KR100919603B1 - Sustained Release of Growth Hormone Using Injectable Hydrogel for Sustained Release of Growth Hormone - Google Patents

Abstract

The present invention relates to an injectable sustained release formulation obtained by using a hydrogel for preparing an injectable sustained release formulation of a bio-growth hormone, and to continuously and uniformly release a growth hormone of a human or animal while maintaining biological activity. In addition, these sustained release formulations can provide hydrogels that not only allow local drug delivery but also significantly reduce the initial release of the drug, with minimal damage to the injection site.

Description

Injectable sustained release formulation obtained by using a hydrogel for the preparation of an injectable sustained release formulation of bio-growth hormones {Sustained Release of Growth Hormone Using Injectable Hydrogel for Sustained Release of Growth Hormone}

The present invention relates to an injectable sustained release formulation obtained using a hydrogel for preparing an injectable sustained release formulation of a biogrowth hormone, wherein the growth hormone maintains biological activity in vivo using a biodegradable and biocompatible polymer, It relates to an injectable sustained release formulation that is encapsulated to be continuously and uniformly released into the body.

The novel hydrogel-based delivery systems are useful for many biomedical applications, including controlled drug delivery and tissue engineering. Of these, injection hydrogel formulations reduce surgical time, minimize damage to muscle contractions, reduce wounds and postoperative pain and allow for rapid recovery in a cost effective manner. In order to transport cells and macromolecules such as therapeutic proteins / peptides, chemical or physical gelation used in injectable formulations employs methods that can be formed in the presence of biomolecules.

One of the major problems in the development of sustained-release systems for such therapeutic proteins is that the protein drug preserves its prototype, which is generally caused in vivo by instability of the protein drug due to proteolytic / hydrolytic decline. vivo ) half-life is short. In addition, because of the rapid clearance through the kidneys, several injections (which can reduce patient adaptability) or increased doses (which can induce local toxicity and cause severe metabolic immune responses) are necessary to maintain therapeutically active concentrations. Needed. In contrast to low molecular weight hydrophobic agents, the three-dimensional structure of a protein is easily broken by external resistances contacted (eg, temperature, pH or organic solvents) in the preparation of the carrier, leading to deamidation, oxidation, denaturation or aggregation of the protein. Which in turn may lead to biochemical inactivation of the protein and further to an immune response. For example, polylactide-glycolide copolymer poly (lactide-co-glicolide) (PLGA) microparticles have been widely used as a sustained release system for hydrophobic drugs of low molecular weight, but have been applied to such protein drugs. When it is done, it shows many problems due to the modification of protein drugs.

Human growth hormone (hGH), on the other hand, is a protein composed of 191 amino acids with a molecular weight of approximately 22 kDa, which promotes bone growth and plays an important role in regulating protein, lipid and carbohydrate metabolism. Thus, the hGH has been used to treat children with late development caused by growth hormone deficiency, Turner syndrome or chronic kidney disease, and has also been applied to growth hormone replacement therapy in adults. Conventional hGH administration is a subcutaneous injection every day or three times a week for many years, has a short half-life, causing renal toxicity or requiring repeated injections. Thus, hGH has been a major target in the development of sustained release drug delivery vehicles. Conventional approaches to making sustained-release drug carriers directly modify hGH with PEG to reduce the rate of clearance or capture it in biodegradable microparticles to allow gradual release of hGH. The latter is focused on encapsulating hGH into carriers made physically of spherical or hydrogel type using PLGA or poloxamer-related systems, so the sustained release of hGH diffuses from biodegradable substrates rather than interacting with carrier substrates. By decomposition. However, such systems do not fully meet medical needs. In particular, in the case of releasing proteins from PLGA microparticles prepared by the double emulsion method, the release pattern has a very large initial release and subsequently leads to incomplete release. In addition, denaturation and aggregation of the added protein occurs at the aqueous / organic interface generated during the process. Recently, a carrier using PLGA microparticles (Nutropin ^Depot® ) has been approved by the US FDA to treat hGH deficient children. However, clinical studies have observed slower growth and increased anti-hGH antibody responses from children treated with it daily than those treated with hGH injections, presumably from high biodegradable materials as well as high initial release. It is believed to be due to induced inflammation.

As related related art, Korean Patent Application No. 10-2001-0042196 (the name of the invention: biodegradable pharmaceutical composition and microsphere formulation capable of sustained release of human growth hormone) includes a biodegradable polymer microsphere formulation for sustained release of human growth hormone, Disclosed is the release of fine particle formulations, hydrogel formulations by diffusion and dissolution or dissolution of the carrier in the carrier. However, there was a disadvantage that does not solve the problem of initial excessive release.

Therefore, the present inventors have made intensive studies to solve the technical problems of the conventional growth hormone delivery system, and developed a hydrogel to be a sustained release delivery system of growth hormone by using an attraction force acting between the biopolymer and the growth hormone. In this way, the protein drug (growth hormone) is effectively stabilized, the initial release is reduced, and the slow release of the protein drug is induced to complete the present invention.

Accordingly, it is an object of the present invention to provide a polysaccharide functionalized hydrogel that can be easily injected into the body while minimizing damage to the transplant site and is excellent in the stability and sustained release of protein drugs.

Another object of the present invention is to provide an encapsulated sustained release formulation that can be enclosed in a hydrogel and can significantly reduce the initial release.

According to the present invention, a hydrogel for preparing a sustained release formulation of a growth hormone; And an effective amount of a biogrowth hormone; wherein the biogrowth hormone has a form entrapped in a hydrogel and is sustained release through continuous binding affinity with a bioactive polysaccharide. Hydrogels for the preparation of sustained release formulations of growth hormone are provided by injectable sustained release formulations of biological growth hormone, characterized in that obtained by gelling heparin and diacrylate polyethylene glycol.

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EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated concretely.

In the present invention, the hydrogel is made of a bioactive polysaccharide having a binding force to the growth hormone.

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In the present invention, the natural polysaccharide interacts with the growth hormone, and thus, all polysaccharides that play a role in stabilizing the hormone by maintaining the three-dimensional structure of the growth hormone, increasing biological activity and inhibiting hydrolysis may be used.

Representative examples of the polysaccharide that can be used in the present invention may include, but are not limited to, heparin, hyaluronic acid, chitosan, alginate or combinations thereof having a thiol functional group represented by Formula 1 below. Among them, it is preferable to use heparin, which is an anionic polysaccharide approved by the US FDA, having weak binding ability and no in vivo toxicity with human growth hormone which is a model protein of the present invention.

Bioactive polysaccharides usable in the present invention is one selected from natural polysaccharides such as heparin, hyaluronic acid, chitosan, dextran, alginate, chondroitin sulfate, and the like. Based on the above materials to form a hydrogel.

More specifically, the one or more selected polysaccharides are functionalized with a functional group selected from acrylates, diacrylates, oligoacrylates, and the like, and multi-thiol functional groups, respectively. Hydrogel is formed by Michael-type addition reaction between functional groups to release biogrowth hormone.

In particular, if heparin-based manufacturing has a weak binding ability with human growth hormone, while maintaining the active structure of the drug without degeneration or irreversible aggregation of the drug to significantly reduce the initial drug release to promote slow release.

In this case, the biological growth hormone is not limited thereto, but is meant to include human growth hormone or animal growth hormone.

The present invention also provides a bioactive polysaccharide-based hydrogel (1) having a functional group capable of Michael-type addition reaction, and (2) sustained release of a protein drug based on a biocompatible polymer and polysaccharides. do.

The hydrogel comprises (1) two or more components having a functional group selected from acrylates, diacrylates, oligoacrylates and the like, and a multi-thiol functional group.

The component consists of a synthetic high molecular material that is biocompatible with the natural polysaccharides. Specifically, the natural polysaccharide is not limited thereto, but as described above, heparin, hyaluronic acid, chitosan, dextran, alginate, chondroitin sulfate, etc. It is selectable.

In particular, if heparin-based manufacturing has a weak binding ability with human growth hormone, while maintaining the active structure of the drug without degeneration or irreversible aggregation of the drug to significantly reduce the initial drug release to promote slow release.

In addition, in the present invention, "biocompatible polymer" refers to a polymer having tissue compatibility and blood compatibility that does not necrolyse or coagulate tissue in contact with living tissue or blood. do.

Biocompatible polymers that can be used in the present invention, and a typical example of a polymer having a functional group capable of crosslinking through a Michael-type addition reaction includes a diacrylated polyethylene glycol of Formula 2 (hereinafter, 'PEGDA'). ') Is used, but is not limited thereto.

The biocompatible synthetic polymer material usable in the present invention includes at least one selected from polyethylene glycol, polyethylene oxide, polyvinyl alcohol, or other synthetic polymers having biocompatibility. It is preferable to use the kind which consists of and does not react with the surrounding protein especially.

Components such as the above-described natural polysaccharides and synthetic polymer materials having biocompatibility are functionalized with the functional groups described above to prepare hydrogels by Michael-type addition reaction to provide sustained release of biological growth hormone.

More specifically, (1) the reaction of a material having a functional group such as acrylates, diacrylates, oligoacrylates and the like having a multi-thiol functional group Gelation is formed by (2) one or more selected from polysaccharides such as heparin, hyaluronic acid, chitosan, dextran, alginate, chondroitin sulfate A hydrogel is formed based on the material, and as a crosslinker, in addition to such polysaccharides, polyethylene glycol, polyethylene oxide, polyvinyl alcohol, or other synthetic compounds having biocompatibility Hydrogels are prepared by functionalizing one or more of the polymers to provide slow release of human or animal growth hormone.

In this case, the biological growth hormone is not limited thereto, but is meant to include human growth hormone or animal growth hormone.

On the other hand, as used herein, "binding capacity" means a physical bond that specifically binds only between the protein drug and the polysaccharide according to the present invention to form a complex in a relatively stable state in vivo. In particular, the concept includes a polysaccharide-protein interaction that stabilizes the protein by maintaining the three-dimensional structure of the protein, increasing biological activity and inhibiting hydrolysis.

 Furthermore, according to the present invention, it is possible to provide a sustained-release drug delivery system comprising a polysaccharide-based hydrogel and an effective amount of the growth hormone.

 In this case, the bio-growth hormone is characterized in that it has a form entraped with a hydrogel without chemically bonding to the hydrogel, specifically, the bio-growth hormone includes a human recombinant growth hormone or an animal recombinant growth hormone. can do.

 According to the sustained-release preparation provided by the present invention, it can be injected by injection so that it can be easily implanted into the body while minimizing damage to the implanted site. It has an advantage that can be greatly reduced. In addition, such sustained-release preparations may be usefully applied to the development of protein drug filling and sustained protein drug delivery systems that maintain activity.

Hereinafter, the content of the present invention will be described in detail through examples. However, the following examples are provided to explain the contents of the present invention and do not limit the scope of the present invention.

For reference, specific information on the materials used in the following examples is as follows.

Heparin (sodium salt from pig intestinal mucosa, Mw 12 kDa) was purchased from Cellsus (Cincinnati, Ohio, US). Poly (ethylene glycol) diacrylate (PEG-DA, Mw 3.4 and 6 kDa, 98%) and tetrafunctional poly (ethylene glycol) (PEG-SH ₄ Mw 10 kDa) from SunBio (Anyang City, Korea) Purchased.

Potassium phosphate monobasic, sodium phosphate dibasic, sodium azide and tris (hydroxymethyl) aminomethane were purchased from Sigma (St. Louis, MO, USA), and sodium chloride and potassium chloride were obtained from Merk (Darmstadt, Germany). ).

PBS with 0.05% NaN ₃ (0.1 mol / L PBS solution with phosphate buffered saline, 0.138 mol / L NaCl and 0.0027 mol / L KCl, pH = 7.4) was prepared with potassium phosphate monobasic and sodium phosphate dibasic. Lysosomes were obtained from Sigma, and bovine serum albumin was obtained from AMRESCO (Solon, Ohio, USA). Human growth hormone (hGH) was donated by LG Biotech (Seoul, Korea).

All chemicals were used without further purification. The sterile syringe filter used ^Whatman® (Florham Park, New Jersey, USA) 0.2 μm.

Example

Example  1: heparin-based Hydrogel  Produce

As shown schematically in FIG. 1, all of the hydrogels prepared in the present invention were formed by the Michael-type addition reaction. First, diacrylate polyethylene glycol (hereinafter, 'PEGDA' molecular weight 6 kDa) was sufficiently dissolved in a solution of Phosphate buffer saline. And thiol-functionalized heparin (hereinafter referred to as 'Hep-SH', thiolization degree) having a thiol functional group so that each functional group (acrylate functional group and thiol functional group) can be crosslinked in a 1: 1 molar ratio. : It melt | dissolved together by adding the diacrylate polyethylene glycol reaction solution which has already melt | dissolved in 40% and molecular weight 12 kDa). The concentration of the final reaction solution thus prepared was 10.0, 15.0% (w / v) and pH was 7.4. The reaction solution formed a hydrogel within 10 minutes under 37 ° C.

Comparative example  1: polyethylene Glycol  Based Hydrogel  Produce

Diacrylate polyethylene glycol (hereinafter 'PEGDA' molecular weight 3.4 kDa) and tetra are used to prepare hydrogels having similar structural characteristics, that is, average molecular weights of crosslinked chains, prepared in Example 1. Tetra-functional polyethylene glycol (hereinafter 'PEG-SH ₄ ', molecular weight 10 kDa) was selected. Polyethylene glycol-based hydrogel was prepared by the same method as the method for preparing heparin-based hydrogel.

Example  2: model protein of human growth hormone Filling  Heparin based Hydrogel  Produce

Human growth is achieved by mixing diacrylate polyethylene glycol with heparin with a thiol functional group in a final reaction solution with final concentrations of 10.0 and 15.0% (w / v) of 1:10 for model protein human growth hormone and precursor mass. Heparin-based hydrogels filled with human growth hormone could be prepared within 10 minutes by filling 1 mg of the hormone and gently mixing and placing it under 37 ° C.

Comparative example  2: model protein of human growth hormone Filling  Polyethylene Glycol  Based Hydrogel  Produce

In Example 2, a polyethylene glycol hydrogel filled with human growth hormone was prepared by the same method of filling human growth hormone.

Experimental Example  1: heparin-based Hydration gel  Polyethylene Glycol Based hydrogel  Elastic modulus comparison measurement

The modulus of elasticity of 10.0, 15.0% (w / v) heparin-based hydrogels and polyethylene glycol-based hydrogels was measured using a rheometer (Gemini, Malvern Instrument, England). 100 μl of the final reaction solution of heparin-based hydrogel and polyethylene glycol-based hydrogel prepared in Example 1 and Comparative Example 1 was placed on a Sandblast parallel plate (diameter 15 mm), and 37 ° C. The change of elastic modulus (G '(Pa)) of the hydrogel was compared by applying a strain of 0.1% at 500 μm gap and frequency of 1 rad / s.

As shown in Fig. 2 (a), the elastic modulus of the same concentration of the hydrogel was almost the same, based on the experimental results, the microstructural characteristics of the heparin-based hydrogel and polyethylene glycol-based hydrogel, that is, It is confirmed that the effective mesh sizes are about the same.

Meanwhile, FIG. 3 shows a photograph of the model of the hGH structure using RasMol software. FIG. 3 (a) shows the basic amino acid residues of hGH (8 lysines, 10 arginine and 3 histidine residues) and FIG. 3 (b) shows the surface-exposed charged residues.

Experimental Example 2: Comparative measurement of affinity with heparin for various proteins

Heparin affinity chromatography confirms whether human growth hormone has affinity with heparin compared to Lysozyme, which has a high affinity with heparin, and bovine serum albumin (BSA), which has no affinity with heparin. It was used to Bovine serum albumin, heparin, and lysozyme with a concentration of 250 μg / mL were treated with heparin using a heparin-filled column (AFpak AHR-894 affinity column; 8.0 mmID> 50 mmL, Shoko, Co., Tokyo, Japan). The degree of affinity of was measured. As mobile phase, sterileized de-ionized water was used as mobile phase A and 10 mM Tris-HCl buffer (pH 7.4) with 300 mM NaCl was used as mobile phase B. The mobile phase was able to gradually increase the salt concentration step by step by time program (10% B after 10 minutes, 20% B after 20.00 minutes, 50% B after 30 minutes, and 100% B after 40 minutes), 0.2 Samples were measured in the wavelength region of 220 nm for 40 minutes at a flow rate of mL / min.

4 is a graph showing that the longer the retention time, the greater the affinity with heparin as a result of the heparin affinity experiment. Lysoso, which is well known for its high affinity with heparin, had the longest retention time expected. And it is noteworthy in Figure 4 was confirmed that when compared with the serum serum albumin and human growth hormone human growth hormone was detected later than the serum serum albumin, from which the human growth hormone has a weak binding force with heparin, heparin It was confirmed that the hydrogel based on the gel could contribute to the slow release of human growth hormone.

Experimental Example  3: From hydration gel  Comparison of cumulative percent release of human growth hormone released

In order to confirm the sustained-release effects of protein drugs on heparin-based hydrogels and polyethylene glycol-based hydrogels filled with human growth hormone prepared in Example 2 and Comparative Example 2, the following in vitro ( in In vitro conditions were measured for the release pattern.

The final reaction solution filled with human growth hormone was injected into a 15 mL centrifuge tube, and then stored at 37 ° C. for 2 hours to form a complete hydrogel, and an excess of PBS (Phosphate buffered saline, pH 7.4) satisfying infinite dilution conditions. , with 0.05% NaN ₃ ) solution was used to recover the released human growth hormone and the amount was quantified using the Micro BCA protein assay. The PBS solution used for human growth hormone recovery was replaced with a fresh PBS solution with each recovery and the samples were stored at −80 ° C. until protein quantification.

FIG. 5A shows humans released from heparin based hydrogels and polyethylene glycol based hydrogels at various mass ratios of human growth hormone and total precursor (hGH: polymer = 1: 10, 1: 5, 1: 2.5) This graph compares the cumulative release of growth hormone (%). As shown in FIG. 5A, in the case of polyethylene glycol-based hydrogels prepared at a mass ratio of 1: 5 and 10.0% (w / v), almost 100% of the amount of human growth hormone filling was released within a week. On the other hand, 10.0% (w / v) heparin-based hydrogels were released until 20 days regardless of mass ratio. Note that the solubility of the human growth hormone decreases as the amount of the human growth hormone is increased compared to the total amount of the precursor, that is, the mass ratio increases from 1:10 to 1: 2.5. Therefore, when the human growth hormone is released through the hydrogel, the solubility is low so that the undissolved human growth hormone must be released after it is dissolved in the hydrogel, that is, it is released in two stages, so it is 1: 2.5 mass ratio compared to 1:10 mass ratio. It was confirmed that the heparin-based hydrogel prepared by the more slowly released.

5b is released from heparin-based hydrogels and polyethylene glycol-based hydrogels according to the degree to which the carboxyl group (COOH) of heparin is modified to thiol group (SH) (heparin thiolation 20, 30, 40, 50%). This is a graph comparing the cumulative release of human growth hormone (%). As shown in FIG. 5B, in the case of polyethylene glycol-based hydrogels prepared at a mass ratio of 1:10 and 10.0% (w / v), almost 100% of the human growth hormone filling amount was released within a week. On the other hand, all of the 10.0% (w / v) heparin-based hydrogels were released until 20 days later. When the hydrogel is manufactured using heparin having a high degree of thiolation, the strength of the hydrogel is increased because of the high degree of crosslinking, and the ability to swell in an aqueous solution environment is reduced. Therefore, it was confirmed that the release of human growth hormone proceeded more slowly when using 50% of thiolated heparin than 20% of thiolized heparin to make hydrogel.

Experimental Example  4: From hydration gel  Observation of Stabilizing Effect of Released Human Growth Hormone

The stabilizing effect of the human growth hormone released from the human growth hormone-filled hydrogels prepared in Example 2 and Comparative Example 2 was observed as follows. First, the human growth hormone released from the hydrogel was recovered. The recovered human growth hormone was structurally preserved, i.e., stabilized, through an aqueous size exclusion PROTEIN KW-800 Series column (SEC, 8.0 mmID> 300 mmL, Shoko, Co., Tokyo, Japan). The effect was analyzed. As a mobile phase, 10 mM phosphate buffer with 30 mM of NaCl was used, and human growth hormone released at a flow rate of 1 mL / min and 220 nm wavelength was analyzed.

6 is a graph comparing the stability of human growth hormone released from heparin-based hydrogel and polyethylene glycol-based hydrogel as a result of the stabilization effect according to the degree of integrity of the monomeric form (monomeric form). As shown in FIG. 6, human growth hormone (hGH, molecular weight 22 kDa) having a lower molecular weight than bovine serum albumin (BSA, molecular weight 66 kDa) was detected later. In comparison with deliberately modified human growth hormone (denatured hGH) and native human growth hormone (native hGH), the dimeric form of the deliberately modified human growth hormone was identified. Based on this, we compared the stabilizing effects of the human growth hormone released from the hydrogel. From both the heparin-based hydrogel and the polyethylene glycol-based hydrogel, the incidence of dimer form was less than 2%. It was confirmed that the packed protein was stabilized.

In conclusion, the sustained release preparation provided by the present invention was released while maintaining the activity of the protein filled in the heparin-based hydrogel, the binding ability with heparin can be confirmed that induced the sustained release of the protein drug of interest. Such sustained release preparations are also injectable with minimal damage to the living body.

According to the sustained-release formulation according to the present invention, it is possible to easily transplant into the body while minimizing damage to the transplantation site, and is excellent in the stability and sustained release of protein drugs, and in particular, has the advantage of greatly reducing the initial release of the drug. In addition, such sustained release formulations may be usefully applied to the development of protein drug filling and sustained protein drug delivery systems that maintain activity.

1 is a diagram schematically showing an ideal network structure of heparin-based hydrogels and PEG-based hydrogels.

2 is a graph showing a comparison of the elastic modulus according to the concentration of heparin-based hydrogel and polyethylene glycol (PEG) -based hydrogel through Rheometer, (a) is time Contrast, (b) is a diagram showing the result of the frequency contrast strength.

Figure 3 is a photograph of the hGH structure using RasMol software, where (a) is the basic amino acid residues of hGH (8 lysine, 10 arginine and 3 histidine residues), and (b) is surface-exposed charge. Residues.

Figure 4 is a graph showing the comparison of the measurement of affinity with heparin by bovine serum albumin (BSA), lysozyme (lysozyme) and human growth hormone (hGH) using a heparin column using a heparin column.

5a is a model released from heparin based hydrogels and polyethylene glycol based hydrogels according to various mass ratios (hGH: polymer = 1: 10, 1: 5, 1: 2.5) between human growth hormone and total precursors It is a graph comparing the cumulative release amount (%) of human growth hormone, a protein,

5b is released from heparin-based hydrogels and polyethylene glycol-based hydrogels according to the degree to which the carboxyl group (COOH) of heparin is modified to thiol group (SH) (heparin thiolation 20, 30, 40, 50%). It is a graph comparing the cumulative release (%) of the model protein human growth hormone.

Figure 6 compares the stability of human growth hormone released from heparin-based hydrogels and polyethylene glycol-based hydrogels using a size exclusion column according to the degree of preservation of the monomeric form. It is a graph.

Claims (10)

delete delete delete delete delete delete delete delete Hydrogel for preparing sustained release formulations of biological growth hormone; And an effective amount of a biogrowth hormone; The biogrowth hormone has a form entrapped in a hydrogel, and is released through continuous binding affinity with a bioactive polysaccharide, Injectable sustained-release formulation of bio-growth hormone, characterized in that the hydrogel for preparing the sustained-release formulation of the biological growth hormone is obtained by gelling heparin and diacrylate polyethylene glycol. The method of claim 9, The bio-growth hormone is an injectable sustained release formulation of bio-growth hormone, characterized in that it comprises human growth hormone or animal growth hormone.