KR102542329B1 - Pharmaceutical composition for anti-inflammation or anti-angiogenesis - Google Patents

Abstract

The present invention is for anti-inflammatory or anti-angiogenic use comprising at least one selected from the group consisting of sulfated hyaluronic acid having an average sulfated substitution rate of 200 to 300% for alcoholic hydroxyl groups per repeating unit of hyaluronic acid, derivatives and salts thereof. It relates to pharmaceutical compositions.

Description

Anti-inflammatory or anti-angiogenic pharmaceutical composition {PHARMACEUTICAL COMPOSITION FOR ANTI-INFLAMMATION OR ANTI-ANGIOGENESIS}

The present invention relates to a pharmaceutical composition for anti-inflammatory or anti-angiogenic use.

Hyaluronic acid (HA) is a non-sulfurized glycosaminoglycan, a natural polysaccharide in which D-glucuronic acid (GlcUA) and N-acetyl-D-glucosamine (GlcNAc) are linked in repeated sequences. The hyaluronic acid is present in various parts of the animal, such as vitreous body, cartilage, synovial fluid, placenta, and skin, and is in the form of mucopolysaccharide.

Hyaluronic acid is involved in absorbing moisture in the extracellular matrix or imparting flexibility between cells, and is involved in cell signaling, wound healing, and morphogenesis. In particular, hyaluronic acid inhibits the phagocytosis of macrophages to exhibit anti-inflammatory activity.

Because of these activities, hyaluronic acid is used as a treatment for arthritis, wound healing, and as an adjunct in eye and middle ear surgery. In addition, hyaluronic acid has excellent biocompatibility and its use in tissue engineering and regenerative medicine is gradually increasing.

On the other hand, sulfated hyaluronic acid is known to have anti-inflammation and anti-VEGF effects. However, anti-inflammatory and anti-VEGF effects can vary greatly depending on the sulfated substitution rate of sulfated hyaluronic acid, and since sulfated hyaluronic acid is not a natural substance, toxicity may appear depending on the substitution rate. In addition, sulfated hyaluronic acid is known to have blood anticoagulant activity similar to heparin, which may appear as an unintended side effect depending on the purpose of use. In order to use sulfated hyaluronic acid as a drug with actual efficacy, it is important that it has excellent anti-inflammatory and anti-angiogenic efficacy and does not show cytotoxicity, and it is important that it does not have blood anticoagulant activity to eliminate unintended side effects. Accordingly, it is necessary to develop a sulfated hyaluronic acid that exhibits anti-inflammatory and anti-VEGF effects and does not show cytotoxicity and blood anticoagulant activity.

Korean Patent Publication No. 2017-0120991

The present invention provides an anti-inflammatory or anti-angiogenic pharmaceutical composition comprising high-potency, low-toxic sulfated hyaluronic acid, which has excellent anti-inflammatory and anti-angiogenic efficacy and does not exhibit cytotoxicity and blood anticoagulant properties, derivatives and salts thereof. It's what you want to do.

The present invention provides an anti-inflammatory pharmaceutical composition comprising at least one selected from the group consisting of sulfated hyaluronic acid having an average sulfated substitution rate of 200 to 300% for alcoholic hydroxyl groups per repeating unit of hyaluronic acid, derivatives and salts thereof. do.

In addition, the present invention is an antiangiogenic agent comprising at least one selected from the group consisting of sulfated hyaluronic acid having an average sulfated substitution rate of 200 to 300% for alcoholic hydroxyl groups per repeating unit of hyaluronic acid, derivatives and salts thereof. A scientific composition is provided.

In addition, the present invention relates to an inflammatory eye disease or blood vessel comprising at least one selected from the group consisting of sulfated hyaluronic acid having an average sulfated substitution rate of 200 to 300% for alcoholic hydroxyl groups per repeating unit of hyaluronic acid, derivatives and salts thereof. It provides a pharmaceutical composition for the treatment or prevention of neoplastic eye diseases.

The present invention is a pharmaceutical agent for the treatment or prevention of arthritis comprising at least one selected from the group consisting of sulfated hyaluronic acid having an average sulfated substitution rate of 200 to 300% for alcoholic hydroxyl groups per repeating unit of hyaluronic acid, and derivatives and salts thereof. A scientific composition is provided.

In addition, the present invention provides an anticancer agent comprising at least one selected from the group consisting of sulfated hyaluronic acid having an average sulfated substitution rate of 200 to 300% for alcoholic hydroxyl groups per repeating unit of hyaluronic acid, derivatives and salts thereof.

According to the present invention, an anti-inflammatory or anti-angiogenic pharmaceutical composition comprising high-potency, low-toxic sulfated hyaluronic acid that exhibits excellent anti-inflammatory and anti-angiogenic efficacy and does not show cytotoxicity and blood anticoagulant properties, derivatives and salts thereof can provide.

The anti-inflammatory or anti-angiogenic pharmaceutical composition according to the present invention can be effectively used as a preventive or therapeutic agent for arthritis, eye diseases, etc., or as an anticancer agent.

1 is a 1H NMR result for confirming the sulfated substitution of sulfated hyaluronic acid (SHA).
Figure 2 is a graph for evaluating the anti-inflammatory efficacy of the sulfated hyaluronic acid of Examples 3 to 4 and Comparative Examples 1 to 4.
Figure 3 is a graph of anti-inflammatory efficacy evaluation according to the molecular weight of the sulfated hyaluronic acid of the present invention.
Figure 4 is a graph showing the degree of MMP13 secretion upon treatment with sulfated hyaluronic acid in Example 1 and Comparative Examples 1 and 3-4.
5 is an optical micrograph for observing the effect of inhibiting tube formation when a VEGF-treated sample of HUVEC cells is treated with sulfated hyaluronic acid.
6 is a graph evaluating the cell proliferation inhibitory effect of HUVEC cells upon treatment with sulfated hyaluronic acid of Examples 2 to 4 and Comparative Examples 1, 3, and 5.
7 is a graph for evaluating the cytotoxicity of sulfated hyaluronic acid in Examples 3 and 4 and Comparative Examples 1 and 4.

Hereinafter, the present invention will be described in more detail to aid understanding of the present invention. At this time, the terms or words used in this specification and claims should not be construed as being limited to ordinary or dictionary meanings, and the inventor appropriately defines the concept of terms in order to explain his/her invention in the best way. It should be interpreted as meaning and concept consistent with the technical idea of the present invention based on the principle that it can be done.

The present invention provides an anti-inflammatory pharmaceutical composition comprising at least one selected from the group consisting of sulfated hyaluronic acid having an average sulfated substitution rate of 200 to 300% for alcoholic hydroxyl groups per repeating unit of hyaluronic acid, derivatives and salts thereof. do. In addition, the present invention is an antiangiogenic agent comprising at least one selected from the group consisting of sulfated hyaluronic acid having an average sulfated substitution rate of 200 to 300% for alcoholic hydroxyl groups per repeating unit of hyaluronic acid, derivatives and salts thereof. A scientific composition is provided.

Conventional sulfated hyaluronic acid, derivatives and salts thereof are known to have anti-inflammatory and anti-VEGF effects, but studies on anti-inflammatory, anti-VEGF effects and toxicity according to the sulfated substitution rate and molecular weight have been lacking, and heparin and the like It is also known that it has a blood anticoagulant effect, so there was a problem that unintended side effects may occur during actual use. Therefore, in the present invention, by setting the average degree of sulfated substitution per repeating unit of hyaluronic acid to be 200 to 300%, it exhibits high efficacy and low toxicity in anti-inflammatory and anti-angiogenesis, and does not exhibit blood anticoagulant activity. . Such high-efficiency and low-toxicity anti-inflammatory, anti-angiogenic pharmaceutical composition can be effectively used as a preventive or therapeutic agent for arthritis, a preventive or therapeutic agent for eye diseases, or an anticancer agent.

The sulfated hyaluronic acid means that at least one alcoholic hydroxyl group of hyaluronic acid is substituted by sulfate, and the sulfated hyaluronic acid derivative means chemically modified sulfated hyaluronic acid or substituted sulfated hyaluronic acid. . For example, the sulfated hyaluronic acid derivative is a sulfated hyaluronic acid-poloxamer derivative, a sulfated hyaluronic acid-polyethylene glycol derivative, a sulfated hyaluronic acid-(CH ₂ ) _x -CH ₃ derivative (x is an integer from 1 to 20) ), sulfated hyaluronic acid-(CH ₂ ) _y -NH ₂ derivative (y is an integer from 1 to 20), sulfated hyaluronic acid-benzyl ester derivative, sulfated hyaluronic acid-chitosan derivative, sulfated hyaluronic acid-PLGA derivative , Sulfated hyaluronic acid-gelatin or sulfated hyaluronic acid-collagen derivatives. The sulfated hyaluronate salt is, for example, sulfated sodium hyaluronate, sulfated potassium hyaluronate, sulfated calcium hyaluronate, sulfated magnesium hyaluronate, sulfated zinc hyaluronate, sulfated cobalt hyaluronate or sulfated hyaluronate It may consist of one or more selected from tetrabutylammonium ronate. Alternatively, the sulfated hyaluronic acid may be represented by, for example, the following chemical formula.

[chemical formula]

In the above formula, R ₁ and R ₂ may each independently be SO ₃ H or H, and n is an integer of 1 or greater.

Hereinafter, for convenience, unless otherwise indicated, sulfated hyaluronic acid refers to both sulfated hyaluronic acid and its derivatives and salts.

Hyaluronic acid has a total of 4 -OH groups, including 1 primary -OH group and 3 secondary -OH groups per repeating unit. The primary -OH group undergoes sulfate substitution first, and as the reaction progresses, the secondary -OH group can undergo sulfate substitution. When all -OH groups in hyaluronic acid are sulfated substitution, the total sulfated substitution rate can be 400%. At this time, having a sulfated substitution rate of 100% or more means that after the highly reactive primary -OH group is sulfated substituted, the secondary -OH group is also sulfated substituted. The present invention relates to sulfated hyaluronic acid having an average sulfated substitution rate of 200 to 300% per repeating unit, that is, sulfated hyaluronic acid in which an average of 2 to 3 of the 4 alcoholic hydroxyl groups per repeating unit of the hyaluronic acid is sulfated substituted. will be. When the average sulfated substitution rate of the sulfated hyaluronic acid was less than 200%, the anti-inflammatory and antiangiogenic efficacy was significantly insufficient, and when it exceeded 300%, cytotoxicity occurred. Specifically, the sulfated substitution rate is 210 to 300%, 210 to 280%, 210 to 260%, 220 to 300%, 220 to 280%, 220 to 260%, 240 to 300%, 240 to 280%, or 240%. to 260%.

The sulfated hyaluronic acid or its derivative or salt may have a weight average molecular weight (Mw) of 500 to 3,000 kDa. More preferably, it may be 1,000 to 2,500 kDa, and more preferably, it may be 1,400 to 2,200 kDa. Sulfated hyaluronic acid, derivatives or salts thereof within the above weight average molecular weight range have excellent anti-angiogenic efficacy, almost no blood anticoagulant reaction, have a long residence time in the body, are suitable for use in actual medicines, and do not have too high a viscosity. may be appropriate for production and use.

As such, the sulfated hyaluronic acid having an average sulfated substitution rate per repeating unit of 200 to 300% according to the present invention has excellent anti-inflammatory or anti-angiogenic efficacy. In addition, it does not exhibit cytotoxic and blood anticoagulant properties. Accordingly, the pharmaceutical composition containing the sulfated hyaluronic acid according to the present invention can be effectively used as a preventive or therapeutic agent for arthritis, an ocular disease preventive or therapeutic agent, or an anticancer agent.

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

In addition, the present invention may provide a method for preventing or treating arthritis in a mammal by administering the pharmaceutical composition containing the sulfated hyaluronic acid. Representative examples that can be treated through the pharmaceutical composition containing the sulfated hyaluronic acid of the present invention include the treatment of osteoarthritis or rheumatoid arthritis, or gout or calcium pyrophosphate deposition disease It can also be used in other inflammatory arthritis, such as calcium pyrophosphate dihydrate deposition disease, or in reducing or preventing adhesions that can form after surgical procedures.

In addition, a method for preventing or treating inflammatory eye diseases and angiogenic eye diseases by administering the pharmaceutical composition containing the sulfated hyaluronic acid of the present invention can be provided. As a representative example, it can be used for inflammatory or angiogenic eye diseases such as dry eye syndrome, uveitis, macular degeneration, and diabetic retinopathy.

The pharmaceutical composition comprising the sulfated hyaluronic acid of the present invention may be particularly useful for treating chronic or acute inflammation.

As used herein, “treatment” refers to stopping, delaying, or mitigating the progression of a disease when used on an object showing symptoms of disease, and “prevention” means to prevent symptoms when used on an object at high risk but does not show symptoms of disease. It means to stop, delay or alleviate.

The following examples are presented to provide those skilled in the art with a complete disclosure and explanation of how the compounds, compositions, and methods provided herein are made and evaluated, and are intended to be purely illustrative. Accordingly, the 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, e.g., component concentrations, desired solvents, solvent mixtures, temperatures, pressures, and other reaction parameters and conditions that may be used to optimize product properties such as purity, yield, etc. . These are also considered within the scope of this application. Any combination of the elements described above in all possible variations is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[Example 1]

After dissolving the HA-TBA derivative in which tetrabutyl ammonium salt was introduced into hyaluronic acid (HA) in dimethylformamide (DMF), an organic solvent, at a concentration of 3 mg/mL, sulfur trioxide pyridine complex complex) was added in an amount of 25 moles of the HA-TBA derivative monomer, reacted with nitrogen gas for 1 hour at 5 ° C, and then purified through ethanol precipitation to obtain 200% sulfated substituted hyaluronic acid.

[Example 2]

Sulfur trioxide pyridine complex was added at 30 mole times the HA-TBA derivative monomer, followed by nitrogen gas and reaction at 5℃ for 0.5 hour, followed by purification through ethanol precipitation to achieve 210% sulfuration substitution The same procedure as in Example 1 was performed except that the sulfated hyaluronic acid was obtained.

[Example 3]

After adding 20 moles of the HA-TBA derivative monomer, sulfur trioxide pyridine complex was reacted with nitrogen gas for 2 hours at 5℃, and then purified through ethanol precipitation to achieve 240% sulfuration substitution The same procedure as in Example 1 was performed except that the sulfated hyaluronic acid was obtained.

[Example 4]

After adding 30 moles of the HA-TBA derivative monomer, sulfur trioxide pyridine complex was reacted for 3 hours at 5℃ with nitrogen gas, and then purified through ethanol precipitation to achieve 280% sulfuration substitution The same procedure as in Example 1 was performed except that the sulfated hyaluronic acid was obtained.

[Comparative Example 1]

Sulfur trioxide pyridine complex was added at 10.6 mole times the HA-TBA derivative monomer, reacted with nitrogen gas for 2 hours at 5℃, and purified through ethanol precipitation to achieve 110% sulfuration substitution The same procedure as in Example 1 was performed except that the sulfated hyaluronic acid was obtained.

[Comparative Example 2]

Sulfur trioxide pyridine complex was added at 18 mole times the HA-TBA derivative monomer, reacted with nitrogen gas for 2 hours at 5℃, and purified through ethanol precipitation to achieve 180% sulfuration substitution The same procedure as in Example 1 was performed except that the sulfated hyaluronic acid was obtained.

[Comparative Example 3]

Sulfur trioxide pyridine complex was added in an amount of 50 moles of the HA-TBA derivative monomer, reacted with nitrogen gas for 3 hours at 5 ° C, and then purified through ethanol precipitation to achieve 320% sulfuration substitution The same procedure as in Example 1 was performed except that the sulfated hyaluronic acid was obtained.

[Comparative Example 4]

Sulfur trioxide pyridine complex was added 100 mole times the HA-TBA derivative monomer, reacted with nitrogen gas for 3 hours at 5℃, and purified through ethanol precipitation to achieve 370% sulfuration substitution The same procedure as in Example 1 was performed except that the sulfated hyaluronic acid was obtained.

[Comparative Example 5]

After adding 16 moles of the HA-TBA derivative monomer, sulfur trioxide pyridine complex was reacted for 2 hours at 5℃ with nitrogen gas, and then purified through ethanol precipitation to achieve 150% sulfuration substitution The same procedure as in Example 1 was performed except that the sulfated hyaluronic acid was obtained.

[Experimental Example 1: Confirmation of Sulfation Substitution Rate]

It was confirmed that the sulfated hyaluronic acid (SHA) prepared in Examples 1 to 4 and Comparative Examples 1 to 5 was synthesized through 1H NMR analysis, and the sulfated substitution rate was confirmed through EA (elemental analysis) and the molecular weight was confirmed through MALLS/RI analysis. The results are shown in Figure 1 (NMR analysis results) and Table 1 (EA and MALLS/RI analysis results).

Figure 1 is the result of NMR analysis for Example 1. Referring to Figure 1, as sulfation progresses, the peak of 3.4ppm disappears, and the peak between 4.1-4.3ppm appears. It was confirmed that a sulfated group was introduced.

Sulfation substitution rate (%) Molecular Weight (kDa) Example 1 200 1,911 Example 2 210 1,730 Example 3 240 2,142 Example 4 280 1,922 Comparative Example 1 110 1,628 Comparative Example 2 180 1,969 Comparative Example 3 320 1,992 Comparative Example 4 370 1,937 Comparative Example 5 150 2,731

Referring to Table 1, it was confirmed that the sulfated hyaluronic acid of Examples 1 to 4 had a sulfated substitution rate in the range of 200 to 300%, and the sulfated hyaluronic acid of Comparative Examples 1 to 5 had a sulfated substitution rate outside the above range. did

[Experimental Example 2: Evaluation of anti-inflammatory efficacy]

Using the sulfated hyaluronic acid (SHA) prepared in Examples 3 to 4 and Comparative Examples 1 to 4, the anti-inflammatory efficacy of two types of cells, chondrocyte and fibroblast-like synoviocyte (FLS), was evaluated.

Chondrocytes and FLS two types of cells were seeded in 24 wells at 5x10 ⁴ and 2x10 ⁴ cells/well, respectively, and treated with 5ng/ml and 2ng/ml of TNF-alpha to activate the cells overnight. After that, the medium was replaced and each sample was treated with 1 mg/ml. After sample treatment and further incubation for 24 hours, the amount of cytokine secretion of cells in the medium was measured using ELISA, and the results are shown in FIG. 2 .

Referring to FIG. 2, it was confirmed that the higher the sulfated substitution rate in both chondrocytes and FLS cells, the better the secretion inhibitory effect of IL-6 and IL-8 cytokines. In the case of Comparative Examples 1 and 2 with a substitution rate of 100%, the anti-inflammatory efficacy was similar to that of HA, but in the case of Examples 3 and 4 and Comparative Examples 3 and 4, which had a substitution rate of 200% or more, both cells had IL-6 and The secretion of IL-8 was inhibited.

In order to confirm the efficacy according to molecular weight in addition to the sulfated substitution rate, the same cell experiment as above was performed using samples in which only the molecular weight was adjusted to be small by thermal decomposition of Examples 3 and 4. The results are shown in Figure 3.

Referring to FIG. 3, at a level of 240-280% sulfated substitution rate, similarly excellent anti-inflammatory efficacy was shown at a molecular weight level of 300-2000 kDa.

In addition to IL-6 and IL-8, the level of secretion of MMP13, a factor related to inflammation, was confirmed by ELISA using the cell medium samples. The results are shown in FIG. 4 .

Referring to FIG. 4, in the case of MMP13, Example 1 with a substitution rate of 200% showed excellent inhibitory efficacy, but in Comparative Examples 3 and 4 with a substitution rate of 300% or more, secretion tended to increase.

[Experimental Example 3: Evaluation of anti-angiogenic efficacy]

Tube formation and proliferation inhibitory effects of HUVEC cells were confirmed.

In order to confirm the angiogenesis inhibitory effect of HUVEC cells, VEGF-treated HUVEC cells were subjected to 3D culture using Matrigel, while sulfated hyaluronic acid samples (Examples 2-3, Comparative Examples 1, 4) was confirmed through microscopic images to determine whether or not tube formation was suppressed when treated. In addition, VEGF-treated HUVEC cells were seeded in 96 wells coated with matrigel, and then treated with the anti-VEGF drug Avastin as a comparative drug for sulfated hyaluronic acid. After incubation for 6 hours, the tube The degree of formation of was confirmed through an optical microscope. The results are shown in FIG. 5 .

Referring to FIG. 5, in the Avastin and hyaluronic acid (HA) treated group, vascular structures were generated at a level similar to that of the control group, but blood vessels were formed in Examples 2 to 3 and Comparative Example 4 in which the sulfate substitution rate was 200% or more. It was found that this rarely happened. However, in the case of Comparative Example 1 having a sulfated substitution rate of less than 200%, a vascular structure was formed.

In order to confirm the proliferation inhibitory effect of HUVEC cells, using a sample group treated with or without VEGF, FGF, and FBS, respectively, as a control, sulfated hyaluronic acid (Examples 2 to 4, Comparative Examples 1, 3, and 5 ) and the anti-VEGF drug, Avastin, was confirmed to inhibit cell proliferation after treatment at each concentration.

HUVEC cells were treated with 7,500 cells/well in 96 wells, and then VEGF was treated with 100 ng/ml, and then each sulfated hyaluronic acid and Avastin sample was treated by concentration. While incubating for 72 hours in Incucyte equipment, the number of cells (cell population) was confirmed. The results are shown in Figures 6 and 7.

Referring to FIG. 6, in the case of the sample treatment group of Examples 2 to 4, cell proliferation tended to be inhibited depending on the concentration, and at high concentrations, the proliferation of HUVEC cells was suppressed to the level of the negative control group that was not treated with VEGF, FGF, and FBS. suppressed. In the case of the anti-VEGF drug, Avastin, even at a relatively low concentration, cell proliferation was inhibited to the level of the control group without VEGF. In the case of the sulfated hyaluronic acid of Examples 2 to 4, a high concentration is required, but the cell proliferation inhibitory effect is superior to that of Avastin by blocking various growth factors. In the case of unsulfated hyaluronic acid, it did not show a cell proliferation inhibitory effect, but rather showed a cell proliferation effect at a high concentration. In the case of Comparative Example 1, the cell confluence appeared at a level similar to that of the VEGF-treated group even at high concentrations, and the effect of inhibiting the proliferation of HUVEC cells was insufficient. In the case of Comparative Example 5, in order to inhibit the proliferation of HUVEC cells, a higher concentration was required than in Examples 2-4, so the cell proliferation inhibitory effect was lower. In the case of Comparative Example 3, cells did not proliferate even at low concentrations.

[Experimental Example 4: Evaluation of cytotoxicity]

The cytotoxicity of two types of cells, HUVEC and chondrocyte, was evaluated using the sulfated hyaluronic acid (SHA) prepared in Examples 3 and 4 and Comparative Examples 1 to 4, respectively.

After seeding two types of cells, HUVEC and chondrocytes, in 96 wells at 7500 cells/well, each sample was treated with 1 mg/ml. While incubating for 70 hours, cell populations were identified using Incucyte equipment. The results are shown in Figure 8.

Referring to FIG. 7 , in the sample groups treated with Examples 3 and 4 and Comparative Examples 1 and 2, cell proliferation rate may be affected, but toxicity is not observed. However, the sample groups treated with Comparative Examples 3 and 4 showed cytotoxicity. Therefore, in order to develop sulfated hyaluronic acid as a therapeutic agent, the substitution rate needs to be 300% or less.

[Experimental Example 5: Evaluation of blood anticoagulation]

A blood anticoagulant test was performed using the sulfated hyaluronic acid of Examples 2 to 3 and Comparative Examples 1, 3, and 4. In particular, the sample of Example 2 was heat-treated to prepare sulfated hyaluronic acid (SHA) with molecular weights of 1,700 kDa, 1,000 kDa, 400 kDa, and 200 kDa, and the effect of molecular weight was also evaluated. Using each of the sulfated hyaluronic acids, the degree of activity inhibition of Factor IIa and Factor Xa was measured by a chromogenic assay. The results are shown in Table 2.

Anti-IIa activity [IU/mg] Anti-Xa activity [IU/mg] Heparin 209 209 Comparative Example 1 0.00 0.05 Example 3 0.13 0.17 Comparative Example 3 0.42 0.29 Comparative Example 4 0.42 0.42 Example 2 1,700 kDa 0.06 0.21 Example 2 1,000 kDa 0.09 0.17 Example 2 400 kDa 0.22 0.17 Example 2 200 kDa 0.22 0.16

Referring to Table 2 and FIG. 8, it can be seen that the Factor IIa and Factor Xa inhibitory activities of sulfated hyaluronic acid are more than 500 times lower than that of heparin. In addition, compared to Comparative Examples 3 and 4 having a high sulfate substitution rate, Factor IIa and Factor Xa inhibitory activities of Example 3 were significantly lower, and it was confirmed that the anticoagulant reaction hardly occurred in Example 3. In addition, in the case of Example 2, it was confirmed that Factor IIa and Factor Xa inhibitory activities were further lowered when the molecular weight was 1,000 kDa or more.

Claims (9)

delete delete An anti-angiogenic pharmaceutical composition comprising at least one selected from the group consisting of sulfated hyaluronic acid having an average sulfated substitution rate of 200 to 300% for alcoholic hydroxyl groups per repeating unit of hyaluronic acid and salts thereof.
According to claim 3,
At least one or more selected from the group consisting of the sulfated hyaluronic acid and salts thereof, the weight average molecular weight (Mw) of 500 to 3,000 kDa anti-angiogenic pharmaceutical composition.
delete delete A pharmaceutical composition for the treatment or prevention of angiogenic eye disease comprising at least one selected from the group consisting of sulfated hyaluronic acid having an average sulfated substitution rate of 200 to 300% for alcoholic hydroxyl groups per repeating unit of hyaluronic acid and salts thereof. composition.
According to claim 7,
The composition is a pharmaceutical composition for the treatment or prevention of dry eye syndrome, macular degeneration or diabetic retinopathy.
An anticancer agent comprising at least one selected from the group consisting of sulfated hyaluronic acid and salts thereof having an average sulfated substitution ratio of 200 to 300% for alcoholic hydroxyl groups per repeating unit of hyaluronic acid.

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