WO2024049279A2 - Sustained-release microspheres containing leuprolide, injectable preparation comprising same, and preparation method therefor - Google Patents

Abstract

The present invention relates to sustained-release microspheres containing a high amount of leuprolide, an injectable preparation comprising same, and a preparation method therefor. During administration, leuprolide is released at a sufficient level in an early stage so that effects of leuprolide are exhibited, thereby allowing exposure to a sufficient amount of a drug while inflammatory reactions and the like, which can be problematic during administration, are minimized, and thus the effects of leuprolide can safely be exhibited at least 1 month.

Description

Sustained-release microspheres containing leuprolide, injectable preparations containing the same, and methods for producing the same

The present invention relates to sustained-release microspheres containing leuprolide, an injectable preparation containing the same, a method for producing the same, and a pharmaceutical composition containing the sustained-release microspheres.

More specifically, sustained-release microspheres that contain a high content of leuprolide in the microspheres and exhibit effective drug release characteristics initially while providing stable drug release characteristics for a long period of time, and a method of producing the same, the leuprolide It relates to an injectable preparation containing sustained-release microspheres containing Lyde.

Luteinizing hormone-releasing hormone (LHRH), also known as gonadotropin-releasing hormone (GnRH), is a hypothalamic decapeptide (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-) that regulates the reproductive system in vertebrates. Pro-Gly-NH ₂ ).

LHRH agonists and antagonists are used for the treatment of endometriosis, fibroids, polycystic ovary, breast, ovarian and endometrial cancer in women, gonadotropic pituitary desensitization during medically assisted birth protocols, benign prostate and polymorphism and prostate cancer in men. It is reported to be effective in the treatment of and treatment of precocious puberty in men or women.

Currently used LHRH agonists (Luteinizing Hormone Releasing Hormone agonists) are peptide compounds that generally must be administered via injection due to low oral bioavailability.

In addition, it is known that LHRH agonists are drugs for chronic diseases that require long-term administration, and that early and rapid exposure to a sufficient amount of the drug is required for drug efficacy to manifest. It is known that for the drug efficacy of leuprolide acetate, one of the LHRH agonists, to be expressed, a sufficient amount of drug exposure to the target area is required at the beginning of administration, and it is known that it is desirable to have a high initial drug release rate.

In addition, in the case of medicines for the treatment of such chronic diseases, preparations capable of self-administration are required to increase patient convenience, and to satisfy this, a high amount of drug encapsulated in microspheres is required. However, in the case of conventional sustained-release microspheres containing leuprolide as an active ingredient, there is a problem in that the encapsulation amount is not sufficient. Generally, a heating process is used to remove residual solvents when manufacturing microspheres, but this heating process When used, a problem occurs in which a large amount of leuprolide is lost. In this case, when a large amount of leuprolide is lost, microspheres containing leuprolide must be administered to the patient to achieve the desired sustained-release effect, and in this case, problems such as pain or inflammation may occur.

The present invention was designed to solve the above-described conventional problems. It can contain a high content of leuprolide in the microspheres, and not only exhibits effective drug release characteristics initially, but also provides stable drug release characteristics for a long period of time. The purpose is to provide sustained-release microspheres containing a high content of leuprolide that minimize pain or inflammatory reactions at the site of administration after administration due to administering a relatively small number of microspheres, and a method for producing the same.

In one aspect of the present invention, an injectable preparation comprising sustained-release microspheres containing leuprolide or a pharmaceutically acceptable salt thereof as an active ingredient and a biocompatible polymer,

The content of the active ingredient in the injection preparation includes 1 to 50 mg/mL as leuprolide,

The injectable preparation may have a number of microspheres of 1,000,000 to 6,000,000/ml.

In one aspect of the present invention, leuprolide or a pharmaceutically acceptable salt thereof in the microspheres may be 9.5 to 40% by weight, 10 to 30% by weight, or 12 to 25% by weight based on the total weight of the microspheres.

In one aspect of the present invention, the biocompatible polymer includes polyethylene glycol-poly(lactide-co-glycolide) block-copolymer, polyethylene glycol-polylactide block-copolymer, and polyethylene glycol-polycaprolactone block-copolymer. It is one or more selected from the group consisting of polymer, polylactide, polyglycolide, poly(lactide-co-glycolide), poly(lactide-co-glycolide)glucose, polycaprolactone, and mixtures thereof. You can.

In one aspect of the present invention, the biocompatible polymer in the microspheres may be 70 to 90% by weight, 75 to 90% by weight, or 80 to 90% by weight based on the total weight of the microspheres.

In one aspect of the present invention, the weight ratio of the active ingredient to the biocompatible polymer in the microspheres may be 1:4 to 1:9, 1:3.5 to 1:9, or 1:3 to 1:9.

In one aspect of the present invention, the biocompatible polymer may be a combination of polymers of the same type having different intrinsic viscosity and/or monomer ratios.

In one aspect of the present invention, the pharmaceutically acceptable salt of leuprolide may be leuprolide acetate.

In one aspect of the present invention, the weight average molecular weight of the biocompatible polymer may be 4,000 to 240,000.

In one aspect of the present invention, the in vitro release rate of the injectable formulation may be 8 to 40%, more specifically 10 to 30%, 24 hours after release of the active ingredient.

In one aspect of the present invention, the injectable formulation may be for a 1-month to 3-month formulation.

In one aspect of the present invention, when the injection preparation is administered within an individual, the cumulative drug area under the curve up to 24 hours after administration (AUC _0-24hrs , Area under the curve) is the cumulative drug area under the curve up to the target administration period (AUC 0-24hrs, Area under the curve) AUC _total , that is, for a 1-month formulation, AUC _total is the area under the cumulative drug curve from 0 to 28 days after administration, and for a 3-month formulation, AUC _total refers to the area under the cumulative drug curve from 0 to 84 days after administration. It may be 0.5 to 55%, 15 to 50%, or 20 to 50% of the total.

In one aspect of the present invention, when the injection preparation is administered within a subject, the ratio of the area under the cumulative drug curve at 0 to 24 hours after administration to the area under the cumulative drug curve at 48 hours to 168 hours after administration (i.e., administration Area under the cumulative drug curve for 0 to 24 hours after administration: Area under the cumulative drug curve for 48 hours to 168 hours after administration) is 2:1 to 10:1, preferably 2:1 to 8:1, more preferably It may be 2:1 to 6:1.

In one aspect of the present invention, the residual solvent in the sustained-release microspheres of the injectable formulation may be less than 1,000 ppm.

In one aspect of the present invention, the C _max of the drug after administration of the injection preparation may be 5,000 to 2,000,000 pg/mL.

In one aspect of the present invention, the particle size (D50) of the sustained-release microspheres in the injection preparation may be 10 to 40 um.

In one aspect of the present invention, the injectable preparation reduces fibroid nuclei and improves symptoms in uterine fibroids accompanied by endometriosis, hypermenorrhea, lower abdominal pain, back pain, and anemia, prostate cancer, premenopausal breast cancer, or central precocious puberty. It may be for prevention or treatment.

In one aspect of the present invention, a method for producing sustained-release microspheres containing leuprolide or a pharmaceutically acceptable salt thereof can be provided, comprising the following steps:

a) (i) prepare a dispersed solution by dissolving the biocompatible polymer and leuprolide or a pharmaceutically acceptable salt thereof in one or more organic solvents, or (ii) prepare an oil phase by dissolving the biocompatible polymer in one or more organic solvents. Prepare a solution for the oil phase, prepare an aqueous solution by dissolving leuprolide or a pharmaceutically acceptable salt thereof in an aqueous solvent, and add the aqueous solution to the oil phase solution to form a water-in-oil (W/O) solution. ) Preparing an emulsion;

b) The dispersion solution or water-in-oil emulsion prepared in step a) is added to an aqueous solution containing a surfactant as a continuous phase to form microspheres, and the dispersion solution or water-in-oil emulsion is injected and stirred to solidify the microspheres. preparing a suspension containing microspheres;

c) extracting the organic solvent by adding an aqueous solution containing ethanol and a surfactant to the suspension containing the solidified microspheres of step b);

d) exchanging the continuous phase in the suspension containing the microspheres of step c) with an aqueous solution containing fresh ethanol and a surfactant and stirring; and

e) Recovering microspheres.

In one aspect of the present invention, a method for producing sustained-release microspheres containing leuprolide or a pharmaceutically acceptable salt thereof can be provided, comprising the following steps:

a') (i) dissolving the biocompatible polymer and leuprolide or a pharmaceutically acceptable salt thereof in one or more organic solvents to prepare a dispersion solution, or (ii) dissolving the biocompatible polymer in one or more organic solvents to prepare a dispersion solution. A solution for the oil phase was prepared, and leuprolide or a pharmaceutically acceptable salt thereof was dissolved in an aqueous solvent to prepare an aqueous solution, and the aqueous solution was added to the oil phase solution to form a water-in-oil type (W/ O) preparing an emulsion;

b') The dispersion solution or water-in-oil emulsion prepared in step a') is added to an aqueous solution containing ethanol and a surfactant as a continuous phase to form microspheres, and the microspheres are solidified after injection of the dispersion solution or water-in-oil emulsion. and preparing a suspension containing microspheres by stirring for organic solvent extraction;

c') exchanging the continuous phase in the suspension containing the microspheres of step b') with an aqueous solution containing fresh ethanol and a surfactant and stirring; and

d') Recovering microspheres.

According to the method for producing sustained-release microspheres containing leuprolide according to the present invention, unlike the conventional production method, it is possible to produce sustained-release microspheres containing leuprolide at a high encapsulation rate, allowing the drug to be contained for a long time. It is possible to manufacture microspheres that maximize the therapeutic effect by maintaining the concentration in the therapeutic range.

Figure 1 is a graph showing the in vivo pharmacokinetics of microspheres for a 1-month formulation according to the present invention (Example 1) and Leuprin 3.75 mg (Comparative Example 1), a reference drug.

Figure 2 is a graph showing the in vivo pharmacokinetics of microspheres for a 3-month formulation (Examples 5 and 6) according to the present invention.

Figures 3a and 3b are scanning electron microscope photographs confirming the shape of microspheres of an example according to the present invention.

Figures 4A to 4C are diagrams showing the histopathology slide samples of Example 4 and Comparative Example 2 showing the infiltration of inflammatory cells at the injection site by extracting tissues on the 3rd, 10th, and 28th days after administration of microspheres.

Hereinafter, the present invention will be described in more detail.

In one embodiment, the present invention is an injectable preparation comprising sustained-release microspheres containing leuprolide or a pharmaceutically acceptable salt thereof as an active ingredient and a biocompatible polymer,

The content of the active ingredient in the injection preparation includes 1 to 50 mg/mL as leuprolide,

The present invention relates to an injectable preparation in which the number of microspheres in the injectable preparation is 1,000,000 to 6,000,000/mL.

Specifically, the content of the active ingredient in the injection preparation is leuprolide, 2 to 50 mg/mL, 2 to 47 mg/mL, 2 to 45 mg/mL, 2 to 30 mg/mL, 2 to 27.5 mg/mL, 2 to 5 mg/mL. It may be 25 mg/mL, 3 to 25 mg/mL, or 3 to 22.5 mg/mL. More specifically, if the injectable preparation is a 1-month preparation, it may be 1 to 15 mg/mL, 1 to 12.5 mg/mL, or 2 to 12.5 mg/mL, and if the injectable preparation is a 3-month preparation, it may be 7 to 30 mg/mL, 9 It may be from 27.5 mg/mL or 10 to 25 mg/mL, and for a 6-month preparation, it may be 20 to 50 mg/mL, 20 to 47.5 mg/mL, 25 to 45 mg/mL, 30 to 50 mg/mL, 30 to 47.5 mg/mL. mL, or 30 to 45 mg/mL.

Specifically, the number of microspheres in the injection preparation is 1,000,000 to 5,000,000/ml, 1,200,000 to 4,500,000/ml, 1,000,000 to 4,000,000/ml, 1,200,000 to 3,500,000/ml or 1,500,000. It may be 0 to 3,000,000/mL. More specifically, if the injectable preparation is a 1-month preparation, it may be 1,000,000 to 2,500,000 units/mL or 1,500,000 to 2,000,000 units/mL, and if the injectable preparation is a 3-month preparation, it may be 2,000,000 to 3,500,000 units/mL or 2,500,000 to 3,000, 000 units/mL , if the injectable preparation is a 6-month preparation, it may be 3,000,000 to 6,000,000 units/mL, 3,500,000 to 5,500,000 units/mL, or 4,000,000 to 5,000,000 units/mL.

The present invention, unlike conventional leuprolide-containing microspheres and preparations containing the same, contains a high content of the active ingredient leuprolide or a pharmaceutically acceptable salt thereof in the microspheres, thereby providing an effective level of effective pharmacological effect. It is characterized by containing a sufficient number of ingredients, while allowing a significantly small number of microspheres per unit volume of the injectable preparation to be included in the injectable preparation. Due to these characteristics, the injectable formulation according to the present invention not only exhibits effective drug release characteristics initially and provides stable drug release characteristics for a long period of time, but also provides stable drug release characteristics for a long period of time, and by administering a relatively small number of microspheres, It can minimize pain or inflammatory reactions.

In the present invention, 'leuprolide' is 5-oxo-L-prolyl-L-histidyl-L-tryptophanyl-L-seryl-L-tyrosyl-D-leucyl-L-leucyl-L -Arginyl-L-prolyl ethylamide is an LHGH agonist. The leuprolide may be expressed as leuprorelin. Additionally, in the present invention, all pharmaceutically acceptable salts of leuprolide can be used.

In the present invention, 'pharmaceutically acceptable' means that it is physiologically acceptable and does not usually cause allergic reactions or similar reactions when administered to humans. In the present invention, 'pharmaceutically acceptable salt' refers to an acid addition salt formed from a pharmaceutically acceptable free acid. Organic acids and inorganic acids can be used as the free acids. The organic acids are not limited thereto, but include citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, fumaric acid, formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, metasulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, Includes glutamic acid and aspartic acid. Additionally, the inorganic acids include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid. In a specific embodiment, the pharmaceutically acceptable salt of leuprolide of the present invention may be leuprolide acetate.

In a specific embodiment, the leuprolide or a pharmaceutically acceptable salt thereof may be used in an amount of 9.5 to 40% by weight, 10 to 30% by weight, or 12 to 25% by weight based on the total weight of the microspheres.

In a specific embodiment, the biocompatible polymer is polyethylene glycol-poly(lactide-co-glycolide) block-copolymer, polyethylene glycol-polylactide block-copolymer, polyethylene glycol-polycaprolactone block-copolymer, At least one selected from the group consisting of polylactide, polyglycolide, poly(lactide-co-glycolide), poly(lactide-co-glycolide)glucose, polycaprolactone, and mixtures thereof. It may be from 3 types, 1 to 2 types, or 2 to 3 types.

“Two or more types of biocompatible polymers” means a combination or blend of different types of biocompatible polymer materials (for example, a blend of poly(lactide-co-glycolide) and polylactide). However, a combination of polymers of the same type with different intrinsic viscosity, molecular weight, and/or monomer ratio (e.g., poly(lactide-co-) with a molar ratio of lactide to glycolide of 40:60 A combination of poly(lactide-co-glycolide) with a molar ratio of lactide to glycolide of 50:50, or poly(lactide-co-glycolide) with an intrinsic viscosity of 0.16 dL/g and 1.7 dL/g of poly(lactide-co-glycolide)), or it may be the same type of polymer with different end groups (for example, an ester end group or an acid end group).

Examples of commercially available biocompatible polymers that can be used in the present invention include RG 502H, RG 503H, RG 504H, RG 502, RG 503, RG 504, and RG 653H from the Resomer series of Evonik Rohm GmbH. , RG 752H, RG 753H, RG 752S, RG 755S, RG 756S, RG 858S, R202H, R203H, R205H, R 202S, R203S, R205S, Cobion's PDL 02A, PDL 02, PDL 04, PDL 05, PDLG 7502A , PDLG 7502, PDLG 7507, PDLG 5002A, PDLG 5002, PDLG 5004A, PDLG 5004, PDLG 5010, PL 10, PL 18, PL 24, PL 32, PL 38, PDL 20, PDL 45, PC 02, PC 04, PC 12 , PC 17, and PC 24 alone or in combination or blend, but are not limited thereto.

The appropriate molecular weight or blending ratio of the biocompatible polymer can be appropriately selected by a person skilled in the art considering the decomposition rate of the biocompatible polymer and the resulting drug release rate. In a specific embodiment, the biocompatible polymer contained in the microspheres according to the present invention is Resomer R203H (i.v. = 0.25-0.35 dL/g; weight average molecular weight: 18,000-24,000, manufacturer: Evonik, Germany), Resomer R202H (i.v. = 0.16-0.24 dL/g; weight average molecular weight: 10,000-18,000, manufacturer: Evonik, Germany), Purasorb PDL02A (i.v = 0.16-0.24 dL/g; weight average molecular weight: 10,000-18,000, manufacturer: Purac, Netherlands), Resomer RG752H (i.v. = 0.14-0.22 dL/g; weight average molecular weight: 10,000-18,000, manufacturer: Evonik, Germany), Purasorb PDLG7502A (i.v. = 0.16-0.24 dL/g; weight average molecular weight: 10,000-18,000, manufacturer: Purac , Netherlands).

The biocompatible polymer may preferably be polylactide.

Additionally, an example of the biocompatible polymer being a combination (blend) of two or more types of polymers may be a blend of polylactide and poly(lactide-co-glycolide). In this case, it is preferable that polylactide is contained at least 50%.

Additionally, in the case of poly(lactide-co-glycolide), the molar ratio of lactide to glycolide in the polymer is 40:60 to 90:10, 45:55 to 85:15, or 50:50 to 75:25, For example, it could be 45:55, 50:50, 75:25, or 85:15.

In addition, the intrinsic viscosity of the poly(lactide-co-glycolide) or polylactide is 0.16 dL/g to 1.7 dL/g, 0.16 dL/g to 1.5 dL/g, and 0.16 dL/g to 1.2 dL/g. , 0.16 dL/g to 0.9 dL/g, 0.16 dL/g to 0.6 dL/g, 0.16 dL/g to 0.4 dL/g, 0.2 dL/g to 1.3 dL/g, 0.2 dL/g to 1.0 dL/g. , 0.2 dL/g to 0.7 dL/g, 0.2 dL/g to 0.5 dL/g, 0.24 dL/g to 1.2 dL/g, 0.24 dL/g to 0.7 dL/g, or 0.24 dL/g to 0.5 dL/. It may be g.

The intrinsic viscosity of poly(lactide-co-glycolide) or polylactide used in the present invention is measured at a concentration of 0.1% (w/v) in chloroform at 25°C using an Ubbelohde viscometer. . If the intrinsic viscosity of poly(lactide-co-glycolide) or polylactide is less than 0.16 dL/g, the molecular weight of the polymer is insufficient, making it difficult to exhibit the sustained-release effect of leuprolide or a pharmaceutically acceptable salt thereof. , if the intrinsic viscosity exceeds 1.7 dL/g, the release of leuprolide or a pharmaceutically acceptable salt thereof may be delayed too much. In addition, when manufacturing microspheres using a polymer with high intrinsic viscosity, there is a problem of having to use an excessive amount of production solvent due to the high viscosity of the polymer, and it is difficult to manufacture reproducible microspheres.

The weight average molecular weight of the biocompatible polymer is not particularly limited, but may have a weight average molecular weight of 4,000 to 240,000. For example, the weight average molecular weight of the biocompatible polymer is 4,000 to 100,000, 7,000 to 50,000, 5,000 to 20,000, 10,000 to 18,000, and 18,000 to 28,000. Includes all lower numerical ranges within the above range, such as average molecular weight.

In a specific embodiment, the biocompatible polymer in the microspheres may be 70 to 90% by weight, 75 to 90% by weight, or 80 to 90% by weight based on the total weight of the microspheres.

In a specific embodiment, the weight ratio of the active ingredient to the biocompatible polymer in the microspheres may be 1:4 to 1:9, 1:35 to 1:9, or 1:3 to 1:9.

In a specific embodiment, the sustained-release microspheres containing leuprolide or a pharmaceutically acceptable salt thereof of the present invention may have a particle diameter (D50) of 10 to 40 μm.

In a specific embodiment, the sustained-release microspheres prepared according to the production method according to the present invention may have a significant amount of residual solvent removed compared to the prior art. For example, the residual solvent in the microspheres may be less than 1000 ppm, less than 900 ppm, or 800 ppm. It may be less than, less than 700 ppm, less than 600 ppm, 1000 to 0.01 ppm, 900 to 0.01 ppm, 800 to 0.01 ppm, 700 to 0.01 ppm, or 600 to 0.01 ppm.

In a specific embodiment, the sustained-release microspheres may be suitable for a 1-month to 3-month formulation.

In a specific embodiment, the sustained-release microspheres may have an in vitro release rate of 8 to 40% or 10 to 30% 24 hours after release of the active ingredient.

In a specific embodiment, the sustained-release microspheres may have a C _max of 20,000 to 2,000,000 pg/mL after being administered to rats as an active ingredient at a dose of 1.5 mg/head for 1 month (4 weeks). Preferably, in the case of a 1-month formulation, the C _max of the drug may be 20,000 to 900,000 pg/mL, and in the case of a 3-month formulation, the C _max of the drug may be 50,000 to 2,000,000 pg/mL.

In a specific embodiment, when administering the sustained-release microspheres within an individual, the cumulative drug area under the curve up to 24 hours after administration (AUC _0-24hrs , Area under the curve) is the cumulative drug area under the curve up to the target administration period (AUC) _total , for a 1-month formulation, the area under the cumulative drug curve up to 28 days after drug administration; for a 3-month formulation, the cumulative area under the drug curve up to 84 days after drug administration; and for a 6-month formulation, the area under the cumulative drug curve up to 168 days after drug administration. It may be 0.5 to 55%, 0.6 to 50%, or 0.7 to 50% of the cumulative drug curve area.

More specifically, in the case of a 1-month formulation, the cumulative area under the drug curve up to 24 hours after administration (AUC _0-24hrs , Area under the _curve ) is 12. It may be from 45% to 45%, 20 to 42% or 30 to 40%.

In the case of a 3-month formulation, the cumulative area under the drug curve up to 24 hours after administration (AUC _0-24hrs , Area under the curve) is 30 to 55% or 35% of the cumulative area under the curve until the target administration period (AUC _total ). It may be from 40 to 53%, or from 40 to 50%.

In the case of a 6-month formulation, the cumulative area under the drug curve up to 24 hours after administration (AUC _0-24hrs , Area under the _curve ) is 0.5 to 55%, 0.6%. It may be from 0.7 to 50% or from 0.7 to 50%.

In a specific embodiment, when the sustained-release microspheres are administered within a subject, the ratio of the area under the cumulative drug curve at 0 to 24 hours after administration to the area under the cumulative drug curve at 48 hours to 168 hours after administration (i.e., after administration Area under the cumulative drug curve for 0 to 24 hours: Area under the cumulative drug curve for 48 hours to 168 hours after administration) is 2:1 to 10:1, preferably 2:1 to 8:1, more preferably 2. :1 to 6:1.

In order for leuprolide to exert its effective drug effect, a high blood concentration of the drug must be secured through a significant level of drug release in the initial stage. The sustained-release microspheres according to the present invention, when administered in vivo as described above, exhibit the ratio of the area under the cumulative drug curve at 0 to 24 hours after administration: the area under the cumulative drug curve at 48 hours to 168 hours after administration, Not only does it ensure sufficient initial release of leuprolide or a pharmaceutically acceptable salt thereof, but also maintains the efficacy of the active ingredient for a desired period of time, for example, 1 month or more, 3 months or more, 1 month to 3 months, etc. Enable continuous and sufficient performance.

As another aspect, the present invention,

A method for producing sustained-release microspheres containing leuprolide or a pharmaceutically acceptable salt thereof,

a) (i) prepare a dispersed solution by dissolving the biocompatible polymer and leuprolide or a pharmaceutically acceptable salt thereof in one or more organic solvents, or (ii) prepare an oil phase by dissolving the biocompatible polymer in one or more organic solvents. Prepare a solution for the oil phase, prepare an aqueous solution by dissolving leuprolide or a pharmaceutically acceptable salt thereof in an aqueous solvent, and add the aqueous solution to the oil phase solution to form a water-in-oil (W/O) solution. ) Preparing an emulsion;

b) The dispersion solution or water-in-oil emulsion prepared in step a) is added to an aqueous solution containing a surfactant as a continuous phase to form microspheres, and the dispersion solution or water-in-oil emulsion is injected and stirred to solidify the microspheres. preparing a suspension containing microspheres;

c) extracting the organic solvent by adding an aqueous solution containing ethanol and a surfactant to the suspension containing the solidified microspheres of step b);

d) exchanging the continuous phase in the suspension containing the microspheres of step c) with an aqueous solution containing fresh ethanol and a surfactant and stirring; and

e) relates to a manufacturing method comprising the step of recovering microspheres.

In a further aspect, the present invention provides another method for producing sustained-release microspheres containing leuprolide or a pharmaceutically acceptable salt thereof,

a') (i) dissolving the biocompatible polymer and leuprolide or a pharmaceutically acceptable salt thereof in one or more organic solvents to prepare a dispersion solution, or (ii) dissolving the biocompatible polymer in one or more organic solvents to prepare a dispersion solution. A solution for the oil phase was prepared, and leuprolide or a pharmaceutically acceptable salt thereof was dissolved in an aqueous solvent to prepare an aqueous solution, and the aqueous solution was added to the oil phase solution to form a water-in-oil type (W/ O) preparing an emulsion;

b') The dispersion solution or water-in-oil emulsion prepared in step a') is added to an aqueous solution containing ethanol and a surfactant as a continuous phase to form microspheres, and the microspheres are solidified after injection of the dispersion solution or water-in-oil emulsion. and preparing a suspension containing microspheres by stirring for organic solvent extraction;

c') exchanging the continuous phase in the suspension containing the microspheres of step b') with an aqueous solution containing fresh ethanol and a surfactant and stirring; and

d') relates to a manufacturing method comprising the step of recovering microspheres.

Conventionally, microsphere preparations using biocompatible polymers to develop sustained-release preparations have developed into a field of active research interest and clinical application. However, in order to manufacture a microsphere preparation for long-term continuous administration, the drug to be contained in the microspheres must be encapsulated in a high amount considering the administration period and dosage.

However, in the case of the present inventors, when producing sustained-release microspheres containing leuprolide or a pharmaceutically acceptable salt thereof as an active ingredient, when using commonly used solvent extraction and evaporation methods, the solvent is removed after production of the microspheres. It was discovered that when heat is applied during the process, the encapsulation rate decreases significantly and unexpectedly.

From this perspective, the present invention is a method for producing sustained-release microspheres containing leuprolide or a pharmaceutically acceptable salt thereof, and is characterized in that ethanol is used without heating to remove the residual solvent of the microspheres.

Hereinafter, the manufacturing method according to the present invention will be described in detail for each step.

The step a) or a') is a step of preparing (i) a dispersion solution or (ii) a water-in-oil emulsion for producing microspheres.

When using the dispersion acid solution of (i) above, the emulsion prepared in step b) or b') below is an O/W emulsion, and when using the water-in-oil emulsion of (ii), step b) or The emulsion prepared in b') may be a W/O/W type emulsion.

To prepare the dispersion acid solution of (i) above, the biocompatible polymer and leuprolide or a pharmaceutically acceptable salt thereof are dissolved in one or more organic solvents to prepare a dispersed phase solution. In a specific embodiment, the one or more organic solvents may be two or more organic solvents, and one of the two or more organic solvents may be used as a co-solvent. In a specific embodiment, the biocompatible polymer is dissolved in one organic solvent, and leuprolide or a pharmaceutically acceptable salt thereof is dissolved in another organic solvent to prepare each solution, and then mixed to form a dispersed phase. (i) can be performed by obtaining a solution.

Step a) or step a') may be performed at 15 to 25°C, or at room temperature.

The organic solvent used in steps (i) and (ii) of step a) or a') is dichloromethane, chloroform, ethyl acetate, methyl ethyl ketone, acetone, acetonitrile, dimethyl sulfoxide, dimethyl formamide, It may be at least one solvent selected from the group consisting of enmethylpyrrolidone, acetic acid, methyl alcohol, ethyl alcohol, propyl alcohol, and benzyl alcohol, or a mixed solvent of two or more of the above solvents.

In a specific embodiment, the amount of leuprolide or a pharmaceutically acceptable salt thereof is 9 to 40% compared to the total weight (i.e., total solid weight) of the biocompatible polymer and leuprolide or a pharmaceutically acceptable salt thereof to be prepared. It may be used in weight percent, 10 to 35 weight percent, or 12 to 25 weight percent.

In a specific embodiment, matters relating to the biocompatible polymer may be applied to the injectable preparation containing the sustained-release microspheres, unless otherwise defined.

The appropriate molecular weight or blending ratio of the biocompatible polymer can be appropriately selected by a person skilled in the art in consideration of the decomposition rate of the biocompatible polymer and the resulting drug release rate. In a specific embodiment, the biocompatible polymer contained in the microspheres according to the present invention is Resomer R203H (i.v. = 0.25-0.35 dL/g; weight average molecular weight: 18,000-24,000, manufacturer: Evonik, Germany), Resomer R202H (i.v. = 0.16-0.24 dL/g; weight average molecular weight: 10,000-18,000, manufacturer: Evonik, Germany), Purasorb PDL02A (i.v = 0.16-0.24 dL/g; weight average molecular weight: 10,000-18,000, manufacturer: Purac, Netherlands), Resomer RG752H (i.v. = 0.14-0.22 dL/g; weight average molecular weight: 10,000-18,000, manufacturer: Evonik, Germany), Purasorb PDLG7502A (i.v. = 0.16-0.24 dL/g; weight average molecular weight: 10,000-18,000, manufacturer: Purac , Netherlands) can be used.

The method for producing leuprolide sustained-release microspheres according to the present invention is b) or b') adding the dispersed phase solution or water-in-oil emulsion prepared in step a) or a') to an aqueous solution containing a surfactant as a continuous phase. This includes forming microspheres, and after completing the injection of the dispersion acid solution or water-in-oil emulsion, stirring to solidify the microspheres to prepare a suspension containing the microspheres.

In step b) or b'), the content of the surfactant in the continuous phase containing the surfactant is 0.01% by weight to 20% by weight, preferably 0.1% by weight to 5% by weight, based on the total volume of the continuous phase. You can. If the surfactant content is less than 0.01% by weight, a dispersed phase or emulsion in the form of droplets may not be formed in the continuous phase, and if the surfactant content exceeds 20% by weight, excessive surfactant may cause the formation of a dispersed phase or emulsion in the continuous phase. After the particulates are formed, it may be difficult to remove the surfactant.

The surfactant in step b) or b') is methylcellulose, polyvinylpyrrolidone, carboxymethylcellulose, lecithin, gelatin, polyvinyl alcohol, polyoxyethylene sorbitan fatty acid ester, and polyoxyethylene castor oil derivatives, and their It may be one or more types selected from the group consisting of mixtures.

The aqueous solvent for preparing the aqueous solution containing the surfactant in step b) or b') is water, or a solvent selected from the group consisting of water and methyl alcohol, ethyl alcohol, propyl alcohol, and ethyl acetate, or a mixed solvent thereof. It may be.

In step b) or b'), the method of homogeneously mixing the biocompatible polymer solution in which leuprolide or a pharmaceutically acceptable salt thereof is dispersed and the continuous phase containing the surfactant is not particularly limited, but includes a high-speed stirrer, It can be performed using an in-line mixer, ultrasonic disperser, static mixer, membrane emulsion method, microfluidics emulsion method, etc. When forming an emulsion using a high-speed stirrer, in-line mixer, ultrasonic disperser, or static mixer, it is difficult to obtain a uniform emulsion, so an additional particle size selection process may be performed between each step described later.

In a specific embodiment, the stirring process in step b) or b') may be performed for 1 to 5 hours, 2 to 4 hours, or 3 hours for solidification. Although not limited thereto, step b) or b') may be performed at 4 to 24°C, 10 to 17°C, or 15°C.

Additionally, in step b'), an aqueous solution further containing ethanol in addition to a surfactant may be used as a continuous phase to extract the organic solvent during the formation and solidification of the microspheres. In step b'), the content of ethanol in the aqueous solution containing ethanol and surfactant is 5 to 40 (v/v)%, 5 to 30 (v/v)%, 5 to 5%, based on the total volume of the aqueous solution. It may be 20(v/v)%, 7 to 40(v/v)%, 7 to 30(v/v)%, 7 to 20(v/v)% or 10 to 20(v/v)%. . If the ethanol content is less than 5(v/v)%, the residual amount of organic solvent may increase, and if it exceeds 30(v/v)%, the encapsulation rate may be reduced or initial release control may be difficult. .

In step c), an aqueous solution containing ethanol and a surfactant is added to the suspension containing the solidified microspheres prepared in step b) to extract the organic solvent in a continuous phase.

In the present invention, in step c), the temperature is lower than that of the prior art, at 4 to 24°C, 10 to 17°C, or 15°C for a certain period of time, for example, 1 hour to 48 hours, 5 to 36 hours, or 7 to 15°C. The organic solvent can be effectively extracted from the microspheres by maintaining or stirring for 24 hours, 10 to 20 hours, or 15 hours. Some of the extracted organic solvent may evaporate from the surface of the microspheres.

The type of surfactant used in step c) may be the same as that used in step b).

Additionally, the solvent in the aqueous solution of step c) may be water or a mixed solvent of water and one or more solvents selected from the group consisting of methyl alcohol, ethyl alcohol, propyl alcohol, and ethyl acetate.

In step c), the content of ethanol in the aqueous solution containing ethanol and surfactant is 5 to 40 (v/v)%, 5 to 30 (v/v)%, 5%, based on the total volume of the aqueous solution. to 20 (v/v)%, 7 to 40 (v/v)%, 7 to 30 (v/v)%, 7 to 20 (v/v)% or 10 to 20 (v/v)%. there is. If the ethanol content is less than 5(v/v)%, the residual amount of organic solvent may increase, and if it exceeds 30(v/v)%, the encapsulation rate may be reduced or initial release control may be difficult. .

In step d) or c'), after extracting the organic solvent in step c) or b'), it is exchanged with an aqueous solution containing fresh ethanol and a surfactant and incubated for a certain period of time, specifically 1 hour to 48 hours, 1 hour to 48 hours. Stir for 24 hours, 1 hour to 12 hours, and 1 hour to 5 hours. Preferably, the holding time may be 3 hours, but is not limited thereto.

Thereafter, in step e) or d'), the microspheres are finally recovered.

In step e) or d'), the method of recovering microspheres according to the present invention may be performed using various known techniques, for example, methods such as filtration or centrifugation may be used. In step e) or d'), the microsphere suspension upon recovery is further washed with a washing liquid (e.g. water, specifically ultrapure water), preferably repeatedly, to remove residual surfactant.

In the production method of the present invention, after step e) or d'), the obtained microspheres are dried using a conventional drying method, for example, freeze-drying, to obtain final dried microspheres.

In one specific embodiment, the sustained-release microspheres containing leuprolide or a pharmaceutically acceptable salt thereof prepared by the production method of the present invention may have a particle diameter (D50) of 10 to 40 μm.

In a specific embodiment, the sustained-release microspheres prepared according to the production method according to the present invention may have a significant amount of residual solvent removed compared to the prior art. For example, the residual solvent in the microspheres may be less than 1000 ppm.

In one aspect of the present invention, the encapsulation rate of the prepared sustained-release microspheres is 60% or more, 60 to 100%, 70% or more, 70 to 100%, 75% or more, 75 to 100%, 75 to 98%, or 75%. It may be from 95% to 95%. Preferably, it may be 80% or more, 80 to 100%, 80 to 98%, 90 to 100%, 95 to 100%, or 80 to 95%.

[Example]

Hereinafter, preferred embodiments are presented to aid understanding of the present invention. However, the following examples are merely illustrative of the present invention, and it is clear to those skilled in the art that various changes and modifications are possible within the scope and spirit of the present invention. It is natural that such variations and modifications fall within the scope of the attached patent claims.

Examples 1 to 7: Preparation of leuprolide microspheres using ethanol in co-solvent and O/W emulsion process

In order to confirm the effect of sustained-release microspheres containing leuprolide or a pharmaceutically acceptable salt thereof according to the present invention, microspheres of leuprolide or a pharmaceutically acceptable salt thereof were prepared.

The dispersed phase is a mixture of biocompatible polymers PLGA or PLA and leuprolide acetate (manufacturer: Polypeptide Laboratories Pvt, Ltd., India) with dichloromethane (manufacturer: J.T Baker, USA) and methyl alcohol (manufacturer: Tedia Company, USA). Alternatively, it was mixed with ethyl acetate (manufacturer: Junsei chemical Co. Ltd., Japan) and methyl alcohol (manufacturer: Tedia Company, USA) and dissolved until it became transparent to the naked eye. A 1.0% (w/v) polyvinyl alcohol (viscosity: 4.8-5.8 mPa·s) aqueous solution was used as the continuous phase, and the continuous phase was connected to an emulsifier equipped with a porous membrane and the prepared dispersed phase was injected to prepare microspheres. did. The temperature of the membrane emulsification device, preparation vessel, and microsphere suspension was maintained at 15°C, and after dispersion phase injection was completed, it was stirred at 200 rpm for 3 hours. Ethanol was added and stirred for 18 hours to extract the organic solvent, and then stirred in the same continuous phase for 3 hours. The microsphere suspension was washed several times with ultrapure water to remove residual polyvinyl alcohol, and the microspheres were recovered by lyophilization.

The biocompatible polymer used as the dispersed phase is PLA or PLGA polymer, Resomer R203H (i.v. = 0.25-0.35 dL/g; weight average molecular weight: 18,000-24,000, manufacturer: Evonik, Germany), Resomer R202H (i.v. = 0.16-0.24 dL) /g; Weight average molecular weight: 10,000-18,000, Manufacturer: Evonik, Germany), Purasorb PDL02A (i.v = 0.16-0.24 dL/g; Weight average molecular weight: 10,000-18,000, Manufacturer: Purac, Netherlands), Resomer RG752H (i.v. = 0.14-0.22 dL/g; weight average molecular weight: 10,000-18,000, manufacturer: Evonik, Germany), Purasorb PDLG7502A (i.v = 0.16-0.24 dL/g; weight average molecular weight: 10,000-18,000, manufacturer: Purac, Netherlands) alone. Or it was used in combination.

The type of polymer used in this preparation example, the theoretical content of the drug, the solvent used, the concentration of ethanol in the continuous phase, and the temperature of the residual solvent removal process are shown in Table 1 below.

		Example	microsphere polymer	TL (%)	Solvent 1	Solvent 2	Surfactants	Residual solvent removal process	Temperature (℃)
Example) Leuprolide microspheres (using ethanol process)	1 month	Example 1	PDLG 7502A	10	DCM	MeOH	PVA	Solvent Extraction (10% EtOH)	15
		Example 2	PDLG 7502A	15	DCM	MeOH	PVA	Solvent Extraction (20% EtOH)	15
		Example 3	PDLG 7502A	20	DCM	MeOH	PVA	Solvent Extraction (20% EtOH)	15
		Example 3-1	PDLG 7502A	15	Ethyl acetate	MeOH	PVA	Solvent Extraction (20% EtOH)	15
	3 months	Example 4	R202H	20	DCM	MeOH	PVA	Solvent Extraction (10% EtOH)	15
		Example 5	R202H	20	DCM	MeOH	PVA	Solvent Extraction (20% EtOH)	15
		Example 6	R202H:R203H=7:3	20	DCM	MeOH	PVA	Solvent Extraction (20% EtOH)	15
		Example 7	R202H	17.5	DCM	MeOH	PVA	Solvent Extraction (20% EtOH)	15

Comparative Examples 1 and 2: Control drug

Currently commercially available leuprolide microsphere control drugs, Leuplin 3.75 mg (manufacturer: Takeda) and Leuplin 11.25 mg (manufacturer: Takeda) were named Comparative Examples 1 and 2, respectively.

Comparative Example 3: Preparation of leuprolide microspheres without using ethanol process for organic solvent extraction 1 (O/W formulation)

Leuprolide microspheres that did not use the ethanol process for organic solvent extraction were prepared by the following method.

The dispersed phase is a mixture of biocompatible polymers PLGA or PLA and leuprolide acetate (manufacturer: Polypeptide Laboratories Pvt, Ltd., India) with dichloromethane (manufacturer: J.T Baker, USA) and methyl alcohol (manufacturer: Tedia Company, USA). It was dissolved until it became transparent to the naked eye. A 0.5% (w/v) polyvinyl alcohol (viscosity: 4.8-5.8 mPa·s) aqueous solution was used as the continuous phase, and the continuous phase was connected to an emulsifier equipped with a porous membrane and the prepared dispersed phase was injected to prepare microspheres. did. The temperature of the membrane emulsification device, preparation vessel, and microparticle suspension was maintained at 25°C, and after the injection of the dispersed phase was completed, the organic solvent was removed while maintaining the temperature of the microparticle suspension at 36°C for 3 hours. After removal of the organic solvent, the temperature of the microparticle suspension was lowered to 25°C. The microsphere suspension was washed several times with ultrapure water to remove residual polyvinyl alcohol, and the microspheres were freeze-dried.

The biocompatible polymer used as the dispersed phase was PLA or PLGA polymer, and Purasorb PDLG7502A (i.v = 0.16-0.24 dL/g; weight average molecular weight: 10,000-18,000, manufacturer: Purac, Netherlands) was used alone.

The type of polymer used in this production example, the theoretical content of the drug, the solvent used, and the temperature of the residual solvent removal process are shown in Table 3 below.

Comparative Example 4: Preparation of leuprolide microspheres without using ethanol process for organic solvent extraction 2 (W/O/W)

Leuprolide microspheres that did not use the ethanol process for organic solvent extraction were prepared by the following method.

The dispersed phase was prepared by dissolving the biocompatible polymers PLGA or PLA and leuprolide acetate (manufacturer: Polypeptide Laboratories Pvt, Ltd., India) in dichloromethane (manufacturer: J.T Baker, USA) and distilled water, respectively, to prepare an oil phase and an aqueous phase. A W/O emulsion was formed by emulsifying at 12,000 rpm using an IKA homogenizer. A 0.5% (w/v) polyvinyl alcohol (viscosity: 4.8-5.8 mPa·s) aqueous solution was used as the continuous phase, and microspheres were prepared by connecting the continuous phase to an emulsifier equipped with a porous membrane and simultaneously injecting the prepared dispersed phase. . The temperature of the membrane emulsification device, preparation vessel, and microparticle suspension was maintained at 25°C, and after the injection of the dispersed phase was completed, the microparticle suspension was heated and maintained for 3 hours to remove the organic solvent. After removal of the organic solvent, the temperature of the microparticle suspension was lowered to 25°C. The microsphere suspension was washed several times with ultrapure water to remove residual polyvinyl alcohol, and the microspheres were freeze-dried.

The biocompatible polymer used as the dispersed phase is PLA or PLGA polymer, Resomer R202H (i.v. = 0.16-0.24 dL/g; weight average molecular weight: 10,000-18,000, manufacturer: Evonik, Germany), Purasorb PDL02A (i.v. = 0.16-0.24 dL/g). g; weight average molecular weight: 10,000-18,000, manufacturer: Purac, Netherlands), and Resomer RG752H (i.v. = 0.14-0.22 dL/g; weight average molecular weight: 10,000-18,000, manufacturer: Evonik, Germany) were used alone.

The type of polymer used in this production example, the theoretical content of the drug, the solvent used, and the temperature of the residual solvent removal process are shown in Table 2 below.

		Comparative example number	microsphere polymer	TL (%)	Solvent 1	Solvent 2	Surfactants	Residual solvent removal process	Temperature (℃)
Comparative example) Leuprolide microspheres (without ethanol process)	1 month	Comparative Example 3	PDLG7502A	20	DCM	MeOH	PVA	Solvent evaporation	36
	3 months	Comparative Example 4	PDL02A	16.0	DCM	Water	PVA	Solvent evaporation	45

Experimental Example 1. Measurement of drug content of leuprolide sustained-release microspheres

To measure the drug content of the microspheres prepared in the above example, 10 mg of microspheres were completely dissolved in 2.5 mL of acetonitrile, and then extracted by adding 7.5 mL of 0.1% (w/w) trifluoroacetic acid aqueous solution. The solution filtered using a 0.45 um filter was used as the test solution. 20uL of the test solution was injected into the HPLC and measured at a detection wavelength of 280nm. The column used in this measurement was Inertsil ODS-3, 5 um, 4.6x150 mm, and the mobile phase was acetonitrile containing 0.1% (w/w) trifluoroacetic acid and 0.1% (w/w) trifluoroacetic acid. Acetic acid aqueous solution was mixed and used at a ratio of 25:75 (v/v). The measured encapsulation amounts are shown in Table 3.

Experimental Example 2. Particle size analysis of sustained-release microspheres

A test was conducted using laser diffraction to quantitatively measure the average particle size, distribution, and uniformity of microspheres.

The particle size measurement results of the sustained-release microspheres prepared in the above examples and comparative examples are shown in Table 3 below.

	duration	Example and Comparative Example No.	Particle size (D50)	Inclusion rate (E.E, (w/w)%))	Drug content in microspheres (D.C, (w/w)%)
Example) Leuprolide microspheres (using ethanol process)	1 month	Example 1	24.77	E.E. 98.1%	DC 9.81%
		Example 2	32.45	E.E 91.2%	DC 13.67%
		Example 3	27.64	E.E 79.6%	DC 15.92%
	3 months	Example 4 (GB-7103-519)	38.09	EE 95.5%	DC 19.09%
		Example 5	38.63	E.E. 93.8%	DC 18.76%
		Example 6	30.05	E.E 99.1%	DC 19.82%
		Example 7	22.48	E.E. 92.6%	DC 16.21%
Comparative example) Control drug (Leuplin)	1 month	Comparative Example 1	11.61	-	DC 7.72%
	3 months	Comparative Example 2	12.49	-	DC 8.49%
Comparative example) Leuprolide microspheres (without ethanol process)	1 month	Comparative Example 3	23.98	E.E 37.2%	DC 7.45%
	3 months	Comparative Example 4	46.26	E.E 59.4%	DC 9.48%

As shown in Table 3 above, it can be seen that a high encapsulation rate is maintained when the solvent is extracted by performing ethanol extraction compared to the microspheres prepared through the solvent evaporation process. In the case of the existing solvent evaporation process, it was confirmed that the main component in the microspheres was lost during the manufacturing process due to the high temperature of the continuous phase, which resulted in a decrease in the encapsulation rate.

Experimental Example 3: In vitro drug initial release measurement of leuprolide microspheres

The following experiment was performed to confirm the initial drug release of the microspheres prepared in the above Examples and Comparative Examples. Place 10 mg of microspheres in an HDPE wide-mouth bottle, fill with 50 mL of release test solution, and store in an incubator at 37°C. After 24 hours, 1 mL of this sample solution was taken and centrifuged, and the obtained supernatant was analyzed for leuprolide content and release rate using HPLC under the same analysis conditions as in Experimental Example 1. The release test solution for this measurement was a pH 7.4 aqueous solution containing phosphate and sodium azide.

The results are shown in Table 4.

		Example number	in vitro
			Initial release rate (%, 1-day)
Example) Leuprolide microspheres (using ethanol process)	1 month	Example 1	18.04
		Example 2	28.99
		Example 3	39.41
	3 months	Example 4	19.14
		Example 5	26.81
		Example 6	9.42
		Example 7	23.11
Comparative example) Control drug (Leuplin)	1 month	Comparative Example 1	13.98
	3 months	Comparative Example 2	8.14
Comparative example) Leuprolide microspheres (solvent evaporation method)	1 month	Comparative Example 3	6.21
	3 months	Comparative Example 4	16.99

Experimental Example 4. Evaluation of drug release profile characteristics of leuprolide microspheres

Pharmacokinetic evaluation was performed on the microspheres of Examples and Preparation Examples using 9-week-old SD (Sprague-Dawly) rats. The microsphere injection preparations of Examples and Preparation Examples in rats were measured at a dose of 1.5 mg/head as an active ingredient for 1 month (4 weeks) in rats, suspended in 0.3 mL dispersion solvent, and then subcutaneously injected into SD rats. . 0.25 to 0.5 mL of blood was collected at pre-planned times, and blood leuprolide concentration was measured using LC-MS/MS. The measurement results are shown in Table 5 and Figures 1 (1-month formulation) and Figure 2 (3-month formulation).

		Example number	In vivo		Cumulative AUC (%)
			Dose (mg/head)	C max (ng/mL)	0-24hrs	0-48hrs	0-168hrs
Example) Leuprolide microspheres (using ethanol process)	1 month	Example 1 (GB-7101-025)	1.5	97.6	22.9	28.2	40.9
	3 months	Example 5	4.5	946.7	46.4	51.1	58.6
		Example 6	4.5	863.0	44.6	50.5	63.8
Comparative example) Control drug (Leuplin)	1 month	Comparative Example 1	1.5	113.6	22.9	28.2	40.9
	3 months	Comparative Example 2	4.5	413.0	49.4	53.4	60.8
Comparative example) Leuprolide microspheres (solvent evaporation method)	3 months	Comparative Example 4	1.5	286.0	50.7	53.2	65.3

Experimental Example 5. Morphological analysis through electron microscopy

The morphological characteristics of the microspheres according to the present invention were analyzed through electron microscopy. The experimental procedure is as follows. 5 mg of microspheres prepared in Examples and Preparation Examples were placed on an aluminum stub with carbon tape attached and coated with platinum using ION-COATER (COXEM, Korea). An aluminum stub was mounted on a scanning electron microscope (COXEM EM-30, Korea), and the morphological characteristics of the microspheres were observed at an acceleration voltage of 10~ kV. The results are shown in Figures 3a (1-month formulation) and 3b (3-month formulation).

As shown in Figures 3a and 3b, it was confirmed that the microspheres according to the present invention maintained their spherical shape well. In the case of the comparative example in which the residual solvent was removed through heating, it underwent relatively rapid solidification and showed a porous surface. On the other hand, the formulations in the example where solidification was gradually carried out at low temperature through ethanol extraction were confirmed to have a smooth surface.

Experimental Example 6. Analysis of residual solvent of leuprolide microspheres

To measure the residual amount of dichloromethane used as a solvent for the dispersion phase, weigh about 100 mg of a microsphere sample, place it in a headspace vial, suspend it in 0.5 mL of purified water, dissolve it in 5 mL of N-methylpyrrolidone, and perform gas chromatography. Residual solvent was measured by (GC). The column used at this time was DB-624 (manufacturer: Agilent, USA) (0.53mm . The temperature of SPL was maintained at 150°C, and the split ratio of the sample was 1:5. Carrier gas used high purity helium gas. The temperature was maintained at 50°C for 5 minutes at a flow rate of 5.0 ml/min, raised to 250°C at a rate of 10°C per minute, and maintained for 5 minutes.

1) Preparation of standard solution

Accurately weigh 120 mg of dichloromethane standard, place in a 100 mL volumetric flask, add approximately 80 mL of N-methylpyrrolidone, mix, and then add N-methylpyrrolidone to make exactly 100 mL. Take exactly 10 mL of this solution, place it in a 100 mL volumetric flask, and add N-methylpyrrolidone to make exactly 100 mL. Accurately take 5 mL of this solution and 0.5 mL of water, place them in a headspace vial, cap and seal, and use the mixed solution as the standard solution.

2) Preparation of test solution

Weigh approximately 100 mg of the sample, place it in a headspace vial, suspend it in 0.5 mL of purified water, and then add 5 mL of N-methylpyrrolidone to dissolve it. Cover with a stopper, seal it, and use it as the sample solution.

As a result of the above experiment, the content of residual dichloromethane in the measured microspheres is shown in Table 6.

		Example number	residual solvent
			Dichloromethane (ppm)
Example) Leuprolide microspheres (using ethanol process)	1 month	Example 1	45.5
		Example 2	261.8
		Example 3	0.0
	3 months	Example 5	473.8
		Example 6	446.4
		Example 7	383.5
Comparative example) Leuprolide microspheres (solvent evaporation method)	1 month	Comparative Example 3	221.3
	3 months	Comparative Example 4	78.9

As shown in Table 6, as the amount of ethanol used increases, the residual amount of dichloromethane in the microspheres decreases.

It is advantageous to ensure the storage stability of sustained-release microspheres containing leuprolide and their safety to the human body as the amount of residual solvent in the microspheres decreases. Therefore, it was confirmed that the microspheres according to the present invention have excellent storage stability and safety to the human body due to the low residual solvent amount of less than 1000 ppm.

Experimental Example 7. Particle number analysis of leuprolide microspheres

The particle number of microspheres according to the present invention was measured as follows. The number of microspheres was confirmed by directly checking the number of particles using an optical microscope. 10 mg of microspheres prepared in Examples and Comparative Examples were weighed and dispersed in 0.5 (w/w)% PVA. At this time, the comparative example containing 15 (w/w)% mannitol was performed by correcting the weight (weight correction was performed by weighing 11.8 mg so that the weight of the microspheres was 10 mg when mannitol was excluded). The microspheres dispersed in 0.5 (w/w)% PVA were diluted with 0.5 (w/w)% PVA so that the concentration was 0.1 mg/mL. 10 μm of the diluted 0.1 mg/mL concentration microsphere solution was taken and the number of particles was confirmed under a microscope.

Afterwards, the administration dose was adjusted so that the content of the active ingredient (API) in the examples and comparative examples was the same, and is shown in Tables 7 and 8. Table 7 shows the 1-month formulation, and Table 8 shows the 3-month formulation.

Based on one administration	Example 2	Comparative Example 1 (Leuplin 3.75mg)
API concentration (mg/mL)	3.75	3.75
Microparticle concentration (mg/mL)	27.4	44.1
Number of microspheres (piece/mg)	67,000	149,300
Number of administered microspheres (number/mL)	1,836,623	6,586,765

Based on one administration	Example 4	Comparative Example 2 (Leuplin 11.25mg)
API concentration (mg/mL)	11.25	11.25
Microparticle concentration (mg/mL)	58.9	130.1
Number of microspheres (piece/mg)	49,700	262,300
Number of administered microspheres (number/mL)	2,927,356	34,114,162

As can be seen from the results in Tables 7 and 8 above, even if they contain the same amount of active ingredients, the conventional injection preparation containing leuprolide has a microparticle concentration that is 1.6 to 2.2 times higher than the injection preparation according to the present invention. With this, it was confirmed that the number of microspheres was also 3.6 to 11.7 times greater. This has the advantage that the injection preparation according to the present invention has a significantly lower number and concentration of microspheres compared to the same active ingredient content, and can reduce various problems that may occur due to a large number of microspheres and a high concentration of microspheres upon administration, such as the level of inflammation. You can have it.

Experimental Example 8: Comparison of inflammation according to the number of microparticles

The degree of inflammation at the administration site when the microspheres prepared in Example 4 and Comparative Example 2 were administered was confirmed. First, 0.5 mL of suspension (Diluent) (0.5(w/w)% NaCMC, 5(w/w)% Mannitol, 0.1(w/w)% Tween 80) was added to the microtube containing the test substance, and the microspheres were added. Mix well until completely dispersed. After taking the suspension using a 1mL syringe (23G needle), the microspheres were administered subcutaneously to the back of SD rats at a dose of 4.5mg/head. Tissues were removed on the 3rd, 10th, and 28th days after administration and analysis of inflammation at the administration site was performed, which is shown in Table 9.

Formulation	Dosage (mg/head as API)	Dosage amount (mL/head)	number of animals
			3-day inflammation analysis	10-day inflammation analysis	28-day inflammation analysis
Example 4 (GB-7103-519)	4.5	0.5	3	3	3
Comparative Example 2 (Leuplin 11.25mg)	4.5	0.5	3	3	3

(SD rat, male 8 weeks old) At each confirmation time, histopathology slide samples of Example 4 and Comparative Example 2 were prepared, stained with H&E (Hematoxylin and Eosin), and inflammatory cell infiltration was confirmed. Photographs of samples stained in this way are shown in Figures 4a (3 days), 4b (10 days), and 4c (28 days). Yellow circles represent infiltrated cells, red arrows represent angiogenesis, and blue arrows represent fibrous tissue formation.

Table 10 is a table that quantifies the degree of inflammatory cell infiltration on the 3rd, 10th, and 28th days after administration of microspheres to SD rats.

division		Example 4	Comparative Example 2 (Leuplin 11.25mg)
Inflammatory cell infiltration	Day 3	2.78±0.19	3.00±0.00
	Day 10	1.87±0.51	2.33±0.34
	Day 28	1.89±0.58	3.00±0.00

* Inflammatory cell infiltration is graded from 0 to 3 by randomly selecting three parts at 400x field of view (HPF), counting the number of cells. - Grade 0 is ≤5 cells per HPF.

- Grade 1 is 6-20 cells per HPF

- Grade 2 is 21-50 cells per HPF

- Grade 3 is >50 cells per HPF

Referring to FIGS. 4A to 4C and Table 11, on the 3rd day after microball administration, the inflammatory response was strong in both Example 4 and Comparative Example 2, so no significant difference could be confirmed, but on the 10th and 28th day after microball administration, the inflammatory response was strong. While the inflammatory response continued in Example 2, it was confirmed that the inflammatory response was relatively reduced in Example 4.

Claims (30)

1. An injectable preparation containing sustained-release microspheres containing leuprolide or a pharmaceutically acceptable salt thereof as an active ingredient and a biocompatible polymer,

   The content of the active ingredient in the injection preparation includes 1 to 50 mg/mL as leuprolide,

   An injectable preparation, wherein the number of microspheres in the injectable preparation is 1,000,000 to 6,000,000/ml.
2. The injectable preparation according to claim 1, wherein the content of the active ingredient is leuprolide, comprising 9.5 to 40% by weight based on the total weight of microspheres.
3. The method of claim 1, wherein the biocompatible polymer is polyethylene glycol-poly(lactide-co-glycolide) block-copolymer, polyethylene glycol-polylactide block-copolymer, and polyethylene glycol-polycaprolactone block-copolymer. , polylactide, polyglycolide, poly(lactide-co-glycolide), poly(lactide-co-glycolide)glucose, polycaprolactone, and mixtures thereof. Injectable preparation.
4. The injectable preparation according to claim 1, wherein the biocompatible polymer in the microspheres is 70 to 90% by weight based on the total weight of the microspheres.
5. The injectable preparation according to claim 1, wherein the weight ratio of the active ingredient to the biocompatible polymer in the microspheres is 1:4 to 1:9.
6. The injectable preparation according to claim 1, wherein the pharmaceutically acceptable salt of leuprolide is leuprolide acetate.
7. The injectable preparation according to claim 1, wherein the biocompatible polymer has a weight average molecular weight of 4,000 to 240,000.
8. The injectable preparation according to claim 1, wherein the residual solvent in the sustained-release microspheres is less than 1000 ppm.
9. The injectable preparation according to claim 1, wherein the in vitro release rate 24 hours after release of the active ingredient is 8 to 40%.
10. The injectable preparation according to claim 1, wherein the AUC _0-24hrs up to 24 hours after administration of the active ingredient is 0.5 to 55% of the cumulative AUC _total during the target administration period.
11. The method of claim 1, wherein, upon intra-subject administration of sustained-release microspheres, the ratio of the area under the cumulative drug curve at 0 to 24 hours after administration to the area under the cumulative drug curve at 48 hours to 168 hours after administration is 2:1 to 2:1. 10:1, injectable formulation.
12. The injectable formulation according to claim 1, wherein the sustained-release microspheres have a D50 of 10 to 40 μm.
13. The injectable preparation according to claim 1, wherein the injectable preparation is for one-month administration, and the number of microspheres in the injectable preparation is 1,000,000 to 2,500,000/ml.
14. The injectable preparation according to claim 1, wherein the injectable preparation is for administration for 3 months, and the number of microspheres in the injectable preparation is 2,000,000 to 3,500,000/ml.
15. The injectable preparation according to claim 1, wherein the injectable preparation is for 6-month administration, and the number of microspheres in the injectable preparation is 3,000,000 to 6,000,000/ml.
16. The method of claim 1, for use in the reduction of fibroid nuclei and improvement of symptoms in uterine fibroids accompanied by endometriosis, hypermenorrhea, lower abdominal pain, back pain, and anemia, and in the prevention or treatment of prostate cancer, premenopausal breast cancer, or central precocious puberty. , injection preparation.
17. a) (i) prepare a dispersed solution by dissolving the biocompatible polymer and leuprolide or a pharmaceutically acceptable salt thereof in one or more organic solvents, or (ii) prepare an oil phase by dissolving the biocompatible polymer in one or more organic solvents. Prepare a solution for the oil phase, prepare an aqueous solution by dissolving leuprolide or a pharmaceutically acceptable salt thereof in an aqueous solvent, and add the aqueous solution to the oil phase solution to form a water-in-oil (W/O) solution. ) Preparing an emulsion;

    b) The dispersion solution or water-in-oil emulsion prepared in step a) is added to an aqueous solution containing a surfactant as a continuous phase to form microspheres, and the dispersion solution or water-in-oil emulsion is injected and stirred to solidify the microspheres. preparing a suspension containing microspheres;

    c) extracting the organic solvent by adding an aqueous solution containing ethanol and a surfactant to the suspension containing the solidified microspheres of step b);

    d) exchanging the continuous phase in the suspension containing the microspheres of step c) with an aqueous solution containing fresh ethanol and a surfactant and stirring; and

    e) A method for producing sustained-release microspheres comprising the step of recovering the microspheres,

    The sustained-release microspheres are sustained-release microspheres containing 9.5 to 40% by weight of leuprolide or a pharmaceutically acceptable salt thereof and a biocompatible polymer as leuprolide relative to the total weight of the microspheres.
18. a') (i) dissolving the biocompatible polymer and leuprolide or a pharmaceutically acceptable salt thereof in one or more organic solvents to prepare a dispersion solution, or (ii) dissolving the biocompatible polymer in one or more organic solvents to prepare a dispersion solution. A solution for the oil phase was prepared, and leuprolide or a pharmaceutically acceptable salt thereof was dissolved in an aqueous solvent to prepare an aqueous solution, and the aqueous solution was added to the oil phase solution to form a water-in-oil type (W/ O) preparing an emulsion;

    b') The dispersion solution or water-in-oil emulsion prepared in step a') is added to an aqueous solution containing ethanol and a surfactant as a continuous phase to form microspheres, and the microspheres are solidified after injection of the dispersion solution or water-in-oil emulsion. and preparing a suspension containing microspheres by stirring for organic solvent extraction;

    c') exchanging the continuous phase in the suspension containing the microspheres of step b') with an aqueous solution containing fresh ethanol and a surfactant and stirring; and

    d') A method for producing sustained-release microspheres comprising the step of recovering the microspheres,

    The sustained-release microspheres are sustained-release microspheres containing 9.5 to 40% by weight of leuprolide or a pharmaceutically acceptable salt thereof and a biocompatible polymer as leuprolide relative to the total weight of the microspheres.
19. The method of claim 17 or 18, wherein the organic solvent in step a) or a') is dichloromethane, chloroform, ethyl acetate, methyl ethyl ketone, acetone, acetonitrile, dimethyl sulfoxide, dimethylformamide, and enmethyl p. A manufacturing method, which is a solvent selected from the group consisting of rolidone, acetic acid, methyl alcohol, ethyl alcohol, propyl alcohol, and benzyl alcohol, or a mixed solvent thereof.
20. The method of claim 17 or 18, wherein the biocompatible polymer is polyethylene glycol-poly(lactide-co-glycolide) block-copolymer, polyethylene glycol-polylactide block-copolymer, polyethylene glycol-polycaprolactone. 1 selected from the group consisting of block-copolymers, polylactide, polyglycolide, poly(lactide-co-glycolide), poly(lactide-co-glycolide)glucose, polycaprolactone, and mixtures thereof A manufacturing method that is more than one species.
21. The manufacturing method according to claim 17 or 18, wherein the biocompatible polymer is a combination of polymers of the same type having different intrinsic viscosity and/or monomer ratios.
22. The method of claim 17 or 18, wherein the organic solvent used in steps (i) and (ii) of step a) or a') is dichloromethane, chloroform, ethyl acetate, methyl ethyl ketone, acetone, acetonitrile, dimethyl A manufacturing method comprising at least one solvent selected from the group consisting of sulfoxide, dimethylformamide, enmethylpyrrolidone, acetic acid, methyl alcohol, ethyl alcohol, propyl alcohol, and benzyl alcohol, or a mixed solvent of two or more of the above solvents.
23. The manufacturing method according to claim 17 or 18, wherein step a) or a') is performed at 15 to 25°C.
24. The method of claim 17 or 18, wherein in step b) or b'), the content of the surfactant in the continuous phase containing the surfactant is 0.01% by weight to 20% by weight based on the total volume of the continuous phase. Phosphorus, manufacturing method.
25. The method of claim 17 or 18, wherein the surfactant in step b) or b') is methylcellulose, polyvinylpyrrolidone, carboxymethylcellulose, lecithin, gelatin, polyvinyl alcohol, polyoxyethylene sorbitan fatty acid ester. and polyoxyethylene castor oil derivatives and mixtures thereof.
26. The manufacturing method according to claim 17 or 18, wherein the stirring process in step b) or b') is performed at 4 to 20° C. for 1 to 5 hours.
27. The method of claim 18, wherein the content of ethanol in the aqueous solution containing ethanol and the surfactant in step b') is 5 to 40 (v/v)% based on the total volume of the aqueous solution. .
28. The manufacturing method according to claim 17 or 18, wherein step c) is performed at 4 to 24°C.
29. The manufacturing method according to claim 17 or 18, wherein the stirring in step d) or c') is performed for 1 to 48 hours.
30. The production method according to claim 17 or 18, wherein the encapsulation ratio of the produced sustained-release microspheres is 60 to 100%.

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