Title of Invention	AMPHIPHILIC BLOCK COPOLYMER MICELLE COMPOSITION CONTAINING TAXANE AND MANUFACTURING PROCESS OF THE SAME
Abstract	A taxane-containing amphiphilic block copolymer micelle composition, including taxane, an amphiphilic block copolymer containing a hydrophilic block and a hydrophobic block, and an osmolality adjusting agent, is described. Also described are a method for preparing the same composition. The composition has excellent stability so that it can prevent rapid release of a drug and can improve a desired pharmacological effect. Additionally, the method enables highly efficient preparation of the composition.

Title of Invention

AMPHIPHILIC BLOCK COPOLYMER MICELLE COMPOSITION CONTAINING TAXANE AND MANUFACTURING PROCESS OF THE SAME

Abstract

A taxane-containing amphiphilic block copolymer micelle composition, including taxane, an amphiphilic block copolymer containing a hydrophilic block and a hydrophobic block, and an osmolality adjusting agent, is described. Also described are a method for preparing the same composition. The composition has excellent stability so that it can prevent rapid release of a drug and can improve a desired pharmacological effect. Additionally, the method enables highly efficient preparation of the composition.

Full Text	Description AMPHIPHBLIC BLOCK COPOLYMER MICELLE COMPOSITION CONTAINING TAXANE AND MANU- FACTURING PROCESS OF THE SAME Technical Field [1] Example embodiments of the present invention relate to an amphiphilic block copolymer micelle composition containing taxane and a process for preparing the same. Background Art [2] Submicronic particulate drug delivery systems using biodegradable polymers have been studied for the purpose of carrying out intravenous administration of drugs. Recently, it has been reported that nanoparticle systems and polymeric micelle systems using biodegradable polymeis are useful technological systems that can modify the in vivo distribution of a drug to reduce undesired side effects and can provide improved efficiency. Additionally, because such systems enable targeted drug delivery, they can achieve controlled drug release to target organs, tissues or cells. In fact, such systems are known to have excellent compatibility with body fluids and to improve the solu- bilization ability of a hardly soluble drug and the bioavailability of a drug. [3] Recently, there has been reported a method for preparing block copolymer micelles by chemically bonding a drug to a block copolymer comprising a hydrophilic segment and a hydrophobic segment. The block copolymer is an A-B type diblock copolymer polymerized from a hydrophilic segment (A) and a hydrophobic segment (B). In the above-mentioned block copolymer, polyethylene oxide is used as the hydrophilic segment (A) and a polyaminoacid or hydrophobic group-bonded polyaminoacid is used as the hydrophobic segment (B). Such drugs as Adriamycin or indomethacin can be physically encapsulated within the cores of the polymeric micelles formed from the block copolymer, so that the block copolymer micelles can be used as drug delivery systems. However, the polymeric micelles formed from the block copolymer cause many problems in the case of in vivo applications, since they cannot be hydrolyzed in vivo but are degraded only by enzymes, have poor biocompatibility, and cause immune responses, or the like. [4] Therefore, many attempts have been made to develop core-shell type drug delivery systems having improved biodegradability and biocompatibility. [5] For example, diblock or multiblock copolymers comprising polyalkylene glycol as a hydrophilic polymer and polylactic acid as a hydrophobic polymer are known to those skilled in the art. More particularly, acrylic acid derivatives are bonded to the end groups of such diblock or multiblock copolymers to form copolymeis. The resultant copolymers are subjected to crosslinking to stabilize the polymeric micelles. However, methods for preparing such diblock or multiblock copolymeis have difficulties in in- troducing croslinkers to the hydrophobic segments of A-B or A-B-A type diblock or triblock copolymeis for the polymers to form stable structures via croslinking. Ad- ditionally, the croslinkeis used in the above methods may not ensure safety in the human body because the crosslinkers have not been applied in the human body as yet. Furthermore, the croslinked polymeis cannot be degraded in vivo, and thus cannot be applied for in vivo use. [6] As another example, a so-called solvent evaporation process has been known as a method for preparing a polymer micelle composition. The solvent evaporation process can be applied as a large-scale process by which taxane derivatives, which are hardly soluble in water, can be encapsulated within amphiphilic block copolymer micelles. However, utilization of the solvent evaporation process is limited with respect to the selection of a solvent, because the solvent should be an organic solvent in which both taxane and the polymer can be dissolved, and should have such a low boiling point that it can be volatilized via evaporation. In addition, the organic solvent should be a phar- maceutically acceptable solvent, whose residue does not adversely affect the human body. Further, the solvent evaporation process essentially includes a step of exposing reagents to high temperature for a long period of time, and thus it may cause such problems as degradation of pharmaceutically active ingredients or decreased pharma- cological effects. Disclosure of Invention Technical Problem [7] Therefore, in an effort to solve the above-described problems associated with the related art, there is provided a taxane-containing amphiphilic block copolymer micelle composition having improved stability. [8] There is also provided a process for preparing a taxane-containing amphiphilic block copolymer micelle composition via simplified steps in short time. Technical Solution [9] In an aspect, there is provided a taxane-containing amphiphilic block copolymer micelle composition comprising taxane, an amphiphilic block copolymer containing a hydrophilic block and a hydrophobic block, and an osmolality adjusting agent. [101. In another aspect, there is provided a process for preparing a taxane-containing am- phiphilic block copolymer micelle composition, comprising: (a) dissolving taxane and an amphiphilic block copolymer into an organic solvent; and (b) adding an aqueous solution containing an osmolality adjusting agent thereto to form polymeric micelles. Advantageous Effects [11] The taxane-containing amphiphilic block copolymer micelle composition according to one embodiment disclosed herein has excellent stability so that it can prevent rapid release of a drug. Additionally, the method for preparing the composition according to another embodiment disclosed herein avoids a need for a separate step of removing an organic solvent, thereby maximizing a desired pharmacological effect and reducing the number of preparation steps and preparation time. Brief Description of Drawings [12] Description will now be given in detail with reference to certain example em- bodiments of a taxane-containing amphiphilic block copolymer micelle composition and a process for preparing the same illustrated in the accompanying drawings which are given hereinbelow by way of illustration only and thus are not limitative, wherein: [13] Fig. 1 is the H NMR spectrum of the diblock copolymer [mPEG-PLA] obtained from Preparation Example 1; and [14] Fig. 2 is the H NMR spectrum of the diblock copolymer [mPEG-PLGA] obtained from Preparation Example 2. Mode for the Invention [15] Hereinafter, reference will now be made in detail to various embodiments, examples of which are illustrated in the accompanying drawings and described below. While the invention will be described in conjunction with example embodiments, it will be understood that the present description is not intended to be limitative. [16] The taxane-containing amphiphilic block copolymer micelle composition according to one embodiment disclosed herein may comprise taxane, an amphiphilic block copolymer containing a hydrophilic block and a hydrophobic block, and an osmolality adjusting agent. The taxane-containing amphiphilic block copolymer micelle composition has excellent biodegradability and biocompatibility, and provides a polymeric micelle structure having relatively improved stability. [17] In the composition according to one embodiment disclosed herein, the taxane may be present in an amount of Q1-30 wt%, and the amphiphilic block copolymer containing a hydrophilic block and a hydrophobic block may be present in an amount of 20-98 wt%, based on the total dry weight of the micelle composition. Additionally, the osmolality adjusting agent may be present in an amount of Q1-50 wt% based on the total dry weight of the composition. [18] The taxane may be in an anhydrous or hydrated state, or amorphous or crystalline state. Additionally, the taxane may be extracted from natural plants, or may be obtained by semi-synthesis or plant cell cultivation. In one embodiment, the taxane may be present in the composition in an amount of Q1-30 wt%, specifically Q5-15 wt%, and more specifically 1-7 wt% based on the total dry weight of the composition. [19] In one embodiment, the taxane includes paclitaxel, docetaxel, 7-epipaclitaxel, t - acetyl paclitaxel, 10-desacetyl-paclitaxel, 10-desacetyl-7-epipaclitaxel, 7-xylosylpaclitaxel, 10-desacetyl-7-glutarylpaclitaxel, 7-N,N-dimethylglycylpaclitaxel, 7-L-alanylpaclitaxel or a mixture thereof. Particularly, paclitaxel or docetaxel may be used. [20] In one embodiment, the amphiphilic block copolymer may comprise a hydrophilic block (A) and a hydrophobic block (B) linked with each other in the form of A-B, A- B-A or B-A-B structure. Additionally, the amphiphilic block copolymer may form core-shell type polymeric micelles in its aqueous solution state, wherein the hy- drophobic block forms the core and the hydrophilic block forms the shell. [21] In one embodiment, the hydrophilic block (A) of the amphiphilic block copolymer may be polyethylene glycol (PEG) or monomethoxypolyethylene glycol (mPEG). Par- ticularly, it may be mPEG. The hydrophilic block (A) may have anumber average molecular weight of 500-20,000 daltons, specifically 1,000-5,000 daltons, and more specifically 1,000-2,500 daltons. [22] The hydrophobic block (B) of the amphiphilic block copolymer may be a water- insoluble, biodegradable polymer. In one embodiment, the hydrophobic block (B) may be polylactic acid (PLA) or poly(lactic-co-glycolic acid) (PLGA). In another embodiment; the hydrophobic block (B) may-have anumberaverage molecular weight of 500-20,000 daltons, specifically 1,000-5,000 daltons, and more specifically 1,000-2,500 daltons. Hydroxyl end groups of the hydrophobic block (B) may be protected with fatty acid groups, and particular examples of the fatty acid groups include acetate, propionate, butyrate, stearate, palmitate groups, and the like. The am- phiphilic block copolymer comprising the hydrophilic block (A) and the hydrophobic block (B) may be present in the composition in an amount of 20-98 wt%, specifically 65-98 wt%, and more specifically 80-98 wt% based on the total dry weight of the composition. [23] In another embodiment, the hydrophilic block (A) and the hydrophobic block (B) may be present in the amphophilic block copolymer in such a ratio that the copolymer comprises 40-70 wt%, specifically 50-60 wt% of the hydrophilic block (A) based on the weight of the copolymer. When the hydrophilic block (A) is present in a proportion less than 40%, the polymer has undesirably low solubility to water, resulting in difficulty in forming micelles. On the other hand, when the hydrophilic block (A) is present in a proportion greater than 70%, the polymer becomes too hydrophilic to form stable polymeric micelles, and thus the composition may not be used as a composition for solubilizing taxane. [24] The osmolality adjusting agent functions to improve the stability of the taxane- containing amphiphilic block copolymer micelle composition. Particularly, the osmolality adjusting agent significantly improves the stability of the composition in its aqueous solution state. One possible mechanism of the function of the osmolality adjusting agent is as follows. [25] The degree of encapsulation of a drug within a polymeric micelle structure is in proportion to the fraction of cores formed from the hydrophobic block of the polymer in an aqueous solution. Additionally, the stability of the polymeric micelles depends on the dynamic equilibrium state formed by the polymeric micelles in an aqueous solution, i.e., on the equilibrium constant between the polymeric micelle state and the unimer state dissolved in water. [26] Although a large amount of poorly soluble drug can be encapsulated within a polymeric micelle structure, the hydrophilic blocks of the polymer micelles may be surrounded with a great amount of water molecules upon the encapsulation of the drug, and thus the interaction between the water molecules and the hydrophilic blocks may weaken the hydrophobic interaction between hydrophobic blocks of the micelles, thereby destabilizing the micelles in a dynamic equilibrium state. Addition of the osmolality adjusting agent causes an electrostatic attraction force between the osmolality adjusting agent and water, resulting in dissociation of water molecules from the hydrophilic blocks of the polymeric micelles. As a result, the hydrophobic in- teraction between the hydrophobic blocks, which otherwise would participate in loose interaction, increases relatively, so that stable micelle structures can be formed. In addition, the osmolality adjusting agent is not removed during the preparation of the composition according to one embodiment disclosed herein but remains in the finished composition. Through the stabilization effect realized by the osmolality adjusting agent, the taxane-containing amphiphilic block copolymer micelle composition has excellent stability. [27] The osmolality adjusting agent is pharmaceutically acceptable one and may be selected from any osmolality adjusting agents as long as it does not cause hemolysis upon the contact with blood. In one embodiment, the osmolality adjusting agent may be an electrolyte, specifically an inorganic salt. Preferably, the osmolality adjusting agent may be at least one selected from the group consisting of sodium chloride, calcium chloride, sodium sulfate and magnesium chloride. More particularly, the osmolality adjusting agent may be sodium chloride or calcium chloride. Especially, it may be sodium chloride. In another embodiment, the osmolality adjusting agent may be present in the composition in an amount of 0.1-50 wt%, specifically 0.5-20 wt%, and more specifically 1-10 wt%, based on the total dry weight of the composition. [28] In another aspect, there is provided a lyophilized composition comprising the taxane- containing amphiphilic block copolymer micelle composition. [29] The lyophilized composition may further comprise a lyophilization aid. In one embodiment, the lyophilization aid may be at least one selected from the group consisting of lactose, mannitol, sorbitol and sucrose. The lyophilization aid is added for the lyophilized composition to maintain a cake form. In addition, the lyophilization aid serves to help the amphiphilic block copolymer micelle composition to form homo- geneously in short time during the reconstitution of the lyophilized composition. In another embodiment, the lyophilization aid may be used in an amount of 1-90 wt%, and more particularly 10-60 wt%, based on the total dry weight of the lyophilized composition. [30] In one embodiment, the lyophilized composition may comprise 0.1-15 wt% of taxane based on the total dry weight of the composition, upon the reconstitution in an aqueous solution. Additionally, upon the reconstitution, the amphiphilic block copolymer may be present at a concentration of 10-150 mg/mL, the osmolality adjusting agent may be present at a concentration of 5-30 mg/mL (specifically, 10-20 mg/mL), and the lyophilization aid may be present at a concentration of 1-100 mg/mL. In another embodiment, the lyophilized composition can have a controlled micelle particle size in a range of 1-400 nm, and more particularly 5-200 nm in an aqueous solution, depending on the molecular weight of the copolymer. [31] In one embodiment, the taxane-containing amphiphilic block copolymer micelle composition may be formulated into the form of an aqueous solution, powder or tablet. In another embodiment, the composition may be an injection formulation. For example, the composition may be reconstituted with distilled water for injection, 0.9% physiological saline, 5% aqueous dextrose solution, and the like. When the composition is reconstituted, at least 95% of taxane is stable for 12 hours or more without precipitation. [32] In still another aspect, there is provided a method for preparing the taxane-containing amphiphilic block copolymer micelle composition. [33] According to one embodiment disclosed herein, the method for preparing the taxane- containing amphiphilic block copolymer micelle composition may comprise: [34] (a) dissolving taxane and an amphiphilic block copolymer into an organic solvent; and [35] (b) adding an aqueous solution containing an osmolality adjusting agent thereto to form polymeric micelles. [36] In another embodiment, the method may further comprise, after step (b): [37] (c) adding a lyophilization aid to the polymeric micelles; and [38] (d) carrying out lyophilization. [39] When taxane is encapsulated with a micelle composition by using an organic solvent via a solvent evaporation process, rapid drug precipitation may occur in the taxane- containing micelle composition after the composition is reconstituted in injection water and is left at room temperature. This is because the organic solvent used in the solvent evaporation process remains in the composition. [40] Therefore, according to one embodiment of the method disclosed herein, drug pre- cipitation may be prevented by using an osmolality adjusting agent and a minimized amount of organic solvent. To minimize the amount of the organic solvent still remaining in the finished composition, the composition needs to be dried at a high temperature of 6CPC or higher under reduced pressure for at least 12 houis. However, such reduced-pressure, high-temperature drying conditions may cause degradation of a drug. Thus, the method for preparing the taxane-containing amphiphilic block copolymer micelle composition according to one embodiment disclosed herein uses a minimized amount of organic solvent so that the finished composition can be directly subjected to lyophilization while avoiding a need for a separate step of removing the organic solvent. [41] The taxane-containing amphiphilic block copolymer micelle composition containing the osmolality adjusting agent and using a minimized amount of organic solvent according to one embodiment disclosed herein can provide a lyophilized composition which is free from precipitation of taxane for 12 hoius or more when reconstituted into an injection formulation. [42] In one embodiment, the organic solvent in step (a) may include at least one selected from the group consisting of acetone, ethanol, methanol, ethyl acetate, acetonitrile, methylene chloride, chloroform, acetic acid and dioxane. The organic solvent may be used in an amount of 0.5-30 wt%, specifically 0.5-15 wt%, and more specifically 1-10 wt% based on the weight of the resultant micelle composition. When the organic solvent is used in an amount less than 0.5 wt%, there may be a difficulty in dissolving a drug. On the other hand, when the organic solvent is used in an amount greater than 30 wt%, drug precipitation may occur upon the reconstitution of the lyophilized composition. [43] In step (b), the osmolality of the aqueous solution containing the osmolality adjusting agent may be adjusted to 30-15,000 mOsm/kg, specifically 100-5,000 mOsm/kg, and more specifically 200-2,590 mOsm/kg. When the osmolality of the aqueous solution is less than 30 mOsm/kg, drug precipitation may occur during the preparation of the composition. On the other hand, when the osmolality is greater than 15,000 mOsm/kg, phase separation may occur in the polymer. In one example embodiment, the osmolality adjusting agent may be at least one selected from the group consisting of sodium chloride, calcium chloride, sodium sulfate and magnesium chloride. In addition, the osmolality adjusting agent may be used in an amount of 0.1-50 wt% based on the total dry weight of the micelle composition. Step (b) may be performed at a temperature of 25°C or lower. [44] In one embodiment, the method for preparing the taxane-containing amphiphilic block copolymer micelle composition may further comprise sterilizing the aqueous polymeric micelle solution obtained from step (c) with a sterilization filter, before step (d) of carrying out lyophilization. [45] The taxane-containing amphiphilic block copolymer micelle composition according to one embodiment disclosed herein may be orally or parenterally administered in the form of an aqueous solution or powder. Parenteral administration includes admin- istration via intravascular, intramuscular, subcutaneous, intraperitoneal, nasal, rectal, ophthalmic, pulmonary or other routes. Oral administration includes administration in the form of tablets or capsules, or aqueous solution itself. [46] In addition, the lyophilized composition according to one embodiment disclosed herein causes little variation in the concentration of docetaxel in a reconstituted composition over time. However, when no osmolality adjusting agent is added, docetaxel concentration decreases after the lapse of one hour. [47] [48] The following examples are not intended to be limitative. [49] [50] Preparation Example 1: Synthesis of Monomethoxypolyethylene glycol-Polylactide (mPEG-PLA) Block Copolymer (A-B Type) [51] First, 5.0 g of monomethoxypolyethylene glycol (number average molecular weight: 2,000 daltons) was introduced into a 100 mL two-neck round-bottom flask, and was heated to 13CPC under reduced pressure (1 mmHg) for 3-4 houis to remove water therefrom. Next, the flask was purged with nitrogen gas, and stannous octoate (Sn(Oct) ) was added thereto as a reaction catalyst using a syringe in an amount of 0.1 wt% (10.13 mg, 25 mmol) based on the weight of d- and 1-lactides. After the reaction mixture was agitated for 30 minutes, it was subjected to depressurization (1 mmHg) at 130oC for 1 hour to remove the solvent (toluene) in which the catalyst was dissolved. Then, 10.13 g of purified lactide was added thereto, and the resultant mixture was heated at 130oC for 18 houis. After heating, the resultant polymer was dissolved into methylene chloride, and was added to diethyl ether to cause precipitation of the polymer. The resultant polymer was dried in a vacuum oven for 48 houis. [52] The copolymer, monomethoxylpolyethylene glycol-polylactide (mPEG-PLA), had a number average molecular weight of 2,000-1,765 daltons. Analysis of the copolymer performed by H-NMR revealed that the copolymer was an A-B type diblock copolymer (see Fig. 1). [53] [54] Preparation Example 2: Synthesis of Monomethoxypolyethylene glycol- Poly(lactic-co-glycolic acid) (mPEG-PLGA) Block Copolymer (A-B Type) [55] A block copolymer was obtained by reacting monomethoxypolyethylene glycol (number average molecular weight: 5,000 daltons), lactide and glycolide in the presence of stannous octoate as a catalyst at 12CPC for 12 houis in the same manner as Preparation Example 1. [56] The copolymer, monomethoxypolyethylene glycol-poly(lactic-co-glycolic acid) (mPEG-PLGA), had a number average molecular weight of 5,000-4,000 daltons and was an A-B type copolymer. Analysis of the copolymer performed by 1H-NMR revealed that the copolymer was an A-B type diblock copolymer (see Fig. 2). [57] [58] Example 1: Preparation of mPEG-PLA Block Copolymer Micelle Composition Containing Sodium Chloride and Docetaxel [59] First, 760 mg of the amphiphilic block copolymer, mPEG-PLA (number average molecular weight: 2,000-1,765 daltons), obtained from Preparation Example 1 was completely dissolved into 0.2 mL of ethanol at 6CPC to provide a clear ethanol solution comprising the copolymer. The ethanol solution was cooled to 25°C, and 20 mg of docetaxel was added thereto and the resultant solution was agitated until docetaxel was completely dissolved. [60] Next, aqueous solutions each containing 0.9 wt% and 1.8 wt% of sodium chloride and having an osmolality of 300 mOsm/kg and 600 mOsm/kg were prepared in separate containers. The osmolality was measured by using a commercially available osmometer (Gonotech GmbH, OSMOMAT030). Each aqueous solution was added to the ethanol solution comprising the copolymer in an amount of 4 mL, and the resultant mixture was agitated at 4CPC for 10 minutes to form an aqueous polymeric micelle solution. [61] Then, 100 mg of d-mannitol was dissolved into each solution, and the resultant solution was filtered through a filter with a pore size of 200 nm to remove undissolved docetaxel, followed by lyophilization. [62] The lyophilized composition was subjected to liquid chromatography as follows to determine the content of docetaxel. Additionally, particle size was measured by a dynamic light scattering (DLS) method. The results are shown in the following Table 1. [63] [Table 1] [64] [65] Liquid Chromatography [66] 1) Column: a stainless steel column having a length of 250 mm and an inner diameter of 4.6 mm and packed with pentafluorophenyl-coated particles having a particle diameter of 5 µm and a pore diameter of 300 A. [67] 2) Mobile Phase: acetonitrile: methanol: water = 26:32:420 [68] 3) Flow Rate: 1.5 mL/min [69] 4) Loading Amount: 20 µl [70] 5) Detector: UV absorption spectrometer (measurement wavelength: 232 nm) [71] [72] Example 2: Preparation of mPEG-PLA Block Copolymer Micelle Composition Containing Calcium Chloride and Paclitaxel [73] First, 100 mg of the amphiphilic block copolymer, mPEG-PLA (number average molecular weight: 2,000-1,765 daltons), obtained from Preparation Example 1 was completely dissolved into 0.1 mL of ethanol at 6CPC to provide a clear ethanol solution comprising the copolymer. The ethanol solution was cooled to 25°C, and 20 mg of paclitaxel was added thereto and the resultant solution was agitated until paclitaxel was completely dissolved. [74] Next, aqueous solutions each containing 0.9 wt% and 1.8 wt% of calcium chloride and having an osmolality of 230 mOsm/kg and 460 mOsm/kg were prepared in separate containers. Each aqueous solution was added to the ethanol solution comprising the copolymer in an amount of 4 mL, and the resultant mixture was agitated at 4CPC for 10 minutes to form an aqueous polymeric micelle solution. [75] Then, 39 mg of d-mannitol was dissolved into each solution, and the resultant solution was filtered through a filter with a pore size of 200 nm to remove undissolved paclitaxel, followed by lyophilization. [76] The lyophilized composition was subjected to the liquid chromatography as described in Example 1 to determine the content of paclitaxel. Additionally, particle size was measured by a DLS method. The results are shown in the following Table 2. [77] [Table 2] [78] [79] Example 3: Preparation of mPEG-PLGA Block Copolymer Micelle Composition Containing Sodium Chloride and Docetaxel [80] First, 760 mg of the amphiphilic block copolymer, mPEG-PLGA (number average molecular weight: 5,000-4,000 daltons), obtained from Preparation Example 2 was completely dissolved into 0.2 mL of acetone at 50oC to provide a clear acetone solution comprising the copolymer. The acetone solution was cooled to 25°C, and 40 mg of docetaxel was added thereto and the resultant solution was agitated until docetaxel was completely dissolved. [81] Next, 8 mL of an aqueous solution containing 0.9 wt% of sodium chloride and having an osmolality of 300 mOsm/kg was added to the acetone solution comprising the copolymer and further containing the drug, and the resultant mixture was agitated at 25°C for 20 minutes to form a homogeneous solution. Once the homogeneous solution was formed, 200 mg of d-mannitol was dissolved into the solution to provide a clear aqueous polymeric micelle solution. Finally, the aqueous polymeric micelle solution was filtered through a filter with a pore size of 200 nm to remove undissolved docetaxel, followed by lyophilization. [82] The lyophilized composition was subjected to the liquid chromatography as described in Example 1 to determine the content of docetaxel. Additionally, particle size was measured by a DLS method. [83] Docetaxel content: 101. 3 wt%. [84] Particle size: 35 nm. [85] [86] Example 4: Preparation of mPEG-PLGA Block Copolymer Micelle Composition Containing Calcium Chloride and Paclitaxel [87] Fust, 100 mg of the amphiphilic block copolymer, mPEG-PLGA (number average molecular weight: 5,000-4,000 daltons), obtained from Preparation Example 2 was completely dissolved into 0.2 mL of acetone at 50oC to provide a clear acetone solution comprising the copolymer. The acetone solution was cooled to 25°C, and 40 mg of paclitaxel was added thereto and the resultant solution was agitated until paclitaxel was completely dissolved. [88] Next, 8 mL of an aqueous solution containing 0.9 wt% of calcium chloride and having an osmolality of 230 mOsm/kg was added to the acetone solution comprising the copolymer and further containing the drug, and the resultant mixture was agitated at 25°C for 20 minutes to form a homogeneous solution. Once the homogeneous solution was formed, 53 mg of d-mannitol was dissolved into the solution to provide a clear aqueous polymeric micelle solution. Finally, the aqueous polymeric micelle solution was filtered through a filter with a pore size of 200 nm to remove undissolved paclitaxel, followed by lyophilization. [89] The lyophilized composition was subjected to high-performance liquid chro- matography (HPLC) to determine the content of docetaxel. Additionally, particle size was measured by a DLS method. [90] Docetaxel content: 101. 1 wt%. [91] Particle size: 35 nm. [92] [93] Comparative Example 1: Preparation of mPEG-PLA Block Copolymer Micelle Composition Containing No Inorganic Salt [94] First, 760 mg of the amphiphilic block copolymer, mPEG-PLA (number average molecular weight: 2,000-1,765 daltons), obtained from Preparation Example 1 was completely dissolved into 0.2 mL of ethanol at 60oC to provide a clear ethanol solution comprising the copolymer. The ethanol solution was cooled to 25°C, and 20 mg of docetaxel was added thereto and the resultant solution was agitated until docetaxel was completely disolved. [95] Next, 4 mL of distilled water for injection (osmolality: 0 mOsm/kg) was added to the ethanol solution comprising the copolymer, and the resultant mixture was agitated at 40°C for 10 minutes to form a homogeneous solution. Once the homogeneous solution was formed, 100 mg of d-mannitol was disolved into the solution to provide a clear aqueous polymeric micelle solution. Finally, the aqueous polymeric micelle solution was filtered through a filter with a pore size of 200 nm to remove undissolved docetaxel, followed by lyophilization. [96] The lyophilized composition was subjected to HPLC to determine the content of docetaxel. Additionally, particle size was measured by a DLS method. [97] Docetaxel content: 100.3 wt%. [98] Particle size: 18 nm. [99] [100] Experimental Example 1: Stability Test [101] The sodium chloride-containing polymeric micelle compositions according to Example 1 were compared with the polymeric micelle composition containing no inorganic salt according to Comparative Example 1 in terms of the stability of the aqueous solution at 37°C. [102] Each of the lyophilized compositions according to Example 1 and Comparative Example 1 was diluted with distilled water for injection to a docetaxel concentration of 1 mg/mL. While each diluted solution was left at 37°C, concentration of docetaxel contained in each micelle structure was measured over time. The results are shown in the following Table 3. [103] [Table 3] [104] [105] As can be seen from the results of Table 3, the compositions according to Example 1 cause no precipitation of docetaxel even after the lapse of 12 hous, while the composition according to Comparative Example 1 shows an amount of docetaxel pre- cipitation of 59% after the lapse of 12 hours. It can be seen from the above results that addition of sodium chloride may increase the docetaxel retainability of a micelle composition by about at least two times. Additionally, it can be also seen that a higher ratio of the amount of the inorganic salt to that of the amphiphilic block copolymer provides the micelle competition with higher stability. [106] Description has been given in detail with reference to example embodiments. However, it will be appreciated by those skilled in the art that changes may be made in these embodiment without departing from the principles and spirit of the taxane- containing amphiphilic block copolymer micelle composition and the method for preparing the same, the scope of which is defined in the accompanying claims and their equivalents. CLAIMS Claim 1 A taxane-containing amphiphilic block copolymer micelle composition, comprising taxane, an amphiphilic block copolymer containing a hydrophilic block (A) and a hydrophobic block (B), and an osmolality adjusting agent, wherein the taxane-containing amphiphilic block copolymer micelle composition comprises 0.1-30 wt% of taxane, 20-98 wt% of an amphiphilic block copolymer containing a hydrophilic block and a hydrophobic block, and 0.1-50 wt% of an osmolality adjusting agent, based on the total dry weight of the composition. Claim 2 (deleted) Claim 3 The composition as defined in claim 1, wherein the amphiphilic block copolymer containing a hydrophilic block (A) and a hydrophobic block (B) is an A-B, A-B-A or B-A-B type block copolymer. Claim 4 The composition as defined in claim 1, wherein the taxane is paclitaxel, docetaxel, 7- epipaclitaxel, t-acetylpaclitaxel, 10-desacetyl-paclitaxel, 10-desacetyl-7-epipaclitaxel, 7-xylosylpaclitaxel, 10-desacetyl-7-glutarylpaclitaxel,7-N,N-dimethylglycylpaclitaxel,7-L-alanylpaclitaxeloramixture thereof. Claim 5 The composition as defined in claim 1, wherein the hydrophilic block (A) has a number average molecular weight of 500-20,000 daltons, and the hydrophobic block (B) has a number average molecular weight of 500-20,000 daltons. Claim 6 The composition as defined in claim 1, wherein the hydrophilic block (A) is polyethylene glycol or monomethoxypolyethylene glycol, and the hydrophobic block (B) is polylactic acid(PLA) or a copolymer of polylactic acid and glycolic acid(PLGA). Claim 7 The composition as defined in claim 1, wherein the osmolality adjusting agent is an inorganic salt. Claim 8 The composition as defined in claim 7, wherein the inorganic salt is one or more selected from the group consisting of sodium chloride, calcium chloride, sodium sulfate and magnesium chloride. Claim 9 The composition as defined in claim 1, wherein the amphiphilic block copolymer micelle composition is a lyophilized composition further comprising a lyophilization aid. Claim 10 The composition as defined in claim 9, wherein the lyophilized composition is characterized that at least 95% of taxane is stable for 12 hours without pre- cipitation, when the composition is reconstituted with distilled water for injection, 0.9% physiological saline and 5% aqueous dextrose solution. [11] The composition as defined in claim 9, wherein the lyophilization aid is used in an amount of 1-90 wt% based on the total dry weight of the lyophilized composition. [12] The composition as defined in claim 9, wherein the lyophilization aid is one or more selected from the group consisting of lactose, mannitol, sorbitol and sucrose. [13] A method for preparing a taxane-containing amphiphilic block copolymer micelle composition, comprising: dissolving taxane and an amphiphilic block copolymer into an organic solvent to provide a polymer solution; and adding an aqueous solution containing an osmolality adjusting agent to the polymer solution to form polymeric micelles. [14] The method as defined in claim 13, further comprising, after adding an aqueous solution containing an osmolality adjusting agent to the polymer solution to form polymeric micelles: adding a lyophilization aid to the polymeric micelles; and carrying out lyophilization. [15] The method as defined in claim 13, wherein the organic solvent is one or more selected from the group consking of acetone, ethanol, methanol, ethyl acetate, acetonitrile, methylene chloride, chloroform, acetic acid and dioxane. [16] The method as defined in claim 13, wherein the organic solvent is used in an amount of 0.5-30 wt% based on the weight of the micelle composition. [17] The method as defined in claim 13, wherein the aqueous solution has an osmolality of 30-15,000 mOsm/kg. [18] The method as defined in claim 13, wherein the osmolality adjusting agent b one or more selected from the group consisting of sodium chloride, calcium chloride, sodium sulfate and magnesium chloride. A taxane-containing amphiphilic block copolymer micelle composition, including taxane, an amphiphilic block copolymer containing a hydrophilic block and a hydrophobic block, and an osmolality adjusting agent, is described. Also described are a method for preparing the same composition. The composition has excellent stability so that it can prevent rapid release of a drug and can improve a desired pharmacological effect. Additionally, the method enables highly efficient preparation of the composition.

Full Text

Description
AMPHIPHBLIC BLOCK COPOLYMER MICELLE
COMPOSITION CONTAINING TAXANE AND MANU-
FACTURING PROCESS OF THE SAME
Technical Field
[1] Example embodiments of the present invention relate to an amphiphilic block
copolymer micelle composition containing taxane and a process for preparing the
same.
Background Art
[2] Submicronic particulate drug delivery systems using biodegradable polymers have
been studied for the purpose of carrying out intravenous administration of drugs.
Recently, it has been reported that nanoparticle systems and polymeric micelle systems
using biodegradable polymeis are useful technological systems that can modify the in
vivo distribution of a drug to reduce undesired side effects and can provide improved
efficiency. Additionally, because such systems enable targeted drug delivery, they can
achieve controlled drug release to target organs, tissues or cells. In fact, such systems
are known to have excellent compatibility with body fluids and to improve the solu-
bilization ability of a hardly soluble drug and the bioavailability of a drug.
[3] Recently, there has been reported a method for preparing block copolymer micelles
by chemically bonding a drug to a block copolymer comprising a hydrophilic segment
and a hydrophobic segment. The block copolymer is an A-B type diblock copolymer
polymerized from a hydrophilic segment (A) and a hydrophobic segment (B). In the
above-mentioned block copolymer, polyethylene oxide is used as the hydrophilic
segment (A) and a polyaminoacid or hydrophobic group-bonded polyaminoacid is
used as the hydrophobic segment (B). Such drugs as Adriamycin or indomethacin can
be physically encapsulated within the cores of the polymeric micelles formed from the
block copolymer, so that the block copolymer micelles can be used as drug delivery
systems. However, the polymeric micelles formed from the block copolymer cause
many problems in the case of in vivo applications, since they cannot be hydrolyzed in
vivo but are degraded only by enzymes, have poor biocompatibility, and cause immune
responses, or the like.
[4] Therefore, many attempts have been made to develop core-shell type drug delivery
systems having improved biodegradability and biocompatibility.
[5] For example, diblock or multiblock copolymers comprising polyalkylene glycol as a
hydrophilic polymer and polylactic acid as a hydrophobic polymer are known to those
skilled in the art. More particularly, acrylic acid derivatives are bonded to the end
groups of such diblock or multiblock copolymers to form copolymeis. The resultant
copolymers are subjected to crosslinking to stabilize the polymeric micelles. However,
methods for preparing such diblock or multiblock copolymeis have difficulties in in-
troducing croslinkers to the hydrophobic segments of A-B or A-B-A type diblock or
triblock copolymeis for the polymers to form stable structures via croslinking. Ad-
ditionally, the croslinkeis used in the above methods may not ensure safety in the
human body because the crosslinkers have not been applied in the human body as yet.
Furthermore, the croslinked polymeis cannot be degraded in vivo, and thus cannot be
applied for in vivo use.
[6] As another example, a so-called solvent evaporation process has been known as a
method for preparing a polymer micelle composition. The solvent evaporation process
can be applied as a large-scale process by which taxane derivatives, which are hardly
soluble in water, can be encapsulated within amphiphilic block copolymer micelles.
However, utilization of the solvent evaporation process is limited with respect to the
selection of a solvent, because the solvent should be an organic solvent in which both
taxane and the polymer can be dissolved, and should have such a low boiling point that
it can be volatilized via evaporation. In addition, the organic solvent should be a phar-
maceutically acceptable solvent, whose residue does not adversely affect the human
body. Further, the solvent evaporation process essentially includes a step of exposing
reagents to high temperature for a long period of time, and thus it may cause such
problems as degradation of pharmaceutically active ingredients or decreased pharma-
cological effects.
Disclosure of Invention
Technical Problem
[7] Therefore, in an effort to solve the above-described problems associated with the
related art, there is provided a taxane-containing amphiphilic block copolymer micelle
composition having improved stability.
[8] There is also provided a process for preparing a taxane-containing amphiphilic block
copolymer micelle composition via simplified steps in short time.
Technical Solution
[9] In an aspect, there is provided a taxane-containing amphiphilic block copolymer
micelle composition comprising taxane, an amphiphilic block copolymer containing a
hydrophilic block and a hydrophobic block, and an osmolality adjusting agent.
[101. In another aspect, there is provided a process for preparing a taxane-containing am-
phiphilic block copolymer micelle composition, comprising: (a) dissolving taxane and
an amphiphilic block copolymer into an organic solvent; and (b) adding an aqueous
solution containing an osmolality adjusting agent thereto to form polymeric micelles.
Advantageous Effects
[11] The taxane-containing amphiphilic block copolymer micelle composition according
to one embodiment disclosed herein has excellent stability so that it can prevent rapid
release of a drug. Additionally, the method for preparing the composition according to
another embodiment disclosed herein avoids a need for a separate step of removing an
organic solvent, thereby maximizing a desired pharmacological effect and reducing the
number of preparation steps and preparation time.
Brief Description of Drawings
[12] Description will now be given in detail with reference to certain example em-
bodiments of a taxane-containing amphiphilic block copolymer micelle composition
and a process for preparing the same illustrated in the accompanying drawings which
are given hereinbelow by way of illustration only and thus are not limitative, wherein:
[13] Fig. 1 is the H NMR spectrum of the diblock copolymer [mPEG-PLA] obtained
from Preparation Example 1; and
[14] Fig. 2 is the H NMR spectrum of the diblock copolymer [mPEG-PLGA] obtained
from Preparation Example 2.
Mode for the Invention
[15] Hereinafter, reference will now be made in detail to various embodiments, examples
of which are illustrated in the accompanying drawings and described below. While the
invention will be described in conjunction with example embodiments, it will be
understood that the present description is not intended to be limitative.
[16] The taxane-containing amphiphilic block copolymer micelle composition according
to one embodiment disclosed herein may comprise taxane, an amphiphilic block
copolymer containing a hydrophilic block and a hydrophobic block, and an osmolality
adjusting agent. The taxane-containing amphiphilic block copolymer micelle
composition has excellent biodegradability and biocompatibility, and provides a
polymeric micelle structure having relatively improved stability.
[17] In the composition according to one embodiment disclosed herein, the taxane may be
present in an amount of Q1-30 wt%, and the amphiphilic block copolymer containing
a hydrophilic block and a hydrophobic block may be present in an amount of 20-98
wt%, based on the total dry weight of the micelle composition. Additionally, the
osmolality adjusting agent may be present in an amount of Q1-50 wt% based on the
total dry weight of the composition.
[18] The taxane may be in an anhydrous or hydrated state, or amorphous or crystalline
state. Additionally, the taxane may be extracted from natural plants, or may be
obtained by semi-synthesis or plant cell cultivation. In one embodiment, the taxane
may be present in the composition in an amount of Q1-30 wt%, specifically Q5-15
wt%, and more specifically 1-7 wt% based on the total dry weight of the composition.
[19] In one embodiment, the taxane includes paclitaxel, docetaxel, 7-epipaclitaxel, t -
acetyl paclitaxel, 10-desacetyl-paclitaxel, 10-desacetyl-7-epipaclitaxel,
7-xylosylpaclitaxel, 10-desacetyl-7-glutarylpaclitaxel, 7-N,N-dimethylglycylpaclitaxel,
7-L-alanylpaclitaxel or a mixture thereof. Particularly, paclitaxel or docetaxel may be
used.
[20] In one embodiment, the amphiphilic block copolymer may comprise a hydrophilic
block (A) and a hydrophobic block (B) linked with each other in the form of A-B, A-
B-A or B-A-B structure. Additionally, the amphiphilic block copolymer may form
core-shell type polymeric micelles in its aqueous solution state, wherein the hy-
drophobic block forms the core and the hydrophilic block forms the shell.
[21] In one embodiment, the hydrophilic block (A) of the amphiphilic block copolymer
may be polyethylene glycol (PEG) or monomethoxypolyethylene glycol (mPEG). Par-
ticularly, it may be mPEG. The hydrophilic block (A) may have anumber average
molecular weight of 500-20,000 daltons, specifically 1,000-5,000 daltons, and more
specifically 1,000-2,500 daltons.
[22] The hydrophobic block (B) of the amphiphilic block copolymer may be a water-
insoluble, biodegradable polymer. In one embodiment, the hydrophobic block (B) may
be polylactic acid (PLA) or poly(lactic-co-glycolic acid) (PLGA). In another
embodiment; the hydrophobic block (B) may-have anumberaverage molecular weight
of 500-20,000 daltons, specifically 1,000-5,000 daltons, and more specifically
1,000-2,500 daltons. Hydroxyl end groups of the hydrophobic block (B) may be
protected with fatty acid groups, and particular examples of the fatty acid groups
include acetate, propionate, butyrate, stearate, palmitate groups, and the like. The am-
phiphilic block copolymer comprising the hydrophilic block (A) and the hydrophobic
block (B) may be present in the composition in an amount of 20-98 wt%, specifically
65-98 wt%, and more specifically 80-98 wt% based on the total dry weight of the
composition.
[23] In another embodiment, the hydrophilic block (A) and the hydrophobic block (B)
may be present in the amphophilic block copolymer in such a ratio that the copolymer
comprises 40-70 wt%, specifically 50-60 wt% of the hydrophilic block (A) based on
the weight of the copolymer. When the hydrophilic block (A) is present in a proportion
less than 40%, the polymer has undesirably low solubility to water, resulting in
difficulty in forming micelles. On the other hand, when the hydrophilic block (A) is
present in a proportion greater than 70%, the polymer becomes too hydrophilic to form
stable polymeric micelles, and thus the composition may not be used as a composition
for solubilizing taxane.
[24] The osmolality adjusting agent functions to improve the stability of the taxane-
containing amphiphilic block copolymer micelle composition. Particularly, the
osmolality adjusting agent significantly improves the stability of the composition in its
aqueous solution state. One possible mechanism of the function of the osmolality
adjusting agent is as follows.
[25] The degree of encapsulation of a drug within a polymeric micelle structure is in
proportion to the fraction of cores formed from the hydrophobic block of the polymer
in an aqueous solution. Additionally, the stability of the polymeric micelles depends on
the dynamic equilibrium state formed by the polymeric micelles in an aqueous
solution, i.e., on the equilibrium constant between the polymeric micelle state and the
unimer state dissolved in water.
[26] Although a large amount of poorly soluble drug can be encapsulated within a
polymeric micelle structure, the hydrophilic blocks of the polymer micelles may be
surrounded with a great amount of water molecules upon the encapsulation of the drug,
and thus the interaction between the water molecules and the hydrophilic blocks may
weaken the hydrophobic interaction between hydrophobic blocks of the micelles,
thereby destabilizing the micelles in a dynamic equilibrium state. Addition of the
osmolality adjusting agent causes an electrostatic attraction force between the
osmolality adjusting agent and water, resulting in dissociation of water molecules from
the hydrophilic blocks of the polymeric micelles. As a result, the hydrophobic in-
teraction between the hydrophobic blocks, which otherwise would participate in loose
interaction, increases relatively, so that stable micelle structures can be formed. In
addition, the osmolality adjusting agent is not removed during the preparation of the
composition according to one embodiment disclosed herein but remains in the finished
composition. Through the stabilization effect realized by the osmolality adjusting
agent, the taxane-containing amphiphilic block copolymer micelle composition has
excellent stability.
[27] The osmolality adjusting agent is pharmaceutically acceptable one and may be
selected from any osmolality adjusting agents as long as it does not cause hemolysis
upon the contact with blood. In one embodiment, the osmolality adjusting agent may
be an electrolyte, specifically an inorganic salt. Preferably, the osmolality adjusting
agent may be at least one selected from the group consisting of sodium chloride,
calcium chloride, sodium sulfate and magnesium chloride. More particularly, the
osmolality adjusting agent may be sodium chloride or calcium chloride. Especially, it
may be sodium chloride. In another embodiment, the osmolality adjusting agent may
be present in the composition in an amount of 0.1-50 wt%, specifically 0.5-20 wt%,
and more specifically 1-10 wt%, based on the total dry weight of the composition.
[28] In another aspect, there is provided a lyophilized composition comprising the taxane-
containing amphiphilic block copolymer micelle composition.
[29] The lyophilized composition may further comprise a lyophilization aid. In one
embodiment, the lyophilization aid may be at least one selected from the group
consisting of lactose, mannitol, sorbitol and sucrose. The lyophilization aid is added
for the lyophilized composition to maintain a cake form. In addition, the lyophilization
aid serves to help the amphiphilic block copolymer micelle composition to form homo-
geneously in short time during the reconstitution of the lyophilized composition. In
another embodiment, the lyophilization aid may be used in an amount of 1-90 wt%,
and more particularly 10-60 wt%, based on the total dry weight of the lyophilized
composition.
[30] In one embodiment, the lyophilized composition may comprise 0.1-15 wt% of taxane
based on the total dry weight of the composition, upon the reconstitution in an aqueous
solution. Additionally, upon the reconstitution, the amphiphilic block copolymer may
be present at a concentration of 10-150 mg/mL, the osmolality adjusting agent may be
present at a concentration of 5-30 mg/mL (specifically, 10-20 mg/mL), and the
lyophilization aid may be present at a concentration of 1-100 mg/mL. In another
embodiment, the lyophilized composition can have a controlled micelle particle size in
a range of 1-400 nm, and more particularly 5-200 nm in an aqueous solution,
depending on the molecular weight of the copolymer.
[31] In one embodiment, the taxane-containing amphiphilic block copolymer micelle
composition may be formulated into the form of an aqueous solution, powder or tablet.
In another embodiment, the composition may be an injection formulation. For
example, the composition may be reconstituted with distilled water for injection, 0.9%
physiological saline, 5% aqueous dextrose solution, and the like. When the
composition is reconstituted, at least 95% of taxane is stable for 12 hours or more
without precipitation.
[32] In still another aspect, there is provided a method for preparing the taxane-containing
amphiphilic block copolymer micelle composition.
[33] According to one embodiment disclosed herein, the method for preparing the taxane-
containing amphiphilic block copolymer micelle composition may comprise:
[34] (a) dissolving taxane and an amphiphilic block copolymer into an organic solvent;
and
[35] (b) adding an aqueous solution containing an osmolality adjusting agent thereto to
form polymeric micelles.
[36] In another embodiment, the method may further comprise, after step (b):
[37] (c) adding a lyophilization aid to the polymeric micelles; and
[38] (d) carrying out lyophilization.
[39] When taxane is encapsulated with a micelle composition by using an organic solvent
via a solvent evaporation process, rapid drug precipitation may occur in the taxane-
containing micelle composition after the composition is reconstituted in injection water
and is left at room temperature. This is because the organic solvent used in the solvent
evaporation process remains in the composition.
[40] Therefore, according to one embodiment of the method disclosed herein, drug pre-
cipitation may be prevented by using an osmolality adjusting agent and a minimized
amount of organic solvent. To minimize the amount of the organic solvent still
remaining in the finished composition, the composition needs to be dried at a high
temperature of 6CPC or higher under reduced pressure for at least 12 houis. However,
such reduced-pressure, high-temperature drying conditions may cause degradation of a
drug. Thus, the method for preparing the taxane-containing amphiphilic block
copolymer micelle composition according to one embodiment disclosed herein uses a
minimized amount of organic solvent so that the finished composition can be directly
subjected to lyophilization while avoiding a need for a separate step of removing the
organic solvent.
[41] The taxane-containing amphiphilic block copolymer micelle composition containing
the osmolality adjusting agent and using a minimized amount of organic solvent
according to one embodiment disclosed herein can provide a lyophilized composition
which is free from precipitation of taxane for 12 hoius or more when reconstituted into
an injection formulation.
[42] In one embodiment, the organic solvent in step (a) may include at least one selected
from the group consisting of acetone, ethanol, methanol, ethyl acetate, acetonitrile,
methylene chloride, chloroform, acetic acid and dioxane. The organic solvent may be
used in an amount of 0.5-30 wt%, specifically 0.5-15 wt%, and more specifically 1-10
wt% based on the weight of the resultant micelle composition. When the organic
solvent is used in an amount less than 0.5 wt%, there may be a difficulty in dissolving
a drug. On the other hand, when the organic solvent is used in an amount greater than
30 wt%, drug precipitation may occur upon the reconstitution of the lyophilized
composition.
[43] In step (b), the osmolality of the aqueous solution containing the osmolality adjusting
agent may be adjusted to 30-15,000 mOsm/kg, specifically 100-5,000 mOsm/kg, and
more specifically 200-2,590 mOsm/kg. When the osmolality of the aqueous solution is
less than 30 mOsm/kg, drug precipitation may occur during the preparation of the
composition. On the other hand, when the osmolality is greater than 15,000 mOsm/kg,
phase separation may occur in the polymer. In one example embodiment, the
osmolality adjusting agent may be at least one selected from the group consisting of
sodium chloride, calcium chloride, sodium sulfate and magnesium chloride. In
addition, the osmolality adjusting agent may be used in an amount of 0.1-50 wt%
based on the total dry weight of the micelle composition. Step (b) may be performed at
a temperature of 25°C or lower.
[44] In one embodiment, the method for preparing the taxane-containing amphiphilic
block copolymer micelle composition may further comprise sterilizing the aqueous
polymeric micelle solution obtained from step (c) with a sterilization filter, before step
(d) of carrying out lyophilization.
[45] The taxane-containing amphiphilic block copolymer micelle composition according
to one embodiment disclosed herein may be orally or parenterally administered in the
form of an aqueous solution or powder. Parenteral administration includes admin-
istration via intravascular, intramuscular, subcutaneous, intraperitoneal, nasal, rectal,
ophthalmic, pulmonary or other routes. Oral administration includes administration in
the form of tablets or capsules, or aqueous solution itself.
[46] In addition, the lyophilized composition according to one embodiment disclosed
herein causes little variation in the concentration of docetaxel in a reconstituted
composition over time. However, when no osmolality adjusting agent is added,
docetaxel concentration decreases after the lapse of one hour.
[47]
[48] The following examples are not intended to be limitative.
[49]
[50] Preparation Example 1: Synthesis of Monomethoxypolyethylene glycol-Polylactide
(mPEG-PLA) Block Copolymer (A-B Type)
[51] First, 5.0 g of monomethoxypolyethylene glycol (number average molecular weight:
2,000 daltons) was introduced into a 100 mL two-neck round-bottom flask, and was
heated to 13CPC under reduced pressure (1 mmHg) for 3-4 houis to remove water
therefrom. Next, the flask was purged with nitrogen gas, and stannous octoate (Sn(Oct)
) was added thereto as a reaction catalyst using a syringe in an amount of 0.1 wt%
(10.13 mg, 25 mmol) based on the weight of d- and 1-lactides. After the reaction
mixture was agitated for 30 minutes, it was subjected to depressurization (1 mmHg) at
130oC for 1 hour to remove the solvent (toluene) in which the catalyst was dissolved.
Then, 10.13 g of purified lactide was added thereto, and the resultant mixture was
heated at 130oC for 18 houis. After heating, the resultant polymer was dissolved into
methylene chloride, and was added to diethyl ether to cause precipitation of the
polymer. The resultant polymer was dried in a vacuum oven for 48 houis.
[52] The copolymer, monomethoxylpolyethylene glycol-polylactide (mPEG-PLA), had a
number average molecular weight of 2,000-1,765 daltons. Analysis of the copolymer
performed by H-NMR revealed that the copolymer was an A-B type diblock
copolymer (see Fig. 1).
[53]
[54] Preparation Example 2: Synthesis of Monomethoxypolyethylene glycol-
Poly(lactic-co-glycolic acid) (mPEG-PLGA) Block Copolymer (A-B Type)
[55] A block copolymer was obtained by reacting monomethoxypolyethylene glycol
(number average molecular weight: 5,000 daltons), lactide and glycolide in the
presence of stannous octoate as a catalyst at 12CPC for 12 houis in the same manner as
Preparation Example 1.
[56] The copolymer, monomethoxypolyethylene glycol-poly(lactic-co-glycolic acid)
(mPEG-PLGA), had a number average molecular weight of 5,000-4,000 daltons and
was an A-B type copolymer. Analysis of the copolymer performed by 1H-NMR
revealed that the copolymer was an A-B type diblock copolymer (see Fig. 2).
[57]
[58] Example 1: Preparation of mPEG-PLA Block Copolymer Micelle Composition
Containing Sodium Chloride and Docetaxel
[59] First, 760 mg of the amphiphilic block copolymer, mPEG-PLA (number average
molecular weight: 2,000-1,765 daltons), obtained from Preparation Example 1 was
completely dissolved into 0.2 mL of ethanol at 6CPC to provide a clear ethanol solution
comprising the copolymer. The ethanol solution was cooled to 25°C, and 20 mg of
docetaxel was added thereto and the resultant solution was agitated until docetaxel was
completely dissolved.
[60] Next, aqueous solutions each containing 0.9 wt% and 1.8 wt% of sodium chloride
and having an osmolality of 300 mOsm/kg and 600 mOsm/kg were prepared in
separate containers. The osmolality was measured by using a commercially available
osmometer (Gonotech GmbH, OSMOMAT030). Each aqueous solution was added to
the ethanol solution comprising the copolymer in an amount of 4 mL, and the resultant
mixture was agitated at 4CPC for 10 minutes to form an aqueous polymeric micelle
solution.
[61] Then, 100 mg of d-mannitol was dissolved into each solution, and the resultant
solution was filtered through a filter with a pore size of 200 nm to remove undissolved
docetaxel, followed by lyophilization.
[62] The lyophilized composition was subjected to liquid chromatography as follows to
determine the content of docetaxel. Additionally, particle size was measured by a
dynamic light scattering (DLS) method. The results are shown in the following Table
1.
[63] [Table 1]
[64]
[65] Liquid Chromatography
[66] 1) Column: a stainless steel column having a length of 250 mm and an inner diameter
of 4.6 mm and packed with pentafluorophenyl-coated particles having a particle
diameter of 5 µm and a pore diameter of 300 A.
[67] 2) Mobile Phase: acetonitrile: methanol: water = 26:32:420
[68] 3) Flow Rate: 1.5 mL/min
[69] 4) Loading Amount: 20 µl
[70] 5) Detector: UV absorption spectrometer (measurement wavelength: 232 nm)
[71]
[72] Example 2: Preparation of mPEG-PLA Block Copolymer Micelle Composition
Containing Calcium Chloride and Paclitaxel
[73] First, 100 mg of the amphiphilic block copolymer, mPEG-PLA (number average
molecular weight: 2,000-1,765 daltons), obtained from Preparation Example 1 was
completely dissolved into 0.1 mL of ethanol at 6CPC to provide a clear ethanol solution
comprising the copolymer. The ethanol solution was cooled to 25°C, and 20 mg of
paclitaxel was added thereto and the resultant solution was agitated until paclitaxel was
completely dissolved.
[74] Next, aqueous solutions each containing 0.9 wt% and 1.8 wt% of calcium chloride
and having an osmolality of 230 mOsm/kg and 460 mOsm/kg were prepared in
separate containers. Each aqueous solution was added to the ethanol solution
comprising the copolymer in an amount of 4 mL, and the resultant mixture was
agitated at 4CPC for 10 minutes to form an aqueous polymeric micelle solution.
[75] Then, 39 mg of d-mannitol was dissolved into each solution, and the resultant
solution was filtered through a filter with a pore size of 200 nm to remove undissolved
paclitaxel, followed by lyophilization.
[76] The lyophilized composition was subjected to the liquid chromatography as
described in Example 1 to determine the content of paclitaxel. Additionally, particle
size was measured by a DLS method. The results are shown in the following Table 2.
[77] [Table 2]
[78]
[79] Example 3: Preparation of mPEG-PLGA Block Copolymer Micelle Composition
Containing Sodium Chloride and Docetaxel
[80] First, 760 mg of the amphiphilic block copolymer, mPEG-PLGA (number average
molecular weight: 5,000-4,000 daltons), obtained from Preparation Example 2 was
completely dissolved into 0.2 mL of acetone at 50oC to provide a clear acetone
solution comprising the copolymer. The acetone solution was cooled to 25°C, and 40
mg of docetaxel was added thereto and the resultant solution was agitated until
docetaxel was completely dissolved.
[81] Next, 8 mL of an aqueous solution containing 0.9 wt% of sodium chloride and
having an osmolality of 300 mOsm/kg was added to the acetone solution comprising
the copolymer and further containing the drug, and the resultant mixture was agitated
at 25°C for 20 minutes to form a homogeneous solution. Once the homogeneous
solution was formed, 200 mg of d-mannitol was dissolved into the solution to provide
a clear aqueous polymeric micelle solution. Finally, the aqueous polymeric micelle
solution was filtered through a filter with a pore size of 200 nm to remove undissolved
docetaxel, followed by lyophilization.
[82] The lyophilized composition was subjected to the liquid chromatography as
described in Example 1 to determine the content of docetaxel. Additionally, particle
size was measured by a DLS method.
[83] Docetaxel content: 101. 3 wt%.
[84] Particle size: 35 nm.
[85]
[86] Example 4: Preparation of mPEG-PLGA Block Copolymer Micelle Composition
Containing Calcium Chloride and Paclitaxel
[87] Fust, 100 mg of the amphiphilic block copolymer, mPEG-PLGA (number average
molecular weight: 5,000-4,000 daltons), obtained from Preparation Example 2 was
completely dissolved into 0.2 mL of acetone at 50oC to provide a clear acetone
solution comprising the copolymer. The acetone solution was cooled to 25°C, and 40
mg of paclitaxel was added thereto and the resultant solution was agitated until
paclitaxel was completely dissolved.
[88] Next, 8 mL of an aqueous solution containing 0.9 wt% of calcium chloride and
having an osmolality of 230 mOsm/kg was added to the acetone solution comprising
the copolymer and further containing the drug, and the resultant mixture was agitated
at 25°C for 20 minutes to form a homogeneous solution. Once the homogeneous
solution was formed, 53 mg of d-mannitol was dissolved into the solution to provide a
clear aqueous polymeric micelle solution. Finally, the aqueous polymeric micelle
solution was filtered through a filter with a pore size of 200 nm to remove undissolved
paclitaxel, followed by lyophilization.
[89] The lyophilized composition was subjected to high-performance liquid chro-
matography (HPLC) to determine the content of docetaxel. Additionally, particle size
was measured by a DLS method.
[90] Docetaxel content: 101. 1 wt%.
[91] Particle size: 35 nm.
[92]
[93] Comparative Example 1: Preparation of mPEG-PLA Block Copolymer Micelle
Composition Containing No Inorganic Salt
[94] First, 760 mg of the amphiphilic block copolymer, mPEG-PLA (number average
molecular weight: 2,000-1,765 daltons), obtained from Preparation Example 1 was
completely dissolved into 0.2 mL of ethanol at 60oC to provide a clear ethanol solution
comprising the copolymer. The ethanol solution was cooled to 25°C, and 20 mg of
docetaxel was added thereto and the resultant solution was agitated until docetaxel was
completely disolved.
[95] Next, 4 mL of distilled water for injection (osmolality: 0 mOsm/kg) was added to the
ethanol solution comprising the copolymer, and the resultant mixture was agitated at
40°C for 10 minutes to form a homogeneous solution. Once the homogeneous solution
was formed, 100 mg of d-mannitol was disolved into the solution to provide a clear
aqueous polymeric micelle solution. Finally, the aqueous polymeric micelle solution
was filtered through a filter with a pore size of 200 nm to remove undissolved
docetaxel, followed by lyophilization.
[96] The lyophilized composition was subjected to HPLC to determine the content of
docetaxel. Additionally, particle size was measured by a DLS method.
[97] Docetaxel content: 100.3 wt%.
[98] Particle size: 18 nm.
[99]
[100] Experimental Example 1: Stability Test
[101] The sodium chloride-containing polymeric micelle compositions according to
Example 1 were compared with the polymeric micelle composition containing no
inorganic salt according to Comparative Example 1 in terms of the stability of the
aqueous solution at 37°C.
[102] Each of the lyophilized compositions according to Example 1 and Comparative
Example 1 was diluted with distilled water for injection to a docetaxel concentration of
1 mg/mL. While each diluted solution was left at 37°C, concentration of docetaxel
contained in each micelle structure was measured over time. The results are shown in
the following Table 3.
[103] [Table 3]
[104]
[105] As can be seen from the results of Table 3, the compositions according to Example 1
cause no precipitation of docetaxel even after the lapse of 12 hous, while the
composition according to Comparative Example 1 shows an amount of docetaxel pre-
cipitation of 59% after the lapse of 12 hours. It can be seen from the above results that
addition of sodium chloride may increase the docetaxel retainability of a micelle
composition by about at least two times. Additionally, it can be also seen that a higher
ratio of the amount of the inorganic salt to that of the amphiphilic block copolymer
provides the micelle competition with higher stability.
[106] Description has been given in detail with reference to example embodiments.
However, it will be appreciated by those skilled in the art that changes may be made in
these embodiment without departing from the principles and spirit of the taxane-
containing amphiphilic block copolymer micelle composition and the method for
preparing the same, the scope of which is defined in the accompanying claims and
their equivalents.
CLAIMS
Claim 1 A taxane-containing amphiphilic block copolymer micelle composition, comprising
taxane, an amphiphilic block copolymer containing a hydrophilic block (A) and a hydrophobic block (B),
and an osmolality adjusting agent, wherein the taxane-containing amphiphilic block copolymer micelle
composition comprises 0.1-30 wt% of taxane, 20-98 wt% of an amphiphilic block copolymer containing a
hydrophilic block and a hydrophobic block, and 0.1-50 wt% of an osmolality adjusting agent, based on the
total dry weight of the composition.
Claim 2 (deleted)
Claim 3 The composition as defined in claim 1, wherein the amphiphilic block copolymer
containing a hydrophilic block (A) and a hydrophobic block (B) is an A-B, A-B-A or B-A-B type block
copolymer.
Claim 4 The composition as defined in claim 1, wherein the taxane is paclitaxel, docetaxel, 7-
epipaclitaxel, t-acetylpaclitaxel, 10-desacetyl-paclitaxel, 10-desacetyl-7-epipaclitaxel, 7-xylosylpaclitaxel,
10-desacetyl-7-glutarylpaclitaxel,7-N,N-dimethylglycylpaclitaxel,7-L-alanylpaclitaxeloramixture thereof.
Claim 5 The composition as defined in claim 1, wherein the hydrophilic block (A) has a number
average molecular weight of 500-20,000 daltons, and the hydrophobic block (B) has a number average
molecular weight of 500-20,000 daltons.
Claim 6 The composition as defined in claim 1, wherein the hydrophilic block (A) is polyethylene
glycol or monomethoxypolyethylene glycol, and the hydrophobic block (B) is polylactic acid(PLA) or a
copolymer of polylactic acid and glycolic acid(PLGA).
Claim 7 The composition as defined in claim 1, wherein the osmolality adjusting agent is an
inorganic salt.
Claim 8 The composition as defined in claim 7, wherein the inorganic salt is one or more selected
from the group consisting of sodium chloride, calcium chloride, sodium sulfate and magnesium chloride.
Claim 9 The composition as defined in claim 1, wherein the amphiphilic block copolymer micelle
composition is a lyophilized composition further comprising a lyophilization aid.
Claim 10 The composition as defined in claim 9, wherein the lyophilized
composition is characterized that at least 95% of taxane is stable for 12 hours without pre-
cipitation, when the composition is reconstituted with distilled water for
injection, 0.9% physiological saline and 5% aqueous dextrose solution.
[11] The composition as defined in claim 9, wherein the lyophilization aid is used in
an amount of 1-90 wt% based on the total dry weight of the lyophilized
composition.
[12] The composition as defined in claim 9, wherein the lyophilization aid is one or
more selected from the group consisting of lactose, mannitol, sorbitol and
sucrose.
[13] A method for preparing a taxane-containing amphiphilic block copolymer
micelle composition, comprising:
dissolving taxane and an amphiphilic block copolymer into an organic solvent to
provide a polymer solution; and
adding an aqueous solution containing an osmolality adjusting agent to the
polymer solution to form polymeric micelles.
[14] The method as defined in claim 13, further comprising, after adding an aqueous
solution containing an osmolality adjusting agent to the polymer solution to form
polymeric micelles:
adding a lyophilization aid to the polymeric micelles; and
carrying out lyophilization.
[15] The method as defined in claim 13, wherein the organic solvent is one or more
selected from the group consking of acetone, ethanol, methanol, ethyl acetate,
acetonitrile, methylene chloride, chloroform, acetic acid and dioxane.
[16] The method as defined in claim 13, wherein the organic solvent is used in an
amount of 0.5-30 wt% based on the weight of the micelle composition.
[17] The method as defined in claim 13, wherein the aqueous solution has an
osmolality of 30-15,000 mOsm/kg.
[18] The method as defined in claim 13, wherein the osmolality adjusting agent b one
or more selected from the group consisting of sodium chloride, calcium chloride,
sodium sulfate and magnesium chloride.

A taxane-containing
amphiphilic block copolymer
micelle composition, including
taxane, an amphiphilic block
copolymer containing a hydrophilic
block and a hydrophobic block,
and an osmolality adjusting agent,
is described. Also described are
a method for preparing the same
composition. The composition
has excellent stability so that
it can prevent rapid release
of a drug and can improve a
desired pharmacological effect.
Additionally, the method enables
highly efficient preparation of the
composition.

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Patent Number

272102

Indian Patent Application Number

2133/KOLNP/2010

PG Journal Number

13/2016

Publication Date

25-Mar-2016

Grant Date

17-Mar-2016

Date of Filing

10-Jun-2010

Name of Patentee

SAMYANG BIOPHARMACEUTICALS CORPORATION

Applicant Address

263, YEONGI-DONG, JONGNO-GU, SEOUL 110-470 REPUBLIC OF KOREA

Inventors:

#	Inventor's Name	Inventor's Address
1	LEE, SA-WON	#503-801, YEOLMAE MAEUL 5 DANJI, UIJOK-DONG, YUSEONG-GU, DAEJEON 305-770 REPUBLIC OF KOREA
2	SEO, MIN-HYO	#204-707, GUKHWA APT., SAMCHEON-DONG, SEO-GU, DAEJEON 302-782 REPUBLIC OF KOREA

PCT International Classification Number

C08K 5/04

PCT International Application Number

PCT/KR2008/006021

PCT International Filing date

2008-10-13

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	10-2007-0141181	2007-12-31	Republic of Korea
2	10-2008-0098521	2008-10-08	Republic of Korea