 3.5 ... When hyaluronic acid can have an elasticity range of about 400 Pa or more, specifically about 400 Pa to about 1,000 Pa, and ornithine comprises 3.5 wt% to 8.7 wt% based on the amount of hyaluronic acid input, and STMP comprises 38 wt% based on the amount of hyaluronic acid input, the cross-linked hyaluronic acid of the present invention can have an elasticity range of about 500 Pa or more, specifically about 500 Pa to about 1,000 Pa, and when ornithine comprises 8.7 wt% based on the amount of hyaluronic acid input, and STMP comprises 38 wt% based on the amount of hyaluronic acid input, the cross-linked hyaluronic acid of the present invention can have an elasticity range of about 700 Pa or more, specifically about 700 Pa to about 1,000 Pa, but is not limited thereto.

[0064]

[0065] In one embodiment, when the cross-linked hyaluronic acid comprises 8.7 wt% of ornithine relative to the amount of hyaluronic acid added, the STMP may comprise 1.25 wt% to 127 wt% relative to the amount of hyaluronic acid added in order to have the desired elasticity.

[0066] For example, when ornithine comprises 8.7 wt% relative to the amount of hyaluronic acid input, and STMP comprises 1.25 wt% to 127 wt% relative to the amount of hyaluronic acid input, the cross-linked hyaluronic acid of the present invention may have an elasticity range of about 600 Pa or more, specifically about 600 Pa to about 1,000 Pa, and when ornithine comprises 8.7 wt% relative to the amount of hyaluronic acid input, and STMP comprises 2.5 wt% to 63.7 wt% relative to the amount of hyaluronic acid input, the cross-linked hyaluronic acid of the present invention may have an elasticity range of about 650 Pa or more, specifically about 650 Pa to about 1,000 Pa, and when ornithine comprises 8.7 wt% relative to the amount of hyaluronic acid input, and STMP comprises 12.7 wt% to 63.7 wt% relative to the amount of hyaluronic acid input, the cross-linked hyaluronic acid of the present invention may have an elasticity range of about 650 Pa or more, specifically about 650 Pa to about 1,000 Pa. The cross-linked hyaluronic acid may have an elasticity range of about 670 Pa or more, specifically, about 670 Pa to about 1,000 Pa, and when ornithine comprises 8.7 wt% of the amount of hyaluronic acid input and STMP comprises 63.7 wt% of the amount of hyaluronic acid input, the cross-linked hyaluronic acid of the present invention may have an elasticity range of about 700 Pa or more, and when ornithine comprises 8.7 wt% of the amount of hyaluronic acid input and STMP comprises 38 wt% of the amount of hyaluronic acid input, the cross-linked hyaluronic acid of the present invention may have an elasticity range of about 750 Pa or more, but is not limited thereto.

[0067] Specifically, it can be seen that controlling the concentration of ornithine and STMP is important in order for the cross-linked hyaluronic acid of the present invention to have the desired elasticity.

[0068]

[0069] In the present invention, the term "about" may be presented before a specific numerical value. The term "about" as used herein encompasses not only the exact number described after the term, but also a range that is or is nearly that number. Whether a number is or is nearly the specific number described can be determined based on the context in which it is presented. For example, the term "about" may refer to a range of -10% to +10% of a numerical value. In another example, the term "about" may refer to a range of -5% to +5% of a given numerical value. However, this is not a limitation.

[0070]

[0071] In addition, the cross-linked hyaluronic acid of the present invention may not be one in which hyaluronic acid, ornithine, and STMP are repeated in a regular unit. Specifically, the cross-linked hyaluronic acid of the present invention may include any one or more of the units of the following chemical formulae 3 to 7, but these may not be repeated in a regular manner but may be included irregularly.

[0072] As an example of implementation, the cross-linked hyaluronic acid of the present invention may include one or more, two or more, three or more, four or more, or five types of units of the following chemical formulae 3 to 7, and may be a composite composition.

[0073] The following chemical formulas 3 to 7 are merely examples of the units forming the cross-linked hyaluronic acid of the present invention, and the cross-linked hyaluronic acid of the present invention may be included without limitation in its specific structure as long as it is double-cross-linked with ornithine and STMP.

[0074]

[0075] [Chemical Formula 3]

[0076]

[0077] [Chemical Formula 4]

[0078]

[0079] [Chemical Formula 5]

[0080]

[0081] [Chemical Formula 6]

[0082]

[0083] [Chemical Formula 7]

[0084]

[0085]

[0086] In the present invention, the term “double-crosslinked hyaluronic acid” means hyaluronic acid that is crosslinked by both ornithine and STMP, rather than by ornithine and STMP being crosslinked with each other.

[0087] In one embodiment of the present invention, the STMP and ornithine may be safe as they do not exhibit cytotoxicity compared to DVS (Divinyl Sulfone) and BDDE (1,4-Butanediol diglycidyl ether), which are widely used in the existing hyaluronic acid cross-linking.

[0088] As an example of implementation, the cross-linked hyaluronic acid of the present invention may have excellent elasticity and thermal stability while maintaining lower cytotoxicity compared to hyaluronic acid cross-linked solely with STMP and hyaluronic acid cross-linked solely with ornithine, but is not limited thereto.

[0089] In addition, the cross-linked hyaluronic acid of the present invention has excellent biocompatibility and can be used as a composition for tissue repair, bioadministration, and filler.

[0090]

[0091] Another aspect of the present invention provides a tissue repair composition comprising the cross-linked hyaluronic acid of the present invention.

[0092] The above cross-linked hyaluronic acid is as described in other aspects.

[0093] In the present invention, the term "tissue repair" means something that can be used for replacing, repairing, and / or reconstructing human tissues and organs, such as blood vessels, heart, septum, fascia, and / or skin.

[0094]

[0095] Another aspect of the present invention provides a filler composition comprising the cross-linked hyaluronic acid of the present invention.

[0096] The above cross-linked hyaluronic acid is as described in other aspects.

[0097] In the present invention, the term "filler" refers to a medical device that is one of the tissue repair materials and is injected into the skin to restore volume, etc., and has an operating principle of maintaining skin volume through physical repair.

[0098] As an example of implementation, the filler composition of the present invention may be a composition for dermal filler.

[0099] As an example of one embodiment, the filler composition of the present invention may be a hyaluronic acid filler composition. Hyaluronic acid fillers are composed of polysaccharides similar to the components of the human body, have few side effects such as skin hypersensitivity, are easy to administer and remove, have excellent elasticity, and maintain moisture in the skin and maintain skin volume and elasticity, making them the most widely used fillers at present. Generally, when manufacturing hyaluronic acid fillers, a cross-linking agent is used to ensure that hyaluronic acid is maintained in the body for a long time without being decomposed. However, DVS (Divinyl Sulfone) and BDDE (1,4-Butanediol diglycidyl ether), which are widely used as existing hyaluronic acid cross-linking agents, have the disadvantage of being biotoxic.

[0100] As an example of implementation, the filler composition of the present invention may use ornithine and STMP, which have significantly lower cytotoxicity and superior biostability compared to DVS (Divinyl Sulfone) and BDDE (1,4-Butanediol diglycidyl ether), as crosslinking agents.

[0101] The filler composition of the present invention may have superior elasticity and thermal stability compared to a filler composition comprising STMP-only cross-linked hyaluronic acid and ornithine-only cross-linked hyaluronic acid, but is not limited thereto.

[0102]

[0103] Another aspect of the present invention provides a composition for bioadministration comprising the cross-linked hyaluronic acid of the present invention.

[0104] The above cross-linked hyaluronic acid is as described in other aspects.

[0105] The term "administration" in the present invention refers to introducing the composition of the present invention into a subject by an appropriate method. Administration may be administered via various routes, including topical application, subcutaneous injection, and intradermal administration, as long as the composition can reach the target tissue. The formulation may be, but is not limited to, topical application, subcutaneous injection, intradermal injection, or intramuscular injection. The preferred dosage of the composition of the present invention may vary depending on the condition of the subject.

[0106]

[0107] The tissue repair composition, filler composition, and bio-administration composition according to the present invention may further comprise one or more compounds selected from the group consisting of anesthetics, polyols, vitamins, amino acids, metals, antioxidants, and mineral salts, and may comprise a buffer, for example, a phosphate buffer, to adjust the pH.

[0108] The tissue repair composition, filler composition, and bio-administrable composition of the present invention may provide an injectable composition. The term "injectable" refers to a material having the properties required for administering the composition to the dermal region of an individual using an injection device having a fine needle.

[0109] The cross-linked hyaluronic acid, tissue repair composition, filler composition, and / or bio-administrable composition of the present invention may exhibit an elastic modulus in an appropriate range that can be injected into tissue, but is not limited thereto.

[0110] In the present invention, the term "elasticity" means a property of a solid when force is applied to an object, that is, a property of changing shape when force is applied but returning to the original shape when force is removed. For example, the elasticity of a filler indicates the degree to which it maintains a desired shape under skin tissue, and the higher the elasticity, the longer it maintains the initially injected shape. This elasticity can be expressed as a storage modulus (G': elastic modulus, storage modulus), and the unit at this time can be Pascal (Pa).

[0111] The higher the elasticity of cross-linked hyaluronic acid or a composition containing it, the more it forms particles, which feel slightly firmer and are therefore better for adding volume. Furthermore, the filler maintains its shape well after a single procedure. Furthermore, higher elasticity tends to prolong the lifespan of the filler. However, excessively high elasticity can lead to an uneven surface, which, depending on the level of skill, can lead to the formation of layers on the skin.

[0112] In one embodiment, the cross-linked hyaluronic acid, tissue repair composition, filler composition, and / or bio-administrable composition of the present invention may exhibit an elastic modulus of about 100 Pa to 1,000 Pa, specifically 100 Pa to 800 Pa. For example, a low elastic modulus of less than 100 Pa may form an unstable cross-linked body, and an excessively high elastic modulus of more than 800 Pa may cause a foreign body reaction in the human body.

[0113] Among these, their low to medium elastic modulus can be used for lips, contour, skin improvement, mid-face, etc., their medium elastic modulus can be used for lower face, etc., and their medium to high elastic modulus can be used for upper face, mid-face, nose, chin, etc., so they can have a wide range of elastic moduli (Carola de la Guardia et al., 2022;38:116-123; and Won Lee et al., 2019; 00:1-9, doi:10.1097 / dss.0000000000001858).

[0114]

[0115] Therefore, it may be important to inject the desired filler by controlling the elasticity in the target tissue.

[0116]

[0117] Another aspect of the present invention provides a composition for producing cross-linked hyaluronic acid comprising ornithine, STMP, and hyaluronic acid, a composition for producing a biomaterial for tissue repair, a composition for producing a filler, and a composition for producing a bio-administrative agent.

[0118] The above ornithine, STMP, hyaluronic acid, cross-linked hyaluronic acid, tissue repair, and filler, etc. are as described in other aspects.

[0119] The composition for manufacturing of the present invention may further include any suitable excipient commonly used in the manufacture of cross-linked hyaluronic acid, filler, biomaterial for tissue repair, and / or composition for bioadministration, and such excipient may be, for example, a preservative, a wetting agent, a dispersing agent, a suspending agent, a buffer, a stabilizer, or an isotonic agent, but is not limited thereto.

[0120]

[0121] Another aspect of the present invention provides a kit for preparing a cross-linked hyaluronic acid composition, a tissue repair composition, a filler composition, and a composition for bioadministration, comprising ornithine, STMP, and hyaluronic acid.

[0122] The above ornithine, STMP, hyaluronic acid, and cross-linked hyaluronic acid are as described in other aspects.

[0123] The above kit may be provided such that hyaluronic acid; ornithine; and STMP are stored in separate containers and mixed at the time of use, but is not limited thereto.

[0124] In addition, the kit of the present invention may include, but is not limited to, one or more other component compositions, solutions or devices for preparing cross-linked hyaluronic acid, tissue repair compositions, filler compositions, and compositions for bioadministration. Examples of the other components may include, but are not limited to, suitable carriers, solubilizers, buffers, stabilizers, etc. The carrier may include, but is not limited to, a soluble carrier, an insoluble carrier, an example of a soluble carrier is a physiologically acceptable buffer known in the art, for example, PBS, and an example of an insoluble carrier may include, but is not limited to, a polymer such as polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, a fluororesin, cross-linked dextran, polysaccharides, magnetic microparticles plated with metal on latex, other paper, glass, metal, agarose, and combinations thereof.

[0125] Additionally, the kit of the present invention may additionally include a user guide describing optimal reaction conditions. The guide is a printed document explaining how to use the kit, such as reaction conditions. The guide includes instructions in the form of a pamphlet or leaflet, a label attached to the kit, and descriptions on the surface of the package containing the kit. The guide also includes information disclosed or provided through electronic media, such as the Internet.

[0126]

[0127] Another aspect of the present invention provides a method for improving skin, comprising administering to a subject a cross-linked hyaluronic acid tissue repair composition, filler composition, and / or bio-administrable composition of the present invention.

[0128] The cross-linked hyaluronic acid, composition, object, and administration, etc. are as described in other aspects.

[0129] The above skin improvement may include, but is not limited to, skin filling, (soft) tissue repair, wrinkle improvement, and skin volume increase.

[0130] In the present invention, the term "subject" means any animal requiring skin improvement, and may mean all mammals including dogs, cows, horses, rabbits, mice, rats, chickens, and humans.

[0131]

[0132] Another aspect of the present invention provides a composite composition comprising at least one of the units of the above chemical formulae 3 to 7.

[0133] In addition, the complex composition of the present invention may contain one or more units of the chemical formulae 3 to 7 in an irregular manner rather than being constantly repeated.

[0134] As an example of implementation, the composite composition of the present invention may include one or more, two or more, three or more, four or more, or five types of units represented by the following chemical formulae 3 to 7. However, the chemical formulae 3 to 7 are merely examples of the units of the present invention and are not limited thereto.

[0135] As an example of implementation, the composite composition may include, but is not limited to, the units being directly or indirectly connected.

[0136] As an example of implementation, the composite composition can be used as a tissue repair composition, a filler composition, and / or a composition for bioadministration.

[0137]

[0138] The present invention is described in more detail below through examples. However, the following examples are merely preferred embodiments intended to illustrate the present invention and are therefore not intended to limit the scope of the present invention. Furthermore, technical details not described herein can be readily understood and implemented by those skilled in the technical field of the present invention or similar fields.

[0139]

[0140] Example 1. Biocompatibility evaluation according to crosslinking agent type

[0141] The biocompatibility of various cross-linkers was evaluated by cytotoxicity using a known method (Chang Hee Jeong, et al., 2021, Toxicology In vitro Feb;70:105034; ISO10993-5). The negative / positive control groups and experimental groups are shown in Table 1 below. Divinyl Sulfone (DVS) and 1,4-Butanediol diglycidyl ether (BDDE), used in the experimental groups, are widely used cross-linkers. In the present invention, ornithine and STMP were evaluated for biocompatibility to confirm their suitability as cross-linkers.

[0142]

[0143] Control group Negative control group: high-density polyethylene film Positive control group: polyurethane film containing 0.1% zinc diethyldithiocarbamate (ZDEC) Experimental group (DVS, BDDE, ornithine, STMP Ornithine+STMP) 0 ppm 5 ppm 25 ppm 100 ppm 500 ppm 1,000 ppm

[0144] Specifically, HDF (human dermal fibroblast) was cultured at a cell density of 1 X 10 4 Cells / well were dispensed into 96-well plates and cultured for 24 hours in a 37°C, 5% CO2 incubator. Afterwards, the control and experimental group samples in Table 1 were treated with 0 to 1,000 ppm and cultured for 24 hours in a 37°C, 5% CO2 incubator, and then treated with WST reagent and reacted for 1 hour in a 37°C, 5% CO2 incubator, and then the absorbance (OD) at 450 nm was measured. 450 ) was measured. Cell viability was derived as follows, and if the cell viability decreased by 30% or more depending on the experimental and control groups, it was considered cytotoxic.

[0145]

[0146] - Viability (%) = (Exp.- Blank) / (Control - Blank)

[0147]

[0148] As a result, as shown in Fig. 1, the previously used known cross-linking agent DVS showed cytotoxicity from 5 ppm, BDDE from 100 ppm, and ornithine alone and STMP alone showed no cytotoxicity up to 1,000 ppm. In addition, the mixed solution of ornithine and STMP also showed no cytotoxicity up to 1,000 ppm.

[0149] Accordingly, it was confirmed that ornithine, STMP, and a mixture of ornithine and STMP have higher biocompatibility than the previously widely used cross-linking agents DVS and BDDE. Based on the above results, experiments were conducted in the present invention using ornithine; STMP; ornithine and STMP as cross-linking agents.

[0150]

[0151] Example 2. Preparation and results of ornithine-only cross-linked hyaluronic acid formulation

[0152]

[0153] 2-1. Manufacturing of ornithine-only cross-linked hyaluronic acid formulation

[0154] Hyaluronic acid (HA) cross-linked solely with ornithine was prepared.

[0155] At this time, hyaluronic acid was used with a molecular weight of 20 kDa to 2,000 kDa, and the initial ornithine concentration was 0.35 wt% to 8.7 wt% of hyaluronic acid. To prepare hyaluronic acid cross-linked solely with ornithine, hyaluronic acid and ornithine were dissolved at pH 4 to 6, NHS (N-Hydroxysuccinimide) was added, and they were mixed for 30 minutes. Then, EDC (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) was added, and mixing and cross-linking were performed for 3 hours. NaOH solution was added to decompose the ester bond, and HCl solution was added to neutralize. Dialysis was performed in PBS for 4 days. Free hyaluronic acid (Free HA) and lidocaine were added or not, and grinding and final concentration adjustment were performed.

[0156]

[0157] 2-2. Check elasticity

[0158] Phase angle is used to analyze the elasticity of a material, and the detailed analysis conditions of the phase angle used in the experiment are as shown in Table 2.

[0159] Oscillation (check 1.0 Hz value)GeometryParallel Plate 20 mmLoading gap1.0 mmFrequency0.1 ~ 10 Hz

[0160] A phase angle of 0° indicates a solid, 100% elastic material, and a phase angle of 90° indicates a liquid, 100% viscous material. To be used as a filler, the phase angle must be 80° or less (WO 2016-096920 A1). The elasticity was confirmed based on the ornithine concentration. Specifically, the elasticity was confirmed at ornithine concentrations of 0.35 wt% and 8.7 wt%.

[0161] As a result, as shown in Fig. 2, it was confirmed that the elasticity value was higher when the ornithine concentration was 8.7 wt%.

[0162]

[0163] Example 3. Preparation and results of STMP-only cross-linked hyaluronic acid formulation

[0164]

[0165] 3-1. Manufacturing of STMP-only cross-linked hyaluronic acid formulation

[0166] Hyaluronic acid cross-linked solely with STMP was prepared as follows.

[0167] At this time, hyaluronic acid having a molecular weight of 20 kDa to 2,000 kDa was used. For the production of hyaluronic acid cross-linked solely with STMP (WO 2014 / 181147 A1), hyaluronic acid dissolved in a 25 M NaOH solution for 2.5 hours and a STMP solution dissolved in a 0.25 M NaOH solution were mixed for 30 minutes and a cross-linking reaction was performed at 50°C for 15 hours.

[0168]

[0169] 3-2. Checking the progress of the cross-linking reaction

[0170] As the crosslinking reaction progressed after STMP injection, the pH decreased, and the pH was measured to be 8.58 after the crosslinking reaction was completed. As crosslinking progresses, free phosphate groups are generated, causing the pH to decrease, so a decrease in pH indicates that the crosslinking reaction is progressing.

[0171]

[0172] 3-3. Confirmation of elastic body non-formation

[0173] The purpose of this study was to confirm whether STMP was formed as an elastic body using the phase angle under the same conditions as those used in the experiment in 2-2.

[0174] As a result, it was confirmed that hyaluronic acid cross-linked solely with STMP did not form an elastomer with a phase angle of 84.9° (Fig. 3). The above results suggest that tissue repair may not be possible in the case of STMP cross-linking alone. Therefore, hyaluronic acid cross-linked solely with STMP did not form an elastomer and thus did not proceed to subsequent steps, such as neutralization using HCl.

[0175]

[0176] Example 4. Preparation and results of ornithine and STMP double-cross-linked hyaluronic acid formulations

[0177]

[0178] 4-1. Preparation of hyaluronic acid formulations according to the sequence of double cross-linking of ornithine and STMP.

[0179] To determine the effect of ornithine and STMP (Sodium Trimetaphosphate) cross-linking order, double-cross-linked hyaluronic acid was prepared by cross-linking with ornithine after STMP cross-linking.

[0180] At this time, hyaluronic acid was used having a molecular weight of 20 kDa to 2,000 kDa, an initial ornithine concentration of 0.1 wt% to 15 wt% relative to hyaluronic acid, and an initial STMP concentration of 0.1 wt% to 250 wt% relative to hyaluronic acid.

[0181]

[0182] 4-1-1. Manufacturing of ornithine-crosslinked double hyaluronic acid formulation after STMP crosslinking

[0183] To prepare hyaluronic acid double-crosslinked with ornithine after STMP crosslinking, hyaluronic acid was dissolved at pH 10 to 12, and STMP was added and dissolved.

[0184] Mixing and crosslinking were performed at room temperature for more than 30 minutes. HCl solution was added to the STMP crosslinker to lower the pH to 4 to 6, ornithine and NHS (N-Hydroxysuccinimide) were added, and mixing was performed for more than 30 minutes. EDC (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) was additionally added, and mixing and crosslinking were performed for more than 30 minutes. NaOH solution was added to break down the incorrect ester bonds, and HCl solution was additionally added to neutralize. Dialysis was performed in PBS for more than 3 days, and free hyaluronic acid (Free HA) and lidocaine were added or not, and the mixture was ground and the final concentration was adjusted.

[0185]

[0186] 4-1-2. Manufacturing of STMP-crosslinked double hyaluronic acid formulation after ornithine crosslinking

[0187] To prepare double cross-linked hyaluronic acid with STMP after ornithine cross-linking, hyaluronic acid and ornithine were dissolved at pH 4 to 6, NHS (N-Hydroxysuccinimide) was added, and they were mixed for more than 30 minutes. Then, 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) was added, and they were mixed and cross-linked for more than 30 minutes. After adding NaOH solution, they were mixed for more than 30 minutes to break down the mis-ester bonds, and STMP was added to proceed with cross-linking. STMP cross-linking was carried out for more than 30 minutes, and HCl solution was added to neutralize. Dialysis was performed in PBS for more than 3 days, and free hyaluronic acid (Free HA) and lidocaine were added or not, and the mixture was ground and the final concentration was adjusted.

[0188]

[0189] 4-2. Analysis of elasticity effects according to ornithine and STMP cross-linking order

[0190] In order to confirm the effect of elasticity according to the crosslinking order of ornithine and STMP, ornithine crosslinking was performed after STMP crosslinking under the conditions of 8.7 wt% ornithine concentration and 38 wt% STMP concentration, which have the best elasticity. As shown in Fig. 4, when STMP was crosslinked first, it was found that the elasticity of the final product was very low and the crosslinked body was not formed well.

[0191] Through this, it is suggested that the double crosslinking of the present invention can produce crosslinked hyaluronic acid having a variety of elasticity ranges by performing STMP crosslinking after ornithine crosslinking in that order.

[0192]

[0193] 4-3. Analysis of elasticity effects by ornithine concentration

[0194] An elasticity analysis was performed after double cross-linking with STMP after ornithine cross-linking. After cross-linking with various ornithine concentrations and a fixed STMP concentration of 38 wt%, elasticity was analyzed, and as shown in Fig. 5, it was confirmed that the composite had a wide elasticity range (approximately 100 Pa to 800 Pa) that could be applied to various parts.

[0195] Specifically, in order to manufacture cross-linked hyaluronic acid having an elasticity of about 100 Pa, it was confirmed that ornithine should be included in an amount of 0.7 wt% or 21 wt% relative to the amount of hyaluronic acid input, and STMP should be included in an amount of 38 wt% relative to the amount of hyaluronic acid input. In addition, in order to manufacture cross-linked hyaluronic acid having an elasticity of about 300 Pa, it was confirmed that ornithine should be included in an amount of 1.7 wt% relative to the amount of hyaluronic acid input, and STMP should be included in an amount of 38 wt% relative to the amount of hyaluronic acid input. In order to manufacture cross-linked hyaluronic acid having an elasticity of about 600 Pa, it was confirmed that ornithine should be included in an amount of 8.7 wt% relative to the amount of hyaluronic acid input, and STMP should be included in an amount of 1.25 wt% to 127 wt% relative to the amount of hyaluronic acid input.

[0196]

[0197] 4-5. Elasticity effect analysis by STMP concentration

[0198] Based on the results of 2-2, we attempted to confirm the elasticity according to the STMP concentration when the ornithine concentration was 8.7 wt%.

[0199] As a result, as shown in Fig. 6, it was confirmed that the elasticity increased when double cross-linked with STMP compared to ornithine single cross-linking, and that the elasticity showed the highest increase rate at 38 wt% STMP. In particular, when the concentration of ornithine was fixed at 8.7 wt%, it was confirmed that detailed elasticity control was possible so that the elasticity increased by 29.2 Pa to 71.5 Pa compared to 8.7 wt% ornithine single cross-linking by varying the concentration of STMP, so that the final elasticity value could be approximately 600 Pa to 800 Pa.

[0200] This suggests that the concentrations of ornithine and STMP can be adjusted to manufacture the cross-linked hyaluronic acid of the present invention to have the desired elasticity, and in particular, the detailed elasticity value can be adjusted by adjusting the concentration of STMP.

[0201]

[0202] Example 5. Elasticity test according to hyaluronic acid molecular weight

[0203]

[0204] It was confirmed that the hyaluronic acid produced by the manufacturing method of the present invention through double crosslinking of ornithine and STMP has a wide range of elasticity that can be applied to various parts, and it was also intended to confirm whether it has an appropriate range of elasticity that can be applied to various hyaluronic acid molecular weights.

[0205] As a result, as shown in Fig. 7, it was confirmed that ultra-low molecular weight (approximately 25 kDa), low molecular weight (approximately 435 kDa), medium molecular weight (approximately 800 kDa), and high molecular weight (approximately 1140 kDa) hyaluronic acid have various elasticity ranges that can be applied to various parts.

[0206] This suggests that even when hyaluronic acids of different molecular weights are applied, cross-linked hyaluronic acids with a wide range of elasticity that can be applied to various areas are well formed.

[0207]

[0208] The above results suggest that the present invention has excellent effects in producing filler compositions and compositions for bioadministration with excellent biocompatibility and physical properties in various areas.

[0209]

[0210] From the above description, those skilled in the art will understand that the present invention can be implemented in other specific forms without altering its technical spirit or essential characteristics. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be interpreted as encompassing all changes or modifications derived from the meaning and scope of the following claims and their equivalent concepts, rather than the detailed description above.

Claims

1. (a) a step of cross-linking hyaluronic acid and ornithine; and (b) A method for producing cross-linked hyaluronic acid, comprising the step of cross-linking hyaluronic acid cross-linked with ornithine in (a) with STMP (Sodium Trimetaphosphate).

2. In paragraph 1, A manufacturing method wherein the above ornithine has a concentration of 0.5 to 25 wt% relative to the amount of hyaluronic acid input.

3. In paragraph 1, A manufacturing method wherein the above STMP has a concentration of 1 to 130 wt% relative to the amount of hyaluronic acid input.

4. In paragraph 1, A manufacturing method comprising at least one of a step of dissolving hyaluronic acid and ornithine in a buffer prior to the step (a); and a step of adding a cross-linking agent.

5. In paragraph 1, A manufacturing method further comprising at least one of a step of adding a basic solution between the steps (a) and (b) to decompose the ester bond between hyaluronic acids; a paste step; and a step of adding STMP.

6. In paragraph 1, (b) A manufacturing method further comprising at least one of a step of neutralizing by adding an acid solution after step; a dialysis step; a grinding step; a final concentration adjustment step; a mixing step; and a sterilization step.

7. In paragraph 4, A manufacturing method, wherein the cross-linking agent comprises at least one of an activator and a coupling reagent.

8. Cross-linked hyaluronic acid containing hyaluronic acid, ornithine and STMP.

9. In the 8th paragraph, the cross-linked hyaluronic acid is cross-linked hyaluronic acid capable of controlling the concentration of hyaluronic acid, ornithine and STMP according to the desired elasticity.

10. A tissue repair composition comprising the cross-linked hyaluronic acid of clause 9.

11. A filler composition comprising the cross-linked hyaluronic acid of clause 9.

12. A composition for bioadministration containing the cross-linked hyaluronic acid of clause 9.

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