TWI782232B - Sulfated hyaluronic acid-based hydrogel and pharmaceutical composition comprising same - Google Patents

Abstract

The present invention relates to a sulfated hyaluronic acid-based hydrogel formed by reacting -COOH group of a crosslinking monomer including at least one selected from the group consisting of sulfated hyaluronic acid, derivatives and salts thereof, with a crosslinking agent.

Description

Hydrogel based on sulfated hyaluronic acid and pharmaceutical composition containing it

The present invention relates to a hydrogel based on sulfated hyaluronic acid and a pharmaceutical composition containing it.

Hyaluronic acid is a polysaccharide molecule with considerable viscoelastic properties. Hyaluronic acid exists in the joint cavity as a basic component of synovial fluid, acts as a lubricant and shock absorber in the joint cavity, and protects chondrocytes from inflammatory cytokines (Asari et al., Arch Histol Cytol, 1995, 58:65-76; Brun et al., Osteoarthr Cartil, 2003, 11:208-16; Stove et al., J Orthop Res, 2002, 20:551-5). Hyaluronic acid (by itself or in a derived form) has long been used as a "viscosupplement" or lubricant in the treatment of degenerative osteoarthritis.

Sulfated hyaluronic acid and its derivatives are known for their anti-inflammatory, anti-VEGF effects. However, in order for sulfated hyaluronic acid to be used as a drug with practical utility, it should have a high residual rate in the body and physical properties suitable for use. The previously known sulphated hyaluronic acid polymer solutions have a disadvantage: their rapid degradation in the body and their physical properties are not easily controlled according to the use.

Therefore, there is a need to develop materials based on sulfated hyaluronic acid, which exhibit anti-inflammatory and anti-VEGF effects and are resistant to degradation, so that their effects will last for a long time in the body and their physical properties can be controlled for each use. [Patent prior art] WO2002-032407

[technical problem]

The present invention provides a novel sulfated hyaluronic acid-based hydrogel for prolonging the effectiveness of sulfated hyaluronic acid in vivo and controlling the physical properties required for its respective use.

In addition, the present invention provides an anti-inflammatory or anti-angiogenic pharmaceutical composition containing sulfated hyaluronic acid-based hydrogel as an active ingredient. [Technical solution]

The present invention provides a hydrogel based on sulfated hyaluronic acid, which is formed by reacting the -COOH group of a crosslinking monomer with a crosslinking agent, the crosslinking monomer comprising sulfated hyaluronic acid, its derivatives and At least one member of the group consisting of salts.

In addition, the present invention provides an anti-inflammatory or anti-angiogenesis pharmaceutical composition, which includes the sulfated hyaluronic acid-based hydrogel as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for preventing or treating arthritis, a pharmaceutical composition for preventing or treating eye diseases, and an anticancer pharmaceutical composition, the pharmaceutical composition comprising the sulfated-based Hydrogel of hyaluronic acid. [Beneficial effect]

The hydrogel based on sulfated hyaluronic acid according to the present invention has anti-inflammatory or anti-angiogenic effects, and can inhibit its degradation in the body, so that the anti-inflammatory and anti-angiogenic effects last for a long time, and the gel can be easily controlled Regarding the physical properties of its use.

The sulfated hyaluronic acid-based hydrogel according to the present invention can be effectively used as an agent for preventing or treating arthritis, eye diseases, etc., and an anticancer agent.

Hereinafter, the present invention will be described in more detail to facilitate understanding of the present invention. Based on the principle that the inventor can properly define the concepts of terms to best explain his invention, the terms or words used in this specification and the scope of the patent application should not be construed as being limited to ordinary or dictionary meanings, and these terms or words It should be interpreted as meanings and concepts consistent with the technical idea of the present invention.

The present invention provides a hydrogel based on sulfated hyaluronic acid, which includes at least one member selected from the group consisting of sulfated hyaluronic acid, its derivatives and salts. The hydrogel based on sulfated hyaluronic acid of the present invention is formed by reacting the -COOH group of a cross-linking monomer with a cross-linking agent. at least one of the group.

Sulfated hyaluronic acid, its derivatives and salts are known for their anti-inflammatory, anti-VEGF effects, but polymer solutions including such sulfated hyaluronic acid, its derivatives and salts, etc. have a weakness: they are rapidly degraded in the body And its physical properties are not easy to control according to usage. Therefore, the present invention provides a hydrogel based on sulfated hyaluronic acid, which includes at least one selected from the group consisting of sulfated hyaluronic acid, its derivatives and salts, thereby inhibiting the degradation of sulfated hyaluronic acid in vivo to prolong its In vivo efficacy and the physical properties of the gel allow easy control of its use. The sulfated hyaluronic acid-based hydrogel according to the present invention has anti-inflammatory or anti-angiogenic effects, and can be effectively used as an agent for preventing or treating arthritis, eye diseases, etc., and an anticancer agent.

The sulfated hyaluronic acid means that at least one of the alcoholic hydroxyl groups of hyaluronic acid is substituted by sulfate, and the sulfated hyaluronic acid derivative means other chemically modified sulfated hyaluronic acid or substituted sulfated hyaluronic acid. hyaluronic acid. For example, the sulfated hyaluronic acid derivatives can be selected from sulfated hyaluronic acid-poloxamer derivatives, sulfated hyaluronic acid-polyethylene glycol derivatives, sulfated hyaluronic acid-(CH ₂ ) _n -CH ₃ derivatives, Composed of one or more of sulfated hyaluronic acid-benzyl derivatives, sulfated hyaluronic acid-polyglucosamine derivatives, sulfated hyaluronic acid-PLGA derivatives, sulfated hyaluronic acid-gelatin or sulfated hyaluronic acid-collagen derivatives. The sulfated hyaluronate may be composed of one or more selected from sulfated sodium hyaluronate, sulfated calcium hyaluronate, sulfated magnesium hyaluronate, sulfated zinc hyaluronate, sulfated cobalt hyaluronate or sulfated tetrabutylammonium hyaluronate. Alternatively, the sulfated hyaluronic acid can be represented by, for example, the following general formula:

[Mode]

Wherein, R ₁ and R ₂ can be independently SO ₃ H or H and n is an integer of 1 or greater.

The weight average molecular weight (Mw) of at least one selected from the group consisting of the sulfated hyaluronic acid, its derivatives and salts may be from 800 to 5,000 kDa. More preferably, it may be from 1,000 to 4,000 kDa and even better, from 1,200 to 3,000 kDa. By using sulfated hyaluronic acid, derivatives and salts thereof within the weight average molecular weight range, the reactivity with the crosslinking agent and the ease of preparing hydrogels can be improved, and the properties of the prepared hydrogels can be improved. physical properties.

Hereinafter, for convenience, unless otherwise indicated, the term "sulfated hyaluronic acid" refers to all sulfated hyaluronic acid, its derivatives and salts.

In the conventional sulfated hyaluronic acid, the primary -OH group, that is, the C6 position of hyaluronic acid is substituted by a sulfate group/radical, and therefore it is difficult to use the sulfated hyaluronic acid as a cross-linking monomer to cross-link each other with a cross-linking agent. There are known methods to form hydrogels based on sulfated hyaluronic acid.

Therefore, the present invention provides a novel hydrogel based on sulfated hyaluronic acid, which is formed by reacting the -COOH group of a cross-linking monomer with a cross-linking agent, the cross-linking monomer comprising sulfated hyaluronic acid, At least one of the group consisting of derivatives and salts thereof.

Herein, the cross-linking monomer can be at least one member selected from the group consisting of sulfated hyaluronic acid, derivatives and salts thereof, in which some or all of the alcoholic hydroxyl groups of hyaluronic acid are substituted by sulfate groups/roots . Hyaluronic acid has a total of four -OH groups, including three secondary -OH groups in addition to the primary -OH group. When the sulfate group/root substitution reaction is carried out, the highly reactive primary -OH group is first replaced by the sulfate group/root, and as the reaction proceeds, the secondary -OH group can be replaced by the sulfate group/root. When all -OH groups of hyaluronic acid are replaced by sulfate groups/radicals, the total substitution ratio can be 400 percent. According to an embodiment of the present invention, at least one cross-linking monomer selected from the group consisting of the sulfated hyaluronic acid, its derivatives and salts is one in which 30-400% of the alcoholic hydroxyl groups of the hyaluronic acid are Sulfate/radical substituents. In other words, like the present invention, a hyaluronic acid-based hydrogel using sulfated hyaluronic acid-COOH cross-linked uses -COOH groups instead of -OH groups for cross-linking, and thus, all primary -OH groups in which are sulfated Salt-substituted sulfated hyaluronic acid can be used as the cross-linking monomer, and furthermore, sulfated hyaluronic acid in which all primary-OH groups and secondary-OH groups are substituted with sulfate groups/radicals can be used. In addition, sulfated hyaluronic acid in which only some of the alcoholic hydroxyl groups of hyaluronic acid are substituted with sulfate groups/radicals may be used, and furthermore, sulfated hyaluronic acid in which only some of the primary -OH groups are replaced with sulfate groups/radicals may also be used. Regarding the sulfated hyaluronic acid according to one embodiment of the present invention, in view of safety, stability, anti-inflammatory effect, anti-angiogenic effect, physical properties, etc., it can be obtained by using sulfated hyaluronic acid having a substitution ratio suitable for various purposes. Hyaluronic Acid maximizes effectiveness and duration.

With respect to the sulfated hyaluronic acid-based hydrogel of the present invention, a diamine-based cross-linking agent can be used as a cross-linking agent. Regarding the sulfated hyaluronic acid-based hydrogel of the present invention, a diamine-based cross-linking agent is used as a cross-linking agent such that the -COOH group of the cross-linking monomer reacts with the cross-linking agent to form a hydrogel. The diamine-based crosslinking agent can be, for example, diaminobutane, diaminohexane, diaminooctane, adipate dihydrazide (ADH), polyethylene glycol diamine (PEG-diamine ), cystamine, etc. Optimally, diaminohexane can be used. Here, as a catalyst, a carbodiimide-based catalyst such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), 1-[3-(di Methylamino)propyl]-3-ethylcarbodiimide methiodide (1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide methiodide)), diisopropylcarbodiimide (DIC), N,N'-Dicyclohexylcarbodiimide (DCC), etc. In addition, to aid the activity of EDC, N-hydroxysuccinimide (NHS), sulfo-N-hydroxysuccinimide (sulfo-NHS), 1-hydroxybenzotriazole (HOBt) can be used ,wait. These catalysts can be used to react the -COOH group of sulfated hyaluronic acid with the _-NH2 of the crosslinker.

Thus, the sulfated hyaluronic acid-based hydrogel according to the present invention has anti-inflammatory or anti-angiogenic effects. In addition, the sulfated hyaluronic acid-based hydrogel according to the present invention can prolong the anti-inflammatory or anti-angiogenic effect by slowing down the degradation rate in vivo. Therefore, the sulfated hyaluronic acid-based hydrogel according to the present invention can be effectively used as an agent for preventing or treating arthritis, eye diseases, an anticancer agent, and the like.

In addition, the present invention provides an anti-inflammatory or anti-angiogenesis pharmaceutical composition, which includes the sulfated hyaluronic acid-based hydrogel as an active ingredient. The pharmaceutical composition according to the present invention can be used for prevention or treatment of arthritis, eye diseases, and anticancer treatment.

The "pharmaceutical composition" may include the sulfated hyaluronic acid-based hydrogel of the present invention and other chemical components, such as diluents, carriers, and the like. Therefore, the pharmaceutical composition may include a pharmaceutically acceptable carrier, diluent, excipient or a combination thereof as required. A pharmaceutical composition facilitates the administration of a compound into an organism. Various methods exist for administering the compounds including, but not limited to, oral, injection, aerosol, parenteral, and topical administration.

Furthermore, the present invention provides a method of preventing or treating arthritis in a mammal by administering a sulfated hyaluronic acid-based hydrogel as an active ingredient. Herein, the hydrogel based on sulfated hyaluronic acid can preferably be administered as an injectable formulation. As representative examples, the sulfated hyaluronic acid based hydrogels of the present invention can be used in the treatment of osteoarthritis or rheumatoid arthritis, or other inflammatory arthritis such as gout or calcium pyrophosphate dihydrate deposition disease, or it can be used in Reduces or prevents adhesions that may form after surgical procedures. The hydrogels based on sulfated hyaluronic acid of the present invention are particularly useful in the treatment of chronic or acute inflammation.

As used herein, the term "treating" means stopping, delaying or ameliorating the progression of the disease in a subject exhibiting disease symptoms. The term "preventing" means stopping, delaying or ameliorating the signs of a disease in an individual at risk of exhibiting symptoms of the disease, even if he or she does not exhibit symptoms.

The following examples are presented to provide a complete disclosure and illustration of how to make and evaluate the compounds, compositions and methods provided herein to those of ordinary skill in the art, and are intended to be purely exemplary. Accordingly, these examples are in no way intended to limit the scope of what the inventors regard as their invention. There are many variations and combinations of reaction conditions, such as component concentrations, desired solvents, solvent mixtures, temperature, pressure, and other reaction parameters and conditions that can be used to optimize product characteristics (eg, purity, yield, etc.). These are also considered to be within the scope of this application. The invention encompasses any combination of all possible variations of the above-described elements unless otherwise indicated herein or otherwise clearly contradicted by context.

[Preparation Example 1] - Synthesis of Sulfated Hyaluronic Acid (Sulfated HA)

Hyaluronic acid (HA)-tetrabutylammonium salt (TBA) derivative was dissolved in dimethylformamide (DMF), which is an organic solvent, at a concentration of 3 mg/mL, where TBA was introduced together with the ion exchange resin In HA. 15 molar times the sulfur trioxide pyridine complex of the HA-TBA derivative was added thereto. After reacting at 5° C. for 2 hours under nitrogen, ethanol precipitation was performed to produce a purified sulfated hyaluronic acid derivative.

Example 1

The sulfated hyaluronic acid (SHA) prepared in Preparation Example 1 was dissolved in water to 6 wt%. Next, 100 mol parts of EDC, 100 mol parts of NHS, and 20 mol parts of diaminooctane were added to 100 mol parts of sulfated hyaluronic acid monomer, and allowed to react overnight at 40°C to prepare hydrogels.

[Experimental example 1: Identification of SHA and hydrogel based on sulfated hyaluronic acid]

The sulfate group/radical substitution degree of the sulfated hyaluronic acid (SHA) prepared in Preparation Example 1 was determined by 1H NMR analysis and elemental analysis. The sulfated hyaluronic acid-based hydrogel prepared in Example 1 was identified by 1H NMR analysis. The results are shown in Figures 1 and 2.

Referring to the NMR data of Fig. 1, from the fact that the peak at 3.4 ppm completely disappears from the data of Fig. 1, it is found that the sulfated hyaluronic acid (SHA) prepared in Preparation Example 1 contains all the primary -OH groups (i.e. hyaluronic acid's C6 position) are sulfated hyaluronic acid (sulfated HA) substituted by sulfate groups/radicals. In the sulfated hyaluronic acid-based hydrogel prepared in Example 1, it was found from the data in Figure 2 that the peak of the cross-linking agent appeared between 1.0 and 1.6 ppm. Thus, it was confirmed that a hydrogel based on sulfated hyaluronic acid was produced.

Example 2

A sulfated hyaluronic acid-based hydrogel was prepared in the same manner as in Example 1, except that diaminohexane was used instead of diaminooctane as the diamine-based crosslinking agent.

Comparative example 1

The sulfated hyaluronic acid (SHA) prepared in Preparation Example 1 was dissolved in NaOH to 20% by weight, and then 1,4 - Butanediol Diglycidyl Ether (BDDE) and allowed to react overnight at 30°C. However, Comparative Example 1, which used sulfated hyaluronic acid (sulfated HA) in which all C6 positions were substituted with sulfate groups/radicals as a crosslinking monomer and BDDE as a crosslinking agent, did not cause hydrogel formation, and even in After the reaction was complete, it still resulted in a solution and thus could not be analyzed.

[Experimental Example 2: Measurement of Viscoelasticity of Hydrogel]

The viscoelasticity of the sulfated hyaluronic acid-based hydrogels prepared in Examples 1 to 2 was measured with an MCR 302 rheometer equipped with a 25 mm steel plate at 25°C, 4% strain, and 2.5 Hz. In addition, the sulfated hyaluronic acid-based hydrogels prepared in Examples 1 to 2 were autoclaved at 121° C. for 10 minutes in an autoclave, and then changes in viscoelasticity were measured. The results are shown in Table 1.

Table 1 Before sterilization After sterilization G'(Pa) G''(Pa) G'(Pa) G''(Pa) Example 1 551 132 221 47 Example 2 679 197 240 63

Referring to Table 1 above, it was found that the sulfated hyaluronic acid-based hydrogels prepared in Examples 1 to 2 can be prepared to have a wide range of viscoelasticity ranging from hard to soft physical properties according to the application purpose. Furthermore, it was found that the sulfated hyaluronic acid-based hydrogels prepared in Examples 1 to 2 could have the desired viscoelastic range even after terminal autoclaving, if necessary.

[Experimental Example 3: Evaluation of Enzyme Resistance]

The enzyme resistance of the SHA-COOH (SCO) gel of Example 2 was compared with hyaluronic acid-COOH hydrogel (HA-COOH gel) and shown in FIG. 3 .

1 g of each hydrogel was mixed with 10 ul of a 500 unit/g solution of hyaluronidase, which is a hyaluronan-degrading enzyme. Next, viscoelasticity was measured with an MCR 302 rheometer equipped with a 25 mm steel plate at 37° C., 4% strain, and 2.5 Hz. The results are shown in Figure 3.

Referring to Figure 3, the hyaluronic acid-COOH hydrogel (HA-COOH gel) prepared under the same conditions degrades rapidly over time. Meanwhile, in the SHA-COOH gel of Example 2, it was found that deterioration of physical properties due to degradative enzymes was significantly improved.

[Experimental Example 4: Evaluation of Cytotoxicity]

Cytotoxicity of two types of cells FLS and chondrocytes was evaluated using the SHA-COOH (SCO) gel of Example 1, hyaluronic acid (HA) and sulfated hyaluronic acid (SHA).

Two types of cells, FLS and chondrocytes, were seeded in 96 wells at concentrations of ^8.0x103 and ^3.4x104 cells/well, respectively, and then grown overnight and treated with 1 mg/ml of each sample. Cell viability was measured by resazaurin assay after sample treatment and incubation for an additional 24 hours. The results are shown in Figure 4.

Referring to Figure 4, all samples of the SHA-COOH (SCO) gel, hyaluronic acid (HA) and sulfated hyaluronic acid (SHA) of Example 1 resulted in both cellular FLS and 80% or greater viability of chondrocytes. Therefore, all samples were found to be non-cytotoxic.

[Experimental Example 5: Determination of HUVEC Antiangiogenic Effect]

The angiogenesis (tube formation) lines resulting from 3D culture using Matrigel of VEGF-treated and non-VEGF-treated HUVEC cell sample groups are shown in FIG. 5 .

In the VEGF-treated group/non-VEGF-treated group, HUVEC cells were seeded in Matrigel-coated 96-wells, and after 16 hours of culture, the formation of blood vessels (tubes) was confirmed with an optical microscope. Furthermore, in order to evaluate the effect of each sample on inhibiting angiogenesis (tube formation), HUVEC cells were seeded in 96 wells coated with Matrigel, and then HUVEC cells were treated with a certain concentration of VEGF, followed by sulfation of Example 1. Hyaluronic acid hydrogel (SCO gel) treatment. After 16 hours of incubation, the formation of blood vessels (tubes) was observed with a light microscope. The results are shown in Figure 5.

Referring to Fig. 5, it was found that the treatment with the SCO gel of Example 1 hardly caused angiogenesis.

[Experimental Example 6: Anti-inflammatory Effect Evaluation]

The anti-inflammatory effect was evaluated in two types of cells, chondrocytes and FLS, using the sulfated hyaluronic acid-based hydrogel (SCO gel), hyaluronic acid (HA) and sulfated hyaluronic acid (SHA) of Example 1.

Two types of cells, chondrocytes and FLS, were seeded in 96 wells at ^3.4x104 and ^8.0x103 cells/well, respectively, and treated with 10 ng/ml and 1 ng/ml of TNF-α to activate the cells overnight, And then cells were treated with 5 mg/ml and 1 mg/ml of each sample. After 24 hours of incubation after sample treatment, the secretion of cytokines by the cells in the medium was measured by using ELISA. The results are shown in Figure 6.

Referring to FIG. 6, compared with the control group not treated with any sample, the sulfated hyaluronic acid-based hydrogel (SCO gel) of Example 1 reduced the secretion of IL-6 and IL-8 of chondrocytes to 60% or less, and reduced the secretion of IL-6 and IL-8 by FLS cells to 30% or less. Therefore, anti-inflammatory effect can be confirmed.

[Experimental Example 7: Evaluation of Anticoagulant Response]

Inhibition of Factor IIa and Factor Xa activity was measured by a chromogenic assay using the sulfated hyaluronic acid-based hydrogel (SCO gel), heparin, and sulfated hyaluronic acid (SHA) of Example 1. The results are shown in Table 2.

Table 2 Anti-IIa activity [unit/mg] Anti-Xa activity [IU/mg] heparin 209 209 SHA 0.13 0.17 Example 1 0.05 0.12

Referring to Table 2 above, all samples of sulfated hyaluronic acid (SHA) inhibited Factor IIa and Factor Xa activities 1200 times or more lower than heparin. In particular, the activities of the sulfated hyaluronic acid-based hydrogels (SCO gels) of Example 1 were all 1500 times or more lower than that of heparin. Therefore, it was confirmed that almost no anticoagulant reaction occurred.

[Fig. 1] 1H NMR results for identification of sulfated substitution of sulfated hyaluronic acid (SHA).

[ Fig. 2 ] 1H NMR results for identifying the sulfated hyaluronic acid-based hydrogel of the present invention.

[ Fig. 3 ] is a graph illustrating the evaluation of enzyme resistance of the sulfated hyaluronic acid-based hydrogel of the present invention.

[ Fig. 4 ] is a graph illustrating the evaluation of the cytotoxicity of the sulfated hyaluronic acid-based hydrogel of the present invention.

[Fig. 5] shows the observation of tube formation in VEGF-treated HUVEC cell samples and non-VEGF-treated HUVEC cell samples, and the effect of sulfated hyaluronic acid-based hydrogel (SCO gel) on VEGF-treated HUVEC cell samples Optical micrographs of the inhibitory effect of tube formation in .

[ Fig. 6 ] A graph illustrating the evaluation of the anti-inflammatory effect of the sulfated hyaluronic acid-based hydrogel of the present invention is shown.

Claims (8)

A hydrogel based on sulfated hyaluronic acid formed by reacting the -COOH group of a crosslinking monomer comprising a crosslinking agent selected from the group consisting of sulfated hyaluronic acid and its salts At least one of them, wherein the sulfated hyaluronic acid is represented by the following formula:

Where R ₁ and R ₂ are each independently SO ₃ H or H and n is an integer of 1 or greater provided that R ¹ and R ² are not H at the same time; 30-400% of the alcoholic hydroxyl groups of hyaluronic acid are via sulfate groups / root substitution; the weight average molecular weight (Mw) of at least one selected from the group consisting of sulfated hyaluronic acid and salts thereof ranges from 800 to 5,000 kDa; and the crosslinking agent is a diamine-based crosslinking agent, Wherein the cross-linking agent based on diamine is at least one selected from the following group: diaminobutane, diaminohexane, diaminooctane, polyethylene glycol diamine (PEG- diamine), and cystamine. The hydrogel based on sulfated hyaluronic acid as claimed in claim 1, which is used for anti-inflammation or anti-angiogenesis. A pharmaceutical composition comprising the sulfated hyaluronic acid-based hydrogel according to claim 1 as an active ingredient. The pharmaceutical composition as claimed in item 3 is used for treating arthritis. The pharmaceutical composition according to claim 3 is used for treating eye diseases, wherein the eye diseases are related to angiogenesis. The pharmaceutical composition according to claim 3 is used for anti-cancer, wherein the cancer is related to angiogenesis. A use of the hydrogel based on sulfated hyaluronic acid as claimed in claim 1 is used to manufacture medicine for treating arthritis in mammals. The pharmaceutical composition as claimed in item 3 is used for anti-inflammation or anti-angiogenesis.

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