KR20180023337A - Sustained release-microsphere comprising oils containing C18:1, C18:1(OH) or C18:2 long chain fatty acid and method for preparing the same - Google Patents

Abstract

The present invention relates to a long-term sustained release microsphere containing a physiologically active substance and a method for producing the same, wherein the long-term sustained release microsphere contains biologically active substances, biocompatible polymers, and oils with long chain fatty acids of C18:1, C18:1(OH) and C18:2 as release inhibitors. The rate of encapsulating the biologically active substances is increased by enclosing the biocompatible polymers where the biologically active substances are encapsulated using the release inhibitor. The long-term sustained release microsphere has excellent release characteristics with minimal initial overdischarge so as to solve problems such as side effects and the like due to initial overdischarge.

Description

[0001] The present invention relates to a sustained-release microsphere employing a release inhibitor including oil containing long chain fatty acids of C18: 1, C18: 1 (OH) or C18: 2 and a method of producing the sustained release microspheres (OH) or C18: 2 long chain fatty acid and method for preparing same same}

The present invention relates to a sustained release microsphere capable of continuously controlling the release of a drug by enclosing a water-soluble drug in a carrier made of a biodegradable polymer, and a method for producing the sustained release microsphere, and more particularly, Unlike the sustained-release preparation containing long-chain fatty acids, specifically long-chain fatty acids of C18: 1, C18: 1 (OH) or C18: 2 as a release inhibitor, The active substance is applied to form a lipid matrix, and it is possible to continuously maintain a constant release rate and effective blood concentration without rapid initial over-release and delayed release of the drug, and thus can be usefully used as a long-term sustained-release preparation of a hydrophilic drug And a method of manufacturing the same.

In the case of drugs for the treatment of chronic diseases, long term release formulations can be used to reduce the frequency of administration, so that only a single administration can maintain the effective drug concentration for a long time and improve the patient's compliance with the medication. Therefore, it is considered that a sustained-release injection formulation is very useful in order to maintain the concentration of the drug for a long time while improving the convenience and compliance of the patient by reducing the frequency of taking the drug.

This sustained-release injectable formulation refers to an injection formulation that is formulated so that the drug can be continuously and uniformly released while maintaining the biological activity in the body during subcutaneous or intramuscular injection.

Conventionally, such conventional methods of producing sustained release injection agents are known as a coacervation method, a melt extrusion method, a spray drying method, and a solvent evaporation method. Among these methods, the emulsion evaporation method, which is classified into a dual emulsion evaporation method (W / O / W; water / oil / water) and a single emulsion evaporation method (O / W;

However, when a water-soluble drug is used in the microsphere using the emulsion evaporation method, the drug is diffused into the external continuous phase during the manufacturing process of the microsphere, so that the efficiency of filling the drug is very poor.

Furthermore, with respect to the microspheres produced according to this multiple emulsion method, the release of the drug encapsulated in the microspheres is characterized by two distinct phases: an initial burst and a subsequent sustained release, The release of the drug may cause side effects due to excessive release of unexpected drug when applied in actual clinical practice, which is a problem that must be solved in the development of formulations. Such a manufacturing method is disadvantageous in that the initial excess release rate of the encapsulated drug is high When the active ingredient is dissolved in an organic solvent or water, there is a problem in that the physical properties and stability of the drug are deteriorated while forming microspheres.

In this regard, Korean Patent Registration No. 10-0442931 discloses a method of dissolving a polymer carrier substance in a suitable organic solvent in which a drug compound is not dissolved, adding an aqueous medium containing an excessive protective colloid and a phase inducing agent, A method of manufacturing a preparation is disclosed. However, such a manufacturing method has a disadvantage in that the procedure is difficult and the yield of the product is extremely low, so that the unit price of the product can be increased.

Further, Korean Patent Registration No. 10-0409413 discloses a method for producing an emulsion by an underwater drying method and then heating and drying the emulsion at a temperature higher than the glass transition temperature (Tg) of the biodegradable polymer to significantly suppress the initial release and minimize the organic solvent , This manufacturing method has a serious disadvantage that the glass transition temperature should be at least 47 캜 or higher and that the physiologically active substance can be denatured by heat.

Korean Patent Registration No. 10-0293882 discloses a novel salt comprising a cation derived from a peptide containing a basic group and an anion derived from a carboxy end polyester and a method for producing these salts and a method for producing these salts in the preparation of a sustained- Describes the method of use. However, in this method, the drug and the polymer are frozen and dried under vacuum to obtain a transparent film, which is then dispersed again in dichloromethane and re-dried. The polymer and the polymer are compressed and molded to have a relatively large diameter (for example, 16 or 18 Gauge) injection needle, which causes the patient to be afraid.

U.S. Patent Nos. 6,419,961, 5,585,460 and 4,652,441 disclose polylactic acid-polyglycolic acid copolymer microspheres containing a peptide-based drug such as leuprorelin acetate using a multiple emulsion method Is disclosed. In particular, in the case of U.S. Patent No. 4,652,441, water-soluble polymers such as gelatin, albumin, pectin, and agar are introduced into the inner aqueous phase together with the drug to increase the viscosity of the internal aqueous phase, A double encapsulation of the polylactic acid-polyglycolic acid copolymer was induced and long-term sustained-release injections could be prepared. However, when gelatin is used to increase the viscosity of the internal aqueous phase, the primary emulsion solution should be heated to a high temperature of 80 DEG C in order to uniformly distribute the drug in the solution during the preparation of the primary emulsion solution, There is a problem in that the manufacturing process of the microspheres is complicated because it has to be cooled to 20 캜 to 30 캜 when re-dispersing in the continuous phase. In addition, the above-described preparation method has a limitation that it can be used only for the production of a drug having stability against heat.

In addition, according to Qingguo Xu et al. (Controlled release of amoxicillin from hydroxyapatite-coated poly (lactic-co-glycolic acid) microspheres, Journal of Controlled Release 127 A method of coating with apatite to inhibit the initial release and releasing the physiologically active substance for a long period of time has been disclosed, but it has a disadvantage that it is difficult to produce by a troublesome coating production method requiring a long time.

Accordingly, the present inventors have found that the microparticles exhibit porosity depending on the temperature, concentration, and stirring speed in the above-described manufacturing process in the conventional single emulsion (W / O emulsion) or double emulsion (W / O / W emulsion) (Initial burst) and to lower the drug loading rate.

Accordingly, the present invention provides a method for producing a long chain fatty acid, which comprises using a release inhibitor of an oil containing a long chain fatty acid of C18: 1, C18: 1 (OH) or C18: 2 to perform a conventional solvent evaporation method or solvent extraction method, In order to manufacture microspheres suppressing the overexertion problem, which is a problem occurring in the emulsion, the micropores are removed from the surface of the microspheres, the surface area is reduced, and the penetration of the external moisture is delayed, Soluble drug-containing sustained-release microspheres having a drug release profile capable of maintaining an effective blood concentration for a long period of time, and a method for producing the same.

In order to achieve the above object, in one aspect, the present invention provides a biocompatible composition comprising a physiologically active substance, a biocompatible polymer and a release inhibitor,

Characterized in that the biocompatible polymer encapsulated with the physiologically active substance is further encapsulated by a release inhibitor and the release inhibitor is an oil containing a long chain fatty acid of C18: 1, C18: 1 (OH) or C18: 2 Release microspheres.

Physiologically active substances applicable to the present invention include physiologically active peptides and proteins, and physiologically active peptides and proteins are composed of two or more amino acids having a molecular weight of about 200 to 100,000, examples of which are peptides and protein drugs The present invention relates to a pharmaceutical composition for inhibiting the growth of human growth hormone, growth hormone releasing hormone, growth hormone releasing peptide, interferon, colony stimulating factor, interleukin, macrophage activator, macrophage peptide, B cell factor, T cell factor, protein A, Alpha-1 antitrypsin, albumin and its fragment polypeptides, apolipoprotein-E, erythropoietin, Factor VII, Factor VIII, Factor IX, Factor IX, Plasminogen activator, urokinase, streptokinase, protein C, C-reactive protein, renin inhibitor, Cole A growth factor, an osteogenic growth factor, an osteogenesis promoting protein, a calcitonin, an insulin, an atriopeptin, a cartilage inducing factor, a connective tissue activating factor, a follicle growth factor, a platelet derived growth factor, Lactating hormone, luteinizing hormone, luteinizing hormone releasing hormone, nerve growth factor, paratroid hormone, lilacsin, sicretin, somatomedin, insulin-like growth factor, adrenocorticotropic hormone, glucagon, cholestystinin, pancreatic poly Peptides, gastrin releasing peptides, corticotropin releasing factors, thyroid stimulating hormone, monoclonal or polyclonal antibodies against various viruses, bacteria, toxins, and various virus-derived vaccine antigens.

In the present invention, the physiologically active substance is not particularly limited, and may be a luteinizing hormone-releasing hormone (LHRH) analogue or a peptide drug or a salt thereof . For example, agonists of LHRH analogs include goserelin, looproide, tryptorelin, buserelin, napalelin, antagonists such as setroelelix, alginate, and other available Examples of the peptide drug include octreotide and the like. These LHRH analogues act on the pituitary gland when administered to the body to inhibit the secretion of luteinizing hormone (in the case of an agonist, it initially promotes secretion but is inhibited when released continuously) Testosterone, which is a sex hormone, is a peptide substance that shows therapeutic effect in hormone-reactive prostate cancer, breast cancer, endometriosis etc. by inhibiting secretion of estrogen. Examples of the above peptide salts include, but are not limited to, acid addition salts of peptides, specifically goserelin acetate.

In the present invention, the term "biocompatible polymer" means a substance also known as a biodegradable polymer. Conventional polymers used in the production of microcapsules may be used as pharmaceuticals. Preferably, It is also possible to use polylactic acid such as Polycaprolactone , Polyaminoacids, Polyhydroxybutyrate, Polyoxyethylene glycolate, Polyanhydrides, Polylactides, Polyglycolide Polyglycolides), poly (lactide-co-glycolide), which is a copolymer thereof, and the like. Preferably, it is poly (Lactic-co-Glycolic Acid, PLGA).

The biocompatible polymer (PLGA) that can be used in the present invention may have a weight average molecular weight of 60,000 or less. For example, poly (lactide-co-glycolide) (50:50) having a molecular weight of about 13,000, poly (lactide-co-glycolide) (50:50) having a molecular weight of about 33,000, poly (Lactide-co-glycolide) (50:50), a poly (lactide-co-glycolide) (75:25) having a molecular weight of about 20,000, a poly (lactide) It is possible to use. Such biocompatible polymers include RG502H, RG503H, RG504H, RG752H and R202H of Boehringer Ingelheim.

In the present invention, the biocompatible polymer may include 70 to 99% by weight, more preferably 80 to 98% by weight, and most preferably 85 to 97% by weight based on the total weight of the final microspheres to be produced. In the present invention, when the biocompatible polymer is contained in an amount of less than 70% by weight, the distribution of the physiologically active substance is relatively increased, and there may be a problem that the drug efficacy can not be maintained during the initial overexpression or a desired period. The amount to be administered to the patient may be too great to be administered or the administration itself may become impossible.

In the microsphere according to the present invention, the biocompatible polymer primarily encapsulates the physiologically active substance and is further encapsulated by the following release inhibitor.

In the present invention, the release inhibitor is included for the purpose of encapsulating the physiologically active substance and controlling the release of the physiologically active substance, thereby forming a lipid matrix surrounding the biopolymer encapsulating the physiologically active substance. Preferably, the release inhibitor has a carbon number of 16 or more Long chain fatty acids, specifically C18: 1, C18: 1 (OH) or C18: 2 fatty acids. Examples of oils containing such long chain fatty acids, i.e. C18: 1 or C8: 2 fatty acids, include linoleic acid (C18: 2) 64%; Palmitic acid (C16: 0) 14%; 10% oleic acid (C18: 1); Linolenic acid (C18: 3) 7%; Soybean lecithin consisting of 4% stearic acid (C18: 0), or linoleic acid (C18: 2) 39.3%; Oleic acid (C18: 1) 33.1%; Palmitic acid (C16: 0) 19.1%; Stearic acid (C18: 0) 1.9%; 0.6% arachidic acid (C20: 0); Cottonseed oil or ricinoleic acid (C18: 1 (OH)) (87%) with myristic acid (C14: 0) 0.3% 7% oleic acid (C18: 1); Linoleic acid (C18: 2) 3%; Palmitic acid (C16: 0) 2%; 58.9% castor oil or linoleic acid (C18: 2) with 1% stearic acid (C18: 0) and the like; 25.8% oleic acid (C18: 1); Palmitic acid (C16: 0) 11.0%; Stearic acid (C18: 0) 1.7%; 66% of corn oil or linoleic acid (C18: 2) having 1.1% linolenic acid (C18: 3) Linolenic acid (C18: 3) 0.5%; 26% oleic acid (C18: 1); Palmitic acid (C16: 0) 4%; 7.5-20.0% of sunflower oil or palmitic acid (C16: 0) with 2% stearic acid (C18: 0) or the like; Palmitoleic acid (C16: 1), 0.3-5.0%; Stearic acid (C18: 0), 0.5-5.0%; Oleic acid (C18: 1), 55.0-83.0%; 40.4% olive oil or linoleic acid (C18: 2) having linoleic acid (C18: 2), 3.5-21.0% and the like; Oleic acid (C18: 1) 45.4%; Palmitic acid (C16: 0) 9.1%; 50-57% of sesame oil or linoleic acid (C18: 2) having 4.3% stearic acid (C18: 0); Linolenic acid (C18: 3) 5-10%; 17-26% oleic acid (C18: 1); Palmitic acid (C16: 0) 9-13%; 2.4% of soybean oil or arachidic acid (C20: 0) with stearic acid (C18: 0) 3-6% and the like; Palmitic acid (C16: 0) 8.3%; 3.1% stearic acid (C18: 0); Linoleic acid (C18: 2) 26.0%; (87%) of ricinoleic acid (C18: 1 (OH)) (87%), and the peanut oil having an oleic acid (C18: ); A castor oil having oleic acid (C18: 1) (7%) is preferred.

In the present invention, the release inhibitor is preferably contained in an amount of 0.1 to 20.0 parts by weight, more preferably 0.1 to 10.0 parts by weight, and most preferably 0.1 to 5.0 parts by weight, based on 100 parts by weight of the biocompatible polymer. If the amount of the release inhibitor is less than 0.1 part by weight, the effect of the surfactant of the release inhibitor may be insufficient and there may be a problem with the drug encapsulation rate. If the amount is more than 20.0 parts by weight, Can be interrupted.

The sustained-release microspheres according to the present invention preferably comprise 1.0 to 30% by weight, more preferably 2.0 to 20% by weight, and most preferably 3.0 to 10% by weight of the physiologically active substance based on the total weight of the microspheres. If the physiologically active substance is contained in an amount of less than 1.0% by weight, the amount of microspheres to be administered to the patient may be too large to be administered, or there may be a problem in administration. If the amount exceeds 30% by weight, There may be a problem that inhibition is difficult.

The sustained-release microspheres according to the present invention have a form in which a physiologically active substance is encapsulated by a biocompatible polymer and the encapsulated biocompatible polymer is further encapsulated by a release inhibitor to form a lipid matrix, And the side effects caused by the initial excessive release of the physiologically active substance are solved.

Furthermore, in another aspect, the present invention relates to a method for producing a biodegradable polymer, which comprises suspending and dispersing a water-soluble physiologically active substance in an organic solvent in which a biodegradable polymer and a release inhibitor are dissolved or dissolving a biodegradable polymer in an aqueous solvent There is provided a method for preparing a sustained release microsphere by mixing a solvent to form a suspension or an emulsion, mixing the release inhibitor and then adding it to an aqueous medium to prepare a microsphere, and removing the organic solvent.

Hereinafter, the production method according to the present invention will be described in detail.

The present invention

i) dissolving the biocompatible polymer in an organic solvent;

Ii) preparing a suspension by suspending or dispersing the physiologically active substance in the organic solvent in which the biocompatible polymer prepared in the step i) is dissolved, or adding a solubilizing agent to the physiologically active substance and mixing the biocompatible polymer prepared in the step i) Preparing a non-aqueous solution by dissolving a physiologically active substance to which the dissolution aid is added in an organic solvent in which

Iii) mixing the release inhibitor into the suspension or non-aqueous solution prepared in step ii);

Iv) adding the solution or emulsion formed in step iii) into an aqueous medium to form microspheres, and then adjusting the particle size; And

And v) removing the organic solvent. The present invention also provides a method for producing sustained-release microspheres.

With regard to the physiologically active substance, the biocompatible polymer and the release inhibitor in the production method according to the present invention, the matters concerning the sustained release microspheres are similarly applied.

The step i) of the present invention is a step of dissolving the biocompatible polymer in an organic solvent in a previous step for preparing an oil dispersion phase.

In addition, the organic solvent which can be used for dissolving the biocompatible polymer in step i) is not particularly limited as long as it can dissolve the biocompatible polymer. For example, at least one solvent selected from the group consisting of methylene chloride, chloroform, acetonitrile, dimethylsulfoxide, dimethylformamide and ethyl acetate may be used.

Further, the step ii) of the present invention may be carried out by preparing a suspension solution by suspending and dispersing the physiologically active substance itself in the organic solvent in which the biocompatible polymer prepared in the step i) is dissolved, or by mixing the biocompatible polymer prepared in i) Together with the dissolved organic solvent, dissolves the physiologically active substance with a solubilizing agent and mixes them with each other to prepare a non-aqueous solution.

 There is no particular limitation on the solubilizing agent in step ii) used when the physiologically active substance is suspended and dispersed in the organic solvent prepared in step i), so long as it can dissolve the physiologically active substance.

The solubilizing agent which can be used in the present invention to dissolve the physiologically active substance is preferably dimethylsulfoxide (DMSO) or N-methyl-2-pyrrolidinone (NMP). The dissolution aid may be used in an amount of 200 to 1,000% by weight based on the weight of the physiologically active substance.

In the step ii) of the present invention, a surfactant may be optionally added to facilitate the formation of a desired physical property or emulsion of the suspension. Surfactants may be used without any particular limitation as long as they are conventionally used in the art. Surfactants include, for example, silicone oil, polysorbate (Tween), sorbitan ester (Span), poloxamer, polyethyleneglycol, and tocopherol ), And the like can be used.

The step iii) of the present invention may be carried out by mixing the substance produced in step i) and step ii) with the release inhibitor to prepare an oil dispersion such as, for example, a transparent emulsion form, Which is capable of delaying or suppressing the release of the physiologically active substance and delaying the release of the physiologically active substance for a desired period of time, Means a component that is used for the purpose of increasing the rate of drug insertion of a sphere.

In the present invention, in step (iv), the aqueous medium may be water for injection, and optionally, a low viscosity polymer may be mixed to suppress the diffusion of the emulsion. Examples of the low-viscosity polymer used herein include polyvinylpyrrolidone (5.5-8.5 mPas of 10% w / v aqueous solutions at 20 ° C), polyvinyl alcohol (4.0-7.0 mPas of 4% w / v aqueous solution at 20 ° C) may be used, but the present invention is not limited thereto.

Further, a co-solvent may optionally be used together with the aqueous medium of step iv). Examples of such a co-solvent include alcohols substituted by a hydrogen atom of a hydrocarbon with hydroxy, -OH, acetic acid and organic acid, or Acetone, or carbolic acid, and most preferably monohydric alcohols such as methanol, ethanol, propanol, butanol, pentanol, hexanol, heptanol, octanol, , More preferably ethanol, but is not limited thereto. When a co-solvent such as monohydric alcohol, acetone, or acetic acid is used, aggregation of microspheres produced can be reduced. However, when the co-solvent is used in an excessive amount, the drug encapsulation rate may be lowered, and in a case of using a small amount, the elution of the drug may be restricted due to the coagulation effect of the microspheres. Thus, the release inhibitor agent to interpolymer ratio is from 1: 1 to 1: 10.000, or preferably from 1:10 to 1: 1,000, and most preferably from 1:20 to 1: 400.

In step iv), the particle size may be controlled by any known method of controlling the size of microspheres without restriction, but it is preferable to control the particle size using a high-pressure homogenizer.

The method for removing the organic solvent in the step v) of the present invention may be any method commonly used in the art. Examples of the method for removing the organic solvent include, but are not limited to, stirring, heating, nitrogen purge (N2 purge), and the like.

In the production method of the present invention, the release inhibitor is mixed with the suspension or emulsion solution, and then the suspension is injected into the aqueous medium to form a water-insoluble or water-insoluble release inhibitor in the microsphere, The initial dissolution rate is reduced by reducing the contact of the active substance with the external aqueous medium.

In a preferred embodiment, the manufacturing method of the present invention may further include the step of freezing the microspheres obtained after removing the organic solvent in the step v). Further, it may further include a step of performing the centrifugal separation and / or a step of dialysis, and / or a filtration step before the freezing step.

That is, the microspheres prepared through the above process increase the inclusion rate of the physiologically active substance, which is a drug, and do not cause excessive initial burst of the drug regardless of the molecular weight of the biocompatible polymer and the content ratio of lactide Thereby giving a zero-order emission characteristic.

The microcapsule of the present invention prepared as described above can be formulated as a long-term sustained-release preparation for 1 month or longer because the concentration of the drug in the blood is different when the drug is administered to an animal, but the drug concentration is maintained at 3ng / ml or more until 28 days.

The sustained-release microspheres according to the present invention provide a method for producing a long-term sustained-release preparation containing a physiologically active substance, particularly a peptide and a salt thereof, Month or more of the physiologically active substance can be released continuously and uniformly.

1 is a graph showing the results of an in vitro elution test of microspheres prepared according to the present invention.
FIG. 2 is a graph showing the experimental results of measuring the goserelin inclusion rate of the microspheres prepared according to the present invention.
3 is a photograph showing the result of morphometric measurement of the microspheres manufactured according to the present invention.

Hereinafter, the present invention will be described in detail by way of examples. It is to be understood, however, that these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

< Comparative Example > General Underwater emulsification method  (O / W) Microsphere  Produce

50 mg of goserelin acetate (USP grade) was dissolved in 0.2 ml of DMSO according to the contents shown in Table 1 and Table 2, and 500 mg of the biocompatible polymer was dissolved in 1 ml of methylene chloride to prepare a transparent emulsion (DP, Dispersion phase). This solution was added to a mixed solution of 90 ml of an aqueous solution of 0.5% polyvinyl alcohol (Mw = 30,000 to 70,000, Sigma) and 10 ml of ethanol (99.5%) stirred vigorously (10,000 rpm) with an L4R mixer (Silverson) at 25 ° C by a syringe pump ). &Lt; / RTI &gt; After 1 minute, the O / W solution was adjusted by controlling the pressure in a high-pressure homogenizer (Buffalo) so that the size of the microspheres was adjusted to 3 ± 0.5 μm, and repeatedly passed through 10 times to prepare a homogeneous O / W solution. The organic solvent was volatilized in the O / W liquid at a speed of 400 rpm for 12 hours in an agitator. Then, the microspheres were centrifuged (10,000 g, 20 minutes) to obtain microspheres, washed twice with distilled water and lyophilized for 72 hours.

< Comparative Example  1> Intrinsic viscosity  Containing goserelin with other biocompatible polymers Microsphere  Produce

Comparative Example 1 -One Comparative Example 1 -2 Comparative Example 1 -3 API filling amount 50 mg 50 mg 50 mg DMSO 0.2ml 0.2ml 0.2ml MC 1ml 1ml 1ml PLGA PLGA 502H ^{1 )}
500 mg PLGA 503H ^{2 )}
500 mg PLGA 504H ^{3 )}
500 mg 0.5% PVA solution 90ml 90ml 90ml EtOH (99.5%) 10ml 10ml 10ml

1) lactide: glycolide = 50:50, Boehringer Ingelheim, iv = 0.20 dl / g, Resomer ^TM

2) lactide: glycolide = 50:50, Boehringer Ingelheim, iv = 0.39 dl / g, Resomer ^TM

3) lactide: glycolide = 50:50, Boehringer Ingelheim, iv = 0.52 dl / g, Resomer ^TM

Comparative Example  2> Lactide / Glycolide  Using biocompatible polymers with different compositions Product design

Comparative Example 2 -One Comparative Example 2 -2 Comparative Example 2 -3 API filling amount 50 mg 50 mg 50 mg DMSO 0.2ml 0.2ml 0.2ml MC 1ml 1ml 1ml PLGA PLGA 502H ^{1 )}
500 mg PLGA 752H ^{2 )}
500 mg PLGA 202H ^{3 )}
500 mg 0.5% PVA solution 90ml 90ml 90ml EtOH (99.5%) 10ml 10ml 10ml

1) lactide: glycolide = 50:50, Boehringer Ingelheim, iv = 0.20 dl / g, Resomer ^TM

2) lactide: glycolide = 75: 25, Boehringer Ingelheim, iv = 0.20 dl / g, Resomer ^TM

3) lactide: glycolide = 100: 0, Boehringer Ingelheim, iv = 0.20 dl / g, Resomer ^TM

Comparative Example  3> Biocompatible polymer ( RG502H ) And release inhibitors (Medium chain triglyceide, MCT )of Oil dispersion phase  ( ODP , Oil Dispersion phase) Microsphere  Produce

50 mg of goserelin acetate (USP grade) was dissolved in DMSO according to Table 14, and 500 mg of the biocompatible polymer was dissolved in 1 ml of methylene chloride. Medium chain triglyceide (MCT) composed of C6: 0 to C12: 0 3 (ODP, Oil Dispersion phase). The following process was carried out in the same manner as in Comparative Example.

Comparative Example 3 -One Comparative Example 3 -2 Comparative Example 3 -3 Comparative Example 3 -4 API filling amount 50 mg 50 mg 50 mg 50 mg DMSO 0.2ml 0.2ml 0.2ml 0.2ml MC 1ml 1ml 1ml 1ml PLGA 502H 500 mg 500 mg 500 mg 500 mg MCT ^* 0.1% 5% 10% 20% 0.5% PVA solution 90ml 90ml 90ml 90ml EtOH (99.5%) 10ml 10ml 10ml 10ml

^* PLGA weight%

Example  1> Biocompatible polymer ( RG502H ) And the oil dispersion phase of the release inhibitor (Lecithin, lecithin) ODP , Oil Dispersion phase) Microsphere  Produce

A clear emulsion was prepared by dissolving 50 mg of goserelin acetate (USP grade) in 0.2 ml of DMSO, dissolving 500 mg of the biocompatible polymer in 1 ml of methylene chloride and dissolving lecithin as a release inhibitor according to the contents shown in Table 4 , Oil Dispersion phase). The following process was carried out in the same manner as in Comparative Example.

Example 1 -One Example 1 -2 Example 1 -3 Example 1 -4 API filling amount 50 mg 50 mg 50 mg 50 mg DMSO 0.2ml 0.2ml 0.2ml 0.2ml MC 1ml 1ml 1ml 1ml PLGA 502H 500 mg 500 mg 500 mg 500 mg Lecithin ^* 0.1% 5% 10% 20% 0.5% PVA solution 90ml 90ml 90ml 90ml EtOH (99.5%) 10ml 10ml 10ml 10ml

^* PLGA weight%

Example  2> Biocompatible polymer ( RG502H ) And the release inhibitor (cottonseed oil) Oil dispersion phase  ( ODP , Oil Dispersion phase) Microsphere  Produce

A transparent emulsion was prepared by dissolving 50 mg of goserelin acetate (USP grade) in 0.2 ml of DMSO, dissolving 500 mg of biocompatible polymer in 1 ml of methylene chloride and dissolving cottonseed oil as a release inhibitor according to the contents shown in Table 5 (ODP, Oil Dispersion phase). The following process was carried out in the same manner as in Comparative Example.

Example 2 -One Example 2 -2 Example 2 -3 Example 2 -4 API filling amount 50 mg 50 mg 50 mg 50 mg DMSO 0.2ml 0.2ml 0.2ml 0.2ml MC 1ml 1ml 1ml 1ml PLGA 502H 500 mg 500 mg 500 mg 500 mg Cottonseed oil ^* 0.1% 5% 10% 20% 0.5% PVA solution 90ml 90ml 90ml 90ml EtOH (99.5%) 10ml 10ml 10ml 10ml

^* PLGA weight%

Example  3> Biocompatible polymer ( RG502H ) And release inhibitor (Castor oil, castor oil) Oil dispersion phase  ( ODP , Oil Dispersion phase) Microsphere  Produce

A transparent emulsion was prepared by dissolving 50 mg of goserelin acetate (USP grade) in 0.2 ml of DMSO, dissolving 500 mg of biocompatible polymer in 1 ml of methylene chloride and dissolving castor oil as a release inhibitor according to the contents shown in Table 6 (ODP , Oil Dispersion phase). The following process was carried out in the same manner as in Comparative Example.

Example 3 -One Example 3 -2 Example 3 -3 Example 3 -4 API filling amount 500 mg 500 mg 500 mg 500 mg DMSO 0.2ml 0.2ml 0.2ml 0.2ml MC 1ml 1ml 1ml 1ml PLGA 502H 500 mg 500 mg 500 mg 500 mg Castor oil ^* 0.1% 5% 10% 20% 0.5% PVA solution 90ml 90ml 90ml 90ml EtOH (99.5%) 10ml 10ml 10ml 10ml

^* PLGA weight%

Example  4> Biocompatible polymer ( RG502H ) And release inhibitor (Corn oil, corn oil) Oil dispersion phase  ( ODP , Oil Dispersion phase) Microsphere  Produce

A transparent emulsion was prepared by dissolving 50 mg of goserelin acetate (USP grade) in 0.2 ml of DMSO, dissolving 500 mg of the biocompatible polymer in 1 ml of methylene chloride and dissolving corn oil as the release inhibitor according to the contents shown in Table 7 (ODP , Oil Dispersion phase). The following process was carried out in the same manner as in Comparative Example.

Example 4 -One Example 4 -2 Example 4 -3 Example 4 -4 API filling amount 500 mg 500 mg 500 mg 500 mg DMSO 0.2ml 0.2ml 0.2ml 0.2ml MC 1ml 1ml 1ml 1ml PLGA 502H 500 mg 500 mg 500 mg 500 mg Corn oil ^* 0.1% 5% 10% 20% 0.5% PVA solution 90ml 90ml 90ml 90ml EtOH (99.5%) 10ml 10ml 10ml 10ml

^* PLGA weight%

Example  5> Biocompatible polymer ( RG502H ) And release inhibitor (Safflower oil, Sunflower oil )of Oil dispersion phase  ( ODP , Oil Dispersion phase) Microsphere  Produce

A transparent emulsion was prepared by dissolving 50 mg of goserelin acetate (USP grade) in 0.2 ml of DMSO, dissolving 500 mg of the biocompatible polymer in 1 ml of methylene chloride and dissolving the sunflower oil according to the contents shown in Table 8 as release inhibitor (ODP , Oil Dispersion phase). The following process was carried out in the same manner as in Comparative Example.

Example 5 -One Example 5 -2 Example 5 -3 Example 5 -4 API filling amount 500 mg 500 mg 500 mg 500 mg DMSO 0.2ml 0.2ml 0.2ml 0.2ml MC 1ml 1ml 1ml 1ml PLGA 502H 500 mg 500 mg 500 mg 500 mg Safflower oil ^* 0.1% 5% 10% 20% 0.5% PVA solution 90ml 90ml 90ml 90ml EtOH (99.5%) 10ml 10ml 10ml 10ml

^* PLGA weight%

Example  6> Biocompatible polymer ( RG502H ) And the release inhibitor (Olive oil) Oil dispersion phase  ( ODP , Oil Dispersion phase) Microsphere  Produce

A transparent emulsion was prepared by dissolving 50 mg of goserelin acetate (USP grade) in 0.2 ml of DMSO, dissolving 500 mg of the biocompatible polymer in 1 ml of methylene chloride and dissolving the olive oil as a release inhibitor according to the contents shown in Table 9 (ODP, Oil Dispersion phase). The following process was carried out in the same manner as in Comparative Example.

Example 6 -One Example 6 -2 Example 6 -3 Example 6 -4 API filling amount 500 mg 500 mg 500 mg 500 mg DMSO 0.2ml 0.2ml 0.2ml 0.2ml MC 1ml 1ml 1ml 1ml PLGA 502H 500 mg 500 mg 500 mg 500 mg Olive oil ^* 0.1% 5% 10% 20% 0.5% PVA solution 90ml 90ml 90ml 90ml EtOH (99.5%) 10ml 10ml 10ml 10ml

^* PLGA weight%

Example  7> Biocompatible polymer ( RG502H ) And the release inhibitor (Sesame oil, sesame oil) Oil dispersion phase  ( ODP , Oil Dispersion phase) Microsphere  Produce

A transparent emulsion was prepared by dissolving 50 mg of goserelin acetate (USP grade) in 0.2 ml of DMSO, dissolving 500 mg of the biocompatible polymer in 1 ml of methylene chloride and dissolving the sesame oil as a release inhibitor according to the contents shown in Table 10 (ODP, Oil Dispersion phase). The following process was carried out in the same manner as in Comparative Example.

Example 7 -One Example 7 -2 Example 7 -3 Example 7 -4 API filling amount 500 mg 500 mg 500 mg 500 mg DMSO 0.2ml 0.2ml 0.2ml 0.2ml MC 1ml 1ml 1ml 1ml PLGA 502H 500 mg 500 mg 500 mg 500 mg Sesame oil ^* 0.1% 5% 10% 20% 0.5% PVA solution 90ml 90ml 90ml 90ml EtOH (99.5%) 10ml 10ml 10ml 10ml

^* PLGA weight%

Example  8> biocompatible polymer ( RG502H ) And release inhibitor (Soybean oil, Soybean oil )of Oil dispersion phase  ( ODP , Oil Dispersion phase) Microsphere  Produce

A transparent emulsion was prepared by dissolving 50 mg of goserelin acetate (USP grade) in 0.2 ml of DMSO, dissolving 500 mg of the biocompatible polymer in 1 ml of methylene chloride and dissolving the soybean oil according to the content shown in Table 11 as a release inhibitor (ODP, Oil Dispersion phase). The following process was carried out in the same manner as in Comparative Example.

Example 8 -One Example 8 -2 Example 8 -3 Example 8 -4 API filling amount 500 mg 500 mg 500 mg 500 mg DMSO 0.2ml 0.2ml 0.2ml 0.2ml MC 1ml 1ml 1ml 1ml PLGA 502H 500 mg 500 mg 500 mg 500 mg Soybean oil ^* 0.1% 5% 10% 20% 0.5% PVA solution 90ml 90ml 90ml 90ml EtOH (99.5%) 10ml 10ml 10ml 10ml

^* PLGA weight%

Example  9> Biocompatible polymers ( RG502H ) And release inhibitor (Peanut oil, Peanut oil )of Oil dispersion phase  ( ODP , Oil Dispersion phase) Microsphere  Produce

A transparent emulsion was prepared by dissolving 50 mg of goserelin acetate (USP grade) in 0.2 ml of DMSO, dissolving 500 mg of the biocompatible polymer in 1 ml of methylene chloride and dissolving the peanut oil according to the content shown in Table 12 as a release inhibitor (ODP , Oil Dispersion phase). The following process was carried out in the same manner as in Comparative Example.

Example 9 -One Example 9 -2 Example 9 -3 Example 9 -4 API filling amount 500 mg 500 mg 500 mg 500 mg DMSO 0.2ml 0.2ml 0.2ml 0.2ml MC 1ml 1ml 1ml 1ml PLGA 502H 500 mg 500 mg 500 mg 500 mg Peanut oil ^* 0.1% 5% 10% 20% 0.5% PVA solution 90ml 90ml 90ml 90ml EtOH (99.5%) 10ml 10ml 10ml 10ml

^* PLGA weight%

< Example  10> acetic acid for at least 3 months continuous release Leuprorelin  Containing biodegradability Microsphere  Produce

50 mg of goserelin acetate (USP grade) was dissolved in 0.2 ml of DMSO, 500 mg of the biocompatible polymer shown in Table 13 was dissolved in 1 ml of methylene chloride, and the release inhibitor was dissolved according to the given contents to prepare a transparent emulsion (ODP, Oil Dispersion phase. The following process was carried out in the same manner as in Comparative Example.

Example 10 -One Example 10 -2 Example 10 -3 API filling amount 50 mg 50 mg 50 mg DMSO 0.2ml 0.2ml 0.2ml MC 1ml 1ml 1ml PLGA 504H ^{1 )} 500 mg PLGA 752H ^{2 )} 500 mg PLGA 858 ³⁾ 500 mg Castor oil ^* 10% 10% 10% 0.5% PVA solution 90ml 90ml 90ml EtOH (99.5%) 10ml 10ml 10ml

^* PLGA weight%

1) lactide: glycolide = 50:50, Boehringer Ingelheim, MW. = 50,000, Resomer ^TM

2) lactide: glycolide = 75: 25, Boehringer Ingelheim, MW. = 20,000, Resomer ^TM

3) Lactide: glycolide = 100: 0, Boehringer Ingelheim, MW. = 220,000, Resomer ^TM

< Example  11> Of the aqueous phase (CP) Co-seller  (Ethanol) Microsphere  Produce

50 mg of goserelin acetate (USP grade) was dissolved in 0.2 ml of DMSO, 500 mg of the biocompatible polymer was dissolved in 1 ml of methylene chloride, and the release inhibitor was dissolved according to the contents shown in Table 14 to prepare a transparent emulsion (ODP, Oil Dispersion phase ). This solution was mixed with an aqueous solution of 0.5% polyvinyl alcohol (Mw = 30,000 to 70,000, Sigma) and ethanol (99.5%), which was agitated (10,000 rpm) vigorously with an L4R mixer (Silverson) at 25 DEG C, After the solvent was formed, the solution was gradually added dropwise using a syringe pump (1 ml / min). The following process was carried out in the same manner as in Comparative Example.

Example 11 -One Example 11 -2 Example 11 -3 Example 11 -4 API filling amount 50 mg 50 mg 50 mg 50 mg DMSO 0.2ml 0.2ml 0.2ml 0.2ml MC 1ml 1ml 1ml 1ml PLGA 502H 500 mg 500 mg 500 mg 500 mg Castor oil ^* 10% 10% 10% 10% 0.5% PVA solution 100ml 99ml 90ml 50ml EtOH (99.5%) 1ml 10ml 50ml

^* PLGA weight%

< Example  12> Of non-aqueous liquid (ODP) To DMSO  by Microsphere  Produce

50 mg of goserelin acetate (USP grade) was dissolved in DMSO according to Table 15, 500 mg of the biocompatible polymer dissolved in 1 ml of methylene chloride, and the release inhibitor dissolved according to the contents shown in Table 15 to prepare a transparent emulsion (ODP, Oil Dispersion phase. The following process was carried out in the same manner as in Comparative Example.

Example 12 -One Example 12 -2 Example 12 -3 Example 12 -4 Example 12 -5 API filling amount 50 mg 50 mg 50 mg 50 mg 50 mg DMSO ^# 0% 190% 400% 1000% 1000.1% MC 1ml 1ml 1ml 1ml 1ml PLGA 502H 500 mg 500 mg 500 mg 500 mg 500 mg Castor oil ^* 10% 10% 10% 10% 10% 0.5% PVA solution 90ml 90ml 90ml 90ml 90ml EtOH (99.5%) 10ml 10ml 10ml 10ml 10ml

^* PLGA weight%

^# API weight%

< Example  13> by the encapsulation of the physiologically active substance Microsphere  Produce

50 mg of the physiologically active substance was dissolved in DMSO according to Table 16, 500 mg of the biocompatible polymer was dissolved in 1 ml of methylene chloride, and the release inhibitor was dissolved according to the content shown in Table 15 to prepare a transparent emulsion (ODP, Oil Dispersion phase). The following process was carried out in the same manner as in Comparative Example.

Example 13 -One Example 13 -2 Example 13 -3 Example 13 -4 Leuproride 50 mg 50 mg Liraglutide 50 mg 50 mg DMSO 0.2ml 0.2ml 0.2ml 0.2ml MC 1ml 1ml 1ml 1ml PLGA 502H 500 mg 500 mg 500 mg 500 mg Castor oil ^* 10% 10% 0.5% PVA solution 90ml 90ml 90ml 90ml EtOH (99.5%) 10ml 10ml 10ml 10ml

^* PLGA weight%

Experimental Example  1> of the drug In vitro in vitro release experiment

50 mg of goserelin acetate (USP grade) was dissolved in 0.2 ml of DMSO, 500 mg of the biocompatible polymer was dissolved in 1 ml of methylene chloride, and the release inhibitor was dissolved according to the content shown in Table 17 to prepare a transparent emulsion (ODP, Oil Dispersion phase ). The following process was carried out in the same manner as in Comparative Example.

Experimental Example 1 -One Experimental Example 1 -2 Experimental Example 1 -3 API filling amount 50 mg 50 mg 50 mg DMSO 0.2ml 0.2ml 0.2ml MC 1ml 1ml 1ml PLGA 502H 500 mg 500 mg 500 mg Castor oil ^* 0.1% 10% 20.1% 0.5% PVA solution 90ml 90ml 90ml EtOH (99.5%) 10ml 10ml 10ml

^* PLGA weight%

In order to confirm that the hydrophilic drug was continuously released from the polymer microspheres prepared, the drug release experiment was carried out in the following in vitro conditions. That is, polymer microspheres prepared by applying 10% of the release inhibitor in Comparative Example 1-1, Experimental Example 1 and Examples 1 to 9 and 50 mg of the polymer microspheres prepared in Example 10, which was a 3-month formulation, were weighed accurately 50 ml of phosphate buffered saline (pH 7.4) was added and sealed in a shaking water bath at a rate of 120 revolutions per minute at 37 ° C, allowing the drug to be continuously released for more than 28 days. 1 ml of the released drug was centrifuged at 20,000 g for 10 minutes, and 100 μl of the supernatant was measured by HPLC quantification as in Experimental Example 1. The remaining drug was redispersed and replenished in the test tube The The measurement results are shown in Fig. 1 (A), and the initial drug release rate of the microspheres without the release inhibitor (Comparative Example 1-1) was 70% The first-order drug release rate of the microspheres using 10% of triglyceride (MCT) (Comparative Example 3-3) was also 50%. However, in Experimental Example 1, the first-order drug release rate of the microspheres into which 0.1% of the release inhibitor (Castor oil) was introduced was about 20%, and the first-day drug release rate of the microsphere introduced with 100% of the release inhibitor was about 10%. However, the 28-day drug release rate of the microsphere with 20.1% release inhibitor was about 85%, indicating that the drug release was not completely completed within 28 days. In addition, according to Example 1-9 shown in Fig. 1 (B), when an oil composed of a long-chain fatty acid was used as a release inhibitor, the initial dissolution rate was considerably small, , Respectively. And FIG. 1 (C) shows that the release rate can be exhibited at least three months as shown in Example 10. FIG.

Experimental Example  2> Inclusion rate  Measurement experiment

20 mg of the prepared polymer microspheres were precisely weighed, placed in a test tube with a lid, sufficiently dissolved in 3 ml of methylene chloride, 5 ml of distilled water was added, and stirred vigorously for 60 minutes. Then, the solution was centrifuged at 3,000 g for 5 minutes, and a certain amount of the supernatant was taken and the drug concentration was measured using high performance liquid chromatography (HPLC) to calculate the inclusion rate of the drug contained in the microspheres. At this time, the column used was Waters C-18 (4.6 x 150 mm), the injection amount was 20 μl, and the detection wavelength was 220 nm. As a mobile phase, water (a) containing 0.1% TFA and acetonitrile (b) were used in a ratio of 75%: 25%.

2 (A) and (B) show that the inclusion rate of the prepared microspheres is increased as the concentration of the release inhibitor increases from 0.1 to 20% The drug loading amount was 50-100%. The inclusion rate of microspheres with 5% or more release inhibitor was 70%. However, the drug loading of the formulation of Comparative Example 1 and the formulation of Comparative Example 3 containing medium chain triglyceride was less than 50%. In addition, castor oil of the release inhibitor exhibited the highest drug encapsulation efficiency by about 10% higher than other release inhibitors.

 2 (C) shows the results of the incorporation rate of the microspheres produced by the co-solvent (ethanol) of the aqueous phase (CP) and the microspheres of DMSO of the non-aqueous liquid phase (ODP) Up to 1: 200, the inclusion rate of 70% or more was not influenced by the increase of the co - solvent in the aqueous solution, but the inclusion rate was 70% or more when the co - solvent was increased to 1: 1,000 or more. In addition, when the content of DMSO in the non-aqueous liquid phase was less than 2% by weight, the main component was not completely dissolved, the inclusion rate was less than 50%, and the content of API by 10% And the enclosing rate was less than 50% when the amount was exceeded. The inclusion rate of microspheres containing various physiologically active substances was less than 50% when the release inhibitor was not introduced. When the release inhibitor was introduced, the inclusion rate was 70 %. &Lt; / RTI &gt;

Experimental Example  3> Microsphere  Particle shape measurement

Approximately 50 mg of microspheres were fixed on an aluminum stub and coated with platinum under a vacuum of 0.1 torr and a high voltage (10 kV) for 15 minutes. Then, the microspheres were mounted on a SEM main body, and the morphology of the microspheres was measured using an image analysis program Respectively. The results of the measurement are shown in FIG. 3. According to this result, many micropores are observed on the surfaces of microspheres using microspheres and medium chain triglycerides (MCT), which are not used with the release inhibitor, while the castor oil is applied The porosity of the prepared microspheres is reduced and is introduced to the surface of the microspheres.

In addition, it can be seen that the coagulation of microspheres was reduced by the ethanol used as a cosolvent in the microsphere introduced with the release inhibitor.

Claims (21)

A biologically active substance, a biocompatible polymer, and a release inhibitor,
Characterized in that the biocompatible polymer encapsulated with the physiologically active substance is further encapsulated by a release inhibitor and the release inhibitor is an oil containing a long chain fatty acid of C18: 1, C18: 1 (OH) or C18: 2 Slow release microsphere.
The method of claim 1, wherein the physiologically active substance is one or a salt thereof selected from the group consisting of octreotide, lanreotide, goserelin, looprolide, tryptolele, histolerein, and desmopressin Gt; microspheres &lt; / RTI &gt;
The sustained release microsphere according to claim 1, wherein the physiologically active substance is contained in an amount of 1.0 to 30% by weight based on the total weight of the microspheres.
The sustained-release microsphere according to claim 2, wherein the physiologically active peptide is goserelin.
The sustained-release microsphere according to claim 1, wherein the biocompatible polymer is polylactide, polylactide-co-glycolide or poly (lactide-co-glycolide) glucose.
The sustained release microsphere according to claim 1, wherein the biocompatible polymer is contained in an amount of 70 to 99% by weight based on the total weight of the microspheres.
The method according to claim 1,
Wherein the release inhibitor is contained in an amount of 0.1 to 20 parts by weight based on 100 parts by weight of the biocompatible polymer.
The oil of claim 7, wherein the oil comprising C18: 1, C18: 1 (OH) or C18: 2 fatty acids is selected from the group consisting of lecithin, cottonseed oil, castor oil, corn oil, sunflower oil, olive oil, sesame oil, Wherein the sustained-release microspheres
The oil spill according to claim 8, wherein the oil containing the C18: 1, C18: 1 (OH) or C18: 2 fatty acid is a castor oil.
2. The sustained release microspheres of claim 1, wherein the sustained release microspheres are in the form of a lipid matrix.
i) dissolving the biocompatible polymer in an organic solvent;
Ii) preparing a suspension by suspending or dispersing the physiologically active substance in the organic solvent in which the biocompatible polymer prepared in the step i) is dissolved, or adding a solubilizing agent to the physiologically active substance and mixing the biocompatible polymer prepared in the step i) Preparing a non-aqueous solution by dissolving a physiologically active substance to which the dissolution aid is added in an organic solvent in which
Iii) mixing the release inhibitor into the suspension or non-aqueous solution prepared in step ii);
Iv) adding the solution or emulsion formed in step iii) into an aqueous medium to form microspheres, and then adjusting the particle size; And
And v) removing the organic solvent.
12. The process according to claim 11, wherein the organic solvent used in step i) is at least one solvent selected from the group consisting of methylene chloride, chloroform, acetonitrile, dimethylsulfoxide, dimethylformamide and ethyl acetate. &Lt; / RTI &gt;
12. The method according to claim 11, wherein the solubility aid used in step ii) is dimethylsulfoxide (DMSO) or N-methyl-2-pyrrolidinone (NMP).
12. The method according to claim 11, wherein the solubilizing agent used in step ii) has a weight of 200 to 1,000% by weight of the physiologically active substance.
12. The method according to claim 11, wherein the aqueous medium in step iv) further comprises a co-solvent.
The method of producing sustained release microspheres according to claim 11, wherein the surfactant is further added in the step ii).
17. The method of claim 16, wherein the surfactant is selected from the group consisting of silicone oil, polysorbate (Tween), sorbitan ester (Span), poloxamer, polyethyleneglycol, Wherein the surfactant is at least one surfactant selected from the group consisting of tocopherol.
16. The method according to claim 15, wherein the co-solvent is at least one selected from the group consisting of alcohol, acetic acid, organic acid, acetone, and carbolic acid.
19. The process according to claim 18, wherein the co-solvent is at least one monohydric alcohol selected from the group consisting of methanol, ethanol, propanol, butanol, pentanol, hexanol, heptanol, octanol, A method of manufacturing a microsphere.
12. The method of claim 11, further comprising lyophilizing the sustained-release microspheres produced after step v).
12. The method of claim 11, further comprising any one step selected from the group consisting of centrifugation, dialysis, and filtration prior to the lyophilization step.

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