WO2018038461A1

WO2018038461A1 - Extended release microsphere to which release inhibitor comprising oil, in which c18:1, c18:1(oh) or c18:2 long-chain fatty acid is contained, is applied and preparation method therefor

Info

Publication number: WO2018038461A1
Application number: PCT/KR2017/008981
Authority: WO
Inventors: 임덕수; 김병기; 김대현; 유지석; 박영준
Original assignee: 영진약품 주식회사
Priority date: 2016-08-25
Filing date: 2017-08-17
Publication date: 2018-03-01
Also published as: KR101961848B1; KR20180023337A

Abstract

The present invention relates to a long-term extended release microsphere containing a bioactive material, and a preparation method therefor, and provides: a long-term extended release microsphere, which comprises a bioactive material, a biocompatible polymer, and as a release inhibitor, an oil containing C18:1, C18:1 (OH) and C18:2 long-chain fatty acids, and resolves problems such as side effects, caused by initial over-release, by having remarkable release characteristics of increased incorporation rate of the bioactive material and minimization of initial over-release since the release inhibitor encompasses the bioactive material-incorporated biocompatible polymer one more time; and a preparation method therefor.

Description

【Specification】

[Name of invention]

Sustained release microspheres using release inhibitors including oils containing C18: l, C18: l (0H) or C18: 2 long chain fatty acids, and methods for preparing the same

Technical Field

The present invention relates to a sustained-release microsphere and a method for preparing the same that can continuously control the release of the drug by encapsulating a water-soluble drug in a carrier made of a biodegradable polymer, more specifically ^' physiological activity encapsulated in a conventional biocompatible polymer Unlike sustained-release preparations that contain substances, oils containing long-chain fatty acids, specifically C18: 1, C18: 1 (0H) or C18: 2 long-chain fatty acids, are included in biocompatible polymers containing release inhibitors. It is characterized by applying the bioactive substance to form a lipid matrix, and can be used as a long-term sustained release preparation of hydrophilic drugs because it can continuously maintain a constant release rate and effective blood concentration without rapid initial overrelease and delayed release of the drug. The present invention relates to a microsphere and a method for manufacturing the same.

Background Art

In the case of drugs for treating chronic diseases, long-term release formulations can be used to reduce the frequency of administration, and thus the effective drug concentration can be maintained for a long time with only one administration, and the patient's drug treatment can be improved. Accordingly, sustained-release injection formulations are considered to be very useful for reducing the frequency of drug use and improving patient comfort and virtues while maintaining the drug concentration for a long time.

This sustained release injectable formulation refers to an injectable formulation formulated so that the drug can be sustained and uniformly released in the body upon subcutaneous or intramuscular injection while maintaining biological activity. Conventionally, such a method of preparing slow-release injection preparations is known as coacervation, melt extrusion, spray drying, and solvent evaporation. Among these methods, dual emulsion evaporation (W / 0 / W; water / oil / water) and single emulsion evaporation (0 / W; oil / water) are classified. Emulsion dog evaporation is most commonly used.

However, in the case of using the water-soluble drug in the microspheres using the emulsion evaporation method, since the drug is diffused into the external continuous phase during the manufacturing process of the microspheres, the encapsulation efficiency of the drug is very poor. Furthermore, with respect to microspheres prepared according to this multiemulsion method, the release of drug encapsulated in the microspheres is characterized by two stages: initial over release and subsequent sustained release. Initial over-release may cause side effects due to unintended over-release of drug when applied in actual clinic, which is a problem that must be solved during formulation development. Such a manufacturing method may result in initial over-release of encapsulated drug. Along with the disadvantage that the active ingredient drug is dissolved in an organic solvent or water, there is a problem in that the physical properties of the drug are changed while the microspheres are formed, and the stability is lowered.

In this regard, Korean Patent Registration No. 10-0442931 discloses a sustained release in the form of a microcapsule by dissolving a polymer carrier material in a suitable organic solvent in which a pharmaceutical compound is not dissolved, and then adding an aqueous medium containing an excess of protective colloid and a phase inducing agent. Methods of preparing the formulations are disclosed. However, such a manufacturing method has a disadvantage that the procedure can be difficult and the yield of the product can be raised because the yield is extremely low.

-Furthermore, Korean Patent Registration No. 10-0409413 discloses a method of preparing an emulsion dog by an underwater drying method and then heating and drying the biodegradable polymer above the glass transition temperature (Tg) to significantly suppress the initial release and minimize the organic solvent. However, this manufacturing method has a glass transition temperature of at least 47 ° C or more, there is a serious disadvantage that the bioactive material may be denatured by heat. In addition, Korean Patent Registration No. 10-0293882 discloses novel salts consisting of cations derived from peptides containing basic groups and anions derived from carboxy-terminated polyesters, methods of preparing these salts, and of these salts in the preparation of sustained-release preparation compositions. A method of use is described. However, this method freezes and drops the drug and polymer in vacuum. After drying to obtain a transparent film, it is dispersed again in dichloromethane, re-dried, compressed and molded, and then administered with a relatively large diameter (for example, 16 or 18 gauge) needle, causing fear to the patient. there is a problem.

In U.S. Pat.Nos. 6,419,961, 5,585,460 and 4,652,441, peptides such as leuprorel in acetate can be obtained using a multiple emulsion dog method. A method for preparing polylactic acid-polyglycolic acid copolymer microspheres containing a) -based drug is disclosed. In particular, U.S. Patent No. 4,652,441 introduces water-soluble polymers such as gelatin, albumin, pectin, and agar together with drugs to increase the viscosity of the aqueous phase. As a result, a double encapsulat ion of gelatin and polylactic acid-polyglycolic acid copolymer was induced to prepare a long-term sustained release injection. However, in the case of using gelatin to increase the viscosity of the inner aqueous phase, the preparation of the primary emulsion solution should be heated to a high temperature of 80 ^o C in order to distribute the drug uniformly in the solution, the primary emulsion solution When redistributing

Since the need to nyaenggak to 20 ° C to about 30 ^o C there is a problem in the production process of the microsphere that complex. In addition, the above manufacturing method has a limitation in that it can be used only for the preparation of drugs having stability against heat. In addition, Qingguo Xu et al., Control led re lease of amoxi ci ll in from hydroxyapatiteite coated po ly (1 act i c-co-glycol ic acid) mi crospheres, Journal of Control led Release 127 (2008); 146 -153) discloses a method of coating the outside of biodegradable microspheres with hydroxyapatite to suppress initial release and to release bioactive substances for a long time, but it is difficult to produce them with a cumbersome coating method that requires a long time. There are disadvantages.

[Detailed Description of the Invention]

[Technical Challenges]

Therefore, the inventors of the present invention described the temperature, concentration and agitation rate in the manufacturing process described above in the conventional single emulsification method (W / 0 emuls ion) or double emulsification method (W / O / W emulsion). Accordingly, the microparticles showed porosity, which was intended to solve the disadvantage of increasing initial initial burst and lowering the drug encapsulation rate. Therefore, the present invention uses a conventional solvent evaporation method, a solvent extraction method, or an emulsification method using a release inhibitor of oils containing a long chain fatty acid of C18: 1, C18: 1 (0H) or C18: 2 ( In order to manufacture microspheres in which the initial over-emission problem, which is a problem in emulsion), is suppressed, a method for preparing a long-term sustained-release microsphere which is more stable by eliminating pores on the surface of the microsphere, reducing the surface area, and delaying the penetration of external moisture is proposed. It is an object of the present invention to provide a water-soluble drug-containing sustained-release microsphere having a drug release profile capable of maintaining an effective blood concentration for a long time, and a method of preparing the same.

Technical Solution

In one aspect, to achieve the above object, the present invention includes a bioactive material, a biocompatible polymer and a release inhibitor,

The biocompatible polymer in which the bioactive material is encapsulated is further encapsulated by a release inhibitor, and the release inhibitor is an oil containing a C18: l, C18: l (0H) or C18: 2 long chain fatty acid. A slow release microsphere.

Advantageous Effects

The present invention provides a sustained release microsphere according to the present invention provides a method for preparing a long-term sustained release formulation containing a biologically active substance, in particular, peptides and salts thereof. It is possible to release the bioactive substance continuously and uniformly for more than a month.

[Brief Description of Drawings]

La is a microsphere of Experimental Examples 1-1, 1-2, 1-3 prepared according to the present invention and the microspheres (Comparative Example 1-1) and Medium chain triglyceride (MCT) without release inhibitor 10 % Microspheres up to 28 days used _. It is a graph which shows the result of the in vitro long-term dissolution test of (comparative example 3-3).

Lb according to Examples 1-3, 2-3, 3-3, 4-3, 5-3, 6-3, 7-3, 8-3 and 9-3 This is a graph showing the long-term dissolution test results of the microspheres up to 28 days.

Lc is a graph showing the results of long-term dissolution experiments up to 84 days of the microspheres prepared according to Examples 10-1, 10-2, and 10-3.

2a and 2b are microspheres prepared according to the present invention (Examples 1-1 to 12-5) and Comparative Examples 1-1, 1-2, 1-3, 2-1, 2-2 and 2-3 It is a graph which shows the test result of goserelin encapsulation rate of the.

Figure 3a is a photograph showing the experimental results confirming the porous surface properties of the microspheres according to the application of Castor oi l as the release inhibitor. Specifically, (A) does not use castor oil, but only 10% ethanol (Comparative Example 1-1), microsphere, (B) uses 10¾) MCT and 10% ethane (Comparative Example 3 —3) Microspheres, and (C) shows the surface properties of the microspheres using (Example 3-3) 10% of Castor Oil and 10% of ethane.

3B is a photograph showing test results confirming the microspheres' magnification according to whether ethane is applied as a cosolvent.

Specifically (C) shows the microsphere coarseness using 10% Castor oi l and 1OT of ethanol (Example 3-3), (D) using 10% Castor oi l and distilled water (Example 11 -1) It is a photograph showing the cohesion of microspheres. [Best form for implementation of the invention]

Hereinafter, the present invention will be described in more detail. Bioactive substances which can be applied to the present invention include bioactive peptides and proteins, and the like. The bioactive peptides and proteins are composed of two or more amino acids and have a molecular weight of about 200 to 100, 000, and these peptides and protein drugs Examples include 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, allergy inhibitor, cell necrosis sugar. Proteins, immunotoxins, lymphotoxins, tumor necrosis factor, tumor suppressor, metastatic growth factor, alpha-1 antitrypsin, albumin and fragment fragments thereof, apolipoprotein-E, erythropoietin, factor VI I, factor VI II ， Factor IX ， Plasminogen activator ， Eurokinase, Streptokinase ， Protein Vaginal C, C-reactive protein, renin inhibitor, collagenase inhibitor, superoxide dismutase, Platelet-derived growth factor, epidermal growth factor, osteogenic growth factor, osteogenic protein, calcitonin, insulin, atriopeptin, cartilage inducer, connective tissue activator, follicle stimulating hormone, luteinizing hormone, luteinizing Hormone-releasing hormone, nerve growth factor, parathyroid hormone, relaxin, secretin, somatomedin, insulin-like growth factor, adrenocorticotropic hormone, glucagon, cholecystokinin, pancreatic polypeptide, gastrin releasing peptide, corticotropin Release factors, thyroid stimulating hormones, monoclonal or polyclonal antibodies against various viruses, bacteria, toxins, various virus derived vaccine antigens, and the like. In the present invention, the physiologically active substance is not particularly limited, and is preferably selected from a luteinizing hormone-release hormone (LHRH) analogue or a peptide drug or salts thereof. For example, the agonist (agoni st) in the LHRH homologue includes goserelin, leuprolide, tryptotellin, buserelin, nafarrelin, and the like. Available peptide drugs include octreotide and the like. The agonists of these LHRH homologues act on the pituitary gland to inhibit the secretion of luteini zing hormone when administered to the body (in the case of an agonist, it promotes secretion initially, but when released continuously) Inhibits the secretion of testosterone, estrogen, which is a sex hormone, and is a peptide substance that has a therapeutic effect in prostate cancer, breast cancer, endometriosis, etc. In addition, examples of the peptide salts include, but are not limited to, acid addition salts of peptides, specifically, gocethelin acetate. In the present invention, the term "biocompatible polymer" refers to materials that are also known as biodegradable polymers, and conventional polymers used in the manufacture of microcapsules may be used as pharmaceuticals. Caprolactone (Polycaprolactone), Polyaminoacids, Polyhydroxybutyrate, Polyoxyethylene glycolate, Polyanhydrides, Polylactide, Polylactide Polyglycol ides, copolymers thereof Poly (lactide-co-glycolide) and the like. Preferably poly (lactide-co-glycolide) (Poly Lactic-co-Glycolic Acid, PLGA). Biocompatible polymers (PLGA) that can be used in the present invention may use those having a weight average molecular weight of 60,000 or less. For example, about molecular weight

13, 000 poly (lactide-co-glycolide) (50:50), poly (lactide-co-glycolide) (50:50) with a molecular weight of about 33,000, poly (lock) Tited-co-glycolide) (50:50), poly (lactide-co-glycolide) with molecular weight οΐ 20,000 (75:25), poly (lactide) with molecular weight οΐ 16'000 (100: 0 And the like. Examples of such biocompatible polymers include RG502H, RG503H, RG504H, RG752H, and R202H from Boehringer Ingelheim. In the present invention, the biocompatible polymer may include 70 to 99 wt%, more preferably 80 to 98 wt%, and most preferably 85 to 97 wt% based on the total weight of the final microspheres prepared. In the present invention, if the biocompatible polymer is included in less than 70% by weight, there may be a problem in that the distribution of the bioactive substance is relatively increased, thereby preventing the overdose from maintaining the drug for a desired period of time. The amount to be administered to the patient may be so high that it may be difficult or impossible to administer.

In the microsphere according to the present invention, the biocompatible polymer is primarily encapsulated with the bioactive material, and is further encapsulated by the following release inhibitors. In the present invention, the release inhibitor is included for the encapsulation of the bioactive material and effective release control, and forms a lipid matrix surrounding the exterior of the biopolymer containing the bioactive material, and preferably has a long chain of 16 or more carbon atoms. Fatty acids, specifically C18: 1, C18: K0H) or C18: difatty acid. Examples of the oils containing the above long-chain fatty acids, ie, C18: 1 or C8: 2 fatty acids, include linoleic acid (C18: 2) 64%; palmitic acid (C16: 0) 14 %; Oleic acid (C18: 1) 10%; linolenic acid (1 inolenic acid) (C18: 3) 7%; stearic acid (C18: 0) «soybean lecithin 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%; arachidic acid (C20: 0) 0.6%; myristic acid Cottonseed oil having 0.3% or the like (C14: 0), or ricinoleic acid (C18: 1 (OH)) (87%); oleic acid (C18: 1) 7%; linoleic acid (C18: 2) 3%; palmitic acid (C16: 0) 2%; castor oil or linoleic acid (C18: 0) with 1% of stearic acid (C18: 0) : 2) 58.9%; oleic acid (C18: 1) 25.8%; Palmitic acid (C16: 0) 11.0%; stearic acid (C18: 0) 1.7%; corn oil or linoleic with 1.1% linolenic acid (C18: 3) ^{acid) (C18: 2) '} 66%; Linolenic acid (C18: 3) 0.5%; oleic acid (C18: 1) 26%; palmitic acid (C16: 0) 4%; stearic acid (C18: 0) Sunflower oil or palmitic acid (C16: 0) 7.5-20.0% having 2% etc .; palmitoleic acid (C16: l), 0.3-5.0%; stearic acid ) (C18: 0), 0.5-5.0%; oleic acid (C18: l), 55.0-83.0%; olive oil or linoleic acid with linoleic acid (C18: 2), 3.5-21.0%, etc. linoleic acid) (C18: 2) 40.4%; Oleic acid (C18: l) 45.4%; palmitic acid (C16: 0) 9.1%; stearic acid (C18: 0) 4.3%, etc. Sesame oil or linoleic acid acid) (C18: 2) 50-57%; linolenic acid (C18: 3) 5-10%; oleic acid (C18: 1) 17-26%; palmitic acid ) (C16: 0) 9-13%; soybean oil or arachidic acid (C20: 0) 2.4%; palmitic acid (palmitic acid) with stearic acid (C18: 0) 3-6%, etc. ic acid) (C16: 0) 8.3%; stearic acid (C18: 0) 3.1%; linoleic acid (1 inoleic acid) (C18: 2) 26.0%; oleic acid (C18: 1) Peanut oil having 56.0% and the like, and is not particularly limited, but the most lyric acid (C18: 1 (OH)) (87%); oleic acid (C18) : l). Castor oil having (7%) is preferred. In the present invention, the release inhibitor may be included in an amount of 0.1 to 2C 0 parts by weight, more preferably 0.1 to 10.0 parts by weight, most preferably 0.1 to 5.0 parts by weight based on 100 parts by weight of the biocompatible polymer. If the amount of release inhibitor included is less than the weight part of the release inhibitor, the surfactant effect of the release inhibitor is insufficient, there may be a problem in the drug encapsulation rate, if exceeding 20.0 parts by weight, the release of the drug is delayed or the microspheres due to excess components May interfere with formation. Sustained release microspheres according to the invention preferably comprise 1.0 to 30 weight ¾, more preferably 2.0 to 20% by weight, most preferably 3.0 to 10% by weight of the bioactive material based on the total microspheres. In the present invention, when the bioactive substance is included in less than 1.0% by weight, the amount of microspheres to be administered to the patient may be too high, which may render the administration impossible or may cause a problem. There may be a problem that is difficult to suppress. Since the sustained-release microsphere according to the present invention has a form in which a bioactive material is encapsulated by a biocompatible polymer, and the encapsulated biocompatible polymer is further enclosed by a release inhibitor to form a lipid matrix, the conventional microsphere Compared to the present invention, it shows an excellent rate of bioactive substance encapsulation and solves problems such as side effects caused by the initial over-release of the bioactive substance.

,

Furthermore, as another aspect, the present invention is an organic solvent in which a water-soluble bioactive substance is suspended or dispersed in an organic solvent in which a biodegradable polymer and a release inhibitor are dissolved, or an organic solvent in which a biodegradable polymer is dissolved. A solvent is prepared to form a suspension or emulsion, and then a release inhibitor is mixed and then added to an aqueous medium to prepare a microsphere, and a method for preparing a sustained release microsphere by removing the organic solvent is provided. Hereinafter, the manufacturing method according to the present invention will be described in detail.

The present invention, i) dissolving the biocompatible polymer in an organic solvent;

ii) in the organic solvent in which the biocompatible polymer prepared in step i) is dissolved A suspension is prepared by suspending or dispersing a bioactive substance, or adding a dissolution aid to the bioactive substance and dissolving the bioactive substance to which the dissolution aid is added in an organic solvent in which the biocompatible polymer prepared in step i) is dissolved. Preparing a non-aqueous solution

iii) mixing the release inhibitor in the suspension or non-aqueous solution prepared in step i i);

iv) injecting the solution or emulsion formed in step iii) into an aqueous medium to form a microsphere, and then adjusting the particle size; And

V) provides a method for producing a slow release microsphere comprising the step of removing the organic solvent. In the production method according to the present invention, the same information regarding the sustained-release microspheres is applied to the bioactive material, the biocompatible polymer and the release inhibitor. Wherein i) step of the present invention is a step of dissolving a biocompatible polymer in an organic solvent to the previous step for the preparation of the oil dispersed phase (oi l di spers ion phase) '. In addition, the organic solvent that can be used to dissolve the biocompatible polymer in step i) is not particularly limited as long as it can dissolve the biocompatible polymer. For example, one or more solvents selected from the group consisting of methylene chloride, chloroform, acetonitrile, dimethyl sulfoxide, dimethylformamide and ethyl acetate may be used. Furthermore, step ii) of the present invention may be prepared by suspending and dispersing the bioactive material itself in an organic solvent in which the biocompatible polymer prepared in step i) is dissolved, or preparing a biocompatible polymer prepared in step i). With the dissolved organic solvent, a dissolution aid is added to the physiologically active substance and dissolved therein, followed by mixing them with each other to prepare a non-aqueous solution. In addition, the dissolution aid of step ii) used in the case of suspending and dispersing the bioactive material in the organic solvent prepared in step i) is not particularly limited as long as it can dissolve the bioactive material. Dissolution aids that can be used to dissolve the bioactive material in the present invention are preferably dimethylsulfoxide (DMS0) or

N-methyl-2-pyridinone (NMP). The dissolution aid may be used in a weight of 200 to 1,000% relative to the weight of the bioactive material. In step ii) of the present invention, a surface active agent may be optionally added to assist in the formation of the desired physical properties or emulsion of the suspension. Surfactants may be used without particular limitation so long as they are conventionally used in the art. Surfactants include, for example, silicone oil, polysorbate (Tween), sorbitan ester (Span), poloxamer, polyethyleneglycol and tocope. One or more surfactants selected from the group consisting of (tocopherol) and the like can be used.

The step iii) of the present invention is a step of mixing the release agent and the material prepared in steps i) and Π) to prepare an oil dispersed phase, for example in the form of a transparent emulsion, wherein the release inhibitor used in the step is water-soluble Micro-fabricated and poorly water-soluble or water-insoluble ingredients that have a function of delaying or inhibiting the release of phosphorus bioactive substances or delaying or releasing excessive release of initial bioactive substances or delaying the release of bioactive substances for a desired period of time. By spice means a component used for the purpose of increasing the drug inclusion. In the present invention, in the step iv), the aqueous medium may be preferably water for injection, and optionally, a low viscosity polymer may be mixed and used to suppress the diffusion of the emulsion. The low viscosity polymers used here include, for example, polyvinylpyridone (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 solut ion at 20 ⁰ C) may be used, but is not limited thereto. Furthermore, in addition to the aqueous medium of step iv), an optional cosolvent may be further used. Examples of such cosolvents include alcohols in which a hydrogen atom of a hydrocarbon is substituted with a hydroxyl group (hydroxy, _0H), or acetic acid and an organic acid, or acetone. Or carbolic acid, most preferably methanol, ethanol, propanol, butanol, pentane, nucleic acid, narrow coal, octane, nonanol, or decanol monovalent alcohol. More preferably ethanol can be mentioned but is not limited thereto. When co-solvents such as monohydric alcohol, acetone, acetic acid, etc. are used, the microspheres produced can be reduced. However, when the co-solvent is used in excess, the encapsulation rate of the drug may be lowered, and in small amounts, the dissolution of the drug may be limited by the microsphere coarse effect. Therefore, at this time, the release inhibitor to cosolvent ratio is 1: 1: 1: 10.000, or preferably 1: 10-1: 1, 000, most preferably 1: 20-1: 400.

In addition, in step iv), the particle size control can be used without limitation the known particle size control method of microspheres, it is preferable to control using a high pressure homogenizer. As the method for removing the organic solvent in the step V) of the present invention, any method commonly used in the art may be used. The organic solvent may be, for example, agitated, heated, or purged with nitrogen (N 2 purge), but is not limited thereto.

In the preparation method of the present invention _, a release inhibitor is mixed with a suspension solution or an emulsion solution, and then added to an aqueous medium to release a water inhibitor which is water insoluble or poorly water soluble in forming a microsphere, and is soluble while being distributed inside or outside the microsphere. The bioactive material reduces the initial dissolution rate by reducing contact with external aqueous media. In a preferred embodiment, the preparation method of the present invention may further comprise the step of freezing the microspheres obtained after removing the organic solvent in the step V). It may also further comprise the step of centrifugation and / or dialysis (Di alysi s) and / or filtration step before the freezing step. That is, the microspheres prepared through the above process increase the encapsulation rate of the bioactive substance as a drug, and there is no excessive initial burst of the drug regardless of the molecular weight and lactide content of the biocompatible polymer used. Gives zero-order emission characteristics.

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

[Form for implementation of invention]

Hereinafter, the present invention will be described in detail by way of examples. However, these examples are only presented to understand the content of the present invention, and the scope of the present invention should not be construed as being limited to these embodiments.

<Comparative Example> Preparation of microspheres by general oil emulsification (0 / W) 50 mg of gocethelin acetate (USP grade) was dissolved in 0.2 ml of DMSO according to the contents shown in Tables 1 and 2, and 500 mg of biocompatible polymer was added to methylene. Dissolve in 1 ml of chloride to give a clear emulsion dog (DP, Dispersion phase). This solution was added to 90 ml of 0.5% polyvinyl alcohol (Mw = 30, 000 ~ 70,000, Sigma) aqueous solution and 10ml mixture of ethanol (99.5%), which was vigorously stirred (10,000 rpm) with L4R mixer (Silverson) at 25 ° C. It was slowly added dropwise using a syringe pump (lml / min). After 1 minute, the 0 / W liquid was adjusted in a high pressure homogenizer (Buffalo) to adjust the size of the microspheres to 3 ± 0.5 μΐΏ and repeatedly passed through 10 times to prepare a homogeneous 0 / W liquid. This 0 / W liquid was volatilized the organic solvent for 12 hours at 400 rpm in the stirrer. After centrifugation (10,000g, 20 minutes) to obtain a microsphere, washed twice with distilled water and freeze-dried for 72 hours.

<Comparative Example 1> Preparation of high cetelin-containing microspheres using biocompatible polymers having different intrinsic viscosity

Table 1

DMSO 0.2ml 0.2ml 0.2ml

MC 1ml 1ml 1ml

PLGA PLGA 502H ¹⁾ PLGA 503H ² PLGA 504H ^3J

500mg 500mg 500mg

0.5% PVA solution 90ml 90ml 90ml

EtOH (99.5%) 10ml 10ml 10ml

1) Lactad: glycolide = 50:50, Boehringer Ingelheim, i.v. = 0.20dl / g, Resoraer ™

2) Lactide: glycolide = 50: 50, Boehringer Ingelheim, i.v. = 0.39dl / g, Resomer ™

3) Lactide: glycolide = 50:50, Boehringer Ingelheim, i.v. = 0.52dl / g, Resomer TM Comparative Example 2> Formulation design using biocompatible polymers with different compositions of lactide / glycolide

Table 2

1) Lactide: glycolide = 50: 50, Boehringer Ingelheim, i.v. = 0.20dl / g, Resomer ™

2) Lactide: glycolide = 75:25, Boehringer Ingelheim, i.v. = 0.20dl / g, Resomer ™

3) Lactide: glycolide = 100: 0, Boehringer Ingelheim, iv = 0.20dl / g, Resomer ™ Comparative Example 3> Biocompatible Polymer (RG502H) and Release Inhibitor (Medium chain) Preparation of Microspheres by Mixing Oil Dispersion Phase (ODP) of triglyceide (MCT)

After dissolving 50 mg of high-cetellin acetate (USP grade) in DMS0 and dissolving 500 mg of biocompatible polymer in 1 ml of methylene chloride, medium chain triglyceide (MCT) composed of C6: 0 ~ C12: 0 as release inhibitor is listed. Dissolved according to the content shown in 3 to clarify the clear emulsion (ODP, Oil Dispersion phase). The following manufacturing process was performed similarly to the comparative example.

[Table 3]

* Example 1> Preparation of Microspheres by Mixing Oil Dispersion Phase (ODP) between Biocompatible Polymer (RG502H) and Release Inhibitor (Lecithin, Lecithin)

50 mg of high-cetellin acetate (USP grade) was dissolved in 0.2 ml of DMSO, and 500 mg of biocompatible polymer was dissolved in 1 ml of methylene chloride, and then as a release inhibitor, lecithin was dissolved according to the contents shown in Table 4 to prepare a transparent emulsion (ODP). , Oil Dispersion phase). The following manufacturing process was performed similarly to the comparative example.

Table 4

Example 2 Preparation of Microspheres by Mixing Oil Dispersion Phase (0DP, Oil Dispersion Phase) of Biocompatible Polymer (RG502H) and Release Inhibitor (Cottonseed Oil Cottonseed Oil)

50 mg of high cetelin acetate (USP grade) was dissolved in 0.2 ml of DMS0, and 500 mg of biocompatible polymer was dissolved in 1 ml of methylene chloride, and as a release inhibitor, cottonseed oil was dissolved according to the contents shown in Table 5 to prepare a transparent emulsion (0DP). , Oi l Di spersion phase). The following manufacturing process was performed similarly to the comparative example.

Table 5

^* % By weight of PLGA Example 3> Preparation of microspheres by mixing the oil-dispersed phase (0DP, Oi l Dispersion phase) between the biocompatible polymer (RG502H) and the release inhibitor (Castor oi l, castor oil)

50 mg of high-cetelin acetate (USP grade) was dissolved in 0.2 ml of DMS0, and 500 mg of biocompatible polymer was dissolved in 1 ml of methylene chloride. As a release inhibitor, castor oil was dissolved according to the contents shown in Table 6 to prepare a transparent emulsion dog (ODP, Oil Dispersion phase). The following manufacturing process was performed similarly to the comparative example.

[Table 6]

Example 4> Preparation of Microspheres by Mixing Oil Dispersion Phase (ODP) of Biocompatible Polymer (RG502H) and Release Inhibitor (Corn Oil, Corn Oil)

50 mg of goserelin acetate (USP grade) was dissolved in 0.2 ml of DMS0, and 500 m _g of biocompatible polymer was dissolved in 1 ml of methylene chloride as a release inhibitor. Corn oil was dissolved according to the contents shown in Table 7 to prepare a clear emulsion. (ODP, Oil Dispersion phase). The following manufacturing process was performed similarly to the comparative example.

[Table 7]

EtOH (99.5%) 10ml 10ml 10ml 10ml

Example 5> Preparation of microspheres by mixing oil dispersion phase (ODP) between biocompatible polymer (RG502H) and release inhibitor (Saf flower oil, sunflower oil)

50 mg of high cetelin acetate (USP grade) was dissolved in 0.2 ml of DMS0, and 500 mg of the biocompatible polymer was dissolved in 1 ml of methylene chloride, and then as a release inhibitor, sunflower oil was dissolved according to the contents shown in Table 8 to prepare a transparent emulsion. ODP, Oil Dispersion phase. The following manufacturing process was performed similarly to the comparative example.

[Table 8]

% By weight of PLGA Example 6> Preparation of microspheres by mixing oil dispersion phase (ODP) of biocompatible polymer (RG502H) and release inhibitor (Olive oil, olive oil)

50 mg of goserelin acetate (USP grade) was dissolved in 0.2 ml of DMS0, and 500 mg of biocompatible polymer was dissolved in 1 ml of methylene chloride, and then, as a release inhibitor, olive oil was dissolved according to the contents shown in Table 9 to prepare a transparent emulsion (ODP, Oil Dispersion phase). The following manufacturing process was performed similarly to the comparative example. Table 9

PLGA% by weight Example> Preparation of microspheres by mixing oil dispersion phase (ODP) of biocompatible polymer (RG502H) and release inhibitor (Sesame oil, sesame oil)

50 mg of high cetelin acetate (USP grade) was dissolved in 0.2 ml of DMSO, and 500 mg of biocompatible polymer was dissolved in 1 ml of methylene chloride, and then sesame oil was dissolved according to the contents shown in Table 10 to prepare a clear emulsion (ODP). , Oil Dispersion phase). The following manufacturing process was performed similarly to the comparative example.

Table 10

^* % By weight of PLGA Example 8> Biocompatible polymer (RG502H) and release inhibitor (Soybean oil) Preparation of Microspheres by Mixing Oil Dispersion Phase (ODP) of Soybean Oil)

50 mg of high-cetellin acetate (USP grade) was dissolved in 0.2 ml of DMSO, and 500 mg of biocompatible polymer was dissolved in 1 ml of methylene chloride, and then, as a release inhibitor, soybean oil was dissolved according to the contents shown in Table 11 to prepare a transparent emulsion (ODP). , Oi l Di spersion phase). The following manufacturing process was performed similarly to the comparative example.

Table 11

^* % Of PLGA Example 9> Preparation of Microspheres by Mixing Oil Dispersion Phase (ODP) of Biocompatible Polymer (RG502H) and Release Inhibitor (Peanut Oil, Peanut Oil)

50 mg of goserelin acetate (USP grade) was dissolved in 0.2 ml of DMSO, and 500 mg of biocompatible polymer was dissolved in 1 ml of methylene chloride, and then as a release inhibitor, peanut oil was dissolved according to the contents shown in Table 12 to prepare a transparent emulsion (ODP). , Oi l Di spers ion phase). The following manufacturing process was performed similarly to the comparative example.

Table 12

Example 10 Preparation of Biodegradable Microspheres Containing Acetate Prolinelin for at least 3 Months Sustained Release

50 mg of goserelin acetate (USP grade) was dissolved in 0.2 ml of DMSO

500 mg of the biocompatible polymer shown in 13 was dissolved in 1 ml of methylene chloride, and then a release inhibitor was dissolved according to the indicated content to prepare a transparent emulsion (0DP, Oil Dispersion phase). The following manufacturing process was performed similarly to the comparative example.

Table 13

^* % Of PLGA weight

1) Lactide: glycolide = 50: 50, Boehringer Ingelheim, 丽. = 50,000 Resomer ™

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

3) Lactide: glycolide = 100: 0, Boehringer Ingelheim, MW. = 220, 000 esomer ^1M

Example 11 Preparation of Microspheres Using Cosolvent (ethane) in Aqueous Solution (CP)

50 mg of goserelin acetate (USP grade) was dissolved in 0.2 ml of DMSO, 500 mg of biocompatible polymer was dissolved in 1 ml of methylene chloride, and then release inhibitor was dissolved according to the contents shown in Table 14 to prepare a transparent emulsion dog (ODP, Oi l). Di spersion phase. This solution is a mixture of 0.5% polyvinyl alcohol (Mw = 30, 000-70, 000, Sigma) and ethanol (99.5%) mixture vigorously stirred (10, 000 rpm) with an L4R mixer (Si lverson) at 25 ° C. To prepare an aqueous solvent according to the content shown in Table 14 and then slowly added dropwise using a syringe pump (lml / min). The following manufacturing process was performed similarly to the comparative example.

Table 14

% Of PLGA weight

Example 12 Preparation of Microspheres by DMSO in Non-Aqueous Liquid Phase (ODP) 50 mg of high-cetellin acetate (USP grade) was dissolved in DMS0 according to Table 15, and 500 mg of biocompatible polymer was dissolved in 1 ml of methylene chloride. Dissolved according to the contents shown in Table 15 to prepare a transparent emulsion dog (0DP ᅳ Di Differentiation phase). The following manufacturing process was performed similarly to the comparative example.

Table 15

^# ¾ of API weight

Example 13 Preparation of Microspheres by Enclosure of Biologically Active Substance 50 mg of the bioactive substance was dissolved in DMS0 according to Table 16, and 500 mg of the biocompatible polymer was dissolved in 1 ml of methylene chloride, and then the release inhibitor was added to the contents shown in Table 15. Were dissolved to prepare a clear emulsion dog (0DP, Oil Dispersion phase). The following manufacturing process was performed similarly to the comparative example.

Table 16

^* PLGA by weight ¾> Experimental Example l> In vitro release of drug

50 mg of high cetelin acetate (USP grade) was dissolved in 0.2 ml of DMSO, 500 mg of biocompatible polymer was dissolved in 1 ml of methylene chloride, and then release inhibitor was dissolved according to the contents shown in Table 17 to prepare a transparent emulsion dog (ODP, Oi l). Di spers ion phase). The following manufacturing process was performed similarly to the comparative example.

Table 17

^* % Of PLGA weight

In order to confirm that the hydrophilic drug is continuously controlled from the prepared polymer microspheres, drug release experiments were conducted under the following in vitro conditions. That is, the polymer microspheres prepared by applying the release inhibitor 10% in Comparative Example 1-1, Experimental Example 1 and Examples 1-9 and 50 mg of the polymer microspheres prepared in Example 10, which is a 3-month formulation, After being placed in a discharge bottle, 50 ml of a phosphate complete solution of pH 7.4 was sealed and placed in a shaking water bath at 120 ° C. at 37 ° C. to continuously release the drug for 28 days or more. The released drugs were centrifuged at 20, 000 g for 10 minutes after taking 1 ml of the released solution, and then 100 ul of the supernatant was measured by HPLC quantitative analysis as in Experiment 1, and the remaining solution was redispersed in the test tube. He was layered. The measurement results are shown in FIG. 1 (A), and the first-day drug release rate of the microspheres (Comparative Example 1-1) in which no release inhibitor was used was 70% at first. Initial bursts were observed, and the initial overdose was released at 50% for the first-day drug release of microspheres (Comparative Example 3-3) using 10% Medium chain triglyceride (MCT). . However, in Experimental Example 1, the microspheres containing 0.1% of the release oil (Castor oil) was about 20%, and the daily drug release rate of the microspheres containing 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 was not completely released within 28 days. In addition, according to Example 1-9, shown in Figure 1 (B), when the oil composed of long-chain fatty acid (long-chain fatty acid) is used as the release inhibitor, the initial dissolution rate is significantly smaller than zero months or more release characteristics It can be seen that it represents. And FIG. 1C shows that the release rate can be at least 3 months as shown in Example 10. . Experimental Example 2 Experiment of Enclosure Rate Measurement of Drugs

20 mg of the prepared polymer microspheres were precisely weighed and placed in a test tube with a lid. After dissolving in 3 ml of methylene chloride, 5 ml of distilled water was added and stirred vigorously for 60 minutes. Thereafter, the solution was centrifuged at 3,000 g for 5 minutes, and a certain amount of supernatant was taken to measure the concentration of the drug using high performance liquid chromatography (HPLC) to calculate the encapsulation rate of the drug encapsulated in the microspheres. The column used was Waters C-18 (4.6x150 (), injection volume was and detection wavelength was 220nm. As the mobile phase, water (a) and acetonitrile (b) containing 0.1% TFA were used in a 75%: 25% ratio.

The encapsulation amount of the prepared microspheres is shown in FIGS. 2 (A) and (B), and according to this, as the concentration of the release inhibitor increased from 0.1 to 20¾>, the encapsulation of the drug was increased, and the drug of the formulation in which the release inhibitor was introduced The loading amount was 50-100%. In addition, the microspheres containing more than 5% of release inhibitors showed a 70% encapsulation rate. However, the amount of drug encapsulation in Comparative Examples 1 to 2 formulation without release inhibitor and Comparative Example 3 formulation in which medium chain triglyceride was introduced is shown within. In addition, castor oil among the release inhibitors showed the highest encapsulation rate of about 10¾> higher than other release inhibitors. Efficiency was shown.

In addition, the results of the preparation of the microspheres by the cosolvent (CP) in the aqueous phase (CP) and the microspheres by the DMS0 in the non-aqueous phase (0DP) are shown in FIG. 2 (C). The ratio of 1 to 200 was not affected by more than 70% of the encapsulation with the increase of the cosolvent in the aqueous solution, but when the co-solvent was increased to more than 1, 000, the encapsulation was less than 70%. In addition, as a result of the encapsulation rate according to the preparation of the microspheres by DMS0, when the DMS0 of the non-aqueous liquid phase was less than 2% of the API weight, the main component was not completely dissolved, and the encapsulation rate was less than 50%. If exceeded, the encapsulation rate is also shown to be less than 50%. As a result of the encapsulation rate according to the preparation of microspheres containing various bioactive substances, the encapsulation rate was less than 50% when no release inhibitor was introduced. The excellent sealing rate which was the above was shown. Experimental Example 3 Particle Morphology Measurement of Microspheres

To observe the appearance of the microparticles, approximately 50 mg of microspheres were fixed on an aluminum stub and vacuum degree O. After coating with platinum at ltorr and high voltage (10kV) for 15 minutes, it was mounted on the SEM body and the morphology of the microspheres was observed using an image analysis program. The measurement results are shown in FIG. 3, which indicates that the microspheres without release inhibitors and the microspheres with medium chain triglyceride (MCT) were found to have a large amount of porosity while the release inhibitors (Castor It can be seen that the porosity of the prepared microspheres is reduced and introduced into the surface of the microspheres.

In addition, it can be seen that the microspheres were reduced by ethane used as a cosolvent in the microspheres into which the release inhibitor was introduced.

Claims

[Range of request]

[Claim 1]

Bioactive substances, biocompatible polymers and release inhibitors,

The biocompatible polymer in which the bioactive material is encapsulated is further encapsulated by a release inhibitor, and the release inhibitor is an oil including a C18: l, C18: l (0H) or C18: 2 long chain fatty acid. Sustained release microspheres.

[Claim 2]

The method according to claim U, wherein the bioactive substance is one or a salt thereof selected from the group consisting of octreotide, lanreotide, goserelin, lupulide, tryptorelin, historelin and desmopressin Characterized by slow release microspheres.

[Claim 3]

According to claim 1, The bioactive material is 1.0 to 30% by weight based on the total microspheres, sustained-release microspheres.

[Claim 4]-The sustained-release microsphere of claim 2, wherein the bioactive peptide is goserelin.

[Claim 5]

The sustained-release microsphere of claim 1, wherein the biocompatible polymer is polylactide, polylactide-co-glycolide or poly (lactide-co-glycolide) glucose.

[Claim 6]

The sustained-release microsphere of claim 1, wherein the biocompatible polymer is included in an amount of 70 to 99 wt ¾> based on the total weight of the macrosphere.

[Claim 7]

In U,

The release inhibitor is based on 100 parts by weight of the biocompatible polymer, 0.1 to 20 Sustained-release microspheres, which are included in parts by weight

[Claim 8]

8. The oil according to claim 7, wherein the C18: l, C18: K0H) or C18: 2 fatty acid is selected from the group consisting of lecithin, cottonseed oil, castor oil, corn oil, sunflower oil, olive oil, sesame oil, soybean oil and peanut oil. Being one or more, slow-release microspheres

[Claim 9]

The sustained-release microsphere of claim 8, wherein the oils containing C18: l, C18: K0H) or C18: 2 fatty acids are castor oil.

[Claim 10]

The sustained release microsphere of claim 1, which is in the form of a lipid matrix.

[Claim 11]

i) dissolving the biocompatible polymer in an organic solvent;

ii) preparing a suspension by suspending or dispersing a bioactive material in an organic solvent in which the biocompatible polymer prepared in step 0 is dissolved, or adding a dissolution aid to the bioactive material and adding the biocompatible polymer prepared in step i). Preparing a non-aqueous solution by dissolving a physiologically active substance added with the dissolution aid in a dissolved organic solvent.

iii) mixing the release inhibitor in the suspension or non-aqueous solution prepared in step Π);

V) A method for producing a slow release microsphere comprising the step of removing the organic solvent

[Claim 12]

The method of claim 11, wherein the organic solvent used in step i) is methylene chloride, A chloroform, acetonitrile, dimethyl sulfoxide, dimethylformamide and ethyl acetate, at least one solvent selected from the group consisting of, a method for producing a sustained-release microsphere.

[Claim 13]

The method of claim 11, wherein the dissolution aid used in step ii) is dimethylsulfoxide (DMS0) or N-methyl ᅳ 2-pyridinone (XP).

[Claim 14]

The method of claim 11, wherein the dissolution aid used in step ii) has a weight of 200 to 1,000% by weight of the bioactive material.

[Claim 15]

The method of claim 11, wherein the aqueous medium of step iv) further comprises a cosolvent.

[Claim 16]

According to claim 11, In the step ii) further adding a surfactant, a method for producing a sustained-release microspheres.

[Claim 17]

The method according to claim 16, wherein the surfactant is silicone oil, polysorbate (Tween), sorbitan ester (Span), poloxamer, polyethyleneglycol and A process for producing sustained-release microspheres, wherein the tocopherol is at least one surfactant selected from the group consisting of:

[Claim 18]

The method of claim 15, wherein the cosolvent is at least one selected from the group consisting of alcohol, acetic acid, organic acid, acetone, and carbolic acid.

[Claim 19]

The method of claim 18, wherein the co-solvent is one or more monohydric alcohols selected from the group consisting of methanol, ethanol, propanol, butanol, pentane, nucleic acid, heptanol, octanol, nonanol and decanol Method for producing the emerging microspheres.

[Claim 20]

The method of claim 11, further comprising lyophilizing the slow-release microspheres prepared after step V).

[Claim 21]

The method of claim 11, further comprising any one selected from the group consisting of centrifugation, dialysis, and filtration before the lyophilization step.