KR100566573B1 - Preparation method of sustained release microspheres containing LHRH analogue - Google Patents

Abstract

The present invention relates to a method for producing sustained-release microspheres made of a biodegradable polymer containing a luteinizing hormone releasing hormone (LHRH) homolog. More specifically, the process of dissolving the LHRH homologue and the biodegradable polymer in acetic acid, formic acid or a mixture thereof without the use of water; Spray drying it to a temperature of 60 to 90 ° C. and a compressed air amount of 300 to 500 NL / h; And it relates to a method for producing LHRH homologue-containing sustained-release microspheres by drying for 10 to 12 hours under a reduced pressure of 0.931 to 1.33 Pa (7 to 10 mTorr). In the method for preparing sustained-release microspheres containing the LHRH homologue of the present invention, the microspheres are prepared by a simple process of spray drying a solution of the LHRH homolog and the biodegradable polymer and then drying under reduced pressure without using any water and a toxic solvent. The microspheres prepared according to the present invention have no problem of residual toxic solvents exhibited in the conventional manufacturing method, and the size of the microspheres can be prepared in a suitable size which is uniform and easy to inject. In addition, the microspheres containing the LHRH homologue prepared according to the present invention continuously releases the LHRH homologue to an effective concentration for a certain period of time when administered to the body, thereby being useful for the treatment of diseases such as prostate cancer, endometriosis and uterine myoma. Can be used.

LHRH homologue, biodegradable polymer, sustained-release microspheres, spray drying

Description

Preparation method of sustained-release microspheres containing LHRH homologues {Preparation method of sustained release microspheres containing LHRH analogue}

1A is an electron micrograph of tryptorelin-containing poly (lactide-co-glycolide) (PLGA, RG503H) microspheres prepared under the conditions of Example 1 of the present invention.

FIG. 1B is an electron micrograph of tryptorelin-containing poly (lactide-co-glycolide) (PLGA, RG503H) microspheres prepared under the conditions of Preparation Example 5. FIG.

FIG. 1C is an electron micrograph of tryptorelin-containing poly (lactide-co-glycolide) (PLGA, RG503H) microspheres prepared under the conditions of Preparation Example 9. FIG.

2 is a male S.D. of the Tryptorelin-containing PLGA microspheres prepared under the conditions of Preparation Example 1 of the present invention as in Test Example 3. Figure 1 shows the testosterone levels in serum after subcutaneous injection to rats (n = 5).

The present invention relates to a method for producing sustained-release microspheres in which LHRH homologue which is a bioactive substance is encapsulated using a biodegradable polymer as a carrier. Such microsphere formulations are sustained release injectable formulations formulated such that subcutaneous or intramuscular injection allows LHRH homologues to remain biologically active and to be released sustained and uniformly in the body.

Physiologically, gonadotropin releasing hormone (GnRH, LHRH) is released in the hypothalamus due to the reaction of hypothalamic releasing hormone in low blood testosterone or estrogen levels. It is transported to the pituitary gland via the hypothalamic-pituitary portal system to synthesize and secrete luteinizing hormone (LH) and follicle stimulating hormone (FSH). Stimulation causes the release of testosterone and estrogen. LHRH analogues, including LHRH agonists and LHRH antagonists, act on the pituitary gland to inhibit the secretion of LH (in the case of agonists, early release and sustained release). In the case of inhibition), it suppresses the secretion of testosterone and estrogen, and thus has a therapeutic effect in prostate cancer, endometriosis, uterine myoma, etc.

However, LHRH homologues are very poor in gastrointestinal stability and can be administered only by injection as in the case of general peptide drugs, and in this case, the bioavailability is very low and daily injection is required. In addition, the treatment period is very long, there are many difficulties in compliance, treatment efficiency and treatment of the patient. For this reason, it has been developed as a microsphere formulation for encapsulating PLGA, a biodegradable polymer, so that LHRH homologue is continuously released when administered to the body for a long time.

Conventional methods for preparing sustained-release microspheres containing LHRH homologues include phase separation (USP 4,673,595, EP 52,510), cryopulverization after melt extrusion (USPs 5,134,122, 5,192,741, 5,225,205, 5,431,348). , 5,439,688, 5,445,832, 5,776,885), double emulsification evaporation method (w / o / w, water / oil / water) , Single emulsion evaporation (o / w, oil / water) (US Patent Nos. 4,389,330, 5,945,126; Shameem M, Lee Hee Yong, DeLuca PP, AAPS Pharmsci., 1 (3) article 7, 1999; Kostanski JW, Pharm Dev. Tech. 5, 585-596, 2000).

Looking at the manufacturing process by the phase separation method in detail as follows. The biodegradable polymer PLGA (1 g) was dissolved in an excess (50 g) of methylene chloride solvent and LHRH homologue (30.4 mg) dissolved in a small amount of water (0.3 ml) was added under stirring, followed by silicone oil (30 ml). It is a method to obtain embryonic microspheres by adding them at a constant speed, solidifying them using a non-solvent such as trichlorofluoromethane (2 l), filtering and drying under reduced pressure to obtain microspheres. As shown in the manufacturing process, an excess of toxic solvents such as methylene chloride can not be completely removed by the vacuum drying process alone, so that a significant amount of residual microsphere formulation remains, which can cause fatal harm when administered to the human body.

In contrast, low temperature pulverization after melt injection can limit the use of toxic solvents. This method is a method of producing a microsphere by melt-injecting a mixture of PLGA and LHRH homologue (or hydrophobic salt thereof) through an injector at high temperature and then grinding it at low temperature. In order to obtain a mixture of PLGA and LHRH homologues, a method of uniformly mixing with a stirrer using a methylene chloride solvent and then removing the organic solvent with a rotary evaporator and a vacuum dryer may be used. A method of mixing the two in a fine powder obtained by sieving after milling at may be used. In the latter case, there is no problem of residual toxic solvents since no toxic solvent is used during the manufacturing process. However, in the present manufacturing method, a grinding process is performed to obtain microparticles, although at low temperatures, the LHRH homologue may be denatured by the heat generated in this process. Difficult to do

The double emulsion evaporation method (w / o / w) is generally used for encapsulation of water-soluble drugs such as peptides and proteins, and disperses an aqueous solution of LHRH homologues in an organic solvent containing PLGA to form a primary emulsion. (water in oil), which is then dispersed in an aqueous phase containing an emulsifier. In addition, single emulsion evaporation (o / w) is a method mainly used for the inclusion of fat-soluble drugs, in which LHRH homologue and PLGA are dissolved together in a mixture of a suitable organic solvent (in this case, methanol and methylene chloride), It is a method to disperse in. In both processes, the organic solvent is removed by extraction or evaporation in the process of dispersing the polymer in the organic phase, so that the solubility of the polymer is reduced, thereby forming microspheres. The technically important part of these two methods is mainly the encapsulation rate of peptide drugs.

Since the LHRH homologue is a water-soluble drug, a large amount of the drug is released during the dispersion in the water phase, and the encapsulation rate is low. To solve this problem, Okada et al. Used gelatin-like materials to increase the viscosity of primary emulsions during the preparation of microspheres by double emulsion evaporation, thereby reducing the diffusion rate of LHRH homologues in secondary emulsions. Drug encapsulation rates have been increased (Okada, H. and Toguchi, H., Crit. Rev. Ther. Drug Carrier Syst., 12, 1-99, 1995). Similarly, in single emulsion evaporation, the inclusion rate of LHRH homologue can be increased by appropriately increasing the concentration of PLGA dissolved in the organic solvent. In general, the microspheres prepared by the double emulsion evaporation method have a higher porosity than the microspheres prepared by the single emulsion evaporation method, thereby increasing the surface area and relatively high initial release rate of the drug.

As described above, all conventional LHRH homolog-containing PLGA microspheres are manufactured using a toxic solvent, and the size of the microspheres varies depending on the method. The present invention is to solve the above problems and to propose a method for producing PLGA microspheres containing a very simple LHRH homolog.

It is an object of the present invention to solve the residual toxic solvent problem seen in conventional luteinizing hormone releasing hormone (LHRH) homolog containing microsphere formulations through a simpler process, to provide microspheres of uniform size suitable for injection, and to provide LHRH homologs. To provide a method for producing a sustained-release microspheres made of a biodegradable polymer containing LHRH homologue, which is continuously released at an effective concentration for a long time.

The inventors dissolve LHRH homologues and biodegradable polymers in acetic acid, formic acid, or mixtures thereof without using water, spray drying at a temperature of 60 ° C. or higher and a compressed air amount of 300 to 500 NL / h, and then drying under reduced pressure. The present invention was completed by confirming that LHRH homologue-containing microspheres as described above were prepared.

The present invention relates to a method for producing sustained-release microspheres composed of biodegradable polymers containing LHRH homologues.

Specifically, the present invention comprises the steps of dissolving the LHRH homologue and the biodegradable polymer in acetic acid, formic acid or a mixture thereof without the use of water; Spray drying it to a temperature of 60 to 90 ° C. and a compressed air amount of 300 to 500 NL / h; And a process for drying for 10 to 12 hours under a reduced pressure of 0.931 to 1.33 Pa (7 to 10 mTorr), the method for preparing LHRH homologue-containing sustained-release microspheres.

As used herein, the term “biodegradable polymer” refers to polylactide (PLA), polyglycolide (PGA), or a copolymer thereof, poly (lactide-co-glycolide) (Polylactide-co- Polyorthoester, Polyanhydride, Polyaminoacid, Polyhydroxybutyric acid, Polycaprolactone, as well as polyesters such as glycolide and PLGA Polyalkylcarbonate, and the like. In particular, polyester series such as PLA, PGA, PLGA are hydrolyzed in the body and metabolized into lactic acid and glycolic acid, which are harmless to the human body, and biocompatibility and stability are recognized.The biodegradation rate is the molecular weight of the polymer and the ratio of the two monomers. Depending on the water affinity, such as a short 1-2 weeks to a length of 1-2 years may be adjusted and a polymer material that is already commercialized and approved by the US FDA, etc., may be preferably used in the present invention.

As used herein, the term "LHRH analogue", when administered to the body, acts on the pituitary gland to inhibit the secretion of LH (in the case of agonists, in the early stages, when they are released and sustained, Inhibits the release of testosterone and estrogens, and means peptides that have a therapeutic effect in prostate cancer, endometriosis, uterine fibroids, etc., which proceed with hormonal reactivity, and specifically, tryptorelin and leuprolide. , LHRH agonists and antides, algitides, orntides, and setlorel, including goserelin, nafarelin, buserelin, salts thereof LHRH antagonists including cetrorelix, salts thereof, and the like. In particular, acetates of the above-mentioned LHRH agonists or LHRH antagonists can be preferably used.

The first step in the preparation of the LHRH homologue-containing biodegradable polymer microspheres according to the present invention is the step of uniformly dissolving the LHRH homologue and the biodegradable polymer in one or more organic solvents containing no water to facilitate spray drying. to be.
Biodegradable polymers such as PLGA are well soluble in relatively nonpolar solvents such as methylene chloride, chloroform, tetrahydrofuran, ethyl acetate, ethyl formate, acetonitrile, acetone, dimethyl sulfoxide, dimethylformamide, and the like. Insoluble in polar solvents such as methanol, ethanol and propyl alcohol. In contrast, LHRH homologues are water soluble peptides that are well soluble in polar solvents such as water and methanol. When water is used to dissolve the peptide, the initial release amount increases considerably as the peptide content increases, which is attributable to the increase in porosity during drying by dissolving the peptide in water at the time of preparation of the microspheres. 6 and 7). When the peptide content is low, the initial release is not large, but polymers such as poly (lactide-co-glycolide) decompose in vivo to form lactic acid and glycolic acid, and high doses cause pain. Preparation Example 5). It is well known that it is desirable to inject a small amount of polymer by encapsulating a large amount of peptide in the biodegradable polymer.

Well-selected solvents for biodegradable polymers and LHRH homologues according to the preparation method of microspheres may be a major factor in determining the properties of the final microsphere formulation. For example, in water solvent evaporation methods such as double emulsion evaporation or single emulsion evaporation, organic solvents having this property, i.e., methylene chloride, ethyl acetate, have to form an emulsion in the aqueous phase. Solvents such as and the like are often used. In addition, in the solvent evaporation method in the aqueous phase, the biodegradable polymer solution must have a certain viscosity to increase the encapsulation rate of the peptide drug, so that the polymer solution should be above a certain concentration (for example, 20% (w / w) or more). It is difficult to dissolve the LHRH homologue with the biodegradable polymer solution and to control the size of the final microspheres. On the other hand, when the microspheres are prepared by spray drying, when the LHRH homologue is dispersed in a powder state in which the biodegradable polymer is not dissolved, the initial release amount from the final microsphere formulation becomes too large. Will occur. Therefore, the first part of the process of preparing microspheres by spray drying is the process of uniformly dissolving the LHRH homologue and biodegradable polymer into a single phase using a suitable solvent or auxiliary solvent.

The present inventors use methylene chloride, acetonitrile, ethyl acetate, ethyl formate, acetic acid, formic acid, etc. to dissolve the biodegradable polymer, and as an auxiliary solvent, the LHRH homolog can be dissolved while mixing well with the above solvent. Using methanol, the optimum composition ratio of the solvent and mixed solvent capable of dissolving the LHRH homologue and the biodegradable polymer in a single phase was investigated. As a result, as shown in Table 1 and Table 2, in the case of acetic acid or formic acid, LHRH homologue and biodegradable polymer could be uniformly dissolved in a single phase even with a single solvent, and the ratio of methanol as a cosolvent was 30% (w / w). It was confirmed that the biodegradable polymer is precipitated when exceeding). In the case of methylene chloride, it was confirmed that the LHRH homologue and the biodegradable polymer can be dissolved in a single phase only when the ratio of the cosolvent methanol is appropriate. That is, when the ratio of methanol was less than 10% (w / w), the LHRH homologue did not dissolve, and when the ratio of methanol exceeded 30% (w / w), it was confirmed that a biodegradable polymer was precipitated (Test Example 2).

Therefore, acetic acid, formic acid, or a mixture thereof is used as a solvent to dissolve the LHRH homologue and biodegradable polymer in the preparation of the LHRH homologue-containing sustained-release microparticles of the present invention. For the purposes of the present invention, acetic acid used to dissolve LHRH homologues and biodegradable polymers means no glacial acetic acid since no water is used to prepare the sustained release particulates. The solvent of acetic acid, formic acid or mixtures thereof may further contain up to 30% (w / w) methanol. Preferably, a solvent further comprising 20% (w / w) or less methanol can be used.

In addition, the present inventors have a great influence on the formation of the particles during spray drying of the concentration of the polymer and the composition and ratio of the mixed solvent used to dissolve the LHRH homologue and the biodegradable polymer when spray drying the mixture of the LHRH homologue and the biodegradable polymer. It was confirmed. If the concentration of the polymer is too thick, 20% (w / w) or more, the spherical particulate formulation is not easily formed only by adjusting the flow rate, the amount of air and the temperature of the mixed solution. For example, as described above, when the ratio of methylene chloride and methanol is appropriate, the LHRH homologue and the biodegradable polymer are homogeneously dissolved in a single phase. In the case of spray drying, the ratio of methanol, which is a nonsolvent to the instantaneous instantaneous polymer, increases in the state of evaporation of methylene chloride first, and thus the spherical particulates do not form as the polymer is continuously sprayed with reduced solubility. It is explained that it is dried in the form of a thread without it. In contrast, when methanol was mixed with acetic acid or formic acid, microspheres were well formed by spray drying, and PLGA microspheres were formed well even when the content of LHRH homologue was high up to 25% (w / w). However, when the content of LHRH homologue is more than 25%, the initial release amount was excessively increased (Test Example 1).

Therefore, the LHRH homolog can be contained in an amount of preferably 0.1 to 25% (w / w) relative to the biodegradable polymer in the preparation of the LHRH homolog-containing sustained-release microparticles of the present invention. More preferably, the LHRH homologue may be contained in an amount of 2 to 15% (w / w). In addition, the biodegradable polymer may be preferably added in an amount of 0.5 to 20% (w / w) to the solvent during the dissolution process in preparing the LHRH homolog-containing sustained-release microparticles of the present invention. More preferably, the biodegradable polymer may be added in an amount of 3.5 to 15% (w / w).

The second step of the manufacturing process of LHRH homologue-containing biodegradable polymer microspheres according to the present invention, after spray-drying a homogeneous solution of LHRH homologue and biodegradable polymer at a temperature of 60 to 90 ℃ and a compressed air amount of 300 to 500 NL / h Drying under reduced pressure. In particular, the spray drying temperature is preferably 60 to 85 ° C.

At low spray temperatures, eg below 50 ° C. and high compressed air volumes, eg above 800 NL / h, too small microspheres are formed, resulting in an increase in the surface area of the formulation itself resulting in high initial release and a desired duration in vivo due to particle size. The drug is released within a shorter period of time (Comparative Preparation Example 8). In addition, when spray drying at a low spray temperature, for example at 50 ° C. while applying a conventional compressed air amount, for example 450 NL / h, the solvent, for example acetic acid, is sufficiently removed due to the low drying temperature during spray drying. Since the microspheres are entangled and have a low pH that can cause pain when injected, they should be dried for a long time and can be dried with distilled water and lyophilized to shorten the drying time. 24 hours) drug loading is lowered to 65%.

Above, the LHRH homologue-containing sustained-release particle preparation method of the present invention is a simple process including only dissolving and spray-drying the LHRH homologue and biodegradable polymer, the formulation prepared therefrom is uniform as shown in Figure 1a It showed a size distribution, exhibited adequate injection pH with no residual residual toxic solvents, and a low initial release, which was characterized by an excellent sustained release formulation that maintained an effective titer for a long period of time, eg, over 49 days.

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

<Example>

<Test Example 1> Solubility test and spray drying test for each solvent of biodegradable polymer

Methylene chloride, acetonitrile, ethyl acetate to prepare microspheres by spray drying using PLGA (RG503H, Boehringer Ingelheim) with a molecular weight of 33,000 with a composition ratio of lactide and glycolide as a biodegradable polymer Solubility in, ethyl formate, acetic acid, formic acid, etc. was investigated.

Biodegradable polymers in all solvents were dissolved even at concentrations of 20% (w / w) or higher, but at 20% (w / w) or higher, the microspheres did not form well due to the form of yarn during spray drying.

Test Example 2 Solubility Test of LHRH Homolog and Biodegradable Polymer

The solubility of LHRH homologue tryptorelin was tested at concentrations below 20% (w / w) of polymer concentrations that readily produced microspheres during spray drying. At this time, the concentration of the polymer was fixed at 15% (w / w) and 5% (w / w) and the content of tryptorelin was fixed at 15% (w / w), and methanol was used as a cosolvent. . Table 1 and Table 2 show the solubility of tryptorelin and polymers according to the ratio of cosolvents when the concentrations of the polymer were 15% (w / w) and 5% (w / w).

As shown in the table above, in the case of acetic acid and formic acid, tryptorelin and 5% or 15% polymers were well dissolved even in the absence of methanol as an auxiliary solvent, and when the concentration of methanol was 30% or less, tryptorelin and 5% Or all 15% polymers were well dissolved.

Preparation Example 1 Tryptorelin-containing RG503H microspheres

To the polymer RG503H, the content of tryptorelin was added to 2% by weight, and acetic acid was added to dissolve homogeneously to make a final solution such that the concentration of the polymer was 5% (w / w). This solution was fed to a spray dryer (Buchi-191) at a flow rate of 2.5 ml / min and the amount of sprayed air was 400 NL / h to prepare microspheres containing tryptorelin. At this time, the temperature of the drying air flowing into the spray dryer was 65 ℃ and the average diameter of the obtained microspheres was 6.2 ㎛. Acetic acid remaining in the obtained microspheres was removed by drying for 12 hours under a pressure of 0.931 to 1.33 Pa (7 to 10 mTorr).

Preparation Example 2 Tryptorelin-containing RG503H microspheres

To the polymer RG503H, tryptorelin was added in an amount of 2% by weight, and then nitric acid containing 20% (w / w) of methanol was added to dissolve homogeneously, resulting in a final solution of 5% (w / w). made. This solution was fed to a spray dryer (Buchi-191) at a flow rate of 2.5 ml / min and the amount of sprayed air was 450 NL / h to prepare microspheres containing tryptorelin. At this time, the temperature of the drying air flowing into the spray dryer was 65 ℃ and the average diameter of the obtained microspheres was 6.4 ㎛. Acetic acid and methanol remaining in the obtained microspheres were removed by drying for 12 hours under a pressure of 0.931 to 1.33 Pa (7 to 10 mTorr).

Preparation Example 3 Tryptorelin-containing RG503 Microspheres

To the polymer RG503 (50:50, molecular weight 38,000) was added so that the content of tryptorelin is 2% by weight, and then homogeneously dissolved by adding acetic acid to make a final solution so that the concentration of the polymer is 5% (w / w). This solution was supplied to the spray dryer (Buchi-191) at a flow rate of 3 ml / min, and the amount of sprayed air was 480 NL / h to prepare microspheres containing tryptorelin. At this time, the temperature of the drying air flowing into the spray dryer was 65 ℃ and the average diameter of the obtained microspheres was 6.7 ㎛. Acetic acid remaining in the obtained microspheres was removed by drying for 12 hours under a pressure of 0.931 to 1.33 Pa (7 to 10 mTorr).

Preparation Example 4 Tryptorelin-containing RG503H microspheres

To the polymer RG503H, the content of tryptorelin was added to 3% by weight, and acetic acid was added to dissolve homogeneously to make a final solution such that the concentration of the polymer was 5% (w / w). This solution was supplied to the spray dryer (Buchi-191) at a flow rate of 3 ml / min, and the amount of sprayed air was 400 NL / h to prepare microspheres containing tryptorelin. At this time, the temperature of the drying air flowing into the spray dryer was 80 ℃ and the average diameter of the obtained microspheres was 6.4 ㎛. Acetic acid remaining in the obtained microspheres was removed by drying for 12 hours under a pressure of 0.931 to 1.33 Pa (7 to 10 mTorr).

Preparation Example 5 RG502 microspheres containing tryptorelin

To the polymer RG502 (50:50, molecular weight 14,500) was added so that the content of tryptorelin 3% by weight, and then homogeneously dissolved by adding acetic acid to make a final solution so that the concentration of the polymer is 5% (w / w). This solution was supplied to the spray dryer (Buchi-191) at a flow rate of 3 ml / min and the amount of sprayed air was 350 NL / h to prepare microspheres containing tryptorelin. At this time, the temperature of the drying air flowing into the spray dryer was 80 ℃ and the average diameter of the obtained microspheres was 6.6 ㎛. Acetic acid remaining in the obtained microspheres was removed by drying for 12 hours under a pressure of 0.931 to 1.33 Pa (7 to 10 mTorr).

Preparation Example 6 RG504 Microspheres Containing Tryptorelin

To the polymer RG504 (50:50, molecular weight 55,000) was added so that the content of tryptorelin to 3% by weight and then homogeneously dissolved by adding acetic acid to make a final solution so that the concentration of the polymer is 5% (w / w). This solution was supplied to the spray dryer (Buchi-191) at a flow rate of 3 ml / min, and the amount of sprayed air was 480 NL / h to prepare microspheres containing tryptorelin. At this time, the temperature of the drying air flowing into the spray dryer was 80 ℃ and the average diameter of the obtained microspheres was 6.5 ㎛. Acetic acid remaining in the obtained microspheres was removed by drying for 12 hours under a pressure of 0.931 to 1.33 Pa (7 to 10 mTorr).

Preparation Example 7 R202H Microspheres Containing Tryptorelin

To the polymer R202H (PLA, molecular weight 17,500) was added to the content of tryptorelin to 3% by weight, and then added acetic acid to dissolve homogeneously to make a final solution so that the concentration of the polymer is 5% (w / w). This solution was supplied to the spray dryer (Buchi-191) at a flow rate of 3 ml / min, and the amount of spray air was 450 NL / h to prepare microspheres containing tryptorelin. At this time, the temperature of the drying air flowing into the spray dryer was 80 ℃ and the average diameter of the obtained microspheres was 6.3 ㎛. Acetic acid remaining in the obtained microspheres was removed by drying for 12 hours under a pressure of 0.931 to 1.33 Pa (7 to 10 mTorr).

Preparation Example 8 RG755 Microspheres Containing Tryptorelin

To the polymer RG755 (75:25, molecular weight 63,000) was added so that the content of tryptorelin 3% by weight, and then homogeneously dissolved by adding acetic acid to make a final solution so that the concentration of the polymer is 5% (w / w). This solution was supplied to the spray dryer (Buchi-191) at a flow rate of 3 ml / min, and the amount of spray air was 450 NL / h to prepare microspheres containing tryptorelin. At this time, the temperature of the drying air flowing into the spray dryer was 80 ℃ and the average diameter of the obtained microspheres was 7.0 ㎛. Acetic acid remaining in the obtained microspheres was removed by drying for 12 hours under a pressure of 0.931 to 1.33 Pa (7 to 10 mTorr).

Preparation Example 9 RG756 Microspheres Containing Tryptorelin

To the polymer RG756 (75:25, molecular weight 90,000) was added so that the content of tryptorelin 3% by weight and then homogeneously dissolved by adding acetic acid to make a final solution so that the concentration of the polymer is 5% (w / w). This solution was supplied to a spray dryer (Buchi-191) at a flow rate of 3 ml / min, and the amount of spray air was 500 NL / h to prepare microspheres containing tryptorelin. At this time, the temperature of the drying air flowing into the spray dryer was 80 ℃ and the average diameter of the obtained microspheres was 7.2 ㎛. Acetic acid remaining in the obtained microspheres was removed by drying for 12 hours under a pressure of 0.931 to 1.33 Pa (7 to 10 mTorr).

Preparation Example 10 RG503H Microspheres Containing Tryptorelin

The polymer RG503H was added so that the content of tryptorelin is 12% by weight, and then homogenized by adding acetic acid to make a final solution so that the concentration of the polymer was 5% (w / w). This solution was fed to a spray dryer (Buchi-191) at a flow rate of 2.5 ml / min and the amount of sprayed air was 450 NL / h to prepare microspheres containing tryptorelin. At this time, the temperature of the drying air flowing into the spray dryer was 65 ℃ and the average diameter of the obtained microspheres was 6.7 ㎛. Acetic acid remaining in the obtained microspheres was removed by drying for 12 hours under a pressure of 0.931 to 1.33 Pa (7 to 10 mTorr).

Preparation Example 11 RG503H Microspheres Containing Tryptorelin

The polymer RG503H was added so that the content of tryptorelin was 15% by weight, and then homogenized by adding acetic acid to make a final solution such that the concentration of the polymer was 5% (w / w). This solution was supplied to the spray dryer (Buchi-191) at a flow rate of 3 ml / min, and the amount of sprayed air was 400 NL / h to prepare microspheres containing tryptorelin. At this time, the temperature of the drying air flowing into the spray dryer was 60 ℃ and the average diameter of the obtained microspheres was 6.6 ㎛. Acetic acid remaining in the obtained microspheres was removed by drying for 12 hours under a pressure of 0.931 to 1.33 Pa (7 to 10 mTorr).

Preparation Example 12 RG503H Microspheres Containing Tryptorelin

The final solution was added to the polymer RG503H so that the content of tryptorelin was 25% by weight, and then homogenized by adding acetic acid to make the polymer concentration 3.5% (w / w). This solution was supplied to the spray dryer (Buchi-191) at a flow rate of 3 ml / min, and the amount of sprayed air was 480 NL / h to prepare microspheres containing tryptorelin. At this time, the temperature of the drying air flowing into the spray dryer was 60 ℃ and the average diameter of the obtained microspheres was 6.3 ㎛. Acetic acid remaining in the obtained microspheres was removed by drying for 12 hours under a pressure of 0.931 to 1.33 Pa (7 to 10 mTorr).

Preparation Example 13 RG502H Microspheres Containing Tryptorelin

The final solution was added to the polymer RG502H (50:50, molecular weight 8,000) so that the content of tryptorelin is 15% by weight, and then homogeneously dissolved by adding acetic acid to make the concentration of the polymer 10% (w / w). This solution was supplied to the spray dryer (Buchi-191) at a flow rate of 2.5 ml / min and the amount of sprayed air was 420 NL / h to prepare microspheres containing tryptorelin. At this time, the temperature of the drying air flowing into the spray dryer was 60 ℃ and the average diameter of the obtained microspheres was 6.6 ㎛. Acetic acid remaining in the obtained microspheres was removed by drying for 12 hours under a pressure of 0.931 to 1.33 Pa (7 to 10 mTorr).

Preparation Example 14 RG502H Microspheres Containing Tryptorelin

The polymer RG502H was added so that the content of tryptorelin was 15% by weight, and then homogenized by adding acetic acid to make a final solution such that the concentration of the polymer was 15% (w / w). This solution was fed to a spray dryer (Buchi-191) at a flow rate of 2.5 ml / min and the amount of sprayed air was 400 NL / h to prepare microspheres containing tryptorelin. At this time, the temperature of the drying air flowing into the spray dryer was 60 ℃ and the average diameter of the obtained microspheres was 6.7 ㎛. Acetic acid remaining in the obtained microspheres was removed by drying for 12 hours under a pressure of 0.931 to 1.33 Pa (7 to 10 mTorr).

Preparation Example 15 Tryprinelin-Containing RG752 Microspheres

To the polymer RG752 (75:25, molecular weight 20,000) was added so that the content of tryptorelin is 15% by weight, and then homogeneously dissolved by adding acetic acid to make a final solution so that the concentration of the polymer is 5% (w / w). This solution was fed to a spray dryer (Buchi-191) at a flow rate of 2.5 ml / min and the amount of sprayed air was 400 NL / h to prepare microspheres containing tryptorelin. At this time, the temperature of the drying air flowing into the spray dryer was 60 ℃ and the average diameter of the obtained microspheres was 6.1 ㎛. Acetic acid remaining in the obtained microspheres was removed by drying for 12 hours under a pressure of 0.931 to 1.33 Pa (7 to 10 mTorr).

Preparation Example 16 RG503H Microspheres Containing Tryptorelin

After the addition of tryptorelin content to the polymer RG503H to 2% by weight, formic acid was added to dissolve homogeneously to make a final solution so that the concentration of the polymer is 5% (w / w). This solution was supplied to the spray dryer (Buchi-191) at a flow rate of 2.5 ml / min and the amount of sprayed air was 350 NL / h to prepare microspheres containing tryptorelin. At this time, the temperature of the drying air flowing into the spray dryer was 70 ℃ and the average diameter of the obtained microspheres was 6.5 ㎛. Formic acid remaining in the obtained microspheres was removed by drying for 12 hours under a pressure of 0.931 to 1.33 Pa (7 to 10 mTorr).

Preparation Example 17 RG503H Microspheres Containing Tryptorelin

The final solution was added to the polymer RG503H so that the content of tryptorelin was 2% by weight, and then homogeneously dissolved by adding formic acid containing 20% (w / w) of methanol to obtain a polymer concentration of 5% (w / w). made. This solution was fed to a spray dryer (Buchi-191) at a flow rate of 2.5 ml / min and the amount of sprayed air was 400 NL / h to prepare microspheres containing tryptorelin. At this time, the temperature of the drying air flowing into the spray dryer was 70 ℃ and the average diameter of the obtained microspheres was 6.5 ㎛. Formic acid and methanol remaining in the obtained microspheres were removed by drying for 12 hours under a pressure of 0.931 to 1.33 Pa (7 to 10 mTorr).

Preparation Example 18 RG503H Microspheres Containing Tryptorelin

The final solution was added to the polymer RG503H so that the content of tryptorelin was 10% by weight and then homogeneously dissolved by adding formic acid to give a concentration of 5% (w / w). This solution was supplied to the spray dryer (Buchi-191) at a flow rate of 3 ml / min, and the amount of spray air was 450 NL / h to prepare microspheres containing tryptorelin. At this time, the temperature of the drying air flowing into the spray dryer was 75 ℃ and the average diameter of the obtained microspheres was 7.2 ㎛. Formic acid remaining in the obtained microspheres was removed by drying for 12 hours under a pressure of 0.931 to 1.33 Pa (7 to 10 mTorr).

Preparation Example 19 RG503H Microspheres Containing Tryptorelin

The final solution was added to the polymer RG503H so that the content of tryptorelin was 15% by weight and then homogeneously dissolved by adding formic acid to give a concentration of 5% (w / w). This solution was supplied to a spray dryer (Buchi-191) at a flow rate of 2.5 ml / min, and the amount of sprayed air was 480 NL / h to prepare microspheres containing tryptorelin. At this time, the temperature of the drying air flowing into the spray dryer was 70 ℃ and the average diameter of the obtained microspheres was 6.8 ㎛. Formic acid remaining in the obtained microspheres was removed by drying for 12 hours under a pressure of 0.931 to 1.33 Pa (7 to 10 mTorr).

Preparation Example 20 RG502H Microspheres Containing Tryptorelin

The final solution was added to the polymer RG502H so that the content of tryptorelin was 2% by weight and then homogeneously dissolved by adding formic acid to give a concentration of 5% (w / w) of the polymer. This solution was supplied to the spray dryer (Buchi-191) at a flow rate of 3 ml / min, and the amount of spray air was 450 NL / h to prepare microspheres containing tryptorelin. At this time, the temperature of the drying air flowing into the spray dryer was 70 ℃ and the average diameter of the obtained microspheres was 6.6 ㎛. Formic acid remaining in the obtained microspheres was removed by drying for 12 hours under a pressure of 0.931 to 1.33 Pa (7 to 10 mTorr).

Preparation Example 21 RG503 Microspheres Containing Tryptorelin

The content of tryptorelin was added to the polymer RG503 to 2% by weight, and formic acid was added to dissolve homogeneously to make a final solution such that the concentration of the polymer was 5% (w / w). This solution was supplied to the spray dryer (Buchi-191) at a flow rate of 2.5 ml / min and the amount of sprayed air was 350 NL / h to prepare microspheres containing tryptorelin. At this time, the temperature of the drying air flowing into the spray dryer was 75 ℃ and the average diameter of the obtained microspheres was 5.9㎛. Formic acid remaining in the obtained microspheres was removed by drying for 12 hours under a pressure of 0.931 to 1.33 Pa (7 to 10 mTorr).

Preparation Example 22 RG504 Microspheres Containing Tryptorelin

The final solution was added to the polymer RG504 so that the content of tryptorelin was 10% by weight and then homogeneously dissolved by adding formic acid to give a concentration of 5% (w / w). This solution was fed to a spray dryer (Buchi-191) at a flow rate of 2.5 ml / min and the amount of sprayed air was 400 NL / h to prepare microspheres containing tryptorelin. At this time, the temperature of the drying air flowing into the spray dryer was 75 ℃ and the average diameter of the obtained microspheres was 6.0 ㎛. Formic acid remaining in the obtained microspheres was removed by drying for 12 hours under a pressure of 0.931 to 1.33 Pa (7 to 10 mTorr).

Preparation Example 23 Tryprinelin-Containing RG752 Microspheres

The polymer RG752 was added so that the content of tryptorelin was 2% by weight, and formic acid was added to dissolve homogeneously to make a final solution such that the concentration of the polymer was 5% (w / w). This solution was fed to a spray dryer (Buchi-191) at a flow rate of 2.5 ml / min and the amount of sprayed air was 450 NL / h to prepare microspheres containing tryptorelin. At this time, the temperature of the drying air flowing into the spray dryer was 70 ℃ and the average diameter of the obtained microspheres was 6.3 ㎛. Formic acid remaining in the obtained microspheres was removed by drying for 12 hours under a pressure of 0.931 to 1.33 Pa (7 to 10 mTorr).

Preparation Example 1 Tryptorelin-containing RG503H microspheres

The polymer RG503H was added so that the content of tryptorelin was 12% by weight, and then homogenized by adding acetic acid to make a final solution so that the concentration of the polymer was 20% (w / w). This solution was supplied to the spray dryer (Buchi-191) at a flow rate of 2.5 ml / min and the amount of sprayed air was 400 NL / h to prepare microspheres containing tryptorelin. At this time, the temperature of the drying air flowing into the spray dryer was 65 ℃.

Unlike in Example 10, the polymer concentration was too thick, and microspheres were not formed well.

Preparation Example 2 Tryptorelin-containing RG503H microspheres

To the polymer RG503H, tryptorelin was added in an amount of 15% by weight, and then methylene chloride was added so that the polymer concentration was 5% (w / w), and methanol was added in 10% (w / w) of methylene chloride. The final solution was prepared by dissolving both polymer and tryptorelin. This solution was fed to a spray dryer (Buchi-191) at a flow rate of 2.5 ml / min and the amount of sprayed air was 450 NL / h to prepare microspheres containing tryptorelin. At this time, the temperature of the drying air flowing into the spray dryer was 70 ℃.

However, the microspheres did not form well during spray drying and became a thread. This is thought to be due to the difference in volatilization rates of methylene chloride and methanol.

Preparation Example 3 Tryptorelin-containing RG503H microspheres

To the polymer RG503H, tryptorelin content is added to 15% by weight, and acetonitrile is added so that the polymer concentration is 5% (w / w) and methanol is 60% (w / w) of acetonitrile. The final solution was prepared by dissolving both polymer and tryptorelin. This solution was fed to a spray dryer (Buchi-191) at a flow rate of 2.5 ml / min and the amount of sprayed air was 400 NL / h to prepare microspheres containing tryptorelin. At this time, the temperature of the drying air flowing into the spray dryer was 70 ℃.

However, the microspheres did not form well during spray drying and became a thread.

Preparation Example 4 Tryptorelin-containing RG502H microspheres

To the polymer RG502H, tryptorelin was added in an amount of 15% by weight, and then methylene chloride was added so that the polymer concentration was 5% (w / w), and methanol was added in 10% (w / w) of methylene chloride. The final solution was prepared by dissolving both polymer and tryptorelin. This solution was fed to a spray dryer (Buchi-191) at a flow rate of 2.5 ml / min and the amount of sprayed air was 450 NL / h to prepare microspheres containing tryptorelin. At this time, the temperature of the drying air flowing into the spray dryer was 60 ℃.

However, the microspheres did not form well during spray drying and became a thread.

Preparation Example 5 Tryptorelin-containing RG503H microspheres

After dissolving 0.06 g of tryptorelin in 2.0 g of distilled water, the solution was mixed with a solution of 1.94 g of polymer RG503H (IV = 0.4 dl / g) in 40 g of acetic acid, and then supplied to a spray dryer to prepare microspheres. . At this time, the drying temperature was 50 ℃, the spray air amount was 800 NL / h.

Preparation Example 6 RG503H Microspheres Containing Tryptorelin

 Preparation Comparative Example 5 A microsphere was prepared using 0.24 g of tryptorelin and 1.76 g of RG503H in the same manner as in Example 5.

Preparation Example 7 RG503H Microspheres Containing Tryptorelin

Preparation Comparative Microspheres were prepared in the same manner as in Example 5, using 0.3 g of tryptorelin and 1.7 g of RG503H.

Preparation Example 8 Tryptorelin-containing RG503H microspheres

Microparticles were prepared by dissolving 0.3 g of tryptorelin and 1.7 g of RG 503H in 40 g of acetic acid and then supplying it to a spray dryer. At this time, the drying temperature was 50 ℃, the spray air amount was 800 NL / h.

Preparation Example 9 RG503H Microspheres Containing Tryptorelin

Microparticles were prepared by dissolving 0.24 g of tryptorelin and 1.76 g of RG503H in 40 g of acetic acid and then supplying it to a spray dryer. At this time, the drying temperature was 50 ℃, the spray air amount was 450 NL / h.

Experimental Example 3 Electron Microscopy Observation of Tryptorelin-Containing Formulations

Electron micrographs of the microspheres prepared according to Preparation Example 1, Preparation Comparative Example 5, and Preparation Comparative Example 9 are shown in FIGS. 1A, 1B, and 1C, respectively.

Formulations prepared by the method of Preparation Example 1 showed a uniform size distribution, but formulations made by the method of Preparation Example 5 had various particle sizes, and a large number of small microspheres were caused by the amount of compressed air when sprayed. Formed. The formulation of Comparative Example 9 exhibited a phenomenon that the particles were not sufficiently dried and entangled with each other.

Test Example 4 Residual Acetic Acid and Drug Loading Rate of Tryptorelin-Containing Formulations

Preparation of the formulation after secondary drying (at the same time under reduced pressure drying) for a certain time to remove residual acetic acid after spray drying of the microspheres prepared according to Preparation Examples 4, 10 and 11, and Comparative Examples 5 to 9 A pH aqueous solution suspension was made to measure the pH of the solution. In addition, the content of the drug in the formulation was measured and the encapsulation rate is shown in Table 3.

The encapsulation rate of the drug was determined by dissolving a certain amount of the formulation in dimethylsulfoxide (DMSO), diluting it with a mobile phase, and then quantifying the drug. Quantitative conditions were used for a C18 (100 mm x mm i.d., 3 μm) column, mobile phase was 26% acetonitrile (ACN), 0.1% trifluoroacetate (TFA) aqueous solution and detected at UV 220 nm.

Formulation Decompression drying time (h) PH of 1% aqueous suspension Theoretical Peptide Content (%) Actual Peptide Content (%) Inclusion Rate (%) Example 4 12 6.9 3 2.94 98 Example 10 12 6.7 12 11.89 99.1 Example 11 12 6.8 15 14.54 96.9 Comparative Example 5 12 6.3 3 2.79 93 Comparative Example 6 12 5.8 12 11.52 96 Comparative Example 7 12 6.3 15 14.7 98 Comparative Example 8 12 6.1 15 13.95 93 Comparative Example 9 12 4.5 12 11.16 93

As can be seen from the above table, Comparative Example 9 exhibited a low pH that may cause pain upon injection. At least 48 hours of drying time were required to achieve a suitable injection pH of 6.0 to 7.5. In order to shorten the drying time, the drying time was reduced to 24 hours by washing with distilled water and lyophilizing, but the drug loading rate was reduced to 65%.

Experimental Example 5 Quantification of Initial Release of Tryptorelin-Containing Formulations

Microspheres prepared according to Preparation Examples 4, 10 and 11, and Comparative Examples 5 to 9 were each suspended in a phosphate buffer of pH 7.4 at a concentration of 10 mg / ml and released as a buffer for 24 hours while maintaining 37 ° C. The amount of tryptorelin was quantified. The characteristics of each formulation, peptide content, and release over 24 hours are summarized in Table 4.

Quantitative conditions were performed using a C18 (100 mm x mm i.d., 3 μm) column, the mobile phase was 26% acetonitrile (ACN), 0.1% trifluoroacetate (TFA) aqueous solution and detected at UV 220 nm.

Formulation Polymer Peptide Content (%) Water / acetic acid (%) Spray Drying Temperature (℃) Atomized air flow rate (NL / h) Emission amount (24h,%) Example 4 503H 3 0 80 400 0.62 Example 10 503H 12 0 65 450 10 Example 11 503H 15 0 60 400 9.98 Comparative Example 5 503H 3 4.8 50 800 1.53 Comparative Example 6 503H 12 4.8 50 800 32 Comparative Example 7 503H 15 4.8 50 800 40 Comparative Example 8 503H 15 0 50 800 35 Comparative Example 9 503H 12 0 50 450 42

As can be seen from the above table, in Comparative Examples 5 to 7 using water to dissolve peptides, the initial release amount increased significantly as the peptide content was increased. In Comparative Preparation Example 8, in which no water was used to dissolve the peptide, too small microspheres formed under low spray temperature (50 ° C.) and high spray flow rate (800 NL / h) resulting in an increase in the surface area of the formulation itself. Initial release was high. In addition, although no water was used to dissolve the peptide, the initial release amount was also high in the preparation of Comparative Example 9 which prepared the microspheres at low spray temperature (50 ° C.) and flow rate (450 NL / h).

Preparation Example 24 Leukrolide Containing RG503H Microspheres

The content of leuprolide was added to the polymer RG503H so that the content of leuprolide was 15% by weight, followed by the addition of acetic acid to dissolve homogeneously to make a final solution such that the polymer concentration was 5% (w / w). This solution was supplied to a spray dryer (Buchi-191) at a flow rate of 2.5 ml / min, and the amount of sprayed air was 480 NL / h to prepare microspheres containing leuprolide. At this time, the temperature of the drying air flowing into the spray dryer was 60 ℃ and the average diameter of the obtained microspheres was 5.8㎛. Acetic acid remaining in the obtained microspheres was removed by drying for 12 hours under a pressure of 0.931 to 1.33 Pa (7 to 10 mTorr).

Preparation Example 25 Rupprolide-Containing RG503 Microspheres

To the polymer RG503, the content of leuprolide was added to 15% by weight, followed by the addition of acetic acid to dissolve homogeneously to make a final solution such that the concentration of the polymer was 5% (w / w). This solution was supplied to a spray dryer (Buchi-191) at a flow rate of 2.5 ml / min, and the amount of sprayed air was 350 NL / h to prepare microspheres containing leuprolide. At this time, the temperature of the drying air flowing into the spray dryer was 80 ℃ and the average diameter of the obtained microspheres was 6.3 ㎛. Acetic acid remaining in the obtained microspheres was removed by drying for 12 hours under a pressure of 0.931 to 1.33 Pa (7 to 10 mTorr).

Preparation Example 26 Rupprolide-Containing RG502H Microspheres

To the polymer RG502H, the content of leuprolide was added to 15% by weight, followed by the addition of acetic acid to dissolve homogeneously to make a final solution such that the concentration of the polymer was 5% (w / w). This solution was supplied to the spray dryer (Buchi-191) at a flow rate of 2.5 ml / min, and the amount of sprayed air was 450 NL / h to prepare microspheres containing leuprolide. At this time, the temperature of the drying air flowing into the spray dryer was 65 ℃ and the average diameter of the obtained microspheres was 5.9㎛. Acetic acid remaining in the obtained microspheres was removed by drying for 12 hours under a pressure of 0.931 to 1.33 Pa (7 to 10 mTorr).

Preparation Example 27 Rupprolide-Containing RG752 Microspheres

The final solution was added to the polymer RG752 so that the content of leuprolide was 15% by weight, and then homogeneously dissolved by adding acetic acid so that the concentration of the polymer was 5% (w / w). This solution was fed to a spray dryer (Buchi-191) at a flow rate of 2.5 ml / min and the amount of sprayed air was 400 NL / h to prepare microspheres containing leuprolide. At this time, the temperature of the drying air flowing into the spray dryer was 70 ℃ and the average diameter of the obtained microspheres was 6.2 ㎛. Acetic acid remaining in the obtained microspheres was removed by drying for 12 hours under a pressure of 0.931 to 1.33 Pa (7 to 10 mTorr).

Preparation Example 28 leuprolide-containing RG503H microspheres

To the polymer RG503H, the content of leuprolide was added to 5% by weight, and formic acid was added to dissolve homogeneously to make a final solution such that the concentration of the polymer was 5% (w / w). This solution was supplied to the spray dryer (Buchi-191) at a flow rate of 3 ml / min, and the amount of sprayed air was 450 NL / h to prepare microspheres containing leuprolide. At this time, the temperature of the drying air flowing into the spray dryer was 70 ℃ and the average diameter of the obtained microspheres was 6.7 ㎛. Formic acid remaining in the obtained microspheres was removed by drying for 12 hours under a pressure of 0.931 to 1.33 Pa (7 to 10 mTorr).

Preparation Example 29 RG503H Microspheres Containing Goserelin

The content of goserelin was added to the polymer RG503H to 15% by weight, and acetic acid was added to dissolve it homogeneously to make a final solution such that the concentration of the polymer was 5% (w / w). This solution was supplied to the spray dryer (Buchi-191) at a flow rate of 2.5 ml / min, and the amount of sprayed air was 480 NL / h to prepare microspheres containing goserelin. At this time, the temperature of the drying air flowing into the spray dryer was 60 ℃ and the average diameter of the obtained microspheres was 6.7 ㎛. Acetic acid remaining in the obtained microspheres was removed by drying for 12 hours under a pressure of 0.931 to 1.33 Pa (7 to 10 mTorr).

Preparation Example 30 Goserelin-containing RG503H microspheres

The content of goserelin was added to the polymer RG503H to 5% by weight, and acetic acid was added to dissolve it homogeneously to make a final solution such that the concentration of the polymer was 5% (w / w). This solution was supplied to the spray dryer (Buchi-191) at a flow rate of 2.5 ml / min and the amount of sprayed air was 450 NL / h to prepare microspheres containing goserelin. At this time, the temperature of the drying air flowing into the spray dryer was 60 ℃ and the average diameter of the obtained microspheres was 6.2 ㎛. Acetic acid remaining in the obtained microspheres was removed by drying for 12 hours under a pressure of 0.931 to 1.33 Pa (7 to 10 mTorr).

Preparation Example 31 Goserelin-Containing RG502H Microspheres

The content of goserelin was added to the polymer RG502H to 15% by weight, and acetic acid was added to dissolve it homogeneously to make a final solution such that the concentration of the polymer was 10% (w / w). This solution was supplied to the spray dryer (Buchi-191) at a flow rate of 2.5 ml / min, and the amount of sprayed air was 400 NL / h to prepare microspheres containing goserelin. At this time, the temperature of the drying air flowing into the spray dryer was 85 ℃ and the average diameter of the obtained microspheres was 6.3 ㎛. Acetic acid remaining in the obtained microspheres was removed by drying for 12 hours under a pressure of 0.931 to 1.33 Pa (7 to 10 mTorr).

Production Example 32 Goserelin-containing RG752 microspheres

The content of goserelin was added to the polymer RG752 to 10% by weight, and acetic acid was added to dissolve homogeneously to make a final solution such that the concentration of the polymer was 5% (w / w). This solution was supplied to the spray dryer (Buchi-191) at a flow rate of 2.5 ml / min and the amount of sprayed air was 450 NL / h to prepare microspheres containing goserelin. At this time, the temperature of the drying air flowing into the spray dryer was 70 ℃ and the average diameter of the obtained microspheres was 6.8 ㎛. Acetic acid remaining in the obtained microspheres was removed by drying for 12 hours under a pressure of 0.931 to 1.33 Pa (7 to 10 mTorr).

Preparation Example 33 RG503H Microspheres Containing Goserelin

The content of goserelin was added to the polymer RG503H to 5% by weight, and formic acid was added to dissolve homogeneously to make a final solution such that the concentration of the polymer was 5% (w / w). This solution was supplied to the spray dryer (Buchi-191) at a flow rate of 2.5 ml / min, and the amount of sprayed air was 400 NL / h to prepare microspheres containing goserelin. At this time, the temperature of the drying air flowing into the spray dryer was 70 ℃ and the average diameter of the obtained microspheres was 6.2 ㎛. Formic acid remaining in the obtained microspheres was removed by drying for 12 hours under a pressure of 0.931 to 1.33 Pa (7 to 10 mTorr).

<Test Example 6>

Tryptorelin-containing PLGA microspheres prepared under the conditions of Example 1 were suspended in suspension (0.5% CMC, 0.1% Tween20, 0.9% NaCl), followed by male S.D. Rats (n = 5, 250 ± 20 g) were injected once subcutaneously at a dose of 3 mg / kg (tryptorelin acetate) under ether anesthesia. Blood was collected from the caudal vein prior to drug administration, for 6 hours, 1 day, 4 days, and thereafter at 3-4 day intervals for up to 48 days. The collected blood was placed in a serum separation tube and allowed to stand at room temperature for 30 minutes, followed by centrifugation at 4 ° C and 3,600 rpm for 5 minutes, and serum was collected and stored at -70 ° C. Testosterone concentrations in serum were quantified using an Enzyme Immunoassay Kit (EIA Kit, DSL-10-4000, Diagnostic Systems Laboratories, Inc., Webster, Texas, USA).

The results are shown in FIG. Tryptorelin drug continued to be released after 48 days.

The biodegradable polymer microspheres containing the LHRH homolog prepared according to the present invention have no problem of residual toxic solvents exhibited in the conventional manufacturing method, and the size of the microspheres is uniform and can be prepared in a suitable size for easy injection. The microspheres prepared according to the present invention can be usefully used for the treatment of diseases such as prostate cancer, endometriosis, myoma and the like by continuously releasing LHRH homologues in effective concentrations for a period of time when administered in the body.

Claims (9)

Dissolving the LHRH homologue and biodegradable polymer in acetic acid, formic acid or mixtures thereof without the use of water; Spray drying it to a temperature of 60 to 90 ° C. and a compressed air amount of 300 to 500 NL / h; And drying at a reduced pressure of 0.931 to 1.33 Pa (7 to 10 mTorr) for 10 to 12 hours, wherein the LHRH homologue-containing sustained-release microspheres are prepared. The method of claim 1, wherein the biodegradable polymer is polylactide, polyglycolide, poly (lactide-co-glycolide), polyorthoester, polyanhydride, polyamino acid, polyhydroxybutyric acid, polycapro A process for producing LHRH homologue-containing sustained-release microspheres, characterized in that at least one selected from the group consisting of lactones and polyalkylcarbonates. The method of claim 1, further comprising up to 30% (w / w) methanol in acetic acid, formic acid, or mixtures thereof. The method of claim 1, wherein the content of the LHRH homologue to the biodegradable polymer is 0.1 to 25% (w / w). The method for producing LHRH homologue-containing sustained-release microspheres according to claim 1, wherein the biodegradable polymer is added to the solvent during the dissolution step in an amount of 0.5 to 20% (w / w). 6. The method of claim 1, wherein the LHRH homologue is selected from the group consisting of LHRH agonists and LHRH antagonists. 7. 7. The method for producing LHRH homologue-containing sustained-release microspheres according to claim 6, wherein the LHRH agonist is selected from tryptorelin, leuprolide, goserelin, naparelin, buserelin and salts thereof. 7. The method for producing LHRH homologue-containing sustained-release microspheres according to claim 6, wherein the LHRH antagonist is selected from antide, algitide, righttide, serorelix and salts thereof. The method of claim 7 or 8, wherein the LHRH agonist or LHRH antagonist is in acetate form.

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