KR100486028B1 - Protein-containing lipid implant for sustained delivery and its preparation method - Google Patents

Abstract

The present invention relates to a protein-containing sustained-release lipid implant comprising a compressed mixture of protein drug and lipid coated with a hydrophilic polymer material and a method for preparing the same. Since the protein-containing sustained-release lipid implant of the present invention is uniformly distributed in the lipid matrix in a stable state, the protein drug is continuously released while maintaining the active state when the body is administered in the body, thereby maintaining a constant protein concentration in the blood. It can be used to effectively treat disease while reducing the number of doses.

Description

Protein-containing sustained-release lipid implants and a method for preparing the same {Protein-containing lipid implant for sustained delivery and its preparation method}

The present invention includes a compressed mixture of protein drug and lipid coated with a hydrophilic polymer material, so that the protein drug can be released into the body continuously and uniformly while maintaining biological activity, and the protein-containing sustained-release lipid implant and its preparation It is about a method.

Protein drugs are most likely to lose their active structure in the acidic environment of the stomach or be destroyed by enzymatic degradation upon oral administration and also have a significantly low rate of absorption from the gastric or intestinal mucosa. For this reason, most protein drugs are administered by parenteral administration, i.e., intravenous injection, subcutaneous injection, intramuscular injection, etc., and have a short half-life in vivo, and thus, the injections should be repeatedly repeated even after administration through this route. In particular, protein drugs require long-term administration for several months, and thus, studies on sustained and sustained-release formulations using biodegradable polymers are being actively conducted. See Heller, J. et al. Controlled release of water-soluble macromolecules from bioerodible hydrogels, Biomaterials, 4, 262-266 (1983); Langer, R., New methods of drug delivery, Science , 249, 1527-1533 (1990)].

The most biodegradable polymers used in sustained-release formulations of protein drugs include polylactide (PLA), polyglycolide (PGA) and their copolymers poly (lactide-co-glycolide) (Poly (lactide-co-glycolide), PLGA), such as polyester (Polyester) synthetic polymers [DeLuca, PP et al. , Biodegradable polyesters for drug and polypeptide delivery, in: El-Nokaly, MA, Piatt, DM, and Charpentier, BA (Eds.), Polymeric delivery systems, properties and applications , American Chemical Society, pp. 53-79 (1993); Park, TG, Degradation of poly (lactic-co-glycolic acid) microspheres: effect of copolymer composition, Biomaterials , 16, 1123-1130 (1995); Anderson, JM and Shive, MS, Biodegradation and biocompatibility of PLA and PLGA microspheres, Adv. Drug. Del. Rev. , 28, 5-24 (1997); Tracy, MA et al. , Factors affecting the degradation rate of poly (lactide-co-glycolide) microspheres in vivo and in vitro, Biomaterials , 20, 1057-1062 (1999)].

In addition to such polyester-based synthetic polymers, lipids, fatty acids, waxes and derivatives thereof, lipids, albumin, gelatin, collagen, fibrin and other proteins, alginic acid, chitin, chitosan, dextran, hyaluronic acid, starch and the like Natural polymers and the like have been studied as a matrix of sustained-release formulations of protein drugs. Regarding sustained release formulations using natural polymers, US Pat. No. 4,880,839 discloses active drugs of aminophylline, theophylline, hydroxyzine, chlordiazepoxide, chlorpromazine hydrochloride, morphine, propranonal, and polysaccharides, alginates and A report has been made on the preparation of multi-matrix sustained release formulations in which the activity of the active compound lasts for a long time using an aqueous / dispersible matrix such as gelatin as a sustained release matrix.

When water-soluble substances such as proteins or polysaccharides are used as the matrix among the polymers, it is very difficult to provide sustained release of protein drugs for days or weeks or more. In contrast, polyester-based synthetic polymers and lipids are water-insoluble, and when used as a matrix, they can provide sustained release of protein drugs for days, weeks, or months.

Coacervation or emulsion to capture protein drugs into polyester polymer matrices such as polylactide (PLA) or poly (lactide-co-glycolide) (PLGA) Liquid phase separation, encapsulation by spray drying and solvent evaporation in organic or aqueous phases can be used. See McGee, JP et al. , Zero order release of protein from poly (D, L-lactide-co-glycolide) microparticles prepared using a modified phase separation technique, J. Controlled Rel. , 34, 77-86 (1995); Gander, B. et al. , Quality improvement of spray-dried, protein-loaded D, L-PLA microspheres by appropriate polymer solvent selection, J. Microencapsul. , 12, 83-97 (1995); O'Donnell, PB and McGinity, JW, Preparation of microspheres by the solvent evaporation technique, Adv. Drug Del. Rel. , 28, 25-42 (1997).

Since most protein drugs are water-soluble, double emulsion solvent evaporation (W / O / W or double emulsion solvent evaporation) has been widely used in the preparation of sustained-release microparticles containing protein drugs. In this method, an aqueous solution in which a protein or a water-soluble drug is dissolved is added to an organic solvent in which the polymer is dissolved, and then a primary emulsion is prepared by using an apparatus such as an ultrasonic grinder or a homogenizer, and then polyvinyl chloride. Secondary emulsions are made by dispersing in a secondary aqueous solution containing a surfactant such as an alcohol. When the organic solvent is removed by heating or depressurizing the system, the polymer solidifies into fine particles. The particles are recovered by centrifugation or filtration and then lyophilized to obtain biodegradable fine particles containing protein drug. do. However, the protein drug is located at the interface between the aqueous solution and the organic solvent in the process of producing such fine particles, and the denaturation and irreversible aggregation of the protein occurs under severe conditions such as ultrasonic grinding or homogenization. As a result, proteins from the resulting fine particles tend to have an initial overrelease of several ten percent of the total drug followed by few releases over several days to several tens of days and then slightly increase in later releases. Kim, HK and Park, TG, Biotechnol. Bioeng. , 65, 659-667 (1999); Crotts, G. and Park, TG, J. Microencapsul. , 15, 699-713 (1998).

Research has been actively conducted to minimize the denaturation and irreversible agglomeration that occurs during the capture of proteins into polyester polymers. As part of this research, there is a method of adding a stabilizer to an aqueous solution of protein, and it has been reported that the protein is stabilized to some extent by using trihalose, mannitol, dextran, polyethylene glycol, and the like. 5,804,557; Cleland, JL and Jones, AJS, Pharm. Res., 13, 1464-1475 (1996); Cleland, J L et al ., Pharm. Res., 14, 420-425 (1997); Pean, J M et al ., Pharm. Res. , 16, 1294-1299 (1999); Sanchez, A. et al ., Int. J. Pharm., 185, 255-266 (1999); Lavelle, EC et al ., Vaccine, 17, 516-529 (1999). These stabilizers form a hydration layer around the protein, which reduces the interaction between the protein and the organic solvent, thus preventing protein denaturation and irreversible aggregation. Other efforts have also been made to minimize protein denaturation during the manufacturing process by dispersing the protein drug in an aqueous solution, instead of dissolving the protein drug in an aqueous solution. Cleland, JL and Jones, AJS, Stable formulations of recombinant human growth hormone and interferon-γ for microencapsulation in biodegradable microspheres, 13, 1464-1475 (1996); Iwata, M. et al., Particle size and loading efficiency of poly (D, L-lactic-co-glycolic acid) multiphase microspheres containing water soluble substances prepared by the hydrous and anhydrous solvent evaporation methods, J. Microencapsul. , 16, 49-58 (1999).

Among the above-mentioned methods, the method using protein powder is a sustained release formulation of human growth hormone prepared by capturing human growth hormone into poly (lactide-co-glycolide) polymer recently under the trade name Lutropin Depot ^™ . Has been licensed by This product disperses a powder of human growth hormone stabilized with metal ions (Zn ²⁺ ) in a methylene chloride solvent in which a poly (lactide-co-glycolide) polymer is dissolved and then sprayed with liquid nitrogen containing ethanol. Subsequently, the methylene chloride solvent was removed using ethanol at low temperature to prepare a sustained-release microsphere formulation to minimize denaturation of the protein drug during the preparation process (US Pat. No. 5,019,400, 5,654,010). However, recent reports have reported that these sustained release formulations have a relatively low bioavailability (33-55%) compared to conventional injectable formulations (Cleland, JL et al., Emerging protein delivery methods). , Current Opinion in Biotechnology, 12, 212-210 (2001). This low bioavailability of Lutropin Depot ^™ can be attributed to the relatively high initial release of human growth hormone in this formulation and also the biodegradation of the polymer used, namely poly (lactide-co-glycolide). The rate is too slow (weeks or months), suggesting that the protein may have been denatured for a long time after being administered in vivo.

As can be seen from the above reports, to date, it is a technically difficult task to capture protein drugs in a stable state into a polymer matrix of polyesters and provide sustained release for days or weeks or more without an initial excessive release. can do.

While comparative studies have been conducted on sustained-release formulations of protein drugs using synthetic polymers of polyesters, studies on sustained-release formulations of protein drugs using lipids containing lipids, fatty acids, waxes and derivatives thereof have been conducted. I didn't lose. Lipids are biomaterials that are mostly present in the body and have good biocompatibility, and there are various kinds of physical properties such as molecular weight, solubility in solvents, hydrophobicity, electrical properties, etc. While it has many advantages as a sustained-release matrix material, it has not been successfully used in sustained-release formulations of protein drugs due to problems such as strong interaction of lipids with proteins, thereby denaturing protein drugs, and difficulty controlling the rate of release of protein drugs. I couldn't.

Although many studies have been conducted on sustained-release formulations of protein drugs using liposomes, which are artificial biofilms using phospholipids as main ingredients, commercialization has not been achieved due to stability of formulations and stability of protein drugs.

The study of lipid implant formulations using protein did not begin until the late 1980s with model drugs such as insulin and animal growth hormone. Paul Yao-Cheng Wang, in US Pat. No. 5,939,380, prepared insulins in the form of discs or pellets using lipids such as fatty acids, cholesterol, triglycerides, and blood glucose concentrations from 20 to 40 days in animal experiments with male wister rats. By reporting a lowering effect, it has been suggested that such lipid implant formulations can be used as sustained release formulations of protein drugs. In the above patent, the solid protein particles such as insulin or porcine growth hormone and the lipid powder are uniformly mixed in an appropriate ratio and then pressurized to obtain a disc-type lipid implant formulation. Although the effect of lowering the glucose concentration of insulin in animal experiments lasted for 20 to 40 days, the amount of insulin administered as already disclosed in the above patent can be effective for 40 to 80 days, so a significant amount of insulin is used as a matrix. It may be denatured by the lipid material, or may not be completely released from the matrix due to the strong interaction with the lipid. These results indicate technically the need for protein drugs to be stably protected from lipid materials in the preparation of sustained release formulations that uniformly distribute protein drugs into the lipid matrix.

US Pat. No. 5,750,100 reports on implant formulations of protein drugs using artificially synthesized polyglycerol fatty acid esters as a matrix. The polyglycerol fatty acid esters used in this patent have increased hydrophilicity compared to the natural lipids such as fatty acids and glycerides mentioned above, which may increase the stability of protein drugs, but the degradation and absorption of the matrix material during the administration of the body is too late. There is a problem that protein denaturation can be increased accordingly.

According to the present invention, a protein drug-containing sustained-release lipid implant formulation, in which a protein solid particle stabilized using a hydrophilic polymer material is uniformly distributed and compressed in a lipid matrix, is slowly released at a constant rate for a long time without denaturation and initial over-release of the protein drug. This invention was completed by confirming that it was possible.

The gist of the invention

The present invention relates to a protein containing sustained release lipid implant, characterized in that it comprises a compressed mixture of protein drug and lipid coated with a hydrophilic polymeric material.

The hydrophilic polymer material used to prepare the protein drug coated with the hydrophilic polymer material preferably has a molecular weight of 2,000 dalton or more, and is polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, polyethyleneimine, dextran, dextran sulfate. , Chondroitin sulfate, dermatan sulfate, heparan sulfate, keratan sulfate, hyaluronic acid, chitosan, albumin, collagen, fibrin and mixtures thereof.

Lipids that are mixed and compressed with a protein drug coated with a hydrophilic polymeric material are preferably solid at room temperature, and are fatty acids, monoglycerides, diglycerides, triglycerides, sorbitan fatty acid esters, phospholipids, sphingolipids, waxes And salts and derivatives thereof.

Protein drugs coated with a hydrophilic polymeric material preferably include additional protein stabilizers.

The additional protein stabilizer described above is preferably selected from sugars, polyols, surfactants, amino acids, inorganic salts and mixtures thereof.

The content of the hydrophilic polymer in the protein drug coated with the hydrophilic polymer is 0.1 to 99.9% by weight.

The content of the lipid material in the protein containing sustained-release lipid implant is 40 to 99% by weight.

The protein-containing sustained-release lipid implant of the present invention is preferably in the form of microparticles or powders obtained from discs, pellets, rods or processes such as spheronalysis, milling, grinding and the like.

In addition, the present invention is formulated by preparing a protein solid powder coated with a hydrophilic polymer material of protein drug, mixing the coated protein solid powder and lipid prepared in the step, and compressing or extruding the mixture of the step. It characterized by comprising a step, to a method for producing a protein-containing sustained-release lipid implant.

The diameter of the protein solid powder coated with the hydrophilic polymer material is 0.1 μm to 200 μm.

The protein solid powder coated with a hydrophilic polymer material is prepared by dissolving the protein molecule and the hydrophilic polymer material and then drying it by a method selected from a spray drying method, a freeze drying method, a spray freezing drying method and a bubble drying method.

Alternatively, the protein solid powder coated with the hydrophilic polymer material is prepared by dispersing the protein microparticles in a solution in which the hydrophilic polymer material is dissolved and then drying by a method selected from a spray drying method, a freeze drying method, a spray freezing drying method and a bubble drying method.

The protein microparticles are prepared by drying the aqueous solution in which the protein is dissolved by a method selected from spray drying, freeze drying, spray freeze drying and bubble drying.

In the preparation of the protein solid powder coated with the hydrophilic polymeric material preferably further protein stabilizers are added.

Further protein stabilizers used are preferably selected from sugars, polyols, surfactants, amino acids, inorganic salts and mixtures thereof.

The sugar used as additional protein stabilizer is preferably selected from sucrose, glucose, inositol, trihalose, maltose, mannitol, lactose, mannose, xylitol, sorbitol, and cyclodextrin.

In one aspect, the present invention provides a protein containing sustained release lipid implant, characterized in that it comprises a compressed mixture of protein drug and lipid coated with a hydrophilic polymeric material.

In the present invention, "protein drug" is meant to include proteins or peptides or drugs containing them as a main component. For the purposes of the present invention, "proteins" that may be included in the protein-containing lipid implants of the present invention include biologically active proteins or peptides or analogs, mutants, and the like, which are naturally occurring, recombinantly engineered or synthesized. It may be prepared by the above, and may also have various modifications such as addition, substitution or deletion, or glycosylation of amino acids or domains, and are not limited to specific ones.

For example, human growth hormone, growth hormone releasing hormone, growth hormone releasing peptide, interferon, colony stimulating factor, interleukin, macrophage activating factor, macrophage peptide, B cell factor, T cell factor, protein A, allergic inhibitory factor, cell necrosis Glycoproteins, immunotoxins, lymphotoxins, tumor necrosis factor, tumor suppressor, metastatic growth factor, alpha-1 antitrypsin, albumin and fragment fragments thereof, apolipoprotein-E, erythropoietin, factor VII, factor VIII, Factor IX, plasminogen activator, urokinase, streptokinase, protein C, C-reactive protein, renin inhibitor, collagenase inhibitor, superoxide dismutase, platelet derived growth factor, epidermal growth factor, osteogenic growth factor , Bone formation promoting protein, calcitonin, insulin, atriopeptin, cartilage inducing factor, connective tissue activator, follicle stimulation Leucine, luteinizing hormone, luteinizing hormone releasing hormone, nerve growth factor, parathyroid hormone, relaxin, secretin, jomatomedin, insulin-like growth factor, adrenocorticotropic hormone, glucagon, cholecystokinin, pancreatic polypeptide, gastrin Release peptides, corticotropin releasing factor, thyroid stimulating hormone, monoclonal or polyclonal antibodies against various viruses, bacteria, toxins, various virus-derived vaccine antigens, and the like, including human serum albumin, human growth hormone, interferon Alpha, erythropoietin, colony stimulating factor and the like can be preferably used.

The "hydrophilic polymer material" used in the lipid implant of the present invention is a polymer material that surrounds the surface of protein particles included in the lipid implant and stably exists between hydrophobic lipid materials. These hydrophilic polymers protect protein particles in all processes until they are released into the body, stabilize them, prevent denaturation, and induce stable release, and protein particles are exposed to body fluids as lipids degrade or absorb in the body. When dissolved, it is released. These hydrophilic polymers prevent protein denaturation not only during the production of lipid implants but also during release after administration in the body. In addition, hydrophilic polymeric materials can protect proteins from denaturation as well as from degradation and aggregation, thereby enhancing in vivo activity and maintaining sustained release.

Regarding the use of hydrophilic polymer materials, a method of physically simply adding polyethylene glycol or using a polyester polymer covalently bonded with polyethylene glycol as a matrix when preparing a sustained-release formulation containing protein drug using a hydrophobic polymer such as polyester is proposed. Although attempted (Schwendeman, SP, Recent advances in the stabilization of proteins encapsulated in injectable PLGA delivery systems, Critical Reviews in Therapeutic Drug Carrier Systems , 19, 71-98 (2002)), protein particles as described herein It has not been described how to prepare a stabilized protein powder by coating it with a hydrophilic polymer material such as polyethylene glycol, and mixing and compressing it with the lipid material to produce a protein-containing sustained-release lipid implant.

In a preferred embodiment, the present invention is a hydrophilic polymeric material, having a molecular weight of about 2,000 dalton or more, polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, polyethyleneimine, dextran, dextran sulfate, chondroitin sulfate, dermatan sulfate, heparan A substance selected from the group consisting of sulfate, keratan sulfate, hyaluronic acid, chitosan, albumin, collagen, fibrin or mixtures thereof is used. More preferably, polyethylene glycol, hyaluronic acid, dextran, dextran sulfate, chondroitin sulfate, dermatan sulfate, keratan sulfate or heparan sulfate are used, most preferably polyethylene glycol or dextran.

By "lipid implant" is meant herein an implant in which the lipid is used as a matrix of the means of transport of the active drug. The lipid implant is preferably of injectable size but can be inserted at the site of administration by surgical surgery as needed. For the purposes of the present invention, the protein-containing sustained-release lipid implants of the present invention take the form of microparticles or powders obtained from discs, pellets, rods or processes such as spheronalysis, milling, grinding and the like. The “lipid” used in the protein containing sustained-release lipid implant of the present invention is a water-insoluble substance that is absorbed in vivo and shows no side effects and is solid at room temperature. Preferably, fatty acids, monoglycerides, diglycerides, triglycerides, sorbitan fatty acid esters, phospholipids, sphingolipids, cholesterol, waxes, salts and derivatives thereof, and the like may be used, but are not limited thereto.

More preferably, lauric acid, myristic acid, palmitic acid, stearyl acid or the like can be used as the fatty acid, and glyceryl laurate, glyceryl myristate, glyceryl palmitate, glyceryl stearate as monoglycerides. Rate and the like, sorbitan myristic acid, sorbitan palmitate, sorbitan stearate, and the like may be used as the sorbitan fatty acid ester, and trilaurin, trimyristin, tripalmitin, tristearin, etc. may be used as the triglyceride. As the phospholipid, phosphatidylcholine, phosphatidylethanolamine, phosphatidyl acid, phosphatidylserine, phosphatidylglycerol, phosphatidyl inositol, cardiolipin, and the like can be used, and as sphingolipid, sphingosine, ceramide, sphinginine and the like can be used. have.

Particularly preferred for use as a lipid matrix in the protein containing sustained-release lipid implants of the present invention are trilaurin, lauric acid, palmitic acid, stearyl acid, glyceryl monostearate or glyceryl myristrate.

Protein drugs coated with a hydrophilic polymeric material included in the protein-containing sustained-release lipid implant of the present invention may include additional protein stabilizers. Such additional protein stabilizers include sucrose, glucose, inositol, trihalose, maltose, mannitol, lactose, mannose, xylitol, sorbitol, cyclodextrins, sugars including glycerol, polyols, surfactants , Amino acids, inorganic salts, mixtures thereof and the like can be used. Particularly preferred as further stabilizers used to prepare protein solid powders stabilized with hydrophilic polymeric materials are trihalose, mannitol, glycine, zinc chloride and the like.

In addition, the lipid matrix may, if desired, include components conventionally used in the preparation of solid pharmaceutical formulations, such as excipients, binders, disintegrants, preservatives and the like. Preservatives include, for example, paraoxybenzoic acid esters, benzyl alcohol, chlorobutanol, thimerosal and the like.

The protein drug in the protein-containing sustained-release lipid implant of the present invention is stabilized by a hydrophilic polymer material, wherein the content of the hydrophilic polymer material in the protein drug stabilized by the hydrophilic polymer material is about 0.1 to 99.9% by weight, preferably Is about 50-95 weight percent.

In the protein containing sustained-release lipid implant of the present invention, the content of the lipid material in such a formulation is about 40 to 99% by weight, preferably about 60 to 95% by weight.

In another aspect, the present invention provides a method for preparing a protein solid powder coated with a hydrophilic polymer material, mixing the coated protein solid powder and lipids prepared in the step, and compressing or extruding the mixture of the steps. It provides a method for producing a protein-containing sustained-release lipid implant, characterized in that it comprises the step of formulating.

The protein solid powder coated with a hydrophilic polymer material may be prepared by uniformly dissolving the protein molecule and the hydrophilic polymer material and then drying it by spray drying, freeze drying, spray freezing drying, bubble drying, or the like.

Alternatively, the protein solid powder coated with a hydrophilic polymer material may be prepared by dispersing the protein microparticles in a solution in which the hydrophilic polymer material is dissolved and then drying by spray drying, freeze drying, spray freezing drying, bubble drying, or the like.

Protein microparticles used to prepare a protein solid powder coated with a hydrophilic polymer material may be prepared by drying an aqueous solution in which protein molecules are dissolved by spray drying, freeze drying, spray freezing drying, bubble drying, or the like.

For example, in the case of spray drying, the protein fine particle dispersion in a solution in which the protein is dissolved, in a solution in which the protein and the hydrophilic polymer are dissolved, or in a solution in which the hydrophilic polymer material is dissolved, is supplied at a flow rate of about 1.0 to 5.0 ml / min The drying air may be supplied to a spray dryer (eg, Buchi-191) having a temperature of 55 to 140 ° C. to be spray dried therefrom. In addition, when freeze-drying, a solution in which the protein is dissolved or a solution in which the protein and the hydrophilic polymer material are dissolved may be freeze-dried at a temperature of minus 70 ° C. The prepared freeze-dried material may separate only particles of a predetermined size (eg, 50 μm or less) after grinding.

In the preparation of the protein-containing sustained-release lipid implant of the present invention, additional protein stabilizers may be added during the preparation of the protein solid powder coated with the hydrophilic polymeric material or during the preparation of the protein microparticles.

As further protein stabilizers as described above, sugars, polyols, surfactants, amino acids, inorganic salts and mixtures thereof and the like can be used. Preferred as sugars among the additional protein stabilizers are sucrose, glucose, inositol, trihalose, maltose, mannitol, lactose, mannose, xylitol, sorbitol, cyclodextrin and the like. Particularly preferred as further stabilizers used to prepare protein solid powders stabilized with hydrophilic polymeric materials are trihalose, mannitol and glycine.

The protein solid powder coated with the hydrophilic polymer material prepared in the method as described above has a diameter of about 0.1 to 200 μm, preferably about 2 to 50 μm.

In the process of the invention, the mixture of protein solid powder and lipid material stabilized with a hydrophilic polymer material is used under conditions where the protein drug is not denatured, for example, a pressure of 0.01 to 50 tons, a temperature of 0 to 80 ° C., 5 minutes or less For a period of time, it is formulated in the form of discs, pellets or rods by methods such as compression, extrusion. Preferably, the tablet is extruded for 1 to 60 seconds at a pressure of 1 to 10 tons using a tablet press and at a pressure of 0.1 to 5 tons at a temperature of 0 to 50 ° C. when using an extruder. It can manufacture. However, depending on the characteristics of the protein can be selected to minimize the denaturation conditions, even if the lipid is melted can be prepared by applying a pressure if the denaturation of the protein.

Protein particles coated with a hydrophilic polymer material or protein particles coated with a hydrophilic polymer material including an additional stabilizer are mixed homogeneously with the lipid material to produce disks or pellets using a tableting machine or a compressor equipped with a suitable mold. Alternatively, a sustained release rapid implant formulation in the form of a rod may be prepared using an extrusion molding machine. In the present invention, the protein particles and the lipid material are homogeneously mixed in the solid state, which is due to the fact that when the protein particles are mixed with the molten lipids, due to high heat denaturation of the protein and the uniformity and compactness of the lipid matrix cannot be maintained. After transplantation, the protein drug is released at one time or little release.

As an example, when compressed and formulated in disk form, a mixture of protein solid powder and lipids is placed in a 13 mm KBr die and pressure of 0.4 tons using a caber laboratory press. It can be compressed for 1 minute to prepare a disk form formulation. In addition, when extrusion is formulated in the form of a rod, using a ram extruder can be prepared in the form of rod by extruding to a thickness of 1 mm while maintaining a temperature of 37 ℃ at a pressure of 0.4 tons.

6 is a micrograph of the rod formulation surface prepared by melting and cooling of Comparative Example 8. FIG. In the case of formulations prepared only by melting and cooling, as in FIG. 6, they exhibit excessive porosity on the surface of the formulation and consequently cannot have sustained release in vivo. In contrast, Figure 4 is a micrograph of the surface and cross-section of the rod formulation prepared by extrusion according to the present invention, where the protein drug is uniformly distributed in the lipid matrix as a result of compacting and mixing the protein solid powder and lipid in the solid state. You can see that it is. This is consistent with the present invention showing sustained release in an in vivo release test by applying pressure to a mixture of lipid and protein solid powder protected by a hydrophilic polymer in the preparation of a protein containing lipid implant formulation.

Protein-containing sustained-release lipid implants of the invention formulated in the form of discs, pellets or rods, for example, in the case of discs, pellets, are administered in sizes of 0.5 to 1.5 mm thick, 1 to 5 mm wide and 1 to 10 mm long The rod is cut to the appropriate size and inserted into the subcutaneously, and the rod is 0.5 to 1.0 mm in diameter and 5 to 15 mm in length. The rod can be inserted subcutaneously using a syringe designed to enter.

In another method, the protein particles coated with hydrophilic polymers are suspended in a solvent in which lipids are dissolved, and then spray-dried, and the resulting lipid-coated microparticles are placed in a pressure tank to apply pressure to the particles to obtain a formulation, or in the form of a disk, pellet, or rod. Formulation of the formulation can be obtained in the form of fine particles or powder through a process such as spherogination, milling, grinding and the like can be injected after dispersing the fine particle formulation in the injection solution.

Hereinafter, the present invention will be described in more detail with reference to Examples and Test Examples. However, this description is for illustrating the present invention and the scope of the present invention is not limited thereto.

Examples 1 to 17 illustrate the preparation of protein particles or protein particles containing stabilizers.

Example 1 Preparation of Human Serum Albumin Fine Particles by Spray Drying

Human serum albumin and trihalose were dissolved in 10 mM ammonium bicarbonate buffer (pH 7.0) at a concentration of 10 mg / ml, respectively, and the solution was supplied to a spray dryer (Buchi-191) at a flow rate of 3.0 ml / min. Microparticles of human serum albumin were prepared. At this time, the temperature of the drying air flowing into the spray dryer was 90 ℃ and the average diameter of the obtained fine particles was 3 ㎛.

Example 2 Preparation of Human Serum Albumin Fine Particles by Spray Drying

Human serum albumin and mannitol were dissolved in 10 mM ammonium bicarbonate buffer (pH 7.0) at concentrations of 10 mg / ml and 10 mg / ml, respectively, and then the solution was sprayed into a spray dryer (Buchi-191) at a flow rate of 3.0 ml / min. Supplyed to prepare microparticles of human serum albumin. At this time, the temperature of the drying air flowing into the spray dryer was 95 ℃ and the average diameter of the obtained fine particles was 2 ㎛.

<Example 3> Preparation of human serum albumin microparticles by freeze drying

Human serum albumin was dissolved in 10 mM ammonium bicarbonate buffer (pH 7.0) at a concentration of 10 mg / ml, and the solution was frozen at minus 70 ° C and then vacuum dried. The obtained powder was finely ground in a crystal mortar and then a particle having a diameter of 50 μm or less was separated using a sonic sifter (Allen-Bradley, USA).

Example 4 Preparation of Human Growth Hormone Fine Particles by Spray Drying

Human growth hormone was dissolved in 10 mM ammonium bicarbonate buffer (pH 7.0) at a concentration of 10 mg / ml, and then the solution was supplied to the spray dryer (Buchi-191) at a flow rate of 3.0 ml / min to obtain microparticles of human growth hormone. Was prepared. At this time, the temperature of the drying air flowing into the spray dryer was 92 ℃ and the average diameter of the obtained fine particles was 3 ㎛.

Example 5 Preparation of Human Growth Hormone Fine Particles by Spray Drying

Human growth hormone and zinc chloride were dissolved in 10 mM ammonium bicarbonate buffer (pH 7.0) at concentrations of 10 mg / ml and 0.01 mg / ml, respectively, and then the solution was spray dried at a flow rate of 3.0 ml / min (Buchi-191). The microparticles of human growth hormone were prepared by supplying the same. At this time, the temperature of the drying air flowing into the spray dryer was 90 ℃ and the average diameter of the obtained fine particles was 2 ㎛.

Example 6 Preparation of Human Growth Hormone Fine Particles by Spray Drying

Human growth hormone and trihalose were dissolved in 10 mM ammonium bicarbonate buffer (pH 7.0) at concentrations of 10 mg / ml and 5 mg / ml, respectively, and the solution was sprayed at a flow rate of 3.0 ml / min (Buchi- 191) to prepare microparticles of human growth hormone. At this time, the temperature of the drying air flowing into the spray dryer was 90 ℃ and the average diameter of the obtained fine particles was 3 ㎛.

Example 7 Preparation of Human Growth Hormone Microparticles by Freeze Drying

Human growth hormone and mannitol were dissolved in 10 mM ammonium bicarbonate buffer (pH 7.0) at concentrations of 10 mg / ml and 5 mg / ml, respectively, and the solution was frozen at -70 ° C and vacuum dried. The obtained powder was finely ground in a crystal mortar and then a particle having a diameter of 50 μm or less was separated using a sonic sifter (Allen-Bradley, USA).

Example 8 Preparation of Interferon Alpha Fine Particles by Spray Drying

Interferon alpha and trihalose were dissolved in 10 mM ammonium bicarbonate buffer (pH 7.0) at a concentration of 1 mg / ml and 5 mg / ml, respectively, and the solution was spray dried at a flow rate of 3.0 ml / min (Buchi-191). ) To prepare microparticles of interferon alpha. At this time, the temperature of the drying air flowing into the spray dryer was 95 ℃ and the average diameter of the obtained fine particles was 2 ㎛.

Example 9 Preparation of Interferon Alpha Fine Particles by Spray Drying

Interferon alpha and glycine were dissolved in 10 mM ammonium bicarbonate buffer at concentrations of 1 mg / ml and 4 mg / ml, respectively, and this solution (pH 7.0) was supplied to the spray dryer (Buchi-191) at a flow rate of 3.0 ml / min. To prepare microparticles of interferon alpha. At this time, the temperature of the drying air flowing into the spray dryer was 95 ℃ and the average diameter of the obtained fine particles was 4㎛.

Example 10 Preparation of Interferon Alpha Fine Particles by Spray Drying

Interferon alpha, trihalose, and glycine were dissolved in 10 mM ammonium bicarbonate buffer at concentrations of 2 mg / ml, 7 mg / ml, and 41 mg / ml, respectively, and the solution (pH 7.0) was flowed at 3.0 ml / min. By supplying to the spray dryer (Buchi-191) to prepare a fine particle of the interferon alpha. At this time, the temperature of the drying air flowing into the spray dryer was 95 ℃ and the average diameter of the obtained fine particles was 3㎛.

Example 11 Preparation of Interferon Alpha Fine Particles by Spray Drying

Interferon alpha and zinc chloride were dissolved in 10 mM ammonium bicarbonate buffer (pH 7.0) at a concentration of 1 mg / ml and 0.06 mg / ml, respectively, and the solution was sprayed into a spray dryer (Buchi-191) at a flow rate of 3.0 ml / min. Supplyed to produce microparticles of interferon alpha. At this time, the temperature of the drying air flowing into the spray dryer was 95 ℃ and the average diameter of the obtained fine particles was 3 ㎛.

Example 12 Preparation of Interferon Alpha Microparticles by Freeze Drying

Interferon alpha and trihalose were dissolved in 10 mM ammonium bicarbonate buffer (pH 7.0) at concentrations of 1 mg / ml and 5 mg / ml, respectively, and the solution was frozen at -70 ° C and vacuum dried. The obtained powder was finely ground in a crystal mortar and then a particle having a diameter of 50 μm or less was separated using a sonic sifter (Allen-Bradley, USA).

Example 13 Preparation of Interferon Alpha Microparticles by Freeze Drying

Interferon alpha and mannitol were dissolved in 10 mM ammonium bicarbonate buffer (pH 7.0) at concentrations of 1 mg / ml and 9 mg / ml, respectively, and the solution was frozen at -70 ° C and vacuum dried. The obtained powder was finely ground in a crystal mortar and then a particle having a diameter of 50 μm or less was separated using a sonic sifter (Allen-Bradley, USA).

Example 14 Preparation of Erythropoietin (EPO) Fine Particles by Spray Drying

Erythropoietin, trihalose, and glycine were dissolved in 10 mM ammonium bicarbonate buffer at concentrations of 1 mg / ml, 5 mg / ml, and 4 mg / ml, respectively, and this solution (pH 7.0) was 2.5 ml / min. Fine particles of erythropoietin were prepared by feeding the spray dryer (Buchi-191) at a flow rate of. At this time, the temperature of the drying air flowing into the spray dryer was 95 ℃ and the average diameter of the obtained fine particles was 2 ㎛.

Example 15 Preparation of Colony Stimulating Factor (G-CSF) Fine Particles by Spray Drying

Colony stimulating factor and zinc chloride were dissolved in 10 mM ammonium bicarbonate buffer (pH 7.0) at a concentration of 1 mg / ml and 0.06 mg / ml, respectively, and the solution was spray dried at a flow rate of 3.0 ml / min (Buchi-191). Supply to to prepare a fine particle of the colony stimulating factor. At this time, the temperature of the drying air flowing into the spray dryer was 95 ℃ and the average diameter of the obtained fine particles was 3 ㎛.

Example 16 Preparation of Erythropoietin Fine Particles by Freeze Drying

Erythropoietin and trihalose were dissolved in 10 mM ammonium bicarbonate buffer (pH 7.0) at concentrations of 1 mg / ml and 5 mg / ml, respectively, and the solution was frozen at minus 70 ° C and vacuum dried. The obtained powder was finely ground in a crystal mortar and then a particle having a diameter of 50 μm or less was separated using a sonic sifter (Allen-Bradley, USA).

Example 17 Preparation of Colony Stimulating Factor Microparticles by Freeze Drying

Colony stimulating factor and mannitol were dissolved in 10 mM ammonium bicarbonate buffer (pH 7.0) at concentrations of 1 mg / ml and 9 mg / ml, respectively, and the solution was frozen at -70 ° C and vacuum dried. The obtained powder was finely ground in a crystal mortar and then a particle having a diameter of 50 μm or less was separated using a sonic sifter (Allen-Bradley, USA).

Examples 18 to 63 illustrate a method of preparing protein particles protected with a hydrophilic polymer using spray drying, freeze drying, and bubble drying.

Example 18 Preparation of Polyethylene Glycol Microparticles Containing Human Serum Albumin by Spray Drying

Human serum albumin and polyethylene glycol (molecular weight: 10,000) were dissolved in 10 mM ammonium bicarbonate buffer (pH 7.0) at concentrations of 10 mg / ml and 10 mg / ml, respectively, and the solution was spray dried at a flow rate of 3.0 ml / min. It was supplied to (Buchi-191) to prepare a polyethylene glycol microparticles containing human serum albumin. At this time, the temperature of the drying air flowing into the spray dryer was 95 ℃ and the average diameter of the obtained fine particles was 3 ㎛.

Example 19 Preparation of Polyethylene Glycol Microparticles Containing Human Serum Albumin by Spray Drying

Human serum albumin and polyethylene glycol (molecular weight: 3,350) were dissolved in 10 mM ammonium bicarbonate buffer (pH 7.0) at concentrations of 10 mg / ml and 10 mg / ml, respectively, and the solution was spray dried at a flow rate of 3.0 ml / min. It was supplied to (Buchi-191) to prepare a polyethylene glycol microparticles containing human serum albumin. At this time, the temperature of the drying air flowing into the spray dryer was 90 ℃ and the average diameter of the obtained fine particles was 2 ㎛.

Example 20 Preparation of Polyethylene Glycol Microparticles Containing Human Serum Albumin by Spray Drying

The human serum albumin microparticles prepared in Example 1 were uniformly dispersed at a concentration of 5 mg / ml in a solution in which polyethylene glycol (molecular weight: 3,350) was dissolved in methylene chloride at a concentration of 10 mg / ml, and then 3.0 ml of this solution. Polyethylene glycol microparticles containing human serum albumin were prepared by feeding the spray dryer (Buchi-191) at a flow rate of / min. At this time, the temperature of the drying air flowing into the spray dryer was 55 ℃ and the average diameter of the obtained fine particles was 3 ㎛.

Example 21 Preparation of Methylcellulose Microparticles Containing Human Serum Albumin by Spray Drying

Human serum albumin and methylcellulose were dissolved in 10 mM ammonium bicarbonate buffer (pH 7.0) at concentrations of 10 mg / ml and 5 mg / ml, respectively, and the solution was spray dried at a flow rate of 3.0 ml / min (Buchi-191). Supplied to human serum albumin-containing methylcellulose microparticles. At this time, the temperature of the drying air flowing into the spray dryer was 95 ℃ and the average diameter of the obtained fine particles was 2 ㎛.

Example 22 Preparation of Hyaluronic Acid Microparticles Containing Human Serum Albumin by Spray Drying

Human serum albumin and hyaluronic acid were dissolved in 10 mM ammonium bicarbonate buffer (pH 7.0) at concentrations of 10 mg / ml and 10 mg / ml, respectively, and the solution was spray dried at a flow rate of 2.5 ml / min (Buchi-191). Supply to human serum albumin-containing hyaluronic acid microparticles were prepared. At this time, the temperature of the drying air flowing into the spray dryer was 100 ℃ and the average diameter of the obtained fine particles was 3 ㎛.

Example 23 Preparation of Human Serum Albumin-Containing Dextran Microparticles by Freeze-Drying

Human serum albumin and dextran (molecular weight: 70,000) were dissolved in 10 mM ammonium bicarbonate buffer (pH 7.0) at concentrations of 10 mg / ml and 90 mg / ml, respectively, and the solution was frozen at -70 ° C and vacuum dried. It was. The obtained powder was finely ground in a crystal mortar and then a particle having a diameter of 50 μm or less was separated using a sonic sifter (Allen-Bradley, USA).

<Example 24> Preparation of human serum albumin-containing polyethylene glycol microparticles by freeze drying

Human serum albumin and polyethylene glycol (molecular weight: 10,000) were dissolved in 10 mM ammonium bicarbonate buffer (pH 7.0) at concentrations of 10 mg / ml and 90 mg / ml, respectively, and the solution was frozen at minus 70 ° C and vacuum dried. It was. The obtained powder was finely ground in a crystal mortar and then a particle having a diameter of 50 μm or less was separated using a sonic sifter (Allen-Bradley, USA).

<Example 25> Preparation of human serum albumin-containing polyethylene glycol microparticles by freeze drying

Human serum albumin and polyethylene glycol (molecular weight: 3,350) were dissolved in 10 mM ammonium bicarbonate buffer (pH 7.0) at concentrations of 10 mg / ml and 90 mg / ml, respectively, and the solution was frozen at minus 70 ° C and vacuum dried. It was. The obtained powder was finely ground in a crystal mortar and then a particle having a diameter of 50 μm or less was separated using a sonic sifter (Allen-Bradley, USA).

Example 26 Preparation of Human Serum Albumin-Containing Dextran and Polyethyleneglycol Microparticles by Lyophilization

Human serum albumin, dextran (molecular weight: 70,000) and polyethylene glycol (molecular weight: 3,350) were dissolved in 10 mM ammonium bicarbonate buffer (pH 7.0) at concentrations of 10 mg / ml, 10 mg / ml and 90 mg / ml, respectively. The solution was then frozen at minus 70 ° C. and then dried in vacuo. The obtained powder was finely ground in a crystal mortar and then a particle having a diameter of 50 μm or less was separated using a sonic sifter (Allen-Bradley, USA).

Example 27 Preparation of Carboxymethyl Cellulose Microparticles Containing Human Serum Albumin by Freeze Drying

Human serum albumin and carboxymethylcellulose were dissolved in 10 mM ammonium bicarbonate buffer (pH 7.0) at concentrations of 10 mg / ml and 20 mg / ml, respectively, and the solution was frozen at -70 ° C and vacuum dried. The obtained powder was finely ground in a crystal mortar and then a particle having a diameter of 50 μm or less was separated using a sonic sifter (Allen-Bradley, USA).

Example 28 Preparation of Dextran Microparticles Containing Human Growth Hormone by Spray Drying

Human growth hormone and dextran (molecular weight: 70,000) were dissolved in 10 mM ammonium bicarbonate buffer (pH 7.0) at concentrations of 5 mg / ml and 25 mg / ml, respectively, and the solution was spray dried at a flow rate of 3.0 ml / min. It was supplied to (Buchi-191) to prepare human growth hormone-containing dextran microparticles. At this time, the temperature of the drying air flowing into the spray dryer was 100 ℃ and the average diameter of the obtained fine particles was 2 ㎛.

Example 29 Preparation of Polyethylene Glycol Microparticles Containing Human Growth Hormone by Spray Drying

Human growth hormone, zinc chloride, and polyethylene glycol (molecular weight: 10,000) were dissolved in 10 mM ammonium bicarbonate buffer (pH 7.0) at concentrations of 10 mg / ml, 0.01 mg / ml, and 10 mg / ml, respectively, followed by 3.0 Human growth hormone-containing polyethylene glycol microparticles were prepared by feeding the spray dryer (Buchi-191) at a flow rate of ml / min. At this time, the temperature of the drying air flowing into the spray dryer was 95 ℃ and the average diameter of the obtained fine particles was 4 ㎛.

Example 30 Preparation of Polyethylene Glycol Microparticles Containing Human Growth Hormone by Spray Drying

Human growth hormone and polyethylene glycol (molecular weight: 3,350) were dissolved in 10 mM ammonium bicarbonate buffer (pH 7.0) at concentrations of 10 mg / ml and 10 mg / ml, respectively, and the solution was spray dried at a flow rate of 3.0 ml / min. (Buchi-191) was supplied to produce polyethylene glycol microparticles containing human growth hormone. At this time, the temperature of the drying air flowing into the spray dryer was 90 ℃ and the average diameter of the obtained fine particles was 3 ㎛.

Example 31 Preparation of Carboxymethyl Cellulose Microparticles Containing Human Growth Hormone by Spray Drying

Human growth hormone and carboxymethylcellulose were dissolved in 10 mM ammonium bicarbonate buffer (pH 7.0) at concentrations of 10 mg / ml and 20 mg / ml, respectively, and the solution was spray dried at a flow rate of 3.0 ml / min (Buchi-191). ) Carboxymethylcellulose microparticles containing human growth hormone were prepared. At this time, the temperature of the drying air flowing into the spray dryer was 98 ℃ and the average diameter of the obtained fine particles was 2 ㎛.

Example 32 Preparation of Methylcellulose Microparticles Containing Human Growth Hormone by Spray Drying

Human growth hormone and methylcellulose were dissolved in 10 mM ammonium bicarbonate buffer (pH 7.0) at concentrations of 10 mg / ml and 5 mg / ml, respectively, and the solution was spray dried at a flow rate of 3.0 ml / min (Buchi-191). Supplied to human serum albumin-containing methylcellulose microparticles. At this time, the temperature of the drying air flowing into the spray dryer was 90 ℃ and the average diameter of the obtained fine particles was 3 ㎛.

Example 33 Preparation of Dextran Sulfate Microparticles Containing Human Growth Hormone by Spray Drying

Human growth hormone and dextran sulfate (molecular weight: 25,000) were dissolved in 10 mM ammonium acetate buffer (pH 4.0) at concentrations of 10 mg / ml and 50 mg / ml, respectively, and then the solution was sprayed at a flow rate of 2.5 ml / min. It was supplied to a dryer (Buchi-191) to prepare human growth hormone-containing dextran sulfate microparticles. At this time, the temperature of the drying air flowing into the spray dryer was 90 ℃ and the average diameter of the obtained fine particles was 3 ㎛.

Example 34 Preparation of Chondroitin Sulfate Microparticles Containing Human Growth Hormone by Spray Drying

Human growth hormone and chondroitin sulfate were dissolved in 10 mM ammonium acetate buffer (pH 4.0) at concentrations of 10 mg / ml and 10 mg / ml, respectively, and then the solution was spray dried at a flow rate of 3.0 ml / min (Buchi-191). Supplied to human growth hormone-containing chondroitin sulfate microparticles. At this time, the temperature of the drying air flowing into the spray dryer was 92 ℃ and the average diameter of the obtained fine particles was 3 ㎛.

Example 35 Preparation of Dermatan Sulfate Microparticles Containing Human Growth Hormone by Spray Drying

Human growth hormone and dermatan sulfate were dissolved in 10 mM ammonium acetate buffer (pH 4.0) at concentrations of 10 mg / ml and 10 mg / ml, respectively, and the solution was sprayed at a flow rate of 3.0 ml / min (Buchi-191). Human growth hormone-containing dermatan sulfate microparticles were prepared. At this time, the temperature of the drying air flowing into the spray dryer was 95 ℃ and the average diameter of the obtained fine particles was 3 ㎛.

Example 36 Preparation of Keratan Sulfate Microparticles Containing Human Growth Hormone by Spray Drying

Human growth hormone and keratan sulfate were dissolved in 10 mM ammonium acetate buffer (pH 4.0) at concentrations of 10 mg / ml and 20 mg / ml, respectively, and the solution was spray dried at a flow rate of 3.0 ml / min (Buchi-191). Human growth hormone-containing keratan sulfate microparticles were prepared. At this time, the temperature of the drying air flowing into the spray dryer was 93 ℃ and the average diameter of the obtained fine particles was 3 ㎛.

Example 37 Preparation of Heparan Sulfate Microparticles Containing Human Growth Hormone by Spray Drying

Human growth hormone and heparan sulfate were dissolved in 10 mM ammonium acetate buffer (pH 4.0) at concentrations of 10 mg / ml and 30 mg / ml, respectively, and the solution was sprayed at a flow rate of 3 ml / min (Buchi-191). Human growth hormone-containing heparan sulfate microparticles were prepared. At this time, the temperature of the drying air flowing into the spray dryer was 90 ℃ and the average diameter of the obtained fine particles was 4 ㎛.

<Example 38> Preparation of polyethylene glycol microparticles containing human growth hormone by freeze drying

Human growth hormone and polyethylene glycol (molecular weight: 10,000) were dissolved in 10 mM ammonium bicarbonate buffer (pH 7.0) at concentrations of 10 mg / ml and 20 mg / ml, respectively, and the solution was frozen at minus 70 ° C and vacuum dried. It was. The obtained powder was finely ground in a crystal mortar and then a particle having a diameter of 50 μm or less was separated using a sonic sifter (Allen-Bradley, USA).

<Example 39> Preparation of human growth hormone-containing polyethylene glycol microparticles by freeze drying

Human growth hormone and polyethylene glycol (molecular weight: 3,350) were dissolved in 10 mM ammonium bicarbonate buffer (pH 7.0) at concentrations of 10 mg / ml and 20 mg / ml, respectively, and the solution was frozen at -70 ° C and vacuum dried. It was. The obtained powder was finely ground in a crystal mortar and then a particle having a diameter of 50 μm or less was separated using a sonic sifter (Allen-Bradley, USA).

<Example 40> Preparation of human growth hormone-containing dextran sulfate microparticles by freeze drying

Human growth hormone and dextran sulfate (molecular weight: 25,000) were dissolved in 10 mM ammonium acetate buffer (pH 4.0) at concentrations of 10 mg / ml and 50 mg / ml, respectively, and the solution was frozen at minus 70 ° C and then vacuumed. Dried. The obtained powder was finely ground in a crystal mortar and then a particle having a diameter of 50 μm or less was separated using a sonic sifter (Allen-Bradley, USA).

Example 41 Preparation of Human Growth Hormone-Containing Dextran Sulfate and Polyethylene Glycol Microparticles

Human growth hormone, dextran sulfate (molecular weight: 25,000) and polyethylene glycol (molecular weight: 3,350) were dissolved in 10 mM ammonium acetate buffer (pH 4.0) at concentrations of 10 mg / ml, 10 mg / ml and 20 mg / ml, respectively. The solution was frozen at minus 70 ° C. and then dried in vacuo. The obtained powder was finely ground in a crystal mortar and then a particle having a diameter of 50 μm or less was separated using a sonic sifter (Allen-Bradley, USA).

Example 42 Preparation of Dextran Microparticles Containing Interferon Alpha by Spray Drying

Interferon alpha, human serum albumin and dextran (molecular weight: 70,000) were dissolved in 10 mM ammonium bicarbonate buffer (pH 7.0) at concentrations of 1 mg / ml, 4 mg / ml and 20 mg / ml, respectively, followed by 2.5 Interferon alpha-containing dextran microparticles were prepared by feeding the spray dryer (Buchi-191) at a flow rate of ml / min. At this time, the temperature of the drying air flowing into the spray dryer was 90 ℃ and the average diameter of the obtained fine particles was 3 ㎛.

Example 43 Preparation of Polyethylene Glycol Microparticles Containing Interferon Alpha by Spray Drying

Interferon alpha and polyethylene glycol (molecular weight: 10,000) were dissolved in 10 mM ammonium bicarbonate buffer (pH 7.0) at concentrations of 1 mg / ml and 9 mg / ml, respectively, and the solution was spray dried at a flow rate of 3.0 ml / min. Buchi-191) to prepare interferon alpha-containing polyethylene glycol microparticles. At this time, the temperature of the drying air flowing into the spray dryer was 95 ℃ and the average diameter of the obtained fine particles was 2 ㎛.

Example 44 Preparation of Polyethylene Glycol Microparticles Containing Interferon Alpha by Spray Drying

Interferon alpha, human serum albumin, and polyethylene glycol (molecular weight: 10,000) were dissolved in 10 mM ammonium bicarbonate buffer (pH 7.0) at concentrations of 1 mg / ml, 4 mg / ml, and 10 mg / ml, respectively, followed by 3.0 Interferon alpha-containing polyethylene glycol microparticles were prepared by feeding the spray dryer (Buchi-191) at a flow rate of ml / min. At this time, the temperature of the drying air flowing into the spray dryer was 95 ℃ and the average diameter of the obtained fine particles was 2㎛.

Example 45 Preparation of Carboxymethylcellulose Microparticles Containing Interferon Alpha by Spray Drying

Interferon alpha and carboxymethylcellulose were dissolved in 10 mM ammonium bicarbonate buffer (pH 7.0) at concentrations of 1 mg / ml and 9 mg / ml, respectively, and the solution was spray dried at a flow rate of 3.0 ml / min (Buchi-191). Was supplied to prepare interferon alpha-containing carboxymethylcellulose microparticles. At this time, the temperature of the drying air flowing into the spray dryer was 98 ℃ and the average diameter of the obtained fine particles was 2㎛.

Example 46 Preparation of Methylcellulose Microparticles Containing Interferon Alpha by Spray Drying

Interferon alpha and methylcellulose were dissolved in 10 mM ammonium bicarbonate buffer (pH 7.0) at concentrations of 1 mg / ml and 3 mg / ml, respectively, and the solution was sprayed into a spray dryer (Buchi-191) at a flow rate of 3.0 ml / min. Supplied to produce methylcellulose microparticles containing interferon alpha. At this time, the temperature of the drying air flowing into the spray dryer was 90 ℃ and the average diameter of the obtained fine particles was 2 ㎛.

Example 47 Preparation of Dextran Sulfate Microparticles Containing Interferon Alpha by Spray Drying

Interferon alpha, human serum albumin and dextran sulfate (molecular weight: 25,000) were dissolved in 10 mM ammonium acetate buffer (pH 4.0) at concentrations of 1 mg / ml, 9 mg / ml and 50 mg / ml, respectively. Interferon alpha-containing dextran sulfate microparticles were prepared by feeding the spray dryer (Buchi-191) at a flow rate of 3 ml / min. At this time, the temperature of the drying air flowing into the spray dryer was 95 ℃ and the average diameter of the obtained fine particles was 3 ㎛.

Example 48 Preparation of Chondroitin Sulfate Microparticles Containing Interferon Alpha by Spray Drying

Interferon alpha and chondroitin sulfate were dissolved in 10 mM ammonium acetate buffer (pH 4.0) at a concentration of 1 mg / ml and 20 mg / ml, respectively, and the solution was sprayed into a spray dryer (Buchi-191) at a flow rate of 3.0 ml / min. Supplied to produce interferon alpha containing chondroitin sulfate microparticles. At this time, the temperature of the drying air flowing into the spray dryer was 92 ℃ and the average diameter of the obtained fine particles was 2 ㎛.

Example 49 Preparation of Dermatan Sulfate Fine Particles Containing Interferon Alpha by Spray Drying

Interferon alpha and dermatan sulfate were dissolved in 10 mM ammonium acetate buffer (pH 4.0) at concentrations of 1 mg / ml and 20 mg / ml, respectively, and then the solution was spray dried at a flow rate of 2.5 ml / min (Buchi-191). It was fed to to prepare interferon alpha-containing dermatan sulfate microparticles. At this time, the temperature of the drying air flowing into the spray dryer was 95 ℃ and the average diameter of the obtained fine particles was 3 ㎛.

Example 50 Preparation of Keratan Sulfate Microparticles Containing Interferon Alpha by Spray Drying

Interferon alpha and keratan sulfate were dissolved in 10 mM ammonium acetate buffer (pH 4.0) at concentrations of 1 mg / ml and 20 mg / ml, respectively, and the solution was spray dried at a flow rate of 3.0 ml / min (Buchi-191). It was supplied to the interferon alpha containing keratan sulfate microparticles. At this time, the temperature of the drying air flowing into the spray dryer was 95 ℃ and the average diameter of the obtained fine particles was 2 ㎛.

Example 51 Preparation of Heparan Sulfate Fine Particles Containing Interferon Alpha by Spray Drying

Interferon alpha and heparan sulfate were dissolved in 10 mM ammonium acetate buffer (pH 4.0) at concentrations of 1 mg / ml and 20 mg / ml, respectively, and then the solution was spray dried at a flow rate of 3 ml / min (Buchi-191). It was fed to to prepare the interferon alpha-containing heparan sulfate microparticles. At this time, the temperature of the drying air flowing into the spray dryer was 85 ℃ and the average diameter of the obtained fine particles was 2㎛.

Example 52 Preparation of Polyethylene Glycol Microparticles Containing Erythropoietin by Spray Drying

The erythropoietin microparticles prepared in Example 14 were uniformly dispersed at a concentration of 5 mg / ml in a solution in which polyethylene glycol (molecular weight: 3,350) was dissolved in methylene chloride at a concentration of 10 mg / ml, and then 3.0 ml of this solution. Polyethylene glycol microparticles containing erythropoietin were prepared by feeding the spray dryer (Buchi-191) at a flow rate of / min. At this time, the temperature of the drying air flowing into the spray dryer was 55 ℃ and the average diameter of the obtained fine particles was 3 ㎛.

Example 53 Preparation of Polyethylene Glycol Fine Particles Containing Colony Stimulating Factors by Spray Drying

The colony stimulating factor microparticles prepared in Example 15 were uniformly dispersed at a concentration of 5 mg / ml in a solution in which polyethylene glycol (molecular weight: 3,350) was dissolved in methylene chloride at a concentration of 10 mg / ml, and then 3.0 ml of this solution. Polyethylene glycol microparticles containing colony stimulating factor were prepared by feeding the spray dryer (Buchi-191) at a flow rate of / min. At this time, the temperature of the drying air flowing into the spray dryer was 55 ℃ and the average diameter of the obtained fine particles was 5 ㎛.

<Example 54> Preparation of interferon alpha-containing dextran microparticles by freeze drying

Interferon alpha, mannitol, and dextran (molecular weight: 70,000) were dissolved in 10 mM ammonium bicarbonate buffer (pH 7.0) at concentrations of 1 mg / ml, 9 mg / ml, and 90 mg / ml, respectively. After freezing in vacuo it was dried in vacuo. The obtained powder was finely ground in a crystal mortar and then a particle having a diameter of 50 μm or less was separated using a sonic sifter (Allen-Bradley, USA).

<Example 55> Preparation of interferon alpha-containing polyethylene glycol microparticles by freeze drying

Interferon alpha, zinc chloride, and polyethylene glycol (molecular weight: 10,000) were dissolved in 10 mM ammonium bicarbonate buffer (pH 7.0) at concentrations of 1 mg / ml, 0.06 mg / ml, and 10 mg / ml, respectively. After freezing at 캜, it was dried in vacuo. The obtained powder was finely ground in a crystal mortar and then a particle having a diameter of 50 μm or less was separated using a sonic sifter (Allen-Bradley, USA).

<Example 56> Preparation of interferon alpha-containing polyethylene glycol microparticles by freeze drying

Interferon alpha and polyethylene glycol (molecular weight: 3,350) were dissolved in 10 mM ammonium bicarbonate buffer (pH 7.0) at a concentration of 1 mg / ml and 10 mg / ml, respectively, and the solution was frozen at -70 ° C and vacuum dried. . The obtained powder was finely ground in a crystal mortar and then a particle having a diameter of 50 μm or less was separated using a sonic sifter (Allen-Bradley, USA).

<Example 57> Preparation of interferon alpha-containing carboxymethylcellulose microparticles by freeze drying

Interferon alpha and carboxymethylcellulose were dissolved in 10 mM ammonium bicarbonate buffer (pH 7.0) at concentrations of 1 mg / ml and 20 mg / ml, respectively, and the solution was frozen at -70 ° C and vacuum dried. The obtained powder was finely ground in a crystal mortar and then a particle having a diameter of 50 μm or less was separated using a sonic sifter (Allen-Bradley, USA).

Example 58 Preparation of Interferon Alpha-Containing Methylcellulose Microparticles by Freeze-Drying

Interferon alpha and methylcellulose were dissolved in 10 mM ammonium bicarbonate buffer (pH 7.0) at concentrations of 1 mg / ml and 5 mg / ml, respectively, and the solution was frozen at minus 70 ° C and vacuum dried. The obtained powder was finely ground in a crystal mortar and then a particle having a diameter of 50 μm or less was separated using a sonic sifter (Allen-Bradley, USA).

Example 59 Preparation of Interferon Alpha-Containing Dextran Sulfate Microparticles by Freeze-Drying

Interferon alpha and dextran sulfate (molecular weight: 25,000) were dissolved in 10 mM ammonium acetate buffer (pH 4.0) at concentrations of 1 mg / ml and 9 mg / ml, respectively, and the solution was frozen at -70 ° C and vacuum dried. It was. The obtained powder was finely ground in a crystal mortar and then a particle having a diameter of 50 μm or less was separated using a sonic sifter (Allen-Bradley, USA).

<Example 60> Preparation of interferon alpha-containing chondroitin sulfate microparticles by freeze drying

Interferon alpha and chondroitin sulfate were dissolved in 10 mM ammonium bicarbonate buffer (pH 7.0) at concentrations of 1 mg / ml and 5 mg / ml, respectively, and the solution was frozen at minus 70 ° C and vacuum dried. The obtained powder was finely ground in a crystal mortar and then a particle having a diameter of 50 μm or less was separated using a sonic sifter (Allen-Bradley, USA).

<Example 61> Preparation of interferon alpha-containing dermatan sulfate microparticles by freeze drying

Interferon alpha and dermatan sulfate were dissolved in 10 mM ammonium bicarbonate buffer (pH 7.0) at concentrations of 1 mg / ml and 5 mg / ml, respectively, and the solution was frozen at minus 70 ° C and vacuum dried. The obtained powder was finely ground in a crystal mortar and then a particle having a diameter of 50 μm or less was separated using a sonic sifter (Allen-Bradley, USA).

<Example 62> Preparation of interferon alpha-containing heparan sulfate microparticles by freeze drying

Interferon alpha and heparan sulfate were dissolved in 10 mM ammonium bicarbonate buffer (pH 7.0) at concentrations of 1 mg / ml and 10 mg / ml, respectively, and the solution was frozen at minus 70 ° C and vacuum dried. The obtained powder was finely ground in a crystal mortar and then a particle having a diameter of 50 μm or less was separated using a sonic sifter (Allen-Bradley, USA).

Example 63 Preparation of Interferon Alpha Fine Particles by Bubble Drying

Interferon alpha, polyethylene glycol, and human serum albumin were dissolved in 10 mM ammonium bicarbonate buffer (pH 7.0) at concentrations of 3.3 mg / ml, 13.3 mg / ml, and 33.4 mg / ml, respectively, and then the solution was flowed at 0.3 ml / min. By supplying to the bubble dryer (BD-500) to prepare a fine particle of the interferon alpha. At this time, the temperature of the drying air flowing into the bubble dryer was 60 ℃ and the average diameter of the obtained fine particles was 1.5 ㎛.

The following Examples 64 to 89 illustrate the preparation of sustained release formulations containing protein particles.

Example 64 Preparation of Sustained-Release Disk Formulations Containing Human Serum Albumin Coated with Hydrophilic Polymer

Trilaurin and the human serum albumin-containing polyethylene glycol (molecular weight: 10,000) particles prepared in Example 18 were mixed well in a ratio of 90:10, then 200 mg of the 13 mm KBr Die (Pike: Human serum albumin-containing discs were prepared by compressing for 1 minute with a force of 0.4 ton using a caber laboratory press.

Example 65 Preparation of Sustained Release Rod Formulations Containing Human Serum Albumin Coated with Hydrophilic Polymer

Trilaurin and the human serum albumin-containing polyethyleneglycol (molecular weight: 3,350) particles prepared in Example 19 were mixed well in a ratio of 90:10, and 200 mg of them were 37 ° C. at a pressure of 0.4 tons using a ram extruder. Human serum albumin containing rods were prepared by extrusion to a thickness of 1 mm at.

Example 66 Preparation of Interferon Alpha-Containing Sustained Release Rod Formulation Coated with Hydrophilic Polymer

Trilaurin and human serum albumin and the interferon alpha-containing polyethyleneglycol (molecular weight: 10,000) particles prepared in Example 43 were mixed well in a ratio of 95: 2: 3, 200 mg of which was 0.4 using a ram extruder. An interferon alpha containing rod was prepared by extrusion to a thickness of 1 mm while maintaining 37 ° C. at a pressure of tons.

Example 67 Preparation of Interferon Alpha-Containing Sustained Release Rod Formulation Coated with Hydrophilic Polymer

Trilaurin and human serum albumin and the interferon alpha-containing polyethyleneglycol (molecular weight: 10,000) particles prepared in Example 43 were mixed well in a ratio of 90: 4: 6, and 200 mg of this was 0.4 using a ram extruder. An interferon alpha containing rod was prepared by extrusion to a thickness of 1 mm while maintaining 37 ° C. at a pressure of tons.

Example 68 Preparation of Interferon Alpha-Containing Sustained Release Rod Formulation Coated with Hydrophilic Polymer

The interferon-containing microparticles prepared in Example 44 were well mixed at a ratio of 90:10, and 200 mg of the particles were taken and a thickness of 1 mm was maintained at 37 ° C. at a pressure of 0.4 tons using a ram extruder. To an interferon alpha containing rod.

Example 69 Preparation of Sustained-Release Disc Formulation Containing Human Serum Albumin

The lauric acid and the human serum albumin-containing polyethyleneglycol particles prepared in Example 18 were mixed well at a ratio of 7: 3, 200 mg of which was taken into a 13 mm KBr die (Pike, USA) and a cabbage laboratory press Human serum albumin containing discs were prepared by compression for 1 minute with a force of 0.4 tons.

Example 70 Preparation of Sustained Release Rod Formulation Containing Human Serum Albumin

Palmitic acid and the human serum albumin-containing polyethyleneglycol particles prepared in Example 20 were mixed well at a ratio of 7: 3, 200 mg of which was mixed with a ram extruder while maintaining 37 ° C. at a pressure of 0.4 tons and having a thickness of 1 mm. Extruded to prepare human serum albumin containing rods.

Example 71 Preparation of Sustained-Release Rod Formulations Containing Human Serum Albumin

Glyceryl monostearate and the human serum albumin-containing methylcellulose particles prepared in Example 21 were mixed well at a ratio of 70:30, taking 200 mg of them, while maintaining the temperature at 37 ° C. at a pressure of 0.4 tons using a ram extruder. Human serum albumin containing rods were prepared by extrusion to a thickness of mm.

Example 72 Preparation of Sustained-Release Disk Formulations Containing Human Growth Hormone

The lauric acid and the human growth hormone-containing polyethylene glycol particles made in Example 38 were mixed well at a ratio of 7: 3, 200 mg of which was taken into a 13 mm KBr die (Pike, USA) and a caber laboratory press was added. Human growth hormone-containing disks were prepared by compression for 1 minute with a force of 0.4 tons.

Example 73 Preparation of Sustained-Release Rod Formulations Containing Human Growth Hormone

Palmitic acid and the human growth hormone-containing dextran sulfate particles made in Example 40 were mixed well in a ratio of 7: 3, 200 mg of which were taken, and a ram extruder was used to maintain 37 ° C. at a pressure of 0.4 tons at 1 mm. Extrusion to thickness produced a human growth hormone containing rod.

Example 74 Preparation of Sustained-Release Rod Formulations Containing Human Growth Hormone

The human growth hormone microparticles prepared in Example 6 were uniformly dispersed at a concentration of 5 mg / ml in a solution in which polyethylene glycol (molecular weight: 3,350) was dissolved in methylene chloride at a concentration of 10 mg / ml, and then 3.0 ml / Human growth hormone-containing polyethylene glycol microparticles were prepared by feeding the spray dryer (Buchi-191) at a flow rate of min. Trilaurin and the particles are mixed well at a ratio of 8: 2, 200 mg of which are extruded to a thickness of 1 mm using a ram extruder while maintaining 37 ℃ at a pressure of 0.4 tons to load a human growth hormone-containing rod Prepared.

Example 75 Preparation of Sustained-Release Disk Formulations Containing Human Growth Hormone

Glyceryl myristrate and the human growth hormone-containing dextran sulfate particles made in Example 40 were mixed well at a ratio of 8: 2, 200 mg of which were taken in a 13 mm KBr die (Pike, USA) and cabbage laboratories. A human growth hormone-containing disk was prepared by compressing for 1 minute with a force of 0.4 ton using a tori press.

Example 76 Preparation of Sustained-Release Rod Formulations Containing Human Growth Hormone

Trilaurin and the human growth hormone-containing dextran sulfate particles made in Example 41 were mixed well at a ratio of 6: 4, 200 mg of which were taken, and 1 mm while maintaining 37 ° C. at a pressure of 0.4 tons using a ram extruder. A human growth hormone containing rod was prepared by extrusion to a thickness of.

Example 77 Preparation of Interferon Alpha-Containing Sustained Release Rod Formulations

The interferon alpha microparticles prepared in Example 8 were uniformly dispersed at a concentration of 5 mg / ml in a solution in which polyethylene glycol (molecular weight: 3,350) was dissolved in methylene chloride at a concentration of 5 mg / ml, and then the solution was 3.0 ml / min. Interferon alpha-containing polyethylene glycol microparticles were prepared by feeding the spray dryer (Buchi-191) at a flow rate of. The interferon alpha-containing rods were prepared by mixing trilaurin and the particles well at a ratio of 8: 2, taking 200 mg of them and extruding them to a thickness of 1 mm using a ram extruder while maintaining 37 ° C. at a pressure of 0.4 tons. .

Example 78 Preparation of Interferon Alpha-Containing Sustained Release Rod Formulations

Glyceryl monostearate and the interferon alpha-containing dextran fine particles prepared in Example 42 were mixed well in a ratio of 70:30, taking 200 mg of them, while maintaining a temperature of 37 ° C. at a pressure of 0.4 tons using a ram extruder. The interferon alpha containing rods were made by extrusion to a thickness of mm.

Example 79 Preparation of Interferonalpha-Containing Sustained-Release Disk Formulations

The lauric acid and the interferon-containing polyethylene glycol particles prepared in Example 43 were mixed well at a ratio of 8: 2, 200 mg of which was taken into a 13 mm KBr die (Pike, USA), and using a caber laboratory press. Interferon alpha containing discs were made by compression for 1 minute with a force of 0.4 tons.

Example 80 Preparation of Interferon Alpha-Containing Sustained Release Rod Formulations

Palmitic acid and the interferon alpha-containing methylcellulose particles prepared in Example 46 were mixed well at a ratio of 70:30, 200 mg of which were taken at a temperature of 0.4 ton using a ram extruder at a thickness of 1 mm. Extruded to produce interferon alpha containing rods.

Example 81 Preparation of Interferon Alpha-Containing Sustained-Release Disk Formulations

Glyceryl myristrate and the interferon alpha-containing chondroitin sulfate particles prepared in Example 48 were mixed well at a ratio of 8: 2, 200 mg of which were taken into a 13 mm KBr die (Pike, USA) and cabbage laboratories. An interferon alpha containing disc was prepared by compressing for 1 minute with a force of 0.4 ton using a Tori press.

Example 82 Preparation of Interferon Alpha-Containing Sustained Release Rod Formulations

The interferon alpha-containing keratan sulfate fine particles prepared in Example 51 were mixed well in a ratio of 6: 4, 200 mg of which was mixed with a ram extruder while maintaining 37 ° C. at a pressure of 0.4 tons at 1 mm. An interferon alpha containing rod was prepared by extrusion to a thickness of.

Example 83 Preparation of Sustained-Release Disk Formulations Containing Interferonalpha

The interferon alpha particles prepared in Example 54 were evenly dispersed at a concentration of 5 mg / ml in a solution in which polyethylene glycol (molecular weight: 3,350) was dissolved in methylene chloride at a concentration of 5 mg / ml, and then the solution was 3.0 ml / min. Interferon alpha-containing polyethylene glycol microparticles were prepared by feeding the spray dryer (Buchi-191) at a flow rate of. The interferon alpha-containing rods were prepared by mixing trilaurin and the particles well at a ratio of 8: 2, taking 200 mg of them and extruding them to a thickness of 1 mm using a ram extruder while maintaining 37 ° C. at a pressure of 0.4 tons. .

Example 84 Preparation of Interferon Alpha-Containing Sustained Release Rod Formulations

Palmitic acid and the interferon alpha-containing polyethylene glycol particles prepared in Example 55 were mixed well at a ratio of 70:30, 200 mg of which was mixed with a ram extruder at a thickness of 1 mm while maintaining 37 ° C. at a pressure of 0.4 tons. Extruded to produce interferon alpha containing rods.

Example 85 Preparation of Interferon Alpha-Containing Sustained-Release Disk Formulations

Glyceryl myristrate and the interferon alpha-containing dextran sulfate particles made in Example 59 were mixed well in a ratio of 8: 2, 200 mg of which was taken into a 13 mm KBr die (Pike, USA) An interferon alpha containing disc was prepared by compressing for 1 minute with a 0.4 ton force using a boror press.

Example 86 Preparation of Interferon Alpha-Containing Sustained Release Rod Formulations

The interferon alpha-containing chondroitin sulfate microparticles prepared in Example 60 were mixed well in a ratio of 6: 4, 200 mg of which was mixed with a ram extruder and maintained at 37 ° C. at a pressure of 0.4 tonnes of 1 mm. Extrusion to thickness produced an interferon alpha containing rod.

Example 87 Preparation of Erythropoietin-Containing Sustained Release Rod Formulations

Trilaurin and the erythropoietin-containing polyethyleneglycol microparticles prepared in Example 52 were mixed well in a ratio of 9: 1, 200 mg of which was mixed with a ram extruder while maintaining 37 ° C. at a pressure of 0.4 tons at 1 mm. An erythropoietin-containing rod was prepared by extrusion to a thickness of.

Example 88 Preparation of Sustained Release Rod Formulation Containing Colony Stimulating Factors

Trilaurin and the colony-stimulating factor-containing microparticles prepared in Example 53 were mixed well in a ratio of 9: 1, 200 mg of which was taken, and a ram extruder was used to maintain 37 ° C. at a pressure of 0.4 tons and a thickness of 1 mm. To an interferon alpha containing rod.

Example 89 Preparation of Interferon Alpha-Containing Sustained Release Rod Formulations

The interferon alpha-containing microparticles prepared in Example 63 were mixed well in a ratio of 85:15, and then 200 mg of them were taken using a ram extruder while maintaining 37 ° C. at a pressure of 0.8 tons at a thickness of 1 mm. To an interferon alpha containing rod.

Comparative Example 1 Preparation of Human Serum Albumin-Containing Disk Formulations

Trilaurin and the human serum albumin particles prepared in Example 1 were mixed well at a ratio of 90:10, 200 mg of them were taken in a 13 mm KBr die (Pike, USA) and the caber laboratory press Human serum albumin containing discs were prepared by compression for 1 minute with a force of 0.4 tons.

Comparative Example 2 Preparation of Human Serum Albumin-Containing Rod Formulations

Trilaurin and human serum albumin particles prepared in Example 1 were mixed well at a ratio of 90:10, and 200 mg of them were 1 mm thick using a ram extruder while maintaining 37 ° C. at a pressure of 0.4 tons. Extruded human serum albumin containing rods were made.

Comparative Example 3 Preparation of Interferon Alpha-Containing Disc Formulations

The interferon alpha particles produced in Example 10 were well mixed with trilaurin at a ratio of 84:11, and 200 mg of them were extruded to a thickness of 1 mm using a ram extruder while maintaining 37 ° C. at a pressure of 0.4 tons. Interferon alpha containing rods were prepared.

Comparative Example 4 Preparation of Interferon-Containing Disc Formulations

The interferon particles produced in Example 10 were well mixed with trilaurin in a ratio of 91: 8, and then 200 mg of them were taken in a 13 mm KBr die (Pike, USA) and 0.4 using a caber laboratory press. Human serum albumin containing discs were prepared by compression for 1 minute with ton force.

Comparative Example 5 Preparation of Interferon Alpha-Containing Rod Formulation Using Melting and Cooling

The pre-melted myristic acid and the interferon alpha microparticles produced in Example 10 are quickly mixed well in a ratio of 19: 1, and then injected into a silicon tube having an inner diameter of 0.8 mm and cooled. A sufficiently cooled rod was removed from the tube to prepare an interferon-containing myristic acid rod.

Comparative Example 6 Preparation of Interferon Alpha-Containing Rod Formulation Using Melting and Cooling

The pre-melted lauric acid and the interferon alpha microparticles prepared in Example 10 are quickly mixed well in a ratio of 19: 1, and then injected into a silicon tube having an internal diameter of 0.8 mm and cooled. A sufficiently cooled rod was separated from the tube to prepare an interferon-containing lauric acid rod.

Comparative Example 7 Preparation of Interferon Alpha-Containing Microspheres Formulation Using Melting and Dispersing

A mixture of the pre-melted diglyceryl palmitostearate and the interferon alpha microparticles prepared in Example 10 at a ratio of 19: 1 was dispersed in a 55% 0.25% polyvinyl alcohol solution and then cooled to contain interferon. Microspheres were prepared.

Comparative Example 8 Preparation of Interferon Alpha-Containing Rod Formulation Using Melting and Cooling

Kepr acid and lauric acid were mixed well at a ratio of 1: 9, and the human serum albumin and interferon alpha-containing polyethylene glycol microparticles prepared in Example 43 were mixed well at a ratio of 85: 13: 2 in a molten solution at a diameter of 0.8 mm. Inject into a silicone tube to cool. A sufficiently cooled rod was removed from the tube to prepare an interferon containing rod.

Comparative Example 9 Preparation of Interferon Alpha-Containing Rod Formulation Simple Mixture of Polyethyleneglycol

2 mg, 8 mg, 20 mg, and 170 mg of interferon alpha, polyethylene glycol, human serum albumin, and trilaurin, respectively, were mixed in a fine mortar and mixed finely, and then mixed with a ram extruder at a pressure of 0.8 tons. An interferon alpha containing rod was prepared by extrusion to a thickness of 1 mm while maintaining 37 ° C.

The following test examples confirm the stability of the sustained release formulation of the present invention, the initial over-release release and sustained sustained effect.

The ex vivo release test was tested in 10 mM PBS (pH 7.0) buffer and quantitatively released using the assay conditions specified in EUROPEAN PHARMACOPOEIA. However, the interferon was detected at a low content of Ex 280 nm and Em 350 nm using a fluorescence detector.

In vivo release testing was performed subcutaneously injecting an interferon alpha containing formulation using SD rats weighing 200-250 g, 6-7 weeks old, and then the concentration of interferon alpha in the blood was quantified using an ELISA kit.

Test Example 1 Protein Stability Test

The interferon alpha extracted from the interferon alpha-containing sustained-release rod prepared in Example 66 and the interferon alpha remaining in the formulation 3 days after the ex vivo release test were extracted to determine the stability of the protein in the formulation after the sustained release formulation preparation and during the release. It was confirmed in grams, and the results are shown in FIG.

FIG. 1A is a chromatogram of interferon alpha extracted from the formulation immediately after preparation of the sustained release formulation, and FIG. 1B is a chromatogram of interferon alpha extracted from the formulation during the release test of the sustained release formulation. This shows that the protein was not denatured during the preparation of the interferon alpha-containing sustained release formulation and during the in vitro release test period.

In contrast, in vitro release test supernatants of Comparative Examples 5 and 6 formulations prepared from mixtures of molten lipids were identified by size exclusion chromatography to confirm denaturation of the protein by heat. In addition, the comparative example 9 formulation prepared by simply mixing the hydrophilic polymer was not destabilized as compared to the formulation of the protein coated with the hydrophilic polymer Example 89 formulation was confirmed that the protein denaturation during the in vitro release test period. The results are shown in Figures 3a, 3b, 3c, 3d and 3e.

Experimental Example 2 Initial In Vitro Release Test

The formulations of Examples 64 to 67 and Comparative Examples 1 to 8 were cut to a suitable size, placed in 10 mM phosphate buffer to the same protein concentration, and left at 37 ° C. for 1 hour to obtain the amount of protein released into the supernatant by liquid chromatography. Quantification

 The results are shown in Table 1 and Table 2.

Initial ex vivo release of human serum albumin containing formulations Formulation Hydrophilic polymer type and content (%) Protein content (%) Formulation form Initial In Vitro Release (%) Example 64 5%, PEG (M.W. 10,000) 5% disk 2.9% Example 65 5%, PEG (M.W. 3,350) 5% road 4% Comparative Example 1 0% 5% disk 14.3% Comparative Example 2 0% 5% road 25%

As shown in Table 1, regardless of the form of the formulation, the initial in vitro release amount could be reduced by protecting the protein particles with a hydrophilic polymer, and the same result can be seen in Table 2.

Initial In Vitro Release of Interferon Alpha-Containing Formulations Formulation Hydrophilic polymer type and content (%) Protein content (%) Formulation form Initial In Vitro Release (%) Example 66 2.7%, PEG (M.W. 10,000) 0.3% road 2.27% Example 67 5.4%, PEG (M.W. 10,000) 0.6% road 0.95% Comparative Example 3 0% 0.64% road 19.2% Comparative Example 5 0% 0.2% road 23.5% Comparative Example 6 0% 0.2% road 22% Comparative Example 7 0% 0.2% road 55% Comparative Example 8 1.8%, PEG (M.W. 10,000) 0.2% road 5.7%

As shown in Table 2, the formulations of Examples 66 and 67, protected by coating interferon alpha particles with a hydrophilic polymer, reduced initial release compared to the formulations of Comparative Examples 3, 5, 6, and 7 which were not coated with hydrophilic polymers. I could see. In addition, even in the formulation of Comparative Example 8 in which the protein particles coated with the hydrophilic polymer material and the molten lipid material were mixed, the initial release amount could be slightly reduced. However, as confirmed in the following in vivo release test, the formulation of Comparative Example 8 did not show sustained release for more than 24 hours due to excessive porosity due to no pressure applied to the lipid matrix.

On the other hand, in the case of Example 67, although the protein content is twice as much as Example 66, it was confirmed that the initial in vitro release amount was further reduced by doubling the amount of the hydrophilic polymer. On the other hand, the hydrophilic polymer-coated protein particles (Example 43) used in the preparation of Example 67 were all dissolved in the buffer within 1 to 2 minutes when suspended in the buffer. From these observations, it can be seen that the effect of coating the protein with a hydrophilic polymer is to separate the protein from the lipid matrix, thereby increasing the stability of the protein and reducing the initial release.

In addition, the sustained release by the lipid forming a dense matrix by the stability and pressure of the protein particles coated with a hydrophilic polymer material through the following in vivo release test.

Test Example 3 In Vivo Release Test (Rat)

The formulation of Example 89 was injected subcutaneously in SD rats such that the dose of interferon alpha was 100 μg / kg, and the formulations of Comparative Examples 4, 5, 6 and 8 were 25 μg / kg, 19 μg / kg, respectively, The rats were injected subcutaneously to 18 μg / kg and 30 μg / kg.

The amount of interferon alpha in blood before and after administration was quantified using an ELISA kit, and the results are shown in FIGS. 2A, 2B and 2C.

The formulation of Comparative Example 4 did not detect a sharp drop in blood interferon concentration after 24 hours, and the formulation of Example 89 showed sustained release for at least 3 days. This is because the lipid matrix of Comparative Example 4 was densely formed by pressure, but because the protein particles were not protected by the hydrophilic polymer, they could not be modified and released in the lipid during the test. In addition, as can be seen in Figure 2c, Comparative Formulations 5, 6 and 8 of the formulations were all slightly higher initial release than the dosage, did not show sustained release for more than 24 hours.

This is because the protein particles that are not protected by the hydrophilic polymer are not released due to denaturation by the lipid matrix or heat, and even though they are protected by the hydrophilic polymer, the formulation preparation method by melting and cooling cannot form a dense lipid matrix. This is because they exhibit excessive initial release and cannot have sustained release.

Test Example 4 In vivo Release Test (Monkey)

Example 89 Injection was subcutaneously in monkeys so that the interferon alpha dosage of the formulation was 100 μg / kg, 250 μg / kg. The amount of blood interferon alpha before and after administration was quantified using an ELISA kit, and the results are shown in FIG. 2D. C _max was increased by 10060 pg / ml, 28657 pg / ml, and AUC was increased by 2.5 and 2.6 times, respectively, to 19260 pgday / ml and 50307 pgday / ml, respectively. As the increase, C _max and AUC were found to be proportional.

Compared with Test Example 3 results of the same formulation, an increase in sustained release over 9 days in monkeys is interpreted as a difference between species.

The test examples confirm that the sustained release formulation of the present invention has no protein denaturation after preparation of the formulation and exhibits therapeutic activity for a prolonged period without initial overrelease of the protein.

While the present invention has been described with reference to specific embodiments, it will be understood by those skilled in the art that the present invention is not limited thereto and that various modifications are possible within the spirit and scope of the present invention.

As described above, the protein drug, which is an active ingredient, is stabilized with a hydrophilic polymer material and then uniformly mixed with the lipid material and compressed to prepare a lipid implant formulation. Thus, the stability of the protein drug appeared in a sustained-release formulation using a hydrophobic matrix such as lipid. The problem could be solved, and when administered into the body, the protein drug was continuously released in active form at an effective concentration for a long time. Therefore, the protein containing sustained-release lipid implant of the present invention can be used to effectively treat a disease while reducing the number of injections.

1 is a high performance reverse phase chromatogram showing protein stability after preparation and during release of the interferon alpha containing sustained release formulation of the present invention.

2 shows in vivo release test results of interferon alpha containing sustained release formulations and comparative formulations.

3 is a size exclusion chromatogram showing protein denaturation of an interferon alpha containing formulation.

4 is a micrograph of the surface and cross section of an interferon alpha containing sustained release formulation of the present invention.

5 is a micrograph of a comparative interferon alpha containing formulation by melting and dispersing.

FIG. 6 is a micrograph of the surface of an interferon alpha containing formulation of Comparative Example by melting and cooling.

Claims (16)

delete delete A compressed mixture of protein drug and lipid coated with a hydrophilic polymeric material; Hydrophilic polymers have a molecular weight of 2,000 Daltons or more, polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, polyethyleneimine, dextran, dextran sulfate, chondroitin sulfate. Dermatan sulfate, heparan sulfate, keratan sulfate, hyaluronic acid, chitosan, albumin, collagen, fibrin and mixtures thereof; Lipids are protein-containing, characterized in that they are selected from fatty acids, monoglycerides, diglycerides, triglycerides, sorbitan fatty acid esters, phospholipids, sphingolipids, cholesterol, waxes and salts and derivatives thereof that are solid at room temperature. Sustained release rapid implants. 4. The protein containing sustained-release lipid implant of claim 3, wherein the protein drug comprises an additional protein stabilizer. 5. The protein containing sustained-release lipid implant of claim 4, wherein the additional protein stabilizer is selected from sugars, polyols, surfactants, amino acids, inorganic salts, and mixtures thereof. The protein-containing sustained-release lipid implant of claim 3, wherein the hydrophilic polymer material is 0.1 to 99.9% by weight in the protein drug coated with the hydrophilic polymer material. The protein containing sustained-release lipid implant according to claim 3, wherein the content of the lipid material in the protein-containing sustained-release lipid implant is 40 to 99% by weight. The protein containing sustained-release lipid implant of claim 3 in the form of a disc, pellet or rod. Protein drugs are polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, polyethyleneimine, dextran, dextran sulfate, chondroitin sulfate. Preparing a protein solid powder coated with a hydrophilic polymer having a molecular weight of at least 2000daltons selected from dermatan sulfate, heparan sulfate, keratan sulfate, hyaluronic acid, chitosan, albumin, collagen, fibrin and mixtures thereof; The coated protein solid powder prepared in the above step, and fatty acids, monoglycerides, diglycerides, triglycerides, sorbitan fatty acid esters, phospholipids, sphingolipids, cholesterol, waxes and salts thereof which are solid at room temperature and Mixing a lipid selected from derivatives; And Compressing or extruding the mixture of the above formulated, characterized in that the method for producing a protein-containing sustained-release lipid implant. The method of claim 9, wherein the protein solid powder coated with the hydrophilic polymeric material has a diameter of 0.1 μm to 200 μm. The method of claim 9, wherein the protein microparticles are dispersed in a solution in which the hydrophilic polymer material is dissolved, and then dried by a method selected from spray drying, freeze drying, spray freezing drying, and bubble drying to prepare a protein solid powder coated with a hydrophilic polymer material. How to. The method of claim 11, wherein the aqueous solution in which the protein is dissolved is dried by a method selected from spray drying, freeze drying, spray freeze drying, and bubble drying to produce protein fine particles. 10. The method of claim 9, wherein the protein molecules and the hydrophilic polymer are dissolved and then dried by a method selected from spray drying, freeze drying, spray freeze drying, and bubble drying to prepare a protein solid powder coated with a hydrophilic polymer. The method of claim 11, comprising adding an additional protein stabilizer. The method of claim 14, wherein the additional protein stabilizer is selected from sugars, polyols, surfactants, amino acids, inorganic salts, and mixtures thereof. The method of claim 15, wherein the sugar is selected from sucrose, glucose, inositol, trihalose, maltose, mannitol, lactose, mannose, xylitol, sorbitol, and cyclodextrin.