SUSTAINED-RELEASE INJECTION COMPRISING DESLORELIN, AND PREPARATION METHOD THEREFOR

Abstract

The present disclosure relates to a sustained-release injection formulation comprising biodegradable polymer microspheres containing deslorelin as an active ingredient, and a preparation method therefor. The sustained-release microsphere injection containing deslorelin, according to the present disclosure, has good administrability and enables, with a single administration, a deslorelin drug to be maintained at an effective concentration for 6 months without rapid temporary release into the blood of an animal.

Claims

1. A sustained-release microsphere containing deslorelin, comprising 5 to 25% by weight of deslorelin based on the total weight of the microsphere, and two or more polymers selected from the group consisting of poly(lactide-co-glycolide) and polylactide polymers.

2. The sustained-release microsphere containing deslorelin according to claim 1, wherein two or more polymers selected from the group consisting of poly(lactide-co-glycolide) and polylactide polymers of the sustained-release microsphere containing deslorelin are contained in one type of microsphere or each independently contained in two or more types of microspheres.

3. The sustained-release microsphere containing deslorelin according to claim 1, wherein when the sustained-release microsphere containing deslorelin is administered to an animal via a subcutaneous or intramuscular route, more than 85% of deslorelin in the microsphere is released for 6 months.

4. The sustained-release microsphere containing deslorelin according to claim 1, wherein an intrinsic viscosity of the poly(lactide-co-glycolide) or polylactide is 0.16 to 1.2 dL/g.

5. The sustained-release microsphere containing deslorelin according to claim 1, wherein an average particle size of the microsphere is 10 μm to 100 μm.

6. The sustained-release microsphere containing deslorelin according to claim 1, wherein a span value of the microsphere is 1.2 or less.

7. The sustained-release microsphere containing deslorelin according to claim 1, wherein a recovery rate upon injection of the microsphere is 80 to 100% when measured using a 1 mL syringe equipped with a 25G injection needle.

8. The sustained-release microsphere containing deslorelin according to claim 3, wherein the microsphere releases 10% or more of the drug within 1 month after administration, and 85% or more of the drug within 6 months after administration.

9. A method for preparing a sustained-release microsphere containing deslorelin, the method comprising the steps of: (a) dissolving deslorelin and two or more polymers selected from the group consisting of poly(lactide-co-glycolide) and polylactide polymers in a mixed organic solvent to prepare a deslorelin-polymer solution (dispersed phase); (b) adding the deslorelin-polymer solution prepared in step (a) to an aqueous solution phase (continuous phase) containing a surfactant to prepare a dispersed phase in an emulsion state; (c) extracting the organic solvent from the dispersed phase in the emulsion state phase prepared in step (b) into a continuous phase and evaporating the organic solvent to form a microsphere; and (d) recovering the microsphere from the continuous phase of step (c) to prepare a deslorelin microsphere.

10. A method for preparing sustained release microspheres including deslorelin, comprising the steps of: preparing two or more different preliminary microspheres using two or more different poly(lactide-co-glycolide) or polylactide polymer and deslorelin; and mixing the two or more different preliminary microspheres, wherein the step of preparing the two or more different preliminary microspheres comprises the following steps: (a) dissolving deslorelin and poly(lactide-co-glycolide) or polylactide polymer in a mixed organic solvent to prepare a deslorelin-polymer solution (dispersed phase); (b) adding the deslorelin-polymer solution prepared in step (a) to an aqueous solution phase (continuous phase) containing a surfactant to prepare a dispersed phase in an emulsion state; (c) extracting the organic solvent from the dispersed phase in the emulsion state prepared in step (b) into a continuous phase and evaporating the organic solvent to form a microsphere; and (d) recovering the microsphere from the continuous phase of step (c) to prepare the preliminary microsphere.

11-12. (canceled)

13. The method according to claim 9, wherein an intrinsic viscosity of the poly(lactide-co-glycolide) or polylactide is 0.16 to 1.2 dL/g.

14. (canceled)

15. The method according to claim 9, wherein the continuous phase of step (b) is one or more mixed solvents selected from the group consisting of water, or water and methyl alcohol, ethyl alcohol, propyl alcohol and ethyl acetate.

16-17. (canceled)

18. The method according to claim 9, wherein a recovery rate upon injection of the prepared sustained-release microsphere containing deslorelin is 80 to 100% when measured using a 1 mL syringe equipped with a 25G injection needle.

19. The method according to claim 9, wherein when the prepared sustained-release microsphere containing deslorelin is administered to an animal via a subcutaneous or intramuscular route, 85% or more of deslorelin in the microsphere is released for 6 months.

20. (canceled)

21. The method according to claim 10, wherein the mixed organic solvent of step (a) is two or more mixed solvents selected from the group consisting of dichloromethane, chloroform, ethyl acetate, methyl ethyl ketone, acetone, acetonitrile, dimethyl sulfoxide, dimethylformamide, N-methylpyrrolidone, acetic acid, methyl alcohol, ethyl alcohol, propyl alcohol and benzyl alcohol.

22. The method according to claim 10, wherein an intrinsic viscosity of the poly(lactide-co-glycolide) or polylactide is 0.16 to 1.2 dL/g.

23. The method according to claim 10, wherein the continuous phase of step (b) is one or more mixed solvents selected from the group consisting of water, or water and methyl alcohol, ethyl alcohol, propyl alcohol and ethyl acetate.

24. The method according to claim 10, wherein a recovery rate upon injection of the prepared sustained-release microsphere containing deslorelin is 80 to 100% when measured using a 1 mL syringe equipped with a 25G injection needle.

25. The method according to claim 10, wherein when the prepared sustained-release microsphere containing deslorelin is administered to an animal via a subcutaneous or intramuscular route, 85% or more of deslorelin in the microsphere is released for 6 months.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0052] FIG. 1 is a photograph of a sustained-release microsphere injection formulation containing deslorelin prepared in Example 3 when observed with an electron microscope.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0053] Hereinafter, the present disclosure will be described in more detail by way of examples. However, these examples are for illustrative purposes only, and the scope of the present disclosure is not limited thereto.

Comparative Example

Comparative Example 1: Preparation 1 of a Sustained-Release Microsphere Injection Formulation Containing Deslorelin Prepared Using a Single Polymer

[0054] The dispersed phase was prepared by mixing 9 g of biocompatible polymer Resomer R203H (manufacturer: Evonik, Germany) and 1 g of deslorelin acetate (manufacturer: Chengdu Kaijie Biopharm Co., Ltd, China) with 32.14 g of dichloromethane (manufacturer: J. T. Baker, USA) and 9.59 ml of N-methylpyrrolidone (manufacturer: JUNSEI, Japan). The dispersed phase was stirred for at least 30 minutes and sufficiently dissolved before use. As the continuous phase, a 1 wt % polyvinyl alcohol aqueous solution (viscosity: 4.8 to 5.8 mPa.Math.s) was used. The dispersed phase was connected and injected to an emulsififcation apparatus equipped with a porous membrane (pore size: 20 μm), and at the same time, the prepared dispersed phase was injected to form an emulsion. The emulsion was stirred at 40° C. at 200 rpm for 3 hours to remove the organic solvent. When the removal of the organic solvent was completed, the temperature of the microsphere suspension was set to 25° C., and the microsphere suspension was repeatedly washed with distilled water several times to remove residual polyvinyl alcohol, and then lyophilized, thereby obtaining a microsphere containing deslorelin.

Comparative Example 1-1: Preparation 2 of a Sustained-Release Microsphere Injection Formulation Containing Deslorelin Prepared Using a Single Polymer

[0055] The dispersed phase was prepared by mixing 8.5 g of biocompatible polymer Resomer R203H (manufacturer: Evonik, Germany) and 1.5 g of deslorelin acetate (manufacturer: Chengdu Kaijie Biopharm Co., Ltd, China) with 30.36 g of dichloromethane (manufacturer: J. T. Baker, USA) and 9.06 ml of N-methylpyrrolidone (manufacturer: JUNSEI, Japan). The dispersed phase was stirred for at least 30 minutes and sufficiently dissolved before use. As the continuous phase, a 1% polyvinyl alcohol aqueous solution (viscosity: 4.8 to 5.8 mPa.Math.s) was used. The dispersed phase was connected and injected to an emulsififcation apparatus equipped with a porous membrane (pore size: 20 μm), and at the same time, the prepared dispersed phase was injected to form an emulsion. The emulsion was stirred at 40° C. at 200 rpm for 3 hours to remove the organic solvent. When the removal of the organic solvent was completed, the temperature of the microsphere suspension was set to 25° C., and the microsphere suspension was repeatedly washed with distilled water several times to remove residual polyvinyl alcohol, and then lyophilized, thereby obtaining a microsphere containing deslorelin.

Comparative Example 1-2: Preparation 3 of a Sustained-Release Microsphere Injection Formulation Containing Deslorelin Prepared Using a Single Polymer

[0056] The dispersed phase was prepared by mixing 2.4 g of biocompatible polymer Resomer R203H (manufacturer: Evonik, Germany) and 0.6 g of deslorelin acetate (manufacturer: Chengdu Kaijie Biopharm Co., Ltd, China) with 8.57 g of dichloromethane (manufacturer: J. T. Baker, USA) and 2.56 ml of N-methylpyrrolidone (manufacturer: JUNSEI, Japan). The dispersed phase was stirred for at least 30 minutes and sufficiently dissolved before use. As the continuous phase, a 1% polyvinyl alcohol aqueous solution (viscosity: 4.8 to 5.8 mPa.Math.s) was used. The dispersed phase was connected and injected to an emulsififcation apparatus equipped with a porous membrane (pore size: 20 μm), and at the same time, the prepared dispersed phase was injected to form an emulsion. The emulsion was stirred at 40° C. at 200 rpm for 3 hours to remove the organic solvent. When the removal of the organic solvent was completed, the temperature of the microsphere suspension was set to 25° C., and the microsphere suspension was repeatedly washed with distilled water several times to remove residual polyvinyl alcohol, and then lyophilized, thereby obtaining a microsphere containing deslorelin.

Comparative Example 1-3: Preparation 4 of a Sustained-Release Microsphere Injection Formulation Containing Deslorelin Prepared Using a Single Polymer

[0057] The dispersed phase was prepared by mixing 7.0 g of biocompatible polymer Resomer R203H (manufacturer: Evonik, Germany) and 3.0 g of deslorelin acetate (manufacturer: Chengdu Kaijie Biopharm Co., Ltd, China) with 25.0 g of dichloromethane (manufacturer: J. T. Baker, USA) and 7.46 ml of N-methylpyrrolidone (manufacturer: JUNSEI, Japan). The dispersed phase was stirred for at least 30 minutes and sufficiently dissolved before use. As the continuous phase, a 1% polyvinyl alcohol aqueous solution (viscosity: 4.8 to 5.8 mPa.Math.s) was used. The dispersed phase was connected and injected to an emulsififcation apparatus equipped with a porous membrane (pore size: 20 μm), and at the same time, the prepared dispersed phase was injected to form an emulsion. The emulsion was stirred at 40° C. at 200 rpm for 3 hours to remove the organic solvent. When the removal of the organic solvent was completed, the temperature of the microsphere suspension was set to 25° C., and the microsphere suspension was repeatedly washed with distilled water several times to remove residual polyvinyl alcohol, and then lyophilized, thereby obtaining a microsphere containing deslorelin.

Comparative Example 2: Preparation 5 of a Sustained-Release Microsphere Injection Formulation Containing Deslorelin Prepared Using a Single Polymer

[0058] The dispersed phase was prepared by mixing 8.5 g of biocompatible polymer Resomer R202H (manufacturer: Evonik, Germany) and 1.5 g of deslorelin acetate (manufacturer: Chengdu Kaijie Biopharm Co., Ltd, China) with 30.36 g of dichloromethane (manufacturer: J. T. Baker, USA) and 9.06 ml of N-methylpyrrolidone (manufacturer: JUNSEI, Japan). The dispersed phase was stirred for at least 30 minutes and sufficiently dissolved before use. As the continuous phase, a 1% polyvinyl alcohol aqueous solution (viscosity: 4.8 to 5.8 mPa.Math.s) was used. The dispersed phase was connected and injected to an emulsififcation apparatus equipped with a porous membrane (pore size: 20 μm), and at the same time, the prepared dispersed phase was injected to form an emulsion. The emulsion was stirred at 40° C. at 200 rpm for 3 hours to remove the organic solvent. When the removal of the organic solvent was completed, the temperature of the microsphere suspension was set to 25° C., and the microsphere suspension was repeatedly washed with distilled water several times to remove residual polyvinyl alcohol, and then lyophilized, thereby obtaining a microsphere containing deslorelin.

Comparative Example 3: Preparation 6 of a Sustained-Release Microsphere Injection Formulation Containing Deslorelin Prepared Using a Single Polymer

[0059] The dispersed phase was prepared by mixing 8.5 g of biocompatible polymer Resomer R205S (manufacturer: Evonik, Germany) and 1.5 g of deslorelin acetate (manufacturer: Chengdu Kaijie Biopharm Co., Ltd, China) with 30.36 g of dichloromethane (manufacturer: J. T. Baker, USA) and 9.06 ml of N-methylpyrrolidone (manufacturer: JUNSEI, Japan). The dispersed phase was stirred for at least 30 minutes and sufficiently dissolved before use. As the continuous phase, a 1% polyvinyl alcohol aqueous solution (viscosity: 4.8 to 5.8 mPa.Math.s) was used. The dispersed phase was connected and injected to an emulsififcation apparatus equipped with a porous membrane (pore size: 20 μm), and at the same time, the prepared dispersed phase was injected to form an emulsion. The emulsion was stirred at 40° C. at 200 rpm for 3 hours to remove the organic solvent. When the removal of the organic solvent was completed, the temperature of the microsphere suspension was set to 25° C., and the microsphere suspension was repeatedly washed with distilled water several times to remove residual polyvinyl alcohol, and then lyophilized, thereby obtaining a microsphere containing deslorelin.

Comparative Example 4: Preparation 7 of a Sustained-Release Microsphere Injection Formulation Containing Deslorelin Prepared Using a Single Polymer

[0060] The dispersed phase was prepared by mixing 9.0 g of biocompatible polymer Resomer RG752H (manufacturer: Evonik, Germany) and 1.0 g of deslorelin acetate (manufacturer: Chengdu Kaijie Biopharm Co., Ltd, China) with 32.14 g of dichloromethane (manufacturer: J. T. Baker, USA) and 9.59 ml of N-methylpyrrolidone (manufacturer: JUNSEI, Japan). The dispersed phase was stirred for at least 30 minutes and sufficiently dissolved before use. As the continuous phase, a 1% polyvinyl alcohol aqueous solution (viscosity: 4.8 to 5.8 mPa.Math.s) was used. The dispersed phase was connected and injected to an emulsififcation apparatus equipped with a porous membrane (pore size: 20 μm), and at the same time, the prepared dispersed phase was injected to form an emulsion. The emulsion was stirred at 40° C. at 200 rpm for 3 hours to remove the organic solvent. When the removal of the organic solvent was completed, the temperature of the microsphere suspension was set to 25° C., and the microsphere suspension was repeatedly washed with distilled water several times to remove residual polyvinyl alcohol, and then lyophilized, thereby obtaining a microsphere containing deslorelin.

Comparative Example 5: Preparation 8 of a Sustained-Release Microsphere Injection Formulation Containing Deslorelin Prepared Using a Single Polymer

[0061] The dispersed phase was prepared by mixing 2.55 g of biocompatible polymer Resomer RG753H (manufacturer: Evonik, Germany) and 0.45 g of deslorelin acetate (manufacturer: Chengdu Kaijie Biopharm Co., Ltd, China) with 9.11 g of dichloromethane (manufacturer: J. T. Baker, USA) and 2.72 ml of methylalcohol (manufacturer: Tedia Company, USA). The dispersed phase was stirred for at least 30 minutes and sufficiently dissolved before use. As the continuous phase, a 1% polyvinyl alcohol aqueous solution (viscosity: 4.8 to 5.8 mPa.Math.s) was used. The dispersed phase was connected and injected to an emulsififcation apparatus equipped with a porous membrane (pore size: 30 μm), and at the same time, the prepared dispersed phase was injected to form an emulsion. The emulsion was stirred at 40° C. at 200 rpm for 3 hours to remove the organic solvent. When the removal of the organic solvent was completed, the temperature of the microsphere suspension was set to 25° C., and the microsphere suspension was repeatedly washed with distilled water several times to remove residual polyvinyl alcohol, and then lyophilized, thereby obtaining a microsphere containing deslorelin.

Comparative Example 6: Preparation 9 of a Sustained-Release Microsphere Injection Formulation Containing Deslorelin Prepared Using a Single Polymer

[0062] The dispersed phase was prepared by mixing 8.5 g of biocompatible polymer Resomer RG653H (manufacturer: Evonik, Germany) and 1.5 g of deslorelin acetate (manufacturer: Chengdu Kaijie Biopharm Co., Ltd, China) with 30.36 g of dichloromethane (manufacturer: J. T. Baker, USA) and 9.06 ml of N-methylpyrrolidone (manufacturer: JUNSEI, Japan). The dispersed phase was stirred for at least 30 minutes and sufficiently dissolved before use. As the continuous phase, a 1% polyvinyl alcohol aqueous solution (viscosity: 4.8 to 5.8 mPa.Math.s) was used. The dispersed phase was connected and injected to an emulsififcation apparatus equipped with a porous membrane (pore size: 20 μm), and at the same time, the prepared dispersed phase was injected to form an emulsion. The emulsion was stirred at 40° C. at 200 rpm for 3 hours to remove the organic solvent. When the removal of the organic solvent was completed, the temperature of the microsphere suspension was set to 25° C., and the microsphere suspension was repeatedly washed with distilled water several times to remove residual polyvinyl alcohol, and then lyophilized, thereby obtaining a microsphere containing deslorelin.

Comparative Example 6-1: Preparation 10 of a Sustained-Release Microsphere Injection Formulation Containing Deslorelin Prepared Using a Single Polymer

[0063] The dispersed phase was prepared by mixing 4.5 g of biocompatible polymer Resomer RG653H (manufacturer: Evonik, Germany) and 0.5 g of deslorelin acetate (manufacturer: Chengdu Kaijie Biopharm Co., Ltd, China) with 16.07 g of dichloromethane (manufacturer: J. T. Baker, USA) and 4.8 ml of N-methylpyrrolidone (manufacturer: JUNSEI, Japan). The dispersed phase was stirred for at least 30 minutes and sufficiently dissolved before use. As the continuous phase, a 1% polyvinyl alcohol aqueous solution (viscosity: 4.8 to 5.8 mPa.Math.s) was used. The dispersed phase was connected and injected to an emulsififcation apparatus equipped with a porous membrane (pore size: 20 μm), and at the same time, the prepared dispersed phase was injected to form an emulsion. The emulsion was stirred at 40° C. at 200 rpm for 3 hours to remove the organic solvent. When the removal of the organic solvent was completed, the temperature of the microsphere suspension was set to 25° C., and the microsphere suspension was repeatedly washed with distilled water several times to remove residual polyvinyl alcohol, and then lyophilized, thereby obtaining a microsphere containing deslorelin.

Comparative Example 7: Preparation 11 of a Sustained-Release Microsphere Injection Formulation Containing Deslorelin Prepared Using a Single Polymer

[0064] The dispersed phase was prepared by mixing 8.5 g of biocompatible polymer Resomer RG503H (manufacturer: Evonik, Germany) and 1.5 g of deslorelin acetate (manufacturer: Chengdu Kaijie Biopharm Co., Ltd, China) with 30.06 g of dichloromethane (manufacturer: J. T. Baker, USA) and 9.06 ml of methylpyrrolidone (manufacturer: JUNSEI, Japan). The dispersed phase was stirred for at least 30 minutes and sufficiently dissolved before use. As the continuous phase, a 1% polyvinyl alcohol aqueous solution (viscosity: 4.8 to 5.8 mPa.Math.s) was used. The dispersed phase was connected and injected to an emulsififcation apparatus equipped with a porous membrane (pore size: 20 μm), and at the same time, the prepared dispersed phase was injected to form an emulsion. The emulsion was stirred at 40° C. at 200 rpm for 3 hours to remove the organic solvent. When the removal of the organic solvent was completed, the temperature of the microsphere suspension was set to 25° C., and the microsphere suspension was repeatedly washed with distilled water several times to remove residual polyvinyl alcohol, and then lyophilized, thereby obtaining a microsphere containing deslorelin.

Comparative Example 8: Preparation 12 of a Sustained-Release Microsphere Injection Formulation Containing Deslorelin Prepared Using a Single Polymer

[0065] The dispersed phase was prepared by mixing 8.5 g of biocompatible polymer Resomer RG858S (manufacturer: Evonik, Germany) and 1.5 g of deslorelin acetate (manufacturer: Chengdu Kaijie Biopharm Co., Ltd, China) with 60.71 g of dichloromethane (manufacturer: J. T. Baker, USA) and 18.12 ml of N-methylpyrrolidone (manufacturer: JUNSEI, Japan). The dispersed phase was stirred for at least 30 minutes and sufficiently dissolved before use. As the continuous phase, a 1% polyvinyl alcohol aqueous solution (viscosity: 4.8 to 5.8 mPa.Math.s) was used. The dispersed phase was connected and injected to an emulsififcation apparatus equipped with a porous membrane (pore size: 20 μm), and at the same time, the prepared dispersed phase was injected to form an emulsion. The emulsion was stirred at 40° C. at 200 rpm for 3 hours to remove the organic solvent. When the removal of the organic solvent was completed, the temperature of the microsphere suspension was set to 25° C., and the microsphere suspension was repeatedly washed with distilled water several times to remove residual polyvinyl alcohol, and then lyophilized, thereby obtaining a microsphere containing deslorelin.

Comparative Example 9: Preparation 1 of a Sustained-Release Microsphere Injection Formulation Containing Deslorelin Prepared Using Two Types of Polymers

[0066] In order to prepare a sustained-release microsphere injection formulation having an average particle size of 100 μm or more using two types of polymers with a high-speed stirrer, a sustained-release microsphere injection formulation containing deslorelin was prepared according to the following method. The dispersed phase was prepared by mixing 1.91 g of biocompatible polymer Resomer R203H (manufacturer: Evonik, Germany), 0.64 g of Resomer RG752H (manufacturer: Evonik, Germany) and 0.45 g of deslorelin acetate (manufacturer: Chengdu Kaijie Biopharm Co., Ltd, China) with 9.11 g of dichloromethane (manufacturer: J. T. Baker, USA) and 2.72 ml of N-methylpyrrolidone (manufacturer: JUNSEI, Japan). The dispersed phase was stirred for at least 30 minutes and sufficiently dissolved before use. As the continuous phase, a 1% polyvinyl alcohol aqueous solution (viscosity: 4.8 to 5.8 mPa.Math.s) was used. The dispersed phase was added to the continuous phase and stirred at 1000 rpm using a high-speed stirrer to form an emulsion, which was stirred at 40° C. for 3 hours to remove the organic solvent. When the removal of the organic solvent was completed, the temperature of the microsphere suspension was set to 25° C., and the microsphere suspension was repeatedly washed with distilled water several times to remove residual polyvinyl alcohol, and then lyophilized, thereby obtaining a microsphere containing deslorelin.

Comparative Example 10: Preparation 2 of a Sustained-Release Microsphere Injection Formulation Containing Deslorelin Prepared Using Two Types of Polymers

[0067] In order to prepare a sustained-release microsphere injection formulation having an average particle size of 100 μm or more using two types of polymers with a high-speed stirrer, a sustained-release microsphere injection formulation containing deslorelin was prepared according to the following method. In order to prepare a sustained-release microsphere injection formulation within an average particle size of 100 μm, the RPM of the high-speed stirrer was relatively increased and the injection formulation was prepared as follows. The dispersed phase was prepared by mixing 1.91 g of biocompatible polymer Resomer R203H (manufacturer: Evonik, Germany), 0.64 g of Resomer RG752H (manufacturer: Evonik, Germany) and 0.45 g of deslorelin acetate (manufacturer: Chengdu Kaijie Biopharm Co., Ltd, China) with 9.11 g of dichloromethane (manufacturer: J. T. Baker, USA) and 2.72 ml of N-methylpyrrolidone (manufacturer: JUNSEI, Japan). The dispersed phase was stirred for at least 30 minutes and sufficiently dissolved before use. As the continuous phase, a 1% polyvinyl alcohol aqueous solution (viscosity: 4.8 to 5.8 mPa.Math.s) was used. The dispersed phase was added to the continuous phase and stirred at 1000 rpm using a high-speed stirrer to form an emulsion, which was stirred at 40° C. for 3 hours to remove the organic solvent. When the removal of the organic solvent was completed, the temperature of the microsphere suspension was set to 25° C., and the microsphere suspension was repeatedly washed with distilled water several times to remove residual polyvinyl alcohol, and then lyophilized, thereby obtaining a microsphere containing deslorelin.

Comparative Example 11: Preparation 3 of a Sustained-Release Microsphere Injection Formulation Containing Deslorelin Prepared Using Two Types of Polymers

[0068] In order to prepare a sustained-release microsphere injection formulation using two types of polymers, wherein the drug introduced for preparing the formulation was used in an amount of 40 wt. % based on the total solid content, and the final content of deslorelin in the sustained-release microsphere injection formulation was as high as 27.3 wt. %, a sustained-release microsphere injection formulation was prepared according to the following method. The dispersed phase was prepared by mixing 1.35 g of biocompatible polymer Resomer R203H (manufacturer: Evonik, Germany), 0.45 g of Resomer RG752H (manufacturer: Evonik, Germany) and 1.2 g of deslorelin acetate (manufacturer: Chengdu Kaijie Biopharm Co., Ltd, China) with 6.43 g of dichloromethane (manufacturer: J. T. Baker, USA) and 1.92 ml of N-methylpyrrolidone (manufacturer: JUNSEI, Japan). The dispersed phase was stirred for at least 30 minutes and sufficiently dissolved before use. As the continuous phase, a 1% polyvinyl alcohol aqueous solution (viscosity: 4.8 to 5.8 mPa.Math.s) was used. The dispersed phase was connected and injected to an emulsififcation apparatus equipped with a porous membrane (pore size: 50 μm), and at the same time, the prepared dispersed phase was injected to form an emulsion. The emulsion was stirred at 40° C. at 200 rpm for 3 hours to remove the organic solvent. When the removal of the organic solvent was completed, the temperature of the microsphere suspension was set to 25° C., and the microsphere suspension was repeatedly washed with distilled water several times to remove residual polyvinyl alcohol, and then lyophilized, thereby obtaining a microsphere containing deslorelin.

Comparative Example 12: Preparation 4 of a Sustained-Release Microsphere Injection Formulation Containing Deslorelin Prepared Using Two Types of Polymers

[0069] In order to prepare a sustained-release microsphere injection formulation using two types of polymers, wherein the drug introduced for preparing the formulation was used in an amount of 5 wt. % based on the total solid content, and the final content of deslorelin in the sustained-release microsphere injection formulation was as low as 4.2 wt. %, a sustained-release microsphere injection formulation was prepared according to the following method. The dispersed phase was prepared by mixing 2.14 g of biocompatible polymer Resomer R203H (manufacturer: Evonik, Germany), 0.71 g of Resomer RG752H (manufacturer: Evonik, Germany) and 0.15 g of deslorelin acetate (manufacturer: Chengdu Kaijie Biopharm Co., Ltd, China) with 10.18 g of dichloromethane (manufacturer: J. T. Baker, USA) and 3.04 ml of N-methylpyrrolidone (manufacturer: JUNSEI, Japan). The dispersed phase was stirred for at least 30 minutes and sufficiently dissolved before use. As the continuous phase, a 1% polyvinyl alcohol aqueous solution (viscosity: 4.8 to 5.8 mPa.Math.s) was used. The dispersed phase was connected and injected to an emulsififcation apparatus equipped with a porous membrane (pore size: 20 μm), and at the same time, the prepared dispersed phase was injected to form an emulsion. The emulsion was stirred at 40° C. at 200 rpm for 3 hours to remove the organic solvent. When the removal of the organic solvent was completed, the temperature of the microsphere suspension was set to 25° C., and the microsphere suspension was repeatedly washed with distilled water several times to remove residual polyvinyl alcohol, and then lyophilized, thereby obtaining a microsphere containing deslorelin.

Comparative Example 13: Preparation 5 of a Sustained-Release Microsphere Injection Formulation Containing Deslorelin Prepared Using Two Types of Polymers

[0070] In order to prepare a sustained-release microsphere injection formulation using two types of polymers, provided that a ratio of PLA to PLGA (PLA:PLGA) was set to 1:3, a sustained-release microsphere injection formulation was prepared according to the following method. The dispersed phase was prepared by mixing 1.06 g of biocompatible polymer Resomer R203H (manufacturer: Evonik, Germany), 3.19 g of Resomer RG752H (manufacturer: Evonik, Germany) and 0.75 g of deslorelin acetate (manufacturer: Chengdu Kaijie Biopharm Co., Ltd, China) with 15.18 g of dichloromethane (manufacturer: J. T. Baker, USA) and 4.53 ml of N-methylpyrrolidone (manufacturer: JUNSEI, Japan). The dispersed phase was stirred for at least 30 minutes and sufficiently dissolved before use. As the continuous phase, a 1% polyvinyl alcohol aqueous solution (viscosity: 4.8 to 5.8 mPa.Math.s) was used. The dispersed phase was connected and injected to an emulsififcation apparatus equipped with a porous membrane (pore size: 20 μm), and at the same time, the prepared dispersed phase was injected to form an emulsion. The emulsion was stirred at 40° C. at 200 rpm for 3 hours to remove the organic solvent. When the removal of the organic solvent was completed, the temperature of the microsphere suspension was set to 25° C., and the microsphere suspension was repeatedly washed with distilled water several times to remove residual polyvinyl alcohol, and then lyophilized, thereby obtaining a microsphere containing deslorelin.

EXAMPLE

[0071] In Examples, when preparing a sustained-release microsphere injection formulation containing deslorelin, two or more types of polymers were used, or two or more formulations prepared using a single polymer were mixed.

Example 1: Preparation 1 of a Sustained-Release Microsphere Injection Formulation Containing Deslorelin Prepared Using Two Types of Polymers

[0072] The dispersed phase was prepared by mixing 6.375 g of biocompatible polymer Resomer R203H (manufacturer: Evonik, Germany), 2.125 g of Resomer RG858S (manufacturer: Evonik, Germany) and 1.5 g of deslorelin acetate (manufacturer: Chengdu Kaijie Biopharm Co., Ltd, China) with 37.95 g of dichloromethane (manufacturer: J. T. Baker, USA) and 11.33 ml of N-methylpyrrolidone (manufacturer: JUNSEI, Japan). The dispersed phase was stirred for at least 30 minutes and sufficiently dissolved before use. As the continuous phase, a 1% polyvinyl alcohol aqueous solution (viscosity: 4.8 to 5.8 mPa.Math.s) was used. The dispersed phase was connected and injected to an emulsififcation apparatus equipped with a porous membrane (pore size: 20 μm), and at the same time, the prepared dispersed phase was injected to form an emulsion. The emulsion was stirred at 40° C. at 200 rpm for 3 hours to remove the organic solvent. When the removal of the organic solvent was completed, the temperature of the microsphere suspension was set to 25° C., and the microsphere suspension was repeatedly washed with distilled water several times to remove residual polyvinyl alcohol, and then lyophilized, thereby obtaining a microsphere containing deslorelin.

Example 2: Preparation 2 of a Sustained-Release Microsphere Injection Formulation Containing Deslorelin Prepared Using Two Types of Polymers

[0073] The dispersed phase was prepared by mixing 7.65 g of biocompatible polymer Resomer R203H (manufacturer: Evonik, Germany), 0.85 g of Resomer RG752H (manufacturer: Evonik, Germany) and 1.5 g of deslorelin acetate (manufacturer: Chengdu Kaijie Biopharm Co., Ltd, China) with 30.36 g of dichloromethane (manufacturer: J. T. Baker, USA) and 9.06 ml of N-methylpyrrolidone (manufacturer: JUNSEI, Japan). The dispersed phase was stirred for at least 30 minutes and sufficiently dissolved before use. As the continuous phase, a 1% polyvinyl alcohol aqueous solution (viscosity: 4.8 to 5.8 mPa.Math.s) was used. The dispersed phase was connected and injected to an emulsififcation apparatus equipped with a porous membrane (pore size: 20 μm), and at the same time, the prepared dispersed phase was injected to form an emulsion. The emulsion was stirred at 40° C. at 200 rpm for 3 hours to remove the organic solvent. When the removal of the organic solvent was completed, the temperature of the microsphere suspension was set to 25° C., and the microsphere suspension was repeatedly washed with distilled water several times to remove residual polyvinyl alcohol, and then lyophilized, thereby obtaining a microsphere containing deslorelin.

Example 3: Preparation 3 of a Sustained-Release Microsphere Injection Formulation Containing Deslorelin Prepared Using Two Types of Polymers

[0074] The dispersed phase was prepared by mixing 6.375 g of biocompatible polymer Resomer R203H (manufacturer: Evonik, Germany), 2.125 g of Resomer RG753H (manufacturer: Evonik, Germany) and 1.5 g of deslorelin acetate (manufacturer: Chengdu Kaijie Biopharm Co., Ltd, China) with 30.36 g of dichloromethane (manufacturer: J. T. Baker, USA) and 9.06 ml of N-methylpyrrolidone (manufacturer: JUNSEI, Japan). The dispersed phase was stirred for at least 30 minutes and sufficiently dissolved before use. As the continuous phase, a 1% polyvinyl alcohol aqueous solution (viscosity: 4.8 to 5.8 mPa.Math.s) was used. The dispersed phase was connected and injected to an emulsififcation apparatus equipped with a porous membrane (pore size: 20 μm), and at the same time, the prepared dispersed phase was injected to form an emulsion. The emulsion was stirred at 40° C. at 200 rpm for 3 hours to remove the organic solvent. When the removal of the organic solvent was completed, the temperature of the microsphere suspension was set to 25° C., and the microsphere suspension was repeatedly washed with distilled water several times to remove residual polyvinyl alcohol, and then lyophilized, thereby obtaining a microsphere containing deslorelin.

Example 4: Preparation 4 of a Sustained-Release Microsphere Injection Formulation Containing Deslorelin Prepared Using Two Types of Polymers

[0075] The dispersed phase was prepared by mixing 4.25 g of biocompatible polymer Resomer R203H (manufacturer: Evonik, Germany), 4.25 g of Resomer RG752H (manufacturer: Evonik, Germany) and 1.5 g of deslorelin acetate (manufacturer: Chengdu Kaijie Biopharm Co., Ltd, China) with 30.36 g of dichloromethane (manufacturer: J. T. Baker, USA) and 9.06 ml of methylalcohol (manufacturer: TEDIA Company, USA). The dispersed phase was stirred for at least 30 minutes and sufficiently dissolved before use. As the continuous phase, a 1% polyvinyl alcohol aqueous solution (viscosity: 4.8 to 5.8 mPa.Math.s) was used. The dispersed phase was connected and injected to an emulsififcation apparatus equipped with a porous membrane (pore size: 20 μm), and at the same time, the prepared dispersed phase was injected to form an emulsion. The emulsion was stirred at 40° C. at 200 rpm for 3 hours to remove the organic solvent. When the removal of the organic solvent was completed, the temperature of the microsphere suspension was set to 25° C., and the microsphere suspension was repeatedly washed with distilled water several times to remove residual polyvinyl alcohol, and then lyophilized, thereby obtaining a microsphere containing deslorelin.

Example 5: Preparation 5 of a Sustained-Release Microsphere Injection Formulation Containing Deslorelin Prepared Using Two Types of Polymers

[0076] The dispersed phase was prepared by mixing 6.375 g of biocompatible polymer Resomer R203H (manufacturer: Evonik, Germany), 2.125 g of Resomer RG653H (manufacturer: Evonik, Germany) and 1.5 g of deslorelin acetate (manufacturer: Chengdu Kaijie Biopharm Co., Ltd, China) with 30.36 g of dichloromethane (manufacturer: J. T. Baker, USA) and 9.06 ml of N-methylpyrrolidone (manufacturer: JUNSEI, Japan). The dispersed phase was stirred for at least 30 minutes and sufficiently dissolved before use. As the continuous phase, a 1% polyvinyl alcohol aqueous solution (viscosity: 4.8 to 5.8 mPa.Math.s) was used. The dispersed phase was connected and injected to an emulsififcation apparatus equipped with a porous membrane (pore size: 20 μm), and at the same time, the prepared dispersed phase was injected to form an emulsion. The emulsion was stirred at 40° C. at 200 rpm for 3 hours to remove the organic solvent. When the removal of the organic solvent was completed, the temperature of the microsphere suspension was set to 25° C., and the microsphere suspension was repeatedly washed with distilled water several times to remove residual polyvinyl alcohol, and then lyophilized, thereby obtaining a microsphere containing deslorelin.

Example 6: Preparation 6 of a Sustained-Release Microsphere Injection Formulation Containing Deslorelin Prepared Using Two Types of Polymers

[0077] The dispersed phase was prepared by mixing 6.375 g of biocompatible polymer Resomer R203H (manufacturer: Evonik, Germany), 2.125 g of Resomer RG503H (manufacturer: Evonik, Germany) and 1.5 g of deslorelin acetate (manufacturer: Chengdu Kaijie Biopharm Co., Ltd, China) with 30.36 g of dichloromethane (manufacturer: J. T. Baker, USA) and 14.73 ml of N-methylpyrrolidone (manufacturer: JUNSEI, Japan). The dispersed phase was stirred for at least 30 minutes and sufficiently dissolved before use. As the continuous phase, a 1% polyvinyl alcohol aqueous solution (viscosity: 4.8 to 5.8 mPa.Math.s) was used. The dispersed phase was connected and injected to an emulsififcation apparatus equipped with a porous membrane (pore size: 50 μm), and at the same time, the prepared dispersed phase was injected to form an emulsion. The emulsion was stirred at 40° C. at 200 rpm for 3 hours to remove the organic solvent. When the removal of the organic solvent was completed, the temperature of the microsphere suspension was set to 25° C., and the microsphere suspension was repeatedly washed with distilled water several times to remove residual polyvinyl alcohol, and then lyophilized, thereby obtaining a microsphere containing deslorelin.

Example 7: Preparation 7 of a Sustained-Release Microsphere Injection Formulation Containing Deslorelin Prepared Using Two Types of Polymers

[0078] The dispersed phase was prepared by mixing 6.375 g of biocompatible polymer Resomer R202H (manufacturer: Evonik, Germany), 2.125 g of Resomer RG858S (manufacturer: Evonik, Germany) and 1.5 g of deslorelin acetate (manufacturer: Chengdu Kaijie Biopharm Co., Ltd, China) with 37.95 g of dichloromethane (manufacturer: J. T. Baker, USA) and 11.33 ml of N-methylpyrrolidone (manufacturer: JUNSEI, Japan). The dispersed phase was stirred for at least 30 minutes and sufficiently dissolved before use. As the continuous phase, a 1% polyvinyl alcohol aqueous solution (viscosity: 4.8 to 5.8 mPa.Math.s) was used. The dispersed phase was connected and injected to an emulsififcation apparatus equipped with a porous membrane (pore size: 20 μm), and at the same time, the prepared dispersed phase was injected to form an emulsion. The emulsion was stirred at 40° C. at 200 rpm for 3 hours to remove the organic solvent. When the removal of the organic solvent was completed, the temperature of the microsphere suspension was set to 25° C., and the microsphere suspension was repeatedly washed with distilled water several times to remove residual polyvinyl alcohol, and then lyophilized, thereby obtaining a microsphere containing deslorelin.

Example 8: Preparation 8 of a Sustained-Release Microsphere Injection Formulation Containing Deslorelin Prepared Using Two Types of Polymers

[0079] The dispersed phase was prepared by mixing 6.375 g of biocompatible polymer Resomer R202H (manufacturer: Evonik, Germany), 2.125 g of Resomer RG752H (manufacturer: Evonik, Germany) and 1.5 g of deslorelin acetate (manufacturer: Chengdu Kaijie Biopharm Co., Ltd, China) with 30.36 g of dichloromethane (manufacturer: J. T. Baker, USA) and 9.06 ml of methylalcohol (manufacturer: TEDIA Company, USA). The dispersed phase was stirred for at least 30 minutes and sufficiently dissolved before use. As the continuous phase, a 1% polyvinyl alcohol aqueous solution (viscosity: 4.8 to 5.8 mPa.Math.s) was used. The dispersed phase was connected and injected to an emulsififcation apparatus equipped with a porous membrane (pore size: 20 μm), and at the same time, the prepared dispersed phase was injected to form an emulsion. The emulsion was stirred at 40° C. at 200 rpm for 3 hours to remove the organic solvent. When the removal of the organic solvent was completed, the temperature of the microsphere suspension was set to 25° C., and the microsphere suspension was repeatedly washed with distilled water several times to remove residual polyvinyl alcohol, and then lyophilized, thereby obtaining a microsphere containing deslorelin.

Example 9: Preparation 9 of a Sustained-Release Microsphere Injection Formulation Containing Deslorelin Prepared Using Two Types of Polymers

[0080] The dispersed phase was prepared by mixing 6.375 g of biocompatible polymer Resomer R202H (manufacturer: Evonik, Germany), 2.125 g of Resomer RG653H (manufacturer: Evonik, Germany) and 1.5 g of deslorelin acetate (manufacturer: Chengdu Kaijie Biopharm Co., Ltd, China) with 30.36 g of dichloromethane (manufacturer: J. T. Baker, USA) and 9.06 ml of N-methylpyrrolidone (manufacturer: JUNSEI, Japan). The dispersed phase was stirred for at least 30 minutes and sufficiently dissolved before use. As the continuous phase, a 1% polyvinyl alcohol aqueous solution (viscosity: 4.8 to 5.8 mPa.Math.s) was used. The dispersed phase was connected and injected to an emulsififcation apparatus equipped with a porous membrane (pore size: 20 μm), and at the same time, the prepared dispersed phase was injected to form an emulsion. The emulsion was stirred at 40° C. at 200 rpm for 3 hours to remove the organic solvent. When the removal of the organic solvent was completed, the temperature of the microsphere suspension was set to 25° C., and the microsphere suspension was repeatedly washed with distilled water several times to remove residual polyvinyl alcohol, and then lyophilized, thereby obtaining a microsphere containing deslorelin.

Example 9-1: Preparation 9-1 of a Sustained-Release Microsphere Injection Formulation Containing Deslorelin Prepared Using Two Types of Polymers

[0081] The dispersed phase was prepared by mixing 2.775 g of biocompatible polymer Resomer R202H (manufacturer: Evonik, Germany), 1.85 g of Resomer RG653H (manufacturer: Evonik, Germany) and 0.375 g of deslorelin acetate (manufacturer: Chengdu Kaijie Biopharm Co., Ltd, China) with 16.52 g of dichloromethane (manufacturer: J. T. Baker, USA) and 4.93 ml of N-methylpyrrolidone (manufacturer: JUNSEI, Japan). The dispersed phase was stirred for at least 30 minutes and sufficiently dissolved before use. As the continuous phase, a 1% polyvinyl alcohol aqueous solution (viscosity: 4.8 to 5.8 mPa.Math.s) was used. The dispersed phase was connected and injected to an emulsififcation apparatus equipped with a porous membrane (pore size: 5 μm), and at the same time, the prepared dispersed phase was injected to form an emulsion. The emulsion was stirred at 40° C. at 200 rpm for 3 hours to remove the organic solvent. When the removal of the organic solvent was completed, the temperature of the microsphere suspension was set to 25° C., and the microsphere suspension was repeatedly washed with distilled water several times to remove residual polyvinyl alcohol, and then lyophilized, thereby obtaining a microsphere containing deslorelin.

Example 9-2: Preparation 9-2 of a Sustained-Release Microsphere Injection Formulation Containing Deslorelin Prepared Using Two Types of Polymers

[0082] The dispersed phase was prepared by mixing 2.25 g of biocompatible polymer Resomer R202H (manufacturer: Evonik, Germany), 1.5 g of Resomer RG653H (manufacturer: Evonik, Germany) and 1.25 g of deslorelin acetate (manufacturer: Chengdu Kaijie Biopharm Co., Ltd, China) with 13.39 g of dichloromethane (manufacturer: J. T. Baker, USA) and 4.00 ml of dimethyl sulfoxide (manufacturer: Samchun Pure Chemical Co., Ktd., South Korea). The dispersed phase was stirred for at least 30 minutes and sufficiently dissolved before use. As the continuous phase, a 1% polyvinyl alcohol aqueous solution (viscosity: 4.8 to 5.8 mPa.Math.s) was used. The dispersed phase was connected and injected to an emulsififcation apparatus equipped with a porous membrane (pore size: 5 μm), and at the same time, the prepared dispersed phase was injected to form an emulsion. The emulsion was stirred at 40° C. at 200 rpm for 3 hours to remove the organic solvent. When the removal of the organic solvent was completed, the temperature of the microsphere suspension was set to 25° C., and the microsphere suspension was repeatedly washed with distilled water several times to remove residual polyvinyl alcohol, and then lyophilized, thereby obtaining a microsphere containing deslorelin.

Example 10: Preparation 10 of a Sustained-Release Microsphere Injection Formulation Containing Deslorelin Prepared Using Two Types of Polymers

[0083] The dispersed phase was prepared by mixing 6.375 g of biocompatible polymer Resomer R202H (manufacturer: Evonik, Germany), 2.125 g of Resomer RG503H (manufacturer: Evonik, Germany) and 1.5 g of deslorelin acetate (manufacturer: Chengdu Kaijie Biopharm Co., Ltd, China) with 30.36 g of dichloromethane (manufacturer: J. T. Baker, USA) and 9.06 ml of N-methylpyrrolidone (manufacturer: JUNSEI, Japan). The dispersed phase was stirred for at least 30 minutes and sufficiently dissolved before use. As the continuous phase, a 1% polyvinyl alcohol aqueous solution (viscosity: 4.8 to 5.8 mPa.Math.s) was used. The dispersed phase was connected and injected to an emulsififcation apparatus equipped with a porous membrane (pore size: 40 μm), and at the same time, the prepared dispersed phase was injected to form an emulsion. The emulsion was stirred at 40° C. at 200 rpm for 3 hours to remove the organic solvent. When the removal of the organic solvent was completed, the temperature of the microsphere suspension was set to 25° C., and the microsphere suspension was repeatedly washed with distilled water several times to remove residual polyvinyl alcohol, and then lyophilized, thereby obtaining a microsphere containing deslorelin.

Example 11: Preparation 11 of a Sustained-Release Microsphere Injection Formulation Containing Deslorelin Prepared Using Two Types of Polymers

[0084] The dispersed phase was prepared by mixing 4.25 g of biocompatible polymer Resomer R203H (manufacturer: Evonik, Germany), 2.125 g of Resomer RG752H (manufacturer: Evonik, Germany), 2.125 g of Resomer RG503H (manufacturer: Evonik, Germany) and 1.5 g of deslorelin acetate (manufacturer: Chengdu Kaijie Biopharm Co., Ltd, China) with 30.36 g of dichloromethane (manufacturer: J. T. Baker, USA) and 9.06 ml of N-methylpyrrolidone (manufacturer: JUNSEI, Japan). The dispersed phase was stirred for at least 30 minutes and sufficiently dissolved before use. As the continuous phase, a 1% polyvinyl alcohol aqueous solution (viscosity: 4.8 to 5.8 mPa.Math.s) was used. The dispersed phase was connected and injected to an emulsififcation apparatus equipped with a porous membrane (pore size: 40 μm), and at the same time, the prepared dispersed phase was injected to form an emulsion. The emulsion was stirred at 40° C. at 200 rpm for 3 hours to remove the organic solvent. When the removal of the organic solvent was completed, the temperature of the microsphere suspension was set to 25° C., and the microsphere suspension was repeatedly washed with distilled water several times to remove residual polyvinyl alcohol, and then lyophilized, thereby obtaining a microsphere containing deslorelin.

Examples 12-22. Preparation of a Formulation Containing Two or More Types of Microspheres Containing Different Polymers

[0085] The sustained-release microsphere formulations containing deslorelin prepared in Comparative Example were mixed based on the polymer and ratio as shown in Table 1 below, and used as Examples 12-22.

TABLE-US-00001 TABLE 1 Micro- Micro- Micro- Category sphere 1 sphere 2 sphere 3 Mixing ratio Example 12 Comparative Comparative — 3 1 — Example 1-3 Example 8 Example 13 Comparative Comparative — 9 1 — Example 1-3 Example 4 Example 14 Comparative Comparative — 3 1 — Example 1-3 Example 5 Example 15 Comparative Comparative — 1 1 — Example 1-3 Example 4 Example 16 Comparative Comparative — 3 1 — Example 1-3 Example 6 Example 17 Comparative Comparative — 3 1 — Example 1-3 Example 7 Example 18 Comparative Comparative — 3 1 — Example2 Example 8 Example 19 Comparative Comparative — 3 1 — Example2 Example 4 Example 20 Comparative Comparative — 3 1 — Example2 Example 6 Example 21 Comparative Comparative — 3 1 — Example2 Example 7 Example 22 Comparative Comparative Comparative 2 1 1 Example 1-3 Example4 Example 7

Example 23: Preparation 12 of a Sustained-Release Microsphere Injection Formulation Containing Deslorelin Prepared Using Two Types of Polymers

[0086] The dispersed phase was prepared by mixing 4.25 g of biocompatible polymer Resomer R207S (manufacturer: Evonik, Germany), 4.25 g of Resomer RG752H (manufacturer: Evonik, Germany) and 1.5 g of deslorelin acetate (manufacturer: Chengdu Kaijie Biopharm Co., Ltd, China) with 42.5 g of dichloromethane (manufacturer: J. T. Baker, USA) and 12.69 ml of N-methylpyrrolidone (manufacturer: JUNSEI, Japan). The dispersed phase was stirred for at least 30 minutes and sufficiently dissolved before use. As the continuous phase, a 1% polyvinyl alcohol aqueous solution (viscosity: 4.8 to 5.8 mPa.Math.s) was used. The dispersed phase was added to the continuous phase and then stirred at 2000 rpm using a high-speed stirrer to form an emulsion, which was stirred at 40° C. for 3 hours to remove the organic solvent. When the removal of the organic solvent was completed, the temperature of the microsphere suspension was set to 25° C., and the microsphere suspension was repeatedly washed with distilled water several times to remove residual polyvinyl alcohol. Then, particles larger than 150 pm and smaller than 25 μm using 100 mesh (150 μm) and 500 mesh (25 μm) sieves were removed, and then lyophilized, thereby obtaining a microsphere containing deslorelin.

Experimental Example

Experimental Example 1. Measurement of Deslorelin Content in Microsphere

[0087] In order to measure the amount of encapsulation of deslorelin in the microspheres prepared in Comparative Examples 1-13 and 1-11, 2 mg of microspheres were completely dissolved in acetonitrile, and then diluted with a mobile phase. 20 μL of the diluted solution was injected into HPLC and measured at a detection wavelength of 280 nm. The column used in this measurement was Gemini C18 5 μm, 4.6×150 mm, and the mobile phase (A) 0.1% TFA aqueous solution and (B) acetonitrile containing 0.1% TFA were used in gradient mode with (B) being 20-50% .

[0088] The content of deslorelin in the microspheres prepared in Comparative Example is shown in Table 2 below.

TABLE-US-00002 TABLE 2 Deslorelin content of the sustained-release microsphere injection formulations containing deslorelin (Comparative Example) Sample Drug content (%) Comparative Example 1 9.7 Comparative Example 1-1 12.2 Comparative Example 1-2 16.2 Comparative Example 1-3 22.0 Comparative Example 2 12.8 Comparative Example 3 10.2 Comparative Example 4 7.6 Comparative Example 5 13.4 Comparative Example 6 15.1 Comparative Example 6-1 8.8 Comparative Example 7 13.3 Comparative Example 8 10.6 Comparative Example 9 12.6 Comparative Example 10 12.1 Comparative Example 11 27.3 Comparative Example 12 4.2 Comparative Example 13 11.5

[0089] In Comparative Examples 1 to 8, a formulation was prepared using a single polymer, wherein the preparation was performed by changing the type of polymer and the target content of drug. Based on the results confirmed in Table 2, it was confirmed that there was a difference in the encapsulation rate of the drug depending on the type of polymer. In general, when the terminal group of the polymer used for preparing the formulation is an ester group, it was confirmed that the drug encapsulation rate tends to be relatively low. In Comparative Examples 9 to 10, examples in which the formulations were prepared using a high-speed stirrer, it was confirmed that the drug content was not significantly affected even if the preparation method was changed. Further, Comparative Examples 11 to 12 are formulations prepared by changing the amount of deslorelin compared to the total solid content added in the preparation of microspheres, and it was confirmed that the content of the final drug was significantly varied depending on the amount of the drug, and it was confirmed that the higher the drug input ratio, the lower the encapsulation efficiency. Comparative Example 13 is an example in which when preparing a formulation using two types of polymers, the mixing ratio is PLA: PLGA=1:3, and it was confirmed that there was no difference in the drug encapsulation efficiency depending on the composition ratio of the polymer.

[0090] The content of deslorelin in the microspheres of Examples 1 to 23 is shown in Table 3 below.

TABLE-US-00003 TABLE 3 Deslorelin content of sustained-release microsphere injection formulations containing deslorelin (Example) Sample Drug content (%) Example 1 12.20 Example 2 12.60 Example 3 12.00 Example 4 12.40 Example 5 13.20 Example 6 12.80 Example 7 12.20 Example 8 12.50 Example 9 13.20 Example 9-1 5.10 Example 9-2 23.30 Example 10 12.90 Example 11 12.80 Example 12 11.80 Example 13 11.74 Example 14 11.05 Example 15 9.90 Example 16 12.93 Example 17 12.48 Example 18 12.25 Example 19 11.50 Example 20 13.38 Example 21 12.93 Example 22 11.63 Example 23 10.70

Experimental Example 2. Morphological Analysis of Sustained-Release Microsphere Injection Formulation Containing Deslorelin Through Electron Microscope

[0091] To analyze the morphological properties of microspheres, a scanning electron microscope observation was performed. The microspheres were coated with platinum using Ion-Coater (COXEM, Korea), and then the morphological properties of the microspheres were observed at an acceleration voltage of 15 kV with a scanning electron microscope (COXEM EM-30, Korea), and the results are shown in FIG. 1.

[0092] FIG. 1 is a photograph of a sustained-release microsphere injection formulation containing deslorelin prepared in Example 3 when observed with an electron microscope. According to FIG. 1, it was confirmed that microspheres having a very uniform particle size were prepared.

Experimental Example 3. Particle Size Analysis of Sustained-Release Microsphere Injection Formulation Containing Deslorelin Using a Laser Diffraction Method

[0093] To quantitatively measure the average particle size, distribution and uniformity of the microspheres, the experiment was performed using a laser diffraction method. The microspheres prepared in Comparative Examples and Examples were mixed with ultrapure water containing a surfactant, mixed with a vortex mixer for 20 seconds, and then placed in an ultrasonic generator and dispersed. This solution was placed in a particle size analyzer (Microtrac Bluewave, Japan) and the particle size was measured.

[0094] As an index of particle size uniformity, the span value was determined by the following Equation 1.

Span Value=(Dv0.9−Dv0.1)/Dv0.5   [Equation 1]

[0095] The particle size measurement results of the sustained-release formulation containing deslorelin prepared in Comparative Examples are shown in Table 4 below.

TABLE-US-00004 TABLE 4 Particle size of sustained-release microsphere injection formulation containing deslorelin (Comparative Example) Sample Dv0.5(μm) Span value Comparative Example 1 33.16 0.78 Comparative Example 1-1 33.32 0.75 Comparative Example 1-2 35.76 0.76 Comparative Example 1-3 38.22 0.97 Comparative Example 2 35.52 0.63 Comparative Example 3 33.05 0.60 Comparative Example 4 28.17 0.59 Comparative Example 5 45.23 0.63 Comparative Example 6 40.35 1.66 Comparative Example 6-1 33.05 0.60 Comparative Example 7 37.22 1.04 Comparative Example 8 31.78 0.63 Comparative Example 9 114.72 0.53 Comparative Example 10 98.21 0.67 Comparative Example 11 63.86 1.33 Comparative Example 12 38.22 0.97 Comparative Example 13 33.23 0.68

[0096] The particle size measurement results of the sustained-release microsphere formulation containing deslorelin prepared in Example are shown in Table 5 below.

TABLE-US-00005 TABLE 5 Particle size of sustained-release microsphere injection formulation containing deslorelin (Example) Sample Dv0.5(μm) Span value Example 1 36.23 0.73 Example 2 38.60 0.82 Example 3 38.26 0.81 Example 4 46.39 1.03 Example 5 36.83 0.63 Example 6 65.85 0.76 Example 7 38.04 0.64 Example 8 38.39 0.63 Example 9 40.40 0.92 Example 9-1 15.95 0.98 Example 9-2 19.20 1.02 Example 10 50.37 0.93 Example 11 53.43 1.10 Example 12 32.94 0.72 Example 13 32.81 0.82 Example 14 32.03 0.71 Example 15 30.75 1.03 Example 16 33.63 0.74 Example 17 34.30 0.83 Example 18 34.59 0.63 Example 19 33.68 0.62 Example 20 35.28 0.65 Example 21 35.95 0.74 Example 22 48.57 1.11 Example 23 95.90 0.83

Experimental Example 4. Injectability Experiment of Sustained-Release Microparticle Injection Formulation Containing Deslorelin

[0097] This experiment was conducted to determine the preferred average particle size of the microspheres through the measurement of the microsphere administration and recovery rate. The specific experimental procedure is as follows.

[0098] 60 mg of the microspheres of Comparative Examples and Examples shown in Table 6 below were weighed, placed in a 1.5 mL vial, and mixed with 0.8 mL of distilled water. Using a 1 mL syringe equipped with a 25G injection needle, the microsphere dispersion was recovered as much as possible, and then the 1.5 mL vial was dried, and the weight of the non-recovered microspheres was measured. The recovery rate was calculated by dividing the weight of the microsphere excluding the measured weight of the non-recovered microsphere by 200 mg of the initial use amount of microsphere.

TABLE-US-00006 TABLE 6 Results of injectability experiment of deslorelin-containing microspheres Sample Recovery rate (%) Example 18 92.1 Example 19 91.8 Comparative Example 6 53.0 Comparative Example 8 93.5 Comparative Example 9 5.6 Comparative Example 10 84.2 Comparative Example 13 94.6

[0099] According to the above results, Example 18, Example 19, Comparative Example 8, Comparative Example 10, and Comparative Example 13 are examples in which the particle size is 100 μm or less and also the span value is 1.2 or less, and in the experiment of recovery rate using a syringe equipped with a 25G injection needle, it showed a high recovery rate from a minimum of 84.2% (w/w) to a maximum of 94.6% (w/w). However, even if it has a low span value as in Comparative Example 9, it was confirmed that when the average particle size exceeded 100 μm, the microspheres blocked the injection needle and were not recovered smoothly. Further, even if the average particle size is 100 μm or less as in Comparative Example 6, when the span value is 1.2 or more, the particle size is not uniform, and the recovery rate of the microsphere was considerably low with about 50% (w/w).

[0100] As a result, both the particle size and the span value affect the injectability, and in particular, when the particle size is 100 μm or less and the span value is 1.2 or less, it was confirmed that it has relatively more excellent injectability than the microspheres that were not so.

Experimental Example 5. Pharmacokinetic Experiment After a Single Subcutaneous Administration in Sprague-Dawley Rat

[0101] After the sustained-release microparticle injection formulation containing deslorelin was administered to rats to evaluate its pharmacokinetics, the concentration of deslorelin in blood over time was measured. The microspheres were measured so that the dose of deslorelin was 4.7 mg/head, dispersed in a 0.3 mL suspension, and then injected subcutaneously into Sprague Dawley rats. 0.5 mL blood was collected at pre-planned time intervals, and the concentrations of deslorelin and testosterone in blood were measured using LC-MS/MS.

[0102] The results of the pharmacokinetic experiments in rats of the formulations prepared in Comparative Examples and Examples were converted into cumulative release rates relative to AUC (area under the curve) and summarized in Table 7 below.

TABLE-US-00007 TABLE 7 Results of pharmacokinetic experiment after a single subcutaneous administration in Sprague-Dawley rat Time (Day) 0 0.042 0.125 1 4 7 Example 2 Cumulative 0 0.347 1.266 4.151 8.143 12.134 Example 4 release rate 0 0.255 1.141 6.136 11.480 15.612 Example 6 of drugs in 0 0.210 0.915 4.411 7.404 10.025 Example 9-2 the body (%) 0 0.403 1.393 3.894 10.454 18.525 Example 19 0 0.462 1.750 6.562 12.379 15.536 Example 22 0 0.416 1.539 5.692 13.045 19.571 Example 23 0 0.308 1.234 5.010 8.342 10.045 Comparative 0 0.536 2.059 8.041 13.241 14.127 Example 1-1 Comparative 0 0.622 2.341 8.693 13.600 13.911 Example 2 Comparative 0 0.461 1.686 5.567 8.061 8.314 Example 5 Comparative 0 0.266 1.082 4.990 13.175 23.950 Example 7 14 21 28 42 56 70 Example 2 Cumulative 19.958 25.190 27.386 30.763 34.668 37.601 Example 4 release rate 25.411 32.112 36.901 46.006 53.106 57.765 Example 6 of drugs in 23.078 37.258 43.747 49.428 51.724 53.522 Example 9-2 the body (%) 33.544 44.246 50.507 57.135 60.682 65.335 Example 19 20.666 26.078 29.792 37.208 48.734 58.450 Example 22 38.017 52.133 56.051 61.574 66.587 71.122 Example 23 12.663 19.381 29.860 49.166 59.514 64.631 Comparative 14.127 14.127 14.127 14.127 14.127 14.661 Example 1-1 Comparative 13.961 13.961 14.608 20.524 34.348 50.133 Example 2 Comparative 10.243 17.375 32.407 68.275 90.115 97.023 Example 5 Comparative 63.174 92.775 97.522 99.517 99.836 100 Example 7 Time (Day) 84 98 112 126 140 154 Example 2 Cumulative 42.562 50.622 60.684 72.052 81.058 86.923 Example 4 release rate 62.261 68.500 75.817 82.523 87.543 91.177 Example 6 of drugs in 57.083 63.385 70.568 77.698 83.425 87.432 Example 9-2 the body (%) 72.183 77.480 80.212 83.635 87.405 90.731 Example 19 63.222 68.066 72.284 76.708 81.117 85.287 Example 22 75.524 79.024 82.515 85.903 88.936 91.823 Example 23 68.537 71.631 74.767 77.616 80.490 83.642 Comparative 20.544 38.713 59.701 74.461 85.119 90.569 Example 1-1 Comparative 61.272 69.617 76.555 82.435 87.069 89.996 Example 2 Comparative 99.494 100 100 100 100 100 Example 5 Comparative 100 100 100 100 100 100 Example 7 Time (Day) 168 182 196 210 224 252 Example 2 Cumulative 91.814 95.803 98.485 99.597 99.805 100 Example 4 release rate 94.135 96.879 98.974 99.709 99.880 100 Example 6 of drugs in 91.414 95.136 97.773 99.094 99.653 100 Example 9-2 the body (%) 93.604 96.241 98.325 99.553 99.940 100 Example 19 90.079 95.223 98.362 99.441 99.968 100 Example 22 94.772 97.743 99.441 99.792 99.903 100 Example 23 86.974 89.750 91.637 93.729 95.988 100 Comparative 93.771 96.331 97.599 98.724 99.550 100 Example 1-1 Comparative 92.736 95.476 97.404 98.857 99.601 100 Example 2 Comparative 100 100 100 100 100 100 Example 5 Comparative 100 100 100 100 100 100 Example 7

[0103] From the above results, it was confirmed that in the case of the biodegradable polymeric microsphere formulation containing deslorelin prepared from a single polymer, the release of drugs in the formulation prepared using poly-lactide-co-glycolide was too short. It was confirmed that the formulations prepared using polylactide have a relatively increased duration of maintaining the blood concentration of drugs , but within the initial 1 month after administration, not only the period during which the drug was hardly released after the initial release occurred, but also the bioavailability was remarkably low.

[0104] However, in the case of the formulation prepared in Examples of the present disclosure to overcome these problems, it was confirmed that the drug was continuously released for 6 months or more after administration. The release pattern of these drugs are not particularly limited, and it was confirmed that preferably, 10% or more of the drug is released within 1 month after administration and 85% or more of the drug is released within 6 months after administration, more preferably, 15% or more of the drug is released within 1 month after administration, 40% to 80% within 3 months after administration, and 85% or more within 6 months after administration, and most preferably, 15% to 70% of the drug is released within 1 month after administration of microsphere, 40% to 80% of the drug is released within 3 months after administration, and 90% or more of the drug is released within 6 months after administration.