KR101110317B1 - Smart releasing scafford of Thymosin beta-4 originated Ac-SDKP peptides for tissue regeneration - Google Patents

Abstract

The present invention relates to a next generation smart release support whose release rate is controlled according to the in vivo activity of the thymosin beta-4-derived Ac-SDKP peptide for tissue regeneration, and more specifically, to the thymosin beta-4-derived Ac-SDKP peptide. The synthetic peptide and the Ac-SDKP peptide smart release support, wherein the peptide and the cysteine amino acid are cleavable by matrix metalloprotease (MMP) or plasmin (plasmin) is immobilized in the N '→ C' direction. To a hydrogel.

According to the present invention, the thymosin beta-4-derived Ac-SDKP peptide having a tissue regeneration effect can be used as a pharmaceutical composition for tissue regeneration such as heart regeneration by controlling the release rate according to in vivo activity.

Description

Smart releasing scafford of Thymosin beta-4 originated Ac-SDKP peptides for tissue regeneration}

The present invention relates to a next generation smart release support whose release rate is controlled according to the in vivo activity of the thymosin beta-4-derived Ac-SDKP peptide for tissue regeneration, and more specifically, to the thymosin beta-4-derived Ac-SDKP peptide. The present invention relates to a hydrogel in which the synthetic peptide is immobilized as a smart release support for the synthetic peptide and the Ac-SDKP peptide, wherein the peptide and the cysteine amino acid are cleavable by MMP or plasmin. .

Tissue engineering and regenerative medicine aim to restore damaged tissue and regeneration through the proper delivery of therapeutic substances, cells and growth factors. Currently, various treatments using cell therapy, especially stem cells, have been performed preclinically, but the simple injection of cells has difficulty in maximizing effects due to low engraftment rate and difficulty in controlling differentiation.

Therefore, various methods using a support have been developed to maximize the engraftment rate and differentiation control capacity of stem cells in vivo. Tissue is a system in which various cells make up complex tissues. Therefore, there is an ideal tissue form or cell composition in regenerating specific tissues, and the supporter plays a role in forming a fused tissue of such cells. In addition, damaged tissues have atypical forms of damage, so simply injecting cells limits the ability to regenerate damaged areas. For example, lesions in burn patients are extensive at the cellular level, and may be a limiting factor in the number of cells that can be applied to treat cells alone, in immobilization, survival, and proliferation of the lesions, and have minimal therapeutic effects. . Therefore, a structural support capable of proliferating and proliferating cells is essential for effective tissue regeneration.

In the development of the first generation of materials, which are simply a function of transferring cells and growth factors, the second generation of third generation materials including biologically active materials to actively support tissue regeneration in vivo is being developed. In particular, recently, techniques for increasing activity in the body and maximizing tissue regeneration ability have been developed by attaching various peptides and growth factors to hydrogel, which is one of the supports. In particular, blood vessels must be generated in damaged tissues for effective tissue regeneration. Various growths such as Vascular endothelial growth factor (VEGF), Fibroblast growth factor (FGF), Platelet-derived growth factor (PDGF), or EGF (Epidermal growth factor) Techniques to promote angiogenesis by including factors have been developed, but have not yet reached a satisfactory level.

Accordingly, the present inventors have made a thorough research to overcome the problems of the prior art, and as a result, developed a scaffold to promote tissue regeneration using Ac-SDKP peptide, which is a key sequence among thymosin beta-4 molecules with high angiogenic activity. In addition, by developing a next-generation smart release support that regulates the degree of release according to the activity in vivo, rather than simply adding a short sequence of peptide in vivo, it works even at low concentrations specifically for damaged tissues rather than a large amount of peptides. Confirmed the effect of the breakfast regeneration and reconstruction of the new release system to complete the present invention.

Accordingly, the main object of the present invention is to develop and apply a next generation smart release support whose release rate is controlled according to the in vivo activity of thymosin beta-4-derived Ac-SDKP peptide for tissue regeneration with excellent angiogenesis. It is to provide a method for producing a hydrogel for tissue regeneration that specifically acts on the tissue.

Another object of the present invention is to provide a pharmaceutical composition for tissue regeneration using a hydrogel to which the Ac-SDKP peptide is immobilized.

According to an aspect of the present invention, the present invention provides a synthetic peptide comprising thymosin beta-4-derived Ac-SDKP peptide, a peptide cleavable by MMP or plasmin, and a cysteine amino acid bound in a N ′ → C ′ direction. To provide.

In the present invention, the term “Thymosin beta-4” is a very small protein present in vivo consisting of 43 amino acids of 4.9 kDa size, wherein the thymosin beta-4 protein is separated from each other. G-actin binds to G-actin, which prevents the formation of G-actin-bound F-actin and converts G-actin and F-actin. By regulating the structure of the actin cytoskeleton (actin cytoskeleton) is a protein that plays a role in regulating the movement of cells. In particular, the main function of thymosin beta-4 is to regulate cell migration to promote cell migration, which causes the migration of vascular endothelial cells, the movement of cells involved in wound healing, hair root cells, and cancer cells. Promotes blood vessel formation, wound healing, hair growth, cancer metastasis. In vivo thymosin proteins are divided into three main groups by their isoelectric points. α-thymosin below pH 5.0, β-thymosin between pH 5.0 and pH 7.0 and γ-thymosin above pH 7.0. Thymosin beta-4 belongs to β-thymosin and is a representative factor for promoting angiogenesis. Ac (Acetyl) -SDKP peptides expressed in vivo in thymosin beta-4 are formed by prolyl endopeptidase or AspN-like protease using thymosin beta-4 as a precursor and are pluripotent hematopoietic Hematopoietic pluripotent stem cells are known to perform the function of inhibiting the cell cycle by blocking the division and division into the S-phase.

In the present invention, the term “MMP” is an enzyme that dissolves extracellular matrix and is one of the four proteases (seminethreonine-, aspartic-, cysteine-, and metalloproteinases) present in the body. It is a substance having. First, it decomposes at least one or more of the extracellular matrix components. Second, Zn ions are essential for enzymatic activity and proteolytic function is inhibited by chelating agents. It has protein resolution through. Fourth, its function is inhibited by TIMP (tissue inhibitor of matrix-metalloproteiase), a selective inhibitor of MMP, and fifth, it contains a common amino acid sequence. The MMP is largely dependent on the specificity of the substrate, the binding site of the inhibitor, the binding site of the substrate and the location of the cell membrane, and the collagenase, stromelysins, gelatinases, and matrilysin ( matrilysin) and MT (membrane type) -MMP attached to the cell membrane.

As used herein, the term "plasmin" is a protease present in the blood, for example, a protease that dissolves fibrin involved in blood coagulation.

In the synthetic peptide of the present invention, the amino acid D (Aspartic acid, Asp) for the peptidase (peptidase) is further included between the thymosin beta-4-derived Ac-SDKP peptide and the peptide cleavable by MMP or plasmin Preferred synthetic peptides. This can be cleaved by prolyl endopeptidase or Asp-N-like protease in the human body to release the Ac-SDKP peptide.

In the present invention, the peptide which is cleavable by MMP or plasmin may be any peptide sequence known in the art as a cleavage site of MMP or plasmin, preferably MMP or GPQGIWGQ, and plasmin LIKMKPM. In particular, the MMP cleavage site may be cleaved by MMP secreted during cardiac remodeling to effectively release the Ac-SDKP peptide to the target site.

In the synthetic peptide of the present invention, the thymosin beta-4-derived Ac-SDKP peptide is preferably characterized in that it is acetylated.

Synthetic peptide of the present invention may have any amino acid sequence as long as it comprises the component peptides, preferably characterized by having the amino acid sequence of SEQ ID NO: 1. Table 1 shows the amino acid sequences of the synthetic peptides.

[Table 1]

According to another aspect of the present invention, the present invention provides a synthetic peptide characterized in that the thymosin beta-4-derived Ac-SDKP peptide, a peptide cleavable by MMP or plasmin, and cysteine amino acids are bonded in a N ′ → C ′ direction. Relates to an immobilized hydrogel.

In the present invention, the hydrogel refers to a water swellable polymer that can be used as a medical material such as drug delivery system, biological tissue synthetic material, such a water swellable polymer has a sufficient amount of water that absorbs water but does not dissolve in water It refers to the hydrophilic polymer, which is a natural polymer such as hyaluronic acid (HA), collagen, polyvinyl alcohol (polyvinyl alcohol, PVA), polyhydroxyethyl methacrylate (poly (hydroxyethymethaacrylate), PHEMA It can be manufactured using a synthetic polymer such as). The hydrogel used in the present invention is preferably a hyaluronic acid based hydrogel. The hyaluronic acid is a biodegradable, biocompatible material which is a natural polymer having a wide molecular weight range of about 1,000 to 10,000,000 g / mole. The hyaluronic acid is a major component of the extracellular matrix (ECM) of connective tissue, and its unique viscoelastic properties and biological functions have been studied as a useful material for the treatment of arthritis, ophthalmic surgery, drug delivery, and tissue engineering. It regulates hydration, forms a hydrated network with collagen fibers in the ECM, and is known to play an important role in wound healing and cell flow and differentiation in the process.

In the present invention, the hyaluronic acid-based hydrogel is preferably acrylated (Acrylate), in particular, the SH group of the cysteine amino acid bonded to the C 'end of the acrylic (Acryl) group of the hydrogel and the synthetic peptide immobilized on the hydrogel Amide is formed by bonding.

In addition, the hyaluronic acid-based hydrogel in the present invention preferably further comprises a polyethylene (poly ethylene glycol). In the present invention, the PEG was used as a crosslinking agent, and since the gel was formed within 10 minutes by Michael type addition reaction in the thiol group of the used PEG (pH 8, 37 degrees), it can be made into an injection support. Injectable scaffolds have the advantage of maintaining the concentration of the drug at the treatment site for a long time by controlling the rate so that the drug is slowly released into the damaged tissue, and locally delivering the drug to the affected area.

According to another aspect of the present invention, the present invention provides a pharmaceutical composition for tissue regeneration containing the synthetic peptide as an active ingredient.

In the pharmaceutical composition for tissue regeneration of the present invention, it may include a conventional tissue regeneration function, preferably tissue regeneration is characterized in that the heart regeneration. In the present invention, thymosin beta-4 consisting of 43 amino acids known to have an effect on heart regeneration by promoting angiogenesis, in particular, it is effective in the target site Ac-SDKP, which is part of the peptides that are active in angiogenesis It is about the technology to release the heart to regeneration. In order to reliably deliver Ac-SDKP, a small peptide fragment, to a target site, a peptide that is secondarily cleaved by peptidase and MMP secreted during cardiac remodeling is designed and finally delivered to a 170 kDa hyaluronic acid hydrogel. Type support. The structure was applied to a myocardial infarction model in a clinic state in which the heart function was significantly reduced.

The compositions of the present invention may be prepared by methods known in the pharmaceutical art for use as therapeutic agents, and may be mixed with pharmaceutically acceptable carriers, excipients, diluents, etc., to powders, granules, tablets, capsules, or injections. It can be prepared and used in the formulation of such. They can also be administered parenterally (eg, applied intravenously, subcutaneously, intraperitoneally or topically) or orally.

The dosage of the therapeutic agent according to the present invention may be appropriately selected according to the age, sex, weight, health condition, symptoms of the disease, administration time, administration method of the patient, preferably 0.1 to 100 mg per day for adults May be administered.

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for illustrating the present invention, and thus the scope of the present invention is not construed as being limited by these embodiments.

Statistical analysis.

All data are mean ± standard deviation (SD). Data analysis was performed using OriginPro 7.5 (OriginLab Co., Northampton, Mass., USA). Comparative analyzes between the two groups were performed using Student's t- test. P <0.05 was interpreted as a significant difference.

Preparation Example 1. Preparation of a solid hydrogel to which a synthetic peptide (Ac-SDKP-D-GPQG-IWGQC) was immobilized.

After dissolving hyaluronic acid (Lifecore Biomedical Co., Chaska, MN, USA) in 40 ml of distilled water to prepare a 0.25 mmole hyaluronic acid solution, EDC (1-Ethyl-3- (3-dimethylaminopropyl) -carbodimide) (Sigma- Adip in the presence of Aldrich, Inc., St. Louis, MO, USA) (0.24 g, 1.25 mmole), 1-hydroxybenzotriazole hydrate (HOBT) (Fluka Chemical Co., Buchs, Switzerland) (0.17 g, 1.25 mmole) It is reacted with an acidic dihydrazide (Fluka Chemical Co., Buchs, Switzerland) (2.2 g, 12.5 mmole) at room temperature for 8 hours. The reaction prepared above was then dialyzed in 100 mM NaCl solution for 2.5 days using a dialysis membrane (MWCO 14,000, SpectraPor; Rancho Dominguez, Calif., USA) and then dialyzed again in distilled water for 1 day. N-Acryloxysuccinimide (NAS) (Polyscience, Inc., Warrington, PA, USA) (0.5 g, 3 mmole) was added to the dialyzed reaction and reacted for 12 hours. The prepared reaction was dialyzed in 100 mM NaCl solution for 2.5 days, and then dialyzed again in distilled water for 1 day. Then, lyophilization for 3 days to obtain a powdered acrylated hyaluronic acid. Thereafter, acrylated hyaluronic acid was dissolved in triethanolamine-buffered solution (TEA; 0.3 M, pH 8) for gel preparation, and then thiolated polyethylene glycol PEG-SH ₄ (MW 10,000), a crosslinking agent, was added. The molar ratio of acrylic group and thiol group is added at 1: 1. Hyaluronic acid based hydrogels are prepared using the Michael-type addition reaction. Subsequently, the peptide solution dissolved in physiological saline for immobilization of the peptide (Ac-SDKP-D-GPQG-IWGQC, MW 1529.6) is mixed with the reaction mixture to form a hydrogel in solution. 1 shows a schematic of a support including peptide design and developed peptides.

Preparation Example 2 Preparation of a Myocardial Infarction Model for In Vivo Experiments.

Six-week-old Sprague-Dawley male rats were anesthetized with enflurane (Choong Wae Pharma Co., Korea) and inserted 2% isoflurane (Choong Wae Pharma Co., Korea) and 0.3 L / min oxygen into the trachea. Maintain anesthesia. The mice were fed a 2.5 mL volume of oxygen to have a breath rate of 80 cycles per minute. The left chest of the animals was thoracic and the end of the approximately 2-3 mm left anterior descending (LAD) end of the coronary artery was tied with a 7-O polypropylene suture. As a result, it was confirmed that myocardial infarction was made by observing color change in the ischemic part of the myocardium. After expanding the lungs again, the chest was sutured with 3-O silk and the muscles and skin were also sutured with 3-O silk. One day after surgery to prevent infection, one dose of 5% cefazolin sodium (Chong Kun Dang Pharm., Korea) was intramuscularly injected. The animals were physically ventilated until they awoke from local anesthesia. Animals for the simulated experiments performed the same procedure except for the LAD suture process.

Example 1 Cardiac Function Analysis Experiments of Myocardial Infarction Model Animals.

In vivo relative to pressure-dose For evaluation, the animals used in the experiment were first divided into three groups (n = 6 for each group).

1. control group: sham operation (SHAM) group and myocardial infarction (MI) group

2. Group injected with 170 kDa hyaluronic acid based hydrogel (170HA)

3. Group injected with 170 kDa hyaluronic acid based hydrogel (Tb4F / 170HA) immobilized with peptide

Four weeks after the myocardial infarction of the animals of each group, the thoracic infarct was synthesized with a hydrogel (total volume; 50 μl) using a 100 (micro) Hamilton syringe and a 26-Gage needle to the pericardium at the edge of the infarct. Was injected. Then 4 weeks after injection, cardiac function was assessed using Miller catheter. Surviving rats of the above-described groups were subjected to local anesthesia in the same manner as in Preparation Example 2. A 2F Micro-tip P-V catheter (Model SPR-838; Millar Instruments Inc, Houston, TX, USA) was then inserted through the right carotid artery into the left ventricle. After the catheter was connected to a pressure conductive system (MPVS-300, Millar Instruments, USA), continuous signals of pressure and volume were monitored in real time using specialized software (Chart 5 v5.4.2, ADInstruments, Australia). , Cardiac Output (CO), Ejection Fraction (EF), Stroke Volume (SV) and Myocardial Elasticity, which are indicators of the contractility of the heart. Parameters of arterial elasticity (Ea) were measured using pressure-dose analysis of differentiated heart (PVAN v3.5, Millar Instruments, USA). After stabilization, the LV pressure-capacitance loop was recorded based on the baseline based on the obtained signal. 2 to 5 show the results of quantitative analysis of left ventricular function. Observation of left ventricular function showed that the MI group was clearly inferior in function to the SHAM group. In addition, since there was no significant difference between the 170HA group and the MI group, only 170 kDa hyaluronic acid-based hydrogel had no significant effect on the recovery of heart function. On the other hand, in the Tb4F / 170HA group injected with hydrogel with peptides cut by MMP protease, all of the indicators except cardiac output showed significant improvement compared to 170HA and MI groups.

Example 2. In vivo tissue regeneration experiment using a hydrogel immobilized peptide.

After cardiac function analysis, each animal's heart was collected for histological evaluation. The heart was stopped by injecting formalin using the cardiac expander. In addition, the stopped heart was cut and then fixed with formalin. Three short axis, 3 mm thick slices were then obtained from the ventricles, then treated with paraffin, and then all slices were cut to 5 μm thick. Left ventricular tissue was obtained by Masson's Trichrome staining. After the image was acquired by optical microscope, quantitative measurement of infarct size and myocardial thickness was performed using computer-aided design (CAD). As a result of observing the left ventricle morphology of each group by Masson's trichrome staining, it was confirmed that the myocardium was significantly thinner and the infarct area was larger in the MI group than the SHAM group as shown in FIG. 6. The 170HA group was not significantly different from the MI group, but the Tb4F / 170HA group was observed to have a significantly thicker myocardium than the MI group.

Infarct size and myocardial thickness were also measured to confirm myocardial regeneration. Infarct Size is a comparison of the size of the infarcts in the left ventricle. The ratio of the lengths of infarcts to the total length of the endocardium and epicardium was averaged. Myocardial thickness was calculated as the ratio of myocardial thickness at the infarct to the thickness of normal myocardium. 7 shows Infact Size (%) measurement results. As shown in FIG. 7, the Tb4F / 170HA group showed a significant difference when compared with the MI group and the 170HA group, and the infarct area was reduced. 8 shows the results of myocardial thickness measurement. As a result of comparing myocardial ratio between normal myocardium and infarct, it was observed that myocardium was thickened in Tb4F / 170HA group compared with MI group and 170HA group.

As described above, according to the present invention, the thymosin beta-4-derived Ac-SDKP peptide having a tissue regeneration effect may be used as a pharmaceutical composition for tissue regeneration such as cardiac regeneration by controlling the release rate according to in vivo activity. .

As described through the above examples, the present invention relates to the development and application of a next generation smart release support whose release rate is controlled according to in vivo activity of thymosin beta-4-derived Ac-SDKP peptide for tissue regeneration. As a result of injecting the hydrogel injection scaffold immobilized with Ac-SDKP peptide into the terminal stage of infarction, which is difficult to regenerate, current tissue reconstruction was found to be significantly better than that of the control group. Could increase. Therefore, the present invention is expected to be used as a pharmaceutical composition for regeneration of tissue containing Ac-SDKP peptide having a tissue regeneration function derived from thymosin beta-4 excellent as an active ingredient.

1 shows a schematic diagram of a support including peptide design and developed peptides.

Figure 2 shows a quantitative analysis of left ventricular function, showing the cardiac output (Cardiac Output, CO) measurement results.

Figure 3 shows the quantitative analysis of the left ventricular function, showing the ejection coefficient (Ejection Fraction, EF) measurement results.

4 shows a quantitative analysis of left ventricular function, and shows the results of single stroke volume (SV) measurement.

5 shows a quantitative analysis of left ventricular function, and shows the results of arterial elasticity (Ea) measurement.

Figure 6 shows the shape of the left ventricle of each group by Masson's Trichrome staining.

Figure 7 shows a quantitative analysis of the left ventricle form, showing the results of Infarct Size (%) measurement.

Figure 8 shows a quantitative analysis of the left ventricle form, showing the results of myocardial thickness measurement.

<110> Korea University Industrial & Academic Collaboration Foundation <120> Smart releasing scafford of Thymosin beta-4 originated Ac-SDKP          peptides for tissue regeneration <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 14 <212> PRT <213> Homo sapiens <400> 1 Ser Asp Lys Pro Asp Gly Pro Gln Gly Ile Trp Gly Gln Cys   1 5 10  

Claims (14)

A synthetic peptide having the amino acid sequence of SEQ ID NO: 1, wherein the thymosin beta-4-derived Ac-SDKP peptide, a peptide cleavable by matrix metalloprotease (MMP), and cysteine amino acids are bonded in a N '→ C' direction. The amino acid sequence of claim 1, further comprising an amino acid (D) for cleavage by a peptidase between the thymosin beta-4-derived Ac-SDKP peptide and a peptide cleavable by MMP. Synthetic peptides. delete The synthetic peptide having the amino acid sequence of SEQ ID NO: 1, wherein the thymosin beta-4-derived Ac-SDKP peptide is acetylated. delete A hydrogel in which the synthetic peptide according to any one of claims 1, 2 and 4 is immobilized. The hydrogel of claim 6, wherein the hydrogel is acrylated. The hydrogel of claim 6, wherein the SH group of the cysteine at the C 'end of the synthetic peptide is amide-bonded with an acryl group of the hydrogel. The hydrogel of claim 6, wherein the hydrogel is an acrylated hyaluronic acid. The hydrogel of claim 6, wherein the hydrogel further comprises PEG (Poly Ethylene Glycol). The hydrogel of claim 6, wherein the hydrogel is injectable. A pharmaceutical composition for myocardial regeneration comprising the synthetic peptide according to any one of claims 1, 2 and 4 as an active ingredient. The pharmaceutical composition for myocardial regeneration according to claim 12, wherein the synthetic peptide is immobilized on a hydrogel. delete

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