MICROSPHERE FOR CONTINUOUS RELEASE AND METHOD FOR MANUFACTURING SAME

Abstract

A method for preparing a microsphere include steps of dissolving an active ingredient and a biodegradable polymer in an organic solvent to prepare a dispersed phase, dissolving a salt in water to prepare a continuous phase, mixing and stirring the dispersed phase and the continuous phase to form an emulsion, removing the organic solvent; and drying.

Claims

1-10. (canceled)

11. A method for preparing a microsphere, comprising: dissolving an active ingredient and a biodegradable polymer in an organic solvent to prepare a dispersed phase; dissolving a salt in water to prepare a continuous phase; mixing and stirring the dispersed phase and the continuous phase to form an emulsion; removing the organic solvent; and drying.

12. The method of claim 11, wherein the salt is selected from the group consisting of sodium chloride (NaCl), potassium chloride (KCl), calcium chloride (CaCl.sub.2)), magnesium sulfate (MgSO4), sodium sulfate (Na.sub.2SO4), mannitol, ammonium, potassium sulfate, disodium phosphate, dipotassium phosphate, trisodium phosphate, disodium citrate, trisodium citrate, sodium succinate, and a combination thereof.

13. The method of claim 12, wherein the salt has a concentration of 1 to 10% (w/v) in the continuous phase.

14. The method of claim 11, wherein the biodegradable polymer is selected from the group consisting of polylactide, poly(lactide-co-glycolide), polyglycolactide, poly(lactide-co-glycolide)glucose, and a combination thereof.

15. The method of claim 11, wherein the continuous phase further comprises a water-soluble polymer.

16. The method of claim 15, wherein the water-soluble polymer is selected from the group consisting of polyvinyl alcohol, polysorbate, poloxamer, polyvinylpyrrolidone, polyvinylmethyl ether, polyvinyl ether, and a combination thereof.

17. The method of claim 11, wherein the active ingredient is a substance acting on the central nervous system.

18. The method of claim 17, wherein the active ingredient is a substance having therapeutic activity against dementia or therapeutic activity against Alzheimer's disease.

19. A microsphere prepared according to the method of claim 11.

20. A microsphere prepared according to the method of claim 12.

21. A microsphere prepared according to the method of claim 13.

22. A microsphere prepared according to the method of claim 14.

23. A microsphere prepared according to the method of claim 15.

24. A microsphere prepared according to the method of claim 16.

25. A microsphere prepared according to the method of claim 17.

26. A microsphere prepared according to the method of claim 18.

27. A sustained release injection comprising the microsphere according to claim 19.

28. A sustained release injection comprising the microsphere according to claim 20.

29. A sustained release injection comprising the microsphere according to claim 22.

30. A sustained release injection comprising the microsphere according to claim 25.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0026] FIGS. 1 to 12 are photographs showing SEM images for confirming the morphology of donepezil microspheres according to each of the examples and comparative examples;

[0027] FIGS. 13 and 14 are photographs showing enlarged SEM images of the surface of microspheres prepared in comparative example 1 and example 1, respectively; and

[0028] FIG. 15 is a graph showing the concentration in the blood obtained for 24 hours after administration of the microspheres prepared in example 1, comparative example 1, comparative example 2, and comparative example 2-1 to SD rats.

BEST MODE FOR CARRYING OUT THE INVENTION

[0029] Hereinafter, examples will be described in detail to help the understanding of the present invention. However, the following examples are merely illustrative of the contents of the present invention, and the scope of the present invention is not limited to the following examples. The examples of the present invention are provided to explain the present invention more completely to those of ordinary skill in the art.

Example 1: Preparation of Microspheres by Adding Polylactide Polymer to NaCl 1% (w/v) Continuous Phase

[0030] 2 g (amount of drug loaded in microspheres (hereinafter, “drug loading”) is 40%) of donepezil (manufacturer: Neuland Laboratories, India) and 3 g of poly D,L-lactide (Resomer R 203 H; manufacturer: Evonik, Germany), which is a biodegradable polymer, were added to 9 g of dichloromethane (manufacturer: Daejung Chemicals & Metals Co., Ltd., South Korea) and completely dissolved by stirring to prepare a polymer solution, which is a dispersed phase. In addition, 6.25 g of polyvinyl alcohol and 12.5 g of NaCl were dissolved in 1.25 L of water to prepare a continuous phase.

[0031] After putting the continuous phase in a double jacket beaker and maintaining the temperature at 10° C. or below using a constant temperature circulating water bath, the dispersed phase (i.e., donepezil-containing polymer solution) was added to the continuous phase (i.e., polyvinyl alcohol and NaCl aqueous solution) and stirred at high speed to form an emulsion.

[0032] Then, in order to remove the organic solvent and obtain solidified microspheres, the organic solvent was volatilized at a temperature of 47° C. for 2 hours, and then slowly cooled to 10° C. for 1 hour. After washing the hardened microspheres with water for injection several times, and going through wet filtration using a sieve and freeze-drying, donepezil-containing microspheres were finally obtained.

Example 1-1: Preparation of Microspheres by Adding Polylactide Polymer to NaCl 1% (w/v) Continuous Phase

[0033] 2.25 g (drug loading: 45%) of donepezil (manufacturer: Neuland Laboratories, India) and 2.75 g of poly D,L-lactide (Resomer R 203 H; manufacturer: Evonik, Germany), which is a biodegradable polymer, were added to 8.25 g of dichloromethane (manufacturer: Daejung Chemicals & Metals Co., Ltd., South Korea) and completely dissolved by stirring to prepare a polymer solution, which is a dispersed phase. In addition, 6.25 g of polyvinyl alcohol and 12.5 g of NaCl were dissolved in 1.25 L of water to prepare a NaCl 1% (w/v) continuous phase. The rest of the preparation method was carried out in the same manner as in example 1 to prepare microspheres.

Example 2: Preparation of Microspheres by Adding Polylactide Polymer to NaCl 5% (w/v) Continuous Phase

[0034] Except that NaCl was added to be 5% (w/v) while preparing the continuous phase, the rest of the preparation method was carried out in the same manner as in example 1 to prepare microspheres.

Example 2-1: Preparation of Microspheres by Adding Polylactide Polymer to NaCl 10% (w/v) Continuous Phase

[0035] Except that NaCl was added to be 10% (w/v) while preparing the continuous phase, the rest of the preparation method was carried out in the same manner as in example 1 to prepare microspheres.

Example 3: Preparation of Microspheres by Adding Polylactide Polymer to KCl 1% (w/v) Continuous Phase

[0036] Except that KCl was added instead of NaCl to be 1% (w/v) while preparing the continuous phase, the rest of the preparation method was carried out in the same manner as in example 1 to prepare microspheres.

Example 3-1: Preparation of Microspheres by Adding Polylactide Polymer to KCl 5% (w/v) Continuous Phase

[0037] Except that KCl was added instead of NaCl to be 5% (w/v) while preparing the continuous phase, the rest of the preparation method was carried out in the same manner as in example 1 to prepare microspheres.

Example 4: Preparation of Microspheres by Adding Poly(D,L-lactide-co-glycolide) Polymer to NaCl 5% (w/v) Continuous Phase

[0038] 2 g (drug loading: 40%) of donepezil (manufacturer: Neuland Laboratories, India) and 3 g of poly D,L-lactide-co-glycolide (PLGA; Resomer RG 753 H; manufacturer: Evonik, Germany), which is a biodegradable polymer, were added to 9 g of dichloromethane (manufacturer: Daejung Chemicals & Metals Co., Ltd., South Korea) and completely dissolved by stirring to prepare a polymer solution, which is a dispersed phase. The rest of the preparation method was carried out in the same manner as in example 1, except that NaCl was added to be 5% by weight while preparing the continuous phase, to prepare microspheres.

Comparative Example 1: Preparation of Microspheres by Adding Polylactide Polymer to a Continuous Phase to which No Salt is Added

[0039] 2 g (drug loading: 40%) of donepezil (manufacturer: Neuland Laboratories, India) and 3 g of poly D,L-lactide (Resomer R 203 H; manufacturer: Evonik, Germany), which is a biodegradable polymer, were added to 9 g of dichloromethane (manufacturer: Daejung Chemicals & Metals Co., Ltd., South Korea) and completely dissolved by stirring to prepare a polymer solution, which is a dispersed phase. In addition, 6.25 g of polyvinyl alcohol was dissolved in 1.25 L of water to prepare a continuous phase (salts such as NaCl or KCl were not added).

[0040] After putting the continuous phase in a double jacket beaker and maintaining the temperature at 10° C. or below using a constant temperature circulating water bath, the dispersed phase (i.e., donepezil-containing polymer solution) was added to the continuous phase (i.e., polyvinyl alcohol aqueous solution) and stirred at high speed to form an emulsion.

[0041] Then, in order to remove the organic solvent and obtain solidified microspheres, the organic solvent was volatilized at a temperature of 47° C. for 2 hours, and then slowly cooled to 10° C. for 1 hour. After washing the hardened microspheres with water for injection several times, and going through wet filtration using a sieve and freeze-drying, donepezil-containing microspheres were finally obtained.

Comparative Example 1-1: Preparation of Microspheres by Adding Polylactide Polymer to a Continuous Phase to which No Salt is Added

[0042] 2.25 g (drug loading: 45%) of donepezil (manufacturer: Neuland Laboratories, India) and 2.75 g of poly D,L-lactide (Resomer R 203 H; manufacturer: Evonik, Germany), which is a biodegradable polymer, were added to 8.25 g of dichloromethane (manufacturer: Daejung Chemicals & Metals Co., Ltd., South Korea) and completely dissolved by stirring to prepare a polymer solution, which is a dispersed phase. The rest of the preparation method was carried out in the same manner as in comparative example 1 to prepare microspheres.

Comparative Example 2: Preparation of Microspheres Using a Continuous Phase to which No Salt is Added, Followed by Washing with EtOH Aqueous Solution

[0043] 2 g of donepezil (manufacturer: Neuland Laboratories, India) and 3 g of poly D,L-lactide (Resomer R 203 H; manufacturer: Evonik, Germany), which is a biodegradable polymer, were added to 9 g of dichloromethane (manufacturer: Daejung Chemicals & Metals Co., Ltd., South Korea) and completely dissolved by stirring to prepare a polymer solution, which is a dispersed phase. In addition, 6.25 g of polyvinyl alcohol was dissolved in 1.25 L of water to prepare a continuous phase (salts such as NaCl or KCl were not added).

[0044] After putting the continuous phase in a double jacket beaker and maintaining the temperature at 10° C. or below using a constant temperature circulating water bath, the dispersed phase (i.e., donepezil-containing polymer solution) was added to the continuous phase (i.e., polyvinyl alcohol aqueous solution) and stirred at high speed to form an emulsion.

[0045] Then, in order to remove the organic solvent and obtain solidified microspheres, the organic solvent was volatilized at a temperature of 47° C. for 2 hours, and then slowly cooled to 10° C. for 1 hour. After washing the hardened microspheres with water for injection several times, and going through first wet filtration using a sieve, microspheres were primarily obtained.

[0046] After washing the primarily obtained microspheres with 20% EtOH aqueous solution at 10° C. for 1 hour, and going through wet filtration again using a sieve and freeze-drying, donepezil-containing microspheres were finally obtained.

Comparative Example 2-1: Preparation of Microspheres Using a Continuous Phase to which No Salt is Added, Followed by Washing with Tween Aqueous Solution

[0047] The preparation method was carried out in the same manner as in comparative example 2 until primarily obtaining donepezil microspheres.

[0048] Then, after washing the primarily obtained microspheres with 3% Tween 20 aqueous solution at 10° C. for 1 hour, and going through wet filtration again using a sieve and freeze-drying, donepezil-containing microspheres were finally obtained.

Comparative Example 3: Preparation of Microspheres by Adding poly(D,L-lactide-co-glycolide) Polymer to a Continuous Phase to which No Salt is Added

[0049] 2 g (theoretical drug loading: 40%) of donepezil (manufacturer: Neuland Laboratories, India) and 3 g of poly(D,L-lactide-co-glycolide) (Resomer RG 753 H; manufacturer: Evonik, Germany), which is a biodegradable polymer, were added to 9 g of dichloromethane (manufacturer: Daejung Chemicals & Metals Co., Ltd., South Korea) and completely dissolved by stirring to prepare a polymer solution, which is a dispersed phase. In addition, 6.25 g of polyvinyl alcohol was dissolved in 1.25 L of water to prepare a continuous phase (salts such as NaCl or KCl were not added).

[0050] After putting the continuous phase in a double jacket beaker and maintaining the temperature at 10° C. or below using a constant temperature circulating water bath, the dispersed phase (i.e., donepezil-containing polymer solution) was added to the polyvinyl alcohol aqueous solution (continuous phase) and stirred at high speed to form an emulsion.

[0051] Then, in order to remove the organic solvent and obtain solidified microspheres, the organic solvent was volatilized at a temperature of 47° C. for 2 hours, and then slowly cooled to 10° C. for 1 hour. After washing the hardened microspheres with water for injection several times, and going through wet filtration using a sieve and freeze-drying, donepezil-containing microspheres were finally obtained.

Experimental Example 1: Observation of Microsphere Morphology Using SEM

[0052] About 20 mg of microspheres obtained in each of the examples and comparative examples was fixed to an aluminum stub using carbon tape, and coated with platinum under a vacuum level of 0.1 torr and high voltage (10 kV) for 3 minutes. Then, the microspheres were mounted on the main body (SEM stage) of SEM (equipment name: SEC-SNE 4500M Plus A, South Korea) to observe the surface morphology of the microspheres using an image analysis program (mini-SEM).

[0053] FIGS. 1 to 14 are SEM images of the microspheres prepared in examples 1, 1-1, 2, 2-1, 3, 3-1 and 4, and comparative examples 1, 1-1, 2, 2-1 and 3, respectively.

[0054] As can be seen in FIGS. 1 to 7, most of the microspheres prepared in the examples did not show drug crystals on the periphery. In addition, as can be seen in FIGS. 10 and 11, the microspheres prepared in comparative example 2 and comparative example 2-1 to which the conventional drug crystal removing process (i.e., ethanol washing process or surfactant washing process) was applied did not show drug crystals on the periphery.

[0055] However, as can be seen in FIGS. 8, 9 and 12, a large amount of drug crystals were seen on the periphery of the microspheres prepared in comparative example 1, comparative example 1-1, and comparative example 3.

[0056] As can be seen in FIG. 13, pores were observed to be formed on the surface of the microspheres prepared in comparative example 1, but as can be seen in FIG. 14, no pores were formed on the surface of the microspheres in example 1 prepared by adding salt to the continuous phase according to the present invention.

Experimental Example 2: Removal of Drug Crystals from the Surface of Microspheres

[0057] Although the presence of drug crystals was roughly confirmed from the SEM photograph images, in order to quantitatively confirm the amount of drug crystals actually generated outside the microspheres without being included therein while preparing microspheres, the microspheres prepared in each of the examples and comparative examples 1, 1-1 and 3 were washed with 20% EtOH aqueous solution at 10° C. for 1 hour and freeze-dried to remove drug crystals from the surface and the periphery of the microspheres.

[0058] Experimental Example 3: Measurement of actual content of donepezil in microspheres, entrapment efficiency, and amount of drug crystals present outside the microspheres

[0059] 100 mg of microspheres prepared in each of examples 1 to 4 and comparative examples 1 to 3 was completely dissolved in acetonitrile and then diluted with a mobile phase. 20 uL of the diluted solution was injected into HPLC and measured at a detection wavelength of 318 nm.

[0060] In addition, 100 mg of microspheres prepared in each of examples 1 to 4 and comparative examples 1, 1-1 and 3 and washed with an aqueous ethanol solution in experimental example 2 was completely dissolved in acetonitrile and then diluted with a mobile phase. 20 uL of the diluted solution was injected into HPLC and measured at a detection wavelength of 318 nm.

[0061] Column: Luna phenyl-Hexyl, C18 5 μm, 4.6×250 mm

[0062] Mobile phase: pH 2.0 tetrahydrofuran, 3:1 mixed solution of triethylamine solution (solution A) and methanol tetrahydrofuran solution (solution B)

[0063] Table 1 shows the measured drug entrapment efficiency (%) and the drug entrapment efficiency in microspheres after washing with ethanol.

TABLE-US-00001 TABLE 1 Measured drug Drug entrapment Calculated amount (%) Theoretical drug entrapment efficiency (%) after of drug crystals loading amount efficiency (%) washing with present outside (%) (A) EtOH (B) microsphere (A − B) Example 1 40% 91.89 90.76 1.13 Example 1-1 45% 96.78 95.54 1.24 Example 2 40% 95.55 94.92 0.63 Example 2-1 40% 95.42 92.61 2.81 Example 3 40% 93.89 93.75 0.14 Example 3-1 40% 90.94 90.82 0.12 Example 4 40% 95.68 95.47 0.21 Comparative 40% 92.71 90.39 2.32 example 1 Comparative 45% 95.58 93.26 2.32 example 1-1 Comparative 40% 93.90 — — example 2 Comparative 40% 94.90 — — example 2-1 Comparative 40% 95.97 93.22 2.75 example 3

Explanation of Terms Used in the Above Table

[0064] Theoretical drug loading amount (%)=Amount of drug introduced while preparing microspheres/(Amount of drug introduced while preparing microspheres+Amount of polymer introduced while preparing microspheres)×100%

[0065] Measured drug entrapment efficiency (%) (A)=Measured amount (mg) of drug actually included in every 100 mg of microspheres immediately after preparation in comparative examples and examples/(100 mg of microspheres×theoretical drug loading amount (%))×100%

[0066] Drug entrapment efficiency after washing with ethanol (%) (B)=Measured amount (mg) of drug actually included in every 100 mg of microspheres after washing in experimental example 2/(100 mg of microspheres×theoretical drug loading amount (%))×100%

Calculated Amount (%) of Drug Crystals Present Outside Microsphere=A-B

[0067] As can be seen in comparative example 1 and comparative example 1-1, the amount of drug crystals present outside exceeded 2% in the microspheres prepared without applying a process of additional washing with ethanol or a surfactant.

[0068] However, in the case of the examples prepared by adding a salt to the continuous phase, the amount of drug crystals confirmed to be present outside the microspheres was very small.

[0069] Therefore, when microspheres are prepared by adding a salt such as NaCl to the continuous phase, the amount of drug crystals that may be present outside the microspheres may be minimized, and thus the actual entrapment efficiency of the drug in the microspheres may be maximized.

Experimental Example 4: Initial In Vitro Release Test of Microspheres

[0070] 10 mg of the microspheres prepared in the examples and comparative examples were respectively added to 100 mL of a pH 7.4 HEPES solution and placed in a constant temperature shaking water bath maintained at 37.0° C. After 24 hours, the supernatant was taken and filtered with a 0.45 μm syringe filter.

[0071] The initial daily dissolution amount of donepezil released from the microspheres was measured using HPLC. The column was XTerra Shield RP18 column 5 μm, 4.6×150 mm, the injection amount was 20 μl, the detection wavelength was 271 nm, and the mobile phase was pH 5.0 phosphate buffer solution and an acetonitrile solution (phosphate buffer solution:acetonitrile=60:40).

[0072] Table 2 below shows the dissolution rate for the 1.sup.st day of donepezil microspheres according to each example and comparative example.

TABLE-US-00002 TABLE 2 Dissolution rate of the 1.sup.st day (%) Example 1 3.25 Example 1-1 7.64 Example 2 6.06 Example 2-1 11.72 Example 3 5.25 Example 3-1 3.26 Example 4 10.25 Comparative example 1 7.78 Comparative example 1-1 10.87 Comparative example 2 5.5 Comparative example 2-1 4.9 Comparative example 3 17.21

[0073] As can be seen in Table 2, example 1 (where NaCl was added in 1% to the continuous phase) and example 2 (where NaCl was added in 5% to the continuous phase) showed a relatively lower dissolution rate compared to comparative example 1 (where NaCl was not added to the continuous phase).

[0074] As in experimental example 3, the smaller the amount of drug crystals present outside the microspheres, the lower the dissolution rate of the 1.sup.st day.

[0075] As can be seen in example 3 and example 3-1, when KCl was used, it was confirmed that the same effect as that of the case of NaCl was exhibited.

[0076] In addition, even when the polymer in dispersed phase was PLGA (Resomer RG 753 H), the initial dissolution rate of microspheres in example 4 was lower than the initial dissolution rate of comparative example 3.

Experimental Example 5: Pharmacokinetic Test of Microspheres Using SD Rats

[0077] In order to confirm the effect of suppressing the initial release of microspheres prepared by adding salt to the continuous phase, the concentration of donepezil in the blood was measured after subcutaneous administration to the back of the neck of rats.

[0078] The microspheres prepared in example 1, comparative example 1, comparative example 2, and comparative example 2-1 were weighed so that the amount of donepezil administered in the microspheres per rat was 25.2 mg/kg, and then dispersed in 0.3 mL suspension and subcutaneously injected into SD rats.

[0079] At regular intervals, 0.3 mL of blood was collected from the jugular vein of the rat, kept in an ice-cooled state, and centrifuged to separate 100 uL of plasma. The separated plasma was analyzed for the concentration of donepezil using LC/MS/MS.

[0080] The measurement results are shown in FIG. 15.

[0081] As can be seen in FIG. 15, it may be confirmed that the microspheres of comparative example 1 to which a washing process with ethanol or a surfactant was not applied showed the highest Cmax of 220.5 ng/mL, whereas the microspheres of comparative example 2 washed with ethanol and the microspheres of comparative example 2-1 washed with a surfactant showed a lower Cmax of 141.03 ng/mL and 90.2 ng/mL, respectively, because drug crystals were removed by washing.

[0082] The microspheres of Example 1 prepared by adding NaCl in a content of 1% to the continuous phase showed the lowest Cmax of 58.9 ng/mL because drug crystals around the microspheres were removed and surface pores were also removed. In other words, it was possible to prevent the release of an excessive amount of drug in the initial stage. This result was similar to the in vitro result.

[0083] When microspheres are prepared by adding NaCl to the continuous phase according to the present invention, the formation of drug crystals around the microspheres is suppressed, and thus a separate process of removing drug crystals may not be additionally introduced and also the drug entrapment efficiency of the microspheres may be improved. In addition, drug crystals are not formed around the microspheres, and thus initial drug release may be suppressed when the microspheres are injected into the body.

INDUSTRIAL APPLICABILITY

[0084] According to the present invention, drug crystals are not formed around the microspheres, and thus initial drug release may be suppressed when the microspheres are injected into the body. In addition, the drug is released continuously, and thus the medicinal effect may be exhibited for a fairly long time with a single injection.