POLYMERIC WATER SOLUBLE PRODRUGS CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application is related to provisional patent application U. S. Serial No.

60/544,694 filed February 13,2004, which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION [0002] The present invention relates to the field of pharmaceutical compositions for the treatment of cancer or other diseases. In particular, the present invention relates to making anti- cancer agents water soluble and less toxic by conjugating the drug to composite polymers.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT [0003] Not applicable.

BACKGROUND OF THE INVENTION [0004] Systemic cancer treatment relies on chemotherapy drugs, which often have poor water solubility and excessive toxicity. For example, a major difficulty in the development of paclitaxel, an anti-cancer drug, for clinical trial use has been its insolubility in water. Current formulation uses ethanol and Cremophor EL to render paclitaxel soluble, which causes allergic reactions and induces side effects such as PVC tube leaching, drug precipitation, and excessive toxicities. Premedications and long infusions are frequently necessary.

[0005] Conjugation of chemotherapeutic agents to polymers has been an attractive approach to reduce systemic toxicity and improve the chemotherapeutic index. Large molecular weight polymer conjugated chemotherapeutic agents do not readily diffuse through normal capillaries and glomerular endothelium, thus sparing normal tissues from irrelevant drug-mediated toxicity (Maeda and Matsumura, 1989; Reynolds, 1995). Accordingly, the tumor specific accumulation of macromolecules by so-called enhanced permeability and retention effect has been used as a means to target tumor passively (Fidler et al., Cancer Chemother. Pharmcol. 34: 465-71,1987).

Additionally, polymer-drug conjugates may act as drug depots for sustained release, producing prolonged drug exposure to tumor cells.

[0006] At present, a variety of synthetic and natural polymers have been examined for their ability to enhance tumor-specific drug delivery (Kopecek, J. Controlled Release 11: 279-90, 1990; Maeda and Matsumura, Rev. Ther. Drug Carrier Systems 6: 193-20,1989). However, only a few are currently undergoing clinical evaluation, including SMANCS in Japan, HPMA-Dox in the United Kingdom, and polyglutamate Paclitaxel in the US (Kopecek and Kopeckova, Pro.

Intl. Symp. Controleld Release Bioactive Material 20: 190-1,1993 ; Li et al. , Cancer Res.

58: 2404-9,1998).

[0007] Prior attempts to obtain water soluble paclitaxel include conjugating moieties such as succinate and amino acids at the 2'-hydroyl group or at the 7-hydroxyl position of paclitaxel (Deutsch et al. , J. Med. Chem. 32: 788-92,1989 ; Mathew et al. , J. Med. Chem. 35: 145-51, 1992). Other attempts include conjugating hydrophilic polyethylene glycol to 2'or 7-hydroxyl <BR> <BR> position of paclitaxel to produce water soluble paclitaxl esters (Greenwald et al. , Bioorganic &amp; Med. Chem. Lett. 4: 2465-70,1994) and using microencapsulation, liposomes, or emulsions as vehicles to deliver paclitaxel (Bartoni and Boitard, J. Microcapsul. 7: 191-7,1990).

[0008] Animal studies and early clinical data showed encouraging results using a conjugate containing paclitaxcel conjugated to polyglutamate to form 2'and 7 position esters (hereinafter <BR> <BR> referred to as PG-paclitaxel) (Li. Et al., Cancer Res. 58: 2404-9,1998 ; Li et al. , Clinical Cancer Res. 5,891-7, 1999; US Pat. No. 5,977, 163; US Pat. No. 6,441, 025). The polymeric formulation was shown to be water soluble and less toxic than unconjugated paclitaxel. However, drug distribution studies revealed high retention of the PG-paclitaxel conjugates in liver, spleen, and kidney (Li et al., Cancer Chemotherapy Pharmcol., 46: 461-22,2000). For example, when the AUC (area under curve) of paclitaxel and PG-paclitaxel are compared, 5-fold higher tumor retention for PG-paclitaxel was observed. In contrast, the retention of PG-paclitaxel was increased by 18,82, and 31 fold for liver, spleen, and kidney, respectively. Such un-proportional increased retention of PG-paclitaxel may cause unforeseen damage to these vital organs. There is therefore a need for new compositions of paclitaxel and other anti-cancer drugs.

[0009] All references, publications, patents, and patent applications disclosed herein are hereby incorporated by reference in their entirety.

BRIEF SUMMARY OF THE INVENTION [0010] The present invention provides a conjugate and use of a conjugate comprising a chemotherapeutic agent (such as an anti-tumor drug) conjugated to a water soluble polyamino acid polymer, wherein the water soluble polyamino acid polymer is conjugated to a hydrophilic polymer such as polyethylene glycol (hereinafter referred to as PEG).

[0011] In some embodiments, the water soluble polyamino acid polymer is selected from the group consisting of a polyglutamic acid polymer, a poly-aspartic acid polymer, and a poly-lysine polymer. In some embodiments, the polyglutamic acid polymer is a copolymer with a polycaprolactone, a polyglycolic acid, a polylactic acid, a polyacrylic acid, a poly (2- hydroxyethyl 1-glutamine), a carboxymethyl dextran, a hyaluronic acid, a human serum albumin, a polyalginic acid or a combination thereof. In some embodiments, the mass ratio between the water soluble polyamino acid polymer and the hydrophilic polymer (such as PEG) is between 20: 80 and 80: 20.

[0012] In some embodiments, the chemotherapeutic drug is an anti-tumor drug. In some embodiments, the anti-tumor drug is paclitaxel. For example, the water soluble polyamino acid polymer may be conjugated to a hydroxyl group of paclitaxel. In some embodiments, the anti- tumor drug is selected from the group consisting of paclitaxel, docetaxel, etopside, teniposide, camptothecin, rapamycin, doxorubicin, estradiol, vinka alkoids, or epothilone.

[0013] In some embodiments, the conjugate comprises an anti-tumor drug conjugated to a water soluble polyamino acid polymer having a molecular weight between about 5000 and about 50,000 Da, wherein said water soluble polyamino acid polymer is conjugated to multiple PEG molecules having a molecular weight between about 500 and about 200,000, wherein said conjugate having a higher water solubility and higher ability to accumulate in a tumor than the unconjugated anti-tumor drug, wherein said anti-tumor drug is selected from the group consisting of paclitaxel, docetaxel, etopside, teniposide, camptothecin, rapamycin, doxorubicin, estradiol, vinka alkoids, or epothilone. In some embodiments, the water soluble polyamino acid polymer is poyglutamic acid. In some embodiments, the anti-tumor drug is paclitaxel.

[0014] In some embodiments, the conjugate comprises a paclitaxel conjugated to a polyglutamic acid, wherein the polyglutamic acid is conjugated to one or more PEG molecules (such as the PEG-PG-paclitaxel described herein).

[0015] The present invention provides a pharmaceutical composition comprising a conjugate of the present invention and a pharmaceutical acceptable carrier.

[0016] The present invention also provides a method of making a conjugate or a pharmaceutical composition described herein, comprising: a) conjugating a chemotherapeutic agent to a water soluble polyamino acid polymer ; and b) conjugating a polyethylene glycol to the water soluble polyamino acid polymer. In some embodiments, these two conjugation steps are carried out simultaneously, for example, by allowing the water soluble polyamino acid polymer and the chemotherapeutic agent to react with each other in the presence of the hydrophilic polymer. In some embodiments, the water soluble polyamino acid polymer is a polyglutamic acid polymer. In some embodiments, the chemotherapeutic agent is paclitaxel.

[0017] In some embodiments, the method of making a conjugate comprises: reacting an antitumor drug with a water soluble polyamino acid polymer and a PEG under a condition that allows conjugation between the antitumor drug and the polyamino acid polymer and conjugation between the polyamino acid polymer and the PEG, and whereby a conjugate comprising the anti- tumor drug conjugated to the water soluble polyamino acid polymer which is conjugated to the PEG is generated.

[0018] The present invention provides a method of treating cancer in a subject comprising administering an effective amount of a pharmaceutical composition described herein to the subject. In some embodiments, the cancer to be treated is selected from the group consisting of breast cancer, ovarian cancer, malignant melanoma, lung cancer, gastric cancer, prostate cancer, colon cancer, head and neck cancer, leukemia, or Kaposi's Sarcoma.

BRIEF DESCRIPTION OF THE DRAWINGS [0019] Figure 1 provides chemical structures of paclitaxel, paclitaxel-polyglutamic acid conjugate (PG-paclitaxel), and a conjugate of PEG with PG-paclitaxel (PE-PG-paclitaxel).

[0020] Figure 2 shows the effects of paclitaxel and PEG-PG-paclitaxel on proliferation of Lewis Lung carcinoma cells.

[0021] Figure 3 shows the effects of paclitaxel and PEG-PG-paclitaxel on proliferation of C26 colon carcinoma cells.

[0022] Figure 4 shows the effects of paclitaxel and PEG-PG-paclitaxel on the body weight of Lewis Lung carcinoma bearing mice.

[0023] Figure 5 shows the anti-tumor effects of paclitaxel and PEG-PG-paclitaxel on OCa-1 carcinoma.

DETAILED DESCRIPTION OF THE INVENTION [0024] The present invention provides a unique approach to reduce problems associated with chemotherapeutic agents (such as anti-tumor drugs) by incorporating hydrophilic polymers (such as PEG) to polymer-chemotherapeutic agent conjugates. Specifically, the present invention provides a conjugate comprising a chemotherapeutic agent (such as an anti-tumor drug) conjugated to a water soluble polyamino acid polymer, wherein the water soluble polyamino acid polymer is conjugated to a hydrophilic polymer (such as PEG).

[0025J One possible reason for excessive retention of PG-paclitaxel conjugate in normal tissues is that the conjugate is uptaken and scavenged by the reticulo-endothelial system (RES), which is designed to clear particular and charged particles like PG-paclitaxel. Without being bound by the theory, the hydrophilic polymer moiety of the conjugate of the present invention can shield the charges on the polyamino acid polymer-chemotherapeutic agent conjugate, thereby reducing the uptake or scavenge of the overall molecule by the RES. The compositions disclosed herein are water soluble, significantly less toxic, and are equal or more effective than unconjugated chemotherapeutic agents in treating tumors or other diseases in animal studies.

The conjugate may also act as a drug depot for sustained release. Furthermore, the compositions described herein avoid the use of organic solvents such as ethanol and Cremophor EL, and thus reduce side effects associated with such solvents.

[0026] The present invention further provides pharmaceutical compositions comprising the conjugate described above, and method of treating cancer by administering an effective amount of the pharmaceutical composition. Methods for making the conjugate and the pharmaceutical composition are also provided.

Conjugate [0027] The present invention provides a conjugate comprising a chemotherapeutic agent (such as an anti-tumor drug) conjugated to a water soluble polyamino acid polymer, wherein said water soluble polymer is conjugated to a hydrophilic polymer (such as PEG)."Conjugate"used herein refers to a complex formed by covalently attaching one moiety to another moiety.

Hydrophilic polymer [0028) In some embodiments, the hydrophilic polymer moiety of the conjugate of the present invention is PEG, including its derivatives such as methoxy-PEG. Other suitable hydrophilic polymers include, but are not limited to, polyethylene oxide (PEO), polyvinyl alcohol (PVA), and polyvinylpyrrolidone (PVP), or derivatives thereof. In some embodiments, the hydrophilic polymer is a polymer that can shield the overall macromolecule from ionic or other molecular interactions.

[0029] In some embodiments, the hydrophilic polymer has a molecular weight of at least about 500 Da, such as between about 500 Da to about 200,000 Da, such as between about 500 to about 20,000 Da, such as between about 3000 to about 8000 Da.

[0030] In some embodiments, the hydrophilic polymer is conjugated to the water soluble polyamino acid polymer via the side chain (s) of the polyamino acid polymer. For example, when the polyamino acid polymer is polyglutamic acid, the hydrophilic polymer can be conjugated to the side chain carboxyl group (s) of the polyglutamic acid. The functional group of the hydrophilic polymer can be-OH, -SH,-COOH, or NH2. The chemical bond formed between the water soluble polyamino acid polymer (such as polygluamate or polyaspatate) and hydrophilic polymer can be stable like amide bond or reversible like ester bond.

[0031] The amount of hydrophilic polymer conjugated to the water soluble polyamino acid polymer may depend on the effective amount of hydrophilic polymer that is needed for shielding the water soluble polyamino acid polymer-chemotherapeutic agent conjugate from being scavenged by the RES system. In some embodiments, the mass ratio between the water soluble polyamino acid polymer and the hydrophilic polymer is between about 20: 80 to about 80: 20, such as between about 30: 70 to about 70: 30, between 40: 60 to about 60: 40, or about 50: 50. In some embodiments, the total mass of hydrophilic polymer molecules per conjugate is about 60,000 to about 70,000 Da.

[0032] The number of hydrophilic polymer molecules to be conjugated to the water soluble polyamino acid polymer may depend on the size of each hydrophilic polymer molecule and possibly the effective total amount that is needed for shielding the water soluble polyamino acid polymer-chemotherapeutic agent conjugate from being scavenged by the RES system. In some embodiments, the conjugate comprises multiple hydrophilic polymer molecules conjugated to a single water soluble polyamino acid polymer molecule. For example, about 2, about 3, about 5, about 6, about 7, about 8 or more hydrophilic polymer molecules may be conjugated to one water soluble polyamino acid polymer molecule. These multiple hydrophilic polymers may be of the same size or be different in sizes.

Chemotherapeutic agents [0033] Suitable chemotherapeutic agents for conjugates of the present invention include, but are not limited to, paclitaxel, doxcetaxel, etopside, teniposide, fludarabine, doxorubicin, daunomycin, emodin, 5-fluorouracil, FUDR, estradiol and derivatives, camptothecin, retinoic acids, verapamil, epothilones, rapamycin, cisplatin, vinka alkoids, and cyclosporins. In some embodiments, the chemotherapeutic agent is an anti-tumor drug.

[0034] The chemotherapeutic agent can be conjugated to a water soluble polyamino acid polymer in various ways known in the art. For example, when the chemotherapeutic agent is paclitaxel, the water soluble polymer can be conjugated to the 2'or 7-hydroxyl group or both of the paclitaxel. When functional groups are used for drug conjugation, as above with the C2'- hydroxyl of paclitaxel, a degradable linkage, in this case, an ester, can be used to ensure that the active drug is released from the polymeric carrier.

[0035] The amount of chemotherapeutic agent present in the conjugate may vary. At the lower end, the conjugate may comprise from about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21% about 22%, about 23%, about 24%, to about 25% (w/w) chemotherapeutic agent relative to the mass of the conjugate. At the high end, the conjugate may comprise from about 26%, about 27%, about 28%, about 29%, about 30%, about 31% about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, to about 40% or more (w/w) chemotherapeutic agent relative to the mass of the conjugate.

[0036] The number of chemotherapeutic agent molecules conjugated to each molecule of water soluble polyamino acid polymer may also vary. At the lower end, the conjugate may comprise from about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, to about 20 or more molecules of chemotherapeutic agent per molecule of water soluble polyamino acid polymer. At the higher end, such a composition may comprise from about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60 about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, to about 75 or more molecules of chemotherapeutic agent per molecule of water soluble polyamino acid polymer.

[0037] In some embodiments, the chemotherapeutic agent is paclitaxel. Paclitaxel is an anti- microtubule agent originally extracted from the needles and barks of the Pacific yew tree, Taxus brevifolia. Paclitaxel has been reported to be active against ovarian, breast, small-cell and non- small cell lung cancer, head and neck cancers, and metastatic melanoma. In some embodiments, the chemotherapeutic agent comprises a free hydroxyl group and can be conjugated to the polymers in a similar way as what is described for paclitaxel. These agents include, but are not limited to, etopside, teniposide, camptothecin, rapamycin, estradil, and epothiliones.

[0038] It is further contemplated that more than one kind of chemotherapeutic agents (or other therapeutic drugs) can be conjugated to a single water soluble polyamino acid polymer molecule. For example, a conjugate comprising paclitaxel conjugated to a polyglutamic acid may also comprise a second lipophilic or poorly soluble anti-cancer agent such as camptothecin, epothilone, cisplaitin, melphalan, Taxotere, etoposide, teniposide, fludarabine, verapamil, or cyclosporine, or sometimes water soluble agents such as 5 flurouracil (5FU), fluorodeoxyuridine (FUDR), doxorubicin or daunomycin.

Water soluble polyamino acid polymer <BR> <BR> [0039] "Water soluble polyamino acid polymer"or"water soluble polymer of amino acid" used herein include, but not limited to, polyglutamic acid, polyaspartic acid, poly-lysine, and amino acid chains comprising mixtures of glutamic acid, aspartic acid, and/or lysine, of either d and/or 1 isomer conformation.

[0040] As used herein, the terms"a polyglutamic acid"or"polyglutamic acids"include "poly (l-glutamic acid), poly (d-glutamic acid), poly (dl-glutamic acid) and the terms"a polyaspartic acid"or"polyaspartic acids"include poly (1-aspartic acid), poly (d-aspartic acid), and poly (dl-aspartic acid). In some embodiments, the water soluble polyamino acid polymer is polyglutamic acid, polyaspartic acid, polylysine, or copolymers of the above-listed polyamino acids with polycaprolactone, polyglycolic acid, polylactic acid, polyacrylic acid, poly (2- hydroxyehyl-1-glutamine), carboxymethyl dextran, hyaluronic acid, human serum albumin and polyalginic acid, or a combination thereof.

[0041] At the lower end of molecular weight, the water soluble polyamino acid polymers of the present invention have a molecular weight of about 1,000, about 2,000, about 3,000, about 4,000, about 5,000, about 6,000, about 7,000, about 8,000, about 9,000, about 10,000, about 11,000, about 12,000, about 13,000, about 14,000, about 15,000, about 16,000, about 17,000, about 18,000, about 19,000, about 20,000, about 21,000, about 22,000, about 23,000, about 24,000, about 25,000, about 26,000, about 27,000, about 28,000, about 29,000, about 30,000, about 31, 000, about 32,000, about 33,000, about 34,000, about 35,000, about 36,000, about 37,000, about 38,000, about 39,000, about 40,000, about 41,000, about 42,000, about 43,000, about 44,000, about 45,000, about 46,000, about 47,000, about 48,000, about 49,000, or about 50,000 Da. At the higher end of molecular weight, the water soluble amino acid polymers of the present invention have a molecular weight of about 51, 000, about 52,000, about 53, 000, about 54,000, about 55,000, about 56,000, about 57,000, about 58,000, about 59,000, about 60,000, about 61,000, about 62,000, about 63,000, about 64,000, about 65,000, about 66,000, about 67,000, about 68,000, about 69,000, about 70,000, about 71,000, about 72,000, about 73,000, about 74,000, about 75,000, about 76,000, about 77,000, about 78,000, about 79,000, about 80,000, about 81,000, about 82,000, about 83,000, about 84,000, about 85,000, about 86,000, about 87,000, about 88,000, about 89,000, about 90,000, about 91,000, about 92,000, about 93,000, about 94,000, about 95,000, about 96,000, about 97,000, about 98,000, about 99,000, or about 100,000 Da. Within these ranges, the ranges of molecular weights for the polymers are preferably about 5,000 to about 100,000 Da, such as about 20,000 to about 80,000 Da, such as about 30,000 to about 60,000 Da. In some embodiments, the molecular weight of the water soluble polyamino acid polymer is between about 5,000 to about 50,000 Da.

[0042] In some embodiments, various substitutions of naturally occurring, unusual, or chemically modified amino acids may be made in the amino acid composition of the water soluble polyamino acid polymer, such as polyglutamic acid, to produce a conjugate of the present invention and still obtain molecules having like or otherwise desirable characteristics of solubility and/or therapeutic efficacy. Naturally occurring, modified, or unusual amino acids that can be incorporated into a water soluble polyamino acid polymer are known in the art, and have been described in U. S. Pat. No. 6,441, 025. Suitable water soluble polyamino acid polymers with various substitutions may be identified by methods known in the art, for example by assaying for improved aqueous solubility of the polymer-chemotherapeutic agent conjugate relative to the unconjugated chemotherapeutic agent. In some embodiments, a polymer-antitumor drug conjugate may be identified by an improved anti-tumor cell activity, relative to that of the unconjugated anti-tumor drug.

[0043] In some embodiments, a water soluble polyamino acid polymer such as polyglutamic acid, poly-aspartic acid, poly-lysine, or water soluble amino acids chain or polymer comprising a mixture of glutamic acid, aspartic acid, and/or lysine, may, at the lower end of the amino acid substitution range, have about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25 or more glutamic acid, aspartic acid, or lysine, residues, respectively, substituted by any of the naturally occurring, modified, or unusual amino acids. In some embodiments, a water soluble polyamino acid polymer such as polyglutamic acid, poly-aspartic acid, poly-lysine, or a polyamino acid chain comprising a mixture of some or all of these three amino acids may, at the lower end, has about 1 %, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, to about 25% or more glutamic acid, aspartic acid, or lysine residues, respectively, substituted by any of the naturally occurring, modified, or unusual amino acids.

[0044] In some embodiments, a water soluble polyamino acid polymer such as polyglutamic acid, poly-aspartic acid, or poly-lysine may, at the high end of the amino acid substitution range, have about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, to about 50% or so of the glutamic acid, aspartic acid, or lysine residues, respectively, substituted by any of the naturally occurring, modified, or unusual amino acids, as long as the majority of residues (such as more than 50%) comprise glutamic acid and/or aspartic acid and/or lysine. In amino acid substitution of the various water soluble amino acid polymers, residues with a hydrophilicity index of +1 or more are preferred.

Pharmaceutical composition [0045] The present invention also provides a pharmaceutical composition comprising a conjugate described above.

[0046] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents and isotonic agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the chemotherapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

The phrase"pharmaceutically acceptable"also refers to molecular entities and compositions that do not produce an allergic or similar undesired reaction when administered to an animal or a human.

[0047] In some embodiments, the present invention provides a pharmaceutical composition suitable for injection in sterile solutions of dispersions. The pharmaceutical composition may be in the form of liquid, or powder to be reconstituted before injection. In some embodiments, the pharmaceutical composition further contains solvents, recipients, preservatives or additives that make the formulation stable, sterile, and suitable for injection.

Method of making the conjugate [00481 The present invention also provides a method of making the conjugate and pharmaceutical composition described herein. Specifically, the method comprises the steps of a) conjugating a chemotherapeutic agent to a water soluble polyamino acid polymer and b) conjugating a hydrophilic polymer (such as PEG) to the water soluble polyamino acid polymer.

Methods of conjugating a chemotherapeutic agent to a water soluble polyamino acid polymer are known in the art. In some embodiments, the two conjugation steps are carried out sequentially (i. e. , step a) is carried out first, then step b) is carried out). In some embodiments, the two conjugation steps are carried out simultaneously. For example, the water soluble polyamino acid polymer and the chemotherapeutic agent can be allowed to react with each other in the presence of the hydrophilic polymer (such as methoxy-PEG). In some embodiments, the present invention provides a method of making a conjugate, said method comprising reacting a chemotherapeutic agent with a water soluble polyamino acid polymer and a hydrophilic polymer (such as PEG) under a condition that allows conjugation between the antitumor drug and the polyamino acid polymer and conjugation between the polyamino acid polymer and the hydrophilic polymer (such as PEG), and whereby a conjugate comprising the anti-tumor agent conjugated to the water soluble polyamino acid polymer which is conjugated to the hydrophilic polymer is generated. The conversion of the chemotherapeutic agent to the desired conjugate can be monitored using methods known in the art (e. g. , by thin layer chromatography). The<BR> conjugate generated may then be purified using any methods known in the art (e. g. , by Sephadex chromatography).

Use of the conjugate for treating diseases [0049] The present invention further provides a method of treating cancer or other diseases in a subject by administering an effective amount of the conjugate or pharmaceutical composition of the present invention. A"subject"is a mammal and includes, but is not limited to, human, bovine, primate, equine, canine, feline, porcine, and ovine animals. An"effective amount"is an amount sufficient to effect beneficial or desired results including clinical results or delaying the onset of the disease. An effective amount can be administered in one or more administrations.

[0050] It is contemplated that the effectiveness of the conjugated chemotherapeutic agent will not be diminished as compared to the unconjugated chemotherapeutic agent. Therefore, the compositions of the present invention are expected to be as effective as the unconjugated chemotherapeutic agent against any diseases for which the unconjugated chemotherapeutic agent is shown to be effective. For example, when the chemotherapeutic agent is an anti-tumor drug (such as paclitaxel), the composition can be used to treat any type of cancer including, but not limited to, breast cancer, ovarian cancer, malignant melanoma, lung cancer, gastric cancer, prostate cancer, colon cancer, head and neck cancer, leukemia, or Kaposi's Sarcoma. As used herein the term"treating"cancer is understood as meaning any medical management of a subject having a tumor. The term would encompass any inhibition of tumor growth or metastasis, or any attempt to inhibit, slow or abrogate tumor growth or metastasis. The method includes killing a cancer cell by non-apoptotic as well as apoptotic mechanism of cell death. The method of treating a tumor may include some prediction of the anti-tumor drug uptake in the tumor prior to administering a chemotherapeutic amount of the drug, by methods that include but are not limited to bolus injection or infusion, as well as intraarterial, intravenous, intraperitoneal, or intratumoral administration of the drug.

[0051] The composition of the present invention may also be administered in conjunction with other drugs, such as other anti-tumor or anti-cancer drugs. For example, the paclitaxel conjugate may, in certain types of treatment, be combined with a platinum drug, an antibiotics such as doxorubicin or daunorubicin, or other drugs that are used in combination with Taxol.

Use of the conjugate for coating implanted devices [0052] The composition of the present invention may also be useful for coating onto the surface of implanted medical devices, such as tubing, shunts, catheters, artificial implants, pins, electrical implants such as pacemakers, and especially for arterial or venous stents, including balloon-expandable stents. The composition of the present invention may be bound to the implantable medical device or be passively adsorbed to the surface of the implantable medical device. For example, stents may be coated with the composition of the present invention by dipping the stent in a solution containing the composition or by spraying the stent with such a solution. Suitable materials for the implantable device should be biocompatible and non-toxic and may be chosen from the metals such as nickel-titanium alloys, steel, or biocompatible polymers, hydrogels, polyurethanes, polyethylenes, ethylenevinyl acetate copolymers, etc. In some embodiments, the composition of the present invention (such as PEG-PG-paclitaxel) is coated onto a stent for insertion into an artery or vein following balloon angioplasty. Such a coated stent may be useful for inhibiting arterial restenosis or arterial occlusion following vascular trauma. Comparing with stent coated with unconjugated agents such as paclitaxel, the present invention provides an improved composition that can reduce inflammation induction and provide controlled long term release of the chemotherapeutic agents.

[0053] Accordingly, in some embodiments, the present invention provides an implantable medical device, wherein the device is coated with a composition comprising a chemotherapeutic agent (such as an anti-tumor drug) conjugated to a water soluble polyamino acid polymer, wherein the water soluble polyamino acid polymer is conjugated to a hydrophilic polymer (such as PEG). The amount of the composition is effective to inhibit smooth muscle cell proliferation.

In some embodiments, the implantable medical device is a stent coated with the composition of the present invention. In some embodiments, the stent is adapted to be used during or after balloon angioplasty and the coating is effective to inhibit restenosis.

[0054] The following examples are provided to demonstrate preferred embodiments of the present invention. It should be appreciated by those of skill in the art that the many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

EXAMPLES Example 1. Synthesis of PEG Shielded PG-paclitaxel [0055] This example provides the synthesis method of PEG-PG-paclitaxel.

[0056] Polyglutamate (PG) sodium salt (MW 21K, Sigma, 100mg) was dissolved in water.

The pH of the aqueous solution was adjusted to 2 using 0.2N HCI. The precipitate was collected, washed with distilled water, and lyophilized to yield 63mg PG.

[0057] 21mg PG (MW 21000, lumol) as prepared above was dissolved in 0. 5ml dry DMF, then 17mg paclitaxel (MW 853, molar ratio PG/paclitaxel=20), 15mg dicyclohexylcarbodiimide (DCC) (75umol) and 0.2mg dimethylaminopyridine (DMAP) in 0. 5ml dry DMF was added.

25mg mPEG-NH2 (MW3400) in 0.1 ml dichloromethane (DCM) was then added to the reaction mixture. The reaction was allowed to proceed at room temperature for overnight. Thin layer chromatography (TLC, silica) showed complete conversion of paclitaxel (Rf=0. 55) to polymer conjugate (PEG-PG-paclitaxel) (Rf=0, mobile phase, CHC13/MeOH=10 : 1).

[0058] The reaction mixture was spinned down to remove insoluble materials and added to 10ml 1M NaHC03. The aqueous solution of PEG-PG-paclitaxel was desalted in water with G25 Sephadex column (HiTrap, Pharmacia). The column was eluted with water and monitored at 276nm. Collected first peak was lyophilized under vacuum.

[0059] The loading of Paclitaxel in PEG-PG-Paclitaxel was measured by ultraviolet spectra at 276nm. In particular, 31 % Paclitaxel was loaded onto the polymer matrix.

Example 2. In Vitro Studies of Anti-Proliferation Activities of PEG-PG-paclitaxel [0060] The effect of PEG-PG-paclitaxel on cell growth was examined by tetrazolium salt (MTS) assay with a commercial kit (Promega).

[0061] Lewis lung carcinoma and CT26 cells were seeded at 10000 cells/well in a 96-well micro titer plate and were treated 4 hours after plating with various concentrations of PEG-PG- paclitaxel, paclitaxel or vehicle. The wells were incubated for 48 hrs and 20ul MTS/PMS mixture was then added to each well and incubated for 1 hour. Optical density at 490nm was measured with a microplate reader (Molecular Device).

[0062] The vehicle treated cells serve as the blanks. For Lewis lung carcinoma cells, the resultant IC50 values were 2.7ng/ml for paclitaxel and 1.8ug PTX equivalent per ml for PEG- PG-Paclitaxel. See Figure 2. For CT26 colon cancer cell line, IC50 values were 0.2ug/ml for Paclitaxel and 18ug/ml for PEG-PG-paclitaxel based on equivalent paclitaxel, shown in Figure 3.

[0063] These in vitro studies show that PEG-PG-paclitaxel is 70-90 times less toxic than paclitaxel.

Example 3 In Vivo Studies of Anti-tumor Activity of PEG-PG-paclitaxel [0064] C57BL6 and C3H/Kam mice were purchased from Harlan SD and maintained in a ventilated facility.

Lewis lung carcinoma model [0065] Lewis lung carcinoma cells were cultured and dispersed in PBS. One million cells are injected subcutaneously on dorsal flank of female C57BL6 mice (6-8 weeks old). Tumor size were monitored and measured with a caliper.

[0066] Mice were grouped arbitrarily 3 days after tumor inoculation and 80% of mice showed appearance of the tumor. Drugs were administrated via tail vein. Mice were divided into three groups and treated with 100ul PBS, 80mg/kg paclitaxel in Cremophor/Ethanol was diluted 4 fold in PBS, and PEG-PG-paclitaxel equivalent to 80mg Paclitaxel/kg in 1 00ul PBS.

[0067] Body weights of the tumor bearing mice were monitored daily and net body weight changes were calculated by deducting the contribution by tumor weight. The tumor volume was calculated as length x width x width x 0.5.

[0068] Figure 4 presents the net body weight changes of tumor bearing mice after treatment.

Paclitaxel reduced body weight significantly and mice were not able to fully recover. In contrast, PEG-PG-paclitaxel on equivalent paclitaxel produced only minor net body weight loss and the mice were able to maintain their body weight throughout the study. This result indicates that PEG-PG-paclitaxel is substantially less toxic than paclitaxel.

[0069] Mice were sacrificed after fifteen days and tumors were excised and weighted. The tumor weight in response to treatment is presented in Table 1.

Table 1 Effect of paclitaxel (PTX) and PEG-PG-PTX on Tumor Weight In Lewis Lung Carcinoma Rx n Tumor weight p vs. V (mg) Vehicle'T'1325. 0414. 3 PTX 5 854. 0122. 8 <0. 01 PEG-PG-PTX 5 810. 0346. 3 <0. 01 [0070] As shown in Table 1, significant tumor inhibition by paclitaxel and PEG-PG-PTX was observed. The tumor inhibiting activities are of equal potency for PTX and PEG-PG-PTX at equivalent paclitaxel dose.

Ocra-l Ovarian Cancer Model [0071] OCa-1 murine ovarian cancer cells were cultured and dispersed in PBS. One million cells are injected into the muscle of right thigh of 6-8 weeks old female C3H/Kam mice. Tumors were monitored, measured with a caliper. Tumor volume was calculated as length x width x width x 0.5.

[0072] Mice were grouped arbitrarily after average tumor volume reached about 500 cubic mm. Drugs were administrated via tail vein. Mice were divided into three groups and treated with 100ul PBS, 80mg/kg paclitaxel in Cremophor/Ethanol diluted 4 fold in PBS, and PEG-PG- paclitaxel equivalent to 160mg paclitaxel/kg in 100ul PBS.

[0073] Tumor dimensions were monitored every other day. Figure 5 presents the tumor volume in different treatment groups. As shown in Figure 5, paclitaxel significantly delayed OCa-1 tumor growth. By contrast, tumor regression in PEG-PG-paclitaxel treated groups was observed.