WO2005040247A1

WO2005040247A1 - Polymeric composition for drug delivery

Info

Publication number: WO2005040247A1
Application number: PCT/KR2003/002259
Authority: WO
Inventors: Sa-Won Lee; Min-Hyo Seo; Myung-Han Hyun; Dong-Hoon Chang; Jeong-Il Yu; Jeong-Kyung Kim; Hye-Jeong Yoon
Original assignee: Samyang Corporation
Priority date: 2003-10-24
Filing date: 2003-10-24
Publication date: 2005-05-06
Also published as: AU2003272127A1

Abstract

Polymeric compositions capable of forming stable micelles in an aqueous solution, comprising an amphiphilic block copolymer of a hydrophilic block and a hydrophobic block wherein the hydroxyl terminal group is substituted with a tocopherol or cholesterol group, and a polylactic acid derivative wherein one end of the polylactic acid is covalently bound to at least one carboxyl group, and wherein the carboxyl group of the polylactic acid derivative may be fixed with a di- or tri-valent metal ion by adding the di- or tri-valent metal ion to the polymeric composition.

Description

POLYMERIC COMPOSITION FOR DRUG DELIVERY

TECHNICAL FIELD This invention relates to a polymeric composition comprising an amphiphilic block copolymer comprised of a hydrophilic block and a hydrophobic block, and a polylactic acid derivative, wherein the hydroxyl terminal group of the hydrophobic block in the amphiphilic block copolymer is substituted with a tocopherol or cholesterol group which has excellent hydrophobicity, and one end of the polylactic acid is covalently bound to at least one carboxyl group and the carboxyl terminal group of the polylactic acid derivative is fixed with a di- or tri-valent metal ion.

BACKGROUND ART When a drug is administered into the body, only a small amount of the drug may reach its target site and most fractions of the administered dose can be distributed in non- targeted sites to cause undesirable side effects. Therefore, in the last two decades, researches have focused on the development of systems efficient for site specific delivery of drugs by the use of appropriate carriers, which include liposomes, small molecular surfactant micelles, polymeric nanoparticles, and polymeric micelles (polymeric nanoparticles made of hardened micelles). The use of liposomes as drug carrier is 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 micelles are easily dissociated when they are diluted with body fluids after administered into the body, and so it is difficult for them to perform a sufficient role as drug carrier.

Recently, polymeric nanoparticles and polymeric micelles using biodegradable polymers have been reported to be extremely useful technologies for overcoming these problems. They change the in vivo distribution of an intravenously administered drug thereby reducing its side effects and improving its efficacy to offer such advantages as specific cell targeting and release control of the drug. They also have good compatibility with body fluids and improve the solubility and bioavailability of poorly water-soluble drugs. Nanometer size drug carriers with hydrophilic surfaces are found to evade recognition and uptake by the reticule-endothelial systems (RES), and thus to circulate in the blood for a long period of time. Another advantage of these hydrophilic nanoparticles is that, due to their extremely small size, the particles extravagate at the pathological sites such as solid tumors through passive targeting mechanism. However, successful drug delivery to the specific target site requires stable retention of the drug by a carrier while in circulation. Since drag 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 rapidly cleared carriers. 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-50 nm. The polymeric micelles have two spherical co-centric regions, a densely packed core of hydrophobic material which is responsible for entrapping the hydrophobic drug, and an outer shell made of hydrophilic material for the evasion of body's RES which permits circulation in the blood for a longer period of time. In spite of their distinct advantages such as small size, high solubility, simple sterilization, and controlled release of drugs, the physical stability of these carriers is a critical issue since the rapid release of the incorporated drug may occur in vivo. The stability of block copolymer micelles includes two different concepts, thermodynamic stability and kinetic stability. Micelles are thermodynamically stable compared to disassembling to single chains in pure water if the total copolymer concentration is above the critical micelle concentration (CMC). Thus, the use of a copolymer system with a low CMC value may increase the in vivo stability of the micelles. The kinetic stability means the rate of disassembly of a micelle. The rate of disassembly depends upon the physical state of the micelle core. Micelles formed from the copolymers containing a hydrophobic block which has a high glass transition temperature will tend to disassemble more slowly than those with a low glass transition temperature. It is also likely to be affected by many of the same factors that affect the rate of unimer exchange between micelles. 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 hydrophilic and hydrophobic blocks. In addition, the evidence also shows that the incorporation of hydrophobic compounds into block copolymer micelles may enhance the micelle stability.

A method for preparing block copolymer micelles by physically entrapping a drag in the block copolymer which is composed of a hydrophilic component and a hydrophobic component was disclosed in EP 0 583,955A2, and JP 206,815/94. The block copolymer employed therein is an A-B type di-block copolymer comprising a polyethylene oxide as the hydrophilic A component and a polyamino acid or derivatives thereof having a hydrophobic functional group as the hydrophobic B component. Polymeric micelles comprising the above block copolymer can physically incorporate a drug, e.g. adriamycin, indomethacin, etc., into the inner core of the polymeric micelles, which can then be used as a drag delivery carrier. However, these polymeric micelles are comprised of block copolymers that cannot readily be degraded in vivo. In addition, the block copolymers have low biocompatibility, which can cause undesirable side effects when administered in vivo.

Great efforts have been devoted to the development of a biodegradable and biocompatible core-shell type drag carrier with improved stability and efficacy, which will entrap a poorly water-soluble drug. A preparation method of chemically fixed polymeric micelles, wherein the polymer is a core-shell type polymer comprising a hydrophilic polyethylene oxide as the shell and a hydrophobic biodegradable polymer that is cross- linked in an aqueous solution as the core, was disclosed in EP 0,552,802A2. However, these polymeric micelles are difficult to prepare because a cross linker must be introduced into the hydrophobic component of A-B type di-block or A-B-A type tri-block copolymer so that the core-forming polymer has a stable structure. A_lso, using a cross linker that has never been applied to the human body causes safety concerns. A micelle forming block copolymer-drag complex was disclosed in US Patent No.

6,080,396. The high molecular block copolymer-drug complex in which the high molecular block copolymer having a hydrophilic polymer segment and a hydrophobic polymer segment forms a micelle having the hydrophilic segment as its outer shell and contains an anthracycline anticancer agent in its hydrophobic inner core. The molecules of the anticancer agent were covalently linked within the micellar core. However, when the drug is covalently linked within the polymeric micelles, it is difficult to control the cleavage rate of the drag linkage.

On the other hand, a report shows that the solubilization of a hydrophobic drag can be achieved by a polymeric micelle composed of a di- or tri-block copolymer comprising a hydrophilic polymer of polyalkylene glycol derivatives and a hydrophobic biodegradable polymer such as fatty acid polyesters or polyamino acids. US Patent No.

5,449,513 discloses 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. indomethacin.

However, the polyamino acid derivative cannot be hydrolyzed in vivo, and thus causes side effects due to immune responses. US Patent No. 5,429,826 discloses a di- or multi-block copolymer comprising a hydrophilic polyalkylene glycol and a hydrophobic polylactic acid. Specifically, this patent describes a method of stabilizing polymeric micelles by micellizing a di- or multi- block copolymer wherein an acrylic acid derivative is bonded to a terminal group of the di- or multi-block copolymer, and then, in an aqueous solution, the polymer is crosslihked in order to form the micelles. The above method could accomplish stabilization of the polymeric micelle, but the crosslinked polymer is not degraded, and thus, cannot be applied in vivo. The above polymeric micelles can solubilize a large amount of poorly water-soluble drag in an aqueous solution with a neutral pH, but have a drawback to release the drug within a short period of time.

Clinical 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 initial treatment, but on recurrence, exhibit an entirely different phenotype. The resistance can be formed to both previously used drugs and new drags with different structures and mechanisms of action. For example, cancer chemotherapy with Taxol^® often fails due to the acquired resistance of cancer cells, which is frequently associated with the overexpression of P-gp and alteration of β-tubulin. Taxol^® resistant cells exhibit cross-resistance to other drags including actinomycin D, doxorabicin, vinblastine, and vincristine. Therefore, the clinical drag resistance is a major barrier to be overcome in order that chemotherapy can be curative for most patients with metastatic cancer.

Drug-resistant cancer cells show higher IC5₀ (50% cell inhibition concentration of drag) than normal ones, and so the chemotherapy using drags is effective with higher concentration for the tumor cells while reduced concentration for the normal cells.

Therefore, longer systemic circulation and specific localization of drags in the tumor tissues are required for guaranteeing the effectiveness against the drug-resistant cancer. In view of the foregoing, the development of an improved polymeric micelle composition for hydrophobic drug delivery that is biocompatible and biodegradable has been appreciated and desired. The present invention provides such an improved polymeric micelle composition which is biocompatible and biodegradable, and can effectively deliver a hydrophobic drag without a decrease in its stability. DISCLOSURE OF THE INVENTION

The present invention relates to a polymeric composition comprising an amphiphilic block copolymer comprised of a hydrophilic block and a hydrophobic block, and a polylactic acid derivative, wherein the hydroxyl terminal group of the hydrophobic block in the amphiphilic block copolymer is substituted with a hydrophobic tocopherol or cholesterol group which has excellent hydrophobicity, and one end of the polylactic acid is covalently bound to at least one carboxyl group and the carboxyl terminal group of the polylactic acid derivative is fixed with a di- or tri-valent metal ion. The compositions of the present invention can form stable polymeric micelles or nanoparticles in body fluids or aqueous solutions. The micelles or nanoparticles formed from the compositions of the present invention have a hydrophilic outer shell and a hydrophobic inner core wherein a large amount of hydrophobic drug can be physically trapped. The drug containing micelles and nanoparticles of the present invention have a prolonged retention time in the bloodstream after administration, and can be utilized to make various pharmaceutical formulations. Additional features and advantages of the invention will be apparent from the detailed description that follows, taken in conjunction with the accompanying drawings, which together illustrate, by way of example, the features of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a schematic diagram of a polymeric micelle formed by monomethoxypolyethylene glycol-polylactide (mPEG-PLA.) in an aqueous environment. Fig. 2 is a schematic diagram of a polymeric micelle formed by sodium carboxylate derivatized D,L-polylactic acid in an aqueous environment. Fig. 3 is a schematic diagram of a polymeric micelle formed by a mixture of monomethoxypolyethylene glycol-polylactide (mPEG-PLA) and sodium carboxylate derivatized D,L-polylactic acid in an aqueous environment. Fig. 4 is a schematic diagram of a Ca²⁺- fixed polymeric micelle of Fig. 3. Fig. 5 is a schematic diagram of a Ca²⁺-fιxed polymeric micelle containing a hydrophobic drag trapped within the hydrophobic core of the micelle. Fig. 6 shows a profile of plasma drag concentration of the paclitaxel-containing polymeric micelles fabricated with various di-block copolymers at various time intervals after administration. Fig. 7 shows the plasma drug concentration of the paclitaxel-containing Ca²⁺- fixed polymeric micelles fabricated with mPEG-PLA-tocopherol and mPEG-PLA-OH at various time intervals after administration. Fig. 8 shows a profile of plasma drug concentration of the paclitaxel-containing Ca²⁺-fixed polymeric micelles, Cremophor EL (Taxol^®), and Tween 80 preparations at various time intervals after administration. Fig. 9 shows the plasma drug concentration of the paclitaxel-containing Ca²⁺- fixed polymeric micelles and Cremophor EL (Taxol^®) at various time intervals after administration. Fig. 10 shows the plasma drag concentration of the docetaxel-containing Ca²⁺- fixed polymeric micelles and Tween 80 preparations (Taxotere") at various time intervals after administration. Fig. 11 shows the plasma drag concentration of the docetaxel-containing Ca²⁺- fixed polymeric micelles and Tween 80 preparations (Taxotere^®) at various time intervals after administration. Fig. 12A shows the anticancer effects of the drug containing Ca²⁺- fixed polymeric micelles in mice using the human breast carcinoma cell line MX-1. Fig. 12B shows the anticancer effects of the drug containing Ca²⁺- fixed polymeric micelles in mice using the human breast carcinoma cell line MDAMB435S. Fig. 12C shows the anticancer effects of the drug containing Ca²⁺-fixed polymeric micelles in mice using the human ovarian carcinoma cell line SKON-3. Fig. 12D shows the anticancer effects of the drug containing Ca²⁺- fixed polymeric micelles in mice using the human ovarian carcinoma cell line SKON-3. Fig. 12E shows the anticancer effects of the drug containing Ca²⁺-fixed polymeric micelles in mice using the human colon carcinoma cell line HT-29. Fig. 12F shows the anticancer effects of the drug containing Ca²⁺-fixed polymeric micelles in mice using the human colon carcinoma cell line HT-29. Fig. 12G shows the anticancer effects of the drug containing Ca²⁺-fixed polymeric micelles in mice using the human prostatic carcinoma cell line PC3. Fig. 12H shows the anticancer effects of the drag containing Ca²⁺-fixed polymeric micelles in mice using the human brain carcinoma cell line U-373MG. Fig. 13 shows the anticancer effects of the drag containing Ca²⁺-fixed polymeric micelles in the animal model with paclitaxel (Taxol ) resistant human cancer.

BEST MODE FOR CARRYING OUT THE INVENTION

Before the present polymeric compositions and methods of using and making thereof are disclosed and described, it should be understood that this invention is not limited to the particular configurations, process steps, and materials disclosed herein, and such configurations, process steps, and materials may be varied. It should be also understood that the terminology employed herein is used for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention which will be limited only by the appended claims and equivalents thereof.

It should be noted that, in this specification and the appended claims, the singular form, "a," "an," or "the", includes plural referents unless the context clearly dictates otherwise. Thus, for example, the reference to a polymer containing "a terminal group" includes the reference to two or more such groups, and the reference to "a hydrophobic drag" includes the reference to two or more such drugs. In describing and claiming the present invention, the following terminology will be used in accordance with the definitions set out below.

As used herein, the term "bioactive agent" or "drag" or any other similar term means any chemical or biological material or compound that is suitable 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 phannacological effect. Such effect may include but is not limited to (1) having a prophylactic effect on the organism and preventing an undesired biological effect such as preventing an infection, (2) alleviating conditions caused by diseases, for example, alleviating pain or inflammation caused as a result of diseases, and/or (3) either alleviating, reducing, or completely eliminating a disease from the organism. The effect may be local, such as providing a local anesthetic effect, or may be systemic.

As used herein, the term "biodegradable" or "biodegradation" is defined as the conversion of materials into less complex intermediates or end products by solubihzation hydrolysis, or by the action of biologically formed entities which can be enzymes or other products of the organism.

As used herein, the term "biocompatible" means materials or the intermediates or end products of materials formed by solubihzation hydrolysis, or by the action of biologically formed entities which can be enzymes or other products of the organism and which cause no adverse effect on the body.

"Poly(lactide)" or "PLA" shall mean a polymer derived from the condensation of lactic acid or formed by the ring opening polymerization of lactide. The terms "lactide" and "lactate" are used interchangeably.

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 be in any medical treatment.

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 systemically. Preferably, the compositions of the present invention are administered by the subcutaneous, intramuscular, transdermal, oral, transmucosal, intravenous, or intraperitoneal routes. Injectables for such use can be prepared in the conventional forms, either as liquid solution or suspension, or in a solid form that is suitable for the preparation as solution or suspension in liquid prior to the injection, or as emulsion. Suitable excipients that can be used for administration include, for example, water, saline, dextrose, glycerol, ethanol, and the like; and if desired, a minor amount 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. 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 illustrated herein, and additional applications of the principles of the present invention as illustrated herein, for one skilled in the relevant art, in connection with this disclosure, should be considered within the scope of the present invention.

The present invention provides a polymeric composition comprising an amphiphilic block copolymer comprised of a hydrophilic block and a hydrophobic block in which the hydroxyl terminal group is substituted with a hydrophobic tocopherol or cholesterol group which has excellent hydrophobicity, and a polylactic acid derivative having the carboxyl terminal group that is bound with a di- or tri-valent metal ion, wherein said composition forms stable polymeric micelles or nanoparticles in an aqueous environment. The present invention also provides a pharmaceutical composition comprising polymeric micelles or nanoparticles formed by the above composition and a hydrophobic drug entrapped therein, wherein said pharmaceutical composition has a prolonged retention time for effective concentration of the drug in the bloodstream after administration. The present invention further provides a pharmaceutical composition having anticancer activity against the anti-cancer drug resistant cancer cell.

The present invention further provides a process for preparing the above pharmaceutical composition.

The amphiphilic block copolymer of the present invention is preferably an A-B type diblock copolymer comprising a hydrophilic A block and a hydrophobic B block. The amphiphilic block copolymer, when placed in an aqueous phase, forms a core-shell type of polymeric micelles wherein the hydrophobic B block forms the core and the hydrophilic A block forms the shell. Preferably, the hydrophilic A block is one selected from the group consisting of polyalkylene glycol, polyvinyl alcohol, polyvinyl pyrrolidone, polyacryl amide, and derivatives thereof. More preferably, the hydrophilic A block is one selected from the group consisting of monomethoxypolyethylene glycol, monoacetoxypolyethylene glycol, polyethylene glycol, polyethylene-co-propylene glycol, and polyvinyl pyrrolidone. Preferably, the hydrophilic A block has a number average molecular weight of 500 to 50,000 Daltons. More preferably, the hydrophilic A block has a number average molecular weight of 1,000 to 20,000 Daltons.

The hydrophobic B block of the amphiphilic block copolymer of the present invention is a highly biocompatible and biodegradable polymer selected from the group consisting of polyesters, polyanhydrides, polyamino acids, polyorthoesters, and polyphosphazine. More preferably, the hydrophobic B block is one selected from the group consisting of polylactides, polyglycolides, polycaprolactone, polydioxan-2-one, polylactic-co-glycolide, polylactic-co-dioxan-2-one, polylactic-co-caprolactone, and polyglycolic-co-caprolactone. The hydrophobic B block is substituted with a hydrophobic tocopherol or cholesterol group which has excellent hydrophobicity at the hydroxyl terminus to increase the hydrophobicity of the hydrophobic B block.

The block copolymer having the hydrophobic block whose hydroxyl terminal group is substituted with tocopherol or cholesterol can be prepared according to methods taught by Korean Application No. 10-2003-0070667, the whole contents of which are incorporated hereinto by reference. In one embodiment, a suitable linker, e.g. a dicarboxylic 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 chemically bound to the hydroxyl terminal group of the hydrophobic B block. Thus, the amphiphilic block copolymer of the present invention may be represented by the following Formula:

Ri.-O-[R₃.]i-[R4']m-[R5']n-C(=OHCH₂) -C(=0)-0-R₂. (F) wherein R_r is CH₃-, or R₂-O-C(=O)-(CH₂)_χ.-C(=O)-[R₅.]_n-[R₄.]m-; R₂- is tocopherol or cholesterol; -CH-CHz- I H₂C C-0 R₃- is -CH₂CH₂-O-, -CH(OH)-CH₂-, -CH(C(=0)-NH₂)-CH₂-, or H₂C — CH₂ . r is -C(=O)-CHZ'-O-, wherein Z' is a hydrogen atom or methyl; R₅. is -C(=O)-CHY"-O-, wherein Y" is a hydrogen atom or methyl, -C(=O)-

CH₂CH₂CH₂CH₂CH₂-O-, or -C(=O)-CH₂OCH₂CH₂-O-; 1' is an integer of 4-1150; m' is an integer of 1-300; n' is an integer of 0-300; and X' is an integer of 0-4.

Preferably, the hydrophobic B block of the amphiphilic block copolymer has a number average molecular weight of 500 to 50,000 Daltons. More preferably, the hydrophobic B block of the amphiphilic block copolymer has a number average molecular weight 1 ,000 to 20,000 Daltons.

The ratio of the hydrophilic A block to the hydrophobic B block of the amphiphilic block copolymer of the present invention is preferably within the range of 3:7 to 8:2, and more preferably within the range of 4:6 to 7:3. If the content of the hydrophilic A block is too low, the polymer may not form polymeric micelles in an aqueous solution, and if the content is too high, the polymeric micelles formed therefrom are not stable. One end of the polylactic acid derivative of the present invention is covalently bound to at least one carboxylic acid or carboxylate salt. The other end of the polylactic acid derivative of the present invention may be covalently bound to a functional group selected from the group consisting of hydroxyl, acetoxy, benzoyloxy, decanoyloxy, and palmitoyloxy. The carboxylic acid or carboxylate salt functions as a hydrophilic group in an aqueous solution of pH 4 or more, and enables the polylactic acid derivative to form polymeric micelles 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 micelles. Therefore, the number average molecular weight of the polylactic acid derivative of the present invention is preferably within the range of 500 to 2,500 Daltons. The molecular weight of the polylactic acid derivative can be adjusted by controlling the reaction temperature, time, and the like, during the preparation process.

The polylactic acid derivative is preferably represented by the following formula: RO-CHZ-[A]_n-[B]_m-COOM (I) wherein A is -COO-CHZ-; B is -COO-CHY-, -COO-CH₂CH₂CH₂CH₂CH₂- or - COO-CH₂CH₂OCH₂; R is a hydrogen atom, or acetyl, benzoyl, decanoyl, palmitoyl, methyl, or ethyl group; Z and Y each are a hydrogen atom, or methyl, or phenyl 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.

One end of the polylactic acid derivative of the present invention is covalently bound to a carboxyl group or an alkali metal salt thereof, preferably, an alkali metal salt thereof. The metal ion in the alkali metal salt which forms the polylactic acid derivative is monovalent, e.g. sodium, potassium, or lithium. The polylactic acid derivative in the metal ion salt form is solid at room temperature, and is very stable because of its relatively neutral pH. More preferably, the polylactic acid derivative is represented by the following formula:

RO-CHZ-[COO-CHX]_p-[COO-CHY']_q-COO-CHZ-COOM (II) wherein X is a methyl group; Y' is a hydrogen atom or phenyl 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).

In addition, polylactic acid derivatives of the following formulas (III) to (N) are also suitable for the present invention:

RO-PAD-COO-W-M' (III)

C00M -C CH₂CO0M cooM wherein W-M' is CH₂C00M _or CH — CH₂CO0M _; the PAD is one selected from the group consisting of D,L-polylactic acid, D-polylactic acid, polymandelic acid, a copolymer of D,L-lactic acid and glycolic 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). S-O-PAD-COO-Q (IN)

wherein S is

j_ is -ΝRi- or -O-; Ri is a hydrogen atom or Ci- _loalkyl; Q is CH₃, CH₂CH₃, CH₂CH₂CH₃, CH₂CH₂CH₂CH₃, or CH₂C₆H₅; a is an integer from 0 to 4; b is an integer from 1 to 10; M is as defined in Formula (I); and PAD is as defined in formula (III).

wherein R' is -PAD-O-C(0)-CH₂CH₂-C(O)-OM and M is as defined in formula

(I); PAD is as defined in formula (III); and a is an integer from 1 to 4, for example, if a=l, 3-arm PLA-COOΝa; if a=2, 4-arm PLA-COOΝa; if a=3, 5-arm PLA-COOΝa; and if a=4, 6-arm PLA-COOΝa. The initiator for synthesis of the polymers (formula N) includes glycerol, erythritol, threltol, pentaerytritol, xylitol, adonitol, sorbitol, and mannitol.

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 total 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 preferably, 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.

The polylactic acid derivatives of the present invention alone can form micelles in an aqueous solution of pH 4 or more, but the polymeric compositions can form micelles 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, preferably at a pH within the range of 4 to 8. The particle size of the micelles or nanoparticles prepared from the polymeric compositions of the present invention may be adjusted to be within the range of 1 to 400 nm, and preferably 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.

As illustrated in Fig.l to Fig.3, the polylactic acid deri-vatives or the amphiphilic block copolymers alone and mixtures thereof may form micelles in an aqueous solution. In the Figures, 1 represents poorly water-soluble drugs; 10 represents monomethoxypolyethylene glycol-polylactide tocopherol (mPEG-PL A- tocopherol); 11 represents monomethoxypolyethylene glycol (mPEG); 12 represents polylactide tocopherol (PLA-tocopherol); 20 represents the sodium salt of D,L-poly(lactic acid); 21 represents D,L-polylactic acid; and 22 represents sodium carboxylate. However, the polymeric compositions of the present invention remarkably improve the drug loading efficiency and stability of the micelles formed in an aqueous solution compared with the micelles formed from the polylactic acid derivatives or the amphiphilic block copolymers alone.

In one embodiment of the present invention, the carboxyl terminal group of the polylactic acid derivative is bound or fixed with a di- or tri-valent metal ion. The metal ion-fixed polymeric composition can be prepared by adding the di- or tri-valent metal ion to the polymeric composition of the amphiphilic block copolymer and the polylactic acid derivative. The polymeric micelles or nanoparticles may be formed by changing the amount of the di- or tri-valent metal ion added for binding or fixing the carboxyl terminal group of the polylactic acid derivative.

The di- or tri-valent metal ion is preferably one selected from the group consisting of Ca²⁺, Mg²⁺, Ba²⁺, Cr³⁺, Fe³⁺, Mn²⁺, Ni²⁺, Cu²⁺, Zn²⁺, and Al³⁺. The di- or tri-valent metal ion may be added to the polymeric composition of the ar phiphilic block copolymer and the polylactic acid derivative in the form of sulfate, chloride, carbonate, phosphate or hydroxylate, and preferably, in the form of CaCl₂, MgCl₂, ZnCl₂, A1C1₃, FeCl₃, CaCO₃, MgCO₃, Ca₃(PO₄)₂, Mg₃(PO₄)₂, AlPO₄, MgSO₄, Ca(OH)₂, Mg(OH)₂, Al(OH)₃, or Zn(OH)₂.

As illustrated in Figs. 4 and 5, when a monovalent metal ion at the carboxyl terminus of the polylactic acid derivative is substituted with a di- or tri-valent metal ion to form a metal ionic bond, the micelles or nanoparticles formed therefrom have improved stability.

Polymeric micelles or nanoparticles can be prepared by changing the equivalents of the metal ion added. Specifically, if a di-valent metal ion is added at 0.5 equivalents or less with respect to the carboxyl terminal groups of the polylactic acid derivative, the metal ion that can form bonds with the carboxyl terminal group is insufficient, and thus polymeric micelles are formed. If a di-valent metal ion is added at 0.5 equivalents or more, the metal ion that can form bonds with the carboxyl terminal group of the polylactic acid derivative is sufficient to firmly fix the micelles, and thus nanoparticles are formed.

In addition, the drag release rate from the polymeric micelles or nanoparticles may be adjusted by changing the amount of equivalents of the metal ion added. If the metal ion is present at 1 equivalent or less with respect to the carboxyl group of the polylactic acid derivative, the number available for bonding to the carboxyl terminal group of the polylactic acid derivative is decreased, and so the drag release rate is increased. If the metal ion is present at 1 equivalent or more, the number available for bonding to the carboxyl terminal group of the polylactic 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 equivalent amount, and to decrease the drag release rate, the metal ion is used in a large equivalent amount.

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 polylactic acid derivative, and 0.01 to 10 equivalents of the di- or tri-valent metal ion with respect to the equivalents of the carboxyl terminal groups of the polylactic acid derivatives. Preferably, they contain 20 to 80wt% of the amphiphilic block copolymer, 20 to 80wt% of the polylactic acid derivative, and 0.1 to 5 equivalents of the di- or tri-valent metal ion. More preferably, they contain 20 to 60wt% of the amphiphilic block copolymer, 40 to 80wt% of the polylactic acid derivative, and 0.2 to 2 equivalents of the di- or tri-valent metal ion.

The present invention also relates to a pharmaceutical composition containing polymeric micelles or nanoparticles formed from the polymeric compositions of the present invention and a poorly water-soluble drag entrapped therein. The pharmaceutical compositions of the present invention provide increased plasma concentrations of hydrophobic drugs and can be used in various pharmaceutical formulations.

As shown in Figs. 3 to 5, a poorly water-soluble drag is mixed with a polymeric composition of an amphiphilic block copolymer and a polylactic acid derivative to form polymeric micelles containing the drug therein. In order to improve its stability, a di- or tri-valent 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 drag-containing polymeric micelles and nanoparticles.

The term "poorly water-soluble drags" or "hydrophobic drags" refers to any drag or bioactive agent which has the water solubility of 100 μg/ml or less. This includes anticancer agents, antibiotics, anti-inflammatory agents, anesthetics, hormones, antihypertensive agents, and agents for the treatment of diabetes, antihyperlipidemic agents, antiviral agents, agents for the treatment of Parkinson's disease, antidementia agents, antiemetics, immunosuppressants, antiulcerative agents, laxatives, and antimalarial agents. The examples of hydrophobic drugs include anticancer agents such as paclitaxel, camptothecin, etoposide, doxorabicin, daunorubicin, epirubicin, idarubicin, ara-C, etc.; and immunosuppressants such as cyclosporine A, etc. Steroidal hormones such as testosterone, estradiol, estrogen, progesterone, triamcinolon acetate, dexamethasone, etc. and anti-inflammatory agents such as tenoxicam, pyroxicam, indomethacin, COX-II inhibitors, etc., which have a very fast excretion rate from the blood, are also the examples of suitable hydrophobic drags that can be used for the present invention.

The content of the poorly water-soluble drug is preferably within the range of 0.1 to 30wt% based on the total weight of the pharmaceutical compositions comprising an amphiphilic block copolymer, a polylactic acid derivative, and a hydrophobic drag. The size of the drug-containing polymeric micelles or nanoparticles may be adjusted from 5 to 400 nm, preferably, from 1O 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 oral or parenteral administration of the poorly water-soluble drug, the drag is entrapped in the polymeric micelles or nanoparticles, and is thereby solubilized. Particularly, the metal ion-fixed polymeric micelles or nanoparticles are retained in the bloodstream for a long period of time, and accumulated in the target lesions. The drag is released from the hydrophobic core of the micelles to exhibit a pharmacological effect while the micelles are degraded.

For parenteral delivery, the drag may be administered intravenously, intramuscularly, intraperitoneally, transnasally, intrarectally, intraocularly, or intrapulmonarily. For oral delivery, the drug is mixed with the polymeric micelles of the present invention, and then administered in the form of tablet, capsule, or aqueous solution.

The particles of the metal ion-fixed polymeric micelles or nanoparticles have an average size of 20-40 nm, as shown in Table 5. The micelles of this size range are suitable for injection formulation and sterile filtration.

The metal ion-fixed polymeric micelles or nanoparticles according to the present invention have excellent stability in aqueous solution. As shown in Table 7, the metal ion- fixed paclitaxel-containing polymeric micelle composition (Composition 1) was more kinetically stable than the metal ion-nontreated composition (Composition 2). The addition of metal ion significantly increased the retention time of drag in the polymeric micelles of the present invention. This is due to the crosslinking electrostatic interaction of carboxylate anion of the polylactic acid derivative which might induce the increase of rigidity of the hydrophobic core. Moreover, the metal ion-fixed polymeric micelles (Composition 1) of the amphiphilic diblock copolymers with hydrophobic moiety (tocopherol succinic acid) substituted for the hydroxyl terminal group of hydrophobic B block had kinetically greater stability than original mPEG-PLA-OH (Composition 4). This result suggests that the increase of hydrophobicity of hydrophobic B block in the amphiphilic polymer results in forming more stable micelles due to stronger interaction between the hydrophobic moiety of the amphiphilic polymer and drug.

The blood concentration of drag in polymeric micelles depends on hydrophobic moiety substituted for the hydroxyl terminal group of hydrophobic B block of the amphiphilic diblock copolymers. As shown in Table 9 and Fig. 6, the polymeric micelles (Composition 5-7) of the amphiphilic diblock copolymers with hydrophobic moiety (tocopherol succinic acid, cholesterol succinic acid, or palmitic acid) substituted for the hydroxyl terminal group of hydrophobic B block had a much longer bloodstream retention time than original mPEG-PLA-OH polymeric micelles (Composition 8). Moreover, mPEG-PLA-tocopherol micelles (Composition 5) were longest circulating in the blood among the polymeric micelles. The second longest circulating composition was mPEG- PLA-cholesterol micelles (Composition 6). This result can be explained by the increased hydrophobicity of tocopherol and cholesterol moiety in the hydrophobic B block compared with palmitic acid moiety.

The metal ion-fixed polymeric micelles (Composition 9) of the amphiphilic diblock copolymers with hydrophobic moiety (tocopherol succinic acid) substituted for the hydroxyl terminal group of hydrophobic B block has a much longer bloodstream retention time than the metal ion- fixed polymeric micelles (Compositions 10) of the original amphiphilic diblock copolymer as shown in Table 11 and Fig. 7. This result also suggests, as demonstrated in Example 10, that the increase of hydrophobicity of hydrophobic B block in the amphiphilic polymer results in forming more stable micelles due to stronger interaction between the hydrophobic moiety of the amphiphilic polymer and drug. As shown in Figs. 8-11, a composition, wherein drag 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 marketed formulations. The polymeric micelle drug composition obtained has greatly improved pharmaceutical efficacy. As shown in Figs 12 A-H, paclitaxel containing Ca²⁺- fixed polymeric micelles has a high inhibition rate on cancer growth, and so exhibits high anticancer activity. Taxol^® (or paclitaxel) as an anti-tubulin drag was widely used in chemo therapeutic treatment of cancer. Differently from other anti-tubulin drugs such as vinblastine and chochicine, Taxol^® can promote the polymerization and stability of microtubules instead of inducing depolymerization; therefore inhibits cell replication by disrupting normal mitotic spindle formation. Though Taxol^® is an effective and useful anti-tumor agent in the clinical chemotherapy, the development of Taxol^®-resistance in the cancer cells always renders the drag to become ineffective. Various mechanisms of Taxol^®-resistance including the overexpression of P-glycoprotein (P-gp) and modification of β-tubulin have been characterized. Among them, the overexpression of P-gp has been a predominant mechanism to explain the multi-drug resistant phenomena, including Taxol^®-resistance. Taxol ^-resistant cancer cells show higher IC₅₀ (50% cell inhibition concentration of drag) than normal ones, and so the chemotherapy with Taxol needs higher concentration of paclitaxel in the tumor cells. Therefore, the specific localization of drug in the tumor tissue is required for guaranteeing the effectiveness against the cancer. The metal ion fixed polymeric micelle had a longer circulating property than the conventional formulations as shown in Figure 8. Thus, it was accumulated more selectively in the tumor tissue by enhanced permeation and retention (EPR) effect than the conventional one (data not shown). To demonstrate the effectiveness of metal ion-fixed polymeric micelles against the Taxol^®-resistant cancer, an animal model for in vivo anti-cancer activity against Taxol^®-resistant cancer was established. When the cancer cells inoculated into mice were exposed by Taxol^® repeatedly, ICso of the drag for Taxol -pretreated cancer cells was increased significantly compared to that of the drug for the native cancer cells. In this animal model, the metal ion-fixed polymeric micelle (Composition ll)-treated group showed a higher inhibition rate than the Cremophor EL formulation (Composition 12)- treated group possibly due to the longer retention for effective concentration of the drag incorporated in the metal ion-fixed polymeric micelle as shown in Fig. 13 and Table 21. Furthermore, the present invention includes a process for preparing the above pharmaceutical composition. Specifically, as shown in Figs. 3 and 5, the amphiphilic block copolymer with a hydrophobic block whose hydroxyl terminal group is substituted with a hydrophobic tocopherol succinic acid or cholesterol succinic acid group which has excellent hydrophobicity, the polylactic acid derivative, and the poorly water-soluble drag are dissolved in an organic solvent, and then the organic solvent is evaporated therefrom. Thereafter, the obtained mixture is added to an aqueous solution to prepare mixed polymeric micelles containing the poorly water-soluble drag. The metal ion-fixed polymeric micelles are prepared by adding a di- or tri-valent metal ion to the polymeric micelles thereby fixing the carboxyl terminal group of the polylactic acid derivative.

The polylactic acid derivative, the amphiphilic block copolymer, and the poorly water-soluble drug at a certain ratio can be dissolved in one or more solvents selected from the group consisting of acetone, ethanol, methanol, ethyl acetate, acetonitrile, methylene chloride, chloroform, acetic acid, and dioxane. The organic solvent can be removed therefrom to prepare a homogenous mixture of the poorly water-soluble drag and the polymer. The homogenous mixture of the poorly water-soluble drag 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 poorly water-soluble drug-containing mixed polymeric micelle aqueous solution. The above drag-containing polymeric micelle aqueous solution can then be lyophilized to prepare the polymeric micelle composition in the form of solid.

An aqueous solution containing 0.001 to 2 M of the di- or tri-valent metal ion is added to the poorly water-soluble drag-containing mixed polymeric micelle aqueous solution. The mixture is slowly stirred at room temperature for 0.1 to 1 hour, and then lyophilized to prepare the metal ion-fixed polymeric micelle or nanoparticle composition in the form of solid.

The following examples will enable those skilled in the art to more clearly 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 following is not intended to limit the scope of the present invention. Other aspects of the invention will be apparent to those skilled in the art to which the invention pertains.

Preparation Example 1 Synthesis 1 of D,L-polyIactic acid (PLA-COOH) One hundred grams of D,L-lactic acid was introduced into a 250 ml 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 the reduced pressure of 25 mmHg for 6 hours. The resulting product was added to 1 liter of distilled water to precipitate the polymer. The precipitated polymer was then added to distilled water to remove the low molecular weight polymer that was soluble in an aqueous solution of pH 4 or less. The precipitated polymer was then added to 1 liter of distilled 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 distilled 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 Daltons as determined by ¹H-NMR spectrum.

Preparation Examples 2 to 4 Synthesis 2 of D,L-poly lactic acid (PLA-COOH) D,L-polylactic acid was obtained according to the same procedure as in Preparation Example 1 except 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 following Table 1.

Table 1

* Yield = (Obtained polymer Used monomer)* 100

Preparation Example 5 Synthesis 1 of the copolymer of D,L-lactic acid and glycolic acid (PLGA-COOH) 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 ml three-neck round-bottomed flask. The same procedure as in Preparation Example 1 was carried out except that the reaction was performed at the temperature of 150 °C and under the reduced pressure of 10 mmHg for 12 hours.

Preparation Example 6 Synthesis 2 of the copolymer of D,L-lactic acid and glycolic acid (PLGA-COOH) Seventy three (73) grams of D,L-lactic acid (0.8 moles) and 27 grams of glycolic acid (0.35 moles) were introduced together into a 250 ml three-neck round-bottomed flask. The same procedure as in Preparation Example 1 was carried out except that the reaction was performed at the temperature of 160 °C and under the reduced pressure of 10 mmHg for 12 hours.

Preparation Example 7 Synthesis 3 of the copolymer of D,L-Iactic acid and glycolic acid (PLGA-COOH) 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 ml three-neck round-bottomed flask. The same procedure as in Preparation Example 1 was carried out except that the reaction was performed at the temperature of 160 °C and under the reduced pressure of 10 mmHg for 12 hours.

Preparation Example 8 Synthesis 4 of the copolymer of D,L-lactic acid and glycolic acid (PLGA-COOH) Seventy three (73) grams of D,L-lactic acid(0.8 moles) and 27 grams of glycolic acid (0.35 moles) were introduced into a 250 ml three-neck round-bottomed flask. The same procedure as in Preparation Example 1 was carried out except that the reaction was performed at the temperature of 180 °C and under the reduced pressure of 5 mmHg for 24 hours. The copolymers synthesized in the above Preparation Examples 5 to 8 are shown in Table 2.

Table 2

Preparation Example 9 Synthesis of the copolymer of D,L-lactic acid and mandelic acid (PLMA-COOH) 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 ml three-neck round- bottomed flask. The same procedure as in Preparation Example 1 was carried out except that the reaction was performed at the temperature of 180 °C and under a reduced pressure of 10 to 20 mmHg for 5 hours. 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 Daltons as determined by Η-NMR spectrum.

Preparation Example 10 Synthesis of acetoxy D,L-polylactic acid derivative (AcO-PLA-COOH) 50 g of D,L-polylactic acid (Mn: 1,140 Daltons), synthesized from Preparation Example 2, and 20 ml of chloracetic acid were introduced together into a 250 ml 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 distillation, and then 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 distilled 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 obtain liquid acetoxy D,L-polylactic acid (46 g, yield: 92%). By Η-NMR, the acetoxy group was identified as a single peak at 2.02 ppm.

Preparation Example 11 Synthesis of palmitoyloxy D,L-polylactic acid derivative (PalmO-PLA-COOH) Twenty (20) grams of D,L-polylactic acid (Mn:l,140 Daltons), synthesized from Preparation Example 2, was introduced into a 250 ml round-bottomed flask. The reactant was completely dehydrated under vacuum in an oil bath of 120 °C. The oil bath was cooled to 50 °C and 50 ml of acetone was added thereto to completely dissolve the polymer. 5 ml of chloropalmitic acid was added thereto, and the reaction was performed at the temperature of 50 °C for 10 hours under nitrogen. The reaction product was washed with an excessive amount of hexane to remove any residual 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 to result in the precipitation of an oligomer. The oligomer was separated and washed twice with distilled 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 to obtain a palmitoyloxy D,L-polylactic acid derivative (19.1 g, yield: 96%). By 1H-NMR, the palmitoyl group was identified as the peaks of 0.88, 1.3, and 2.38 ppm.

Preparation Example 12 Synthesis of 3arm polylactic acid (3arm PLA-COOH) One (1) gram of glycerol (0.01 lmol) was introduced into a 100 ml three-neck round-bottomed flask. The flask was equipped with a stiner, and heated in an oil bath to 80 °C. The reaction was performed for 30 min under the pressure reduced to 25 mmHg by a vacuum aspirator to remove excessive moisture. A reaction catalyst, tin octoate (Tin (Oct) 2), dissolved in toluene was added into 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 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 distilled water, isolated and dried under reduced pressure to obtain powder (3arm PLA-OH).

One hundred (1O0) gram of 3arm PLA-OH (0.033 mol) was 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. 19.8 g of succinic anhydride (0.198 mol) was added thereto, and the mixture was heated to 125 °C for 6 hours. The polymer formed was dissolved in acetone, and distilled water was added dropwise thereto to precipitate the polymer. The precipitated polymer was dissolved in 0.2N NaHC0₃ aqueous solution at 60 °C. The undissolved polymer was removed by filtration. 2 N HC1 aqueous solution was added dropwise thereto to precipitate the polymer. The precipitated polymer was washed five or six times with distilled water, isolated and dried under reduced pressure to obtain powder (3 arm PLA-COOH). The number average molecular weight of the polymer was 3,000 Daltons as determined by 1H-NMR spectrum.

Preparation Example 13 Synthesis of 5arm polylactic acid (5arm PLA-COOH) One (1) gram of xylitol (0.0066mol) was introduced into a 100 ml three-neck round-bottomed flask. The flask was equipped with a stiner, and heated in an oil bath to 80 °C. The reaction was performed for 30 min under the pressure reduced to 25 mmHg by a vacuum aspirator to remove excessive moisture. A reaction catalyst, tin octoate (Tin (Oct) 2), dissolved in toluene was added into 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 (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 NaHCO₃ aqueous solution was added dropwise thereto to precipitate the polymer. The precipitated polymer was washed three or four times with distilled water, isolated and dried under reduced pressure to obtain powder (5 arm PLA-OH).

One hundred (100) gram of 5arm PLA-OH (0.033 mol) was 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. 33.0 g of succinic anhydride (0.33 mol) was added thereto, and the mixture was heated to 125 °C for 6 hours. The polymer formed was dissolved in acetone, and distilled water was added dropwise thereto to precipitate the polymer. The precipitated polymer was dissolved in 0.2 N NaHCO aqueous solution at 60 °C. The undissolved polymer was removed by filtration. 2 N HC1 aqueous solution was added dropwise thereto to precipitate the polymer. The precipitated polymer was washed five or six times with distilled water, isolated and dried under reduced pressure to obtain powder (3arm PLA-COOH). The number average molecular weight of the polymer was 3,000 Daltons as determined by 1H-NMR spectrum.

Preparation Example 14 Synthesis 1 of sodium salt of polylactic acid (PLA-COONa) D,L-polylactic acid (Mn: 540 Daltons) 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 pH 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. 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 to leave the sodium salt of D,L-polylactic acid (yield: 96%) in white solid. As shown in Fig. 2, a hydrogen peak adjacent to the carboxylic acid group was observed at 4.88 ppm by 1H-NMR, and the polymer when dissolved in water had a pH of 6.5 to 7.5.

Preparation Example 15 Synthesis 2 of the sodium salt of polylactic acid (PLA-COONa) The sodium salt of polylactic acid (yield: 95%) was synthesized according to the same procedure as the above Preparation Example 14 except that D,L-polylactic acid (Mn: 1,140 Daltons) synthesized from Preparation Example 2 and an aqueous solution of sodium carbonate were used.

Preparation Example 16 Synthesis of the sodium salt of acetoxy-D,L-polylactic acid (AcO-PLA-COONa) The sodium salt of acetoxy-D,L-polylactic acid (yield: 95%) was synthesized according to the same procedure as Preparation Example 14 except that acetoxy-D,L- polylactic acid (Mn: 1,140 Daltons) synthesized from Preparation Example 10 and an aqueous solution of sodium carbonate were used.

Preparation Example 17 Synthesis 1 of the sodium salt of palmitoyloxy D,L-polylactic acid (PalmO-PLA- COONa) The palmitoyloxy D,L-polylactic acid (Mn: 1,140 Daltons) synthesized from Preparation Example 11 was completely dissolved in an aqueous solution of acetone

(28.6v/v%). 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 (I N) was added thereto for nutralization.

The solution was stirred slowly at room temperature and sodium hydrogen carbonate solution (I N) was slowly added thereto to reach pH 7. Anhydrous magnesium sulfate was added thereto to remove excessive moisture. The solution obtained was filtered, and the acetone solution was evaporated with a solvent evaporator. White solid was obtained therefrom. The solid was dissolved in acetone and the solution was filtered to remove any insoluble in acetone. The acetone was evaporated and the sodium salt of palmitoyloxy D,L-polylactic acid was obtained as white solid (yield: 96%).

Preparation Example 18 Synthesis of the potassium salt of polylactic acid (PLA-COOK) The potassium salt of polylactic acid (yield: 98%) was synthesized according to the same procedure as Preparation Example 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.

Preparation Example 19 Synthesis 3 of the sodium salt of polylactic acid (PLA-COONa) The sodium salt of polylactic acid (yield: 95%) was synthesized according to the same procedure as Preparation Example 14 except that D,L-lactic acid (Mn: 2,100 Daltons) synthesized from Preparation Example 4 was used. Preparation Example 20 Synthesis 1 of the sodium salt of the copolymer of D,L-lactic acid and glycolic acid (PLGA-COONa) The sodium salt of the copolymer of D,L-lactic acid and glycolic acid (yield: 98%) was synthesized according to the same procedure as Preparation Example 14 except that a copolymer of D,L-lactic acid and glycolic acid (Mn: 920 Daltons) synthesized from Preparation Example 5 and an aqueous solution of sodium carbonate were used.

Preparation Example 21 Synthesis 2 of the sodium salt of the copolymer of D,L-lactic acid and glycolic acid (PLGA-COONa) The sodium salt of the copolymer of D,L-lactic acid and glycolic acid (yield: 93%) was synthesized according to the same procedure as Preparation Example 14 except that a copolymer of D,L-lactic acid and glycolic acid (Mn: 1,040 Daltons) synthesized from Preparation Example 6 was used.

Preparation Example 22 Synthesis of the potassium salt of the copolymer of D,L-lactic acid and glycolic acid (PLGA-COOK) The potassium salt of the copolymer of D,L-lactic acid and glycolic acid (yield: 92%) was synthesized according to the same procedure as Preparation Example 14 except that a copolymer of D,L-lactic acid and glycolic acid (Mn: 1,180 Daltons) synthesized from Preparation Example 7 and an aqueous solution of potassium carbonate were used.

Preparation Example 23 Synthesis 3 of the sodium salt of the copolymer of D,L-lactic acid and glycolic acid (PLGA-COONa) The sodium salt of the copolymer of D,L-lactic acid and glycolic acid (yield: 98%) was synthesized according to the same procedure as Preparation Example 14 except that the copolymer of D,L-lactic acid and glycolic acid (Mn: 1,650 Daltons) synthesized from Preparation Example 8 was used.

Preparation Example 24 Synthesis of the sodium salt of the copolymer of D,L-lactic acid and mandelic acid (PLMA-COONa) The sodium salt of the copolymer of D,L-lactic acid and mandelic acid (yield:

96%) was synthesized as white solid according to the same procedure as Preparation Example 14 except that the copolymer of D,L-lactic acid and mandelic acid synthesized from Preparation Example 9 (Mn: 1,096 Daltons) was used. Preparation Example 25 Synthesis of the sodium salt of 3arm polylactic acid (3arm PLA-COONa) The sodium salt of 3 arm polylactic acid was synthesized as white solid according to the same procedure as Preparation Example 14 except that the copolymer of 3-arm D,L- lactic acid (Mn: 3,000 Daltons) synthesized from Preparation Example 12 was used.

Preparation Example 26 Synthesis of the sodium salt of 5arm polylactic acid (5arm PLA-COONa) The sodium salt of 5 arm polylactic acid was synthesized as white solid according to the same procedure as Preparation Example 14 except that the copolymer of 5-arm D,L- lactic acid (Mn: 3,000 Daltons) synthesized from Preparation Example 13 was used.

The carboxylate salts of the polylactic acid derivatives synthesized from the above Preparation Examples 14 to 26 are shown in Table 3.

Table 3

Preparation Example 27 Polymerization of monomethoxypolyethylene glycol-polylactide (mPEG-PLA) block copolymer (AB type) Five (5) grams of monomethoxypolyethylene glycol (Mn: 2,000 Daltons) was introduced into a 100 ml 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 filled with dried nitrogen, and a reaction catalyst, stannous octoate (Sn(Oct)₂), was injected with 1.0 mol% (10.13 mg, O.025 mmol) of polyethylene glycol monomethyl ether 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 catalyst. 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 diethyl ether was added thereto to precipitate the polymer. The polymer obtained was dried in a vacuum oven for 48 hours. The mPEG-PLA obtained had a number average molecular weight of 2,000- 1,765 Daltons, and was confirmed to be of the AB type by 1H-NMR. Preparation Example 28

Polymerization of monomethoxypolyethylene glycol-poly(lactic-co-glycolide) (mPEG- PLGA) block copolymer (AB type) To synthesize mPEG-PLGA block copolymer, monomethoxypolyethylene glycol

(Mn: 5,000 Daltons) was reacted with lactide and glycolide in the presence of the catalyst of stannous octoate at 120 °C for 12 hours according to the same procedure as Preparation

Example 27. The mPEG-PLGA obtained had a number average molecular weight of

5,000-4,000 Daltons, and was confirmed to be of the AB type by Η-NMR.

Preparation Example 29 Polymerization of monomethoxypolyethylene glycol-poly(lactic-co-p-dioxan-2-one) (mPEG-PLDO) block copolymer (AB type) To synthesize mPEG-PLDO block copolymer, monomethoxypolyethylene glycol

(Mn: 12,000 Daltons) was reacted with lactide and p-dioxan-2-one in the presence of the catalyst of stannous octoate at 110 °C for 12 hours according to the same procedure as Preparation Example 27. The mPEG-PLDO obtained had a number average molecular weight of 12,000-10,000 Daltons, and was confirmed to be of the AB type by 1H-NMR.

Preparation Example 30 Polymerization of monomethoxypolyethylene glycol-polycaprolactone (mPEG-PCL) block copolymer (AB type) To synthesize mPEG-PCL block copolymer, monomethoxypolyethylene glycol

(Mn: 12,000 Daltons) was reacted with caprolactone in the presence of the catalyst of stannous octoate at 130 °C for 12 hours, according to the same procedure as Preparation

Example 27. The mPEG-PCL obtained had a number average molecular weight of 12,000- 5,000 Daltons, and was confirmed be of the AB type by 1H-NMR. The block copolymers synthesized from the above Preparation Examples 27 to 30 are shown in the following Table 4.

Table 4

Preparation Example 31 Polymerization of monomethoxypolyethylene glycol-polylactide tocopherol (mPEG- PLA-tocopherol) block copolymer (AB type) Ten (10) grams of mPEG-PLA synthesized from Preparation Example 27 and 1.83 grams of tocopherol succinate were dissolved in 50 ml of acetonitrile in a round-bottomed flask. Reaction catalysts, 0.71 gram of dicyclohexylcarbodiimide (DCC) and 0.042 gram of 4-(dimethylamino)pyridine (DMAP), were added thereto, and the mixture was stirred at room temperature for 24 hours. The mixture was filtered using a filter paper to remove dicyclohexylcarbourea, a byproduct. The mixture was added into a cosolvent composed of n-hexane/diethyl ether (v/v=7/3) for recrystalization to obtain purified mPEG-PLA- tocopherol (10 g; yield=87.5%).

Preparation Example 32 Polymerization of monomethoxypolyethylene glycol-polylactide cholesterol (mPEG- PLA-cholesterol) block copolymer (AB type) 7.6 grams of cholesterol and 2.36 grams of succinic anhydride were dissolved in 100 ml of 1,4-dioxane in a round-bottomed flask. A reaction catalyst, 2.9 grams of 4- (dimethylamino)pyridine (DMAP), was added thereto, and the mixture was stirred at room temperature for 24 hours. The reaction mixture was introduced into HCl solution to precipitate the cholesterol succinate (9.1 g; yield=95%). Ten (10) grams of mPEG-PLA synthesized from Preparation Example 27 and 1.68 grams of cholesterol succinate were dissolved in 50 ml of acetonitrile in a round-bottomed flask. Reaction catalysts, 0.71 gram of dicyclohexylcarbodiimide (DCC) and 0.042 gram of 4-(dimethylamino)pyridine (DMAP), were added thereto, and the mixture was stirred at room temperature for 24 hours. The mixture was filtered using a filter paper to remove dicyclohexylcarbourea, a byproduct. The mixture was added into a cosolvent composed of n-hexane/diethyl ether (v/v=6/4) for recrystahzation to obtain purified mPEG-PLA- cholesterol (lOg; yield=88.6%).

Preparation Example 33 Polymerization of monomethoxypolyethylene glycol-poly(lactic-co-glycolide) tocopherol (mPEG-PLGA-tocopherol) block copolymer (AB type) Ten grams of mPEG-PLGA synthesized from Preparation Example 28 and 1.767 grams of tocopherol succinate were dissolved in 50 ml of acetonitrile in a round-bottomed flask. Reaction catalysts, 0.3 gram of dicyclohexylcarbodiimide (DCC) and 0.018 gram of 4-(dimethylamino)pyridine (DMAP), were added thereto, and the mixture was strrred at room temperature for 24 hours. The mixture was filtered using a filter paper to remove dicyclohexylcarbourea, a byproduct. The mixture was added into a cosolvent composed of n-hexane/diethyl ether (v/v=7/3) for recrystahzation to obtain purified mPEG-PLGA- tocopherol (10 g; yield=87.5%).

Preparation Example 34 Polymerization of monomethoxypolyethylene glycol-poly (lactic-co-glycolide) cholesterol (mPEG-PLGA-cholesterol) block copolymer (AB type) 7.6 grams of cholesterol and 2.36 grams of succinic anhydride were dissolved in

100 ml of 1,4-dioxane in a round-bottomed flask. A reaction catalyst, 2.9 grams of 4-

(dimethylamino)ρyridine (DMAJP), was added thereto, and the mixture was stirred at room temperature for 24 hours. The reaction mixture was introduced into HCl solution to precipitate the cholesterol succinate (9.1 g; yield=95%). Ten (10) grams of mPEG-PLGA synthesized from Preparation Example 28 and 0.70 grains of cholesterol succinate were dissolved in 50 ml of acetonitrile in a round- bottomed flask. Reaction catalysts, 0.3 gram of dicyclohexylcarbodiimide (DCC) and 0.018 gram of 4-(dimethylamino)pyridine (DMAP), were added thereto, and the mixture was stirred at room temperature for 24 hours. The mixture was filtered using a filter paper to remove dicyclohexylcarbourea, a byproduct. The mixture was added into a cosolvent composed of n-hexane/diethyl ether (v/v=7/3) for recrystahzation to obtain purified mPEG-PLGA-cholesterol (10 g; yield=88.6%).

Preparation Example 35 Polymerization of monomethoxypolyethylene glycol-poIy(Iactic-co-p-dioxan-2-one) tocopherol (mPEG-PLDO-tocopherol) block copolymer (AB type) Ten (10) grams of mPEG-PLDO synthesized from Preparation Example 29 and 0.314 grams of tocopherol succinate were dissolved in 50 ml of methylene chloride in a round-bottomed flask. Reaction catalysts, 0.122 gram of dicyclohexylcarbodiimide (DCC) and 0.007 gram of 4-(dimethylamino)pyridine (DMAP), were added thereto, and the mixture was stirred at room temperature for 24 hours. The mixture was filtered using a filter paper to remove dicyclohexylcarbourea, a byproduct. The mixture was added into a cosolvent composed of n-hexane/diethyl ether (v/v=7/3) for recrystahzation to obtain purified mPEG-PLDO-tocopherol (10 g; yield=87.5%).

Preparation Example 36 Polymerization of monomethoxypolyethylene glycol-poly(lactic-co-dioxan-2-one) cholesterol (mPEG-PLDO-cholesterol) block copolymer (AB type) 7.6 grams of cholesterol and 2.36 grams of succinic anhydride were dissolved in

(dimethylamino)pyridine (DMAP), was added thereto, and the mixture was stirred at room temperature for 24 hours. The reaction mixture was introduced into HCl solution to precipitate the cholesterol succinate (9.1 g; yield=95%). Ten (10) grams of mPEG-PLDO synthesized from Preparation Example 29 and 0.288 grams of cholesterol succinate were dissolved in 50 ml of acetonitrile in a round- bottomed flask. Reaction catalysts, 0.122 gram of dicyclohexylcarbodiimide (DCC) and 0.007 gram of 4-(dimethylamino)pyridine (DMAP), were added thereto, and the mixture was stirred at room temperature for 24 hours. The mixture was filtered using a filter paper to remove dicyclohexylcarbourea, a byproduct. The mixture was added into a cosolvent composed of n-hexane/diethyl ether (v/v=7/3) for recrystahzation to obtain purified mPEG-PLDO-cholesterol (10 g; yield=88.6%).

Preparation Example 37 Polymerization of monomethoxypolyethylene glycol-polycaprolactone tocopherol (mPEG-PCL-tocopherol) block copolymer (AB type) Ten (10) grams of mPEG-PCL synthesized from Preparation Example 30 and 0.406 grams of tocopherol succinate were dissolved in 50 ml of acetonitrile in a round- bottomed flask. Reaction catalysts, 0.158 gram of dicyclohexylcarbodiimide (DCC) and 0.009 gram of 4-(dimethylamino)pyridine (DMAP), were added thereto, and the mixture was stirred at room temperature for 24 hours. The mixture was filtered using a filter paper to remove dicyclohexylcarbourea, a byproduct. The mixture was added into a cosolvent composed of n-hexane/diethyl ether (v/v=7/3) for recrystahzation to obtain purified mPEG-PCL-tocopherol (10 g; yield=87.5%).

Preparation Example 38 Polymerization of monomethoxypolyethylene glycol-polycaprolactone cholesterol (mPEG-PCL-cholesterol) block copolymer (AB type) 7.6 grams of cholesterol and 2.36 grams of succinic anhydride were dissolved in

(dimethylamino)pyridine (DMAP), was added thereto, and the mixture was stirred at room temperature for 24 hours. The reaction mixture was introduced into HCl solution to precipitate the cholesterol succinate (9.1 g; yield=95%). Ten (10) grams of mPEG-PLA synthesized from Preparation Example 30 and 0.372 grams of cholesterol succinate were dissolved in 50 ml of acetonitrile in a round- bottomed flask. Reaction catalysts, 0.158 gram of dicyclohexylcarbodiimide (DCC) and 0.009 gram of 4-(dimethylammo)pyridine (DMAP), were added thereto, and the mixture was stirred at room temperature for 24 hours. The mixture was filtered using a filter paper to remove dicyclohexylcarbourea, a byproduct. The mixture was added into a cosolvent composed of n-hexane/diethyl ether (v/v=7/3) for recrystahzation to obtain purified mPEG-PCL-cholesterol (10 g; yield=88.6%).

Preparation Example 39 Preparation of ionically fixed polymeric micelles Step 1: Preparation of the polymeric micelles of D,L-PLA-COONa and mPEG- PLA-tocopherol block copolymers 248.1 mg (0.218 rnmol) of D,L-PLA-COONa (Mn: 1,140) from Preparation

Example 15 and 744.3 mg of mPEG-PLA-tocopherol (Mn: 2,000-1,800 Daltons) from Preparation Example 31 were completely dissolved in 5 ml of ethanol to obtain a clear solution. Ethanol was removed therefrom to prepare a polymeric composition. Distilled water (6.2 ml) was added thereto and the mixture was stirred for 30 minutes at 60 °C to prepare the polymeric micelle aqueous solution.

Step 2: Fixation with the di-valent metal ion 0.121 ml (0.109 mol) of a 0.9 M aqueous solution of anhydrous calcium chloride was added to the polymeric micelle 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 the pore size of 200 nm, and then was lyophilized. The particle size measured according to the Dynamic Light Scattering (DLS) Method was 25 nm.

Example 1 Preparation of Ca²⁺-fϊxed paclitaxel-containing micelles of D,L-PLA-COONa and mPEG-PLA-tocopherol block copolymers Step 1: Preparation of paclitaxel-containing polymeric micelles of D,L-PLA- COONa and mPEG-PLA-tocopherol block copolymers 248.1 mg (0.218 mmol) of D,L-PLA-COONa (Mn: 1,140) from Preparation Example 15, 7.5 mg of paclitaxel, and 744.3 mg of mPEG-PLA-tocopherol (Mn: 2,000- 1,800 Daltons) from Preparation Example 31 were completely dissolved in 5 ml of ethanol to obtain a clear solution. Ethanol was removed therefrom to prepare a paclitaxel- containing polymeric composition. Distilled water (6.2 ml) was added thereto and the mixture was stirred for 30 minutes at 60 °C to prepare a paclitaxel-containing polymeric micelle aqueous solution.

Step 2: Fixation with the divalent metal ion 0.121 ml (0.109 mmol) of a 0.9 M aqueous solution of anhydrous calcium chloride was added to the polymeric micelle 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 the pore size of 200 nm, and then was lyophilized. The content and solubility of paclitaxel were measured by HPLC and the particle size was measured according to the Dynamic Light Scattering (DLS) Method. D,L-PLA-COONa/mPEG-PLA-tocopherol (weight ratio): 1/3 Content of Paclitaxel: 0.75wt% Particle Size: 29 nm

Example 2 Preparation of Mg²⁺-fixed paclitaxel-containing polymeric micelles of D.L-PLMA- COONa and mPEG-PLA-tocopherol block copolymers A Mg²⁺-fixed paclitaxel-containing polymeric micelle composition was prepared according to the same procedure as Example 1 except that 248.1 mg (0.226 mmol) of D,L-

PLMA-COONa (Mn: 1,096) from Preparation Example 24, 7.5 mg of paclitaxel, 744.3 mg of mPEG-PLA-tocopherol (Mn: 2,000-1,800 Daltons) from Preparation Example 31, and 0.230 ml (0.113 mmol) of the 0.5 M aqueous solution of magnesium chloride 6 hydrate (Mw:203.31) were used . D,L-PLMA-COONa/mPEG-PLA-tocopherol (weight ratio): 1/3 Content of Paclitaxel: 0.75 wt% Particle Size: 30 nm

Example 3 Preparation of Ca²⁺-fixed paclitaxel-containing polymeric micelles of D,L-PLMA- COONa and mPEG-PLA-tocopherol block copolymers A Ca²⁺-fixed paclitaxel-containing polymeric micelle composition was prepared according to the same procedure as Example 1 except that 248.1 mg (0.226 mmol) of D,L- PLMA-COONa (Mn: 1,096) from Preparation Example 24, 7.5 mg of paclitaxel, 744.4 mg of mPEG-PLA-tocopherol (Mn: 2,000-1,800 Daltons) from Preparation Example 31, and 0.126 ml (0.113 mmol) of the 0.9 M aqueous solution of anhydrous calcium chloride were used . D,L-PLMA-COONa/mPEG-PLA-tocopherol (weight ratio): 1/3 Content of Paclitaxel: 0.75 wt% Particle Size: 34 nm

Example 4 Preparation of Ca²⁺-fϊxed paclitaxel-containing polymeric micelles of D.L-PLA- COOK and mPEG-PLA-cholesterol block copolymers A Ca²⁺-fιxed paclitaxel-containing polymeric micelle composition was prepared according to the same procedure as Example 1 except that 248.1 mg (0.160 mmol) of D.L- PLA-COOK (Mn: 1,550) from Preparation Example 18, 7.5 mg of paclitaxel, 744.4 mg of mPEG-PLA-cholesterol (Mn: 2,000-1,800 Daltons) from Preparation Example 32, and 0.089 ml (0.080 mmol) of the 0.9 M aqueous solution of anhydrous calcium chloride were used . D,L-PLMA-COONa/mPEG-PLA-cholesterol (weight ratio): 1/3 Content of Paclitaxel: 0.75 wt% Particle Size: 34 nm Example 5 Preparation of Ca²⁺-fϊxed paclitaxel-containing polymeric micelles of D,L-PLMA- COONa and mPEG-PLA-cholesterol block copolymers A Ca²⁺-fixed paclitaxel-containing polymeric micelle composition was prepared according to the same procedure as Example 1 except that 248.1 mg (0.226 mmol) of D,L- PLMA-COONa (Mn: 1,096) from Preparation Example 24, 7.5 mg of paclitaxel, 744.4 mg of mPEG-PLA-cholesterol (Mn: 2,000-1,800 Daltons) from Preparation Example 32, and 0.126 ml (0.113 mmol) of the 0.9 M aqueous solution of anhydrous calcium chloride were used . D,L-PLMA-COONa/mPEG-PLA-cholesterol (weight ratio): 1/3 Content of Paclitaxel: 0.75wt% Particle Size: 34 nm

Example 6 Preparation of Ca²⁺-fixed paclitaxel-containing polymeric micelles of 3 arm PLA- COONa and mPEG-PLA-tocopherol block copolymers A Ca²⁺-fixed paclitaxel-containing polymeric micelle composition was prepared according to the same procedure as Example 1 except that 248.1 mg (0.0827 mmol) of 3 arm PLA-COONa (Mn: 3,000) from Preparation Example 25, 7.5 mg of paclitaxel, 744.4 mg of mPEG-PLA-tocopherol (Mn: 2,000-1,800 Daltons) from Preparation Example 31, and 0.1377 ml (0.124 mmol) of the 0.9 M aqueous solution of anhydrous calcium chloride were used. 3 arm PLACOONa/mPEG-PLA-tocopherol (weight ratio): 1/3 Content of Paclitaxel: 0.75 wt% Particle Size: 29 nm

Example 7 Preparation of Ca²⁺-fixed paclitaxel-containing polymeric micelles of 5 arm PLA- COONA and mPEG-PLA-tocopherol block copolymers A Ca²⁺-fixed paclitaxel-containing polymeric micelle composition was prepared according to the same procedure as Example 1 except that 248.1 mg (0.0827 mmol) of 5 arm PLA-COONa (Mn: 3,000) from Preparation Example 26, 7.5 mg of paclitaxel, 744.4 mg of mPEG-PLA-tocopherol (Mn: 2,000-1,800 Daltons) from Preparation Example 31, and 0.2295 ml (0.207 mmol) of the 0.9 M aqueous solution of anhydrous calcium chloride were used. 5 arm PLACOONa mPEG-PLA-tocopherol (weight ratio): 1/3 Content of Paclitaxel: 0.75 wt% Particle Size: 29 nm Example 8

Preparation of doxorubicin-containing polymeric micelles of D,L-PLMA-COONa and mPEG-PLA-tocopherol block copolymers mPEG-PLA-tocopherol (Mn: .2,000- 1,800), D,L-PLMA-COONa (Mn: 969), and doxorabicin HCl 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 clear solution. Methanol was removed therefrom using a vacuum evaporator to prepare a doxorubicin-containing polymeric composition. Distilled water (4 ml) was added thereto, and the mixture was stirred for 10 minutes at 60 °C to prepare a polymeric micelle aqueous solution containing doxorabicin. The mixture was passed through a filter with the pore size of 200 nm, and than was lyophilized. D,L-PLMA-COONa mPEG-PLA-tocopherol (weight ratio): 1/4.56 Content of doxorabicin: 1.03 t% Particle Size: 35 nm Example 9

Preparation of epirubicin-containing polymeric micelles of D,L-PLMA-COONa and mPEG-PLA-tocopherol block copolymers mPEG-PLA-tocopherol (Mn: 2,000-1,800), D,L-PLMA-COONa (Mn: 969), and epirabicin HCl 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 clear solution. Methanol was removed therefrom using a vacuum evaporator to prepare an epirabicin-containing polymeric composition. Distilled water (4 ml) was added thereto and the mixture was stirred for 10 minutes at 60 °C to prepare a polymeric micelle aqueous solution containing doxorabicin. The mixture was passed through a filter with the pore size of 200 nm, and than was lyophilized. D,L-PLMA-COONa/mPEG-PLA-tocopherol (weight ratio): 1/4.56 Content of epirubicin: 1.03wt% Particle Size: 30 nm Example 10 Particle size for the Ca²⁺-fixed polymeric micelles To determine the particle size of Ca²⁺-fixed polymeric micelles, the polymeric micelle compositions were prepared as follows. mPEG-PLA (Mn: 2,000-1,800) and D,L-PLMA-COONa (Mn: 866, 994, 1,156, 1,536) were admixed at the equivalent ratio of 1 :1, and then the mixture was dissolved in 5 ml of anhydrous ethanol to prepare a clear solution. Ethanol was removed therefrom using a vacuum evaporator to prepare a polymeric composition. Distilled water was added thereto and the mixture was stireed for 10 minutes at 60 °C to prepare a polymeric micelle aqueous solution containing paclitaxel. To the above polymeric micelle solution was added a CaCl₂ aqueous solution (concentration: 100 mg/ml) of the same equivalent as the D,L-PLMA-COONa, 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 PBS buffer of pH 7.4 was added thereto to dilute the mixture to make 40 mg/ml of the polymers. The particle size was measured with a photon correlation particle size analyzer after filtration using 0.22 um membrane filter.

Table 5

As shown in Table 5, the particle of Ca -2+ -fixed polymeric micelles had an average size of 20-40 nm. The micelles of this size range are suitable for injection formulation and sterile filtration. As the molecular weight of D,L-PLMA-COONa increased from 866 to 1536, the particle size increased slightly in both Ca treated and non-treated micelles. The particle size of Ca²⁺ -fixed polymeric micelles was bigger than the micelles non-treated with Ca²⁺ by approximately 10 nm.

Example 11 Kinetic Stability for the Ca²⁺-fixed paclitaxel-containing polymeric micelles To test the stability of the nanoparticle composition, the polymeric micelle compositions were prepared as follows. (Composition 1) Paclitaxel, mPEG-PLA-Tocopherol (Mn: 2,000-1,800), and D,L- PLMA-COONa (Mn: 1,096) were admixed at the equivalent ratio of 1:3:3, and then the mixture was dissolved in 5 ml of anhydrous ethanol to prepare a clear solution. Ethanol was removed therefrom using a vacuum evaporator to prepare a paclitaxel-containing polymeric composition. Distilled water (4 ml) was added thereto, and the mixture was stirred for 10 minutes at 60 °C to prepare a polymeric micelle aqueous solution containing paclitaxel. To the above polymeric micelle solution was added a CaCl₂ aqueous solution (concentration: 100 mg/ml) of the same equivalent as the D,L-PLMA-COONa, 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.

(Composition 2) Paclitaxel, mPEG-PLA-Tocopherol (Mn: 2,000-1,800) and D,L- PLMA-COONa (Mn: 1,096) were admixed at the equivalent ratio of 1:3:3 and then the mixture was dissolved in 5 ml of anhydrous ethanol to prepare a clear solution. Ethanol was removed therefrom using a vacuum evaporator to prepare a paclitaxel-containing polymeric composition. Distilled water (4 ml) was added thereto and the mixture was stirred for 10 minutes at 60 °C to prepare a polymeric micelle aqueous solution containing paclitaxel. The mixture was passed through a filter with the pore size of 200 run, and then was lyophilized.

(Composition 3) Paclitaxel and mPEG-PLA-Tocopherol (Mn: 2,000-1,800) were admixed at the equivalent ratio of 1:3, and then the mixture was dissolved in 5 ml of anhydrous ethanol to prepare a clear solution. Ethanol was removed therefrom using a vacuum evaporator to prepare a paclitaxel-containing polymeric composition. Distilled water (5 ml) was added thereto, and the mixture was stirred for 10 minutes at 60 °C to prepare a polymeric micelle aqueous solution containing paclitaxel. The mixture was passed through a filter with the pore size of 200 nm, and then was lyophilized.

(Composition 4) Paclitaxel, mPEG-PLA (Mn: 2,000-1,765), and D,L-PLMA- COONa (Mn: 1,096) were admixed at the equivalent ratio of 1:3:3, and then the mixture was dissolved in 5 ml of anhydrous ethanol to prepare a clear solution. Ethanol was removed therefrom using a vacuum evaporator to prepare a paclitaxel-containing polymeric composition. Distilled water (4 ml) was added thereto, and the mixture was stirred for 10 minutes at 60 °C to prepare a polymeric micelle aqueous solution containing paclitaxel. To the above polymeric micelle solution was added a CaCl₂ aqueous solution (concentration: 100 mg/ml) of the same equivalent as the D,L-PLMA-COONa, 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.

Table 6

PBS buffer of pH 7.4 was added to the lyophilized compositions to make 1.0 mg/ml of paclitaxel. The mixture was allowed to stand at 37 °C and the concentration of paclitaxel over the lapse of time was measured by HPLC. The results are shown in Table 7.

Table 7

As shown in Table 7, the Ca -2+ -fixed paclitaxel-containing polymeric micelle composition (Composition 1) was more kinetically stable than the Ca²⁺-nontreated composition (Composition 2). The addition of Ca²⁺ significantly increased the retention of paclitaxel in the polymeric micelles of the present invention. This is due to the crosslinking electrostatic interaction of D,L-PLA-COO^" and Ca²⁺ which might induce the increase of rigidity of the hydrophobic core. The Ca -fixed polymeric micelles (Composition 1) of the amphiphilic diblock copolymers with hydrophobic moiety (tocopherol succinic acid) substituted for the hydroxyl terminal group of hydrophobic B block had a much longer retention time than the Ca²⁺-fixed polymeric micelles (Compositions 4) of native mPEG-PLA-OH. This result also suggests that the increase of hydrophobicity of hydrophobic B block in the amphiphilic polymer results in forming more stable micelles due to the stronger interaction between the hydrophobic moiety of the amphiphilic polymer and drug.

Example 12 Pharmacokinetics for the paclitaxel-containing polymeric micelles of the amphiphilic diblock copolymers conjugated with the hydrophobic moiety To evaluate the effect of hydrophobic moiety substituted for the hydroxyl terminal group of hydrophobic B block of the amphiphilic diblock copolymers (mPEG-PLA, Mn 2000-1765) on the bloodstream retention time of the paclitaxel-containing polymeric micelles, the compositions were prepared as follows. Paclitaxel and the amphiphilic diblock copolymer were admixed in the weight ratio of 1 :99, and then the mixture was dissolved in 5 ml of anhydrous ethanol to prepare a clear solution. Ethanol was removed therefrom using a vacuum evaporator to prepare a paclitaxel-containing polymeric composition. Distilled water (4 ml) was added thereto, and the mixture was stirred for 10 minutes at 60 °C to prepare a polymeric micelle aqueous solution containing paclitaxel. The mixture was passed through a filter with the pore size of 200 nm, and then was lyophilized. The above composition and the drugs content are summarized in Table 8.

Table 8

For the animal experiments, male Sprague-Dawley rats weighing 250-300 g were cannulated in the vena femoralis and aorta femoralis. Compositions 5, 6 and 7 were injected into the vena femoralis at the dose of 5 mg/kg over 15 seconds. After the injection, 0.3 ml of the whole blood was taken from the aorta femoralis in 1, 5, 15, 30 minutes, and in 1, 2, 3, 4, 6 hours, and then centrifuged to obtain clear supernatant plasma.

Furthermore, to analyze the plasma concentration of drug, 0.1 ml of the plasma was introduced into a covered glass tube, and 0.1 ml of an acetonitrile solution containing the internal standard substance was added thereto. 10 ml 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 ml of a 40%(v/v) acetonitrile solution, and the mixture was vigorously stirred for 30 seconds, and then subjected to HPLC. The conditions for HPLC were as follows: Injection Volume: 0.075 ml Flow Rate: l.O ml/min Wavelength: 227 nm 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 Column: 4.6x50 nm (C 18, Vydac, USA).

The analysis results of plasma concentrations of the drugs are shown in the following Table 9 and Fig. 6. Table 9

As shown in Table 9 and Fig. 6, the polymeric micelles (Composition 5-7) of the amphiphilic diblock copolymers with hydrophobic moiety (tocopherol succinic acid, cholesterol succinic acid, or palmitic acid) substituted for the hydroxyl terminal group of hydrophobic B block had a much longer bloodstream retention time than native mPEG- PLA-OH polymeric micelles (Compositions 8). This result suggests that the increase of hydrophobicity of hydrophobic B block in the amphiphilic polymer results in forming more stable micelles due to the stronger interaction between the hydrophobic moiety of the amphiphilic polymer and drug. mPEG-PLA-tocopherol micelles (Composition 5) were longest circulating in the blood among the polymeric micelles. The second longest circulating composition was mPEG-PLA-cholesterol micelles (Composition 6).

Example 13 Pharmacokinetics

paclitaxel-containing polymeric micelles To evaluate the effect of hydrophobic moiety substituted for the hydroxyl terminal group of hydrophobic B block of the amphiphilic di-block copolymers (mPEG-PLA, Mn 2000-1765) on the bloodstream retention time of the Ca²⁺-fixed paclitaxel-containing polymeric micelles, the compositions were prepared as follows.

Paclitaxel, mPEG-PLA-tocopherol (Mn: 2,000-1,800) or mPEG-PLA-OH, and D,L-PLMA-COONa (Mn: 1,004) were admixed in a weight ratio of 74.25:24.75:1.00, and then the mixture was dissolved in 5 ml of anhydrous ethanol to prepare a clear solution. Ethanol was removed therefrom using a vacuum evaporator to prepare a paclitaxel- containing polymeric composition. Distilled water (4 ml) was added thereto, and the mixture was stirred for 10 minutes at 60 °C to prepare a polymeric micelle aqueous solution containing paclitaxel. To the above polymeric micelle solution was added a CaCl₂ aqueous solution (concentration: 100 mg/ml) of the same equivalent as the D,L-PLMA- COONa, 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 above composition and the drug contents are summarized in Table 10.

Table 10

For the animal experiments, male Sprague-Dawley rats weighing 220-270 g were cannulated in the vena femoralis and aorta femoralis. Compositions 9 and 10 were injected into the vena femoralis at a dose of 5 mg/kg over 15 seconds. After the injection, 0.3 ml of the whole blood was taken from the aorta femoralis in 1, 5, 15, and 30 minutes, and in 1, 2, 3, 4, and 6 hours, and then centrifuged to obtain clear supernatant plasma.

Furthermore, the plasma drug concentration was analyzed according to the same process as Example 10, and the analysis results on the plasma concentrations of the drugs are shown in the following Table 11 and Fig. 7.

Table 11

As shown in Table 11 and Fig. 7, the Ca²⁺ -fixed polymeric micelles (Composition 9) of the amphiphilic di-block copolymers with hydrophobic moiety (tocopherol succinic acid) substituted for the hydroxyl terminal group of hydrophobic B block had a much longer bloodstream retention time than the Ca²⁺-fixed polymeric micelles (Compositions 10) of native mPEG-PLA-OH. This result also suggests, as demonstrated in Example 10, that the increase of hydrophobicity of hydrophobic B block in the amphiphilic polymer results in forming more stable micelles due to the stronger interaction between the hydrophobic moiety of the amphiphilic polymer and drag.

E.xample 14 Pharmacokinetics for the Ca .2+ -fixed paclitaxel-containing polymeric micelles To compare the bloodstream retention time of the Ca²⁺-fiχed paclitaxel-containing polymeric micelles with that of the formulations containing other carriers, the compositions were prepared as follows. (Composition 11) Ca²⁺-fixed paclitaxel-containing polymeric micelles Paclitaxel, mPEG-PLA-tocopherol (Mn: 2,000-1,800), and D,L-PLMA-COONa (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 clear solution. Ethanol was removed therefrom using a vacuum evaporator to prepare a paclitaxel-containing polymeric composition. Distilled water (4 ml) was added thereto, and the mixture was stirred for 10 minutes at 60 °C to prepare a polymeric micelle aqueous solution containing paclitaxel. To the above polymeric micelle solution was added a CaCl₂ aqueous solution (concentration: 100 mg/ml) of the same equivalent as the D,L-PLMA-COONa, 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 particle size of the polymeric micelles was 34 nm.

(Composition 12) Composition containing paclitaxel, Cremophor EL, and anhydrous ethanol Paclitaxel (30 mg) was dissolved in 5 ml of a mixed solution (50:50 v/v) of

Cremophor EL and anhydrous ethanol to obtain a clear solution. The solution was passed through a filter having the pore size of 200 nm.

(Composition 13) Composition containing paclitaxel, polysorbate 80 (Tween 80), and anhydrous ethanol Paclitaxel (30 mg) was dissolved in 5 ml of a mixed solution (50:50 v/v) of polysorbate 80 and anhydrous ethanol to obtain a clear solution. The solution was passed through a filter having the pore size of 200 nm. The above composition and the drag contents are summarized in Table 12. Table 12

For the animal experiments, male Sprague-Dawley rats weighting 230-250 g were cannulated in the vena femoralis and aorta femoralis. Compositions 11, 12 and 13 were injected into the vena femoralis at a dose of 5 mg/kg over 15 seconds. After the injection, 0.3 ml of the whole blood was taken from the aorta femoralis in 1, 5, 15, 30 minutes, and in 1, 2, 3, 4, 6 hours, and then centrifuged to obtain clear supernatant plasma. Furthermore, the plasma drag concentration was analyzed according to the same process as Example 10, and the analysis results of the plasma concentrations of the drags are shown in the following Table 13 and Fig. 8.

Table 13

As shown in Table 13 and Fig. 8, the Ca .2+ -fixed polymeric micelles (Composition 11) had a longer bloodstream retention time than the injections containing other surfactants (Compositions 12 and 13). Since the Ca ⁺-fixed polymeric micelles (Composition 11) of the present invention had a longer bloodstream retention time than the marketed formulation Taxol^® (Composition 12), the present invention could increase the drug retention time in the bloodstream over Taxol^® by using the biodegradable and biocompatible polymers.

Example 15 Pharmacokinetics for the Ca²⁺-fϊxed paclitaxel-containing polymeric micelles To compare the bloodstream retention time of the Ca²⁺-fixed paclitaxel-containing polymeric micelles with that of the formulations containing other carriers, the compositions were prepared as follows.

(Composition 14) Ca²⁺-fϊxed paclitaxel-containing polymeric micelles Paclitaxel, mPEG-PLA-tocopherol (Mn: 2,000-1,800), and 5arm PLA-COONa (Mn: 3,000) 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 clear solution. Ethanol was removed therefrom using a vacuum evaporator to prepare a paclitaxel-containing polymeric composition. Distilled water (4 ml) was added thereto, and the mixture was stirred for 10 minutes at 60 °C to prepare a polymeric micelle aqueous solution containing paclitaxel. To the above polymeric micelle solution was added a CaCl₂ aqueous solution (concentration: 100 mg/ml) of the same equivalent as the 5arm PLA-COONa, 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 particle size of the polymeric micelles was 32 nm.

(Composition 12) Composition containing paclitaxel, Cremophor EL, and anhydrous ethanol Paclitaxel (30 mg) was dissolved in 5 ml of a mixed solution (50:50 v/v) of Cremophor EL and anhydrous ethanol to obtain a clear solution. The solution was passed through a filter having the pore size of 200 nm. The above composition and the drug contents are summarized in Table 14. Table 14

For the animal experiments, male Sprague-Dawley rats weighing 230-250 g were camiulated in the vena femoralis and aorta femoralis. Compositions 14 and 12 were injected into the vena femoralis at a dose of 5 mg/kg over 15 seconds. After the injection, 0.3 ml of the whole blood was taken from the aorta femoralis in 1, 5, 15, 30 minutes, and in 1, 2, 3, 4, 6 hours, and then centrifuged to obtain clear supernatant plasma. Furthermore, the plasma drug concentration was analyzed according to the same process as Example 10, and the analysis results of the plasma concentrations of the drags are shown in the following Table 15 and Fig. 9.

Table 15

As shown in Table 15 and Fig. 9, the Ca .2+ -fixed polymeric micelles (Composition 14) had a longer bloodstream retention time than the injections containing other surfactants (Compositions 12). Since the Ca²⁺-fϊxed polymeric micelles (Composition 14) of the present invention had a longer bloodstream retention time than the marketed formulation Taxol^® (Composition 12), the present invention could increase the drug retention time in the bloodstream over Taxol^® by using the biodegradable and biocompatible polymers. Example 16 Pharmacokinetics for the Ca²⁺-fixed docetaxel-containing polymeric micelles To compare the bloodstream retention time of the Ca²⁺-fixed docetaxel-containing polymeric micelles with that of the formulations containing other carriers, the compositions were prepared as follows.

(Composition 15) Ca²⁺- fixed docetaxel-containing polymeric micelles Docetaxel, mPEG-PLA-Tocopherol (Mn: 2,000-1,800), and 3 arm PLA-COONa (Mn: 3,000) 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 clear solution. Ethanol was removed therefrom using a vacuum evaporator to prepare a docetaxel-containing polymeric composition. Distilled water (4 ml) was added thereto, and the mixture was stirred for 10 minutes at 60 °C to prepare a polymeric micelle aqueous solution containing docetaxel. To the above polymeric micelle solution was added a CaCl₂ aqueous solution (concentration: 100 mg/ml) of the same equivalent as the 3 arm-PLA-COONa, 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 particle size of the polymeric micelles was 30 nm.

(Composition 16) Composition containing docetaxel, polysorbate 80 (Tween 80), and anhydrous ethanol Docetaxel (20 mg) and Tween 80 (520 mg) were dissolved in 1.5 ml of 13% (v/v) ethanol aqueous solution to obtain a clear solution. The solution was passed through a filter having the pore size of 200 nm.

The above composition and the drag contents are summarized in Table 16.

Table 16

For the animal experiments, male Sprague-Dawley rats weighing 210-240 g were cannulated in the vena femoralis and aorta femoralis. Compositions 15 and 16 were injected into the vena femoralis at a dose of 10 mg/kg over 15 seconds. After the injection, 0.3 ml of the whole blood was taken from the aorta femoralis in 5, 15, 30 minutes, and in 1, 2, 3, 6, 8 hours, and then centrifuged to obtain clear supernatant plasma.

Furthermore, the plasma drug concentration was analyzed according to the same process as Example 10, and the results of the plasma drag concentrations are shown in Table 17 and Fig.10

As shown in Table 17 and Fig. 10, the Ca²⁺- fixed polymeric micelles (Composition 15) had a longer bloodstream retention time than the injections containing Tween 80 (Compositions 16). Since the Ca²⁺- fixed polymeric micelles (Composition 15) of the present invention had a longer bloodstream retention time than the marketed formulation Taxotere^® (Composition 16), the present invention could increase the drug retention time in the bloodstream over Taxotere by using the biodegradable and biocompatible polymers.

Example 17 Pharmacokinetics for the Ca .2+ -fixed docetaxel-containing polymeric micelles To compare the bloodstream retention time of the Ca 2+ -fixed docetaxel-containing polymeric micelles with that of the formulations containing other carriers, the compositions were prepared as follows.

(Composition 17) Ca²⁺-fixed docetaxel-containing polymeric micelles Docetaxel, mPEG-PLA-tocopherol (Mn: 2,000-1,800), and D,L-PLA-COONa (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 clear solution. Ethanol was removed therefrom using a vacuum evaporator to prepare a docetaxel-containing polymeric composition. Distilled water (4 ml) was added thereto, and the mixture was stirred for 10 minutes at 60 °C to prepare a polymeric micelle aqueous solution containing docetaxel. To the above polymeric micelle solution was added a CaCl₂ aqueous solution (concentration: 100 mg/ml) of the same equivalent as the D,L-PLA-COONa, and the mixture was stirred for 20 minutes at room temperature. The mixture was passed through a filter with the pore size of 20O nm, and then was lyophilized. The hydrodynamic particle size of the polymeric micelles was 32 nm.

(Composition 16) Composition containing docetaxel, Tween 80, and 13%> ethanol Docetaxel (20 mg) and Tween 80 (520 mg) were dissolved in 1.5 ml of 13%. (v/v) ethanol aqueous solution to obtain a clear solution. The solution was passed through a filter having the pore size of 200 nm.

The above composition and the drug contents are summarized in Table 17.

Table 17

For the animal experiments, male Sprague-Dawley rats weighing 230-250 g were cannulated in the vena femoralis and aorta femoralis. Compositions 17 and 16 were injected into the vena femoralis at a dose of 5 mg/kg over 15 seconds. After the injection, 0.3 ml of the whole blood was taken from the aorta femoralis in 1, 5, 15, 30 minutes, and in 1, 2, 3, 4, 6 hours, and then centrifuged to obtain clear supernatant plasma.

Furthermore, the plasma drug concentration was analyzed according to the same process as Example 10, and the results of the plasma concentrations of the drugs are shown in the following Table 18 and Fig. 11.

Table 18

As shown in Table 18 and Fig. 11, the Ca -fixed polymeric micelles (Composition 17) had a longer bloodstream retention time than the injections containing Tween 80 (Compositions 16). Since the Ca²⁺-fixed polymeric micelles (Composition 17) of the present invention had a longer bloodstream retention time than the marketed formulation Taxotere^® (Composition 16), the present invention could increase the drug retention time in the bloodstream over Taxotere by using the biodegradable and biocompatible polymers.

Example 18 Maximum tolerated dose of the Ca²⁺-fϊχed paclitaxel-containing polymeric micelles 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. injections through the tail vein on a 0-, 1-, 2- day schedule of the Ca²⁺-fixed paclitaxel-containing polymeric micelle solution (Composition 11) at each dose of 16, 20, 25, 30 mg/kg. Mice survival and variation in the body weights were observed daily over 30 days in all the groups. 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. injections through the tail vein on a 0-, 2-, 4- day schedule of the Ca²⁺-fLxed paclitaxel-containing polymeric micelle solution (Composition 11) at each dose of 20, 25, 30, 35 mg/kg. Mice survival and variation in the body weights were observed daily over 30 days in all the groups.

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. injections through the tail vein on a 0-, 2-, 4-, 6-day schedule of the Ca²⁺-fixed paclitaxel-containing polymeric micelle solution (Composition 11) at each dose of 20, 25, 30 mg/kg. Mice survival and variation in the body weights were observed daily over 30 days in all the groups.

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-, 4-, 8- day schedule of the Ca²⁺-fixed paclitaxel-containing polymeric micelle solution

(Composition 11) at each dose of 25, 28, 30, 35, 39 mg/kg. Mice survival and variation in the body weights were observed daily over 30 days in all groups. The MTD was defined as the allowance of a median body weight loss of approximately 10-20%) of the control, to cause neither death due to toxic effects nor remarkable change in the general signs within 2 weeks after the drug administration. As shown in Table 16, the MTD in each dosing schedule was in a range of 20-30 mg/kg. A vehicle toxicity study was also done. The animals receiving drag-free Ca²⁺-fixed polymeric micelles grew rapidly, and gained slightly more weight than the animals receiving saline or not having injection. This was attributed to the calorie contents of the formulation. Table 19

Example 19 Anticancer activity of Ca²⁺-fixed paclitaxel-containing polymeric micelles Cells were taken from the storage in liquid nitrogen, and established as an in vitro cell culture. After the harvesting, cells were washed in sterile phosphate buffered saline (PBS), and the numbers of viable cells were determined. Cells were re-suspended in sterile PBS at the approximate concentration of 7^χ10⁷ cells/ml. Healthy 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 7><10⁶ human cancer cells (MX-1, SKOV-3, MDAMB435S, HT29, PC-3, U373MG). 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 to 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 100-300 mm³, the drag 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 micelles (Composition 11) and the Cremophor EL formulation (Composition 12) were administered at various doses of paclitaxel through the tail vein, and the volumes of the cancers were measured at different time intervals. The volumes of the cancers were calculated by the formula (W^"χL)/2 wherein W is a short axis, and L is a long axis. For the evaluation of treatment, tumor volumes were calculated as follows: Tumor volumes (TV) = 0.5 L> W² (L : long axis, W: short axis) Relative tumor volume (RTN) - (N_t/No) ^x 100% (Nt : TV on day t, V0: TV on day 0) Treatment efficacy was determined by 3 criteria used in parallel: mean tumor growth curves, optimal growth inhibition (T/C%>), and specific growth delay (SGD) The optimal growth inhibition at a particular day within 4 weeks after the last injection was calculated from the mean RTV values of treated versus control groups multiplied by 100% (T/C%) The SGD was calculated over one and two doubling times as follows: Specific Growth Delay (SGD) : SGD=(T_D treated-T_D control)/T_D control T_D : Tumor-doubling time The levels of activity are defined as follows: τ/c% SGD (+) <50 or >1.0 + <50 and >1.0 ++ <40 and >1.5 +++ <25 and >2.0 ++++ <10 and >3.0

According to NCI standards, a T/C ≤ 42% is the minimum level for activity. A T/C < 10%) is considered as a high anti-tumor activity level which justifies further development.

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 treatment, the minimum tumor diameter was 4 mm or the volume of 30 mm . The animals dying within 2 weeks after the final drug administration were considered as toxic deaths, and were excluded 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 complete recovery was considered not evaluable for antitumor efficacy.

As shown in Figs. 12a -12h and Table 20, both the metal ion-fixed polymeric micelle-treated group and the Cremophor EL formulation-treated group showed a considerable inhibition rate on cancer growth compared with the control group, and particularly, the metal ion-fixed polymeric micelle (Composition 1 l)-treated group showed a higher inhibition rate than the Cremophor EL formulation (Composition 12)-treated group.

Table 20

Example 20 Anticancer activity

paclitaxel-containing polymeric micelles against Taxol^® resistant cancer animal model Cells were taken from the storage in liquid nitrogen, and established as an in vitro cell culture. After the harvesting, cells were washed in sterile phosphate buffered saline (PBS), and the numbers of viable cells were determined. Cells were re-suspended in sterile PBS at the approximate concentration of 7 10⁷ cells/ml. Healthy 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 7xl0⁶ human cancer cells (HT29). After the cancers reached a certain size, they were xeno grafted three times to form xenograft fragments of 3-4 mm. The xenograft fragments were subcutaneously injected to 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 a certain size, the paclitaxel (Cremophor EL formulation, Taxol^®) was administered at the dose of 20 mg/kg/day under the dosing schedule of qldX5 through the tail vein. After 3 weeks, the drag 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 subcutaneously injected to 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 100-300 mm³, the drag was admimstered, 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 micelles (Composition 11) and the Cremophor EL formulation (Composition 12) were administered at various doses of paclitaxel through the tail vein, and the volumes of the cancers were measured at different time intervals.

As described in the above experiment, to demonstrate the effectiveness of metal ion-fixed polymeric micelles against the Taxol -resistant cancer, an animal model for in vivo anti-cancer activity against Taxol^®-resistant cancer was established. When cancer cells inoculated into mice were exposed by Taxol repeatedly, IC₅₀ of paclitaxel for Taxol^φ-pretreated cancer cells was increased significantly compared to that of paclitaxel for the native cancer cells (data not shown). In this animal model, the metal ion-fixed polymeric micelle (Composition 1 l)-treated group showed a higher inhibition rate than the Cremophor EL formulation (Composition 12)-treated group possibly due to the longer retention of effective concentration of the drag incorporated in the metal ion-fixed polymeric micelle as shown in Fig. 13 and Table 21.

Table 21

INDUSTRIAL APPLICABILITY

The compositions of the present invention can form stable polymeric micelles or nanoparticles in body fluids or aqueous solutions. The micelles or nanoparticles formed from the compositions of the present invention have a hydrophilic outer shell and a hydrophobic inner core wherein a large amount of hydrophobic drug can be physically trapped. The drag containing micelles and nanoparticles of the present invention have a prolonged retention time in the bloodstream after administration, and can be utilized to make various pharmaceutical formulations.

It is to be understood that the above-described embodiments are only illustrating the applications of the principles of the present invention. Numerous modifications and alternative 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 fully 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 will be apparent to those of ordinary skill 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 claims.

Claims

WHAT IS CLAIMED IS:

1. A polymeric composition for drag delivery, said composition comprising an amphiphilic block copolymer of a hydrophilic block and a hydrophobic block wherein the hydroxyl terminal group is substituted with a tocopherol or cholesterol group, and a polylactic acid derivative wherein one end of the polylactic acid is covalently bound to at least one carboxyl group.

2. The polymeric composition of Claim 1, wherem the polylactic acid derivative is represented by the following formula:

RO-CHZ-[A]_n-[B]_m-COOM (I) wherein A is -COO-CHZ-; B is -COO-CHY-, -COO-CH₂CH₂CH₂CH₂CH₂- or - COO-CH₂CH₂OCH ; R is a hydrogen atom, or acetyl, benzoyl, decanoyl, palmitoyl, methyl, or ethyl group; Z and Y each are hydrogen atoms, or methyl or phenyl 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.

3. The polymeric composition of Claim 1, wherein the polylactic acid derivative is represented by the following formula:

RO-CHZ-[COO-CHX]p-[COO-CHY']_q-COO-CHZ-COOM (II) wherein X is a methyl group; Y' is a hydrogen atom or phenyl 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 acetyl, benzoyl, decanoyl, palmitoyl, methyl or ethyl group; Z is a hydrogen atom, or methyl or phenyl group; and M is H, Na, K, or Li.

4. The polymeric composition of Claim 1, wherein the polylactic acid derivative is represented by the following formula:

RO-PAD-COO-W-M' (III)

COOM C CHzCOOM cooM I I wherein W-M' is CH COOM _or CH — CH₂C00M_; PAD is one selected from the group consisting of D,L-polylactic acid, D-polylactic acid, polymandelic acid, a copolymer of D,L-lactic acid and glycolic 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 is a hydrogen atom, or acetyl, benzoyl, decanoyl, palmitoyl, methyl or ethyl group; and M is H, Na, K, or Li.

5. The polymeric composition of Claim 1, wherein the polylactic acid derivative is represented by the following formula:

S-O-PAD-COO-Q (IV)

wherein S is

_loalkyl; Q is CH₃, CH₂CH₃, CH₂CH₂CH₃, CH₂CH₂CH₂CH₃, or CH₂C₆H₅; a is an integer from 0 to 4; b is an integer from 1 to 10; M is H, Na, K, or Li; and PAD is one selected from the group consisting of D,L-polylactic acid, D-polylactic acid, polymandelic acid, a copolymer of D,L-lactic acid and glycolic 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.

6. The polymeric composition of Claim 1, wherein the polylactic acid derivative is represented by the following formula: (V)

wherein R' is -PAD-0-C(O)-CH₂CH₂-C(O)-OM and PAD is one selected from the group consisting of D,L-polylactic acid, D-polylactic acid, polymandelic acid, a copolymer of D,L-lactic acid and glycolic 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 1 ,4-dioxan-2-one; M is as defined in formula (I); and a is an integer from 1 to 4.

7. The polymeric composition of Claim 1, wherein the hydrophilic block is one selected from the group consisting of polyalkylene glycols, polyvinyl pyrrolidone, polyvinyl alcohols, an polyacryl amides, and the hydrophobic block is one selected from the group consisting of polylactides, polyglycolides, polydioxan-2-one, polycaprolactone, polylactic-co-glycolide, polylactic-co-caprolactone, polylactic-co- dioxan-2-one, and derivatives thereof wherein the hydroxyl terminal group is substituted with a tocopherol or cholesterol group.

8. The polymeric composition of Claim 7, wherein the amphiphilic block copolymer is represented by the following formula: R_r-O-[R₃,]_r-[R_4>]_m.-[R₅>]_n.-C(=O)-(CH₂)_x-C(=O)-O-R₂. (F) wherein R_r is CH₃-, or R_2>-O-C(=O)-(CH₂)_x,-C(=O)-[R₅.]_n.-[R₄.]_m>-; R₂> is tocopherol or cholesterol;

R4' is -C(^:=O)-CHZ'-O-, wherein Z' is a hydrogen atom or methyl; R₅- is -C(=O)-CHY"-O-, wherein Y" is a hydrogen atom or methyl, -C(=0)- CH₂CH₂CH₂CH₂CH₂-O-, or -C(=O)-CH₂OCH₂CH₂-O-; 1' is an integer of 4-1150; m' is an integer of 1-300; n' is an integer of 0-300; and X' is an integer of 0-4.

9. The polymeric composition of Claim 7, wherein the hydrophilic and hydrophobic blocks have a number average molecular weight within the range of 500 to 50,000 Daltons, respectively.

10. The polymeric composition of Claim 1, wherein the ratio of the hydrophilic block to the hydrophobic block in the amphiphilic block copolymer is 3:7 to 8:2.

11. The polymeric composition Claim 1, comprising 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 total weight of the composition.

12. The polymeric composition of Claim 1, wherein the polylactic acid derivative has a number average molecular weight of 500 to 2,500 Daltons.

13. The polymeric composition of Claim 1, wherein the polylactic acid derivative is in the form of sodium or potassium salt obtained by a condensation reaction in the absence of catalyst followed by neutralization with sodium carbonate, sodium hydrogen carbonate, potassium hydrogen carbonate, or potassium carbonate.

14. A polymeric composition for drag delivery, comprising a polymeric composition of one of Claims 1 to 13, and 0.01 to 10 equivalents of the di- or tri-valent metal ion with respect to 1 equivalent of the carboxyl terminal group of the polylactic acid derivative.

15. The polymeric composition of Claim 14, wherein the di- or tri-valent metal ion is one selected from the group consisting of Ca²⁺, Mg²⁺, Ba²⁺, Cr³⁺, Fe³⁺, Mn²⁺, Ni²⁺, Cu²⁺, Zn²⁺, and Al³⁺.

16. A micelle or nanoparticle prepared from the polymeric composition of Claims 1 to 15.

17. The micelle or nanoparticle of Claim 16, wherein the particle size of micelle or nanoparticle is within the range of 1 to 400 nm.

18. A pharmaceutical composition comprising 70 to 99.9 wt % of the polymeric composition of Claim 14 and 0.1 to 30 wt% of a poorly water-soluble drug.

19. A process for preparing a polymeric composition containing a poorly water-soluble drug, comprising the steps of dissolving an amphiphilic block copolymer of a hydrophilic block and a hydrophobic block wherein the hydroxyl terminal group is substituted with a tocopherol or cholesterol group, a polylactic acid derivative wherein one end of the polylactic acid is covalently bound to at least one carboxyl group, and a poorly water-soluble drag in an organic solvent, evaporating the organic solvent, and adding an aqueous solution to form the poorly water-soluble drug-containing polymeric micelles.

20. The process of Claim 19, further comprising the step of adding a di- or tri-valent metal ion to the poorly water-soluble drug-containing polymeric micelles to fix the carboxyl terminal group of the polylactic acid derivative.

21. The process of Claim 19, wherein the organic solvent is one or more selected from the group consisting of acetone, ethanol, methanol, ethyl acetate, acetonitrile, methylene chloride, chloroform, acetic acid, and dioxane.