KR20200055645A - Sulfated hyaluronic acid derivatives, method of preparing the same and pharmaceutical composition for preventing or treating musculoskeletal joint disease comprising the same - Google Patents

Abstract

The present invention relates to a sulfated hyaluronic acid derivative, a preparation method thereof, and a pharmaceutical composition for preventing or treating musculoskeletal joint disease comprising the sulfated hyaluronic acid derivative. According to the present invention, the sulfated hyaluronic acid derivative and the pharmaceutical composition for preventing or treating skeletal joint disease comprising the same have a buffering effect, and are expected to significantly improve the effect of treating arthritis in patients in clinical practices by controlling pain through relief of inflammation and inhibition of angiogenesis.

Description

Sulfated hyaluronic acid derivatives, method of preparing the same and pharmaceutical composition for preventing or treating musculoskeletal joint disease comprising the same}

The present invention relates to a sulfated hyaluronic acid derivative, a method for manufacturing the same, and a pharmaceutical composition for preventing or treating musculoskeletal joint disease comprising the sulfated hyaluronic acid derivative.

Hyaluronic acid is a linear polysaccharide composed of glucuronic acid and acetylglucosamine, which makes up the extracellular matrix (ECM), joint fluid (synovial fluid), and cartilage. It is one of the glycosamino-glycan (GAG) present in the support. Hyaluronic acid also plays an important role as a signaling molecule in cell motility, cell differentiation, wound healing and cancer metastasis. In terms of immunity, hyaluronic acid is a substance derived from living organisms, so it is widely used in cosmetic and tissue engineering and drug delivery systems because there is no problem of adverse immune reactions.

Hyaluronic acid has a three-dimensional structure in solution, which results in a wide range of internal hydrogen bonds, limited fluidity of the polymer chain, and unique helical and coiled coil structures. In particular, it is possible to form a hydrophilic polymer through cross-linking and absorb water in an amount in the range of several hundred times the dry weight. Hydrogels can be applied in various forms in the medical and pharmaceutical fields due to their excellent biocompatibility and hydrophilic properties. Therefore, hyaluronic acid having a high molecular weight or a pharmaceutically acceptable salt thereof as a therapeutic agent such as degenerative arthritis or rheumatoid arthritis has been clinically used as an injection in the joint cavity. Hyaluronic acid or its salt is usually in the form of a liquid injection, which is directly administered to the joints of the affected knee, shoulder, etc., and the visco-elasticity hyaluronic acid injected directly into the joint cavity is used to recover the lost cartilage tissue of the patient with arthritis. Instead, it has been reported to have a function of relieving shock during joint exercise and helping lubrication to reduce joint pain and normalize functions, as well as improve functional disorders and reduce pain caused by arthritis. Currently, cartilage injection using hyaluronic acid hydrogel with superior buffering effect has been developed to further improve the inflammation and pain of the joint, and is marketed for the treatment of joint disease by injecting it into the patient's joint (Sinobian Injection, LG Life Insurance) Science). These cartilage injections are made of a gel type through cross-linking of hyaluronic acid and have a high viscosity, thus exhibiting a buffering effect and alleviating degenerative arthritis. However, the effect of controlling inflammation associated with degenerative arthritis is almost insignificant, so there is no therapeutic effect in controlling arthritis and pain caused by it. In addition, hyaluronic acid hydrogel cartilage injection has the disadvantage that it is directly decomposed by matrix metalloproteinase in the joint cavity when injected into a highly inflamed joint.

       In addition, as another method of using hyaluronic acid, when a hydrophobic substituent is introduced into the carboxylic acid present in the hyaluronic acid monomer, it is possible to impart hydrophobicity to the hydrophilic properties of hyaluronic acid, thereby forming an amphiphilic molecular structure. Amphiphilic hyaluronic acid combined with hydrophobic groups can be manufactured in the form of nanoparticles (several nm to hundreds of nm) through the process of encapsulating hydrophobic or poorly soluble drugs, thereby reducing side effects such as anticancer drugs and maximizing the therapeutic effect. It has been reported to be used as a material.

On the other hand, United States Patent Application Publication (US 2004/0053885) the sulfate group to a hydroxyl group (-OH) present in the hyaluronic acid (SO ₃ ^-) as a result of ever applied to inflammatory rheumatoid arthritis by using the sulfated hyaluronic ronsanreul prepared by transformation, sulfated It has been reported by observing a remarkable effect of alleviating inflammation in comparison with unhyaluronic acid. In European Patent Publication (WO 96/24362), 40 kDa to 1,300 kDa sulfated hyaluronic acid has reported anti-inflammatory effects on inflammation from various causes, and the minimum molecular weight is 40 kDa and the maximum molecular weight is 1,300 kDa. There has been reported a significant improvement in anti-inflammatory effect compared to hyaluronic acid which is not sulfated. Korean Patent Registration No. 0591960 has reported the effect of inhibiting angiogenesis by antisulfuration of polysaccharides derived from natural products, and its use as an anti-inflammatory agent. Korean published patent (10-2009-0014359) reported oral and intra-articular preparations based on sulfated hyaluronic acid effective for treating degenerative osteoarthritis. However, in these prior arts, there has been no report on the anti-inflammatory effect, the inhibitory effect on angiogenesis, etc. according to the degree of sulfation of the four hydroxyl groups (-OH) contained in the monomer of hyaluronic acid. According to a recent report published in Biomaterials (January 2016, 130-138), when adjusting the degree of sulfation of four hydroxyl groups of hyaluronic acid, angiogenesis factors (VEGF165a), which are particularly involved in angiogenesis Methods for selectively inhibiting bays have been reported.

Therefore, when the hydroxyl group present in hyaluronic acid is modified with a sulfuric acid group and then prepared in the form of a hydrogel or nanoparticles, as described above, a buffering effect for the treatment of arthritis and an effect of simultaneously alleviating inflammation accompanying arthritis can be expected. Can be.

However, in order to prepare hyaluronic acid as a sulfated hyaluronic acid, a large amount of a salt containing SO ₃ ^- ions having strong acidity is added, and the hydroxyl group of hyaluronic acid undergoes a process of sulfation, so that hyaluronic acid is decomposed to have a molecular weight of about 1 It decreases to / 10, and the physical properties of hyaluronic acid change rapidly because the sulfuric acid group is a functional group having a higher polarity than the hydroxyl group. In particular, changes in water solubility in water, formation of negative charges, and rapid decrease in viscosity are expected.

Therefore, the sulfated hyaluronic acid is significantly different from the hyaluronic acid in which the physical properties are not sulfated. In particular, a hydrogel-type derivative of sulfated hyaluronic acid has not been reported because hydrophilicity increases rapidly and viscosity decreases rapidly, making it difficult to prepare hydrogel of sulfated hyaluronic acid. Furthermore, it is impossible to predict at all whether the angiogenic and anti-inflammatory effects observed in sulfated hyaluronic acid will be maintained due to chemical structural modifications.

In addition, even when a hydrophobic substituent is added to the sulfated hyaluronic acid to prepare nanoparticles (several tens to hundreds of nanometers), the sulfated hyaluronic acid in which the hydrophobic group is introduced is converted into nanoparticles due to the change in the physical properties of the sulfated hyaluronic acid. Manufacturing possibilities are unpredictable.

Therefore, the present inventors tried various crosslinking methods to overcome the above-mentioned problems, and as a result, adjusted the amount of crosslinking and the reaction equivalent through amide bonds using carboxylic acids present in the sulfated hyaluronic acid, and hydrogel sulfated hyaluronic acid. The present invention was completed by confirming that a viscosity of the same level as that of hyaluronic acid hydrogel without sulfate was obtained. In addition, as a result of confirming the treatment effect of the sulfated hyaluronic acid hydrogel in an animal experiment, it was confirmed that it has a remarkable arthritis treatment effect compared to the non-sulfated hyaluronic acid hydrogel.

In addition, the present invention was completed by confirming that it is possible to form spherical nanoparticles corresponding to tens to hundreds of nanometers when a hydrophobic substituent designated as cholanic acid (5 beta-cholanic acid) is added to sulfated hyaluronic acid.

It is an object of the present invention to provide a sulfated hyaluronic acid derivative capable of overcoming the low viscosity of sulfated hyaluronic acid and nanoparticles and a method for preparing the same.

In addition, an object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of musculoskeletal joint disease comprising the sulfated hyaluronic acid derivative.

The present invention to solve the above problems,

It is represented by the following [Formula 1], to provide a sulfated hyaluronic acid derivative, characterized in that to form a hydrogel by crosslinking between sulfated hyaluronic acid monomers:

[Formula 1]

In the above [Formula 1],

R ₁ to R ₄ or R ₅ to R ₈ are the same as or different from each other, and each independently hydrogen or SO ₃ ^- X ⁺ , wherein R ₁ to R ₄ Medium and R ₅ to R ₈ is at least one of SO ₃ ^-, and X ^+, wherein X is a cation,

n1 and n2 are each 1 or more,

The crosslinking between the sulfated hyaluronic acid monomers is that of at least one (m> 1) -CH ₂ group connected through an amide bond or a covalent bond of an acyclic material.

The present invention also, by adding and reacting an active ester forming reagent and a crosslinking agent to the sulfated hyaluronic acid monomer represented by the following [Formula 2] to activate the carboxyl group of the sulfated hyaluronic acid monomer represented by the following [Formula 2], It provides a method for producing a sulfated hyaluronic acid derivative comprising; inducing cross-linking between monomers to prepare the compound represented by [Formula 1] of claim 1;

[Formula 2]

In the above [Formula 2],

R ₁ to R ₄ are the same as or different from each other, and each independently hydrogen or SO ₃ ^- X ⁺ , wherein R ₁ to R ₄ At least one of them is SO ₃ ^- X ⁺ , X is a cation, n is an integer of 1 or more.

In this case, the active ester forming reagent may be at least one of N- (3-Dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) and 1-Hydroxybenzotriazole (HOBT).

In addition, the crosslinking agent may be hexamethyldiamine (HMDA), adipic dihydrizide, amine polyethylene glycol, or a mixture thereof.

The present invention is also represented by the following [Formula 3], and provides a sulfated hyaluronic acid derivative in which a hydrophobic substance is bound to the hyaluronic acid monomer:

[Formula 3]

In the above [Formula 3],

R ₁ to R ₄ are the same as or different from each other, and each independently hydrogen or SO ₃ ^- X ⁺ , wherein R ₁ to R ₄ Of at least one or more is SO ₃ ^-, and X ^+, wherein X is a cation, R ₆ is -CH ₂ and at least one ring or non-ring closure comprising a hydrophobic material, n and m are integers of 1 or more.

At this time, the hydrophobic material is 5-β cholanic acid (5-β cholanic acid), cholic acid (cholic acid), kenodeoxycholic acid (chenodeoxycholic acid), glycolic acid (glycocholic acid), taurocholic acid (taurocholic acid), de It may be one or more selected from the group consisting of deoxycholic acid, lithocholic acid and 7-oxo-lithocholic acid.

In addition, the sulfated hyaluronic acid derivative represented by [Formula 3] may be represented by the following [Formula 4]:

[Formula 4]

In the above [Formula 4],

R ₁ to R ₄ are the same as or different from each other, and each independently hydrogen or SO ₃ ^- X ⁺ , wherein R ₁ to R ₄ At least one of SO ₃ ^- X ⁺ , X is a cation, and n and m are integers of 1 or more.

In addition, the present invention is also a sulfuric acid hyaluronic acid monomer represented by the following [Formula 5] by adding and reacting an active ester forming reagent and a compound containing a ring or acyclic hydrophobic group containing at least one -CH ₂ group, sulfuric acid Activating the carboxyl group of the fluorinated hyaluronic acid monomer, and inducing the binding of the monomer and the hydrophobic group to prepare a compound represented by [Formula 3] of claim 4; provides a method for producing a sulfated hyaluronic acid derivative comprising:

[Formula 5]

In the above [Formula 5],

R ₁ to R ₄ are the same as or different from each other, and each independently hydrogen or SO ₃ ^- X ⁺ , wherein R ₁ to R ₄ At least one of them is SO ₃ ^- X ⁺ , X is a cation, m is an integer of 1 or more.

In this case, the active ester forming reagent may be at least one of N- (3-Dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) and 1-Hydroxybenzotriazole (HOBT).

In addition, the hydrophobic group is 5-β cholanic acid (5-β cholanic acid), cholic acid (cholic acid), kenodeoxycholic acid (chenodeoxycholic acid), glycolic acid (glycocholic acid), taurocholic acid (taurocholic acid), deoxy At least one member selected from the group consisting of deoxycholic acid, lithocholic acid, and 7-oxo-lithocholic acid, and the compound containing the hydrophobic group is additionally introduced with an amine group. May be

The present invention also provides a pharmaceutical composition for the prevention or treatment of musculoskeletal joint disease comprising the sulfated hyaluronic acid derivative.

According to the present invention, the sulfated hyaluronic acid derivative contained in the composition in the composition can inhibit inflammation by inhibiting angiogenesis by inhibiting angiogenesis factors.

In addition, according to the present invention, the sulfated hyaluronic acid derivative contained in the composition can relieve inflammation by inhibiting the secretion and cell proliferation of inflammatory cytokines and chemokines of synovial fibroblasts and immune cells.

According to the present invention, the musculoskeletal joint disease is degenerative arthritis (Osteoarthritis), rheumatoid arthritis, meniscus injury, adhesive capsulitis, tendonitis inflamed tendon (Tendinopathy) ), Gouty arthritis and other inflammatory arthritis.

The present invention can overcome the hydrophilicity and low viscosity of sulfated hyaluronic acid to provide a derivative that can be prepared in the form of hydrogels and nanoparticles and a method for manufacturing the same. In addition, the sulfated hyaluronic acid derivative according to the present invention and a pharmaceutical composition for the prevention or treatment of skeletal joint diseases comprising the same, not only have a buffering effect, but also control inflammation through suppression of inflammation and formation of new blood vessels, thereby realizing arthritis of patients in clinical practice. It is expected to greatly enhance the therapeutic effect.

1 schematically shows a method of synthesizing a hydrogel-type sulfated hyaluronic acid derivative according to the present invention, steps 1 and 2 of FIG. 1 show a process of synthesizing sulfated hyaluronic acid from hyaluronic acid, and step 3 Denotes a method for synthesizing a sulfated hyaluronic acid hydrogel (sulfated hyaluronic acid derivative according to the present invention).
Figure 2 shows the results of the Fourier transform infrared spectroscopy analysis to confirm the molecular structure of the sulfated hyaluronic acid derivative in the form of a hydrogel synthesized according to the present invention.
3 is a photograph showing a hyaluronic acid hydrogel and a process for preparing a sulfated hyaluronic acid hydrogel according to the present invention.
4 is a result of animal experiments showing the anti-inflammatory effect of hyaluronic acid hydrogels and sulfated hyaluronic acid hydrogels according to the present invention.
5 is an experimental result confirming the anti-inflammatory effect of hyaluronic acid hydrogel and rheumatoid arthritis synovial fibroblasts of sulfated hyaluronic acid hydrogel according to the present invention.
6 is an electron microscope image of nanoparticles prepared from a sulfated hyaluronic acid derivative in which a hydrophobic functional group is introduced according to the present invention.

Hereinafter, the present invention will be described in more detail.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which the present invention pertains. In general, the nomenclature used herein is well known and commonly used in the art.

As used herein, the term "composition" is considered to include any product that is made directly or indirectly by the combination of a particular component, as well as products that contain a particular component.

The term used in the present invention In the present invention, the term "prevention" refers to all actions to suppress or delay the development of arthritis by administration of the pharmaceutical composition according to the present invention, and "treatment" refers to the administration of the pharmaceutical composition By means all the actions of suspected arthritis and the symptoms of the affected individual are improved or beneficially altered.

In the present invention, the pharmaceutical composition may be characterized by containing a pharmaceutically acceptable carrier, said carrier being an ion exchange resin, alumina, aluminum stearate, lecithin, serum protein, buffer substance, water, salt, Characterized by one or more selected from the group consisting of electrolyte, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substrate, polyethylene glycol, sodium carboxymethylcellulose, polyarylate, wax, polyethylene glycol and wool paper Can be.

In the present invention, the pharmaceutical composition is formulated for intravenous, intraperitoneal, intramuscular, intraarterial, oral, intracardiac, intramedullary, intrathecal, transdermal, intestinal, subcutaneous, sublingual or topical administration. It can be characterized by further containing any one or more adjuvants selected from the group consisting of buffers, antimicrobial preservatives, surfactants, antioxidants, tonicity adjusting agents, preservatives, thickeners and viscosity modifiers, solutions, suspensions, emulsions , It may be characterized by having a formulation selected from the group consisting of gels and powders.

Suitable dosages of the pharmaceutical compositions of the present invention vary depending on factors such as severity of symptoms, patient's weight, age, sex, mode of administration and administration time, etc., and usually, a skilled doctor is effective for the desired treatment or prevention. The dosage can be easily determined.

The present invention is represented by the following [Chemical Formula 1], and provides a sulfated hyaluronic acid derivative characterized by forming a hydrogel by crosslinking between sulfated hyaluronic acid monomers:

[Formula 1]

In the above [Formula 1],

R ₁ to R ₄ or R ₅ to R ₈ are the same as or different from each other, and each independently hydrogen or SO ₃ ^- X ⁺ , wherein R ₁ to R ₄ Medium and R ₅ to R ₈ is at least one of SO ₃ ^-, and X ^+, wherein X is a cation,

n1 and n2 are each 1 or more,

The crosslinking between the sulfated hyaluronic acid monomers is that of at least one (m> 1) -CH ₂ group connected through an amide bond or a covalent bond of an acyclic material.

In addition, X may be any material in the cation state, but may preferably be a monovalent cation, more preferably Na.

As a specific example of the sulfated hyaluronic acid derivative, the sulfated hyaluronic acid derivative may be a compound represented by the following [Formula 6]:

[Formula 6]

.

.

The present invention also, by adding and reacting an active ester forming reagent and a crosslinking agent to the sulfated hyaluronic acid monomer represented by the following [Formula 2] to activate the carboxyl group of the sulfated hyaluronic acid monomer represented by the following [Formula 2], It provides a method for producing a sulfated hyaluronic acid derivative comprising; inducing cross-linking between monomers to prepare the compound represented by [Formula 1] of claim 1;

[Formula 2]

In the above [Formula 2],

R ₁ to R ₄ are the same as or different from each other, and each independently hydrogen or SO ₃ ^- X ⁺ , wherein R ₁ to R ₄ At least one of them is SO ₃ ^- X ⁺ , X is a cation, n is an integer of 1 or more.

In addition, X may be any material in the cation state, but may preferably be a monovalent cation, more preferably Na.

The specific production process of the sulfated hyaluronic acid derivative represented by [Formula 1] is as shown in [Reaction Scheme 1].

[Scheme 1]

Step 1 in [Scheme 1] is a step of making a carboxyl group of a sulfated hyaluronic acid monomer into an active ester, and step 2 is adding a reagent having an amine group at both ends to enable crosslinking, thereby producing sulfated hyaluronic acid. This is the step of preparing a hydrogel.

At this time, the active ester forming reagent used to form the active ester from the carboxyl group of the sulfated hyaluronic acid monomer is not necessarily limited thereto, but N- (3-Dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride (EDC), N It is preferred that at least one of -hydroxysuccinimide (NHS) and 1-Hydroxybenzotriazole (HOBT) be used.

In addition, the crosslinking agent may be applicable to any compound having an amine group at the terminal, but is preferably hexamethyldiamine (HMDA), adipic dihydrizide, amine polyethylene glycol, or a mixture thereof.

In addition, the step reaction and the two-step reaction of [Scheme 1] may be performed by separating each step, but it is preferable that the reaction proceeds regardless of the step by simultaneously adding a reagent that forms an active ester and a reagent that causes crosslinking. . For example, the reaction may be performed by sequentially adding HMDA, EDC, and HOBT reagents and stirring for 12 hours or more.

In [Reaction Scheme 1], it is preferable to add EDC at least 1 times as much as the weight of the sulfated hyaluronic acid monomer, and HOBT is preferably used in an amount corresponding to 70% of the EDC weight. HMDA can be used in the range of 10% to 20% by weight of the sulfated hyaluronic acid monomer, but is most preferably used in the range of 12 to 17%.

The present invention is also represented by the following [Formula 3], and provides a sulfated hyaluronic acid derivative in which a hydrophobic substance is bound to the hyaluronic acid monomer:

[Formula 3]

In the above [Formula 3],

R ₁ to R ₄ are the same as or different from each other, and each independently hydrogen or SO ₃ ^- X ⁺ , wherein R ₁ to R ₄ Of at least one or more is SO ₃ ^-, and X ^+, wherein X is a cation, R ₆ is -CH ₂ and at least one ring or non-ring closure comprising a hydrophobic material, n and m are integers of 1 or more.

In addition, X may be any material in the cation state, but may preferably be a monovalent cation, more preferably Na.

At this time, the hydrophobic material (substituent) is a hydrophobic cholanic acid, and includes primary, secondary and tertiary cholanic acids or derivatives thereof having cholanic acid as a basic skeleton, for example 5-β cholanic acid (5-β cholanic acid), cholic acid, kenodeoxycholic acid, glycolic acid, taurocholic acid, deoxycholic acid, lithocholic acid acid) and 7-oxo-lithocholic acid (7-oxo-lithocholic acid) may be one or more selected from the group consisting of.

In addition, the sulfated hyaluronic acid derivative represented by [Formula 3] may be represented by the following [Formula 4]:

[Formula 4]

In the above [Formula 4],

R ₁ to R ₄ are the same as or different from each other, and each independently hydrogen or SO ₃ ^- X ⁺ , wherein R ₁ to R ₄ At least one of SO ₃ ^- X ⁺ , X is a cation, and n and m are integers of 1 or more.

In addition, X may be any material in the cation state, but may preferably be a monovalent cation, more preferably Na.

In addition, the present invention is also a sulfuric acid hyaluronic acid monomer represented by the following [Formula 5] by adding and reacting an active ester forming reagent and a compound containing a ring or acyclic hydrophobic group containing at least one -CH ₂ group, sulfuric acid Activating the carboxyl group of the fluorinated hyaluronic acid monomer, and inducing the binding of the monomer and the hydrophobic group to prepare a compound represented by [Formula 3] of claim 4; provides a method for producing a sulfated hyaluronic acid derivative comprising:

[Formula 5]

In the above [Formula 5],

R ₁ to R ₄ are the same as or different from each other, and each independently hydrogen or SO ₃ ^- X ⁺ , wherein R ₁ to R ₄ At least one of them is SO ₃ ^- X ⁺ , X is a cation, m is an integer of 1 or more.

In addition, X may be any material in the cation state, but may preferably be a monovalent cation, more preferably Na.

The specific production process of the sulfated hyaluronic acid derivative represented by [Formula 3] is as shown in [Reaction Scheme 2].

[Scheme 2]

In [Scheme 2], step 1 is a step of making a carboxyl group of a sulfated hyaluronic acid monomer into an active ester, and step 2 is a sulfated hyaluron having a hydrophobic substituent bonded through an amide bond with a compound containing a hydrophobic group. This is a step for preparing a ronic acid derivative.

At this time, the active ester forming reagent used to form the active ester from the carboxyl group of the sulfated hyaluronic acid monomer is not necessarily limited thereto, but N- (3-Dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride (EDC), N It is preferred that at least one of -hydroxysuccinimide (NHS) and 1-Hydroxybenzotriazole (HOBT) be used.

In addition, the hydrophobic group is preferably a hydrophobic cholanic acid compound to impart hydrophobicity to the hydrophilic hyaluronic acid to form an autoaggregate in water to provide an amphiphilic complex, and the hydrophobic cholanic acid is based on cholanic acid. Skeletal primary, secondary and tertiary cholanic acid or derivatives thereof, and may include, for example, 5-β cholanic acid (5-β cholanic acid), cholic acid (cholic acid), kenodeoxycholic acid (chenodeoxycholic) group consisting of acid), glycolic acid, taurocholic acid, deoxycholic acid, lithocholic acid and 7-oxo-lithocholic acid It may be one or more selected from.

In addition, as shown in [Reaction Scheme 2], in order to combine the sulfated hyaluronic acid with a hydrophobic group, the compound containing the hydrophobic group may be additionally introduced with an amine group to form an amide bond with the carboxyl group of the sulfated hyaluronic acid. For this, an aminoethylamide derivative of hydrophobic cholanic acid may be used, for example, aminoethyl 5-β cholanamide in which an amine group is introduced into 5-β cholanic acid.

The present invention also provides a pharmaceutical composition for the prevention or treatment of musculoskeletal joint disease comprising the sulfated hyaluronic acid derivative.

According to the present invention, the sulfated hyaluronic acid derivative contained in the composition in the composition can inhibit inflammation by inhibiting angiogenesis by inhibiting angiogenesis factors.

In this case, the angiogenesis factor is not necessarily limited to this, but may be, for example, VEGF165a.

In addition, according to the present invention, the sulfated hyaluronic acid derivative contained in the composition can relieve inflammation by inhibiting the secretion and cell proliferation of inflammatory cytokines and chemokines of synovial fibroblasts and immune cells.

In this case, the immune cells may be T cells.

According to the present invention, the musculoskeletal joint disease is degenerative arthritis (Osteoarthritis), rheumatoid arthritis, meniscus injury, adhesive capsulitis, tendonitis inflamed tendon (Tendinopathy) ), Gouty arthritis and other inflammatory arthritis.

[Example]

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples. Therefore, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Example  One. Sulfated  Preparation of hyaluronic acid

A sodium hyaluronate aqueous solution (eg, 150 kDa, 500 mg in 100 mL distilled water) was mixed with DOWEX 50WX8-400 ion exchange resin (5.0 g, cations in resin exchanged with TBA) and stirred for 3 hours. The mixture was filtered with filter paper to remove the ion exchange resin and freeze dried to give an off-white solid (TBA-HA).

TBA-HA (grey-white powder, 50 mg) was dissolved in 10 mL of N, N-dimethylformamide (DMF) and various amounts of sulfur trioxide pyridine complex (0.076 g, 0.15 g, 0.38 g and 0.61 dissolved in 2.0 mL of DMF) g) to react with s-HA-1 (R ₄ -OH of the sulfated hyaluronic acid monomer represented by [Formula 2] or [Formula 4] is sulfated), s-HA-2, s-HA-3 and s-HA was prepared having four different degrees of sulfation, referred to as s-HA-4 (all of the OH of the monomer is sulfated). The reaction proceeded for 1 hour while maintaining a temperature of 0 to 5 ° C under a nitrogen atmosphere. The reaction was stopped by adding 10 mL of water, and the pH of the solution was adjusted to 8.5-9.0 with a 1.0 M NaOH solution to produce a sulfated sodium hyaluronate salt. Samples were dialyzed for 3 days using distilled water. Finally, the samples were lyophilized to give off-white solids (s-HA-1 (36 mg), s-HA-2 (42.8 mg), s-HA-3 (47.8 mg), and s-HA-4 (51.5 mg). Obtained.

Sulfurization of the hyaluronic acid occurs at the positions R ₁ , R ₂ , R ₃ and R ₄ of the hyaluronic acid monomer represented by [Formula 2], where R may be H or SO ₃ ⁻ . The sulfation reaction proceeds first at the R ₄ position corresponding to the most reactive hydroxyl group of hyaluronic acid (see steps 1 and 2 of FIG. 1).

Example  2. Hyaluronic acid Hydrogel  Produce

For comparison with the sulfated hyaluronic acid hydrogel according to the present invention, a hyaluronic acid hydrogel was prepared from the unsulfurized hyaluronic acid monomer. Specifically, 9.67 mg of N- (3-Dimethylaminopropyl) -N′-ethylcarbodiimide hydrochloride (EDC) and 6.73 mg of 1-Hydroxybenzotriazole (HOBT) were added to a solution of 50 mg (molecular weight 3000 KDa) of hyaluronic acid dissolved in 2 ml of distilled water. After dissolving, 1.16 mg of Hexamethyldiamine (HMDA) was added to the solution, stirred at room temperature to dissolve completely, and then stirred and reacted at 40 ° C for 18 hours to obtain a viscous liquid. Next, 10 mL of ethanol was added and the process of removing was repeated 3 to 5 times to remove unreacted reagents, followed by lyophilization to obtain a solid material, and 1 mL of distilled water was added to prepare a hyaluronic acid hydrogel (FIG. 1). Step 3 reaction and Figure 3).

Example  3. According to the invention Sulfated  Preparation of hyaluronic acid hydrogel (compound represented by [Formula 6])

58 mg of N- (3-Dimethylaminopropyl) -N′-ethylcarbodiimide hydrochloride (EDC) and 40 mg of a solution of 50 mg (s-HA-2) of sulfated hyaluronic acid prepared from Example 1 in 2 ml of distilled water After dissolving 1-Hydroxybenzotriazole (HOBT) in, 7 mg of HMDA was added to the solution, and the mixture was completely dissolved by stirring at room temperature, and then stirred and reacted at 40 ° C for 18 hours to obtain a viscous liquid. Next, 10 mL of ethanol was added and the removal process was repeated 3 to 5 times to remove unreacted reagents, followed by lyophilization to obtain a solid material, and 1 mL of distilled water was added to prepare a sulfated hyaluronic acid hydrogel ( 3 step reaction of FIG. 1 and FIG. 3).

Example  4. According to the invention Sulfated  Preparation of hyaluronic acid hydrogel (compound represented by [Formula 6])

By the same method as in Example 3, 68 mg of N- (3-Dimethylaminopropyl) -N′-ethylcarbodiimide hydrochloride (EDC) and 47 mg of 1-Hydroxybenzotriazole (HOBT) and 8 mg of HMDA sulfated hyaluronic acid were used. A lactic acid hydrogel was prepared.

Example  5. According to the invention Sulfated  Preparation of hyaluronic acid hydrogel (compound represented by [Formula 6])

By the same method as in Example 3, 77 mg of N- (3-Dimethylaminopropyl) -N′-ethylcarbodiimide hydrochloride (EDC) and 54 mg of 1-Hydroxybenzotriazole (HOBT) and 9 mg of HMDA sulfated hyaluronic acid were used. A lactic acid hydrogel was prepared.

Example  6. According to the invention Sulfated  Preparation of hyaluronic acid hydrogel (compound represented by [Formula 6])

By the same method as in Example 3 above, 87 mg of N- (3-Dimethylaminopropyl) -N′-ethylcarbodiimide hydrochloride (EDC) and 60 mg of 1-Hydroxybenzotriazole (HOBT) and 10 mg of HMDA were used for sulfated hyaluronic acid. A hydrochloric acid was prepared.

Example  7. Hyaluronic acid With hydrogel , According to the invention Sulfated  Hyaluronic acid Hydrogel  Viscosity comparison analysis

The hydrogels according to Examples 2 to 6 were measured for viscosity with a rheometer, and the results are shown in Table 1 below.

[Table 1]

Sulfurized hyaluronic acid, unlike hyaluronic acid, has a strong hydrophilicity and requires a wider cross-linking than hyaluronic acid because its molecular weight decreases in the manufacturing process. Therefore, in order to have a satisfactory viscosity, the sulfated hyaluronic acid hydrogel has a satisfactory viscosity. Proper use of HMDA, EDC, and HOBT reagents is an important factor.

As shown in Table 1, when Example 4 is followed, it exhibits a viscosity equivalent to that of a hyaluronic acid hydrogel, whereas when the amount of the reagent used is small, as in Example 3, crosslinking has a narrow range and low viscosity. As in Examples 5 and 6, when the amount of the reagent used was excessive, it was confirmed that the crosslinking was excessively formed, so that the hydration property of the hydrogel was rather decreased, so that the viscosity was lowered. As a result, it was confirmed that it is preferable to use a reagent in the weight range according to Example 4, and in particular, the weight ratio of HMDA is most preferably used in an amount corresponding to the range of 14 to 16% by weight of hyaluronic acid sulfate.

Example  8. Sulfated  Hyaluronic acid improves arthritis

After dissolving type 2 collagen in 0.1N acetic acid, the type 2 collagen solution was mixed with the same concentration of complete Freund's adjuvant (CFA) at a ratio of 1: 1. 0.1 mL of 2 mg / ml collagen mixture (100 μg of type 2 collagen) was slowly injected through the intradermal of the mouse tail. The first injection was given and the second injection was performed 21 days later. As in the first phase, collagen mixed with incomplete Freund's adjuvant (IFA) instead of CFA was injected into the basal skin of the tail in the same amount as the first injection. Simultaneously with the second injection, 25 mg / kg of the hyaluronic acid hydrogel according to Example 2 and 25 mg / kg of the sulfated hyaluronic acid hydrogel according to Example 4 were injected intraperitoneally, respectively. Arthritis was observed for 30 days after the drug injection was administered 6 times 3 times a week for 2 weeks. As a result, it was confirmed that arthritis was significantly improved in mice administered with the sulfated hyaluronic acid hydrogel according to the present invention (FIG. 4). Experimental population: NC (n = 2), PC (n = 7), 25 mg / kg HA-hydrogel (n = 3), 25 mg / kg s-HA hydrogel (n = 3).

Example  9. Sulfated  Hyaluronic acid Hydrogel  Cytokines and Chemokine  Secretion reduction effect

After extracting synovial fibroblasts from synovial tissue of a patient with rheumatoid arthritis, 20 µg / ml of hyaluronic acid hydrogel according to Example 2 and 20 µg / ml of sulfated hyaluronic acid hydrogel according to Example 4 were respectively 10 ng / ml It was incubated for 24 hours after treatment with TNF-α. As a result of obtaining a cell culture solution and performing an ELISA analysis to confirm the secretion amount of IL-6 and IL-8, as a result of processing TNF-α and simultaneously jfl the sulfated hyaluronic acid hydrogel according to the present invention, IL, an inflammatory cytokine It was confirmed that the secretion of IL-6, a chemokine and -6, was significantly reduced (Mean ± SD of triplicates) (FIG. 5).

Example  10. Hydrophobicity Functional acid (Cholanic acid) Combined Sulfated  Hyaluronic acid derivatives and using them I Synthesis of old particles

(1) In order to prepare sulfated hyaluronic acid in the form of nanoparticles, functional groups having hydrophobic properties must be bound to sulfated hyaluronic acid through covalent bonding to have amphiphilic properties. To this end, a hydrophobic functional group is first bonded through activation of a carboxyl group present in glucuronic acid of a monomer of hyaluronic acid sulfate represented by [Formula 2] or [Formula 4] and a terminal amine group introduced into the hydrophobic group. A sulfated hyaluronic acid derivative represented by [Formula 3] was synthesized, and the specific manufacturing process is as follows.

10 g of cholanic acid (Sigma, USA) was dissolved in 600 ml of methanol, and 1.6 ml of hydrochloric acid was added, followed by stirring at 60 ° C for 6 hours. Thereafter, the solution was concentrated to 100 ml using a rotary evaporator, precipitated in 2 L of distilled water, filtered, washed several times with distilled water, and freeze-dried. After drying, 60 ml of ethylenediamine was added to 10 g of methyl-5-β colanate obtained, followed by stirring at 130 ° C. for 8 hours. Diluted with 100 ml methanol at room temperature and precipitated in 2 L distilled water. It was filtered, washed several times with distilled water and freeze-dried. 1 g of sulfated hyaluronic acid (s-HA-2) prepared from Example 1 was added to 160 ml distilled water and stirred to dissolve. 1.57 g of 1-ethyl-3- (3-dimethyl-aminopropyl) carbodiimide (EDC) was added to the solution and 0.941 g of N-hydroxysuccinimide (NHS) was added to 1 ml dimethylformimide. After dissolving in (DMF), the solution was added to the solution and stirred to activate the carboxyl group of sulfated hyaluronic acid. After dissolving 125 mg of the prepared aminoethyl 5-β collanoamide in 320 ml DMF, a hyaluronic acid solution was added dropwise while stirring in a reactor. At this time, the reaction temperature was 60 ° C and the reaction time was 3 days.

       Hyaluronic acid sulfate-cholanic acid complex was obtained in powder form while dripping the mixed solution after the reaction was completed with a precipitation solvent. Isopropyl alcohol and isopropyl ether were mixed 1: 1 and used as a precipitation solvent, and the ratio of the precipitation solvent and the reaction solution was 4: 1. After dropping and stirring the reaction solution in the precipitation solvent, it was washed 3 times with the same solvent as the precipitation solvent and dried under vacuum. The hyaluronic acid-cholanic acid complex obtained after vacuum drying was purified. In order to remove impurities and residual solvent, the complex was dissolved in 100 ml distilled water, put in a separator, and added and purified by adding 100 ml dichloromethane. At this time, the pH of the distilled water was set to 8 or more, and after repeating the above process several times, only the aqueous layer was taken and freeze-dried to obtain a sulfated hyaluronic acid derivative (sulfuric acid hyaluronic acid-cholanic acid complex) having a hydrophobic functional group.

(2) 90 mg of the obtained sulfated hyaluronic acid-cholanic acid complex was dissolved in 10 ml distilled water. After dissolving 10 mg of hydrophobic anti-cancer agent (paclitaxel) in 30 ml ethanol, the mixture was mixed with the aqueous solution of the complex and stirred in a round flask. The solvent was evaporated using a rotary evaporator and a film was formed around the round flask. After confirming that the film was completely formed, 50 ml of distilled water was added to hydrate. After confirming that the film was completely hydrated, the nanoparticles were dispersed using an ultrasonic grinder and analyzed by an electron microscope, and it was confirmed that tens to hundreds of nanometer spherical particles were successfully formed (FIG. 6).

Since the specific parts of the present invention have been described in detail above, it is obvious that for those skilled in the art, this specific technique is only a preferred embodiment, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (8)

It is represented by the following [Formula 1], sulfated hyaluronic acid derivatives characterized in that to form a hydrogel by cross-linking between sulfated hyaluronic acid monomers:
[Formula 1]

In the above [Formula 1],
R ₄ And R ₈ is SO ₃ ^- X ⁺ , R ₁ or R ₅ are the same or different from each other, and each independently hydrogen or SO ₃ ^- X ⁺ ,
R ₂ to R ₃ or R ₆ to R ₇ are the same as or different from each other, and each independently hydrogen or SO ₃ ^- X ⁺ , wherein R ₂ to R ₃ At least one of SO ₃ ^- X ⁺ , wherein R ₆ to R ₇ At least one of them is SO ₃ ^- X ⁺ ,
X is a cation, n1 and n2 are each 1 or more,
The crosslinking between the sulfated hyaluronic acid monomers is a linkage through a covalent bond of a ring or acyclic material comprising at least one (CH ₂ CH ₂ ) m (m≥1) group linked through an amide bond. An active ester-forming reagent and a crosslinking agent are added to and reacted with the sulfated hyaluronic acid monomer represented by the following [Formula 2] to activate the carboxyl group of the sulfated hyaluronic acid monomer represented by the following [Formula 2], and crosslinking between monomers Induction to prepare a compound represented by [Formula 1] of claim 1; Method for producing a sulfated hyaluronic acid derivative comprising:
[Formula 2]

In the above [Formula 2],
R ₄ is SO ₃ ^- X ⁺ , R ₁ is hydrogen or SO ₃ ^- X ⁺ ,
R ₂ to R ₃ are Are the same as or different from each other, and each independently hydrogen or SO ₃ ^- X ⁺ , wherein R ₂ to R ₃ At least one of them is SO ₃ ^- X ⁺ , X is a cation, n is an integer of 1 or more. According to claim 2,
The active ester forming reagent is one or more of N- (3-Dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) and 1-Hydroxybenzotriazole (HOBT). Manufacturing method. According to claim 2,
The crosslinking agent is hexamethyldiamine (HMDA), adipic dihydrizide, amine polyethylene glycol or a method for producing a sulfated hyaluronic acid derivative, characterized in that a mixture thereof. A pharmaceutical composition for the prevention or treatment of musculoskeletal joint disease comprising the sulfated hyaluronic acid derivative according to claim 1. The method of claim 5,
A pharmaceutical composition for the prevention or treatment of musculoskeletal joint disease, characterized in that the sulfated hyaluronic acid derivative contained in the composition relieves inflammation by inhibiting angiogenesis factors and thereby inhibiting angiogenesis. The method of claim 5,
A pharmaceutical composition for the prevention or treatment of musculoskeletal joint disease characterized in that the sulfated hyaluronic acid derivative contained in the composition relieves inflammation by inhibiting the secretion and cell proliferation of inflammatory cytokines and chemokines of synovial fibroblasts and immune cells. The method of claim 5,
The musculoskeletal joint disease is degenerative arthritis (Osteoarthritis), rheumatoid arthritis, meniscus injury, adhesive capsulitis, tendonitis (Tendinopathy), gouty arthritis (gout) Gouty arthritis) and other pharmaceutical compositions for the prevention or treatment of musculoskeletal joint disease, characterized in that selected from the group consisting of inflammatory arthritis.

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