KR100329336B1 - Hyaluronate microparticles for sustained release of a protein drug - Google Patents

Abstract

The present invention provides a solid fine particle formulation having an average particle size of 0.1 to 200 μm, wherein the solid surface of hyaluronic acid or an inorganic salt thereof containing a protein or peptide drug is coated with a lipophilic substance. Long-term sustained release while maintaining biological activity. The fine particle formulation of the present invention has excellent dispersibility in a non-aqueous solution due to the lipophilic surface, and furthermore, in the oil-in-water type due to the lipophilic property of the solid particle surface when redispersed in the aqueous solution again dispersed in the non-aqueous solution ( spontaneously forming oil-in-water emulsions. Therefore, it is characterized by the inclusion of fine particles in the solid phase in the non-aqueous (oil) dispersed phase of the emulsion to form a low viscosity dispersion can be formulated into an easy injection.

Description

Hyaluronate microparticles for sustained release of a protein drug

The present invention relates to sustained release fine particle formulations formulated to sustain release of biologically active protein and peptide drugs in the body and to injections comprising the same. More specifically, the present invention includes a microparticle formulation having an average particle diameter of 0.1 to 200 μm and a formulation having a lipophilic substance coated on the surface of the microparticles of a solid phase containing a protein or a peptide drug in hyaluronic acid or an inorganic salt thereof. To a non-aqueous injection and oil-in-water emulsion injection, the fine particle formulation of the present invention is easy to inject due to the excellent dispersibility of the non-aqueous solution and oil-in-water emulsion, the drug in the body after administration It has the property of being released continuously for a long time while maintaining.

Protein or peptide drugs have a short duration of activity in the body and a low absorption rate by administration methods other than injection, so long-term administration of these drugs requires repeated injections of these drugs. For example, human growth hormone, which is administered for the treatment of pituitary deficiency in children, is continuously injected for 6 months or more every day or every other day. To alleviate this inconvenience, there is a need for sustained release formulations that continuously release the drug in a single dose.

For protein or peptide drugs, such as human growth hormone, microparticles are produced that surround these drugs with a biodegradable polymer matrix, which causes the drug to be released slowly as its matrix material is slowly degraded and removed in the body upon its administration. There is an active research on sex formulations. That is, biolactic and biodegradable polyester-based polylactide, polyglycolide, a copolymer thereof polylactide-co-glycolide (poly (lactide-co-glycolide)), By using micro-ortho-ester, polyanhydride, etc. to prepare microparticles containing protein drugs, protein drugs are slowly released as the polymer material is hydrolyzed during in vivo administration. (M. Chasin and R. Langer, ed., Biodegradable Polymers as Drug Delivery Systems , Marcel Dekker (1990); J. Heller, Adv. Drug Del. Rev. , 10 , 163 (1993)). Sustained release formulations of protein drugs using such synthetic polymers have slow release rates of the polymer and thus release the drug for weeks to months.

On the other hand, sustained-release formulations containing a drug using natural polymer materials have been studied. Examples thereof include gelatin, collagen, chitosan, carboxymethyl cellulose, alginate, and hyaluronic acid. These natural polymers generally absorb water easily to form gels, and in gels with very high viscosity, slow-release effects of protein drugs are obtained by the sustained release effect. Since the degradation enzymes of these natural polymers are present in the body, the drug release duration of the formulations using the natural polymers is usually less than a few days.

Since hyaluronic acid or its inorganic salt exists in vivo as a natural polymer material, it can be extracted from an animal or obtained by fermentation using microorganisms, and they are generally dissolved in water in the form of inorganic salts such as sodium to make a gel to provide an ophthalmic surgical aid, In addition to being used as a therapeutic agent for arthritis, the formed gel may be used as a sustained release formulation due to its very high viscosity, which reduces the diffusion rate of the drug. For example, U. S. Patent No. 5,416, 017 discloses a sustained release injection of erythropoietin using a gel having a concentration of hyaluronic acid of 0.01 to 3%, and Japanese Patent Application Publication No. 1-287041 (1989) discloses insulin Sustained release injections contained in gels with a concentration of 1%, and Japanese Patent Laid-Open No. 2-213 (1990) describe a sustained release formulation containing calcitonin, elkatonin, or human growth hormone in a 5% concentration of hyaluronic acid. Sustained release formulations containing granulocyte-colony stimulating factor (G-CSF) in a concentration of 0.5-4% hyaluronic acid gel have been reported (James Meyer, et al., J. Controlled). Rel ., 35 , 67 (1995).

In such a formulation, a protein drug that is dissolved in the gel of hyaluronic acid, so the viscosity is passed through the large gel matrix at a slower rate, but may indicate a sustained release effect, the hyaluronic acid gel in a few percent concentration of 10 ^{5 to} 10 ⁷ centipoise degree Since it has a high viscosity of, it is not easy to administer by injection, such as the use of a large diameter needle. In addition, since both the protein drug and hyaluronic acid in the gel form of hyaluronic acid formulation are already dissolved in water prior to the injection, the gel is easily diluted by the body fluid after the injection, so that the release of the drug does not last more than one day. For example, as reported in Japanese Patent Application Laid-Open No. 1-287041 (1989), when a sustained-release injection formulation containing insulin in a gel of 1% hyaluronic acid was injected into a rabbit, the effect of insulin on blood glucose concentration was lowered. Does not last more than 24 hours, and the formulation containing granulocyte-colony stimulator in a hyaluronic acid 2% gel (James Meyer, et al., J. Controlled Rel ., 35 , 67 (1995)) and interferon-α For formulations mixed together in a 1.5% gel of hyaluronic acid (US Pat. No. 5,416,017) the concentration of these protein drugs in experimental animal plasma drops rapidly to less than 1/10 of the initial concentration within 24 hours when administered to the experimental animals. In addition, there is no significant difference in the plasma concentration of the protein drug of the animal administered with the hyaluronic acid gel formulation compared with the control animal administered with the protein drug in the aqueous solution without hyaluronic acid.

The above reports do not show the possibility of denaturation of protein or peptide drug in the natural hyaluronic acid gel formulation, but the defect is that the gel is diluted by the body fluid during administration in the body and the release period does not last more than 1 day.

Natural type hyaluronic acid or its inorganic salt is soluble only in water, which forms a gel with a significantly high viscosity even at low concentrations. Due to this property, there has been no case of preparing a sustained release formulation in the form of a solid fine particle using a natural hyaluronic acid or an inorganic salt thereof.

On the other hand, synthetic hyaluronic acid-benzyl ester HYAFF, in which benzyl alcohol is reacted with a carboxyl group of natural hyaluronic acid^TMIs insoluble in water and soluble in organic solvents such as dimethyl sulfoxide. There are examples in which solid particles are prepared by emulsion solvent extraction using such hydrophobic acid derivatives (N.S. Nightlinger, et al., Proceed. Intern. Symp. Control. Rel. Bioact. Mater., 22nd, Paper No. 3205 (1995); L. Ilum, et al.,J. Controlled Rel.,29, 133 (1994). Briefly describing this method, protein drug particles are dispersed in a dimethylsulfoxide solution in which hyaluronic acid-benzyl ester is dissolved, and the dispersion is added to mineral oil to form an emulsion. After adding a solvent such as ethyl acetate mixed with dimethyl sulfoxide to the emulsion, dimethyl sulfoxide is extracted to obtain fine particles composed of protein drug particles and hyaluronic acid-benzyl ester.

In the case of microparticles using hyaluronic acid-benzyl ester, an organic solvent should be used in the preparation of the particles.In this case, the protein may come into contact with a hydrophobic organic solvent and may be denatured. This is high. In fact, in the release experiment of GM-CSF sustained-release microparticles using hyaluronic acid-benzyl ester, when benzyl ester was formed in 100% of the carboxyl group of hyaluronic acid, about 25% of GM-CSF was released from the microparticles during the first 1-2 days. No longer released until day (NS Nightlinger, et al., Proceed. Intern. Symp. Control. Rel. Bioact. Mater ., 22nd, Paper No. 3205 (1995)). In this case, the fact that the release does not persist despite the swelling of 75% of the water content in the microparticles within 3 hours indicates that the protein drug is denatured due to the interaction between the protein drug and hyaluronic acid-benzyl ester or the protein and the organic solvent. It suggests the possibility.

It is an object of the present invention to provide a sustained release fine particle formulation, injectable and aerosol formulation comprising the same, which are formulated to maintain the biological activity of the protein drug in the body and to continuously release the drug.

Figure 1 shows the results of in vitro release of the sustained release solid microparticle formulation of the present invention comprising human growth hormone (hGH).

Figures 2a and 2b is the result of observing the drug stability of the sustained-release solid microparticle formulation of the present invention comprising hGH by reverse phase high performance liquid chromatography (RP-HPLC), Figure 2a is released in the fine particle formulation of the present invention HGH is shown, and FIG. 2B shows the hGH in aqueous solution which is a raw material used to prepare the formulation of the present invention.

3A and 3B are the results of size exclusion chromatography (SEC) observation of the drug stability of the sustained-release solid microparticle formulation of the present invention comprising hGH, Figure 3a shows the hGH released in the fine particle formulation of the present invention 3B shows hGH in aqueous solution which is a raw material used for preparation of the formulation of the present invention.

In accordance with the above object, the present invention is a formulation in which a solid particle containing a protein or peptide drug in natural hyaluronic acid or an inorganic salt thereof is coated with a lipophilic material, and the drug is maintained for a long time while maintaining biological activity. Sustained release solid phase fine particle formulations having an average particle diameter of 0.1 to 200 μm, which are dispersed in a solution for injection, are provided.

In the solid microparticle formulation of the present invention, the surface of the solid microparticles is composed of a lipophilic substance, thereby providing a low viscosity injection formulation having excellent dispersibility in non-aqueous solutions such as vegetable oil. In addition, the addition of a buffered aqueous solution for injection in the state dispersed in the non-aqueous solution is characterized by the coating of the non-aqueous solution on the surface of the solid particles due to the affinity of the lipophilic material and the non-aqueous solution on the surface of the solid particles (coating) . This results in the formation of an oil-in-water emulsion and the inclusion of solid fine particles containing the drug in the non-aqueous phase, so that the solid particles contain the drug even when the injection is prepared in an aqueous buffer solution for injection. They do not have direct contact with water, and thus have longer drug release duration than conventional sustained-release formulations using hyaluronic acid gel, and are very viscous and easy to administer. In addition, due to the characteristics of the fine particle formulation it can be easily administered by making an aerosol formulation, the surface is composed of a lipophilic material is characterized by protection from moisture.

In the formulation of the present invention, since the drug is not in direct contact with the hydrophobic substance, the protein does not denature when hydrophobic substances are dissolved in an organic solvent such as hyaluronic acid-benzyl ester, and thus the drug is not denatured until 100% of the drug is released. There is this.

In order to prepare the fine particles of the present invention, the primary solid phase fine particles having an average particle size of 0.1 to 40 μm, preferably 1 to 10 μm, in which a protein or peptide drug is included in a natural type hyaluronic acid or an inorganic salt thereof, are prepared. Primary solid fine particles are prepared by a method such as spray drying or freeze drying, and includes a stabilizer that enhances the stability of the drug as necessary. Thereafter, the primary solid fine particles composed of the drug and hyaluronic acid (or inorganic salt thereof) are coated with a lipophilic substance to have an average particle diameter of 0.1 to 200 µm, preferably 1 to 100 µm, more preferably 1 to 50 µm, most Preferably, the solid fine particles of 1 to 20 ㎛ are prepared. The size of the finally prepared solid phase fine particles can be arbitrarily adjusted according to the content of the lipophilic substance.

Lipophilic coating materials that can be used in the present invention include lipids such as lecithin, phosphatidylcoline, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol and derivatives of these lipids; Fatty acids such as myristic acid, palmitic acid, stearic acid and salts of these fatty acids; Ester derivatives of fatty acids such as glyceryl stearate, sorbitan palmitate and sorbitan stearate; Surfactants such as poloxamer and waxes.

Drugs that can be used for the fine particles of the solid phase of the present invention include human growth hormone, bovine growth hormone, pig growth hormone, growth hormone releasing hormone, growth hormone releasing peptide, granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage- Granulocyte macrophage-colony stimulating factor (GM-CSF), macrophage-colony stimulating factor (M-CSF), erythropoietin, bone morphogenic protein, interferon (interferon), insulin, atriopeptin-III, monoclonal antibody, tumor necrosis factor (TNF), macrophage activating factor, interleukin, Tumor degenerating factor, insulin-like growth factor, epidermal growth factor, tissue plasminogen activator inogen activator) and urokinase.

Drug stabilizers include carbohydrates such as mannitol, proteins such as plasma albumin, amino acids such as glycine, lipids such as phosphatidylcholine, fatty acids such as stearic acid, inorganic salts such as phosphate, surfactants such as Tween ^R , and polyethylene Glycol or mixtures thereof may be used.

In the solid phase microparticles using the natural type hyaluronic acid of the present invention, the content of the protein or peptide drug ranges from 1% to 90% by weight based on the total weight of the solid particles coated with the lipophilic material, and the content of the stabilizer is lipophilic. The weight ranges from 1% to 90% by weight based on the total weight of the solid particles coated with the material. In addition, the content of the lipophilic material constituting the surface of the solid fine particles is in the range of 1% to 90% by weight based on the total weight of the solid particles coated with the lipophilic material.

Solid phase microparticle formulations using natural hyaluronic acid according to the present invention can be prepared in a sustained release injection by dispersing in a solution for injection. Injectable solutions include injectable aqueous solutions such as distilled water for injection and buffer for injection; And edible oils, mineral oils, squalene, squalene, squalane, cod liver oil, mono-, di- and tri-glycerides Or non-aqueous injectable solutions such as mixtures thereof. Examples of cooking oils include corn oil, olive oil, soybean oil, safflower oil, cotton seed oil, peanut oil, sesame oil, or these. And mixtures thereof. In addition, if necessary, the injection solution may further include a dispersant or a preservative.

Specifically, the injection preparation of the present invention is prepared by dispersing the solid fine particle formulation in a non-aqueous solution such as vegetable oil, or by adding a buffered aqueous solution for injection in the state dispersed in the non-aqueous solution (oil-in-oil emulsion) It can be prepared by forming an in-water emulsion.

On the other hand, the microparticle formulation of the solid phase according to the present invention may also be prepared as an aerosol preparation to be absorbed through the nasal mucosa or bronchial mucosa using conventional excipients.

The present invention will be described in more detail with reference to the following examples. However, the scope of the present invention is not limited only to the following examples.

Example 1 Fine Particles Containing Human Growth Hormone

To a 5 mM phosphate buffer solution in which human growth hormone was dissolved at a concentration of 2 mg / ml, Tween 80, a surfactant, was added in an amount of 0.01% by weight based on the weight of the buffer solution. In this solution, sodium hyaluronate having a molecular weight of 1,000,000 was dissolved at a concentration of 2 mg / ml to form a final solution. This final solution was supplied to the spray dryer (Buhi 190) at a flow rate of 3 ml / min to prepare a fine particle. At this time, the temperature of the drying air flowing into the spray dryer was 85 ^° C. and the average particle diameter of the obtained fine particles was 3 ㎛. 5 g of sodium hyaluronate containing human growth hormone was dispersed in 500 ml of ethanol dissolved in 10 mg / ml of lecithin and supplied to the spray dryer (Buchi 190) to obtain a lecithin-coated average particle size of 7 μm. The fine particles of were prepared.

Example 2 Fine Particles Containing Human Growth Hormone

0.01 wt% of Tween 80, a surfactant, was added to a 5 mM phosphate buffer solution in which human growth hormone was dissolved at a concentration of 1 mg / ml. The final solution was prepared by dissolving sodium hyaluronate having a molecular weight of 2,000,000 at a concentration of 1 mg / ml. The final solution was supplied to a spray dryer (Buchi 190) at a flow rate of 3 ml / min to prepare a particle. At this time, the temperature of the drying air flowing into the spray dryer was 85 ^° C. and the average particle diameter of the obtained fine particles was 2 ㎛. 5 g of sodium hyaluronate containing human growth hormone was dispersed in 500 ml of ethanol dissolved in 10 mg / ml of lecithin and supplied to a spray dryer (Buchi 190) to give a lecithin-coated average particle size of 5 μm. The fine particles of were prepared.

Example 3 Fine Particles Containing Human Growth Hormone

0.01 wt% of Tween 80, a surfactant, was added to a 5 mM phosphate buffer solution in which human growth hormone was dissolved at a concentration of 0.1 mg / ml. Sodium hyaluronate having a molecular weight of 2,000,000 in this solution was dissolved at a concentration of 0.9 mg / ml to form a final solution. This final solution was supplied to the spray dryer (Buchi 190) at a flow rate of 3 ml / min to prepare fine particles. At this time, the temperature of the drying air flowing into the spray dryer was 85 ^° C. and the average particle diameter of the obtained fine particles was 2 ㎛. 5 g of sodium hyaluronate containing human growth hormone was dispersed in 500 ml of ethanol dissolved in 10 mg / ml of lecithin and supplied to a spray dryer (Buchi 190) to give a lecithin-coated average particle size of 5 μm. The fine particles of were prepared.

Example 4 Fine Particles Containing Bovine Growth Hormone

0.01 wt% of Tween 80, a surfactant, was added to 5 mM phosphate buffer in which bovine growth hormone (bST) was dissolved at a concentration of 2 mg / ml. Here, a final solution was prepared by dissolving sodium hyaluronate having a molecular weight of 2,000,000 at a concentration of 2 mg / ml, and then supplying it to a spray dryer (Buchi 190) at a flow rate of 3 ml / min to prepare fine particles. At this time, the temperature of the drying air flowing into the spray dryer was 85 ^° C. and the average particle diameter of the obtained fine particles was 3 ㎛. 10 g of sodium hyaluronate containing bovine growth hormone was dispersed in 500 ml of ethanol dissolved in 10 mg / ml of lecithin and supplied to a spray dryer (Buchi 190) to obtain a lecithin-coated average particle size of 7 μm. The fine particles of were prepared.

Example 5 Porcine Growth Hormone-Containing Fine Particles

0.01 wt% of Tween 80, a surfactant, was added to 5 mM phosphate buffer in which porcine growth hormone (pST) was dissolved at a concentration of 2 mg / ml. Here, a final solution was prepared by dissolving sodium hyaluronate having a molecular weight of 2,000,000 at a concentration of 2 mg / ml, and then supplying it to a spray dryer (Buchi 190) at a flow rate of 3 ml / min to prepare fine particles. At this time, the temperature of the drying air flowing into the spray dryer was 85 ^° C. and the average particle diameter of the obtained fine particles was 3 ㎛. 5 g of sodium hyaluronate containing porcine growth hormone was dispersed in 500 ml of ethanol in which lecithin was dissolved at a concentration of 10 mg / ml. The dispersion was supplied to a spray dryer (Buchi 190) to obtain an average particle diameter of 7 μm. The fine particles of were prepared.

Example 6 Fine Particles Containing Granulocyte Macrophage-Colony Stimulator ( GM-CSF)

0.01 wt% of Tween 80, a surfactant, was added to 5 mM phosphate buffer in which GM-CSF was dissolved at a concentration of 0.4 mg / ml. Here, a final solution was prepared by dissolving sodium hyaluronate having a molecular weight of 2,000,000 at a concentration of 1.6 mg / ml, and then supplying it to the spray dryer (Buchi 190) at a flow rate of 3 ml / min to prepare fine particles. At this time, the temperature of the drying air flowing into the spray dryer was 85 ^° C. and the average particle diameter of the obtained fine particles was 3 ㎛. 5 g of sodium hyaluronate containing GM-CSF was dispersed in 500 ml of ethanol dissolved in 10 mg / ml of lecithin and supplied to a spray dryer (Buchi 190) to obtain a lecithin-coated average particle size of 7 μm. The fine particles of were prepared.

Example 7: Erythropoietin-containing Fine Particles

0.01 wt% of Tween 80, a surfactant, was added to a 5 mM phosphate buffer solution in which erythropoietin (EPO) was dissolved at a concentration of 1000 IU / ml and plasma albumin at 0.5 mg / ml. Here, a final solution was prepared by dissolving sodium hyaluronate having a molecular weight of 2,000,000 at 2.5 mg / ml, and then supplying it to the spray dryer (Buchi 190) at a flow rate of 3 ml / min to prepare fine particles. At this time, the temperature of the drying air flowing into the spray dryer was 85 ^° C. and the average particle diameter of the obtained fine particles was 3.5 ㎛. 5 g of sodium hyaluronate fine particles containing EPO were dispersed in 500 ml of ethanol dissolved in 10 mg / ml of lecithin and supplied to a spray dryer (Buchi 190) to give a lecithin-coated fine particle size of 7 μm. Particles were prepared.

Example 8: Interferon-alpha-Containing Fine Particles

To a solution containing interferon-α 2 × 10 ⁵ IU / ml, 0.2 mg / ml D-mannitol and 0.2 mg / ml plasma albumin, 0.01 wt% of Tween 80 surfactant was added. Here, a final solution was prepared by dissolving sodium hyaluronate having a molecular weight of 2,000,000 at 2.5 mg / ml, and then supplying it to the spray dryer (Buchi 190) at a flow rate of 3 ml / min to prepare fine particles. At this time, the temperature of the drying air flowing into the spray dryer was 105 ^° C. and the average particle diameter of the obtained fine particles was 3.5 ㎛. Dispersion of 5 g of sodium hyaluronate containing interferon-alpha in 500 ml of ethanol dissolved in 10 mg / ml of lecithin was supplied to the spray dryer (Buchi 190) to obtain a lecithin-coated average particle size of 7 μm. The fine particles of were prepared.

Example 9: Interferon-Gamma-containing Fine Particles

To a solution containing interferon-gamma 2 × 10 ⁵ IU / ml, 0.2 mg / ml glycine and 0.2 mg / ml plasma albumin, 0.01 wt% of Tween 80 surfactant was added. Here, a final solution was prepared by dissolving sodium hyaluronate having a molecular weight of 2,000,000 at 2.5 mg / ml, and then supplying it to the spray dryer (Buchi 190) at a flow rate of 3 ml / min to prepare fine particles. At this time, the temperature of the drying air flowing into the spray dryer was 105 ^° C. and the average particle diameter of the obtained fine particles was 3.5 ㎛. 5 g of sodium hyaluronate containing interferon-gamma was dispersed in 500 ml of ethanol dissolved in 10 mg / ml of lecithin and supplied to a spray dryer (Buchi 190) to obtain a lecithin-coated average particle size of 7 μm. The fine particles of were prepared.

Example 10 Insulin-Containing Fine Particles

0.01 wt% of Tween 80, a surfactant, was added to 10 mM phosphate buffer in which insulin was dissolved at a concentration of 20 IU / ml. Here, a final solution was prepared by dissolving sodium hyaluronate having a molecular weight of 2,000,000 at 2 mg / ml, and then supplying it to the spray dryer (Buchi 190) at a flow rate of 3 ml / min to prepare fine particles. At this time, the temperature of the drying air flowing into the spray dryer was 85 ^° C. and the average particle diameter of the obtained fine particles was 3 ㎛. 5 g of sodium hyaluronate containing insulin was dispersed in 500 ml of ethanol dissolved in 10 mg / ml of lecithin and supplied to a spray dryer (Buchi190) to give a lecithin-coated fine particle of 7 탆 in average particle size. Was prepared.

Example 11 Fine Particles Containing IGF-I

0.01 wt% of Tween 80, a surfactant, was added to 5 mM phosphate buffer in which insulin-like growth factor-I was dissolved at a concentration of 2 mg / ml. Here, a final solution was prepared by dissolving sodium hyaluronate having a molecular weight of 2,000,000 at 2 mg / ml, and then supplying it to the spray dryer (Buchi 190) at a flow rate of 3 ml / min to prepare fine particles. At this time, the temperature of the drying air flowing into the spray dryer was 85 ^° C. and the average particle diameter of the obtained fine particles was 3 ㎛. 5 g of sodium hyaluronate containing insulin-like growth factor-I was dispersed in 500 ml of ethanol dissolved in 10 mg / ml of lecithin and supplied to a spray dryer (Buchi 190) to give an average particle diameter of lecithin. 7 μm sized fine particles were prepared.

Example 12: Human Growth Hormone-Containing Fine Particles

0.01 wt% of Tween 80, a surfactant, was added to a 5 mM phosphate buffer solution in which human growth hormone was dissolved at a concentration of 1 mg / ml. The final solution was prepared by dissolving sodium hyaluronate having a molecular weight of 2,000,000 at a concentration of 1 mg / ml. This final solution was supplied to the spray dryer (Buchi 190) at a flow rate of 3 ml / min to prepare fine particles. At this time, the temperature of the drying air flowing into the spray dryer was 85 ^° C. and the average particle diameter of the obtained fine particles was 2 ㎛. 5 g of sodium hyaluronate containing human growth hormone was dispersed in 500 ml of methylene chloride (CH ₂ Cl ₂ ) in which stearic acid was dissolved at a concentration of 5 mg / ml, and the dispersion was supplied to a spray dryer (Buchi 190). Fine particles having a coated average particle size of 6 μm were prepared.

Example 13: Fine particles containing human growth hormone

0.01 wt% of Tween 80, a surfactant, was added to a 5 mM phosphate buffer solution in which human growth hormone was dissolved at a concentration of 1 mg / ml. The final solution was prepared by dissolving sodium hyaluronate having a molecular weight of 2,000,000 at a concentration of 1 mg / ml. This final solution was supplied to the spray dryer (Buchi 190) at a flow rate of 3 ml / min to prepare fine particles. At this time, the temperature of the drying air flowing into the spray dryer was 85 ^° C. and the average particle diameter of the obtained fine particles was 2 ㎛. A dispersion obtained by dispersing 5 g of sodium hyaluronate containing human growth hormone in 500 ml of iso-propyl alcohol dissolved at 5 mg / ml by Span 60 (sorbitan monostearate; obtained from ICI Americas) was spray dried (Buchi 190) to prepare a fine particle having a mean particle size of 7 ㎛ coated with a span 60.

Example 14: Human Growth Hormone-Containing Fine Particles

0.01 wt% of Tween 80, a surfactant, was added to a 5 mM phosphate buffer solution in which human growth hormone was dissolved at a concentration of 1 mg / ml. The final solution was prepared by dissolving sodium hyaluronate having a molecular weight of 2,000,000 at a concentration of 1 mg / ml. This final solution was supplied to the spray dryer (Buchi 190) at a flow rate of 3 ml / min to prepare fine particles. At this time, the temperature of the drying air flowing into the spray dryer was 85 ^° C. and the average particle diameter of the obtained fine particles was 2 ㎛. A dispersion obtained by dispersing 5 g of sodium hyaluronate containing human growth hormone in 500 ml of methylene chloride (CH ₂ Cl ₂ ) in which glyceryl mono-stearate is dissolved at a concentration of 5 mg / ml It was fed to a spray dryer (Buchi 190) to prepare fine particles having a mean particle size of 7 μm coated with glyceryl mono-stearate.

Example 15: Cottonseed Oil Dispersion of Fine Particles Containing Human Growth Hormone

1 ml of cottonseed oil was added to 50, 100, 200, 360 and 500 mg of the fine particles containing the human growth hormone prepared in Example 2, and the fine particles were dispersed in the cottonseed oil with a vortex mixer. Cottonseed oil dispersions having concentrations of 50, 100, 200, 360 and 500 mg / ml were prepared, respectively.

Example 16: Edible oil dispersion of fine particles containing human growth hormone

100 mg of fine particles containing human growth hormone prepared in Example 2 were dispersed in 1 ml of soybean oil, jade oil, and sesame oil to prepare soybean oil, jade oil, and sesame oil dispersion having a fine particle concentration of 100 mg / ml.

Example 17: Edible oil dispersion of microparticles containing bovine growth hormone

360 mg of fine particles containing bovine growth hormone prepared in Example 4 were dispersed in 1 ml of cottonseed oil, soybean oil, jade oil, and sesame oil, thereby preparing a dispersion of cottonseed oil, soybean oil, jade oil, and sesame oil with a fine particle concentration of 360 mg / ml. .

EXAMPLE 18: Edible Oil Dispersion of Fine Particles Containing Porcine Growth Hormone

360 mg of fine particles containing porcine growth hormone prepared in Example 5 were dispersed in 1 ml of cottonseed oil, soybean oil, jade oil, and sesame oil, thereby preparing a dispersion of cottonseed oil, soybean oil, jade oil, and sesame oil having a fine particle concentration of 360 mg / ml. .

Example 19: Edible oil dispersion of fine particles containing interferon-alpha

360 mg of the fine particles containing the interferon-alpha prepared in Example 8 were dispersed in 1 ml of cottonseed oil, soybean oil, jade oil, and sesame oil to prepare a cottonseed oil, soybean oil, apricot oil, and sesame oil dispersion having a fine particle concentration of 360 mg / ml. .

Example 20: Emulsion Dispersion of Fine Particles Containing Human Growth Hormone

0.9% aqueous NaCl solution was added to each 1 ml of the cottonseed oil dispersion prepared in Example 15 having concentrations of 50, 100, 200, 360 and 500 mg / ml of human growth hormone, respectively, 9, 4, 3, 2, 1.5 ml each, the solid dispersion concentration of the final dispersion is 5, 20, 50, 120, 200 mg / ml, respectively, the composition of the dispersion of cottonseed oil / water ratio of 1/9, 1/4, 1/3 , 1/2, 2/3. Shaking the final dispersion by hand forms a homogeneous, white, opaque oil-in-water emulsion and the fine particles in the solid phase are contained in the oil droplets in the dispersed phase. That is, due to the lipophilic property of the solid particle surface, cottonseed oil droplets containing the solid particles are stably generated in the aqueous solution. The oil-in-water emulsion dispersion thus prepared was preserved in a homogeneous phase even after standing for two weeks or more at room temperature.

Example 21: Emulsion Dispersion of Fine Particles Containing Human Growth Hormone

4 ml of 0.9% aqueous NaCl solution was added to 1 ml of soybean oil, jade oil, and sesame oil dispersion, each of which had a concentration of 100 mg / ml fine particles containing human growth hormone, prepared in Example 16. The solid particle concentration was 20 mg / ml in the liquid phase of 4. The final dispersion was shaken by hand to prepare a white, opaque, oil-in-water emulsion dispersion of a homogeneous phase.

Example 22: Emulsion Dispersion of Fine Particles Containing Human Growth Hormone

100 mg of the human growth hormone-containing fine particles prepared in Example 12 were dispersed in 1 ml of soybean oil, and 4 ml of 0.9% aqueous NaCl solution was added to the dispersion, and a white opaque oil-in-water emulsion of a homogeneous phase was shaken by hand. A dispersion was prepared.

Example 23: Emulsion Dispersion of Fine Particles Containing Human Growth Hormone

100 mg of the human growth hormone-containing fine particles prepared in Example 13 were dispersed in 1 ml of soybean oil, and 4 ml of 0.9% aqueous NaCl solution was added to the dispersion, and a white opaque oil-in-water emulsion of a homogeneous phase was shaken by hand. A dispersion was prepared.

Example 24: Emulsion Dispersion of Fine Particles Containing Human Growth Hormone

100 mg of the human growth hormone-containing fine particles prepared in Example 14 were dispersed in 1 ml of soybean oil, and 4 ml of 0.9% aqueous NaCl solution was added to the dispersion, and a white opaque oil-in-water emulsion of a homogeneous phase was shaken by hand. A dispersion was prepared.

Example 25: Emulsion Dispersion of Microparticles Containing Bovine Growth Hormone

2 ml of a 0.9% aqueous solution of NaCl was added to 1 ml of cottonseed oil, soybean oil, jade oil, and sesame oil dispersion each having a concentration of 360 mg / ml fine particles containing bovine growth hormone, prepared in Example 17, and the ratio of cooking oil / water was The solid particle concentration was 120 mg / ml in 1/2 the liquid phase. The final dispersion was shaken by hand to prepare a white, opaque, oil-in-water emulsion dispersion of a homogeneous phase.

Example 26: Emulsion Dispersion of Porcine Growth Hormone-Containing Fine Particles

2 ml of a 0.9% aqueous solution of NaCl was added to 1 ml of cottonseed oil, soybean oil, jade oil, and sesame oil dispersions each having a concentration of 360 mg / ml, wherein the fine particles containing pork growth hormone were prepared in Example 18. The solid particle concentration was 120 mg / ml in the / 2 liquid phase. The final dispersion was shaken by hand to prepare a white, opaque, oil-in-water emulsion dispersion of a homogeneous phase.

Example 27: Emulsion Dispersion of Interferon-alpha-Containing Fine Particles

2 ml of a 0.9% aqueous solution of NaCl was added to each 1 ml of cottonseed oil, soybean oil, jade oil, and sesame oil dispersion having a concentration of 360 mg / ml fine particles containing interferon-alpha prepared in Example 19, and thus the ratio of cooking oil / water The solid particle concentration was 120 mg / ml in 1/2 the liquid phase. The final dispersion was shaken by hand to prepare a white, opaque, oil-in-water emulsion dispersion of a homogeneous phase.

Comparative Example 1: Fine Particles of a Human Growth Hormone-containing Hydrophilic Surface

0.01 wt% of Tween 80, a surfactant, was added to a 5 mM phosphate buffer solution in which human growth hormone was dissolved at a concentration of 1 mg / ml. The final solution was prepared by dissolving sodium hyaluronate having a molecular weight of 2,000,000 at a concentration of 1 mg / ml. This final solution was supplied to the spray dryer (Buchi 190) at a flow rate of 3 ml / min to prepare fine particles. At this time, the temperature of the drying air flowing into the spray dryer was 85 ^° C. and the average particle diameter of the obtained fine particles was 2 ㎛. The produced fine particles have a hydrophilic surface.

Comparative Example 2: Cottonseed Oil Dispersion of Fine Particles on Human Growth Hormone-containing Hydrophilic Surfaces

100 mg of human growth hormone-containing fine particles prepared in Comparative Example 1 were dispersed in 1 ml of cottonseed oil to prepare a cottonseed oil dispersion.

Comparative Example 3: Microparticle O / W Dispersion of Hydrophilic Surface Containing Human Growth Hormone

To 1 ml of a cottonseed oil dispersion containing human growth hormone prepared in Comparative Example 2, 4 ml of an aqueous NaCl 0.9% injectable aqueous solution was added, and an oil / water dispersion was prepared by shaking the container by hand. The dispersion did not form an oil-in-water emulsion, cottonseed oil separated into the upper layer, and solid particles were dispersed in both the upper cottonseed oil and the lower water. Mixing the oil / water mixture dispersion with a vortex mixer did not form an oil-in-water emulsion.

Comparative Example 4: Fine Particles of an Interferon-alpha-Containing Hydrophilic Surface

To a solution containing interferon-α 2 × 10 ⁵ IU / ml, 0.2 mg / ml D-mannitol and 0.2 mg / ml plasma albumin, 0.01 wt% of Tween 80 surfactant was added. Here, a final solution was prepared by dissolving sodium hyaluronate having a molecular weight of 2,000,000 at 2.5 mg / ml, and then supplying it to a spray dryer (Buchi 190) at a flow rate of 3 ml / min to prepare particles. At this time, the temperature of the drying air flowing into the spray dryer was 105 ^° C. and the average particle diameter of the obtained fine particles was 3.5 ㎛.

Comparative Example 5: Microparticle Cottonseed Oil Dispersion on Interferon-alpha-containing Hydrophilic Surfaces

100 mg of fine particles containing interferon-alpha prepared in Comparative Example 4 were dispersed in 1 ml of cottonseed oil to prepare a cottonseed oil dispersion.

Comparative Example 6: Fine Particle O / W Dispersion of Interferon-alpha-containing Hydrophilic Surfaces

To 1 ml of the cottonseed oil dispersion containing interferon-alpha prepared in Comparative Example 5, 4 ml of NaCl 0.9% injectable aqueous solution was added, and the container was shaken by hand to prepare an oil / water dispersion. The dispersion did not form an oil-in-water emulsion, cottonseed oil separated into the upper layer, and solid particles were dispersed in both the upper cottonseed oil and the lower water.

Test Example 1: In vitro release test of human growth hormone-containing microparticles

Hyaluronic acid microparticles containing the human growth hormone prepared in Examples 1, 2 and 3 in the pH 7.4 buffer (sodium chloride 150 mM, phosphate 10 mM, sodium azide 0.05%) of human growth hormone concentration of 1.0 mg / Disperse to ml. This dispersion was put into a 37 ^° C. stirrer and subjected to a release test of human growth hormone. After a certain reaction time, the dispersion was centrifuged at 800 g for 10 minutes, and then 1/10 of the total dispersion was taken from the supernatant, the same amount of the buffer solution was added, and the release test was continued at 37 ^° C. again.

The concentration of human growth hormone in the supernatant was measured by the Laurie method and high performance liquid chromatography (HPLC) method, and the release amount of human growth hormone was determined over time. As shown in Figure 1, the higher the molecular weight of hyaluronic acid and the smaller the content of human growth hormone, the slower the rate of release of human growth hormone in the hyaluronic acid formulation. It can be seen that the release duration can be controlled according to the conditions such as molecular weight and protein content of hyaluronic acid. In addition, the pattern of release by in vitro release test shows a constant release rate until there is no initial rapid release and about 70% release.

Test Example 2: Stability test of human growth hormone-containing microparticles

In order to determine whether the human growth hormone in the hyaluronic acid microparticle formulation has the same activity as the human growth hormone in the aqueous solution used to prepare the microparticle formulation, the human growth hormone-containing of Example 2 in the in vitro release test of Test Example 1 Human growth hormone released up to 48 hours from the fine particle formulation was analyzed using reverse phase HPLC, size exclusion chromatography (SEC) method. In reversed phase HPLC, denaturation by oxidation and deamidation can be confirmed, and in SEC, denaturation due to aggregation can be confirmed.

2 is a result of observing the drug stability of the sustained-release solid microparticles containing the hGH of the present invention by reverse phase high performance liquid chromatography (RP-HPLC), Figure 2a is a result for the hGH released from the fine particles of the present invention 2b shows the results for the raw material hGH in aqueous solution used in the formulation preparation of the present invention. 3 is a result of observing the drug stability of the sustained-release solid microparticles containing the hGH of the present invention by size exclusion chromatography (SEC), Figure 3a shows the results for the hGH released from the fine particles of the present invention, 3b shows the result of the raw material hGH in aqueous solution used to prepare the formulation of the invention. As shown in Figures 2 and 3, the human growth hormone released in the microparticle formulation of the present invention is the same as the RP-HPLC results as in the aqueous solution used as a raw material, the SEC content is even more than 95% It can be seen that there is no denaturation of the protein during the preparation of the fine particles and the release at 37 ^° C.

Test Example 3: In Vitro Release and Stability Testing of Protein Drug-Containing Fine Particles

Example 4 (Bovine Growth Hormone), Example 5 (Swine Growth Hormone), Example 6 (Granulocyte Macrophage-Colony Stimulating Factor), Example 7 (Erythropoietin), Example 8 (Interferon-alpha), Example In vitro release in the same manner as in Test Example 1 using fine particle formulations of the various protein drugs prepared in 9 (interferon-gamma), Example 10 (insulin) and Example 11 (insulin-like growth factor-I) The test was carried out to confirm that the released drug was denatured as in Test Example 2.

Table 1 shows the cumulative release at 10 and 72 hours, and the monomer content of the protein in the cumulative release sample up to 72 hours.

Formulation 10 hours release (%) 72 hours release rate (%) Monomer Content (%) Example 4 45 92 94 Example 5 47 87 89 Example 6 44 89 97 Example 7 32 76 95 Example 8 54 92 94 Example 9 49 88 95 Example 10 60 95 95 Example 11 38 93 97

From the results in Table 1, it was confirmed that the drug was continuously released from the formulation of the present invention and the released drugs were not denatured.

Test Example 4: Injectability test of cottonseed oil dispersion of human growth hormone-containing microparticles

The fine particle formulation of the present invention has the advantage of being uniformly dispersed in a mixture of cooking oil and cooking oil and water, which can be administered with a thin needle. In this test, the force (injection capacity) required to push an injection-filled syringe at a constant rate of 80 mm / min was measured. The injection needle used a 26 gauge (gauge), the scanning capacity for the 0.9% NaCl aqueous solution, cottonseed oil, the cottonseed oil dispersion of Example 15, the emulsion dispersion of Example 20, the dispersion of Comparative Example 2 and the dispersion of Comparative Example 3 2 is shown.

Formulation Solid particle concentration (mg / ml) Injection capacity (kg _f ) 0.9% NaCl aqueous solution 0 0.1 Cottonseed oil 0 1.6 Example 15 Cottonseed Oil Dispersion 100 1.7 Example 20 Emulsion Dispersion 120 0.8 Comparative Example 2 Cottonseed Oil Dispersion 100 10.5 Comparative Example 3 O / W Dispersion 20 23.3

As can be seen in Table 2, since the fine particle formulation of the present invention is composed of a lipophilic material and has excellent dispersibility in cottonseed oil, the injection performance of the formulation of Example 15 (cottonseed oil dispersion) is inferior to that of cottonseed oil itself. Almost equal In addition, the fine particle formulation of the present invention, when dispersed in cottonseed oil due to the lipophilic characteristics of the surface and then redispersed in water, cottonseed oil is dispersed in water while forming droplets surrounding the particle surface to form an emulsion. Due to the formation of this homogeneous emulsion, the formulation of Example 20 (emulsion dispersion) has a lower injectability than the cottonseed oil, despite the highest solid concentration.

On the other hand, since the fine particles of sodium hyaluronate whose surface is not composed of a lipophilic substance are inferior in dispersibility to cottonseed oil, the formulation of Comparative Example 2 (cottonseed oil dispersion) is 6 times higher than when injected with cottonseed oil or the formulation of Example 15. More power is required. In addition, since the surface is hydrophilic, when the particles are dispersed in cottonseed oil and then redispersed in water, cottonseed oil does not cover the surface of the particles and phase separation occurs, and swelling occurs from the surface of the fine particles in contact with water. The dissolution of sodium hyaluronate increases the viscosity of the water layer. Accordingly, the formulation of Comparative Example 3 (O / W dispersion) requires twice as much as the cottonseed oil dispersion of Comparative Example 2, and 14 and 30 times greater injection capacity, respectively, than the formulations of Examples 15 and 20, which are inventive formulations. do.

Test Example 5: Injectability test of cottonseed oil dispersion of human growth hormone-containing microparticles

Oil-in-water emulsion dispersions of the human growth hormone-containing microparticles of Examples 22, 23 and 24, wherein the surface of the microparticles comprising human growth hormone consists of stearic acid, span 60, and glyceryl mono-stearate, respectively The scanning ability of was measured by the method in Test Example 4. As a result, the injection capacity of soybean oil was 1.4 kg _f , and the injection powers of the emulsion dispersions of Examples 22, 23, and 24 were all 0.3 to 0.5 kg _f , which is close to the force required for injection of 0.9% aqueous NaCl solution. Since the surface of the formulation is all affinity with soybean oil, since the soybean oil is coated on the solid particle surface, a uniform oil-in-water emulsion is formed, so that the resistance when passing through the needle is almost as small as water.

Test Example 6: Animal Test of Human Growth Hormone-Containing Microparticle Formulations

The effect of the sustained-release human growth hormone microparticle formulation of the present invention was confirmed using a dwarf rat, 7 weeks old, at about 100 g body weight, as a test animal. Dwarf mice genetically lower growth hormone secretion than normal mice, and human growth hormone administration increases growth and weight.

The dispersions prepared in Examples 15 and 20 were administered to each individual so that the amount of human growth hormone was 350 μg and the weight gain was observed for 6 days. In order to compare the effect of the formulation of the present invention, the commercially available aqueous formulation of LG Chem Eutrophin ^R formulation, the formulation of Comparative Example 2 and the formulation of Comparative Example 3, the amount of human growth hormone 350 ㎍ Weight gain was also measured in the groups each administered as much as possible and the control group not administered the growth hormone. The population of each group was 10. Table 3 shows the mean cumulative weight gain (g) after administration.

Test group 1 day 2 days 3 days 4 days 5 days 6 days Control 0.9 2.7 3.6 4.7 6.3 7.5 Utropin ^R group 4.7 4.2 5.3 6.4 7.1 8.5 Comparative Example 2 Formulation Group 5.0 5.7 7.2 8.5 10.2 11.5 Comparative Example 3 Formulation Administration Group 4.3 4.9 3.6 5.4 6.7 7.8 Example 15 Formulation Administration Group 5.5 6.6 7.3 8.7 11.4 12.3 Example 20 Formulation Administration Group 5.3 6.8 8.1 9.2 11.3 13.0

As can be seen in Table 3, the aqueous phase formulation Eutropin^RAfter 1 day of administration, the group gained weight, but after 2 days, the weight decreased, and after that, the increase was almost similar to that of the control group.

The group administered with the formulation of Comparative Example 2 in which the fine particles of which surface was not composed of lipophilic material in cottonseed oil showed a steady weight gain, but the group administered with the formulation of Comparative Example 3 was similar to the aqueous solution formulation. At 3 days, the weight was decreased, and the weight gain was similar to that of the aqueous solution. The formulation of Comparative Example 3 has almost the same effect as the aqueous solution formulation because the surface of the particles is not coated with cottonseed oil due to the hydrophilicity of the fine particle surface and the drug is dissolved in water.

In contrast, the group administered with the formulation of Example 15 prepared by dispersing the formulation of the present invention in cottonseed oil and the group administered with the formulation of Example 20 prepared by dispersing the formulation of the present invention in an emulsion of cottonseed oil / water have a weight It increased steadily for 6 days, and the increase rate was also higher than the formulation administration group of Comparative Example 2. This is because not only the surface of the fine particles is coated with cottonseed oil even after injection, but also the lecithin coating delays the rate of water absorption of the fine particles, resulting in a longer drug release duration.

Test Example 7: Animal testing of microparticle formulations containing bovine growth hormone

Formulation of Example 17, prepared by dispersing solid fine particles in soybean oil using female dwarf rats at 7 weeks of age, weighing about 100 g, and soybean oil and water. The formulation of Example 25 prepared by dispersing in an emulsion was administered to each subject so that the amount of bovine growth hormone was 12.5 mg and the weight gain was observed. Table 4 shows the mean cumulative weight gain (g) after administration of the sustained-release microparticle formulation administration group and the no-administration control group.

Test group 1 day 2 days 3 days 4 days 5 days 6 days 8th 10 days Control 1.4 2.9 4.6 6.1 6.5 7.2 8.8 10.4 Example 17 Formulation Administration Group 10.3 10.8 14.9 18.1 19.4 20.6 22.2 22.7 Example 25 Formulation Administration Group 9.7 11.5 14.6 17.7 19.9 20.8 22.5 22.9

As can be seen in Table 4, the group administered with the formulation of Example 17 and the group administered with the formulation of Example 25 gained weight steadily for 6 days and gained daily weight gain without the daily weight gain of the control group. Greater than In addition, the soybean oil dispersion formulation of Example 17 and the emulsion dispersion formulation of the soybean oil / water formulation of Example 25 can be seen that the release rate of the drug does not last more than 8 days since the weight gain rate is rapidly decreased after 8 days. The terms are almost equal.

Test Example 8: Animal Testing of Interferon-alpha-Containing Fine Particle Formulations

As follows, the formulation of the present invention containing interferon-alpha was administered to the rabbit to confirm that the titer of the drug lasted for a long time. The oil-in-water emulsion dispersion prepared in Example 27 was administered to a 5 month old rabbit weighing 2.5 kg so that the amount of interferon-alpha was 300 μg. The control group was administered a cottonseed oil dispersion of hyaluronic acid solid particles, the surface of which was not coated with a lipophilic substance, prepared in Comparative Example 5 so that the amount of interferon-alpha was 300 μg, and the other group was treated with an aqueous interferon-alpha formulation. The amount of alpha was administered to 300 μg.

The blood drug concentration was measured by cytopathic effect inhibition test. In brief, it is a titer test method to determine the degree of inhibition of cell lesions by adding virus after treatment with interferon-alpha in test cells. As the test cells, male calf kidney cells (MDBK CATCC CCL-22) were used, and the test virus was a vesicular stomatitis virus (ATCC VR 158). Table 5 below shows the titer (IU / ml) of interferon-alpha in the blood for 5 days.

Test group 7 hours 1 day 2 days 3 days 4 days 5 days Aqueous formulation 1.4 x 10 ³ 1.2 x 10 ¹ Not detected Not detected Not detected Not detected Comparative Example 5 Formulation Administration Group (Control) 2.1 x 10 ³ 2.6 x 10 ³ 9.1 x 10 ² 3.4 x 10 ² 1.7 x 10 ^{2 1.5} x 10 ¹ Example 27 Formulation Administration Group 1.7 x 10 ³ 2.2 x 10 ³ 1.1 x 10 ³ 4.6 x 10 ² 2.8 x 10 ² 8.7 x 10 ¹

As shown in Table 5, the titer of interferon-alpha in the blood was high in the formulation-administered groups of Comparative Example 5 and Example 27 formulated with solid fine particles for 5 days, but in the group administered with the aqueous solution of interferon-alpha in 1 day Thereafter no titer is measured. In the hyaluronic acid solid particle formulation administration group of Comparative Example 5, the surface of which is not made of a lipophilic substance, a titer of about one-tenth of the highest titer appearing on the first day appears on about 3-4 days, and the formulation of the present invention is Example 27 In the formulation administration of, the titer at the level of 1/10 of the highest titer on day 1 appears on day 4-5. That is, in the formulation of the present invention, the surface lipophilic effect has an effect of prolonging the release duration in the body.

Comparative Test Example 1 In Vitro Release of Hyaluronic Acid Gel Formulation

In a 5 mM phosphate buffer solution in which human growth hormone was dissolved at a concentration of 2.3 mg / ml, sodium hyaluronate having a molecular weight of 2,000,000 was dissolved at 20 mg / ml. It was prepared, using the same as in Test Example 1 in vitro release test.

In vitro release experiment of hyaluronic acid gel formulation revealed that 100% of human growth hormone was released into supernatant within 1 hour. That is, it can be seen that the gel formulation is easily diluted by water so that the duration of extracorporeal release is much shorter than the microparticles in the solid phase.

Comparative Test Example 2: Animal Experiment of Hyaluronic Acid Gel Formulation

In a 5 mM phosphate buffer solution in which human growth hormone was dissolved at a concentration of 1.5 mg / ml, sodium hyaluronate having a molecular weight of 2,000,000 was dissolved at 20 mg / ml to form a gel without fluidity. The hyaluronic acid gel formulation was administered to a dwarf rat so that the amount of human growth hormone was 150 μg and the cumulative weight gain (g) was observed for 6 days, and the results are shown in Table 6 below. For comparison, a group in which an aqueous solution formulation, utropin ^R , was administered so that the amount of human growth hormone was 150 µg and a control group were administered.

Test group 1 day 2 days 3 days 4 days 5 days 6 days Control 1.6 2.4 4.1 4.8 6.2 8.1 Hyaluronic Acid Gel Formulation 3.2 3.6 3.0 6.1 6.7 7.7 Utropin ^R group 3.3 2.6 4.2 6.4 7.8 8.3

Contrary to the result of continuous weight gain for six days when the dwarf was administered to rats with the formulation of the present invention containing the test substance of human growth hormone in Test Example 6, the hyaluronic acid gel dosage group was administered with the aqueous solution of eutrophin ^R. The group shows a similar tendency. That is, since the weight is reduced after 2 or 3 days after administration and the weight is about the same as the no-administration control group, it can be seen that the gel formulation does not last a long time for more than one day.

Comparative Test Example 3

Particle formulations containing hGH were prepared in hyaluronic acid-benzyl ester prepared by chemical reaction of natural hyaluronic acid and benzyl alcohol. First, a solution prepared by adding 0.01 wt% of Tween 80, a surfactant, to a 5 mM phosphate buffer solution in which hGH was dissolved at a concentration of 2 mg / ml was supplied to a spray dryer (Buchi 190) at a flow rate of 3 ml / min. Particles were prepared. At this time, the temperature of the drying air flowing into the spray dryer was 85 ^° C. and the average particle diameter of the obtained fine particles was 2.5 ㎛. The prepared hGH microparticles are dispersed in a dimethyl sulfoxide (DMSO) solution in which hyaluronic acid-benzyl ester is dissolved at a concentration of 6%, and the dispersion is a mineral containing a surfactant (Aracel A ^™ ; obtained from Atlas Chemical Ind.). After addition to oil, a microemulsion was made using a homogenizer. The formed microemulsion is composed of mineral oil in continuous phase and hyaluronic acid-benzylester / DMSO solution in which hGH microparticles are dispersed in dispersed phase. When ethyl acetate is added to the microemulsion and stirred, DMSO is extracted with ethyl acetate and the hyaluronic acid-benzyl ester is cured to obtain hyaluronic acid-benzyl ester particles containing hGH particles. The final particle obtained had a size of 5.5 μm and the content of hGH was 45 wt%.

In vitro release test of the particles in the same manner as in Test Example 1 and the results are shown in Table 7.

Elapsed time 0 One 3 5 7 24 48 72 144 Emission amount (%) 0 15 21 23 25 27 28 30 30

As shown in Table 7, the release of the drug hGH in the microparticle formulation prepared using hyaluronic acid-benzyl ester imparting hydrophobicity to the natural type hyaluronic acid is seldom after the initial 5 hours. The reason why the hGH is not released is because the interaction between the hGH and the hyaluronic acid-benzyl ester matrix is too large.

In addition, the cumulative weight (g) of the weight increase observed for 7 days by dwarf mice administered a solution obtained by dispersing the prepared fine particles so that the amount of hGH to 300 μg in cottonseed oil is shown in Table 8 below.

Test group 1 day 2 days 3 days 4 days 5 days 6 days 7 days No administration 1.2 2.3 3.6 5.7 6.6 7.3 8.2 Hyaluronic acid-benzyl ester dosage group 3.6 2.7 5.4 6.3 7.1 8.4 8.0

As shown in Table 8, it can be seen that the effect of the hyaluronic acid-benzyl ester particle formulation has little effect after 1 day.

The sustained release microparticle formulation of the solid phase of the present invention using hyaluronic acid or an inorganic salt thereof has a longer duration of drug release in the body than the sustained release formulation using a hyaluronic acid gel, and the solid particle surface is composed of a lipophilic substance. It is excellent in dispersibility in solution and can be made into a low viscosity non-aqueous injection, as well as dispersing the non-aqueous injection in a buffered aqueous solution for injection can produce a water-in-oil emulsion dispersion injection. The fine particle formulation of the present invention whose surface is composed of a lipophilic substance can be conveniently administered as an aerosol formulation. The formulation of the present invention has the advantage that no denaturation of the drug occurs until all of the drug is released.

Claims (14)

The particle surface of the solid mixture of the protein or peptide drug and hyaluronic acid or an inorganic salt thereof is coated with a lipophilic substance selected from the group consisting of lipids, lipid derivatives, fatty acids, salts of fatty acids, ester derivatives of fatty acids, fatty acid derivatives, surfactants and waxes. Fine particle formulation in solid phase having an average particle size of 0.1 to 200 μm. The method of claim 1, Formulations further comprising a stabilizer of the drug. The method of claim 2, And said stabilizer is selected from the group consisting of carbohydrates, proteins, amino acids, lipids, fatty acids, inorganic salts, surfactants, polyethylene glycols and mixtures thereof. The method of claim 1, Formulation of the fine particles having an average particle diameter of 1 to 50 ㎛. The method of claim 1, The drug includes human growth hormone, bovine growth hormone, pig growth hormone, growth hormone releasing hormone, growth hormone releasing peptide, granulocyte-colony stimulating factor, granulocyte macrophage-colony stimulating factor, macrophage-colony stimulating factor, erythropoietin, bone morphogenic protein, interferon, insulin, atriopeptin III), monoclonal antibodies, tumor necrosis factor, macrophage activating factor, interleukin, tumor degenerating factor, insulin-like growth factor (insulin-like growth factor), epidermal growth factor, tissue plasminogen activator, euro Formulation characterized in that selected from the group consisting of xylanase (urokinase). The method of claim 1, Wherein said inorganic salt of hyaluronic acid is a salt of sodium, potassium, ammonium, calcium, magnesium, zinc, copper or cobalt of hyaluronic acid. The method of claim 1, The lipid is lecithin, phosphatidylcoline, phosphatidylethanolamine, phosphatidyl-serine or phosphatidylinositol, and the fatty acid is myristic acid or palmitic acid. palmitic acid or stearic acid, and the ester derivative of the fatty acid is glyceryl stearate, sorbitan palmitate or sorbitan stearate, and the surfactant is Formulation characterized in that it is a poloxamer. An injection prepared by dispersing the formulation of claim 1 in a solution for non-aqueous injection. The method of claim 8, The non-aqueous injectable solution is cooking oil, mineral oil (mineral oil), squalene (squalene), squalane (squalane), cod liver oil, mono-, di- and tri-glycerides ( tri-glyceride) or a mixture thereof. The method of claim 9, The cooking oil is corn oil, olive oil, soybean oil, soybean oil, safflower oil, cotton seed oil, peanut oil, sesame oil, or mixtures thereof. Injectable, characterized in that. The injection of claim 8 further comprising a preservative or dispersant. An oil-in-water emulsion injection prepared by adding an aqueous solution for injection to the injection of claim 3. The method of claim 12, The injection solution is an injection, characterized in that the distilled water for injection, buffer for injection, or a preservative or dispersant is further included in these. An aerosol formulation comprising the formulation of any one of claims 1 to 7.