WO2008130137A1

WO2008130137A1 - Anionic lipid nanosphere and preparation method of the same

Info

Publication number: WO2008130137A1
Application number: PCT/KR2008/002174
Authority: WO
Inventors: Byung Cheol Shin; Suk Hyun Jung; Deok Hwi Lim; Hasoo Seong; Sun Hang Cho
Original assignee: Korea Research Institute Of Chemical Technology
Priority date: 2007-04-20
Filing date: 2008-04-17
Publication date: 2008-10-30
Also published as: KR20080094473A

Abstract

Disclosed is an anionic lipid nanosphere having negative surface charge and a method of its preparation, more particularly to a lipid nanosphere prepared by introducing polyethylene glycol (PEG) containing polymers on the surface of particles formed of anionic phospholipids to provide negative charge on the surface, thereby increasing encapsulation efficiency of a poorly water soluble drug in an aqueous medium, reducing toxicity of highly toxic drug against normal cells by encapsulating therewith, and increasing duration of circulation in the body, and a method of its preparation.

Description

ANIOIC LIPID NANOSPHERE AND PREPARATION METHOD

OF THE SAME

Technical Field

[1] The present invention relates to an anionic lipid nanosphere having negative surface charge and a method of its preparation, more particularly to a lipid nanosphere prepared by introducing polyethylene glycol (PEG) containing polymers on the surface of particles formed of anionic phospholipids to provide negative charge on the surface, thereby increasing encapsulation efficiency of a poorly water soluble drug in an aqueous medium, reducing toxicity of highly toxic drug against normal cells by encapsulating therewith, and increasing duration of circulation in the body, and a method of its preparation. Background Art

[2] Amphotericin B is a polyene antifungal drug effective in treating almost all fungal infections, particularly systemic fungal infections. Therefore, amphotericin B is used for the treatment of severe life-threatening infections in patients with cancer, bone-marrow transplantation, neutropenia, immune compromise or immune deficiency. Amphotericin B associates with ergosterol, a membrane chemical of fungi, forms a pore that leads to relocation of ion passage, interferes with osmosis control of fungal cells, thereby providing antifungal and antibiotic therapeutic effect.

[3] However, when administered intraveneously, amphotericin B also associates with cholesterol of normal cells, thereby becoming toxic to normal cells and tissues, and accompanied by side effects such as shaking chills, fever, tissue necrosis, renal toxicity, and the like. A special care is essential in its use and medication because amphotericin B is not easily discharged by hemodialysis. Particularly, a good care is needed when used for children, the elderly or patients with weak immunity because of its strong renal toxicity.

[4] Amphotericin B is insoluble in water at pH 6 to 7, and hardly soluble at pH 2 or pH

11, with a very low solubility of 0.1 mg/mL. For intraveneous injection, it is made soluble by preparing into salt, micelle, emulsion, nanosphere or liposome.

[5] US Patent No. 4,822,777 discloses a method for improving solubility of amphotericin

B by preparing it into a salt formulation. Specifically, an amphotericin B composition is prepared from particles of amphotericin B and cholesterol sulfate having particle sizes between about 100 and 400 nm, thereby improving solubility in an aqueous medium. Although solubility of amphotericin B was improved by the method in the above patent, circulation time in the blood was still relatively short, administration dose is limited, and toxicity to normal cells could not be avoided.

[6] US Patent No. 5,059,591 teaches a method of reducing the toxicity of amphotericin B by using a complex of amphotericin B and cholesterol-polyethylene glycol (PEG). By using the complex of amphotericin B and cholesterol-PEG, the time for its circulation in the blood was increased. However, its toxicity is more serious as compared to that of formulated amphotericin B.

[7] US Patent No. 4,981,690 discloses a method of preparing amphotericin B encapsulated within a liposome. According to the patent, phospholipid and cholesterol are used to prepare a pharmaceutical composition in multilamellar liposomal form. However, its circulation time in the blood may be relatively short because a substance that assists consistent circulation is not contained.

[8] US Patent No. 5,965,156 discloses a method of encapsulating amphotericin B into liposome. According to the patent, phosphatidylglycerol is acidified in an acidic organic solvent and amphotericin B is added to the acidified phosphatidylglycerol and a complex is formed between the phosphatidylglycerol and amphotericin B, thereby improving encapsulation efficiency and reducing toxicity. However, use of an acidic solution for ionic bonding of phosphatidylglycerol with amphotericin B causes a problem of increasing the loss of amphotericin due to the increased decomposition. Further, stability of the resulting preparation is deteriorated due to the decomposition by the acid catalyst, which results in burst release of amphotericin B.

[9] Accordingly, there is a need for the development of a novel drug delivery system capable of improving solubility of amphotericin B in an aqueous medium, reducing toxicity of the drug, increasing circulation time in the blood, and being applicable to commercial- scale production. Disclosure of Invention

[10] The inventors of the present invention have made various efforts to solve the aforementioned problems associated with the prior art. As a result, they have succeeded in inventing an anionic lipid nanosphere for encapsulating a poorly water soluble drug such as amphotericin B and a method of its preparation. By modifying the surface of the lipid nanosphere having superior bioaffinity with an anionic material and encapsulating the highly toxic drug therein, it is possible to reduce toxicity to normal cells and provide sustained release of the drug.

[11] Accordingly, an object of the present invention is to provide an anionic lipid nanosphere for encapsulating a poorly water soluble drug in which the surface of a particle formed of anionic phospholipids is modified with a polyethylene glycol (PEG) containing polymer.

[12] Another object of the present inventionto provide a preparation method of the anionic lipid nanosphere modified with a PEG containing polymer. Brief Description of the Drawings

[13] FIG. 1 is a graph showing the pharmacokinetic test result of Test Example 1.

[14] FIG. 2 is a graph showing the toxicity test result of Test Example 2.

Best Mode for Carrying Out the Invention

[15] In one aspect, the present invention relates to an anionic lipid nanosphere for encapsulating a poorly water soluble drug in which a polyethylene glycol (PEG) containing polymer is coated on the surface of particles formed of anionic phospholipids.

[16] In another aspect, the present invention relates to a preparation method of an anionic lipid nanosphere in which a PEG containing polymer is introduced on the surface of a lipid nanosphere formed of anionic phospholipids either by mixing a PEG containing polymer with a phospholipid (A) or by forming an ion complex of phospholipid and PEG having a terminal amine group (B).

[17] In a preferred embodiment, the present invention relates to a method of preparing an anionic lipid nanosphere for encapsulating a poorly water soluble drug, comprising the steps of:

[18] (A-I) mixing 100 parts by weight of a lipid, which is prepared by mixing phosphatidylcholine, anionic phospholipid and sterol with a weight ratio of 40-70 : 5-20 : 10-40, with 10 to 30 parts by weight of a PEG containing polymer, and dissolving in an organic solvent to obtain a lipid- PEG mixture solution

[19] (A-2) dissolving a poorly water soluble drug in a Ci-C₆ linear or branched alcohol to obtain a drug solution

[20] (A-3) mixing the lipid- PEG mixture solution of step (A-I) with the drug solution of step (A-2) with a volume ratio of 1 : 1 to 1 :9 to obtain a lipid-PEG-drug mixture solution

[21] (A-4) dispersing the mixture solution of step (A-3) in an aqueous medium with a volume ratio of 2:1 to 1:10 to form lipid nanospheres and

[22] (A-5) distilling the lipid nanosphere solution of step (A-4) at 20 to 5O⁰C under reduced pressure, removing the organic solvent, and filtering to obtain anionic lipid nanospheres with uniform size in which the drug is encapsulated.

[23] In another preferred embodiment, the present invention provides a preparation methodof an anionic lipid nanosphere for encapsulating a poorly water soluble drug, comprising the steps of:

[24] (B-I) dissolving a lipid prepared by mixing phosphatidylcholine, anionic phospholipid and sterol with a weight ratio of 40-70 : 5-20 : 10-40 in an organic solvent to obtain a lipid mixture solution

[25] (B-2) dissolving a poorly water soluble drug in a Ci-C₆ linear or branched alcohol to obtain a drug solution

[26] (B-3) mixing the lipid mixture solution of step (B-I) with the drug solution of step

(B-2) with a volume ratio of 1:1 to 1:9 to obtain a lipid-drug mixture solution

[27] (B-4) dispersing the mixture solution of step (B-3) in an aqueous medium with a volume ratio of 2: 1 to 1 : 10 to form lipid nanospheres

[28] (B-5) distilling the lipid nanosphere solution of step (B-4) at 20 to 5O⁰C under reduced pressure, removing the organic solvent, and filtering to obtain anionic lipid nanospheres with uniform size in which the drug is encapsulated and

[29] (B -6) mixing with a PEG having terminal amine groups with a weight ratio of the lipid of step (B-I) to the PEG having terminal amine groups being 100:10 to 100:30 to form lipid-PEG ion complexes.

[30] Hereunder is given a more detailed description of the present invention.

[31] The present invention relates to an anionic lipid nanosphere for encapsulating a poorly water soluble drug in which a PEG containing polymer is introduced on the surface of particles formed of anionic phospholipidsto increase encapsulation efficiency of a poorly water soluble drug in an aqueous medium, and the highly toxic drugis encapsulated in a lipid nanosphere with superior bioaffinity to reduce toxicity to normal cells and increase circulation time in the blood, and a method of its preparation.

[32] Particularly, the present invention is characterized in that an anionic phospholipid is used to prepare a lipid nanosphere for encapsulating a poorly water soluble drug. Preferably, the anionic phospholipid is phosphatidic acid having a hydrophobic Ci₄-Ci₈ alkyl chain. More preferably, it is selected from dimyristyl glycerophosphate (DMPA), dipalmitoyl glycerophosphate (DPPA), dimyristyl glycerophosphate (DMPG), disteroyl glycerophosphate (DSPA), disteroyl glycerophosphoglycerol (DSPG), dipalmitoyl glycerophosphoglycerol (DPPG), dimyristyl glycerophosphoserine (DMPS), dipalmitoyl glycerophosphoserine (DPPS), disteroyl glycerophosphoserine (DSPS) and a mixture thereof. When the anionic phospholipid has less than 14 carbon atoms, stability of the lipid nanosphere in vivo decreases as the phase transition temperature is below the body temperature. In contrast, when the anionic phospholipid has more than 18 carbon atoms, encapsulation efficiency of the poorly water soluble drug decreases because of weak binding to the drug, and particle size of the lipid nanosphere increases. Preferably, the anionic lipid is contained in an amount of 5 to 20 weight % based on the entire lipid composition constituting the lipid nanosphere.

[33] As the lipid for forming the lipid nanosphere of the present invention, it is preferable to use either hydrogenated phosphatidylcholine or phosphatidylcholine. Soybean phosphatidylcholine, egg yolk phosphatidylcholine or bovine phospholipid may be used. More preferably, one having a hydrophobic Ci₆-Ci₈ alkyl chain is used. For example, dipalmitoyl phosphatidylcholine or distearoyl phosphatidylcholine, etc. may be used. A strong binding to amphoteric amphotericin B is attained even when the number of carbons is less than 16, but stability of the lipid nanosphere in vivo decreases as the phase transition temperature is below the body temperature. When the number of carbons is larger than 18, encapsulation efficiency of the poorly water soluble drug decreases because of its weak binding to the drug, and particle size of the lipid nanosphere increases. Preferably, the phosphatidylcholine is contained in an amount of 40 to 70 weight % based on the entire lipid composition constituting the lipid nanosphere.

[34] Further, sterol is used as a lipid for forming the lipid nanosphere. Examples of preferred sterol include cholesterol, cholesterol hexasuccinate, 3β-[/V-(/V,./V '- dimethylaminoethane)carbamoyl]cholesterol, ergosterol, stigmasterol, lanosterol, etc. Preferably, the sterol is contained in an amount of 10 to 40 weight % based on the entire lipid composition constituting the lipid nanosphere.

[35] In accordance with the present invention, a PEG containing polymer is introduced to the lipid nanosphere in order to modify the surface of the lipid nanosphere in which the drug is encapsulated. To this end, a PEG containing polymer selected from disteroyl glycerophosphoethanolamine methyloxy ethylene glycol (DSPE- mPEG), poly- oxyethylene sorbitan monopalmitate (Tween), polyethylene polypropylene glycol (poloxamer) and a mixture thereof may be used. Preferably, the polymer is contained in an amount of 10 to 30 parts by weight based on 100 parts by weight of the total lipid. When the content of the PEG containing polymer is below 10 parts by weight, the surface of the lipid nanosphere may not be sufficiently modified with PEG. In contrast, when the content of the PEG containing polymer exceeds 30 parts by weight, the surface of the lipid nanosphere is not further modified because the surface area is limited. With the introduction of PEG, an ion complex is formed by the anionic groups on the surface of the lipid nanosphere and the terminal amine groups of the PEG. Alternatively, PEG is introduced on the surface of the lipid nanosphere by the lipophilic binding between the liposoluble moieties of the PEG containing lipid, or other lipid, phospholipid and cholesterol.

[36] And, the "drug" means a poorly water soluble drug which is not easily encapsulable in conventional drug delivery systems. Amphotericin B is a typical example, but the present invention is not limited thereto.

[37] The present invention provides a "sustained release" lipid nanosphere which is stable in the blood, the surface of which being modified with anions or PEG. As used herein "surface modification" means a coating by mixing or ionic bonding of anions or PEG capable of extending circulation in the blood. And, as used herein, a "sustained release" lipid nanosphere refers to a formulation that remains in the bloodstream for at least 24 hours, whereas typical formulations disappear from the bloodstream within several hours after administration.

[38] The lipid nanosphere of the present invention, in which amphotericin B is encapsulated and the surface of which is modified with anions, has an average particle size of 50 to 300 nm, preferably 100 to 150 nm. When the average particle size of the lipid nanosphere is larger than 300 nm, the lipid nanosphere may be uptaken by the reticuloendothelial system of such organs as liver or spleen during circulation in blood. In contrast, when the average particle size of the lipid nanosphere is smaller than 50 nm, the amount of the drug reaching the target site (drug payload) may not be sufficient.

[39] Hereunder, each step of the preparation method of the lipid nanosphere in which a drug is encapsulated and the surface of which is modified with anions is described in detail.

[40] First, the method (A) of mixing a PEG containing lipid with a lipid is as follows.

[41] In step (A-I), phosphatidylcholine, anionic phospholipid and sterolare mixed with a weight ratio of 40-70 : 5-20 : 10-40 to obtain a lipid. A lipid nanosphere is not formed easily when the content of phosphatidylcholine is less than the aforementioned range, and stability of the lipid nanosphere decreases when the content exceeds the aforementioned range. The size of the lipid nanosphere may increase and lipid nanospheres may coagulate with each other when the content of anionic phospholipid is less than the aforementioned range, and particle size may increase because of coagulation of lipid nanospheres with PEG when the content exceeds the aforementioned range. Further, encapsulation efficiency of the drug may decrease when the content of cholesterol is less than the aforementioned range, and stability of the lipid nanosphere may decrease when the content exceeds the aforementioned range. Encapsulation efficiency of the drug increases as the weight ratio of sterol increases. But, aforementioned range is preferred because particle size increases when the content of sterol exceeds the aforementioned range.

[42] One hundred parts by weight of thus prepared lipid is mixed with 10 to 30 parts by weight of a PEG containing polymer and dissolved in an organic solvent to obtain a lipid-PEG mixture solution.

[43] Coating of PEG may not be performed easily when the content of the PEG containing polymer is less than 10 parts by weight, and particle size becomes too large when the content the content exceeds 30 parts by weight. Hence, the aforementioned range is preferred to be kept. The lipid for forming the lipid nanosphere is dissolved in an organic solvent capable of dissolving lipids, such as chloroform, methanol, toluene, and the like.

[44] In step (A-2), a poorly water soluble drug is dissolved in a Ci-C₆ linear or branched alcoholto obtain a drug solution. Examples of the Ci-C₆ linear or branched alco- holinclude methanol, ethanol, propanol, butanol, isobutanol, isopropanol, and the like. Preferably, the poorly water soluble drug is dissolved in the alcohol to a concentration of 0.1 to 1 mg/mL. When the poorly water soluble drug, preferably amphotericin B, is contained less than 0.1 mg/mL, the concentration of the encapsulated drug decreases. In contrast, when the concentration exceeds 1 mg/mL, the drug may not be completely dissolved in the alcohol. And, in case the drug is dissolved in an organic solvent other than alcohol, e. g., dimethyl sulfoxide (DMSO), dimethylformamide (DMF), etc., removal of the solvent may be complicated and biocompatibility may decrease.

[45] In step (A-3), the lipid-PEG mixture solution of step (A-I) and the drug solution of step (A-2) are mixed with a volume ratio of 1:1 to 1:9 to obtain a lipid-PEG-drug mixture solution. When the amount of the solution of step (A-I) exceeds 50 (v/v)%, concentration of the drug decreases. In contrast, when the amount is below 10 (v/v)%, formulation may be difficult because the quantity of the phospholipid is too small.

[46] In step (A-4), the lipid-PEG-drug mixture solution of step (A-3) is dispersed in an aqueous medium with a volume ratio of 2:1 to 1:10, more preferably 1:1 to 1:3, to obtain lipid nanospheres. For the aqueous medium, distilled water, phosphate buffer, saline solution, sugar solution, e.g., sucrose solution, maltose solution, mannitol solution, and the like, or isotonic solution may be used. When the volume of the aqueous medium is less than the aforementionedrange, the dispersed lipid nanosphere particles may coagulate, thereby resulting in increase in particle size of the final lipid nanospheres. In contrast, when the volume of the aqueous medium exceeds the aforementioned range, a concentration process may be required. Preferably, the dispersion for forming the lipid nanospheres is performed by dispersing the lipid-PEG-drug mixture solution of step (A-3) in water by tip sonication at a rate of 1 to 5 mL/min using a syringe. Particle size may increase when the dispersion rate exceeds the aforementioned range, and it is difficult to attain smaller particle size even when the dispersion rate is lower than the aforementioned range. Hence, the aforementioned range is preferred to be kept.

[47] In step (A-5), the lipid nanosphere solution of step (A-4) is distilled at 20 to 5O⁰C under reduced pressure to remove the organic solvent, and filtered to obtain lipid nanospheres with uniform particle size and having PEG groups, in which the drug is encapsulated. When the temperature during the distillation under reduced pressure is below 2O⁰C, it takes longer to remove the organic solvent and it is difficult to completely remove the organic solvent. In contrast, when the temperature exceeds 5O⁰C, the lipid nanospheres may be damaged or the drug may be denatured. Hence, it is preferable to maintain the aforementioned temperature range. For perfect removal of the organic solvent, it is preferable to remove, along with the organic solvent, 0.5 to 5 times the volume of water during the distillation under reduced pressure. The purified lipid nanosphere solution is subjected to an injection molding machine to obtain a solution of lipid nanospheres having a uniform particle size distributed between 0.1 and 0.5 μm. Preferably, the filter membrane used in the injection molding machine has a pore size of 0.1 to 0.5 μm, identical to the particle size of the lipid nanospheres. When the pore size is larger than 0.5 μm, the particle size of the lipid nanosphere becomes larger than 0.5 μm, thus resulting in blockage at capillaries or uptake by reticuloendothelial cells during intraveneous injection, and consequent abrupt decrease of circulation time in the blood. In contrast, when the pore size is smaller than 0.1 μm, most of the particles pass through semipermeable membranes, thereby resulting in abrupt decrease of availability.

[48] Additionally, dialysis, gel permeation chromatography, filtration at high pressure, and the like may be performed to remove free PEGs remaining without being used to modify the lipid nanospheres. More preferably, gel permeation chromatography is performed to remove them along with phospholipids, drug, etc., remaining without being included in the lipid nanosphere.

[49] As another preparation method of the anionic lipid nanosphere according to the present invention, the method (B) of forming an ion complex comprising lipid and PEG having terminal amine groups is as follows.

[50] In step (B-I), phosphatidylcholine, anionic phospholipid and sterol are mixed with a weight ratio of 40-70 : 5-20 : 10-40 to obtain a lipid mixture solution, as in step (A-I).

[51] In step (B -2), a poorly water soluble drug is dissolved in a Ci-C₆ linear or branched alcohol to obtain a drug solution, as in step (A-2).

[52] In step (B-3), the lipid mixture solution of step (B-I) and the drug solution of step

(B-2) are mixed with a volume ratio of 1:1 to 1:9 to obtain a lipid-drug mixture solution, as in step (A-3).

[53] In step (B-4), the mixture solution of step (B-3) is dispersed in an aqueous medium with a volume ratio of 2: 1 to 1:10, more preferably 1 : 1 to 1 :3, to obtain lipid nanospheres, as in step (A-4).

[54] In step (B-5), the lipid nanosphere solution of step (B-4) is distilled at 20 to 5O⁰C under reduced pressure to remove the organic solvent, and filtered to obtain lipid nanospheres with uniform particle size, in which the drug is encapsulated, as in step (A-5).

[55] In step (B-6), the lipid of step (B-I) is mixed with a PEG having terminal amine groups with a weight ratio of 100:10 to 100:30 and, after adjusting pH to 1 to 4, heating is performed at 40 to 65⁰C for 10 to 30 minutes, so that the lipid and the PEG form an ion complex on the surface of the anionic lipid nanospheres, to obtain lipid nanospheres on which PEG is coated. That is, the PEG is coated on the surface of the lipid nanospheres as an ion complex is formed by the bonding of the anionic groups present on the surface of the lipid nanospheres with the terminal amine groups of the PEG.

[56] An acidic pH condition is selected to facilitate the formation of an ion complex.

When the reaction temperature is below 5O⁰C, the reaction requires a longer time. In contrast, when reaction temperature is above 65⁰C, stability of the lipid nanospheres may be deteriorated. Specific examples of the PEG having terminal amine groups include aminopolyethylene glycol, diaminopolyethylene oxide, amino(polyethylene glycol) methyl ether, and other polyethylene glycol or polyethylene oxide having terminal amine groups.

[57] Additionally, dialysis, gel permeation chromatography, filtration at high pressure, and the like may be performed to remove free PEGs remaining without being used to modify the lipid nanospheres. More preferably, gel permeation chromatography is performed to remove them along with phospholipids, drug, etc., remaining without being included in the lipid nanosphere.

[58] Thus prepared anionic lipid nanospheres have improved encapsulation efficiency of a poorly water soluble drug in an aqueous medium and increase circulation time in blood because the surface of the lipid nanospheres is modified by introducing the PEG containing polymer thereon. Accordingly, they are expected to be useful in solubilizing various poorly water soluble drugs, including amphotericin B.

[59]

Mode for the Invention

[60] The present invention is explained further with the following examples buth they should not be construed as limiting the scope of the present invention.

[61]

[62] Preparation Example 1

[63] 60 mg of phosphatidylcholine (PC) selected from dilauroyl phosphatidylcholine

(DLPC), dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC) and distearoyl phosphatidylcholine (DSPC) and 20 mg of cholesterol (CHOL) were dissolved in 2 mL of chloroform to obtain a lipid mixture solution.

[64] Amphotericin B (AmB) was dissolved in methanol to a concentration of 0.5 mg/mL.

2 mL of the lipid mixture solution was mixed with 8 mL of the amphotericin B solution to obtain 10 mL of an AmB-lipid mixture solution.

[65] The AmB-lipid mixture solution, in the amount of 10 mL, was dispersed in 20 mL of distilled water by tip sonication at a rate of 2 mL/min using a syringe to form lipid nanospheres. The organic solventand distilled water, in the amount of 10 mL, respectively, were removed at 35⁰C by distillation under reduced pressure until the volume of the solution decreased to 10 mL. Particle size distribution of the lipid nanospheres was made uniform by passing them through a 0.2 μm semipermeable membrane using an extruder.

[66] The particle size of thus prepared lipid nanospheres was measured with an elec- trophoretic light scattering spectrophotometer (ELS-Z, Otsuka Electronics, Japan). The result is given in the following Table 1.

[67] Table 1 [Table 1] [Table ]

[68] Preparation Example 2: Preparation of lipid nanospheres comprising anionic phos- pholipids [69] Anionic lipid nanospheres were prepared in the same manner as in Preparation Example 1, except for adding anionic phospholipids dipalmitoyl glycerophosphate (DPPA) or disteroyl glycerophosphoglycerol (DSPG) in order to improve the encapsulation of Sample 3, which has a particle size smaller than 150 nm and T_g of 41⁰C, to 90% or better.

[70] Changes of particle size, zeta potential and encapsulation efficiency of the lipid nanospheres depending on the contents of DPPA and DSPG were measured. The result is given in the following Table 2 and Table 3.

[71] Table 2

[Table 2] [Table ]

[72] Table 3 [Table 3] [Table ]

[73] Preparation Example 3: Preparation of PEG coated lipid nanospheres (A) [74] Anionic lipid nanospheres were prepared in the same manner as in Preparation Example 2, except for adding 0 to 80 parts by weight of a DSPE-mPEG2000 solution to 100 parts by weight of the lipid mixture solution (Sample 8). Change of particle size depending on the content of DSPE-mPEG2000 is given in the following Table 4.

[75] Table 4 [Table 4] [Table ]

[76] Preparation Example 4: Preparation of PEG coated lipid nanospheres (B) [77] Surface of anionic lipid nanospheres was coated with PEG by adding a solution of PEG having terminal amine groups (mPEG-NH₂2000) to the lipid mixture solution (Sample 8) with a weight ratio of 0 to 80. After adjusting pH to 2, heating was performed at 55⁰C for 20 minutes to form an ion complex. Change of particle sizedepending on the content of mPEG-NH₂is given in the following Table5.

[78] Table 5 [Table 5] [Table ]

Composition (weight ratio) Particle size Enc ap s ulat ion

DPPC CHOL DPPA InPEG-NH₂ (run) efficiency (%)

Sample S 60 20 20 0 108.6 96.9

Sample 17 60 20 20 10 109.5 96.9

Sample 18 60 20 20 20 109.7 96.9

Sample 19 60 20 20 40 128.1 96.9

Sample 20 60 20 20 80 184.7 96.9

[79] Test Example 1 : Circulation time in blood [80] Sodium deoxycholate was added to Fungizone (Bristol Myers-Squibb), AmBisome liposome (NeXstar Pharmaceuticals), Sample 8, Sample 14 and Sample 18 to solubilize amphotericin B. Thus prepared injections were administered to SD rats through the tail vein. Blood was taken from the rats at different times to measure the concentration of the drug encapsulated in the lipid nanospheres. Pharmacokinetic parameters and circulation time in the blood were calculated from the measurement result. Concentrationof drug in the lipid nanospheres was measured as follows. The blood sample was diluted with heparin solution and centrifuged. The supernatant was diluted with a solution of 0.5 μg/mL of l-amino-4-nitronaphthalene in methanol. Ab- sorbance was measured using a UV spectrometer at a wavelength of 408 nm.

[81] The result is shown in FIG. 1.

[82] As can be seen from FIG. 1, the PEG coated lipid nanospheres according to the present invention [Sample 14 (Preparation Example 3) and Sample 18 (Preparation Example 4)] exhibited much longer circulation time in blood than Fungizone, and longer circulation time in blood than AmBisomeuntil 3 hours after injection. This demonstrates that the injections including the PEG coated anionic lipid nanospheres according to the present invention (Sample 14 and Sample 18) provide improved circulation time in blood over existing amphotericin B formulations.

[83]

[84] Test Example 2: Toxicity test

[85] MTT test was carried out using human kidney 293 cells. 293 cells were cultured on a

96- well plate, with a concentration of 1 10⁴ cells/mL, in a CO₂ incubator of 37⁰C for 24 hours. To each well plate, amphotericin B was added with concentrations of 6.25, 12.5, 25, 50 and 100 μg/mL, and, after adding each of Fungizone, Sample 8, Sample 14 and Sample 18, the cells were further cultured in the CO₂ incubator for 12 hours. After adding MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] reagent, the cells were further cultured in the CO₂ incubator at 37⁰C for 4 hours to form formazan crystals. After removing 200 μg/mL of solution was from each well plate, the formazan crystals were dissolved by adding 150 μg/mL dimethyl sulfoxide solution. Enzyme-linked immunosorbent assay (ELISA) was carried out and the result was measured at 590 nm.

[86] The toxicity test result is shown in FIG. 2.

[87] As can be seen from FIG. 2, the lipid nanospheres according to the present invention

(Sample 14 and Sample 18) exhibited higher cell viability than Fungizone. It is believed that the toxicity of amphotericin B was decreased by the use of biocompatible phospholipid, cholesterol and PEG. This demonstrates that cell toxicity can be decreased by encapsulating amphotericin B in lipid nanospheres using biocompatible substances. Industrial Applicability

[88] As described above, the present invention relates to an anionic lipid nanosphere having negative surface charge prepared by introducing PEG containing polymers on the surface of particles formed of anionic phospholipids to provide negative charge on the surface, thereby increasing encapsulation efficiency of a poorly water soluble drug in an aqueous medium, reducing toxicity of highly toxic drug against normal cells by encapsulating, and increasing the duration of systemic circulation, and a method of its preparation. The present invention will be very useful for solubilization poorly water soluble drugs such as amphotericin B in an aqueous medium for injection, reduction toxicity thereof and increaseof circulation time in blood.

[89] The invention has been described in detail with reference to preferred embodiments thereof. However, it will be appreciated by those skilled in the art that changes may be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims

[1] An anionic lipid nanosphere for encapsulating a poorly water soluble drug wherein a polyethylene glycol (PEG) containing polymer is coated on the surface of particlesformed of anionic phospholipid.

[2] The lipid nanosphere according to claim 1, wherein the anionic phospholipid is contained in an amount of 5 to 20 weight % based on the entire lipid composition of the lipid nanosphere.

[3] The lipid nanosphere according to claim 1, wherein the poorly water soluble drug is amphotericin B.

[4] The lipid nanosphere according to claim 1, wherein the anionic phospholipid is phosphatidic acid having a hydrophobic Ci₄-Ci₈ alkyl chain.

[5] The lipid nanosphere according to claim 1, wherein the anionic phospholipid is selected from dimyristyl glycerophosphate (DMPA), dipalmitoyl glycerophosphate (DPPA), dimyristyl glycerophosphate (DMPG), disteroyl glycerophosphate (DSPA), disteroyl glycerophosphoglycerol (DSPG), dipalmitoyl glycerophosphoglycerol (DPPG), dimyristyl glycerophosphoserine (DMPS), dipalmitoyl glycerophosphoserine (DPPS), disteroyl glycerophosphoserine (DSPS) and a mixture thereof.

[6] The lipid nanosphere according to claim 1, wherein the PEG containing polymer is selected from disteroyl glycerophosphoethanolamine methyloxyethylene glycol (DSPE-mPEG), polyoxyethylene sorbitan monopalmitate (T ween), polyethylene polypropylene glycol (poloxamer) and a mixture thereof

[7] A preparation method of an anionic lipid nanosphere for encapsulating a poorly water soluble drug, comprising the steps of:

1) mixing 100 parts by weight of a lipid, which is prepared by mixing phosphatidylcholine, anionic phospholipid and sterol with a weight ratio of 40-70 : 5-20 : 10-40, with 10 to 30 parts by weight of a PEG containing polymer, and dissolving in an organic solvent to obtain a lipid-PEG mixture solution

2) dissolving a poorly water soluble drug in a Ci-C₆ linear or branched alcohol to obtain a drug solution

3) mixing the lipid-PEG mixture solution of step 1) with the drug solution of step 2) with a volume ratio of 1 : 1 to 1 :9 to obtain a lipid-PEG-drug mixture solution

4) dispersing the mixture solution of step 3) in an aqueous medium with a volume ratio of 2: 1 to 1 : 10 to form lipid nanospheres and

5) distilling the lipid nanosphere solution of step 4) at 20 to 50°Cunder reduced pressure, removing the organic solvent, and filtering to obtain anionic lipid nanospheres with uniform size in which the drug is encapsulated.

[8] A preparation method of an anionic lipid nanosphere for encapsulating a poorly water soluble drug, comprising the steps of:

1) dissolving a lipid prepared by mixing phosphatidylcholine, anionic phospholipid and sterol with a weight ratio of 40-70 : 5-20 : 10-40 in an organic solvent to obtain a lipid mixture solution

3) mixing the lipid mixture solution of step 1) with the drug solution of step 2) with a volume ratio of 1 : 1 to 1 :9 to obtain a lipid-drug mixture solution

4) dispersing the mixture solution of step 3) in an aqueous medium with a volume ratio of 2: 1 to 1 : 10 to form lipid nanospheres

5) distilling the lipid nanosphere solution of step 4) at 20 to 5O⁰C under reduced pressure, removing the organic solvent, and filtering to obtain anionic lipid nanospheres with uniform size in which the drug is encapsulated and

6) mixing with a PEG having terminal amine groups with a weight ratio of the lipid of step 1) to the PEG having terminal amine groups being 100:10 to 100:30 to form lipid-PEG ion complexes.

[9] The preparation method of a lipid nanosphere according to claim 8 or 9, wherein the drug is amphotericin B. [10] The preparation method of a lipid nanosphere according to claim 8 or 9, wherein, in step 2), the drug is dissolved in the Ci-C₆ linear or branched alcohol to a concentration of 0.1 to 1 mg/mL. [11] The preparation method of a lipid nanosphere according to claim 8 or 9, wherein, in step 4), the mixture solution of step 3) is dispersed by tip sonication at a rate of 1 to 5 mL/min using a syringe. [12] The preparation method of a lipid nanosphere according to claim 8 or 9, wherein, in step 5), along with the organic solvent, 0.5 to 5 times the volume of water is removed during the distillation under reduced pressure. [13] The preparation method of a lipid nanosphere according to claim 8 or 9, wherein, in step 5), the filtration is performed using a filter membrane having a pore size ofO.l to 0.5 μm in order to attain uniform particle size distribution. [14] The preparation method of a lipid nanosphere according to claim 9, wherein, in step 6), pH is adjusted to 1 to 4 to forman ion complex. [15] The preparation method of a lipid nanosphere according to claim 9, wherein, in step 6), reaction temperature is maintained at 40 to 65⁰C to form an ion complex.