Title of Invention

AMPHIPHILIC BLOCK COPOLYMER AND POLYMERIC COMPOSITION COMPRISING THE SAME FOR DRUG DELIVERY

Abstract

The present invention relates to an amphiphilic block copolymer of a hydrophilic block and a hydrophobic block with a terminal hydroxyl group wherein the terminal hydroxyl group of the hydrophobic bock is substituted with a tocopherol or cholesterol group. It also relates to polymeric compositions capable of forming stable micelles in an aqueous solution, comprising the amphiphilic block copolymer and a polylactic acid derivative wherein one or more ends of the polylactic acid are covalently bound to at least one carboxyl group.

Full Text

Description AMPHIPHILIC BLOCK COPOLYMER AND POLYMERIC COMPOSITION COMPRISING THE SAME FOR DRUG DELIVERY [i] [2] TECHNICAL FIELD [3] [4] This invention relates to an amphiphilic Hock copolymer comprising a hydrophilic A Hock and a hydrophobic B Hock with a terminal hydroxyl group, wherein said termini hydroxyl group of the hydrophobic Hock is substituted with a tocopherol or cholesterol group. The invention further relates to a polymeric composition comprising said amphiphilic block copolymer and a polylactic acid derivative wherein one or more ends of the polylactic acid are covalently bound to at least one carboxyl group. It stil further relates to a metal ion-fixed polymeric composition, wherein the carboxyl terming group of the polylactic acid derivative is fixed with a di- or tri-valent metal ion. [5] [6] BACKGROUND ART [7] [8] When a drug is administered into the body, only a small amount of the drag may reach its target site and most of the administered dose is distributed to non-targeted sites and may cause undesirable side effects. Therefore, in the last two decades, research has focused on the development of systems efficient for site specific delivery of drugs by the use of appropriate earners, which include liposomes, small molecular surfactant miceles, polymeric nanopartides, and polymeric miceles (polymeric nanopartides made of hardened miceles). The use of liposomes as drug carriers has been found to be limited mainly due to such problems as low entrapment efficiency, drug instability, rapid drug leakage, and poor storage stability. Small molecular surfactant miceles are easily dissociated when they are diluted in body fluids after having been administered into the body, and therefore it is difficult for them to sufficiently act as drug carriers. [9] Recently, polymeric nanopartides and polymeric miceles using biodegradable polymers have been reported to be extremely useful technologies for overcoming these problem. They change the in vivo distribution of an intravenously administered drug thereby reducing its side effects and improving its efficacy thereby offering such advantages as eel specific targeting and control of the release of the drug. They also have good compatibility with body fluids and improve the solubility and bioavailability of pooriy water-soluble drugs. [10] Nanometer size drug carriers with hydrophilic surfaces have been found to evade recognition and uptake by the reticulo-endothelid systems(RES), and thus to circulate in the Hood for a long period of time. Another advantage of these hydrophilic nanopartides is that, due to their extremely small size, the partides extravagate at the pathological sites, such as solid tumors, through a passive targeting mechanism. However, successful drug delivery to the specific target site requires stable retention of the drug by a carrier while in the circulation. Since drug targeting appears to require a long circulation time and the carrier is exposed to blood components for a long period of time, the stability of a drug-carrier association needs to be improved over that of carriers that are rapidly deared. [11] Among the nanometer size drug carriers with hydrophilic surfaces, polymeric micelles usually consist of several hundreds of block copolymers and have a diameter of about 20 nm to 50 nm. The polymeric miceles have two sphericd co-centric regions, a densely packed core of hydrophobic material which is responsible for entrapping the hydrophobic drug, and an outer shel made of hydrophilic materid for evasion of the body's RES which permits circulation in the Hood for a longer period of time. In spite of their distinct advantages such as small size, high solubility, simple sterilization, and controlled drug release, the physical stability of these carriers is a critical issue since the rapid release of the incorporated drug may occur in vivo. [12] Miceles are thermodynamically stable if the total copolymer concentration is above the critical micelle concentration (CMC). Thus, the use of a copolymer system with a low CMC vdue may increase the in vivo stability of the micelles. The kinetic stability means the rate of disassembly of a micele. The rate of disassembly depends upon the physicd state of the micele core. Miceles formed from copolymers cont aining a hydrophobic block which has a high glass transition temperature will tend to disassemble more slowly than those with a low glass transition temperature. They are also likely to be affected by many of the same factors that affect the rate of unimer exchange between miceles. The unimer exchange rate has been found to be dependent on many factors such as the content of solvent within the core, the hydrophobic content of the copolymer, and the lengths of both the hydrophilic and hydrophobic blocks. [13] Great efforts have been devoted to the development of a biodegradatle and bio- compatible core-shel type drug carrier with improved stability and efficacy, which wil entrap a poody water-solutie drug. A preparation method of chemicaly fixing polymeric miceles, wherein the polymer is a core-shel type polymer comprising a hy-drophilic polyethjiene oxide as the shel and a hydrophobic biodegradable polymer that is cross-linked in an aqueous solution as the core, was disdosed in EP 0,552,802A2. However, these polymeric miceles are difficult to prepare because a cross linker must be introduced into the hydrophobic component of the A-B type di-block or A-B-A type tri-block copolymer so that the core-forming polymer has a stable structure. Also, using a cross linker that has never been used before in a human body raises safety concerns. [14] A micele forming block copolymer-drug complex was disdosed in US Patent No. 6,080,396. The high molecular block copolymer-drug complex in which the high molecular weight block copolymer, having a hydrophilic polymer segment and a hydrophobic polymer segment, forms a micele having the hydrophilic segment as its outer shel and contains an anthracydine anticancer agent in its hydrophobic inner core. The molecules of the anticancer agent are covaiently linked within the micelar core. However, when the drug is covaiently linked within the polymeric miceles, it is difficult to control the deavage rate of the drug-copolymer linkage. [15] [16] On the other hand, a report shows that the solubilization of a hydrophobic drug can be achieved by a polymeric micele composed of a di- or tri-block copolymers comprising a hydrophilic polymer of polyaklene gjycol derivatives and a hydrophobic biodegradatle polymer such as fatty acid polyesters or polyamino acids. US Patent No. 5,449,513 disdoses a di-block copolymer comprising polyethylene glycol as the hydrophilic polymer, and a polyamino acid derivative, e.g. polybenzyl aspartic acid, etc., as the hydrophobic polymer. This di-block copolymer can solubilize hydrophobic anticancer agents, e.g. doxorubicin, or anti-inflammatory agents, e.g. in-domethacin. However, the polyamino acid derivative cannot be hydrolyzed in vivo, and thus causes side effects due to immune responses that are triggered. [17] One approach to improve the stability of polymeric miceles is to increase the hy- drophobicity of the polymer. To do so, the molecular weight or the concentration of the polymer should be adjusted. However, as the molecular weight is increased, its biodegradability is decreased, and so the polymer is poody excreted from the body and accumulates in organs causing toxic effects therein. US Patent No. 5,429,826 disdoses a di- or multi-block copolymer comprising a hydrophilic polyskylene glycol and a hydrophobic polyiactic acid. Specificaly, this patent describes a method of stabilizing polymeric miceles by micelizing a di- or multi-block copolymer wherein an acrylic acid derivative is bonded to a terming group of the di- or multi-block copolymer, and then, in an aqueous solution, the polymer is crosslinked in order to form the miceles. The above method could accomphsh stabilization of the polymeric micele, but the crosslinked polymer is not degraded, and thus, cannot be apjiied for in vivo use. The above polymeric miceles can solubilize a large amount of pooily water-soluHe drug in an aqueous solution with a neutral pH, but have the drawback that the drug is released within a short period of time. Also, in US Patent No. 6,458,373, a poody water-soluHe drug is solubilized into the form of an emulsion with a-tocopherol. According to this patent, to stabilize the emulsion, PEGylated vitamin E is used as a surfactant. PEG)iated vitamin E has a similar structure to the amphiphilic block copolymer comprised of a hydrophilic block and a hydrophobic block, and the highly hydrophobic tocopherol increases the copolymer's affinity with a poody water-soluble drug, and thus, it can solubilize the poody water-soluble drug. However, polyethylene gycol used as the hydrophilic polymer has a limited molecular weight, and so PEG).ated vitamin E done can solubilize a hydrophobic drug such as paditaxel only up to 2.5 mg/raL At 2.5 mg/ni or more, unstaHe miceles are formed, and the drug crystds are likely to form precipitates. Clinicd tumor resistance to chemotherapy can be inherent or acquired. Inherent resistance is present in the tumors that fail to respond to the first-line chemotherapy at the time of diagnosis. Acquired resistance occurs in the tumors that are often highly responsive to the initid treatment, but on recurrence, exhibit an entirely different phenotype. The resistance can be formed to both previously used drugs and new drugs with different structures and mechanisms of action. For example, cancer chemotherapy with Taxol often fails due to the acquired resistance of cancer eels, which is frequently associated with the overexpression of P-gp and iteration of b -tubulin. ® Taxol resistant cells exhibit cross-resistance to other drugs including actinomycin D, doxorubicin, vinHastine, and vincristine. Therefore, dinical drug resistance is a major barrier to be overcome before chemotherapy can be curative for patients with metastatic cancer. Drug-resistant cancer eels show a higher YZ (50% eel inhibition concentration of drug), and so for chemotherapy to be effective a higher concentration of drugs is needed for the tumor cells while reduced drug concentration is desired for the normal eels. Therefore, longer systemic circulation and specific localization of drugs in the tumor tissues are required for improving the effectiveness against drug-resistant cancers. [20] In view of the foregoing, the development of an improved polymeric micele composition for hydrophobic drug delivery that is biocompatiUe and biodegradaUe has been appreciated and desired. The present invention provides such an improved polymeric micele composition which is biocompatiUe and biodegradaUe, and can effective!/ deliver a hydrophobic drug without a decrease in its stability. [21] [22] SUMMARY OF THE INVENTION [23] [24] One aspect of the present invention relates to an amphiphilic block copolymer comprising a hydrophilic A Uodc and a hydrophobic B block with a termind hydroxyl group, wherein said terminal hydroxy; group of the hydrophobic block is substituted with a tocopherol or cholesterol group, and to a preparation process thereof. The amphiphilic block copolymer of the present invention has remarkally increased hy-drophobicity of the hydrophobic block while maintaining almost the same molecular weight as the native polymer. Also, the hydrophobic functional group remarkaUy improves affinity with a poorly water-soluHe drug, and thus polymeric miceles formed from the polymer are more staUe in aqueous solutions, and can maintain the pooriy water-soluHe drug solubilized therein at an increased plasma concentration for an extended period of time. Furthermore, the amphiphilic block copolymer may be mixed with other polymers, and be prepared into a polymeric composition for drug delivery. [25] Another aspect of the present invention relates to a polymeric composition comprising an amphiphilic block copolymer of a hydrophilic A block and a hydrophobic B block with a terminal hydroxyl group, and a polylactic acid derivative, wherein said terminal hydroxy terminal group of the hydrophobic B block is substituted with a tocopherol or cholesterol group, and at least one end of the polylactic acid derivative is covdenfly bound to at least one carboxyl group . [26] The third aspect of the present invention relates to a polymeric composition comprising an amphiphilic block copolymer of a hydrophilic A block and a hydrophobic B block with a terminal hydroxjl group, and a polylactic acid derivative, wherein said terminal hydroxyl terminal group of the hydrophobic B block is substituted with a tocopherol or cholesterol group, and at least one end of the polylactic acid derivative is covdently bound to at least one carboxyl group, wherein the carboxyl terminal group of the polylactic acid derivative is fixed with a di- or tri-vaJent metal ion. [27] The polymeric compositions of the present invention can form stable polymeric miceles or nanopartides in body fluids or aqueous solutions. The miceles or nanopartides formed from the compositions of the present invention have a hy-drophilic outer shel and a hydrophobic inner core wherein a large amount of hydrophobic drug can be physicdly trapped. The drug-containing miceles and nanopartides of the present invention have a prolonged retention time in the hoodstream after administration, and can be utilized to make various pharmaceutic^ formulations. The anticancer drug-containing polymeric miceles prepared from the composition of the present invention can be efficiently transferred to, and effectively act on, anticancer drug-resistant cancer cells. Additional features and advantages of the invention will be apparent from the detailed description that folows, taken in conjunction with the accompanying drawings, which together ilustrate, by way of example, the features of the present invention. [28] [29] BRIEF DESCRIPTION OF THE DRAWINGS [30] [31] Additions! features and advantages of the invention will be apparent from the detailed description which follows, taken in conjunction with the accompanying drawings, which together ilustrate, by way of example, features of the invention; and, wherein: [32] Fig. 1 is a schematic diagram of a polymeric micele formed by monomethoxy- polyethylene gycol-polylaectide-hydrophobic moitety (mPEG-ILA-hydrophobic moiety) in an aqueous environment; [33] Fig. 2 is a schematic diagram of a polymeric micele formed by sodium carboxylate derivatized D,L-polylaectic acid in an aqueous environment; [34] Fig. 3 is a schematic diagram of a polymeric micele formed by a mixture of monomethoxypolyethylene glycol-polylactide-hydrophobic moitety (mPEG-ILA-hydrophobic moitety) and sodium carboxylate derivatized D,L-polyIactic acid in an aqueous environment; 2+ [35] Fig. 4 is a schematic diagram of the Ca -fixed polymeric micele of Fig. 3; 2+ [36] Fig. 5 is a schematic diagram of a Ca -fixed polymeric micele containing a hy- drophobic drug trapped within the hydrophobic core of the micele; [37] Fig. 6 is an H-NMR spectrum of mPEG-ILA-cholesterol (Example 1); [38] Fig. 7 is an 'H-NMR spectrum of mPEG-LA-tocopherol (Example 7); [39] Fig. 8 shows the profile of fiasma drug concentration of paditaxel-containing polymeric miceles fabricated with various di-block copolymers at various time intervds after administration; 2+ [40] Fig. 9 shows the jiasma drug concentration of paditaxel-containing Ca -fixed polymeric miceles fabricated with mPEG-ELA-tocopherol and mPEG-ILA-OH at various time intervds after administration; [41] Fig. 10 shows the profile of plasma drug concentration of paditaxel-containing Ca -fixed polymeric miceles, Cremophor EL (Taxol ), and Tween 80 preparations at various time intervals after administration; 2+ [42] Fig. 11 shows the plasma drug concentration of paditaxel-containing Ca -fixed polymeric miceles and Cremophor EL (Taxol ) at various time intervds after administration; [43] Fig. 12 shows the jiasma drug concentration of docetaxel-containing Ca +-fixed polymeric miceles and Tween 80 preparations (Taxotere ) at various time intervds after administration; [44] Fig. 13 shows the plasma drug concentration of the docetaxel-containing Ca + - fixed polymeric miceles and Tween 80 preparations (Taxotere ) at various time intervals after administration; 2+ [45] Fig. 14 shows the anticancer effects of the drug containing Ca -fixed polymeric miceles in mice using the human breast carcinoma µm line MX-1; 2+ [46] Fig. 15 shows the anticancer effects of the drug containing Ca -fixed polymeric miceles in mice using the human breast carcinoma cell line MDAMB435S; 2+ [47] Fig. 16 shows the anticancer effects of the drug containing Ca -fixed polymeric miceles in mice using the human ovarian carcinoma cell line SKOV-3; 2+ [48] Fig. 17 shows the anticancer effects of the drug containing Ca -fixed polymeric miceles in mice using the human ovarian carcinoma cell line SKOV-3; 2+ [49] Fig. 18 shows the anticancer effects of the drug containing Ca -fixed polymeric miceles in mice using the human colon carcinoma µm line HT-29( 3 cydes); 2+ [50] Fig. 19 shows the anticancer effects of the drug containing Ca -fixed polymeric miceles in mice using the human colon carcinoma µm line HT-29; 2+ [51] Fig. 20 shows the anticancer effects of the drug containing Ca -fixed polymeric miceles in mice using the human prostatic carcinoma µm line PC3; 2+ [52] Fig. 21 shows the anticancer effects of the drug containing Ca -fixed polymeric miceles in mice using the human brain carcinoma Cell line U-373MG; [53] Fig. 22 shows the anticancer effects of the drug containing Ca2+-fixed polymeric miceles in the animd model with paditaxel (Taxol ) resistant human cancer; and 2+ [54] Fig. 23 shows the anticancer effects of the drug containing Ca -fixed polymeric miceles in an animd model with doxorubicin (Adriamycin?) resistant human cancer. [55] Reference wil now be made to the exemplary embodiments ilustrated, and specific language wil be used herein to describe the same. It wil nevertheless be understood that no limitation of the scope of the invention is thereby intended. [56] [57] DETAILED DESCRIPTION OF THE INVENTION [58] [59] Before the present polymeric compositions and methods of using and making thereof are disdosed and described, it should be understood that this invention is not limited to the particular configurations, process steps, and materials disdosed herein, and such configurations, process steps, and materials may be varied. It should be dso understood that the terminology emjioyed herein is used for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention which wil be limited only by the appended claims and equivdents thereof. [60] It should be noted that, in this specification and the appended daims, the singular form, "a," "an," or "the", indudes plursi referents unless the context deady dictates otherwise. Thus, for example, the reference to a polymer containing "a termini group" indudes reference to two or more such groups, and reference to "a hydrophobic drug" indudes reference to two or more such drugs. Further, reference to an amphiphilic dock copolymer indudes mixtures of block copolymers provided that the compositions of each A and B dock, the respective ratios of each dock, and weight or number average molecular weight of each dock and/or the overd dock polymeric composition fal within the limitations defined herein. [61] In describing and daiming the present invention, the folowing terminology wil be used in accordance with the definitions set out below. [62] As used herein, the term "bioactive agent" or "drug" or any other similar term means any chemicd or biological material or compound that is suitatie for administration in view of the methods previously known in the art and/or the methods taught in the present invention and that induces a desired biological or pharmacological effect. Such effects may indude but are not limited to (1) having a prophylactic effect on the organism and preventing an undesired biologicd effect such as preventing an infection, (2) deviating conditions caused by diseases, for example, deviating pain or inflammation caused as a result of diseases, and/or (3) either deviating, reducing, or comjletely eliminating a disease from the organism. The effect may be locd, such as providing a locd anesthetic effect, or may be systemic. [63] As used herein, the term "biodegradable" or "biodegradation" is defined as the conversion of materials into less comjlex intermediates or end products by solubilization hydrolysis, or by the action of biologicdy formed entities which can be enzymes or other products of the organism. [64] As used herein, the term "biocompatible" means materials or the intermediates or end products of materials formed by solubilization hydrolysis, or by the action of bio-logicdy formed entities which can be enzymes or other products of the organism and which cause no adverse effect on the body. [65] As used herein, an "effective amount" means the amount of bioactive agent that is sufficient to provide the desired local or systemic effect at a reasonable risk/benefit ratio as would attend any medical treatment. [66] As used herein, "administering" and similar terms mean delivering the composition to an individual being treated such that the composition is capable of being circulated systemicdy. Preferably, the compositions of the present invention are administered by the subcutaneous, intramuscular, transdermal, oral, transmucosd, intravenous, or intraperitoneal routes. Injections for such use can be prepared in conventional forms, either as a liquid solution or suspension, or in a solid form that is suitable for preparation as a solution or suspension in liquid prior to injection, or as an emulsion. Suitable excipients that can be used for administration indude, for example, water, saline, dextrose, glycerol, ethanol, and the like; and if desired, minor amounts of auxiliary substances such as wetting or emulsifying agents, buffers, and the like. For oral administration, they can be formulated into various forms such as solutions, tablets, capsules, etc. [67] Below, the exemplary embodiments are shown and specific language will be used herein to describe the same. It should nevertheless be understood that no limitation of the scope of the invention is thereby intended. Alterations and further modifications of the inventive features ilustrated herein, and additional applications of the principle of the present invention as ilustrated herein, for one sidled in the relevant art, in connection with this disdosure, should be considered within the scope of the present invention. [68] In one aspect, the present invention provides an amphiphilic block copolymer comprising a hydrophilic A block and a hydrophobic B block with a terminal hydroxjl group, wherein said termind hydroxyl group of the hydrophobic block is substituted with a tocopherol or cholesterol group. [69] The present invention also provides a process for preparing the amphiphilic block copolymer, e.g. process variants I to m below: [70] [71] Process variant I: [72] [73] A process comprising the steps of: [74] 1) carboxjiating a hydrophobic compound having a tocopherol or cholesterol group;and [75] 2) reacting an amphiphilic Hodc copolymer comprised of a hydrophilic A block and a hydrophobic B block having a termind hydroxyl group with said carboxylated hydrophobic compound, in the presence of dicydohexycarbodiimide (DCC) as an initiator, so that the carboxylated hydrophobic compound is chemicdly bound to the terminal hydroxyl group of the hydrophobic B block. [76] [77] Process variant n: [78] [79] A process comprising the steps of: [80] 1) carboxylating a hydrophobic compound having a tocopherol or cholesterol group and activating the resulting carboxylated hydrophobic compound with oxaf>4 chloride; and [81] 2) reacting an amphiphilic block copolymer comprised of a hydrophilic A block and a hydrophobic B block having a terminal hydroxy! group with said activated carboxylated hydrophobic compound, so that the carboxylated hydrophobic compound is chemicdy bound to the terminal hydroxyl group of the hydrophobic B block. [82] [83] Process variant m : [84] [85] A process comprising the steps of: [86] 1) mixing a hydrophobic compound having a tocopherol or cholesterol group with a dichloride compound as a linkage agent; [87] 2) adding an amphiphilic block copolymer comprising a hydrophilic A block and a hydrophobic B block having a termind hydroxyl group to the reaction mixture of step 1, so that the hydrophobic compound is chemiraly bound to the termind hydroxyl group of the hydrophobic B block; and [88] 3) dissolving and precipitating the block copolymer obtained in step 2). [89] [90] The term "a carboxjlated hydrophobic compound" refers to a hydrophobic compound with a hydroxyl group to which a carboxyl group is bound, and the carboxyl group may be derived from succinate, mdonate, gutarate, or adipate. [91] The present invention also provides a drug carrier comprising the amphiphilic block copolymer of the present invention. It also provides a pharmaceutical composition capade of forming polymeric miceles in a body fluid or an aqueous solution, comprising said amphiphilic dock copolymer and a poorly water-solude drug. [92] The amphiphilic dock copolymer of the present invention is preferady an A-B type didock copolymer or B-A-B type tridock copolymer comprising a hydrophilic A dock and a hydrophobic B dock, and the terminal group of the hydrophobic block: is a hydroxyl group. The amphiphilic dock copolymer of the present invention, when place ed in an aqueous environment, forms a core-shel type of polymeric micele wherein the hydrophobic B dock forms the core and the hydrophilic A dock forms the shel. Preferady, the hydrophilic A dock is a member selected from the group consisting of polyalklence glycol, polyvinyl alcohol, polyvinyl pyrrolidone, polyacryl amide, and derivatives thereof. More preferably, the hydrophilic A dock is a member selected from the group consisting of monomethoxypolyethyiene glyycol, monoacetoxy-polyethylene gycol, polyethylene glycol, polyethylene-co-propylene glycol, and polyvinyl pyrrolidone. Preferady, the hydrophilic A dock has a number average molecular weight of 200 to 50,000 Dions . More preferady, the hydrophilic A block has a number average molecular weight of 1,000 to 20,000 Dions . [93] The hydrophobic B dock of the amphiphilic dock copolymer of the present invention is a highly biocompatide and biodegradable polymer selected from the group consisting of polyesters, polyanhydrides, polyamino acids, polyorthoesters and polyphosphate. More preferady, the hydrophobic B dock is one or more selected from the group consisting of polylactides, polyglyycolides, polycaprolactone, polydioxan-2-one, pol)iactic-co-glycolide, polyiactic-co-dioxan-2-one, polylactic-co-caprolactone and polyglycolic-co-caprolactone. Preferady, the hydrophobic B dock of the amphiphilic dock copolymer has a number average molecular weight of 50 to 50,000 Dions . More preferady, the hydrophobic B dock of the amphiphilic dock copolymer has a number average molecular weight 200 to 20,000 Dalons . [94] The hydrophobic B Hock has a hydroxyl termind group, and the hydroxyl terming group is substituted with tocopherol or cholesterol both having excelent hy-drophobicity, which increases the hydrophobicity of the hydrophobic B Hock. When placed in an aqueous solution, the hydrophobic Hock of the amphiphilic Hock copolymer of the present invention avoids contact with water, and forms an inner core, to form a sphericd polymeric micele. Thus, when a pooriy water-soluHe drug is introduced into the amphiphilic Hock copolymer, the pooriy water-soluHe drug is surrounded by the hydrophobic polymer, the inner core of the polymeric micele, and thus, can be entrapped within the micele. The stability of the formed micele depends on the hydrophobicity of the hydrophobic Hock and its affinity with the drug. Therefore, in the present invention, in order to increase the hydrophobicity of the hydrophobic Hock while maintaining its molecular weight, a functional group with excelent hydrophobicity, e.g. tocopherol, cholesterol, etc. is chemicaly bound thereto using a linkage agent. Tocopherol and cholesterol are biological compatiHe and hydrophobic compounds having a ring structure, which can increase the affinity of the Hock copolymer with a pooriy water-soluHe drug. [95] The ratio of the hydrophilic A Hock to the hydrophobic B Hock of the amphiphilic Hock copolymer of the present invention is preferably within the range of 30:70 to 97:3 by weight, and more preferaHy within the range of 4:6 to 7:3. If the co ntent of the hydrophilic A block is too low, the polymer may not form polymeric miceles in an aqueous solution, and if the content is too high, the polymeric miceles formed therefrom are not stable. [96] In one embodiment, the amphiphilic Hock copolymer of the present invention may C(=0)CH CH CH CH CH -O, or C(=0)CH OCH CH -0-; 22222 222 [102] 1' is an integer from 4-1150; [103] m' is an integer from 1-300; [104] n' is an integer from 0-300; and [105] X' is an integer from 0-4. [106] As compared with prior amphiphilic block copolymers, the copolymer with the hy- drophobic group-substituted hydrophobic block of the present invention has increased hydrophobicity, a decreased critical micele concentration (CMC), and increased affinity with a pooriy water-soluble drug, and thus, contains the drug in a staUe environment. Furthermore, the size of the miceles formed in an aqueous solution is increased due to the functional group bound at the end, and thus, a sufficient amount of drug can be contained in the micele. Therefore, the amphiphilic dock copolymer can be efficiently used as a drug carrier. The function^ group with strong hydrophobicity introduced in the present invention has a high molecular-weight; thus it can remarkaUy increase both the hydrophobicity and the affinity of the block copolymer with the drug and thus significantly stabilize the drug containing miceles. [107] In addition, the polymeric micele formed from the amphiphilic block copolymer of the present invention has a prolonged in vivo retention time. The Hood concentration of the drug in the polymeric miceles depends on hydrophobic moiety substituted for the hydroxy; termind group of hydrophobic B block of the amphiphilic diblock copolymers. As shown in TaUe 6 and Fig. 8, the polymeric miceles (Compositions 1-2) of the amphiphilic block copolymers with a hydrophobic moiety (tocopherol or cholesterol) substituted for the hydroxyl terminal group of hydrophobic B block had a much longer bloodstream retention time than the original mPEG-ELA-OH polymeric miceles (Composition 3). Moreover, mPEG-ILA-tocopherol miceles (Composition 1) circulated longest in the Uood among al the polymeric miceles. This result can be explained by the increased hydrophobicity of tocopherol and cholesterol moiety in the hydrophobic B block. [108] The block copolymer having the hydrophobic block whose hydroxyl termini group is substituted with tocopherol or cholesterol can be prepared according to the folowing methods. In one embodiment, a suitaUe linker, e.g. a dicarboxjiic acid such as succinic acid, malonic acid, glutaric acid or adipic acid, is introduced into the hydroxyl group of tocopherol or cholesterol, and the carboxylated tocopherol or cholesterol is chemicaly bound to the hydroxyl terminal group of the hydrophobic B block. [109] In one embodiment, according to the method of US Patent No. 6,322,805, the am- phiphilic block copolymer (mPEG-ILA) comprised of monomethoxypolyethylene glycol (mPEG; Mn=20,000) and polylactide (ILA; Mn=l,750) is weighed, and dehydrated using a vacuum pump at 120 °C, and then dissolved in acetonitrile or methjiene chloride. Thereto is added tocopherol succinate or cholesterol succinate, and dicydohexjfcarbodiimide (DCC) and 4-dimethyIarrrinopyridine (DM\P) are weighed and added thereto as an initiator and a catalyst, respectively, and the reaction is performed at room temperature. The reactant becomes opaque due to dicydo-hexjiurea (DCU) formed in the reaction between the termind -OH of mPEGJLA and ■COOH of the hydrophobic compound. After 24 hours, DCU is removed by using a glass filter, and DMAP is extracted and removed with a hydrochloric acid aqueous solution. To this purified product solution is added MgSO to remove any residual 4 moisture, and then, precipitates are formed in a hexane/diethji ether solvent in order to obtain the amphiphilic block copolymer to which tocopherol succinji or cholesterol succinyl is bound, mPEGJLA-tocopherol or mPEGJLA-cholesterol (in which tocopherol or cholesterol is bound to ELA via succinic acid diester). The precipitated polymeric product is filtered, and then dried under vacuum to obtain the polymer as white partides. [110] In another embodiment, a carboxjlated hydrophobic compound is activated with oxayl chloride without any catdyst, and bound to the end of mPEG-ILA. That is, tocopherol (or cholesterol) succinate is reacted with oxayl chloride, and then, excessive oxaty chloride is removed under vacuum at room temperature. The mPEG-HLA is weighed and added thereto, and the reaction is performed at 100 °C for 12 hours to obtain mPEG-RA-tocopherol (or cholesterol). The synthesized polymer is dissolved in acetonitrile or methylene chloride, precipitated in hexane/diethji ether, and filtered. [Ill] In the above two preparation processes, tocopherol (or cholesterol) malonate, tocopherol (or cholesterol) glutarate, or tocopherol (or cholesterol) adipate, etc. can be used instead of tocopherol (or cholesterol) succinate. [112] In another embodiment, tocopherol or cholesterol is bound to the end of mPEG- ILA by using a dichloride compound as a linkage agent. Specificaiy, tocopherol or cholesterol is weighed and dehydrated by using a vacuum pump at 50 °G Excessive linkage agent is added thereto, and the reaction is performed for 12 hours. After the reaction is competed, the excessively added linkage agent is removed under vacuum at 100 °C Thereto is added weighed mPEG-HLA, and the reaction is performed at 100 °C for 12 hours. The synthesized polymer is dissolved in methylene chloride, and precipitated in hexane/diethyl ether in order to obtain the amphiphilic block copolymer in which tocopherol or cholesterol is bound to ILA via succinic acid diester, i.e. mPEG-ELA-tocopherol or mPEGJLA-cholesterol. The precipitated polymeric product is filtered, and dried under vacuum to obtain the polymer as white partides. The linkage agent which can be used in the reaction may be selected from such dichloride compounds as succinyl chloride, oxalyl chloride, malonyl chloride, glutaryl chloride, adipo chloride, etc. [113] The block copolymer synthesized as above may be mixed with a poorly water- soluUe drug in order to obtain a polymeric micele composition. That is, the dock copolymer (10-200 mg) and the drug (1-50 mg) are dissolved in an organic solvent, e.g. acetonitrile, methylene chloride, etc. The solution is mixed by stirring, and dried under vacuum at 60 °C to prepare a matrix. The matrix of the pooriy water-solutie drug and the polymer is dissolved in distiled water, and then lyophilized to obtain the drug-introduced polymeric micele composition. The above polymeric micele composition may be diluted with an aqueous solution, e.g. physiological sdine, and be used as an injectatie formulation. [114] The term "pooriy water-soluble drugs" or "hydrophobic drugs" refers to any drug or bioactive agent which has the water solubility of 33.3mg/ni or less. This indudes anticancer agents, antibiotics, anti-inflammatory agents, anesthetics, hormones, antihypertensive agents, and agents for the treatment of diabetes, antihyperiipidemic agents, antiviral agents, agents for the treatment of Parkinson's disease, antidementia agents, antiemetics, immunosuppressants, antiulcerative agents, laxatives, and antimalarial agents. Examples of hydrophobic drugs indude paditaxel, ketoconazole, itraconazole, cydosporine, cisapride, acetaminophen, aspirin, acetyl salicylic acid, indomethacin, naproxen, wafarin, papaverine, thiabendazole, miconazole, cinarizine, doxorubicin, omeprazole, choleccaiferol, mefchalan, nifedipine, digoxin, benzoic acid tryptophan, tyrosine, phen)l Canine, azthreonam, ibuprofen, phenoxymethy|>enicilin, thdidomide, methyl testosterone, prochlorperazine, hydrocortisone, dideoxypurine nudeoside, vitamin D2, sulfonamide, sulfonylurea, para-aminobenzoic acid, melatonin, benzyl penicilin, chlorambucil, diazepine, digitoxin, hydrocortisone butyrate, metronidazole benzoate, tobutamide, prostaglandin, fludrocortisone, griseofulvin, miconazole nitrate, leukotriene B4 inhibitor, propranolol, theophyline, flubiprofen, sodium benzoate, benzoic acid, riboflavin, benzodiazepine, phenobarbital, glyburide, sulfadiazine, sulfaethyl, thiadiazole, didofenac sodium, phenyroin, hioridazine hydrochloride, bropyrimie, hydrochlorothiazide, fluconazole, etc. [115] The above poody water-soluHe drug may be added to the block copolymer in a weight-by-weight ratio of 0.1-20.0:80.0-99.9, to be appropriately contained in the inner core of the micele formed from the amphiphilic block copolymer of the present invention. [116] In another embodiment, the present invention provides a polymeric composition comprising an amphiphilic block copolymer of a hydrophilic A block and a hydrophobic B block with a terminal hydroxji group, and a polylactic acid derivative wherein said termind hydroxy termind group of the hydrophobic B block is substituted with a tocopherol or cholesterol group, and at least one end of the polylactic acid derivative is covdenfly bound to at least one carboxyl group. [117] The amphiphilic block copolymer comprised of a hydrophilic A block and a hy- drophobic B block, wherein the hydroxyl termind group of the hydrophobic block is substituted with a hydrophobic tocopherol or cholesterol group, which has excelent hydrophobicity, is as described above. [118] One or more ends of the polylactic acid derivative of the present invention are covdenfly bound to at least one carboxyiic acid or carboxylate salt. The non-bound end of the polylactic acid derivative of the present invention may be covdently bound to a functiond group selected from the group consisting of hydroxji, acetoxy, benzoyloxy, decanoyloxy, and pdmitoloxy. The carboxjlic acid or carboxylate sal functions as a hydrophilic group in an aqueous solution of pH 4 or more, and enaHes the polylactic acid derivative to form polymeric miceles therein. When the polylactic acid derivative of the present invention is dissolved in an aqueous solution, the hydrophilic and hydrophobic components present in the polylactic acid derivative should be balanced in order to form polymeric miceles. Therefore, the number average molecular weight of the polylactic acid derivative of the present invention is preferaHy within the range of 50 to 50,000 Ddtons . The molecular weight of the polylactic acid derivative can be adjusted by controling the reaction temperature, time, and the like, during the preparation process. [119] The polylactic acid derivative is preferaHy represented by the folowing formula: [120] ROCHZ-[A] -[B] COOM(I) [121] wherein A is COOCHZ-; B is COOCHY-, COOCH CH CH CH CH - or - 2 2 2 2 2 COOCH CH OCH ; R is a hydrogen atom, or acetyl, benzoyl, decanoyl, pdmitoyl, 2 2 2 methyl, or eth)l group; Z and Y each are a hydrogen atom, or methyl, or phen)i group; M is H, Na, K, or Li; n is an integer from 1 to 30, and m is an integer from 0 to 20. [122] One or more ends of the polylactic acid derivative of the present invention are covdently bound to a carboxji group or an akali metd salt thereof, preferaHy, an akdi metal sal thereof. The metd ion in the akali metd salt forms of the polylactic acid derivative is monovdent, e.g. sodium, potassium, or lithium. The polylactic acid derivative in the metd ion salt form is solid at room temperature, and is very stable because of its relatively neutral pH. [123] More preferaHy, the poljlactic acid derivative is represented by the folowing formula: [124] ROCHZ-fCOOCHX] -[COOCHY'] COCXHZCOOM (II) p q [125] wherein X is a methyl group; Y* is a hydrogen atom or phenjl group; p is an integer from 0 to 25; q is an integer from 0 to 25, provided that p+q is an integer from 5 to 25; R, Z and M are each as defined in Formula (I). [126] In addition, polylactic acid derivatives of the folowing formulas (HI) to (V) are also suitable for the present invention: [127] RO-PADCOO-WM' (HI) [128] [129] the B\D is a member selected from the group consisting of D,L-polylactic acid, D- polylactic acid, polymandelic acid, a copolymer of D,L-lactic acid and gjjycolic acid, a copolymer of D,L-lactic acid and mandelic acid, a copolymer of D,L-Lactic acid and caprolactone, and a copolymer of D,L-lactic acid and l,4-dioxan-2-one; R and M are each as defined in formula (I). [130] S-OIADCOO-Q (IV) [131] *ji;QisCH , CH CH , CH CH CH , CH CH CH CH , orCH C H ; aisan 10 3232232223 265 integer from 0 to 4; b is an integer from 1 to 10; M is as defined in Formula (I); and BVD is as defined in formula (EI). [132] [ 134] wherein R' is mD-OC(O)CH[ CH C(O)-OM and M is as defined in formula 2 2 (I); R\D is as defined in formula (HI); and a is an integer from 1 to 4, for exanqie, if a=l, 3-arm ILACOONa; if a=2,4-arm ILACOONa; if a=3, 5-arm ILACOONa; and if a=4, 6-arm ILACOONa. [135] The initiator for synthesis of the polymers (formula V) includes glycerol, erythritol, threlol, pentaerytritol, xjiitol, adonitol, sorbitol, and mannitol. [136] The polymeric composition of the present invention may contain 0.1 to 99.9 wt% of the amphiphilic block copolymer and 0.1 to 99.9 wt% of the polylactic acid derivative based on the totd weight of the amphiphilic block copolymer and the polylactic acid derivative. Preferably, the polymeric composition of the present invention contains 20 to 95 wt% of the amphiphilic block copolymer and 5 to 80 wt% of the polylactic acid derivative. More preferaHy, the polymeric composition of the present invention contains 50 to 90 wt% of the amphiphilic block copolymer and 10 to 50 wt% of the polylactic acid derivative. [137] The polylactic acid derivatives of the present invention done can form miceles in an aqueous solution of pH 4 or more, but the polymeric compositions can form miceles in an aqueous solution irrespective of the pH of the solution. Since the biodegradable polymer is usually hydrolyzed at a pH of 10 or more, the polymeric compositions of the present invention may be used at a pH within the range of 1 to 10, preferaHy at a pH within the range of 4 to 8. The partide size of the miceles or nanopartides prepared from the polymeric compositions of the present invention may be adjusted to be within the range of 1 to 400 nm, and preferaHy from 5 to 200 nm, depending on the molecular weight of the polymers and the ratio of the polylactic acid derivative to the amphiphilic block copolymer. [138] As ilustrated in Fig.l to Fig.3, the polylactic acid derivatives or the amphiphilic block copolymers done and mixtures thereof may form miceles in an aqueous solution, and the miceles formed from the polymeric compositions of the amphiphilic block copolymers and the polylactic acid derivatives in an aqueous solution show higher drug entrapping rates and stability than those from the polyiactic acid derivatives or the amphiphilic block copolymers done. In the Figures, 1 represents pooriy water-soluble drugs; 10 represents monomethoxypolyethylenegycol-polylactide hydrophobic moiety (mPEG-ILA- hydrophobic moiety); 11 represents monomethoxypolyethylene glycol (mPEG); 12 represents polyiactide hydrophobic moiety (TLA- hydrophobic moiety); 20 represents the sodium salt of D,L-poly(lactic acid); 21 represents D,L-polyiactic acid; and 22 represents sodium carboxjiate. However, the polymeric compositions of the present invention remarkably improve the drug loading efficiency and stability of the miceles formed in an aqueous solution compared with the miceles formed from the polylactic acid derivatives or the amphiphilic block copolymers alone. [139] In one embodiment of the present invention, there is provided a polymeric composition comprising an amphiphilic dock copolymer comprised of a hydrophilic A block and a hydrophobic B block with a terminal hydroxy! group and a polylactic acid derivative, wherein said termind hydroxyl group is substituted with a hydrophobic tocopherol or cholesterol group, and at least one end of the poljiactic acid derivative is covdenfly bound to at least one carboxyl group, wherein said carboxji group is fixed with a di- or tri-vdent metal ion. [140] The metal ion-fixed polymeric composition can be prepared by adding the di- or tri-vdent metal ion to the polymeric composition of the amphiphilic block copolymer and the po^actic acid derivative. The polymeric miceles or nanopartides may be formed by changing the amount of di- or tri-vdent metal ion added for binding or fixing the carboxyl termind group of the polylactic acid derivative. [141] The di- or tri-vdent metal ion is preferaHy one selected from the group consisting of Ca +, Mg , Ba +, Cr +, Fe , Mn , Ni , Cu +, Zn , and Al . The di- or tri-vdent metal ion may be added to the polymeric composition of the amphiphilic block copolymer and the polylactic acid derivative in the form of sulfate, chloride, carbonate, phosphate or hydroxylate, and preferaHy in the form of CaCl , MgCl, ZnCl, A1C1, FeCl , CaCO , MgCO, Ca (PO ), Mg (PO ), A1PO , MgSO , Ca(OH), Mg(OH), 3 3 3342342 4 4 2 2 Al(OH) , or Zn(OH) . 3 2 [142] As ilustrated in Figs. 4 and 5, when a monovdent metal ion at the carboxyl terminus of the poljiactic acid derivative is substituted with a di- or tri-valent metal ion to form a metal ionic bond, the miceles or nanopartides formed therefrom may have improved stability. [143] Polymeric miceles or nanopartides can be prepared by changing the equivdents of the metal ion added. Specifically, if a di-vdent metal ion is added at 0.5 equivdents or less with respect to the carboxji termind groups of the polyiactic acid derivative, the metal ion that can form bonds with the carboxji terminal group is insufficient, and thus polymeric miceles are formed. If a di-vdent metal ion is added at 0.5 equivdents or more, the metal ion that can form bonds with the carboxji termind group of the polyiactic acid derivative is sufficient to firmly fix the miceles, and thus nanopartides are formed. [144] In addition, the drug release rate from the polymeric miceles or nanopartides may be adjusted by changing the amount of equivdents of the metal ion added. If the metal ion is present at 1 equivdent or less with respect to the carboxji group of the polyiactic acid derivative, the number available for bonding to the carboxji termind group of the polyiactic acid derivative is decreased, and so the drug release rate is increased. If the metal ion is present at 1 equivdent or more, the number availatte for bonding to the carboxji termind group of the polyiactic acid derivative is increased, and so the drug release rate is decreased. Therefore, to increase the drug release rate in the blood, the metal ion is used in a small equivdent amount, and to decrease the drug release rate, the metal ion is used in a large equivdent amount. [145] The metal ion-fixed polymeric compositions of the present invention may contain 5 to 95wt% of the amphiphilic block copolymer, 5 to 95wt% of the polyiactic acid derivative, and 0.01 to 10 equivdents of the di- or tri-vdent metal ion with respect to the number of equivdents of the carboxji termind groups of the polyiactic acid derivatives. Preferably, they contain 20 to 80wt% of the amphiphilic dock copolymer, 20 to 80wt% of the polyiactic acid derivative, and 0.1 to 5 equivdents of the di- or tri-vdent metal ion. More preferably, they contain 20 to 60wt% of the amphiphilic block copolymer, 40 to 80wt% of the polyiactic acid derivative, and 0.2 to 2 equivdents of the di- or tri-vdent metal ion. [146] The polymeric composition comprising an amphiphilic block copolymer comprised of a hydrophilic block and a hydrophobic block in which the hydroxyl termind group is substituted with a hydrophobic tocopherol or cholesterol group having excelent hy-drophobicity, and a polyiactic acid derivative in which the end of the polyiactic acid is covdently bound to at least one carboxji group, and the metal ion-fixed polymeric composition thereof may form stade polymeric miceles or nanopartides in an aqueous environment. Therefore, the present invention dso relates to a pharmaceuticd composition containing polymeric miceles or nanopartides formed from the polymeric compositions of the present invention with a poody water-solude drug entrapped therein. The above composition has a prolonged retention time of effective drug concentration in the Hoodstream after administration. The pharmaceutical compositions of the present invention provide increased plasma concentrations of hydrophobic drugs and can be used in various pharmaceutic^ formulations. [147] As shown in Figs. 3 to 5, a poody water-solutie drug is mixed with a polymeric composition of an amphiphilic dock copolymer and a polylactic acid derivative to form polymeric miceles containing the drug therein. A di- or tri-vdent metal ion may be added to form a metal ionic bond with the carboxyl terminal group of the polylactic acid derivative and thereby to form drug-containing polymeric miceles and nanopartides with increased stability. [148] The content of the poody water-solutie drug is preferaHy within the range of 0.1 to 30wt% based on the totd weight of the pharmaceutical compositions comprising an amphiphilic block copolymer, a polylactic acid derivative, and a hydrophobic drug. The size of the drug-containing polymeric miceles or nanopartides may be adjusted from 5 to 400 nm, preferaHy, from 10 to 200 nm, depending on the molecular weight of the polymers and the ratio of the amphiphilic block copolymer to the polylactic acid derivative. For an example, the partides of the metal ion-fixed polymeric miceles or nanopartides have an average size of 20-40 nm, as shown in TaHe 7. The miceles of this size range are suitatie for injection formulations and sterile filtration. [149] The non-metal ion-treated polymeric composition or metal ion-fixed polymeric miceles or nanopartides according to the present invention have high stability, and particulaiiy, the metal ion-fixed ones have much higher stability in an aqueous solution. As shown in TaHe 9, the drug containing polymeric micele compositions (Compositions 4 & 5) were kineticdly stable and the metal ion-fixed paditaxel-containing polymeric micele composition were even more kineticdly staHe. The addition of a metal ion can significantly increase the retention time of drug in the polymeric miceles of the present invention. This is due to the crosslinking electrostatic interaction of the carboxylate anion of the polylactic acid derivative which might induce an increase in the rigidity of the hydrophobic core. [150] Moreover, the metal ion-fixed polymeric miceles (Composition 4) of the am- phiphilic diblock copolymers with a hydrophobic moiety (tocopherol succinic acid) substituted for the hydroxyl termind group of the hydrophobic B block had kineticdfy greater stability than the origind mPEG-LA-OH (Composition 7). This result suggests that the increase of hydrophobicity of hydrophobic B block in the amphiphilic polymer results in the formation of more staHe miceles due to stronger interactions between the hydrophobic moiety of the amphiphilic polymer and drug. [151] The metal ion-fixed polymeric miceles (Composition 8) of the amphiphilic diblock copolymers with a hydrophobic moiety (tocopherol succinic acid) substituted for the hydroxyl terminal group of the hydrophobic B block has a much longer Hoodstream retention time than the metal ion-fixed polymeric miceles (Composition 9) of the original amphiphilic diblock copolymer as shown in Table 11 and Fig. 9. This result dso suggests, as demonstrated in Examjie 36, that the increase of hy-drophobicity of the hydrophobic B block in the amphiphilic polymer results in the formation of more staHe miceles due to stronger interactions between the hydrophobic moiety of the amphiphilic polymer and drug. [152] As shown in Figs. 10-13, a composition, wherein the drug is entrapped in the metal ion-fixed polymeric composition has a longer retention time of drug in the bloodstream, and so maintains an effective plasma drug concentration for a longer period of time as compared with the currently marketed formulations. [153] The present invention dso provides a pharmaceutical composition for use as an anticancer agent. In a preferable embodiment, it provides a pharmaceutical composition for use as an anticancer agent, comprising an amphiphilic block copolymer of a hydrophilic A block and a hydrophobic B block with a terminal hydroxy! group, and a poljiactic acid derivative, wherein said termind hydroxji termind group of the hydrophobic B block is substituted with a tocopherol or cholesterol group, and at least one end of the polylectic acid derivative is covdently bound to at least one carboxyl group , and an anticancer drug. T he carboxji terminal group of the polylactic acid derivative can be further fixed with a di- or tri-vdent metal ion. [154] Examjies of the anticancer drugs indude, but are not limited to, taxoids, taxines or taxanes like paditaxel and docetaxel; phodophylotoxins; camptothecins like camptothecin, 9-nitrocamptothecin, 9-aminocamptothecin, camptothecin-11, topodecane; anthracydines like doxorubicin, epirubicin, adarubicin, idarubicin, pyrarubicin; vinca Adoids like vincristine, vinorebine, vindesine, vintripole, vinsdtine; eposilones, platinum, etoposide, methotrexate, carmustine, 5-fluorouracil, retinoic acid, retinol, tamoxifen, mitomycin B, mitomycin C, amonafide, iludin S, etc. [155] The polymeric micele-pharmaceuticd composition obtained has greatly improved pharmaceuticd efficacy. As a specific example, as shown in Figs. 14 to 21, paditaxel 2+ containing Ca -fixed polymeric miceles has a high inhibition rate on cancer growth, and dso inhibits the growth of anticancer drug-resistant cancer Cells (Figs. 22 & 23). 7 [156] Taxol (or paditaxel), doxorubicin, etc. are widely used in chemotherapeutic treatment of cancer. These anticancer drugs are effective and useful in chemotherapy, but the development of anticancer drug-resistance in cancer Cells dways renders the drugs ineffective. Various mechanisms of Taxol -resistance including the over-expression of P-$ycoprotein (P-gp) and modification of b -tubulin have been characterized- Among them, the overexpression of F-gp has been a predominant mechanism to explain the multi-drug resistant phenomena, including Taxol -resistance. Anticancer drug-resistant cancer Cells show higher IC (50% Cell inhibition con- 50 centration of drug) than normal ones, and so chemotherapy with the anticancer drug requires a higher concentration of drug in the tumor Cells. Therefore, specific lo-cdization of the drug in the tumor tissue is required for guaranteeing effectiveness. The metal ion fixed polymeric micele had a longer circulation time than the con-ventiond formulations as shown in Figure 10. Thus, it accumulated more selectively in the tumor tissue by an enhanced permeation and retention (EPR) effect compared to the conventional formulations. To demonstrate the effectiveness of metal ion-fixed polymeric raiceles against anticancer drug-resistant cancer, an animal model for in ? vivo anti-cancer activity against Taxol -resistant cancer was establiished. When the cancer Cells which had been inoculated into mice were exposed repeatedly to Taxol , the IC of the drug for Taxol -pretreated cancer Cells was increased significantly compared to that of the drug for the native cancer Cells. In this animal model, the raetd ion-fixed polymeric micele (Composition 10) treated group showed a higher inhibition rate than the Cremophor EL formulation (Composition 11) treated group, possiUy due to the longer retention time for an effective concentration of the drug incorporated in the metal ion-fixed polymeric micele as shown in Fig. 22 and Table 22. The same effect could be obtained from the doxorubicin-resistant cancer animal model (Fig. 23) [157] Therefore, the present invention provides a pharmaceutical composition for treating a drug-resistant cancer comprising an amphiphilic dock copolymer of a hydrophilic A block and a hydrophobic B block with a terminal hydroxyl group, and a polylactic acid derivative, wherein said termind hydroxyl terminal group of the hydrophobic B block is substituted with a tocopherol or cholesterol group, and at least one end of the polyiactic acid derivative is covaienlly bound to at least one carboxyl group, and an anticancer drug. The carboxyl terminal group of the polylactic acid derivative of the above composition can be also fixed with a di- or tri-valent metal ion. [158] Furthermore, the present invention indudes a process for preparing the above phar- maceuticd composition. Specified , as shown in Figs. 3 and 5, the polylactic acid derivative, the amphiphilic block copolymer, and the poonly water-soluble drug at a certain ratio can be dissolved in one or more solvents selected from the group consisting of acetone, ethanol, methanol, ethji acetate, acetonitrile, meth)iene chloride, chloroform, acetic acid, and dioxane. The organic solvent can be removed therefrom to prepare a homogenous mixture of the pooriy water-soluUe drug and the polymer. The homogenous mixture of the pooriy water-solutie drug and the polymeric composition of the present invention can be added to an aqueous solution of pH 4 to 8, at 0 to 80 °C resulting in a pooriy water-soluble drug-containing mixed polymeric micele aqueous solution. The above drug-containing polymeric micele aqueous solution can then be lyophilized to prepare the polymeric micele composition in a solid form. [159] An aqueous solution containing 0.001 to 2 M of the di- or tri-vdent metal ion is added to the pooriy water-soluUe drug-containing mixed polymeric micele aqueous solution to form metal ion-fixed polymeric miceles. The mixture is slowly stirred at room temperature for 0.1 to 1 hour, and then lyophilized to prepare the metal ion-fixed polymeric micele or nanopartide composition in a solid form. [160] Polymeric miceles or nanopartides of the present invention wherein pooriy water- soluble drug is entrapped and solubilized can be administered ordy or parenteraly. The drug is released from the hydrophobic core of the miceles to exhibit a pharmacologics! effect while the miceles are degraded. Particulariy, the metal ion-fixed polymeric miceles or nanopartides are retained in the bloodstream for a long period of time, and accumulate in the target lesions. [161] For parenteral delivery, polymeric miceles or nanopartides may be administered intravenously, intramuscularly, intraperitonedy, transnasdy, intrarectal, in-traoculariy, or intrapubnonarily. For ord delivery, the drug is mixed with the polymeric miceles of the present invention, and then administered in the form of tablet, capsule, or aqueous solution. [162] The dose of the polymeric miceles or nanopartides used in the present invention can be changed over a wide range according to various conditions such as patient's symptoms, age and body weight, and the like. [163] The following examples wil enable those sidled in the art to more dearry understand how to practice the present invention. It should be understood that though the invention has been described in conjunction with the preferred specific embodiments thereof, the folowing is not intended to limit the scope of the present invention. Other aspects of the invention will be apparent to those skiled in the art to which the invention pertains. [164] [ 165] Preparation Example 1 [166] Synthesis 1 of DJ.-polylactic acid (PLA-COOH) [167] One hundred grams of D,L-lactic acid were introduced into a 250 ni three-neck round-bottomed flask. The flask was equipped with a stirrer, and heated in an oil bath to 80 °C The reaction was performed for 1 hour under the pressure reduced to 25 mmHg by a vacuum aspirator to remove excessive moisture. The reaction was then performed at a temperature of 150 °C under a reduced pressure of 25 mmHg for 6 hours. The resulting product was added to 1 liter of distiled water to precipitate the polymer. The precipitated polymer was then added to distiled water to remove the low molecular weight polymer that was soluble in an aqueous solution with a pH of 4 or less. The precipitated polymer was then added to 1 liter of distiled water, and the pH of the aqueous solution was adjusted to 6 to 8 by the addition of sodium hydrogen carbonate portionwise thereto to dissolve the polymer. The water-insoluble polymer was separated and removed by centrifugation or filtration. A 1 N hydrochloric acid solution was added dropwise thereto and the polymer was precipitated in the aqueous solution. The precipitated polymer was washed twice with distiled water, isolated and dried under reduced pressure to obtain a highly viscous liquid (78 g of D,L-polylactic acid, yield: 78%). The number average molecular weight of the polymer was 540 Dalons as determined by H-NMR spectrum. [168] [169] Preparation Examples 2 to 4 [170] Synthesis 2 of D,L-poIylactic acid (PLA-COOH) [171] D,L-polylactic acid was obtained according to the same procedure as in Preparation Examjie 1 except for the control of the reaction temperature, pressure, and time as set forth in Table 1. The number average molecular weight and the yield of D,L-polylactic acid synthesized from the above Preparation Examples 1 to 4 are shown in the folowing TaUe 1. [172] [173] Table 1 [175] [176] Preparation Example 5 [177] Synthesis 1 of the copolymer of D,L-lactic acid and glycolic acid (PLGA-COOH) [178] Fifty-five (55) grams of D,L-lactic acid (0.6 moles) and 45 grams of glycolic acid (0.6 moles) were introduced together into a 250 ni three-neck round-bottomed flask. The same procedure as in Preparation Examjie 1 was carried out except that the reaction was performed at a temperature of 150 °C and under a reduced pressure of 10 mmHg for 12 hours. [179] [180] Preparation Example 6 [181] Synthesis 2 of the copolymer of D^L-lactic acid and glycolic acid (PLGA-COOH) [182] Seventy-three (73) grams of D,L-lactic acid (0.8 moles) and 27 grams of gjycolic acid (0.35 moles) were introduced together into a 250 nl three-neck round-bottomed flask. The same procedure as in Preparation Example 1 was carried out except that the reaction was perfoimed at atemperature of 160 °C and under a reduced pressure of 10 mmHg for 12 hours. [183] [184] Preparation Example 7 [185] Synthesis 3 of the copolymer of D,L-lactic acid and glycolic acid (PLGA-COOH) [186] Ninety-one (91) grams of D,L-lactic acid (1.0 mole) and 9 grams of glycolic acid (0.12 moles) were introduced together into a 250 ni three-neck round-bottomed flask. The same procedure as in Preparation Examjie 1 was carried out except that the reaction was performed at a temperature of 160 °C and under a reduced pressure of 10 mmHg for 12 hours. [187] [188] Preparation Example 8 [189] Synthesis 4 of the copolymer of D,L-lactic acid and glycolic acid (PLGA-COOH) [190] Seventy-three (73) grams of D,L-lactic acid(0.8 moles) and 27 grams of gjlycolic acid (0.35 moles) were introduced into a 250 ni thre^-neck round-bottomed flask. The same procedure as in Preparation Examjie 1 was carried out except that the reaction was performed at a temperature of 180 °C and under a reduced pressure of 5 mmHg for 24 hours. [191] The copolymers synthesized in the above Preparation Examjies 5 to 8 are shown in TaUe 2. [192] [194] [195] Preparation Example 9 [196] Synthesis of a copolymer of D,L-lactic acid and mandelic acid (PLMA-COOH) [197] [198] Seventy-five (75) grams of D, L-lactic acid (0.83 moles) and 25 grams of D,L-mandelic acid (0.16 moles) were introduced together into a 250 ni three-neck round-bottomed flask. The same procedure as in Preparation Example 1 was carried out except that the reaction was performed at a temperature of 180 °C and under a reduced pressure of 10 to 20 mmHg for 5 hours. Fifty-four (54) g (yield: 54%) of a copolymer of D, L-lactic acid and mandelic acid were obtained. The molar ratio of D,L-lactic acid to mandelic acid was 85/15. The number average molecular weight of the polymer was 1,096 Dions as determined by H-NMR spectrum. [199] [200] Preparation Example 10 [201] Synthesis of an acetoxy D^L-polylactic acid derivative (AcO-PLA-COOH) [202] Fifty (50) g of D,L-potylactic acid (Mn: 1,140 Ddtons ), synthesized from Preparation Example 2, and 20 id of chloracetic acid were introduced together into a 250 ill round-bottomed flask. The flask was equipped with a refrigerator, and the reaction mixture was refluxed under nitrogen flow for 4 hours. Excessive chloracetic acid was removed by distilation, and the reaction product was added to a mixture of ice and water. The whole mixture was stirred slowly to precipitate the polymer. The precipitated polymer was separated, washed twice with distiled water, and then dissolved in anhydrous acetone. Anhydrous magnesium sulfate was added thereto to remove excessive moisture. The product obtained was filtered to remove the magnesium sulfate. Acetone was removed using a vacuum evaporator, thereby to obtaining liquid acetoxy D,L-pollylactic acid (46 g, yield: 92%)L By H-NMR, the acetoxy group was identified as a single peak at 2.02 ppm. [203] [204] Preparation Example 11 [205] Synthesis of a palmitoyloxy D,L-polylactic acid derivative (PalmO-PLA-COOH) [206] Twenty (20) grams of D,L-polylactic acid (Mn: 1,140 Dions ), synthesized from Preparation Examjie 2, was introduced into a 250 ml round-bottomed flask. The reactant was comjietely dehydrated under vacuum in an oil bath at 120 °C The oil bath was cooled to 50 °C and 50 rri of acetone was added thereto to comfletely dissolve the polymer. Five (5) ml of chloropdmitic acid was added thereto, and the reaction was performed at a temperature of 50 °C for 10 hours under nitrogen. The reaction product was washed with an excessive amount of hexane to remove any residud reactant. The product was then dissolved in acetone, and the solution was added to a mixture of ice and water. The whole mixture was stirred slowly resulting in the precipitation of an oligomer. The oligomer was separated and washed twice with distiled water, and then dissolved in anhydrous acetone. Anhydrous magnesium sulfate was added to the solution to remove excessive moisture. The product obtained was filtered to remove the magnesium sulfate. Acetone was removed with a vacuum evaporator, thereby obtaining a pdmitojioxy D,L-pollylactic acid derivative (19.1 g, yield: 96%). By H-NMR, the pdmitoji group was identified as the peaks of 0.88,1.3, and 2.38 ppm. [207] [208] Preparation Example 12 [209] Synthesis of 3arm polylactic acid (3ann PLA-COOII) [210] [211] One (1) gram of glycerol (0.01 lmol) was introduced into a 100 ni three-neck round-bottomed flask. The flask was equipped with a stirrer, and heated in an oil bath to 80 °C The reaction was performed for 30 min with the pressure reduced to 25 mmHg by a vacuum aspirator to remove excessive moisture. A reaction catdyst, tin octoate (Tin (Oct) 2), dissolved in toluene was added to the glycerol. The reaction mixture was stirred for 30 minutes, and the pressure was reduced to 1 mmHg at 110 °C for 1 hour to remove the solvent (toluene) dissolving the catalyst. Purified lactide (35.8 g, 0.249 mol; lOwt %) was added thereto, and the mixture was heated to 130 °C under a reduced pressure of 25 mmHg for 6 hours. The polymer formed was dissolved in acetone, and 0.2 N NaHCO aqueous solution was added dropwise thereto to precipitate the polymer. The precipitated polymer was washed three or four times with distiled water, isolated and dried under a reduced pressure to obtain a powder (3arm RA-OH) [212] One hundred (100) grams of 3arm RA-OH (0.033 mol) were introduced into a 100 ni one-neck round-bottomed flask. The reaction was performed for 30 min with the pressure reduced to 25 mmHg by a vacuum aspirator to remove excessive moisture. 19.8 grams of succinic anhydride (0.198 mol) were added thereto, and the mixture was heated to 125 °C for 6 hours. The polymer formed was dissolved in acetone, and distiled water was added dropwise thereto to precipitate the polymer. The precipitated polymer was dissolved in a 0.2N NaHCO aqueous solution at 60 °C The 3 undissolved polymer was removed by filtration. A 2N HC1 aqueous solution was added dropwise thereto to precipitate the polymer. The precipitated potymer was washed five or six times with distiled water, isolated and dried under reduced pressure to obtain a powder (3arm ELACOOHX The number average molecular weight of the polymer was 3,000 Dafcons as determined by H-NMR spectrum. [213] [214] Preparation Example 13 [215] Synthesis of Sarm polylactic add (5arm PLA-COOH) [216] One (1) gram of xylitol (0.0066mol) was introduced into a 100 ni three-neck round-bottomed flask. The flask was equipped with a stirrer, and heated in an oil bath to 80 °C The reaction was performed for 30 min with the pressure reduced to 25 mmHg by a vacuum aspirator to remove excessive moisture. A reaction catdlyst, tin octoate (Tin (Oct) 2), dissolved in toluene was added into the gycerol. The reaction mixture was stirred for 30 minutes, and the pressure was reduced to 1 mmHg at 110 °C for 1 hour to remove the solvent (toluene) dissolving the catalyst. Purified lactide (31.7 g, 0.151 mol; 10wt%) was added thereto, and the mixture was heated to 130 °C under the reduced pressure of 25 mmHg for 6 hours. The polymer formed was dissolved in acetone, and 0.2 N NaHCO aqueous solution was added dropwise thereto to precipitate the polymer. The precipitated polymer was washed three or four times with distiled water, isolated and dried under reduced pressure to obtain powder (5arm ILA-OH> [217] One hundred (100) grams of 5arm ILA-OH (0.033 mol) were introduced into a 100 ,ml one-neck round-bottomed flask. The reaction was performed for 30 min under the pressure reduced to 25 mmHg by a vacuum aspirator to remove excessive moisture. Thirty-three (33.0) grams of succinic anhydride (0.33 mol) were added thereto, and the mixture was heated to 125 °C for 6 hours. The polymer formed was dissolved in acetone, and distiled water was added dropwise thereto to precipitate the polymer. The precipitated polymer was dissolved in 0.2 N NaHCO aqueous solution 3 at 60 °C The undissolved polymer was removed by filtration. A 2 N HC1 aqueous solution was added dropwise thereto to precipitate the polymer. The precipitated polymer was washed five or six times with distiled water, isolated and dried under reduced pressure to obtain a powder (3arm ILA-COOH)L The number average molecular weight of the polymer was 3,000 Dakons as determined by H-NMR spectrum. [218] [219] Preparation Example 14 [220] Synthesis 1 of sodium salt of polylactic acid (PLA-COONa) [221] D,L-polylactic acid (Mn: 540 Dions) synthesized from Preparation Example 1 was dissolved in acetone. The solution was introduced into a round-bottomed flask, and the flask was equipped with a stirrer. The solution was stirred slowly at room temperature, and a sodium hydrogen carbonate solution (1 N) was slowly added thereto to reach a pH of 7. Anhydrous magnesium sulfate was added thereto, and excessive moisture was removed therefrom. The mixture obtained was filtered, and the acetone was evaporated with a solvent evaporator. A white solid was obtained therefrom. The solid was dissolved in anhydrous acetone, and the solution was filtered to remove the insoluble portion. Acetone was evaporated leaving the sodium salt of D,L-polylactic acid (yield: 96%) in a white solid. As shown in Fig. 2, a hydrogen peak adjacent to the carboxylic acid group was observed at 4.88 ppm by H-NMR, and the polymer when dissolved in water had a pH of 6.5 to 7.5. [222] [223] Preparation Example IS [224] Synthesis 2 of the sodium salt of polylactic acid (PLA-COONa) [225] The sodium sal of polylactic acid (yield: 95%) was synthesized according to the same procedure as in the above Preparation Examjle 14 except that D,L-polyiactic acid (Mn: 1,140 Ddtons) synthesized from Preparation Examjie 2 and an aqueous solution of sodium carbonate were used. [226] [227] Preparation Example 16 [228] Synthesis of the sodium salt of acetoxy-D,L-polylactic acid (AcO-PLA-COONa) [229] The sodium salt of acetoxy-D,L-polliactic acid (yield: 95%) was synthesized according to the same procedure as in Preparation Examfie 14 except that acetoxy-D,L-polylactic acid (Mn: 1,140 Dakons ) synthesized from Preparation Examjie 10 and an aqueous solution of sodium carbonate were used. [230] [231] Preparation Example 17 [232] Synthesis 1 of the sodium salt of palmitoyloxy D,L-poIyIactic acid (PalmO-PLA-COONa) [233] The pdmitojioxy D,L-polyiactic acid (Mn: 1,140 Dakons) synthesized from Preparation Example 11 was completely dissolved in an aqueous solution of acetone (28.6v/v%X The solution was introduced into a round-bottomed flask, and the flask was equipped with a stirrer. The solution was stirred slowly at room temperature, and then an aqueous solution of sodium hydrogen carbonate (1 N) was added thereto for nutrdization. The solution was stirred slowly at room temperature and a sodium hydrogen carbonate solution (IN) was slowly added thereto to reach a pH of 7. Anhydrous magnesium sulfate was added thereto to remove excess moisture. The solution obtained was filtered, and the acetone solution was evaporated with a solvent evaporator. A white solid was obtained therefrom. The solid was dissolved in acetone and the solution was filtered to remove any insolutle partides. The acetone was evaporated and the sodium salt of palmitoyloxy D,L-polylactic acid was obtained as a white solid (yield: 96%). [234] [235] Preparation Example 18 [236] Synthesis of the potassium salt of polylactic acid (PLA-COOK) [237] The potassium safe of polylactic acid (yield: 98%) was synthesized according to the same procedure as Preparation Examjie 14 except that D,L-lactic acid (Mn: 1,550 Daltons ) synthesized from Preparation Example 3 and an aqueous solution of potassium hydrogen carbonate were used. [238] [239] Preparation Example 19 [240] Synthesis 3 of the sodium salt of polylactic acid (PLA-COONa) [241] The sodium salt of polylactic acid (yield: 95%) was synthesized according to the same procedure as in Preparation Examjie 14 except that D,L-lactic acid (Mn: 2,100 Dalons ) synthesized from Preparation Example 4 was used. [242] [243] Preparation Example 20 [244] Synthesis 1 of the sodium salt of a copolymer of D,L-lactic acid and glycolic add (PLGA-COONa) [245] The sodium sal of a copolymer of D,L-lactic acid and glycolic acid (yield: 98%) was synthesized according to the same procedure as in Preparation Examjie 14 except that a copolymer of D,L-lactic acid and gylycolic acid (Mn: 920 Dakons) synthesized from Preparation Examjie 5 and an aqueous solution of sodium carbonate were used. [246] [247] Preparation Example 21 [248] Synthesis 2 of the sodium salt of a copolymer of D,L-lactic acid and glycolic acid (PLGA-COONa) [249] The sodium sal of a copolymer of D,L-lactic acid and ^ycolic acid (yield: 93%) was synthesized according to the same procedure as in Preparation Examjie 14 except that a cojx)lymer of D,L-lactic acid and gjjycolic acid (Mn: 1,040 Ddtons ) synthesized from Preparation Examjie 6 was used. [250] [251] Preparation Example 22 [252] Synthesis of the potassium salt of a copolymer of D^L-lactic acid and glycolic acid (PLGA-COOK) [253] The potassium sdt of a copolymer of D,L-lactic acid and gjycolic acid (yield: 92%) was synthesized according to the same procedure as in Preparation Examjie 14 except that a copolymer of D,L-lactic acid and glycolic acid (Mn: 1,180 Daftons ) synthesized from Preparation Examjie 7 and an aqueous solution of j>otassium carbonate were used. [254] [255] Preparation Example 23 [256] Synthesis 3 of the sodium salt of a copolymer of D,L-Iactic acid and glycolic acid (PLGA-COONa) [257] The sodium sdt of a copolymer of D,L-lactic acid and glycolic acid (yield: 98%) was synthesized according to the same procedure as in Preparation Example 14 except that the copolymer of D,L-lactic acid and glycolic acid (Mn: 1,650 Dattons ) synthesized from Preparation Example 8 was used. [258] [259] Preparation Example 24 [260] Synthesis of the sodium salt of a copolymer of D^L-lactic acid and mandelic acid (FLMA-COONa) [261] The sodium salt of a copolymer of D,L-lactic acid and mandelic acid (yield: 96%) was synthesized as white solid according to the same procedure as in Preparation Example 14 except that the copolymer of D,L-lactic acid and mandelic acid synthesized from Preparation Example 9 (Mn: 1,096 Dions) was used. [262] [263] Preparation Example 25 [264] Synthesis of the sodium salt of 3arm polylactic acid (3arm PLA- COONa) [265] The sodium salt of 3 arm polylactic acid was synthesized as a white solid according to the same procedure as in Preparation Example 14 except that the copolymer of 3-arm D,L-lactic acid (Mn: 3,000 Datons ) synthesized from Preparation Example 12 was used. [266] [267] Preparation Example 26 [268] Synthesis of the sodium salt of 5arm polylactic acid (5arm FLA- COONa) [269] The sodium sat of 5 arm polylactic acid was synthesized as a white solid according to the same procedure as in Preparation Example 14 except that the copolymer of 5-arm DJL-lactic acid (Mn: 3,000 Datons) synthesized from Preparation Example 13 was used. [270] The carboxjiate sis of the polylactic acid derivatives synthesized from the above Preparation Examples 14 to26 are shown in TaUe 3. [271] [272] Table 3 [274] Preparation Example 27 [275] Polymerization of a monomethoxypolyethylene glycol-polylactide (mPEG-PLA) block copolymer (AB type) [276] Five (5) grams of monomethoxypolyethylene glycol (Mn: 2,000 Dions) were introduced into a 100 ni two-neck round-bottomed flask, and the mixture was dehydrated by heating to 100 °C under reduced pressure (1 mmHg) for 2 to 3 hours. The reaction flask was filed with dried nitrogen, and a reaction catdyst, stannous octoate (Sn(Oct)), was injected at 0,1 wt% (5 mg) of the lactide by using a syringe. The reaction mixture was stirred for 30 minutes, and the pressure was reduced to 1 mmHg at 110 °C for 1 hour to remove the solvent (toluene) dissolving the catdyst. Purified lactide (5 g) was added thereto, and the mixture was heated to 130 °C for 12 hours. The polymer formed was dissolved in ethanol, and diethjl ether was added thereto to precipitate the polymer. The polymer obtained was dried in a vacuum oven for 48 hours. The mPEG-ILA obtained had a number average molecular weight of 2,000-1,765 Dions , and was confirmed to be of the AB type by 'H-NMR. [277] [278] Preparation Example 28 [279] Polymerization of a monomethoxypolyethylene glycol- polyQactic-co-glycolide) (mPEG-PLGA) block copolymer (AB type) [280] To synthesize an mPEG-ILGA block copolymer, monomethoxypotyethylene $ycol (Mn: 5,000 Dions) was reacted with lactide and gjycolide in the presence of the catdjyst stannous octoate, at 120 °C for 12 hours according to the same procedure as in Preparation Example 27. The mPEG-ILGA obtained had a number average molecular weight of 5,0004,000 Dions , and was confirmed to be of the AB type by 'H-NMR. [281] [282] Preparation Example 29 [283] Polymerization of a monomethoxypolyethylene glycol- polyQactic-ro-p-dioxan-2-one) (mPEG-PLDO) block copolymer (AB type) [284] To synthesize an mPEG4LDO block copolymer, monomethoxypolyethylene $ycol (Mn: 12,000 Dions) was reacted with lactide and p-dioxan-2-one in the presence of the catdlyst, stannous octoate, at 110 °C for 12 hours according to the same procedure as in Preparation Examjie 27. The mPEG-ILDO obtained had a number average molecular weight of 12,000-10,000 Ddtons , and was confirmed to be of the AB type by ^NMR. [285] [286] Preparation Example 30 [287] Polymerization of a monomethoxypolyethylene glycol-polycaprolactone (mPEG-PCL) block copolymer (AB type) [288] To synthesize an mPEG-PCL block copolymer, monomethoxypolyethjiene $ycol (Mn: 12,000 Dakons) was reacted with caprolactone in the presence of the catetyst, of stannous octoate, at 130 °C for 12 hours, according to the same procedure as in Preparation Examjie 27. The mPEG-POL obtained had a number average molecular weight of 12,000-5,000 Ddtons, and was confirmed be of the AB type by 'H-NMR. [289] The block copolymers synthesized from the above Preparation Examples 27 to 30 are shown in the folowing TaHe 4. [290] [291] Tatie4 monomethoxypolyethylene glycol (PLA-niPEG-PLA) block copolymer (BAB type) [295] ILA-mPEG-ELA was obtained according to the same procedure as in Preparation Example 27 except that 25 g of methoxypotyethylene glycol (MW=2,000) and 50 g of D,L-lactide were used. The ELA-mPEG-ILA obtained had a number average molecular weight of 1,765-2,000-1,765 Daltons, and was confirmed to be the BAB type by 'ftNMR. [296] [297] Example 1 [298] Polymerization 1 of mPEG-PLA-cholesterol [299] a) Synthesis of cholesterol succinate [300] 7.6 grams of cholesterol and 2.36 grams of succinic anhydride were dissolved in 100 ni of 1,4-dioxane in a round-bottomed flask. A reaction catalyst, 2.9 grams of 4-(dimeth)lamino)pyridine (DM\P), was added thereto, and the mixture was stirred at room temperature for 24 hours. The reaction mixture was introduced into an HC1 solution to precipitate the cholesterol succinate (9.1 g; yield=95%X [301] [302] b) Binding of mPEGJLA and cholesterol succinate [303] Ten (10) grams of mPEGJLA synthesized from Preparation Examjie 27 and 1.55 grams (L2-fold moles of the polymer) of cholesterol succinate were dissolved in 50 ni of acetonitrile in a round-bottomed flask. The reaction catdysts, 0.76 gram of dicydo-hexyfcaibodiimide (DCC) and 0.045 gram of 4-(dimethylamino)pyridine (DM\P), were added thereto, and the mixture was stirred at room temperature for 24 hours. Upon comjietion of the reaction, the mixture was filtered using a gass filter to remove dicydohexylcarbourea, a byproduct. The residual catalyst was removed by extraction with a hydrochloric acid aqueous solution. To the purified product solution was added magnesium sulfate to remove any residual moisture, and the mixture was added into a cosolvent composed of n-hexane/diethji ether (v/v=7/3) for recrystalization to obtain a purified mPEG-ELA-cholesterol (10 g; yield=88.6%)i Its NMR spectrum is as shown in Fig. 6. [304] [305] Example 2 [306] Polymerization 2 of mPEG-PLA-cholesterol [307] a) Synthesis of cholesterol succinate [308] 7.6 grams of cholesterol and succinyl chloride (twice moles of cholesterol) were introduced into a flask, and dissolved in 50 ml of acetonitrile. The reaction was performed at 50 °C for 12 hours to bind the succinate group to the hydroxy! group of cholesterol, and then, precipitates were formed in an HC1 aqueous solution to obtain cholesterol succinate (8.2 g: yield 92%). [309] [310] h) Binding of mPEGJLA and cholesterol succinate [311] mPEGJLA-cholesterol (9.52 g: yield 85%) was obtained according to the same procedure as in Example lb) except that 10 grams of mPEG-LA and cholesterol succinate synthesized from Example 2a) (1.2-fold moles of the polymer) were used. [312] [313] Examples 3 to 5 [314] Polymerizations 3 to 5 of mPEG-PLA-cholesterol [315] mPEGJLA-cholesterol was obtained according the same procedure as in Example 2 except that malonyl chloride (Example 3), glutaryl chloride (Example 4), and adipoyi chloride (Example 5) each were used at twice the moles of the polymer. [316] [317] Examples 6 to 9 [318] Polymerizations 1 to 4 of mPEG-FLA-tocopherol [319] mPEGJLA-tocopherol was obtained according the same procedure as in Example 2 except that 8.5 g of tocopherol, and malonyl chloride (Example 6), succinyl chloride (Example 7), glutaryl chloride (Example 8), and adipoyi chloride (Example 9) each were used at twice the moles of the polymer. Its NMR spectrum is as shown in Fig. 7 (for Example 7). [320] [321] Example 10 [322] Polymerization of a monomethoxypolyethylene glycol- poly(lactic-co-glycolide) tocopherol (mPEG-PLGA-tocopherol) block copolymer (AB type) [323] Purified mPEG^LGA-tocopherol (10 g; yield=87.5%) was obtained according to the same procedure as in Examjie lb) except a 10 g of mPEG-ILGA synthesized from Preparation Examjie 28 and 1.767 grams of tocopherol succinate was used. [324] [325] Example 11 [326] Polymerization of a monomethoxypolyethylene glycol-poly Oactic-co-glycolide) cholesterol (mPEG-PLGA-cholesterol) block copolymer (AB type) [327] Purified mPEGJLGA-cholesterol (10 g; yield=88.6%) was obtained according to the same procedure as in Example lb) except that 10 g of mPEG-ILGA synthesized from Preparation Example 28 and 0.70 g of cholesterol succinate was used. [328] [329] Example 12 [330] Polymerization of a monomethoxypolyethylene glycol- poly(lactic-co-p-dioxan-2-one) tocopherol (mPEG-PLDO-tocopherol) block copolymer (AB type) [331] Purified mPEG-ILDO-tocopherol (10 g; yield=87.5%) was obtained according to the same procedure as in Example lb) except that 10 g of mPEG-ILDO synthesized from Preparation Examjie 29 and 0.314 g of tocopherol succinate were used. [332] [333] Example 13 [334] Polymerization of a monomethoxypolyethylene glycol- poIy(lactic-co-dioxan-2-one) cholesterol (mPEG-PLDO-cholesterol) block copolymer (AB type) [335] Purified mPEGJLDO-cholesterol (10 g; yield=88.6%) was obtained according to the same procedure as in Examjie lb) except that 10 g of mPEG-ELDO synthesized from Preparation Examjie 29 and 0.288 g of cholesterol succinate were used. [336] [337] Example 14 [338] Polymerization of a monomethoxypolyethylene glycol-polycaprolactone tocopherol (mPEG-PGL-tocopherol) block copolymer (AB type) [339] Purified mPEG-PCL-tocopherol (10 g; yield=87.5%) was obtained according to the same procedure as in Examjie lb) except that 10 g of mPEG-P(X synthesized from Preparation Examjie 30 and 0.406 g of tocopherol succinate were used. [340] [341] Example 15 [342] Polymerization of a monomethoxypolyethylene glycol-polycaprolactone cholesterol (mPEG-PGL-cholesterol) block copolymer (AB type) [343] Purified mPEG^CL-cholesterol (10 g; yield=88.6%) was obtained according to the same procedure as in Example lb) except that 10 g of mPEG-PCL synthesized from Preparation Exampie30 and 0.372 grams of cholesterol succinate were used. [344] [345] Example 16 [346] Polymerization 6 of mPEG-PLA-cholesterol [347] Four (4) grams of cholesterol was weighed, and dehydrated using a vacuum pump at 50 °C Thereto was added succinyi chloride (3.0 g; 2.0-fold moles of cholesterol) and the reaction was performed for 12 hours. After the reaction was completed, the excess succinji chloride was removed under vacuum at 100 °C Thereto was added mPEG^LA (36 g; 0.95-fold mole of cholesterol), and the reaction was performed for 12 hours. The synthesized polymer was dissolved in methylene chloride, and then, precipitated in a hexane/diethji ether solvent to obtain the amphiphilic block copolymer with the cholesterol group, mPEGHLA-cholesterol. The precipitated polymeric product was filtered, and then dried under vacuum to obtain the polymer (35 g; yield 88%) as white partides. [348] [349] Examples 17 to 20 [350] Polymerizations 7 to 10 of mPEG-PLA-cholesterol [351] mPEG-ELA-cholesterol was obtained according to the same procedure as in Example 16 except that oxafyi chloride (Example 17), malonyl chloride (Example 18), gjutarjl chloride (Example 19), and adipo>i chloride (Example 20) were used at 2-fold moles of cholesterol, respectively. [352] [353] Examples 21-25 [354] Polymerizations 5 to 9 of mPEG-PLA-tocopherol [355] mPEG-ELA-tocopherol was obtained according to the same procedure as in Example 16 except that 4.3 g of tocopherol was used, and oxalyl chloride (Example 21), mdonyl chloride (Example 22), succinyl chloride (Example 23), glutaryl chloride (Example 24) and adipxpoyl chloride (Example 25) were used at 2-fold moles of tocopherol, respectively. [356] [357] Example 26 [358] Polymerization 11 of mPEG-FLA-cholesterol [359] Cholesterol succinate (4.9 g) and chloride (2.53 g; 2-fold moles of cholesterol succinate) were weighed, and reacted at 50 °C for 6 hours. After the reaction was completed, excess oxslyl chloride was removed under vacuum. mPEG-ELA (36 g; 0.95-fold moles of cholesterol succinate) was weighed and added thereto. The reaction temperature was set at 100 °C, and the reaction was performed for 12 hours. The synthesized polymer was dissolved in methylene chloride, and then precipitated in hexane/diethji ether, and filtered. The product was dried under vacuum to obtain mPEGJLA-cholesterol (34.6 g; yield 91%). [360] [361] Examples 27-29 [362] Polymerizations 12 to 14 of mPEG-PLA-cholesterol [363] mPEG-ILA-cholesterol was obtained according to the same procedure as inExamjie 26 except using cholesterol mdonate (Example 27), cholesterol $utarate (Example 28) and cholesterol adipate (Example 29). [364] [365] Examples 30-33 [366] Polymerizations 10 to 13 of mPEG-FLA-tocopherol [367] mPEG-HLA-tocopherol was obtained according to the same procedure as inExamjie 26 except that tocopherol mdonate (Example 30), tocopherol succinate (Example 31), tocopherol ^utarate (Example 32), and tocopherol adipate were used(Exam]ie 33)L [368] [369] Example 34 [370] Preparation of tocopherol-PLA-mPEG-PLA-tocopherol [371] Tocopherol -JLAnmPEG-ILA-tocopherol (yield = 92.4%) was obtained according to the same procedure as in Example lb) except that 10 g of ILA-mPEG-ILA synthesized from Preparation Example 31 and tocopherol succinate (2.4-fold moles of the polymer) were used. [372] [373] Example 35 [374] Preparation of cholesterol-PLA-mPEG-PIA-cholesterol [375] Cholesterol-ILA-PEGflA-cholesterol (yield = 94.2%) was obtained according to the same procedure as in Example lb) except that 10 g of ILA-mPEG-LA synthesized from Preparation Example 31 was used. [376] [377] Example 36 [378] Pharmacokinetics for the paclitaxel-containing polymeric micelles of the amphiphilic diblock copolymers conjugated with the hydrophobic moiety [379] To evaluate the effect of a hydrophobic moiety being substituted for the hydroxji termini group of the hydrophobic B block of the amphiphilic dittock copolymers (mPEG-ILA, Mn 2000-1765) on the bloodstream retention time of the paditaxel-containing polymeric miceles, the compositions were prepared as folows. Paditaxel and the amphiphilic diblock copolymer of Examjie 1, 7, or Preparation Example 27, were admixed in a weight ratio of 1:99, and then the mixture was dissolved in 5 ml of anhydrous ethanol to prepare a dear solution. Ethanol was removed therefrom using a vacuum evaporator to prepare a paditaxel-containing polymeric composition. Distiled water (4 rd) was added thereto, and the mixture was stirred for 10 minutes at 60 °C to prepare a potymeric micele aqueous solution containing paditaxel. The mixture was passed through a filter with a pore size of 200 nm, and was then lyophilized. [380] The above composition and the drug content are summarized in Table 5. [381] [383] [384] For the animal experiments, male Sprague-Dawley rats weighing 250-300 g were cannulated in the vena femordis and aorta femordis. Compositions 1 to 3 were injected into the vena femordis at a dose of 5 mg/kg over 15 seconds. After the injection, 0.3 rri of the whole blood was taken from the aorta femordis at 1, 5,15, and 30 minutes, and in 1, 2, 3,4, and 6 hours, and then centrifuged to obtain dear supernatant plasma. [385] To analyze the plasma concentration of drug, 0.1 ml of the plasma was introduced into a covered gjass tube, and 0.1 nl of an acetonitrile solution containing the internal standard substance was added thereto. Ten (10) ni of ethyl acetate was added to the above solution, and the mixture was vigorously stirred for 30 seconds, and then centrifuged at 2,500 rpm for 10 minutes. The whole ethyl acetate layer was taken and transferred to a test tube, and then the organic solvent was completely evaporated at 40 °C under nitrogen flow. Thereto was added 0.1 nl of a 40%(v/v) acetonitrile solution, and the mixture was vigorous^ stirred for 30 seconds, and then subjected to HLC The conditions for HLC were as folows: [386] Injection Volume: 0.075 ni [387] Flow Rate: 1.0 ni/min [388] Wavelength: 227 nm [389] Mobile Phase: 24% aqueous acetonitrile solution for 5 minutes, increased to 58% for 16 minutes, increased to 70% for 2 minutes, decreased to 34% for 4 minutes, and maintained for 5 minutes [390] Column: 4.6x50 nm (C18, Vydac , USA ) [391] The micele size and analysis of the results of the plasma concentrations of the drugs are shown in the folowing Table 6 and Fig. 8. [392] [393] TaUe 6 [394] [395] As shown in TaUe 6 and Fig. 8, the polymeric miceles (Compositions 1 and 2) of the amphiphilic ditiock copolymers with a hydrophobic moiety (tocopherol succinic acid or cholesterol succinic acid) substituted for the hydroxple terminal group of the hydrophobic B block had a much longer Uoodstream retention time than the native mPEG-ILA-OH polymeric miceles (Composition 3) This result suggests that an increase of hydrophobicity of the hydrophobic B block in the amphiphilic polymer results in formation of more stable miceles due to stronger interactions between the hydrophobic moiety of the amphiphilic polymer and drug. [396] In addition, it was confirmed that the mPEG-ILA-tocopherol miceles (Composition 1) were circulated longer than the mPEG-ILA-cholesterol miceles (Composition 2) in the Hood. [397] [398] Example 37 [399] Preparation of ionically fixed polymeric micelles [400] Step 1: Preparation of the polymeric miceles of D.L-ELACOONa and mPEG- rLA-tocopherol block copolymers [401] 248.1 mg (0.218 mmol) of D,LJLACOONa (Mn: 1,140) from Preparation Example 15 and 744.3 mg of mPEG-,LA-tocopherol (Mn: 2,000-1,800 Ddtons ) from Example 7 were complete^ dissolved in 5 ml of ethanol to obtain a dear solution. Ethanol was removed therefrom to prepare a polymeric composition. Distiled water (6.2 iri) was added thereto and the mixture was stirred for 30 minutes at 60 °C to prepare the polymeric micele aqueous solution. [402] [403] Step 2: Fixation with the di-vdent metal ion [404] 0.121 iri (0.109 mmol) of a 0.9 M aqueous solution of anhydrous calcium chloride was added to the polymeric micele aqueous solution prepared in Step 1, and the mixture was stirred for 20 minutes at room temperature. The mixture was passed through a filter having a pore size of 200 nm, and then was lyophilized. The partide size measured according to the Dynamic Light Scattering (DLS) Nfethod was 25 nm. [405] [406] Example 38 [407] Preparation of Ca -fixed paclitaxel-containing micelles of D,L-PLA-COONa and mPEG-PLA-tocopherol block copolymers [408] Step 1: Preparation of paditaxel-containing polymeric miceles of D.L-LACOONa and mPEG-LA-tocopherol block copolymers [409] 248.1 mg (0.218 mmol) of D ,L.-ILACOONa (Mn: 1,140) from Preparation Example 15, 7.5 mg of paditaxel, and 744.3 mg of mPEG-FLA-tocopherol (Mn: 2,000-1,800 Ddtons) from Examjie 7 were completely dissolved in 5 ni of ethanol to obtain a dear solution. Ethanol was removed therefrom to prepare a paditaxel-containing polymeric composition. Distiled water (6.2 iri) was added thereto and the mixture was stirred for 30 minutes at 60 °C to prepare a paditaxel-containing polymeric micele aqueous solution. [410] [411] Step 2: Fixation with the divdent metal ion [412] 0.121 ni (0.109 mmol) of a 0.9 M aqueous solution of anhydrous calcium chloride was added to the polymeric micele aqueous solution prepared in Step 1, and the mixture was stirred for 20 minutes at room temperature. The mixture was passed through a filter having a pore size of 200 nm, and then was lyophilized. The content and solubility of paditaxel were measured by HILC and the partide size was measured according to the Dynamic Light Scattering (DLS) method. [413] D,LJLACOONa/mPEGJLA-tocopherol (weight ratio): 1/3 [414] Content of Paditaxel: 0.75wt% [415] Partide Size: 29 nm [416] [417] Example 39 [418] Preparation of Mg + -fixed paditaxel-containing polymeric micelles of D,L-PLMA-COONa and mPEG-PLA-tocopherol block copolymers 2+ [419] AMg -fixed paditaxel-containing polymeric micele composition was prepared according to the same procedure as in Example 38 except that 248.1 mg (0.226 mmol) of D,L,LM\COONa (Mn: 1,096) from Preparation Example 24, 7.5 mg of paditaxel and 744.3 mg of mPEG,lLA-tocopherol (Mn: 2,000-1,800 Dalons) from Example 7, and 0.230 ml (0.113 mmol) of the 0.5 M aqueous solution of magnesium chloride 6 hydrate (Mw:203.31) were used . [420] D,L-LMA.COONa/mPEG^LA-tocopherol (weight ratio): 1/3 [421] Content of Paditaxel: 0.75wt% [422] Partide Size: 30 nm [423] [424] Example 40 [425] Preparation of Ca -fixed paclitaxel-containing polymeric micelles of D,L-PLMA-COONa and mPEG-PLA-tocopherol block copolymers 2+ [426] A Ca -fixed paditaxel-containing polymeric micele composition was prepared according to the same procedure as inExamfie 38 except that 248.1 mg (0.226 mmol) of D,LJLMACOONa (Mn: 1,096) from Preparation Examjie 24,15 mg of paditaxel and 744.4 mg of mPEGJLA-tocopherol (Mn: 2,000-1,800 Dions) from Example 7, and 0.126 ni (0.113 mmol) of the 0.9 M aqueous solution of anhydrous cdrium chloride were used . [427] D,L-ILM\COONa/mPEG4LA-tocopherol (weight ratio): 1/3 [428] Content of Paditaxel: 0.75 wt% [429] Partide Size: 34 run [430] [431] Example 41 [432] Preparation of Ca+ -fixed paclitaxel-containing polymeric micelles of DjL-PLA-COOK and mPEG-PLA-cholesterol block copolymers 2+ [433] A Ca -fixed paditaxel-containing polymeric micele composition was prepared according to the same procedure as in Example 38 except that 248.1 mg (0.160 mmol) of DJLJLACOQK (Mn: 1,550) from Preparation Example 18, 7.5 mg of paditaxel and 744.4 mg of mPEGflLA-cholesterol (Mn: 2,000-1,800 Dafcons) from Example 1, and 0.089 rri (0.080 mmol) of the 0.9 M aqueous solution of anhydrous cafcium chloride were used. [434] DJLJLMACOONa/mPEG4LAK:hoIesterol (weight ratio): 1/3 [435] Content of Paditaxel: 0.75 wt% [436] Partide Size: 34 nm [437] [438] Example 42 [439] Preparation of Ca2+ -fixed paditaxel-containing polymeric micelles of D,L-PLMA-COONa and mPEG-PLA-cholesterol block copolymers 2+ [440] A Ca -fixed paditaxel-containing polymeric micele composition was prepared according to the same procedure as in Examde 38 except that 248.1 rag (0.226 mmol) of D.L-FLMACOONa (Mn: 1,096) from Preparation Examde 24,15 mg of paditaxel and 744.4 mg of mPEG-ELA-choIesterol (Mn: 2,000-1,800 Ddtons) from Examde 1, and 0.126 ml (0.113 mmol) of the 0.9 M aqueous solution of anhydrous calcium chloride were used . [441] D,L-H.MACOONa/mPEG4LA-cholesterol (weight ratio): 1/3 [442] Content of Paditaxel: 0.75wt% [443] Partide Size: 34 nm [444] [445] Example 43 [446] Preparation of Ca2+-fixed paclitaxel-containing polymeric micelles of 3 arm PLA-COONa and mPEG-PLA-tocopherol block copolymers 2+ [447] A Ca -fixed paditaxel-containing polymeric micele composition was prepared according to the same procedure as in Example 38 except that 248.1 mg (0.0827 mmol) of 3 arm ILACOONa (Mn: 3,000) from Preparation Examde 25, 7.5 mg of paditaxel and 744.4 rag of mPEG-LA-tocopherol (Mn: 2,000-1,800 Ddtons) from Example 7, and 0.1377 ml (0.124 mmol) of the 0.9 M aqueous solution of anhydrous cdcium chloride were used. [448] 3arm ELACOONa/mPEG^LA-tocopherol (weight ratio): 1/3 [449] Content of Paditaxel: 0.75 wt% [450] Partide Size: 29 nm [451] [452] Example 44 [453] Preparation of Ca2+-fixed paclitaxel-containing polymeric micelles of 5 arm PLA-COONA and mPEG-PLA-tocopherol block copolymers 2+ [454] A Ca -fixed paditaxel-containing polymeric micele composition was prepared according to the same procedure as in Examfie 38 except that 248.1 mg (0.0827 mmol) of 5 arm ILACOONa (Mn: 3,000) from Preparation Examjie 26, 7.5 mg of paditaxel and 744.4 mg of mPEG-ILA-tocopherol (Mn: 2,000-1,800 Dakons) from Example 7, and 0.2295 iri (0.207 mmol) of the 0.9 M aqueous solution of anhydrous calcium chloride were used. [455] 5arm HACOONa/mPEG^LA-tocopherol (weight ratio): 1/3 [456] Content of Paditaxel: 0.75 wt% [457] Partide Size: 29 nm [458] [459] Example 45 [460] Preparation of doxorubicin-containing polymeric micelles of D^-PLMA-COONa and mPEG-PLA-tocopherol block copolymers [461] mPEG-LA-tocopherol (Mn: 2,000-1,800), D-L-ILMACOONa (Mn: 969), and doxorubicin HC1 were admixed in a weight ratio of 78.62:17.24:1.00, and then the mixture was dissolved in 5 iri of anhydrous methanol to prepare a dear solution. Methanol was removed therefrom using a vacuum evaporator to prepare a doxorubicin-containing polymeric composition. Distiled water (4 ml) was added thereto, and the mixture was stirred for 10 minutes at 60 °C to prepare a polymeric micele aqueous solution containing doxorubicin. The mixture was passed through a filter with a pore size of 200 nm, and then was tyophilized. [462] D,LflLMVCOONa/mPEGJLA-tocopherol (weight ratio): 1/4.56 [463] Content of doxorubicin: 1.03wt% [464] Partide Size: 35 nm [465] [466] Example 46 [467] Preparation of epirubidn-containing polymeric micelles of D^-PLMA-COONa and mPEG-PLA-tocopheroI block copolymers [468] mPEG4LA-tocopherol (Mn: 2,000-1,800), D,L-lLM\COONa (Mn: 969), and epirubicin HC1 were admixed in a weight ratio of 78.62:17.24:1.00, and then the mixture was dissolved in 5 ml of anhydrous methanol to prepare a dear solution, methanol was removed therefrom using a vacuum evaporator to prepare an epirubicin-containing polymeric composition. Distiled water (4 ni) was added thereto and the mixture was stirred for 10 minutes at 60 °C to prepare a polymeric micele aqueous solution containing doxorubicin. The mixture was passed through a filer with a pore size of 200 nm, and than was lyophilized. [469] D,Lfl.M\COONa/mPEGJLA-tocopherol (weight ratio): 1/4.56 [470] Content of epirubicin: 1.03wt% [471] Partide Size: 30 nm [472] [473] Example 47 [474] Particle size for the Ca -fixed polymeric micelles 2+ [475] To determine the partide size of the Ca -fixed polymeric miceles, the polymeric micele compositions were prepared as folows. [476] mPEGJLA (Mn: 2,000-1,800) and D,L-ILMACOONa (Mn: 866, 994,1,156, 1,536) were admixed in an equivalent ratio of 1:1, and then the mixture was dissolved in 5 ni of anhydrous ethanol to prepare a dear solution. Ethanol was removed therefrom using a vacuum evaporator to prepare a polymeric composition. Distiled water was added thereto and the mixture was stirred for 10 minutes at 60 °C to prepare a polymeric micele aqueous solution containing paditaxel. To the above polymeric micele solution was added a CaCl aqueous solution (concentration: 100 mg/iri) of the 2 same number equivdents as the D,L-LM\COONa solution, and the mixture was stirred for 20 minutes at room temperature. The mixture was passed through a filter with a pore size of 200 nm, and then PBS buffer at a pH of 7.4 was added thereto to dilute the mixture to make a 40 mg/iri concentration of the polymers. The partide size was measured with a photon correlation partide size analyzer after filtration using a 0.22 um membrane filter. [477] [478] TaHe 7 2+ [480] As shown in Tatfe 7, the partide size of the Ca -fixed polymeric miceles had an average size of 2040 nm. Miceles of this size range are suitaHe for injection formulations and sterile filtration. As the molecular weight of the D,L-ILM\COONa 2+ increased from 866 to 1536, the partide size increased slightly in both the Ca treated 2+ and non-treated miceles. The partide size of the Ca -fixed polymeric miceles was 2+ larger by approximately 10 nm than the miceles not treated with Ca . [481] [482] Example 48 [483] Kinetic Stability for the Ca -fixed paclitaxel-containing polymeric micelles [484] To test the stability of the nanopartide composition, the polymeric micele com- positions were prepared as folows. [485] (Composition 4) Paditaxel, mPEGJLA-Tocopherol (Mn: 2,000-1,800), and D,LJLM\COONa (Mn: 1,096) were admixed at an equivdent ratio of 1:3:3, and then the mixture was dissolved in 5 ni of anhydrous ethanol to prepare a dear solution. Ethanol was removed therefrom using a vacuum evaporator to prepare a paditaxel-containing polymeric composition. Distiled water (4 ml) was added thereto, and the mixture was stirred for 10 minutes at 60 °C to prepare a polymeric micele aqueous solution containing paditaxel. To the above polymeric micele solution was added a CaCl aqueous solution (concentration: 100 mg/ni) of the same number of 2 equivalents as the D,L-ILM\COONa, and the mixture was stirred for 20 minutes at room temperature. The mixture was passed through a filter with a pore size of 200 nm, and then was lyophilized. [486] (Composition 5) Paditaxel, mPEGJLA-Tocopherol (Mn: 2,000-1,800) and DJL-BLMACOONa (Mn: 1,096) were admixed at an equivdent ratio of 1:3:3 and then the mixture was dissolved in 5 ni of anhydrous ethanol to prepare a dear solution. Ethanol was removed therefrom using a vacuum evaporator to prepare a paditaxel-containing polymeric composition. Distiled water (4 ml) was added thereto and the mixture was stirred for 10 minutes at 60 °C to prepare a polymeric micele aqueous solution containing paditaxel. The mixture was passed through a filer with a pore size of 200 nm, and then was lyophilized. [487] (Composition 6) Paditaxel and mPEGJLA-Tocopherol (Mn: 2,000-1,800) were admixed at an equivdent ratio of 1:3, and then the mixture was dissolved in 5 id of anhydrous ethanol to prepare a dear solution. Ethanol was removed therefrom using a vacuum evaporator to prepare a paditaxel-containing polymeric composition. Distiled water (5 ni) was added thereto, and the mixture was stirred for 10 minutes at 60 °C to prepare a polymeric micele aqueous solution containing paditaxel. The mixture was passed through a filer with a pore size of 200 nm, and then was lyophilized. [488] (Composition 7) Paditaxel, mPEG-FLA (Mn: 2,000-1,765), and D,LJLM\COONa (Mn: 1,096) were admixed at an equivdent ratio of 1:3:3, and then the mixture was dissolved in 5 ni of anhydrous ethanol to prepare a dear solution. Ethanol was removed therefrom using a vacuum evaporator to prepare a paditaxel-containing polymeric composition. Distiled water (4 ni) was added thereto, and the mixture was stirred for 10 minutes at 60 °C to prepare a polymeric micele aqueous solution containing paditaxel. To the above polymeric micele solution was added a CaCl aqueous solution (concentration: 100 mg/ni) of the same number of 2 equivalents as the D,L-ILM\COONa, and the mixture was stirred for 20 minutes at room temperature. The mixture was passed through a filter with a pore size of 200 nm, and then was lyophilized. [489] [490] TaHe 8 [491] [492] PBS buffer of a pH of 7.4 was added to the lyophilized compositions to make a 1.0 mg/iri concentration of paditaxel. The mixture was slowed to stand at 37 °C and the concentration of paditaxel over the lapse of time was measured by HBLC The results are shown in Table 9. [493] [494] Table 9 [495] 2+ [496] As shown in Tade 9, the Ca -fixed paditaxel-containing polymeric micele 2+ composition (Composition 4) was kineticdly more stafcle than the Ca -nontreated 2+ composition (Composition 5). The addition of Ca significant increased retention of the paditaxel in the polymeric miceles of the present invention. This is due to the 2+ crosslinking electrostatic interaction of D,L-LACOO and Ca which might induce 2+ an increase in the rigidity of the hydrophobic core. The Ca -fixed polymeric miceles (Composition 4) of the amphiphilic ditiock copolymers with a hydrophobic moiety (tocopherol succinic acid) substituted for the hydroxjl termind group of the hydrophobic B block had a much longer retention time than the Ca +-fixed polymeric miceles (Composition 7) of native mPEG-HLA-OH. This result also suggests that the increase of hydrophobicity of the hydrophobic B block in the amphiphilic polymer results in formation of more stable miceles due to stronger interactions between the hydrophobic moiety of the amphiphilic block copolymer and drug. [497] [498] Example 49 [499] Pharmacokinetics for Ca -fixed paditaxel-containing polymeric micelles [500] To evaluate the effect of a hydrophobic moiety substituted for the hydroxy! termind group of the hydrophobic B block of the amphiphilic di-block copolymers (mPEG-ELA, Mn 2000-1765) on the bloodstream retention time of the Ca +-fixed paditaxel-containing polymeric miceles, the compositions were prepared as folows. [501] Paditaxel, mPEGJLA-tocopherol (Mn: 2,000-1,800) or mPEGJLA-OH, and DJLJLM\COONa (Mn: 1,004) were admixed in a weight ratio of 74.25:24.75:1.00, and then the mixture was dissolved in 5ml of anhydrous ethanol to prepare a dear solution. Ethanol was removed therefrom using a vacuum evaporator to prepare a paditaxel-containing polymeric composition. Distiled water (4 ni) was added thereto, and the mixture was stirred for 10 minutes at 60 °C to prepare a polymeric micele aqueous solution containing paditaxel. To the above polymeric micele solution was added a CaCl aqueous solution (concentration: 100 mg/rri) of the same equivdents as the D,L-HlM\COONa, and the mixture was stirred for 20 minutes at room temperature. The mixture was passed through a filter with a pore size of 200 nm, and then was lyophilized. [502] The above composition and the drug contents are summarized in TaUe 10. [503] [504] TaUe 10 [505] [506] For the animd experiments, male Sprague-Dawley rats weighing 220-270 g were cannulated in the vena femorsiis and aorta femoraiis. Compositions 8 and 9 were injected into the vena femoraiis at a dose of 5 mg/kg over 15 seconds. After the injection, 0.3 ni of whole Hood was taken from the aorta femordis in 1,5,15, and 30 minutes, and in 1, 2, 3,4, and 6 hours, and then centrifuged to obtain dear supernatant plasma. [507] The jiasma drug concentration was analyzed according to the same process as in Example 36, and analysis of the results on the fiasma concentrations of the drugs are shown in the folowing Table 11 and Fig. 9. [508] [509] TaUe 11 [510] 2+ [511] As shown in TaUe 11 and Fig. 9, the Ca -fixed polymeric miceles (Composition 8) of the amphiphilic di-block copolymers with a hydrophobic moiety (tocopherol succinic acid) substituted for the hydroxyl terminal group of the hydrophobic B block 2+ had a much longer bloodstream retention time than the Ca -fixed polymeric miceles (Composition 9) of native rnPEGJLA-OH. This result suggests, as demonstrated in Example 36, that the increase of hydrophobicity of the hydrophobic B block in the am-phiphilic polymer results in formation of more stable miceles due to stronger interactions between the hydrophobic moiety of the amphiphilic polymer and drug. [512] [513] Example 50 [514] Pharmacokinetics for the Ca -fixed paditaxel-containing polymeric micelles 2+ [515] To compare the Hoodstream retention time of the Ca -fixed paditaxel-containing polymeric miceles with that of the formulations containing other carriers, the compositions were prepared as folows. 2+ [516] (Composition 10) Ca -fixed paditaxel-containing polymeric miceles [517] Paditaxel, mPEGflLA-tocopherol (Mn: 2,000-1,800), and D,L,LMVCOONa (Mn: 1,004) were admixed in a weight ratio of 99.25:33.08:1.00, and then the mixture was dissolved in 5 ml of anhydrous ethanol to prepare a dear solution. Ethanol was removed therefrom using a vacuum evaporator to prepare a paditaxel-containing polymeric composition. Distiled water (4 id) was added thereto, and the mixture was stirred for 10 minutes at 60 °C to prepare a polymeric micele aqueous solution containing paditaxel. To the above polymeric micele solution was added a CaCl aqueous solution (concentration: 100 mgml) of the same equivdents as the D,L4LM\COONa, and the mixture was stirred for 20 minutes at room temperature. The mixture was passed through a filer with a pore size of 200 nm, and then was lyophilized. The hydrodynamic partide size of the polymeric miceles was 34 nm. [518] (Composition 11) Composition containing paditaxel, Cremophor EL, and anhydrous ethanol [519] Paditaxel (30 mg) was dissolved in 5 nl of a mixed solution (50:50 v/v) of Cremophor EL and anhydrous ethanol to obtain a dear solution. The solution was passed through a filter having a pore size of 200 nm. [520] (Composition 12) Composition containing paditaxel, polysorbate 80 (Tween 80), and anhydrous ethanol [521] Paditaxel (30 mg) was dissolved in 5 ni of a mixed solution (50:50 v/v) of polysorbate 80 and anhydrous ethanol to obtain a dear solution. The solution was passed through a filter having a pore size of 200 nm. [522] The above composition and the drug contents are summarized in Table 12. [523] [525] [526] For the animd experiments, male Sprague-Dawley rats weighting 230-250 g were cannulated in the vena femordis and aorta femordis. Compositions 10,11 and 12 were injected into the vena femordis at a dose of 5 mg/kg over 15 seconds. After the injection, 0.3 ni of the whole blood was taken from the aorta femordis in 1, 5,15, and 30 minutes, and in 1,2, 3, 4, and 6 hours, and then centrifuged to obtain dear supernatant fiasma. [527] The flasma drug concentration was analyzed according to the same process as in E example 36, and andysis of the results of the plasma concentrations of the drugs are shown in the folowing Table 13 and Fig. 10. [528] [529] Table 13 [530] 2+ [531] As shown in TaUe 13 and Fig. 10, the Ca -fixed polymeric miceles (Composition 10) had a longer bloodstream retention time than the injections containing other 2+ surfactants (Compositions 11 and 12). Since the Ca -fixed polymeric miceles (Composition 10) of the present invention had a longer bloodstream retention time 7 than the marketed formulation, Taxol (Composition 11), the present invention could increase the drug retention time in the bloodstream over Taxol by using the biodegradable and biocompatible polymers of the present invention. [532] [533] Example 51 [534] Pharmacokinetics for the Ca -fixed paditaxel-containing polymeric micelles 2+ [535] To compare the Hoodstream retention time of the Ca -fixed paditaxel-containing polymeric miceles with that of the formulations containing other carriers, the compositions were prepared as folows. 2+ [536] (Composition 13) Ca -fixed paditaxel-containing polymeric miceles [537] Paditaxel, mPEGftA-tocopherol (Mn: 2,000-1,800), and 5arm BLACOONa (Mn: 3,000) were admixed in a weight ratio of 99.25:33.08:1.00, and then the mixture was dissolved in 5 ni of anhydrous ethanol to prepare a dear solution. Ethanol was removed therefrom using a vacuum evaporator to prepare a paditaxel-containing polymeric composition. Distiled water (4 ni) was added thereto, and the mixture was stirred for 10 minutes at 60 °C to prepare a polymeric micele aqueous solution containing paditaxel. To the above polymeric micele solution was added a CaCl 2 aqueous solution (concentration: 100 mg/ni) of the same equivdents as the 5arm ILACOONa, and the mixture was stirred for 20 minutes at room temperature. The mixture was passed through a filter with a pore size of 200 nm, and then was lyophilized. The hydrodynamic partide size of the polymeric miceles was 32 nm. [538] (Composition 11) Composition containing paditaxel, Cremophor EL, and anhydrous ethanol [539] Paditaxel (30 mg) was dissolved in 5 nl of a mixed solution (50:50 v/v) of Cremophor EL and anhydrous ethanol to obtain a dear solution. The solution was passed through a filer having a pore size of 200 nm. [540] The above composition and the drug contents are summarized in Table 14. [541] [542]Tatie 14 [543] [544] For the animal experiments, male Sprague-Dawley rats weighing 230-250 g were cannulated in the vena femordis and aorta femordis. Compositions 13 and 11 were injected into the vena femordis at a dose of 5 mg/kg over 15 seconds. After the injection, 0.3 ni of the whole Hood was taken from the aorta femorsiis in 1,5,15, and 30 minutes, and in 1, 2, 3, 4, and 6 hours, and then centrifuged to obtain dear supernatant fiasma. [545] The plasma drug concentration was analyzed according to the same process as in Example 36, and andysis of the results of the plasma concentrations of the drugs are shown in the folowing Table 15 and Fig. 11. [546] [547] TaUe 15 [548] [549] As shown in Tatie 15 and Fig. 11, the Ca +-fixed polymeric miceles (Composition 13) had a longer bloodstream retention time than the injections containing other 2+ surfactants (Composition 11). Since the Ca -fixed polymeric miceles (Composition 13) of the present invention had a longer bloodstream retention time than the marketed formulation, Taxol (Composition 11), the present invention could increase the drug retention time in the bloodstream over Taxol by using the biodegradable and biocompatible polymers of the present invention. [550] [551] Example 52 [552] Pharmacokinetics for the Ca -fixed docetaxel-containing polymeric micelles 2+ [553] To compare the bloodstream retention time of the Ca -fixed docetaxel-containing polymeric miceles with that of the formulations containing other carriers, the compositions were prepared as folows. 2+ [554] (Composition 14) Ca -fixed docetaxel-containing polymeric miceles [555] Docetaxel, mPEGJLA-Tocopherol (Mn: 2,000-1,800), and 3 arm ELACOONa (Mn: 3,000) were admixed in a weight ratio of 99.25:33.08:1.00, and then the mixture was dissolved in 5 ni of anhydrous ethanol to prepare a dear solution. Ethanol was removed therefrom using a vacuum evaporator to prepare a docetaxel-containing polymeric composition. Distiled water (4 ni) was added thereto, and the mixture was stirred for 10 minutes at 60 °C to prepare a polymeric micele aqueous solution containing docetaxel. To the above polymeric micele solution was added a CaCl 2 aqueous solution (concentration: 100 mg/ni) of the same equivalents as the 3 arm-RACOONa, and the mixture was stirred for 20 minutes at room temperature. The mixture was passed through a filter with the pore size of 200 nm, and then was lyophilized. The hydrodynamic partide size of the polymeric miceles was 30 nm. [556] (Composition 15) Composition containing docetaxel, polysorbate 80 (Tween 80), and anhydrous ethanol [557] Docetaxel (20 mg) and Tween 80 (520 mg) were dissolved in 1.5 nl of 13% (v/v) ethanol aqueous solution to obtain a dear solution. The solution was passed through a filer having a pore size of 200 nm. [558] The above composition and the drug contents are summarized in Talie 16. [559] [560] TaUe 16 [561] [562] For the animal experiments, male Sprague-Dawley rats weighing 210-240 g were cannulated in the vena femordis and aorta femordis. Compositions 14 and 15 were injected into the vena femordis at a dose of 10 mg/kg over 15 seconds. After the injection, 0.3 ni of the whole Hood was taken from the aorta femordis in 5,15, and 30 minutes, and in 1,2, 3, 6, and 8 hours, and then centrifiiged to obtain dear supernatant plasma. [563] The plasma drug concentration was analyzed according to the same process as in Examjle 36, and the results of the plasma drug concentrations are shown in Talie 17 and Fig. 12. [564] [565] Talie 17 [566] 2+ [567] As shown in Table 17 and Fig. 12, the Ca -fixed polymeric miceles (Composition 14) had a longer Hoodstream retention time than the injections containing Tween 80 2+ (Composition 15). Since the Ca -fixed polymeric miceles (Composition 14) of the present invention had a longer Hoodstream retention time than the marketed formulation, Taxotere (Composition 15), the present invention could increase the drug retention time in the Hoodstream over Taxotere by using the biodegradaHe and bio- compatiHe polymers of the present invention. [568] [569] Example 53 [570] Pharmacokinetics for the Ca -fixed docetaxel-containing polymeric micelles 2+ [571] To compare the bloodstream retention time of the Ca -fixed docetaxel-containing polymeric miceles with that of the formulations containing other carriers, the compositions were prepared as folows. 2+ [572] (Composition 16) Ca -fixed docetaxel-containing polymeric miceles. [573] Docetaxel, mPEG-LA-tocopherol (Mn: 2,000-1,800), and D,L-LACOONa (Mn: 1,700) were admixed in a weight ratio of 75.0:25.0:1.0, and then the mixture was dissolved in 5 ml of anhydrous ethanol to prepare a dear solution. Ethanol was removed therefrom using a vacuum evaporator to prepare a docetaxel-containing polymeric composition. Distiled water (4 ml) was added thereto, and the mixture was stirred for 10 minutes at 60 °C to prepare a polymeric micele aqueous solution containing docetaxel. To the above polymeric micele solution was added a CaCl aqueous solution (concentration: 100 mg/ml) of the same equivalents as the D,LlLACOONa, and the mixture was stirred for 20 minutes at room temperature. The mixture was passed through a filter with a pore size of 200 nm, and then was lyophilized. The hydrodynamic partide size of the polymeric miceles was 32 nm. [574] (Composition 15) Composition containing docetaxel, Tween 80, and 13% ethanol [575] Docetaxel (20 mg) and Tween 80 (520 mg) were dissolved in 1.5 ni of 13% (v/v) ethanol aqueous solution to obtain a dear solution. The solution was passed through a filter having a pore size of 200 nm. [576] The above composition and the drug contents are summarized in Table 18. [577] [578] Table 18 [579] [580] For the animd experiments, mde Sprague-Dawley rats weighing 230-250 g were cannulated in the vena femoidis and aorta femordis. Compositions 16 and 15 were injected into the vena femordis at a dose of 5 mg/kg over 15 seconds. After the injection, 0.3 nl of the whole Hood was taken from the aorta femoralis in 1, 5, 15, and 30 minutes, and in 1,2, 3, 4, and 6 hours, and then centrifiiged to obtain dear supernatant plasma. [581] The fiasma drug concentration was analyzed according to the same process as in Example 36, and the results of the plasma concentrations of the drugs are shown in the folowing Table 19 and Fig. 13. [582] [583] Tatie 19 [584] 2+ [585] As shown in Tatie 19 and Fig. 13, the Ca -fixed polymeric miceles (Composition 16) had a longer tioodstream retention time than the injections containing Tween 80 2+ (Composition 15). Since the Ca -fixed polymeric miceles (Composition 16) of the present invention had a longer tioodstream retention time than the marketed formulation, Taxotere (Composition 15), the present invention could increase the drug retention time in the tioodstream over Taxotere by using the biodegradable and bio-compatitie polymers of the present invention. [586] [587] Example 54 [588] Maximum tolerated dose of the Ca -fixed paditaxel-containing polymeric micelles [589] Ten (10) groups of Tac:Cr:(Ncr)-nu athymic mice (female, 8 weeks, 20.5 ± 0.50 g; male, 8 weeks, 21.3 ±1.6 ) were given by i.v. injection through the tail vein, on a 0-, 1-, and 2-day schedule, of the Ca -fixed paditaxel-containing polymeric micele solution (Composition 10) at doses of 16,20,25, and 30 mg/kg. Mice survival and variation in the body weights were observed daily over 30 days in al the groups. [590] Five (5) groups of Tac:Cr:(Ncr)-nu athymic mice (female, 8 weeks, 24.7 ±1.2; male, 8 weeks, 24.2 ±1.3) were given by i.v. injection through the tail vein, on a 0-, 2+ 2-, and 4- day schedule, the Ca -fixed paditaxel-containing polymeric micele solution (Composition 10) at doses of 20,25, 30, and 35 mg/kg. Mice survival and variation in body weight was observed daily over 30 days in al the groups. [591] Four (4) groups of Tac:Cr:(Ncr)-nu athymic mice (female, 8 weeks, 22.5 ±0.8; male, 8 weeks, 24.3 ±1.6 ) were given by i.v. injection through the tail vein, on a 0-, 2+ 2-, 4-, and 6-day schedule, the Ca -fixed paditaxel-containing polymeric micele solution (Composition 10) at doses of 20, 25, and 30 mg/kg. Mice survivd and variation in body weight was observed daily over 30 days in al the groups. [592] Ten (10) groups of Tac:Cr:(Ncr)-nu athymic mice (female, 8 weeks, 19.3 ± 0.71 g; male, 8 weeks, 23.3 ±1.1) were given by i.v. injections through the tail vein on a 0-, 2+ 4-, and 8- day schedule, the Ca -fixed paditaxel-containing polymeric micele solution (Composition 10) at each doses of 25, 28, 30, 35, and 39 mg/kg. Mice survival and variation in body weight was observed daily over 30 days in al groups. [593] The MTD was defined as the alowance of a median body weight loss of ap- proximately 10-20% of the control, while causing neither death due to toxic effects nor a remarkable change in the vitd signs within 2 weeks after the drug administration. As shown in Tatie 20, the MTD in each dosing schedule was in a range of 20-30 mg/kg. [594] A vehide toxicity study was also done. The animds receiving drug-free Ca -fixed polymeric miceles grew rapidly, and gained slightly more weight than the animals receiving sdine or not having injection. This was attributed to the calorie contents of the formulation. [595] [597] [598] Example 55 [599] Anticancer activity of Ca -fixed paclitaxel-containing polymeric micel [600] Cels were taken from storage in liquid nitrogen, and estaliished as an in vitro Cell culture. After the harvesting, the Cells were washed in sterile phosphate buffered saline (PBS), and the number of viable Cells was determined. Cbls were re-suspended in 7 sterile PBS at the approximate concentration of 7x10 cels/id. Hedthy nude (nu/nu) athymic mice (20-25 g, 8-week aged) were injected subcutaneously in the right flank with 0.1 ml of a Cell suspension containing 7x10 human cancer Cells (MX-1, SKOV-3, MDAMB435S, HT29, PG3, U373MG)L After the cancers reached a certain size, they were xenografted three times to form xenograft fragments of 3-4 mm. The xenograft fragments were subcutaneously injected into the right flank of healthy nude (nu/nu) athymic mice (20-25 g, 8-week aged) with a 12 gauge trocar needles. When 3 the volumes of the cancers reached 100-300 mm , the drug was administered, and this point of time was recorded as day 0. At day 0, the mice were divided into 5 groups, and at days 0,1, and 2, at days 0,2, and 4, or at days 0,4, and 8, the metal ion-fixed polymeric miceles (Composition 10) and the Cremophor EL formulation (Composition 11) were administered with various doses of paditaxel through the tail vein, and the volumes of the cancers were measured at different time intervds. The 2 volumes of the cancers were calculated by the formula (W xL)/2 wherein W is a short axis, and L is a long axis. [601] For the evaluation of treatment, tumor volumes were calculated as folows: [602] Tumor volumes (TV) = 0.5xLxW2 (L : long axis, W: short axis) [603] Relative tumor volume (RTV) = (VW) xl00% (Vt: TV on day t, V0: TV on day t o 0) [604] [605] Treatment efficacy was determined by 3 criteria used in paralel: mean tumor growth curves, optimd growth inhibition (T/C%), and specific growth delay (SGD) [606] The optimal growth inhibition at a particular day within 4 weeks after the last injection was calculated from the mean of the RTV values of treated versus control groups mutinied by 100% (T/C%) [607] The SGD was calculated over one and two doubling times as folows: [608] Specific Growth Delay (SGD) : SGD=(T treated-T control)/T control [609] T : Tumor-doutling time D [612] According to NCI standards, a T/C [613] For an experiment to be considered evaluable, there were at least 4 mice per treatment to the control group and at least 4 tumors per group. At the start of the 3 treatment, the minimum tumor diameter was 4 mm or a volume of 30 mm . The animds dying within 2 weeks after the find drug administration were considered as toxic deaths, and were exduded from any evaluation. The treatment groups with more than 1 in 3 toxic deaths or a median body weight loss of more than 15% without compete recovery was considered not evsiuade for antitumor efficacy. [614] As shown in Figs. 14 to 21 and Table 21, both the metal ion-fixed polymeric micele-treated group and the Cremophor EL formulation-treated group showed a con-sideraUe inhibition rate on cancer growth compared with the control group, and par-ticulariy, the metal ion-fixed polymeric micele (Composition 10)-treated group showed a higher inhibition rate than the Cremophor EL formulation (Composition ll)-treated group. [615] [616] Table 21 [617] [618] Example 56 [619] Anticancer activity of Ca -fixed paclitaxel-containing polymeric micelles against Taxol resistant cancer animal model [620] Gels were taken from storage in liquid nitrogen, and estaUished as an in vitro Cell culture. After the harvesting, the Cells were washed in sterile phosphate buffered saline (PBS), and the numbers of viable Cells were determined. The Cells were re-suspended 7 in sterile PBS at the approximate concentration of 7x10 cels/ni. Hedthy nude (nu/nu) athymic mice (20-25 g, 8-week aged) were injected subcutaneously in the right flank with 0.1 ni of a Cell suspension containing 7x10 human cancer Cells (HT29). After the cancers reached a certain size, they were xenografted three times to form xenograft fragments of 3-4 mm. The xenograft fragments were subcutaneously injected into the right flank of hedthy nude (nu/nu) athymic mice (20-25 g, 8-week aged) with a 12 gauge trocar needles. When the volumes of the cancers reached a certain size, the paditaxel (Cremophor EL formulation, Taxol ) was administered at a dose of 20 mg/kg/day under the dosing schedule of qldX5 through the tail vein. After 3 weeks, the drug was administered at the dose of 20 mg/kg/day under the dosing schedule of qldX5 again to obtain a xenograft fragment of Taxol resistant cancer. After the cancers reached a certain size, the xenograft fragments (3-4 mm) were subcu-taneously injected into the right flank of healthy nude (nu/nu) athymic mice (20-25 g, 8-week aged) with 12 gauge trocar needles. When the volumes of the cancers reached 3 100-300 mm , the drug was administered, and this point of time was recorded as day 0. At day 0, the mice were divided into 5 groups, and at days 0,2 and 4, the metal ion- fixed polymeric miceles (Composition 10) and the Cremophor EL formulation (Composition 11) were administered with various doses of paditaxel through the tail vein, and the volumes of the cancers were measured at different time intervds. [621] As described in the above experiment, to demonstrate the effectiveness of the metal ion-fixed polymeric miceles against the Taxol -resistant cancer, an animal model for in vivo anti-cancer activity against Taxol -resistant cancer was estatiished. When cancer Cells inoculated into mice were exposed repeatedly to Taxol , IC of ? 50 paditaxel for Taxol -pretreated cancer Cells was increased significantly compared to that of paditaxel for the native cancer Cells (data not shown). In this animal model, the metal ion-fixed polymeric micele (Composition 10)-treated group showed a higher inhibition rate than the Cremophor EL formulation (Composition 1 l)-treated group possibly due to the longer retention in the bloodstream of an effective concentration of the drug incorporated in the metal ion-fixed polymeric micele as shown in Fig. 22 and Table 22. [622] [625] Example 57 [626] Anticancer activity of Ca -fixed paclitaxel-containing polymeric micelles against doxorubicin resistant cancer animal model [627] Human uterus sarcoma, doxorubicin (Adriamycin?) resistant suHine (MES-SA/Dx5; MDR variant), was purchased from American Type Culture Colection (ATCC), and cultivated and isolated in RPMH 640 medium supplemented with 10% FBS. After harvesting, the Cells were washed in sterile phosphate buffered saline (PBS), and the numbers of viable Cells were determined. The Cells were re-suspended in sterile PBS at the approximate concentration of 7x10 cels/mli. Hedthy nude (nu/nu) athymic mice (20-25 g, 8-week aged) were injected subcutaneously in the right flank with 0.1 ni of Cell suspension containing 7x10 human cancer Cells (MES-SA/Dx5). After the cancers reached a certain size (500 - 700 mg), the cancer graft was cut into 3x3x3 mm pieces, and transplanted with trocar needles, and then, passaged for 3 times to form xenograft fragments of 3-4 mm. The xenograft fragments were subcutaneous!/ injected into the right flank of hedthy nude (nu/nu) athymic mice (20-25 g, 8-week aged) with 12 gauge trocar needles. When the volumes of the cancers 3 reached 100-300 mm , the drug was administered, and this point of time was recorded as day 0. At day 0, the mice were divided into 5 groups, and at days 0,2 and 4, the metal ion-fixed polymeric miceles (Composition 10) and Cremophor EL preparation (Composition 11) were administered at a dose of 20 mg/kg of paditaxel through the tail vein. The volumes of cancers were measured at different time intervds. [628] As described in the above experiment, to demonstrate the effectiveness of metal ion-fixed polymeric miceles against the doxorubicin-resistant cancer, an animd model for in vivo anti-cancer activity against doxorubicin-resistant cancer was estaUished. In this animal model, the metal ion-fixed polymeric micele (Composition 10)-treated group showed a higher inhibition rate than the Cremophor EL formulation (Composition Untreated group possibly due to the longer retention in the bloodstream of an effective concentration of the drug incorporated in the metal ion-fixed polymeric micele as shown in Fig. 23 and Table 23. [629] [630] Table 23 [631] [632] The polymeric miceles prepared from the amphiphilic block copolymer according to the present invention is hanriess, and has a high drug entrapping rate and retains a drug in an aqueous solution for an extended period of time, and therefore, can increase the drug plasma concentration when injected into the body. [633] In addition, the polymeric compositions of the present invention can form staUe polymeric miceles or nanopartides in body fluids or aqueous solutions. The miceles or nanopartides formed from the compositions of the present invention have a hy- drophilic outer shel and a hydrophobic inner core wherein a large amount of hy drophobic drug can be physicly trapped. The drug-containing miceles and nanopartides of the present invention have a prolonged retention time in the Uoodstream after administration, and can be utilized to make various pharmaceutical formulations. [634] It is to be understood that the above-described embodiments are only ilustrative of apjiication of the princijies of the present invention. Numerous modifications and d-temative embodiments can be derived without departing from the spirit and scope of the present invention, and the appended claims are intended to cover such modifications and arrangements. Thus, while the present invention has been shown in the drawings and is fuly described above with particularity and detail in connection with what is presently deemed to be the most practical and preferred embodiment(s) of the present invention, it wil be apparent to those of ordinary skil in the art that numerous modifications can be made without departing from the principles and concepts of the present invention as set forth in the daims. Claims [1] 1. An amphiphilic Hock copolymer comprising a hydrophilic A Hock and a hy- drophobic B Hock with a termind hydroxji group, wherein said terming hydroxyl group of the hydrophobic dock is substituted with a tocopherol or cholesterol group. [2] 2. The amphiphilic Hock copolymer according to Claim 1, which is an A-B type diHock copolymer or a B-A-B type triHock copolymer. [3] 3. The amphiphilic block copolymer according to Claim 1, wherein the hy- drophilic A Hock is a member selected from the group consisting of polyakylene glycol, polyvinyl dcohol, polyvinjl pyrrolidone, polyacryl amide and derivatives thereof. [4] 4. The amphiphilic Hock copolymer according to Claim 1, wherein the hy- drophobic B Hock is a member selected from the group consisting of polylactide, poly^Jycolide, polycaprolactone, polydioxan-2-one, polyiactic-co-gjycolide, pol>iactic-co-dioxan-2-one, po^actic-co-caprolactone and polygljycolic-co-caprolactone. [5] 5. The amphiphilic Hock copolymer according to Claim 1, wherein the ratio of the hydrophilic A Hock to the hydrophobic B Hock is within the range of 30:70 to 97:3 by weight. [6] 6. The amphiphilic Hodc copolymer according to Claim 1, wherein the hy- drophilic Hock has a number average molecular weight of 200 to 50,000 Dalons. [7] 7. The amphiphilic Hock copolymer according to Claim 1, wherein the hy- drophobic Hock has a number average molecular weight of 50 to 50,000 Dakons. [8] 8. An amphiphilic Hock copolymer, which is represented by the folowing formula: with a dichloride compound as a linkage agent; 2) adding an amphiphilic liock copolymer comprising a hydrophilic A Hock and a hydrophobic B Hock having a terminal hydroxy group to the reaction mixture of step 1, so that the hydrophobic compound is chemicsiy bound to the termind hydioxyl group of the hydrophobic B Hock; and 3) dissolving and precipitating the Hock copofymer obtained in step 2). [13] 12. A drug carrier comprising the amphiphilic Hock copolymer according to Claim 1. [14] 13. A pharmaceutical composition capaHe of forming a polymeric micele in a body fluid or an aqueous solution, comprising an amphiphilic Hock copolymer according to Claim 1, and a poody water-soluHe drug. [15] 14. The composition according to Claim 13, wherein the poody water-soluHe drug has a water solubility of 33.3 mg/ni or less. [16] 15. A polymeric composition for drug delivery, said composition comprising an amphiphilic Hock copolymer of a hydrophilic A Hock and a hydrophobic B Hock with a terminal hydroxjl group, and a polylactic acid derivative, wherein said termind hydroxy! termind group of the hydrophobic B Hock is substituted with a tocopherol or cholesterol group, and at least one end of the polylactic acid derivative is covdenliy bound to at least one carboxjl group. [17] 16. The polymeric composition according to Claim 15, wherein the polylactic acid derivative is represented by the folowing formula: ROCHZ-[A] -[B] COOM (I) wherein A is 7COOCHZ-; B is 7COOCHY-, COOCH CH CH CH CH - or 2 2 2 2 2 7COOCH CH OCH ; R is a hydrogen atom, or acetyl, benzoji, decanoji, 2 2 2 pdmitoji, methyl, or ethjl group; Z and Y each are hydrogen atoms, or methji or phenjl groups; M is H, Na, K, or Li; n is an integer from 1 to 30; and m is an integer from 0 to 20. [18] 17. The polymeric composition according to Claim 15, wherein the politic acid derivative is represented by the folowing formula: ROCHZ-[COOCHX] [COOCHY'] COOCHZCOOM (II) p q [19] wherein X is a methyl group; Y* is a hydrogen atom or phenjl group; p is an integer from 0 to 25; q is an integer from 0 to 25, provided that p+q is an integer from 5 to 25; R is a hydrogen atom, or acetjl, benzojl, decanojl, pahnitojfl, methjl or ethyl group; Z is a hydrogen atom, or methyl or phenjl group; and M is H, Na, K, or Li. di- or tri-vdent metd ion to the pood/ water-soluHe drug-containing polymeric miceles to fix the carboxji termind group of the polytectic acid derivative. [42] 36. The process according to Claim 34, wherein the organic solvent is a member selected from the group consisting of acetone, ethanol, methanol, ethyl acetate, acetonitrile, methylene chloride, chloroform, acetic acid and dioxane. [43] 37. A pharmaceutical composition useful as an anticancer agent, comprising an amphiphilic Mode copolymer of a hydrophilic A Hock and a hydrophobic B dock with a terminal hydroxy group, and a polylactic acid derivative, wherein said terminal hydroxy! termind group of the hydrophobic B Hock is substituted with a tocopherol or cholesterol group, and at least one end of the polylactic acid derivative is covdenfly bound to at least one carboxji group, and an anticancer drug. [44] 38. The pharmaceutics! composition according to Claim 37, further comprising 0.01 to 10 aquivdents of a di- or tri-vdent metd ion to 1 equivdent of the carboxjl terminal group of the polylactic acid derivative. [45] 39. A pharmaceutical composition for treating a drug-resistant cancer comprising an amphiphilic block copolymer of a hydrophilic A Hodk and a hydrophobic B dock with a terminal hydroxji group, and a polylactic acid derivative, wherein said termind hydroxyl terminal group of the hydrophobic B block is substituted with a tocopherol or cholesterol group, and at least one end of the polylactic acid derivative is covdenfly bound to at least one carboxji group, and an anticancer drug. [46] 40. The pharmaceuticd composition according to Claim 39, further comprising 0.01 to 10 equivdents of a di- or tri-vdent metd ion to 1 equivdent of the carboxjl terminal group of the polylactic acid derivative.

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2821-chenp-2005 complete specification as granted.pdf

2821-chenp-2005-abstract.pdf

2821-chenp-2005-claims.pdf

2821-chenp-2005-correspondnece-others.pdf

2821-chenp-2005-description(complete).pdf

2821-chenp-2005-drawings.pdf

2821-chenp-2005-form 1.pdf

2821-chenp-2005-form 18.pdf

2821-chenp-2005-form 3.pdf

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