Method for preparing biodegradable microspheres using stabilized single-phase mixed solution

Abstract

The present invention relates to a method for preparing biodegradable microspheres using a single-phase mixed solution containing water, alcohol, and dichloromethane. Provided is a method for preparing a biodegradable microsphere having a uniform drug loading efficiency by preparing and using a single-phase mixed solution in which no phase separation occurs without using a thickener and a surfactant. The preparation method of the present invention has the feature of keeping the content of a loaded drug uniform until a final biodegradable microsphere is prepared, by using a single-phase mixed solution in which no phase separation by a solvent occurs in the preparation process. Thus, the preparation method of the present invention is remarkably useful for the preparation of biodegradable microspheres.

Claims

1. A method of preparing biodegradable microspheres having uniform drug loading efficiency, comprising the steps of: i) dissolving a drug in a mixed solvent in which water and alcohol are mixed in a volume ratio of 1:4 to 1:9 to form a drug solution; ii) adding the drug solution obtained in step i) to a non-polar organic solvent in which a biodegradable polymer is dissolved, thus forming a single-phase mixed solution without forming an emulsion; iii) dispersing the single-phase mixed solution in an aqueous phase in which a surfactant is dissolved, thus forming a microsphere emulsion; iv) evaporating the non-polar organic solvent in the microsphere emulsion formed in step iii), and then hardening the microspheres; and v) obtaining the microsphere powder by a freeze-drying, wherein the alcohol is methanol or ethanol, wherein the drug is a water-soluble drug, wherein the biodegradable polymer is poly(lactic-co-glycolic acid) or poly(lactic acid), wherein the non-polar organic solvent is dichloromethane or chloroform, and wherein in step ii), the volume ratio at which the drug solution and the non-polar organic solvent in which the biodegradable polymer is dissolved are mixed is in a range of 1:1 to 1:12.

2. The method of claim 1, wherein the surfactant is one or more selected from the group consisting of Polysorbate 20, Polysorbate 60, Polysorbate 80, and polyvinyl alcohol.

3. The method of claim 1, wherein the drug is a peptide.

4. The method of claim 3, wherein the peptide is leuprorelin or a salt thereof.

5. A method of preparing a single-phase mixed solution usable for preparing biodegradable microspheres, comprising the steps of: a) dissolving a drug in a mixed solvent in which water and alcohol are mixed in a volume ratio of 1:4 to 1:9 to form a drug solution; and b) adding the drug solution obtained in step a) to a non-polar organic solvent in which a biodegradable polymer is dissolved, thus forming a single-phase mixed solution, wherein, in step b), the volume ratio at which the drug solution and the non-polar organic solvent in which the biodegradable polymer is dissolved are mixed is in a range of 1:1 to 1:12, wherein the alcohol is methanol or ethanol, wherein the drug is a water-soluble drug, wherein the biodegradable polymer is poly(lactic-co-glycolic acid) or poly(lactic acid), wherein the non-polar organic solvent is dichloromethane or chloroform, and wherein the single-phase mixed solution is stabilized and does not undergo phase separation.

6. The method of claim 5, wherein the drug is a peptide.

7. The method of claim 6, wherein the peptide is leuprorelin or a salt thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a photograph showing whether phase separation occurred in a single-phase mixed solution of the present invention (Example 1) and a conventional W/O emulsion (Comparative Example 1).

(2) FIG. 2 is a graph showing the results of analyzing whether phase separation occurred over time in a single-phase mixed solution of the present invention (Example 1) and a conventional W/O emulsion (Comparative Example 1).

(3) FIG. 3 shows scanning electron microscope images showing the shapes of biodegradable microspheres prepared by a preparation method of the present invention.

(4) FIG. 4 is a graph for comparing changes in drug loading efficiency of biodegradable microspheres prepared by a preparation method of the present invention (Example 3) and W/O/W-type biodegradable microspheres prepared by a conventional method (Comparative Example 2).

(5) FIG. 5 is a graph showing the results of testing an accelerated drug release rate of biodegradable microspheres prepared by a preparation method of the present invention.

EMBODIMENTS OF THE INVENTION

(6) Hereinafter, the present invention will be described in more detail through Examples. However, Preparation Examples and Examples are merely illustrative of the present invention, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited to Examples.

PREPARATION EXAMPLE 1: PREPARATION OF SINGLE-PHASE MIXED SOLUTION

Example 1

(7) 0.28 g of leuprorelin acetate (polypeptide) was added to a mixture of 0.14 mL of water (injectable water) and 1.0 mL of ethanol, stirred, and completely dissolved, and the solution was added to 3.11 mL of dichloromethane (Daejung Chemicals & Metals Co., Ltd.) in which 1.72 g of poly(D,L-lactic-co-glycolic acid) (Evonik, RG 653 H, Select 5050 DLG 2A) was dissolved and vortexed, and thereby a single-phase mixed solution was obtained.

Example 2

(8) A single-phase mixed solution was prepared in the same manner as in Example 1 except that poly(D,L-lactic acid) (Evonik, R 202 H) was used as a biodegradable polymer instead of poly(D,L-lactic-co-glycolic acid).

Comparative Example 1

(9) A W/O emulsion was prepared by completely dissolving 0.28 g of leuprorelin acetate (polypeptide) in 1.14 mL of water (injectable water) and then adding the solution to 3.11 mL of dichloromethane (Daejung Chemicals & Metals Co., Ltd.) in which 1.72 g of poly(D,L-lactic-co-glycolic acid) (Evonik, RG 653 H, Select 5050 DLG 2A) was dissolved and vortexing.

PREPARATION EXAMPLE 2: PREPARATION OF BIODEGRADABLE MICROSPHERES

Example 3

(10) All of the single-phase mixed solution of Example 1 was injected into 500 mL of a 0.7 wt % aqueous polyvinyl alcohol (PVA) solution at a rate of 0.36 mL/min using a syringe pump while performing homogenization at 7,000 rpm using a homogenizer, and thereby a microsphere emulsion was formed. The formed microsphere emulsion was stirred at 36° C. and 500 rpm for two hours using a mechanical stirrer to evaporate dichloromethane, and the remainder was hardened at 25° C. for 30 minutes. Subsequently, hardened microspheres were filtered using a 75 μm sieve and a 5 μm sieve and then freeze-dried for 48 hours, and thereby biodegradable microsphere powders were obtained.

Example 4

(11) Biodegradable microspheres were obtained in the same manner as in Example 3 except that the single-phase mixed solution of Example 2 was used.

Comparative Example 2

(12) W/O/W-type biodegradable microspheres were obtained in the same manner as in Example 3 except that the W/O emulsion of Comparative Example 1 was used.

Test Example 1: Determination of Occurrence of Phase Separation

(13) The single-phase mixed solution of Example 1 and the W/O emulsion of Comparative Example 1, which were prepared in Preparation Example 1, were visually examined and compared to determine whether phase separation had occurred, and results thereof are shown in the photograph of FIG. 1.

(14) As shown in FIG. 1, it can be seen that in the case of the W/O emulsion of Comparative Example 1, since an interface is generated between the two solutions and phase separation occurred, the W/O layer was present in the form of microdroplets, and thus the W/O emulsion was formed as an opaque solution, but in the case of the single-phase mixed solution of Example 1, since an interface was not generated and phase separation did not occur, the single-phase mixed solution was formed as a transparent solution.

Test Example 2: Analysis of Phase Separation Over Time

(15) Each of the single-phase mixed solution of Example 1 and the W/O emulsion of Comparative Example 1, which were prepared in Preparation Example 1, was input in a glass vial, and a change in transmittance over time at room temperature was determined through measurement with a spectrophotometer and using the following equation, and thereby the occurrence of phase separation was analyzed, and results thereof are shown in FIG. 2:
Change in transmittance=T.sub.h/T.sub.0

(16) T.sub.0: Transmittance measured immediately after preparation of Example 1 or Comparative Example 1

(17) T.sub.h: Transmittance measured at time (h) after preparation of Example 1 or Comparative Example 1

(18) As shown in FIG. 2, unlike in the case of the W/O emulsion of Comparative Example 1 in which T.sub.h was gradually increased compared to T.sub.0 as water was phase-separated from the emulsion over time and moved to an upper layer portion due to a difference in density, in the case of the single-phase mixed solution of Example 1, transmittance of the solution did not change over time because a phase separation phenomenon did not occur.

Test Example 3: Confirmation of Shapes of Biodegradable Microspheres

(19) The biodegradable microspheres of Examples 3 and 4, which were prepared in Preparation Example 2, were photographed with an SEM to examine shapes thereof, and the images are shown in FIG. 3.

(20) As shown in FIG. 3, the biodegradable microspheres prepared according to a preparation method of the present invention had particle sizes falling within a pharmaceutically acceptable range for injections, and it can be seen that such biodegradable microspheres were obtainable using various types of biodegradable polymers.

Test Example 4: Measurement of Change in Drug Loading Efficiency of Biodegradable Microspheres

(21) Degrees of change in the leuprorelin loading efficiency of the biodegradable microspheres of Example 3 and Comparative Example 2, which were prepared in Preparation Example 2, according to the time elapsed after the microspheres had been prepared were calculated by the following equation, and results thereof are shown in FIG. 4.
Degree of change in loading efficiency (%)=EE.sub.h/EE.sub.0×100

(22) EE.sub.h: Drug loading efficiency at h minutes after microsphere preparation

(23) EE.sub.0: Drug loading efficiency immediately after microsphere preparation
Drug loading efficiency (%)=Actual drug loading amount per gram of microspheres/Amount of drug added during preparation per gram of microspheres×100

(24) As shown in FIG. 4, it can be seen that unlike the case of the W/O/W-type biodegradable microspheres of Comparative Example 2 where drug loading efficiency greatly changed over time, in the case of the biodegradable microspheres of Example 3, drug loading efficiency was uniform and hardly changed over time.

Test Example 5: Analysis of Drug Release of Biodegradable Microspheres

(25) 100 mg of the biodegradable microspheres of Example 4 prepared in Preparation Example 2 was taken and added to 100 mL of a 0.02% aqueous Polysorbate 80 solution, and an accelerated drug release test was carried out while shaking the microspheres at 50±0.5° C. and 125 rpm.

(26) In order to evaluate the release of the drug, 1 mL of an eluate was taken at 1, 4, 7, 24, and 30 hours, followed by measurement of absorbance at 280 nm using a spectrophotometer and calculation of a release rate, and results thereof are shown in FIG. 5.

(27) As shown in FIG. 5, it can be seen that the biodegradable microspheres prepared according to the present invention released a drug in a sustained manner

Test Example 6: Testing of Optimum Solvent Mixing Ratio for Preparation of Single-Phase Mixed Solution

(28) Single-phase mixed solutions were prepared in the same manner as in Example 1 of Preparation Example 1 while using methanol as an example of alcohol and varying a mixing ratio of water and methanol and a mixing ratio of the mixture of water and methanol and dichloromethane (DCM; non-polar solvent) as shown in Table 1 to identify optimum solvent mixing ratios, and the mixed solutions were examined to determine whether phase separation had occurred, and results thereof are shown in Table 1.

(29) TABLE-US-00001 TABLE 1 Water/methanol mixture:DCM Water/Methanol 1:1 1:2 1:3 1:4 1:5 1:6 1:7 1:8 1:9 1:10 1:11 1:12 1:13 1:9 X X X X X X X X X X X X ◯ 1:6 X X X ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ 1:4 X ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ 1:3 ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ (◯: phase separation occurred, X: phase separation did not occur)

(30) As shown in Table 1, it can be seen that in a preparation method of the present invention, an optimum mixing ratio of water and alcohol in a mixture thereof is in the range of 1:4 to 1:9, and that an optimum mixing ratio of the water/alcohol mixture and the non-polar organic solvent (in which a biodegradable polymer is dissolved) is in the range of 1:1 to 1:12.