KR20070050586A - Transforming growth factor-chitosan complexes and preparation method thereof - Google Patents

Abstract

The present invention is a transition growth factor-chitosan complex formed by covalently bonding a functional group present in amino acid residues of a transforming growth factor and an amine group present in chitosan, a preparation method thereof, and a pharmaceutical composition for treating arthritis including the same It is about. In the complex of the present invention, the stability and half-life of the transfer growth factor in vivo and in vitro are greatly increased, and thus the biological activity of the transfer growth factor is greatly increased. Moreover, the complexes themselves have no cytotoxicity and thus are very safe for the living body.

Chitosan, metastatic growth factor, arthritis, complex

Description

Transforming growth factor-chitosan complexes and preparation method

1 is a graph showing the cytotoxicity test results of the transition growth factor-chitosan complexes;

FIG. 2 is a graph showing stability measurement results of proteolytic degradation by trypsin of a transition growth factor and a transition growth factor-chitosan complex; FIG.

FIG. 3 is a graph showing the healing effect of the metastatic growth factor-chitosan complex against arthritis compared to the natural metastatic arthritis ability of the pure metastasis growth factor and rabbit;

4 is a photograph showing the articular surface of an animal experimental rabbit 9 weeks after administration of the metastatic growth factor-chitosan complex; And,

5 is a graph showing the metastatic growth factor-chitosan complex in the histological quantitative analysis showing a higher healing effect on the articular sac compared to the control.

The present invention relates to a metastasis growth factor-chitosan complex, and more particularly, to a metastasis growth factor-chitosan complex, a preparation method thereof, and a pharmaceutical composition for treating arthritis including the same.

Growth Factor acts to promote cell growth, and in particular, one of the growth factors, Transforming Growth Factor (TGF), is a polypeptide that transmits signals that stimulate chondrocyte activity. Is one of them. Therefore, due to the movement of molecules such as peptides and steroids, one cell and neighboring cells can interact, and further control cell function as a whole.

Metastasis growth factor (TGF-β) is a single polypeptide consisting of amino acids, and has been clinically applied to experiments on arthritis and chondrocyte differentiation, cancer cell metastasis inhibition, and growth inhibitory effects on most cell lines including epithelial cells. However, these growth factors have many difficulties in use because of their short active half-life.

Previously reported water soluble CM-cellulose and amylase (Wykes, J. et al., Biochemica et Biophysica Acta ( BBA ) , 250: 522-529 (1971)) and water-soluble dextran and lysozyme, β-glucosidase (Vegarud, G. et al., Biochemica et Biophysica By forming covalent bonds between Acta ( BBA ) , 266: 431-452 (1975)) or β-amylase, enzyme-sugar complexes stabilized against physical external factors such as pH, temperature, heat, oxygen, and the like have been developed. In addition, when the dextran-enzyme complex was injected into the venous blood vessels of rats as a therapeutic agent, the residual activity of the enzyme was measured. Was reported.

On the other hand, chitosan is a cationic polysaccharide in which glucose amine and N-acetylglucosamine are 1,4-β-linked, and can be obtained by deacetylating chitin obtained from crabs or other sources. Chitin is slowly degraded in vivo, and chitin and its degradation products are safe natural products. In the field of pharmaceuticals, chitosan has been used as a carrier for delayed release of drugs (Hou et al., Chem Pharm Bull 33 (9): 3986-3992 (1985)), chitin itself has been woven and used as a dressing for wound healing.

Throughout this specification, numerous citations and patent documents are referenced and their citations are indicated. The disclosures of cited documents and patents are incorporated herein by reference in their entirety, so that the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.

The present inventors have conducted continuous research to develop a transition growth factor that has the greatest stability (especially in vivo stability) and applicability of the transition growth factor. As a result, chitosan, a biocompatible biodegradable material having self-sterilization, has been developed. In the case of using the growth growth factor in the form of a complex prepared by covalently attaching the growth growth factor to the growth factor, the stability and the half-life of the growth growth factor can be greatly increased, thereby increasing the biological activity of the growth factor. The present invention was completed by confirming that the present condition exists.

Therefore, an object of the present invention is to prepare a novel transition growth factor-chitosan complex.

It is an object of the present invention to provide a method for preparing a transition growth factor-chitosan complex.

Still another object of the present invention is to provide a pharmaceutical composition for treating arthritis, including a metastasis growth factor-chitosan complex.

Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

According to an aspect of the present invention, the present invention provides a transition growth factor-chitosan complex formed by covalent bonding of a functional group present in amino acid residues of a transforming growth factor (TGF) and an amine group present in chitosan.

The present inventors have conducted continuous research to develop a transition growth factor that has the greatest stability (especially in vivo stability) and applicability of the transition growth factor. As a result, chitosan, a biocompatible biodegradable material having self-sterilization, has been developed. In the case of using the growth growth factor in the form of a complex prepared by covalently attaching the growth growth factor to the growth factor, the stability and the half-life of the growth growth factor can be greatly increased, thereby increasing the biological activity of the growth factor. It was confirmed that there is. Moreover, the thus developed metabolic growth factor-chitosan is cytotoxic and therefore very safe for the living body.

As used herein, the term “transition growth factor” refers to metastasis growth factor-β (TGF-β) unless otherwise specified.

In the complex of the present invention, the transfer growth factor and chitosan are covalently bonded. This covalent bond is formed between the amine group of the chitosan and the functional group present in the amino acid residues of the transition growth factor. According to a preferred embodiment of the present invention, the functional group of the amino acid bonded to the amine group of chitosan is a carboxyl group or a sulfhydryl group, most preferably a carboxyl group. The inventors have found that the carboxyl groups present at the amino acid residues of the transition growth factor are the binding functional groups most suitable for the purposes of the present invention.

If the complex is prepared by combining the amine group of chitosan and the sulfhydryl group of the amino acid residue of the transfer growth factor, m-maleimidobenzoyl-N-hydroxysuccinimide ester hetero-functional linker such as m-maleimidobenzoyl-N-hydroxy sulfosuccinimide ester or N-maleimidobutyryloxysuccinamide ester TGF-chitosan complex is prepared using a (heterobifunctional linker).

One of the features of the present invention is the selection of the carboxyl group of the amino acid residue of the transition growth factor and the amine group of chitosan as the conjugation partner in preparing TGF-chitosan conjugation. The linkage between the functional groups can be carried out through various heterobifunctional linkers, preferably a carbodiimide-containing compound, ASBA (4- [p-azidosalicylamido] butylamine) or AEDP (3 Bonds are formed by-[(2-aminoethyl) dithio] propionic acid). More preferably, the covalent linkage between the carboxyl group of the amino acid residue of the transition growth factor and the amine group of the chitosan is carried out by a water-soluble carbodiimide-containing compound, most preferably EDC (1-ethyl- (dimethylaminopropyl) carbodiimide) Is formed by. The inventors finally confirmed that EDC is the most suitable heterobifunctional linker for preparing TGF-chitosan complexes suitable for the purposes of the present invention. In other words, in preparing a complex by covalently binding TGF with chitosan, EDC is an optimal linker that can stably bind with chitosan to significantly improve the in vivo stability of TGF without compromising the intrinsic activity of TGF. It is selected. It is also a feature suitable for the present invention that the EDC is a zero-length linker.

The chitosan bound to TGF is not greatly limited, but preferably has a molecular weight of 5-100 kDa, more preferably 5-50 kDa, most preferably 10-25 kDa. If the molecular weight is less than 10 kDa, there is a problem that the molecular weight is too small to properly protect the functional groups of the protein that acts as a growth factor of TGF. If the molecular weight exceeds 25 kDa, chitosan protecting the TGF is decomposed to decompose TGF. It takes a long time to stimulate the chondrocytes by exposing the functional groups has a drawback that the growth factor arthritis target effect cannot be exerted in time.

Since the TGF-chitosan complex of the present invention exhibits improved stability in vivo and is cell non-toxic, it can be applied to various diseases or disorders that can be prevented or treated by the activity of TGF. For example, the disease or condition that can be treated or prevented by the complex of the present invention is the regeneration of damaged cartilage tissue due to degenerative arthritis, but has a wider range of application to cartilage cell damage in addition to in vitro cartilage cell culture or degenerative arthritis. It can be applied as a treatment for all related diseases.

The TGF-chitosan complex of the present invention may be prepared by various methods, but preferably (a) hydrolyzing chitosan to obtain a chitosan fraction having a molecular weight of 10-25 kDa; And (b) reacting chitosan having a molecular weight of 10-25 kDa with a transition growth factor in the presence of a water-soluble carbodiimide-containing compound to obtain a transition growth factor-chitosan complex. It is prepared according to the preparation method of the composite.

According to a specific embodiment of the manufacturing method of the present invention will be described in detail as follows:

A low molecular weight chitosan fraction is prepared by performing a chitosan hydrolysis reaction. At this time, it is possible to increase the yield of the desired molecular weight fraction by adjusting the acid concentration, the reaction time and the reaction temperature. For example, 4% (w / w) chitosan acetic acid solution is neutralized titrated using aqueous NaOH solution and left at room temperature until no white precipitate is produced near pH 7.0. Thereafter, the precipitate was washed, dried and purified, HCl was added and stirred in a cold bath, and then reacted at 50 ° C. and 83 ° C. for 0.5-5 hours, distilled water was added to the reaction solution to remove the precipitate, and methanol was added thereto. The precipitate is again dissolved in distilled water and ultrafiltered to yield a low molecular weight (about 10-25 kDa) chitosan fraction.

The low molecular weight chitosan fractions obtained are added with the water-soluble carbodiimide-containing compound (preferably EDC) in an excess of about 50 times the number of moles of TGF addition. For example, 250 μl of 84.348 ng / ml EDC reagent is added, then 500 μl of 30 ng / ml TGF is added and shaken at 4 ° C. for about 2 minutes, and 250 μl of 23.2 μg / ml hydrolyzed chitosan is added. After reacting at room temperature for 4 hours. Thereafter, 100 µl of 0.13 M glycine solution is added to the solution, followed by further shaking for 90 minutes. After 90 minutes, the dialysis membrane cut-off is dialyzed at 12,000 using a 12,000 day, and the filtration membrane is used to obtain the transition growth factor-chitosan complex.

The yield of transition growth factor-chitosan complexes prepared according to the method was 60.84-73.5%, which was much higher than the reported yield of epithelial growth factor-dextran complexes using CDAP crosslinkers, 30-40%. It did not appear to increase in proportion to the amount of growth factor. Therefore, it can be seen that the yield of the production method of the present invention is very excellent.

As described in detail in the following Examples, there was no cytotoxicity of the transition growth factor-chitosan complex of the present invention, after 3 days showed a higher cell growth sustaining effect than the pure transition growth factor was found to increase the activity half-life, It is also more stable than pure transition growth factor. In addition, when the transfer growth factor-chitosan complex was treated at the rabbit arthritis site, the site where the transfer growth factor-chitosan complex was prepared in an amount of only 50% of the pure transfer growth factor amount was treated with the pure transfer growth factor. Achieve better levels of wound healing. Therefore, the use of the metastasis growth factor-chitosan complex as a therapeutic agent for arthritis can provide higher economic benefits than the use of the pure metastasis growth factor as a therapeutic agent for arthritis.

According to another aspect of the present invention, the present invention provides a pharmaceutical composition comprising (a) a pharmaceutically effective amount of the above-described metabolic growth factor-chitosan complex of the present invention; And (b) provides a pharmaceutical composition for treating arthritis comprising a pharmaceutically acceptable carrier.

Since the pharmaceutical composition of the present invention uses the above-described transition growth factor-chitosan complex as an active ingredient, the common content between the two is omitted in order to avoid excessive complexity of the present specification.

The TGF-chitosan complex included in the composition of the present invention exhibits improved stability in vivo and is non-toxic to cells, and therefore is particularly effective for treating arthritis.

As used herein, the term “pharmaceutically effective amount” means an amount sufficient to achieve an arthritis therapeutic effect.

Pharmaceutically acceptable carriers included in the compositions of the present invention are those commonly used in the formulation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, silicic acid Calcium, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oils, and the like. It is not. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention is preferably parenteral, and may be administered using, for example, intravenous administration, intraperitoneal administration, intramuscular administration, subcutaneous administration, or topical administration. Especially preferably, it is intraarticular injection which directly injects into the generated joint.

Suitable dosages of the pharmaceutical compositions of the invention vary depending on factors such as the formulation method, mode of administration, age, weight, sex of the patient, degree of disease symptom, food, time of administration, route of administration, rate of excretion and response to reaction. In general, the skilled practitioner can readily determine and prescribe a dosage effective for the desired treatment. The most preferred dose of the pharmaceutical composition of the present invention is 1 ml in a single dose, in which case the amount of TGF is 15 ng.

The pharmaceutical composition of the present invention is prepared in unit dose form by being formulated using a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or may be prepared by incorporating into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or an aqueous medium, or may be in the form of extracts, powders, granules, tablets or capsules, and may further include a dispersant or stabilizer.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example  One: EDC Condensation reagent  Used TGF Preparation of Chitosan Complex

A 2% (w / w) chitosan (Ja-Kwang, Korea) acetic acid solution was prepared and neutralized titrated using an aqueous NaOH solution. It was left at room temperature until no more white precipitate formed near pH 7.0. After that, the precipitate was washed with distilled water, ethanol and ether, and then purified by drying. The purified chitosan powder was dissolved in a 2% acetic acid aqueous solution at room temperature for about 12 hours to prepare a chitosan solution. Here Conc. HCl was added to run the reaction at 50 ° C. for about 30 minutes. After 30 minutes, one volume of distilled water of the hydrolysis solution was added to induce precipitation of the unreacted product. 1 volume of methanol was added to the hydrolysis liquid from which the precipitate was removed to obtain a precipitate. The precipitate was again dissolved in distilled water and ultrafiltration was performed using YM10 (dialysis membrane cut-off 10 kDa) and YM3 (dialysis membrane cut-off 3 kDa) membranes. The molecular weight of the chitosan fraction finally obtained through the reaction was 23 kDa and 10 kDa.

The low molecular weight (10,535 Da) chitosan obtained in the above process was added with EDC (1-ethyl- (dimethylaminopropyl) carbodiimide; Sigma, USA) at 50 and 10 times the molar excess of TGF added, respectively. That is, 250 [mu] l of 6.76 nmol / ml EDC solution was added to the reactor. To this was added 500 μl of 2000 ng / ml TGF (human recombinant, Sigma) solution at 4 ° C. After shaking for about 120 seconds, 250 μl of a 0.16 μmol / mL hydrolyzed chitosan (10,535 Da) solution was added, followed by reaction at room temperature for 4 hours. Thereafter, 100 µl of 0.13 M glycine solution was added to the solution, followed by further shaking for 90 minutes. After 90 minutes, dialyzed at 4 ° C. for 1 day using a dialysis membrane cut-off of 12,000 to obtain a TGF-chitosan complex. The yield of transition growth factor-chitosan complexes prepared according to the method was 60.84-73.5%, which was much higher than the reported yield of epithelial growth factor-dextran complexes using CDAP crosslinkers, 30-40%. It did not appear to increase in proportion to the amount of growth factor.

Example  2: EGF -Cytotoxicity Test of Chitosan Complex

To assess the cytotoxicity of the metastatic growth factor-chitosan complex, 20 [mu] l of the metastatic growth factor-chitosan complex in the range of 1-4 [mu] g / ml concentration was added to human chondrocytes at 1 × 10 ⁴ cells / well concentration in DMEM (serum free). was added to a 96-well plate containing human chondrocytes, and cytotoxicity was determined by MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (Sigma) analysis for 12 hours. Measurements were made over two days and three days (FIG. 1). As can be seen in Figure 1, the transition growth factor-chitosan complex was found to be cytotoxic. In Figure 1 TGF-LMC represents the TGF-chitosan complex prepared in Example 1.

Example  3: Evaluation of the Stability of Proteolytic Enzyme of Transition Growth Factor-chitosan Complex

The cell growth sustaining effect of the metastatic growth factor-chitosan complex was measured by the following method. The transfer growth factor-chitosan complex and the pure transfer growth factor were trypsinized, a representative proteolytic enzyme at 37 ° C. (FIG. 2). As can be seen in Figure 2, the pure transfer growth factor (denoted TGF) rapidly decreased to less than 30% of the initial activity, while the transfer growth factor-chitosan complex (denoted TGF-LMC) was maintained up to 80% of the initial activity. It was confirmed. From this, it can be seen that the TGF of the transition growth factor-chitosan complex has a higher protective effect on proteolytic enzymes than the pure TGF.

Example  4: Measurement of the Wound Healing Effect of Metastasis Growth Factor-chitosan Complex

Thirty New Zealand White Rabbits, weighing 3-4 kg and over 12 weeks old, whose growth plate was closed, were treated with Yoshioka et al. ( Osteoarthritis cartilage 4: 87-98 (1996)). After inducing artificial osteoarthritis, the first experimental group (metastatic growth factor-chitosan complex injection group, N = 10) and the second experimental group (pure metastatic growth factor injection group, N = 10) and the third experimental group (no administration group) , N = 10), divided into three experimental groups 4 weeks after the procedure to inject the anti-arthritis healing substance corresponding to the experimental group to the arthritis site, and 9 weeks after the procedure to sacrifice the rabbit to the arthritis healing effect of the metastatic growth factor-chitosan complex Compared with control. Experimental results are shown in FIGS. 3, 4 and 5.

3 is a result of measuring the grade of arthritis generated in each control group showed that the experimental group administered the metastatic growth factor-chitosan complex showed a lower grade of arthritis than the other control group. It can be seen that has a higher effect in the treatment of arthritis than other factors compared. In Figure 3, the numbers in the bars represent the populations, and the percentages represent 10% of the populations tested.

4 is a comparison of cartilage tissue on the surface of the rabbit joint immediately after the procedure and the metastasis growth factor-chitosan complex, and compared to the cartilage tissue on the joint surface of the rabbit 9 weeks after the procedure. This picture shows the effect. When confirmed by the coronary phase, it was confirmed that the growth factor-chitosan-administered group was rehabilitated more normally than the other group.

5 is a graph showing the results of histomorphometry-quantitative analysis. The thickness of the synovial membrane cell layer was measured by averaging 100 samples, and the Wilcoxon signed rank test, a non-parametric method, was used to analyze the difference between the two measurements in each group. In comparing the differences between the groups, one-way batch variance analysis was used as a parametric method, and Duncun test and Turkey b test were used for the post test, and the Kruskall-Wallis test and Mann-Whiteney U test for post-mortem test. Was used. Statistical analysis was performed using the SPSS program (SPSS for Windows Release 11.5; Chicago, Illinios) with a confidence interval of 95%.

The thickness of the synovial cell layer was significantly increased compared to the opposite side (p <0.05). In the growth factor-chitosan complex injection group, the mean value of the opposite side and difference was the highest with 10.2 ± 2.7 ㎛, followed by the pure growth factor injection group with an average of 5.2 ± 1.6 ㎛ and the control group with an average of 4.5 ± 2.0 ㎛. Showed. The growth factor-chitosan complex injection group showed significant difference from the other two groups. Therefore, it can be seen that the metastatic growth factor-chitosan complex-administered group produces a thicker synovial cell layer than other experimental groups and has a high arthritis healing effect.

Based on the results of the above animal experiments, it can be seen that the metastatic growth factor-chitosan complex exhibits a significantly improved arthritis healing effect than the pure metastatic growth factor or rabbit's own natural arthritis treatment effect.

As described in detail above, the present invention provides a metastatic growth factor-chitosan complex, a method for preparing the same, and a pharmaceutical composition for treating arthritis, including the same. In the complex of the present invention, the stability and half-life of the transfer growth factor in vivo and in vitro are greatly increased, and thus the biological activity of the transfer growth factor is greatly increased. Moreover, the complexes themselves have no cytotoxicity and thus are very safe for the living body.

Claims (9)

A transition growth factor-chitosan complex formed by covalent bonding of a functional group present in amino acid residues of a transforming growth factor and an amine group present in chitosan. The transition growth factor-chitosan complex according to claim 1, wherein the functional group of the amino acid residue of the transition growth factor covalently bonded to the amine group of chitosan is a carboxyl group. The covalent bond between the carboxyl group of the amino acid residue of the transition growth factor and the amine group of chitosan is carbodiimide-containing compound, ASBA (4- [p-azidosalicylamido] butylamine) or AEDP (3). -[(2-aminoethyl) dithio] propionic acid), characterized in that the transition growth factor-chitosan complex. 4. The transition growth factor-chitosan complex according to claim 3, wherein the covalent bond between the carboxyl group of the amino acid residue of the transition growth factor and the amine group of chitosan is formed by a water-soluble carbodiimide-containing compound. 4. The transition growth factor-chitosan complex according to claim 3, wherein the water-soluble carbodiimide-containing compound is EDC (1-ethyl- (dimethylaminopropyl) carbodiimide). The transition growth factor-chitosan complex according to claim 1, wherein the chitosan has a molecular weight of 10-25 kDa. Pharmaceutical composition for treating arthritis, comprising the following. (a) a pharmaceutically effective amount of the metastatic growth factor-chitosan complex of any one of claims 1 to 6; And (b) a pharmaceutically acceptable carrier. Method for producing a transition growth factor-chitosan complex comprising the following steps: (a) hydrolyzing chitosan to obtain a chitosan fraction with a molecular weight of 10-25 kDa; And (b) reacting the chitosan with a molecular weight of 10-25 kDa with a transition growth factor in the presence of a water-soluble carbodiimide-containing compound to obtain a transition growth factor-chitosan complex. The method of claim 8, wherein the water-soluble carbodiimide-containing compound is EDC (1-ethyl- (dimethylaminopropyl) carbodiimide).

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