MXPA98007442A - Soluble paclitaxel profarmacos in a - Google Patents

Abstract

Water-soluble compositions of paclitaxel, and docetaxel formed by conjugating paclitaxel or docetaxel to a water soluble chelator, polyethylene glycol or polymer such as poly-1-glutamic acid or poly-1-aspartic acid are described. Methods for using the compositions for the treatment of tumors, autoimmune disorders such as rheumatoid arthritis and for the prediction of paclitaxel uptake by tumors and imaging of radiolabelled DTPA-paclitaxel tumors are also described. Other modalities include the coating of implantable stent masses for the prevention of restenoses

Description

PACLITAXEL DEVICES FOR SOLUBLE WATER FIELD OF THE INVENTION The present invention relates generally to the fields of pharmaceutical compositions that will be used in the treatment of cancer, autoimmune diseases and restenosis. The present invention also relates to the field of pharmaceutical preparations of anticancer agents such as paclitaxel (Taxol) and docetaxel (Taxotere), in particular by forming water-soluble paclitaxel by conjugating the drug with water-soluble portions. BACKGROUND OF THE INVENTION Paclitaxel, an anti-microtubular agent extracted from needles and Pacific yew tree bark, Taxus brevifolia, has shown a marked anti-neoplastic effect in human cancer in Phase I studies and early Phase II trials and III (Horwitz et al., 1993).

It has mainly reported advanced ovarian and breast cancer. Significant activity has been documented in small cell and non-small cell lung cancer, head and neck cancers and in metastatic melanoma. However, a major difficulty in the development of paclitaxel for use in clinical trials has been its insolubility in water. Docetaxel was produced semisynthetically from 10-desacetyl baccatin II I, a non-cytotoxic precursor extracted from the needles of Taxus baccata and esterified with a chemically synthesized side chain (Cortes and Pazdur, 1995). It has been shown that several cancer cell lines, including breast, lung and colorectal cancers and melanomas, respond to docetaxel. In clinical trials docetaxel has been used to achieve complete or partial responses in cancers of the breast, ovaries, head and neck, and 5 malignant melanoma. Paclitaxel is normally formulated as a concentrated solution containing 6 mg of paclitaxel per milliliter of Cremophor EL (polyoxyethylated castor oil) and dehydrated alcohol ? (50% v / v) and also must be diluted before administration (Goldspiel, 1994). The amount of Cremophor EL needed to deliver the required doses of paclitaxel is significantly higher than that administered with another drug that is formulated in Cremophor. Several severe toxic effects have been attributed to Cremophor, including vasodilation, dyspnea and hypotension. East The vehicle has also been shown to cause serious hypersensitivity in laboratory animals and humans (Weiss et al., 1990). fr In fact, the maximum dose of paclitaxel that can be administered in mice by i.v. the bolus is dictated by the acute lethal toxicity of the Cremophor vehicle (Eiseman et al., 1994). Also I know knows that Cremophor EL, a surfactant agent. leaches phthalate plasticizers such as di (2-ethylhexyl) phthalate (DEH P) from polyvinyl chloride bags and intravenous tubes. It is known that DEHP causes hepatotoxicity in animals and is carcinogenic in rodents. This The preparation of paclitaxel has also been shown to form particulate matter over time and therefore filtration is necessary during administration (Goldspiel, 1994). Therefore, special provisions are necessary for the preparation and administration of paclitaxel solutions to ensure the safe delivery of the drug to patients and these provisions inevitably lead to higher costs. Previous attempts to obtain water-soluble paclitaxel have included the preparation of prodrugs of paclitaxel by placing solubilizing portions such as succinate and amino acids in the 2'-hydroxyl group or in the 7-hydroxyl position (Deutsch et al. (1989; Mathew et al. 1992), however, it has not been proven that these prodrugs are chemically stable enough for their development.For example, Deutsch et al. (1989) report a derivative of paclitaxel-2'-succinate, but the solubility of the water of the Sodium salt is only about 0.1% and the salts of triethanolamine and N-methylglucamine were only soluble at 1%., the amino acid esters are reported as unstable. Similar results were reported by Mathew et al. (1992). Greenwaid et al. Reported the synthesis of glycol esters of 2 'and 7'-polyethylene of taxol highly soluble in water (Greenwaid et al., 1994), however, no data were reported in reference to the antitumor activity in vivo of these compounds (Greenwaid et al., 1995). Other attempts to solve these problems have involved the microencapsulation of paclitaxel in both liposomes and nanospheres (Bartoni and Boitard, 1990). The liposome formulation was reported to be effective as free paclitaxel, however, only formulations and liposomes containing less than 2% paclitaxel were physically stable (Sharma and Straubinger, 1994). Unfortunately, the nanosphere formulation proved to be toxic. Therefore, there is still a need for a water soluble paclitaxel formulation that can deliver effective amounts of paclitaxel and docetaxel without the disadvantages caused by the insolubility of the drug. Another obstacle to the use of paclitaxel diffusers is the limited resources from which paclitaxel is produced, making paclitaxel therapy expensive. For example, a course of treatment may cost several thousand dollars. There is also the additional disadvantage that not all tumors respond to paclitaxel therapy, and this may be because paclitaxel does not enter the tumor. Therefore, there is an immediate need for effective formulations of paclitaxel and related drugs that are water soluble with long serum storage lives for treatment of tumors, autoimmune diseases such as rheumatoid arthritis, as well as for the prevention of restenosis of subject vessels. to traumas, such as angioplasty and Stent mass implants. SUMMARY OF THE INVENTION The present invention seeks to overcome these and other drawbacks inherent in the prior art by providing compositions comprising a chemotherapeutic and antiangiogenic drug, such as paclitaxel or docetaxel conjugated to a water soluble polymer such as, for example, a polyglutamic acid or a polyaspartic acid, or a metal chelator soluble in water. It is shown herein that these compositions are surprisingly effective as anti-tumor agents against illustrative tumor models and are expected to be at least as effective as paclitaxel or docetaxel against any of the diseases or conditions for which taxanes are known to be effective. taxoids. The compositions of the invention provide water soluble taxoids to overcome the drawbacks associated with the insolubility of the drugs themselves, and also provide the advantages of controlled release so that the tumors shown herein are eradicated in animal models after a single administration. intravenous The methods described herein can also be used to form conjugates of water-soluble polymers of other therapeutic agents, contrast agents and drugs, including etopsids, teniposides, fludarabine, doxorubicin, daunomycin, emodin, 5-fluorouracil, FUDR, estradiol, camptothecin, retinoic acids, verapamil, epothilones and cyclosporin. In particular, those agents with a free hydroxyl group could be conjugated to the polymers by similar chemical reactions as described herein for paclitaxel. Such conjugation could be within the experience of a routine practitioner of the chemical technique, just as it could fall within the scope of the claimed invention. Those agents could include, but not be limited to, etopside, teniposide, camptothecin and the epothilones.

As used herein, conjugated to a soluble polymer in Water means the covalent binding of the drug to the polymer or chelator. It is also understood that the water-soluble conjugates of the present invention can be administered together with other drugs, including other anti-tumor or anti-cancer drugs. Such combinations are known in the art. Paclitaxel or Water-soluble docetaxel of the present invention, in certain types of treatment, can be combined with a platinum drug, an antibiotic such as doxorubicin or daunorubicin, for example, or other drugs that are used in combination with Taxol. The conjugation of chemotherapeutic drugs to polymers is an attractive approach to reduce systemic toxicity and improve the therapeutic index. Polymers with larger molecular mass at 30 kDa do not diffuse easily through normal capillaries and glomerular endothelium, thus depriving normal tissue of drug-mediated toxicity (Maeda and Matsumura, 1989; Reynolds, 1995). On the other hand, it is well established that malignant tumors often have disordered capillary endothelium and greater permeability than normal tissue vasculature (Maeda and Matsumura, 1989; Fidler et al., 1987). Therefore, a polymer drug conjugate that could normally remain in the The vasculature can be selectively spilled from the blood vessels in tumors, resulting in tumorai accumulation of the active therapeutic drug. Additionally, polymer-drug conjugates can act as drug depots for sustained release, producing prolonged drug exposure to tumor cells. Finally, water-soluble polymers can be used to stabilize drugs, as well as to solubilize other insoluble compounds. Currently, a variety of synthetic and natural polymers have been examined for their ability to increase tumor-specific drug delivery (Kopecek, 1990, Maeda and Matsumura, 1989). However, only a few currently undergo clinical evaluation, including SMANCS in Japan and HPMA-Dox in the United Kingdom (Maeda, 1991, Kopeckova, 1993). In the present description, a taxoid is understood to mean those compounds that include paclitaxel and docetaxel and other chemicals that have the taxane skeleton (Cortes and Pazdur, 1995), and can be isolated from natural sources such as the yew tree, or cell culture, or chemically synthesized molecules, and a chemical of the general chemical formula, C 7H51 NO 14, including, ester 6, 12b, bis (acetyloxy-12- (benzoyloxy) -2a, 3, 4, 4a, 5 is preferred , 6, 9,10,11, 12, 12a, 12b-dodecahydro-4,11-dihydroxy-4a-8,13,13-tetramethyl-5-oxo-7,11-methane-1 H-cyclodeca [3, 4] benz- [1,2-b) oxet-9-yl of the acid [2aR- [2aa-4β, 4aβ, 9a (aR *, ßS *), 11a, 12a, 12aa, 12ba,]] - ß- (Benzoylamino) -a-hydroxybenzenepropanoic acid. It is understood that paclitaxel and docetaxel are each more effective than the other against certain types of tumors and that in the practice of the present invention, those tumors that are more susceptible to a particular toxoid could be treated with the water soluble taxoid conjugate. . In embodiments in which paclitaxel is conjugated to a water-soluble metal chelator, the composition may further comprise a chelated metal ion. The chelated metal ion of the present invention can have any ionic form of one of aluminum, boron, calcium, chromium, cobalt, copper, dysprosium, erbium, europium, gadolinium, gallium, germanium, holmium, indium, iridium, iron, magnesium, manganese, nickel, platinum, rhenium, rubidium, ruthenium, samarium, sodium, technetium, thallium, tin, yttrium or zinc. In certain preferred embodiments, the chelated metal ion will be a radionuclide, i.e., a radioactive isotope of one of the metals listings. Preferred radionuclides include, but are not limited to, 67Ga, 68Ga, 111ln, 99mTc, 90Y, 114mln and 193mPt. Preferred water-soluble chelators that will be used in the practice of the present invention include, but are not limited to, diethylenetriaminepentaacetic acid (DTPA), acid Ethylenediaminetetraacetic acid (EDTA), 1, 4,7, 10-tetraazacyclododecane-N, N \ N ", N" '- tetraacetate (DOTA), tetraazacyclotetradecane-N, N \ N ", N"' -tetraacetic acid (TETA) , hydroxyethylidene diphosphate (HEDP), dimercaptosuccinic acid (DMSA), diethylenetriaminetetramethylenephosphonic acid (DTTP) and 1- (p-25 aminobenzyl) -DTPA acid, 1,6-diamino hexan-N, N, N ', N'- acid teracetic acid 9f DPDP and ethylenebis (oxyethylenitrile) -tetraacetic acid, DTPA being the most preferred. A preferred embodiment of the present invention can also be a composition comprising "" in-DTPA-paclitaxel In certain embodiments of the present invention, paclitaxel or docetaxel can be conjugated with a water-soluble polymer and preferably the polymer is conjugated With 2 'or 7-hydroxy or both of paclitaxel or docetaxel, therefore, when functional I groups are used for drug conjugation, as before With the C2'-hydroxyl of paclitaxel, a degradable ligature is used, in this case, an ester, to ensure that the active drug is released from the polymer vehicle. Preferred polymers include, but are not limited to, polyethylene glycol, poly-1-glutamic acid, poly-d-glutamic acid, poly-d-glutamic acid, poly-1-aspartic acid. poly-d-aspartic acid, poly-d1-aspartic acid, polyethylene glycol. copolymers of the polyamino acids listed above with glycol of - polyethylene, polycaprolactone, polyglycolic acid and polyalactic acid. as well as polyacrylic acid, poly-2-hydroxyethyl-1-glutamine. carboxymethyl dextran, hyaluronic acid, human serum albumin and polyethylene glycol alginic acid, polyaspartic acids and polyglutamic acids being the most particularly preferred polyglutamic acids or polyaspartic acids of the present invention preferably have a molecular weight of from about 5,000 to about 100,000 with about 20,000 to about 80,000, or even about 30,000 to about 60,000 being more preferred. It is understood that the compositions of the present invention may be dispersed in a pharmaceutically acceptable carrier as described below. Said solution could be sterile or aseptic and may include water, pH buffer solutions, isotonic agents or other ingredients known to those of ordinary skill in the art, which could cause a non-allergic or harmful reaction when administered to a subject animal or human. Therefore, the present invention can also be described as a pharmaceutical composition comprising a chemotherapeutic or anti-cancer drug such as paclitaxel or docetaxel conjugated to a high molecular weight water soluble polymer or a chelator. The pharmaceutical composition can include polyethylene glycol, polyglutamic acids, polyaspartic acids or a chelator, preferably DTPA. It is also understood that a radionuclide can be used as an antitumor agent or drug, and that the present pharmaceutical composition can include a therapeutic amount of a radioactive isotope. chelated. In certain embodiments, the present invention can be described as a method for determining the absorption of a chemotherapeutic drug such as paclitaxel or docetaxel by tumor tissue. This method can include obtaining a The conjugate of the drug and a metal chelator with a chelated metal ion, contacting the tumor tissue with the composition and detecting the presence of the metal ion chelated in the tumor tissue indicates the absorption by the tumor tissue. The chelated metal ion can be a radionuclide and the detection can be by means of the graphic scintillation record. The tumor tissue can also be contained in an animal or a human subject and the composition could then be administered to the subject. The present invention can also be described in certain embodiments as a method for treating cancer in a subject. This method includes obtaining a composition comprising a chemotherapeutic drug such as paclitaxel or docetaxel conjugated to a water soluble or chelating polymer and dispersing in a pharmaceutically acceptable solution and administering the solution to the subject in an amount effective to treat the tumor. Preferred compositions comprise paclitaxel or docetaxel conjugated to polyglutamic acids or polyaspartic acids and more preferably to poly-1-glutamic acid or poly-1-aspartic acid. It is understood that the compositions of the invention are effective against any type of cancer for which it is shown that the unconjugated taxoid is effective and could include, but not limited to, breast cancer, ovarian cancer, malignant melanoma, lung cancer, gastric cancer, colon cancer, head and neck cancer or leukemia. The method of treating a tumor may include some prediction of the absorption of paclitaxel or docetaxel into the tumor before administering a therapeutic amount of the drug or prodrug. This method can include any of the training techniques * images treated before in which a subject is administered a paclitaxel, chelator, chelated metal and detected in a tumor. This step provides a cost-effective way to determine that a particular tumor could not be expected to respond to DTPA-paclitaxel therapy in those cases where the drug does not enter the tumor. It is contemplated that if an imaging technique can be used to predict the response of paclitaxel and to identify patients who are unlikely to respond, greater expense and time for the patient can be saved. The assumption is that if there is not a reasonable amount of chemotherapeutic agent deposited in the tumor, the probability of tumor response to that agent is relatively small. In certain embodiments, the present invention may described as a method to obtain a body image of a subject. The body image is obtained by administering an effective amount of a chelated radioactive metal ion to a paclitaxel-chelating conjugate conjugated to a subject and measuring the signals of the scintillation graphic record of the radioactive metal. to get an image. The present invention can also be described in certain broad aspects as a method for decreasing at least one symptom of a systemic autoimmune disease comprising administering to a subject having an autoimmune disease.

Systemic, an effective amount of a composition comprising paclitaxel or docetaxel conjugated with poly-1-glutamic acid or acid * poly-1-aspartic. Of particular interest in the context of the present disclosure is the treatment of rheumatoid arthritis, which is known to respond in some cases to taxol when administered in the normal Cremophor formulation (U.S. Patent No. 5,583,153). As in the treatment of tumors, it is contemplated that the effectiveness of the water-soluble taxoids of the present invention will not be diminished by the conjugation of a water-soluble portion, and that the water-soluble prodrug may act as a release formulation. controlled that releases the active drug for a while. Therefore, it is expected that the compositions of the present invention will be as effective as Taxol against rheumatoid arthritis, for example, but will offer the advantage of controlled release. It is also understood that the taxoid compositions of the present invention can be used in combination with other drugs, such as an angiogenesis inhibitor (AGM-1470 (Oliver et al, 1994) or methotrexate.The finding that paclitaxel also inhibits restenosis under balloon angioplasty indicates that the placitaxeles and docetaxeles The water-soluble compounds of the present invention will find a variety of applications beyond direct parenteral administration (WO 9625176). For example, it is contemplated that water-soluble paclitaxel will be useful as a coating for implanted medical devices, such as tubes, shunts, catheters, implants artificial, pins, electrical implants such as pacemakers and especially for arterial or venous Stent mass, including * Stent mass expandable by balloon. In these embodiments, it is contemplated that water soluble paclitaxel can be attached to an implantable medical device, or alternatively water soluble paclitaxel 5 can be passively absorbed onto the surface of the implantable device. For example, Stent masses may be coated with polymer-drug conjugates by immersing the Stent masses in polymer-drug solution or by spraying Stent masses with said solution. The right materials for the The implantable device should be biocompatible and non-toxic and can be chosen from metals such as nickel-titanium alloys, steel or biocompatible polymers, hydrogels, polyurethanes, polyethylenes, ethylene vinyl acetate copolymers, etc. In a preferred embodiment, water soluble paclitaxel, especially a conjugate of PG-paclitaxel, is coated in a Stent mass for insertion into an artery or vein following balloon angioplasty. The invention, therefore, can be described in certain broad aspects as a method to inhibit arterial restenosis or arterial occlusion following vascular trauma comprising administration to a subject in need thereof, a composition comprising paclitaxel or docetaxel conjugated to poly-1-glutamic acid or poly-1-aspartic acid. In the practice of the method, the subject may be a patient for example, coronary conduit, vascular surgery, organ transplant or coronary or arterial angioplasty, and the The composition can be administered directly, intravenously, or still coated on a Stent mass and the Stent mass * Implants if there are signs of vascular trauma. In one embodiment of the invention, therefore, it is an implantable medical device, wherein the device is coated with a composition comprising paclitaxel or docetaxel conjugated to polyglutamic acids or polyaspartic acids in an amount effective to inhibit muscle cell proliferation. smooth. A preferred device is a stent mass coated with the compositions of the invention as described herein, and in In certain preferred embodiments, the Stent mass is adapted to be used after balloon angioplasty and the coating is effective in inhibiting restenosis. In certain preferred embodiments, the invention can be described as a composition comprising acids Polyglutamic conjugates to the 2 'or 7-hydroxyl or both of paclitaxel, or even a composition comprising conjugated polyaspartic acid £? To 2 'or 7-hydroxyl or both of paclitaxel. As used herein, the terms "polyglutamic acid" or "polyglutamic acids" include poly-1-glutamic acid, poly-d-glutamic acid, and poly-1-glutamic acid and the terms "polyaspartic acid" or "polyaspartic acid" "polyaspartic acids" include poly-1-aspartic acid, poly-d-aspartic acid and d-α-aspartic acid. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by someone with experience in the matter to which this invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are now described. BRIEF DESCRIPTION OF THE DRAWINGS FIGURE 1A. Chemical structure of paclitaxel, PEG-paclitaxel and DTPA-paclitaxel. FIGURE 1B. Chemical structure and reaction scheme for the production of PG-paclitaxel. FIGURE 2. Effect of paclitaxel, PEG-paclitaxel and DTPA-paclitaxel on the proliferation of B16 melanoma cells. FIGURE 3. Anti-tumor effect of DTPA-paclitaxel on mammary tumors of MCa-4. FIGURE 4. Mean time (days) to reach the tumor diameter of 12 mm after treatment with paclitaxel, DTPA-paclitaxel and PEG-paclitaxel. FIGURE 5. Graphical gamma scintillation logs of mice having MCa-4 tumors after intravenous injection of pTn-20 DTPA-paclitaxel and '"in-DTPA The arrow indicates the tumor FIGURE 6. Hydrolytic degradation of PG- paclitaxel as determined in PBS at pH 7.4 at 37 ° C. - D-- represents percentage of paclitaxel remaining bound to soluble PG, -? - represents percentage of paclitaxel released, --O-- represents the percentage of matabolito-1 produced.

™ FIG U RA 7A. The anti-tumor effect of PG-paclitaxel on rats having murine breast tumor (13762F). --D-- represents the response of a single dose of i.v. of PG (0.3 g / kg); -? - represents the response to paclitaxel (40 mg / kg), -O- represents the response to PG-paclitaxel (60 mg equiv Paclitaxel / kg). FIG U RA 7B. The anti-tumor effect of PG-paclitaxel and pacl itaxel on mice that have OCa-1 tumors. -D- represents the response to a single dose of i.v. of PG (0.8 g / kg); -? - represents the response to paclitaxel (80 mg / kg), - • - represents the response to 10 PG-paclitaxel (80 mg equiv Paclitaxel / kg), -O- represents response to PG-paclitaxel (160 mg equiv Paclitaxel / kg). FIG U RA 7C. The anti-tumor effect of PG-paclitaxel on mice that have mammary carcinoma tumors MCa-4. -D- represents the response to a floor of i. v. of saline, -? - 15 represents the response to a single dose of i. v. of PG (0.6 g / kg); - + - represents the response to PG-paclitaxel (40 mg / kg), -0- represents the response to PG-paclitaxel (60 mg equiv Paclitaxel / kg), -O- represents the response to PG-paclitaxel (120 mg / kg). FIGU RA 7D. The anti-tumor effect of PG-paclitaxel against soft tissue sarcoma tumor (FSa-1) in mice. -D- represents the response to a single dose of i. v. of saline, - 0- represents the response to a single dose of i. v. of PG (0.8 g / kg); -O- represents the response to paclitaxel (80 mg / kg), -? - represents PG-paclitaxel response (160 mg equiv. Paclitaxel / kg).

FIGURE 7E. The anti-tumor effect of PG-paclitaxel against syngeneic hepatocarcinoma tumor (HCa-1) in mice. -D- represents the response to a single dose of i.v. of saline, -? - represents the response to a single dose of i.v. of PG (0.8 g / kg); -O- represents a response to PG-paclitaxel (80 mg / kg), -? - represents PG-paclitaxel response (160 mg equiv. Paclitaxel / kg). FIGURE 8. Release profile of paclitaxei from PEG-paclitaxel in phosphate buffer (pH 7.4). Paclitaxel, -X-: PEG-paclitaxel, -O- 10 FIGURE 9. The antitumor effect of PEG-paclitaxel on mammary tumors MCa-4. -D- represents the response to a single injection i.v. with PEG exit solution (60 mg / ml), - »- represents the response to Cremophor / alcohol vehicle, -O- represents a single dose of 40 mg / kg body weight of paclitaxel, - • - 15 represents PEG- paclitaxel at 40 mg equiv. Paclitaxel / kg body weight. DETAILED DESCRIPTION OF THE INVENTION The present invention arises from the discovery of novel water-soluble formulations of paclitaxel and docetaxel and to the surprising efficacy of these formulations against tumor cells in vivo. Paclitaxel conjugated poly-1-glutamic acid (PG-paclitaxel) administered to mice having ovarian carcinoma (OCa-l) caused a significant tumor growth retardation compared to the same dose of paclitaxel without PG. Mice treated with paclitaxel alone or with a combination of free paclitaxel and PG showed initially delayed tumor growth, but the tumors regrown to levels comparable to an untreated control group after ten days. In addition, at the maximum tolerated dose (DMT) of the PG-paclitaxel conjugate. (160 5 mg equiv Paclitaxel / kg), tumor growth was completely suppressed, tumors shrunk and mice were observed for two months after treatment remained tumor free (DMT: defined as the maximum dose that produced % or less loss of body weight within two weeks after a single injection i.v.). In a parallel study, the anti-tumor activity of PG-paclitaxel in rats with mammary adenocarcinoma in rats (13762F) was examined. Again, complete eradication of the tumor was observed at 40-60 mg equiv. Paclitaxel / g of PG-paclitaxel. These amazing results demonstrate that the polymer-drug conjugate, PG-paclitaxel. Successfully eradicates well-established solid tumors in both mice and rats after a single intravenous injection. In addition, with a half-life of 40 days at pH 7.4, PG-paclitaxel is one of the most stable water-soluble paclitaxel derivatives known (Deutsch, and others, 1989; Mathew and others, 1992; Zhao and Kingston, 1991). DTPA-paclitaxel is also shown to be as effective as paclitaxel in an in vitro antitumor potency assay using a B16 melanoma cell line. DTPA-25 paclitaxel showed no significant difference in the antitumor effect compared to paclitaxel against a mammary tumor of MCa-4 at a dose of 40 mg / kg body weight in a single injection. In addition, it was shown that DTPA-labeled paclitaxel with 1 1 1 lndium accumulates in the MCa-4 tumor as demonstrated by gamma scintigraphy, demonstrating that the anti-tumoral drugs conjugated with chelant of the present invention are useful and effective for the formation of images. The novel compounds and methods of the present invention provide significant advances over previous methods and compositions, since water-soluble paclitaxel is projected to improve the efficacy of paclitaxel-based anti-cancer therapy, providing water-soluble paclitaxel-derived compositions and of controlled liberation. Said compositions eliminate the need for the solvents to be associated with the side effects seen with the prior paclitaxel compositions. further, radiolabelled paclitaxel, which is shown to retain anti-tumor activity, will also be useful in tumor imaging. In addition, the present invention allows one to determine if a paclitaxel will be absorbed by a particular tumor by scintillation graphic recording, single photon emission computer tomography (TCEFS) or positron emission tomography (PET). Then this determination can be used to decide the effectiveness of anti-cancer treatment. This information may be useful in guiding the practitioner in the selection of patients who will undergo paclitaxel therapy.

Paclitaxel can be rendered soluble in water in two ways: conjugating paclitaxel to water-soluble polymers that serve as drug vehicles and derivatizing the antitumor drug with water-soluble chelating agents. The latter approach also provides an opportunity to mark with radionuclides (eg, 11 ln, 9o? I66H? J 68Qa 99mTc) for nuclear imaging and / or radiotherapy studies. The structures of paclitaxel, polyethylene glycol-paclitaxel (PEG-paclitaxel), conjugate of glutamic acid-paclitaxel (PG-paclitaxel) and diethylenetriaminpentaacetic acid-paclitaxel (DTPA-paclitaxel) are shown in FIGURE 1. In certain embodiments of the present invention, DTPA-paclitaxel or other paclitaxel-chelating agent conjugates, such as EDTA-paclitaxel, DTTP-paclitaxel or DOTA-paclitaxel, for example, can be prepared in the form of water-soluble salts (sodium salt, potassium salt, tretrabutylammonium salt, calcium salt, ferric salt, etc.). These salts will be useful as therapeutic agents for tumor treatment. Second, place DTPA-paclitaxel or other paclitaxel-chelating agents will be useful as diagnostic agents, which when marked with radionuclides such as 111ln or 99mTc, can be used as radio tracer to detect certain tumors in combination with imaging techniques nuclear It is understood that in addition to paclitaxel (taxol) and docetaxel (taxotere), other taxane derivatives may be adapted for use in the compositions and methods of the present invention and that all compositions and methods could be encompassed by the appended claims. Toxicity, pharmacokinetics and tissue distribution studies of DTPA-paclitaxel have shown that in mice the LD50 (lethal dose at 50%) of DPTA-paclitaxel observed with a single intravenous (iv) injection is approximately 100 mg / kg of weight bodily. Direct comparison with paclitaxel is difficult to perform due to the dose-volume restrictions imposed by the limited solubility of paclitaxel and vehicle toxicity associated with iv administration. However, in view of the present disclosure, one skilled in the art of chemotherapy could determine the effective and maximum tolerated doses in a clinical study for use in human subjects. In certain embodiments of the invention, a Stent mass coated with polymer-paclitaxel conjugates can be used to prevent restenosis, the closing of arteries after balloon angioplasty. Recent results in clinical trials using balloon expandable stent masses in coronary angioplasty have shown a significant benefit in openings and reduced restenosis compared to balloon angioplasty (Serruys et al., 1994). According to the injury response hypothesis, neointimal formation is associated with increased cell proliferation. Currently, popular opinion holds that the critical process that leads to vascular lesions in both spontaneous and accelerated arteriosclerosis is the proliferation of smooth muscle cells (CML) (Phillips-Hughes and Kandarpa, 1996). Since the phenotypic proliferation of CML after arterial damage mimics that of neoplastic cells, it is possible that anti-cancer drugs may be useful to prevent the accumulation of neointimal CML. The 5 Stent masses coated with polymer-bound anti-proliferative agents that are capable of releasing these agents for a prolonged time with sufficient concentration will therefore prevent the growth of the intima and media hyperplastic in the lumen thus reducing restenosis. Because it has been shown that paclitaxel suppresses collagen-induced arthritis in a mouse model (Oliver et al, 1994), the formulations of the present invention are also contemplated as being useful in the treatment of autoimmune and / or inflammatory diseases. such as rheumatoid arthritis. The The binding of paclitaxel to tubulin changes the equilibrium to stable microtubule polymers and makes this drug a strong inhibitor of eukaryotic cell replication by blocking cells in the late mitotic stage of G2. Several mechanisms may be involved in the suppression of arthritis by paclitaxel. For example, the effects Paclitaxel-specific phase-specific cytotoxins can rapidly affect the proliferation of inflammatory cells and in addition paclitaxel inhibits cell mitosis, migration, chemotaxis, intracellular transport and production of H2O2 from neutrophils. In addition, paclitaxel may have anti-angiogenic activity blocking the coordinated endothelial cell migration (Oliver et al., 1994).

Therefore, the prodrugs conjugated with polymers of the * present invention are contemplated to be useful as free paclitaxel in the treatment of rheumatoid arthritis. The conjugated polymer formulation described herein also offers the advantages of delayed or sustained release of the drug and increased solubility. It is also an aspect of arthritis treatment that the formulations can be injected or implanted directly into the affected joint areas. Pharmaceutical preparations of paclitaxel or docetaxel Suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid for injection. It must be stable under the conditions of manufacture and storage and must conserve against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium, containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and a liquid polyethylene glycol and the like), suitable mixtures thereof, and oils. vegetables. The prevention of the action of microorganisms can be carried out by various antibacterial agents and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride.

* Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with several of the other ingredients listed above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the Preferred methods of preparation are vacuum drying and freeze drying techniques that give a powder of the active ingredient plus any additional desired ingredients from a previously sterile filtered solution thereof. As used herein, "pharmaceutically vehicle "Acceptable" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents and isotonic agents and the like The use of such pharmaceutically active agent and agent means is well known in the art. conventional medium or The agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. The supplemental active ingredients can also be incorporated into the compositions. The phrase "pharmaceutically acceptable" also refers to entities and molecular compositions that do not produce an allergic or similar breakthrough reaction when administered to an animal or a human being. For parenteral administration in an aqueous solution, for example, the solution must be suitably regulated, if necessary, and the liquid diluent first rendered isotinic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous and intraperitoneal administration. In connection with this, the sterile aqueous medium that may be employed will be known to those of ordinary skill in the art in view of the present disclosure. The following examples are included to demonstrate preferred embodiments of the invention. It will be appreciated by those skilled in the art that the techniques described in the examples that follow represent techniques discovered by the inventor to function well in the practice of the invention and therefore can be considered to constitute preferred modes for their practice. However, those of experience in the art, in view of the present description should appreciate that many changes can be made in the specific embodiments described and still obtain a similar result without departing from the spirit and scope of the invention. DTPA-Paclitaxel Synthesis of DTPA-paclitaxel: To a solution of paclitaxel (100 mg, 0.117 mmol) in dry DM 25 (2.2 ml) was added diethylenetrieminepentaacetic acid anhydride (DTPA A) (210 mg, 0.585 mmol) at 0 ° C. The reaction mixture was stirred at 4 ° C overnight. The suspension was filtered (Millipore 0.2 μm filter) to remove the unreacted DTPA anhydride. The filtrate was poured into distilled water, stirred at 4 ° C for 20 min, and the precipitate was recovered. The crude product was purified by preparative CLF on C? 8 silica gel plates and developed in acetonitrile / water (1: 1). Paclitaxel had an Rf value of 0.34. The band above the paclitaxel with an Rf value of 0.65 to 0.75 was removed by scraping and eluted with a mixture of acetonitrile / water (1: 1) and the solvent was removed to give 15 mg of DTPA-paclitaxel as product ( performance 10.4%): pf: >226 ° C desc. The UV spectrum (sodium salt in water) showed maximum absorption at 228 nm which is also characteristic for paclitaxel. Mass spectrum: (FAB) m / e 1229 (M + H) +, 1251 (M + Na), 1267 (M + K). In the 1 H NMR spectrum (DMSO-de) the resonance of NCH2CH2N and CH2COOH of DTPA appeared as a complex series of signals at d 2.71 - 2.96 ppm, and it is a multiplet at d 3.42 ppm, respectively. The resonance of C7-H at 4.10 ppm in paclitaxel changed to 5.51 ppm, suggesting esterification at position 7. The rest of the spectrum was consistent with the paclitaxel structure. The sodium salt of DTPA-paclitaxel was also obtained by adding a solution of DTPA-paclitaxel in ethanol in an equivalent amount of 0.05 M NaHCO3, followed by lyophilization to a water soluble solid powder (solubility> 20 mg paclitaxel equivalent / ml ). Hydrolytic stability of DTPA-paclitaxel: The hydrolytic stability of paclitaxel from DTPA-paclitaxel was studied under accelerated conditions. Briefly, 1 mg of DTPA-paclitaxel was dissolved in 1 ml of aqueous 0.5 M NaHCO3 solution (pH 9.3) and analyzed by HPLC. The CLAR system consisted of a Nova-Pak column of 150 x 3.9 (id) mm of Waters filled with 4μm of C18 silica gel, a Perkin-10 Elmer CL-Socratic pump, a 900 series interface of PE Nelson, a UV / Vis detector from Spectra-Physics and a data station. The eluent (acetonitrile / methanol / 0.02M ammonium acetate = 4: 1: 5) was run at 1.0 ml / min with UV detection at 228 nm. The retention times of DTPA-paclitaxel and paclitaxel were 1.38 and 8.83 min, respectively. The peak areas were quantified and compared with normal curves to determine the concentrations of DTPA-paclictaxel and paclitaxel. The calculated average life of DPTA-paclitaxel in 0.5 M NaHCO3 solution is approximately 16 days at room temperature. 20 Effects of DTPA-paclitaxel on the growth of melanoma cells of B16 mice in vitro: Cells were seeded in 24-well plates at a concentration of 2.5 x 10 4 cells / ml and grown in Dulbecco's modified minimal essential medium of 50: 50 (DEM) and half F12 containing 10% bovine calf serum at 37 ° C for 24 hours in a humidified atmosphere of 97% 5.5% CO2. The medium was replaced with fresh medium containing paclitaxel or DTPA-paclitaxel in concentration ranging from 5 x 10"9 M to 75 x 10 -9 M. After 40 hours, the cells were released by trypsinization and 5 were counted in a Coulter counter. The final concentrations of DMSO (used to dissolve paclitaxel) and 0.05 M sodium bicarbonate solution (used to dissolve DTPA-paclitaxel) in the cell medium was less than 0.01%. This amount of solvent has no effect on the growth of the cells as determined by the control studies. The effects of DTPA-paclitaxel on the growth of B16 melanoma cells are presented in FIGURE 2. After an incubation of 40 hours at various concentrations, DTPA-paclitaxel and paclitaxel were compared for cytotoxicity. The IC5o for paclitaxel and DTPA-paclitaxel are 15 nM and 7.5 nM, respectively. Antitumor effect on mammary carcinoma tumor model (MCa-4): Female C3Hf / Kam mice were inoculated with mammary carcinoma (MCa-4) in the muscles of the right extremity (5 x 105 cells / mouse). When tumors developed at 8 mm (approximately 2 weeks) a single dose of paclitaxel or DTPA-paclitaxel was n at 10, 20 and 40 mg paclitaxel equivalent / kg body weight. In control studies, saline and absolute alcohol / Cremophor 50/50 diluted with saline (1: 4) were used.

Tumor growth was determined daily by midiend, three orthogonal diameters of the tumor. When the tumor size reached 12 mm in diameter, the growth retardation was calculated. The mice were sacrificed when the tumors reached approximately 15 mm. The curve of tumor growth is shown in FIGURE 3. Compared with controls, both paclitaxel and DTPA-paclitaxel showed antitumor effect at a dose of 40 mg / kg. The data were also analyzed to determine the average number of days for the tumor to reach 12 mm in diameter. The analysis Statistical analysis showed that DTPA-paclitaxel retarded tumor growth significantly compared to the control treated with saline at a dose of 40 mg / kg (p <0.01). The mean time for the tumor to reach 12 mm in diameter was 12.1 days for DTPA-paclitaxel compared to 9.4 days for paclitaxel (FIGURE 4).

Radiolabelling of DTPA-paclitaxel with 111 ln In a 12 ml ampule, 40 μl of 0.6 M sodium acetate pH buffer solution (pH 5.3) was successively added; 40 μl of 0.06 M sodium citrate pH buffer solution (pH 5.5). 20 μl solution of DTPA-paclitaxel in ethanol (2% w / v) and 20 μl of solution of 111lnCI3 (1.0 mCi) in sodium acetate buffer (pH 5.5). After an incubation period of 30 minutes at room temperature, the labeled 111ln-DTPA-paclitaxel was purified by passing the mixture through a Sep-Pac C18 cartridge using saline subsequently as the mobile phase The free 111I-DTPA (<3%) was removed by the saline solution, whereas 111In-DTPA-paclitaxel was recovered in the ethanol wash. The ethanol was evaporated under nitrogen gas and the labeled product reconstituted in saline. Radiochemical yield: 84%. Analysis of 111ln-DTPA-paclitaxel: 5 HPLC was used to analyze the reaction mixture and purity of 111ln-DTPA-paclitaxel. The system consisted of a LDC binary pump, a Waters column 100 x 8.0 mm (i.d.) filled with 5 μm of ODS silica gel. The column was eluted to a regime of flow of 1 ml / min with a gradient mixture of water and methanol (gradient from 0% to 85% methanol for 15 minutes). The gradient system was monitored with a Nal crystalline detector and a Spectra-Physics UV / Vid detector. As evidenced by the CLAR analysis, purification by the Sep-Pak cartridge removed most of the 111ln-DTPA, which had a detection time of 2.7. min. 111ln-DTPA was probably derived from traces of DTPA contaminant in DTPA-paclitaxel. A radio- »111ln-DTPA-paclitaxel chromatogram correlated with its UV chromatrogram, indicating that the peak at 12.3 min was also the target compound. Under the same chromatographic conditions. paclitaxel had a retention time of 17.1 min. The radiochemical purity of the final preparation was 90% as determined by HPLC analysis. Graphical representation of whole body scintillation Female C3Hf / Kam mice were inoculated with carcinoma mammary (MCa-4) in the muscles of the right extremity (5 x 10 = cells). When the tumors had grown to 12 mm in diameter, the mice were divided into two groups. In group I, the mice were anesthetized by intraperitoneal injection of sodium pentobarbital, followed by 111ln-DTPA-paclitaxel (100-200 mCi) via the tail vein. A camera ? equipped with a medium energy collimator was placed on the mice (3 per group). A series of 5 minute acquisitions were recovered at 5, 30, 60, 120, 240 min and 24 hours after the injection. In group II, the same procedures were followed except that the mice were injected with 111ln-DTPA as a control. FIGURE 5 shows graphic gamma scintigraphic fegs of animals injected with 111ln-DTPA and 111ln-DTPA-paclitaxel. 111ln-DTPA is characterized by rapid plasma clearance, rapid and high excretion in the urine with minimal retention in the kidney and negligible retention in the tumor, liver, intestine and other organs or body parts. In contrast, 11ln-DTPA-paclitaxel exhibited a pharmacological profile that resembles that of paclitaxel (Eiseman et al., 1994). The radioactivity in the brain was negligible. The liver and kidney had the highest proportions of tissue: plasma. Hepatobiliary excretion of rediomarked DTPA-paclitaxel or its metabolisms was one of the main routes for the elimination of the drug from the blood. Unlike paclitaxel, a significant amount of 11ln-DTPA-paclitaxel was also excreted through the kidney, which only played a minor role in the elimination of paclitaxel. The tumor had significant absorption of 111 In-DTPA-paclitaxel. These results show that 111! In-DTPA-paclitaxel is able to detect certain tumors and to quantify the absorption of 111ln-DTPA-paclitaxel in tumors, which in turn can help the selection of patients for paclitaxel treatment. Example 2 Poly-glutamic Acid-Paclitaxel The present example demonstrates the conjugation of paclitaxel to a water-soluble polymer, poly-1-glutamic acid (PG). The potential of water-soluble polymers used as drug vehicles is well established (Kopecek, 1990; Maeda and Matsumura, 1989). In addition to its ability to solubilize other insoluble drugs, the drug-polymer conjugate also acts as a slow-release depot for the release of the controlled drug. Synthesis of PG-Paclitaxel PG was selected as a vehicle for paclitaxel since it can be easily degraded by liposomal enzymes, is stable in plasma and contains sufficient functional groups for drug binding. Several antitumor drugs, including Adriamycin 20 (Van Heeswijk et al., 1985; Hoes et al. 1985), cyclophosphamide (Hirano et al., 1979) and Ara-C (Kato et al., 1984) have been conjugated to PG. The sodium salt of PG (MW 34 K, Sigma, 0.35 g) was dissolved in water. The pH of the aqueous solution was adjusted to 2 using 0.2 M HCl.

It was analyzed against distilled water and lyophilized to give 0.29 g of PG. To a solution of PG (75 mg, repeat unit of FW 170, 0.44 mmol) in dry DMF (1.5 mL) was added 20 mg of 5 paclitaxel (0.023 mmol, molar ratio of PG / paclitaxel = 19), 15 mg of dicyclohexylcarbodiimide (DCC) (0.073 mmol) and a trace amount of dimethylaminopyridine (DMAP). The reaction was allowed to proceed at room temperature for 4 hours. Thin layer chromatography (CLF, síl ice) showed the complete conversion of paclitaxel (Rf = 0.55) to a polymer conjugate (Rf = 0, mobile phase, CHCl3 / MeOH = 10: 1). The reaction mixture was poured into chloroform. The resulting precipitate was recovered and dried in a vacuum to give 65 mg of the polymer-drug conjugate. By changing the weight ratio of paclitaxel to PG in the starting materials, the polymer conjugates of various concentrations of paclitaxel can be synthesized. The sodium salt of the PG-paclitaxel conjugate was obtained by dissolving the 0.5 M NaHCO3 product. The aqueous solution of PG-paclitaxel was dialyzed against distilled water (MWCO 1, 000) to remove low molecular weight and excess salt contaminants.

NaHCO3. The lyophilization of the dialysate gave 88.6 mg of white powder. The content of paclitaxel in this polymer conjugate was determined by UV (described below) as 21% (w / w). Yield (conversion to polymer bound to paclitaxel, UV): 93%. PG-paclitaxel with higher paclitaxel content (up to 35%) can be synthesized by this method by simply increasing the ratio of paclitaxel to PG used. 1 H-NMR (GN 500 spectrometer model GE, 500 MHz, in D 2 O): d = 7.75 to 7.36 ppm (aromatic components of paclitaxel); d = 5 6.38 ppm (C10-H), 5.97 ppm (C13-H), 5.63 and 4.78 ppm (C2'-H), 5.55- 5.36 ppm (C3'-H and C2-H, m), 5.10 ppm ( C3-H), 4.39 ppm (C7-H), 4.10 (C10-H), 1.97 ppm (OCOCH3), and 1.18-1.20 ppm (C-CH3) are assigned to the aliphatic components of paclitaxel. Other resonances of paclitaxel were obscured by PG resonances. The resonances of PG at 4.27 ppm (H-a), 2.21 ppm (H-?), And 2.04 ppm (H- ß) agree with the spectrum of PG. The conjugated polymer paclitaxel couplings are poorly solved to be measured with sufficient precision. The solubility in water was > 20 mg paclitaxel / ml. Characterization of PG-paclitaxel ^ Ultraviolet (UV) spectra were obtained on a Beckman DU-640 spectrophotometer (Fullerton, CA). The content of paclitaxel conjugated to PG was estimated by UV based on a normal curve generated with known concentrations of paclitaxel in metal (? = 228 nm), assuming that the polymer conjugate in water and free drug in methanol had the same molar extinction coefficients and that both followed the law of Lambert Beer As shown by its UV spectrum, PG-paclitaxel had paclitaxel absorption characteristic with two changes of? from 228 to 230 nm. The concentration of paclitaxel in PG-paclitaxel was calculated based on the normal curve generated with known concentrations of paclitaxel in methanol at an absorption of 228 nm, assuming that the polymer conjugate in water at 230 nm and the free drug in methanol at 228 nm have the same molar extinction and both follow Lambert Beer's law. Gel Permeation Chromatrography Studies of PG-Paclitaxel. The relative molecular weight of PG-paclitaxel was characterized by? ^ 'Gel permeation chromatography (CPG). The CPG system consisted of two LDC model III pumps coupled with the main LDC gradient, a CPG column from PL and a Waters 990 photodiode array detector. The eluent (DMF) was run at 1.0 ml / min with detection of UV set at 270 nm. The conjugation of paclitaxel to PG resulted in an increase in weight Molecule of PG-paclitaxel, as indicated by the change in L time of retention of 6.4 min of PG at 5.0 min of the PG-paclitaxel conjugate as analyzed by CPG. The calculated molecular weight of PG-paclitaxel containing 15-25% paclitaxel (w / w) is in the range of 45-55 kDa. The crude product contained a heavy contaminant Small molecule (retention time 8.0 to 10.0 min and 11.3 min), which can be effectively removed by converting PG-paclitaxel to its sodium salt, followed by dialysis. Hydrolytic degradation of PG-paclitaxel conjugate. PG-paclitaxel was dissolved in buffer solutions of pH phosphate (PBS, 0.01 M) at pH 6.0, pH 7.4 and pH 9.6 at an equivalent concentration of paclitaxel of 0.4 mM. The solutions were incubated at 37 ° C with moderate agitation. At selected time intervals, aliquots (100 μl) were removed, mixed with an equal volume of methanol and analyzed by high performance liquid chromatography (HPLC). The CLAR system consisted of a reverse phase silica column (Nova-Pac, Waters, CA), a methanol-water mobile phase (2: 1, v / v) supplied at a flow rate of 1.0 ml / min. , and a photodiode detector. The concentration of paclitaxel bound to PG, free paclitaxel and other degradation products in Each sample was calculated by purchasing the peak areas with a normal curve obtained separately prepared from paclitaxel, assuming that the molar extinction coefficient of each peak at 228 nm is the same as that of paclitaxel. The half-life of the conjugate, calculated to be 132, 40 and 4 days at pH 6.0, 7.4 and 9.6 respectively, is determined by a linear least squares regression analysis. The CLAR analysis revealed the incubation of PG-paclitaxel in PBS solutions that produced paclitaxel and other different species including one that is more hydrophobic than paclitaxel (metalito-1). In fact, the amount of metabolite-1, which more was probably 7-epipaclitaxel, recovered in PBS at pH 7.4, exceeding that of paclitaxel after 100 hours of incubation (FIGURE 6). In vitro studies The aliquots obtained from PBS solution at pH 7.4 are underwent analysis by a tubulin polymerization analysis.

The tubulin assembly reaction was performed at 32 ° C in pH PEM pH buffer solution (pH 6.9) at a concentration of tubulin (bovine brain, Cytleton Inc., Boulder, CO) of 1 mg / ml (10 μM) in presence of the test samples (1.0 μM equiv Paclitaxel) and 1.0 mM GTP. Polymerization of tubulin was followed by measuring the absorbance of the solution at 340 nm over time. After 15 min, calcium chloride (125 mM) was added to measure the CaCl2-induced depolymerization of the microtubules. While PG-paclitaxel recently dissolved in PBS was inactive in the production of microtubules, the aliquots of PG-paclitaxel were incubated for three days resulting in the polymerization of tubulin. The formed miototubules were stable against the depolymerization induced by CaCl2. The effect of PG-paclitaxel on cell growth was also examined by tetrazolium salt (MTT) analysis (Mosmann, 1983). The MCF-7 cells or 13762F cells were seeded at 2 x 10 4 cells / ml in a 96-well microtiter plate treated 24 hours later with various concentrations of PG-paclitaxel, paclitaxel or PG, and incubated for an additional 72 hours. . The MTT solution (20 μl, 5 mg / ml) was then added to each well and incubated for 4 hours. The supernatant was aspirated and the MTT formazan formed by metabolically viable cells was measured by a microplate fluorescence reader at a wavelength of 590 nm. During the three-day period, PG-paclitaxel inhibited proliferation of tumor cells to a degree similar to that of paclitaxel. For the MCF-7 breast tumor cell line, the resulting IC50 values were 0.59 μM for paclitaxel and 0.82 μM for PG-paclitaxel (measured in equivalent units of paclitaxel). Against the 13762F cell line, the sensitivity for PG-paclitaxel (IC50 = 1.86 μM) was comparable to that of paclitaxel (ICso = 6.79 μM). For both cell lines, the IC50 of PG alone was greater than 100 μM. Antitumor activity in vivo All animal work was carried out in the animal facility in M.D. Anderson Cancer Center according to the institutional guidelines. The C3H / Kam mice were bred and maintained in a pathogen-free facility in the Department of Experimental Radiation Oncology. Solitary tumors were produced in the muscle of the right limb of C3H / Kam mice (25-30g) by injecting 5 x 10s murine ovarian carcinoma cells (OCa-l), mammary carcinoma (Mca-4). hepatocarcinoma (HCa-l) or fibrous sarcoma (FSa-ll). In a parallel study, 344 female Fischer rats (125-150 g) were injected with 1.0 x 105 viable 13762F tumor cells in 0.1 ml of PBS. Treatments were initiated when the tumors in the mice grew to 500 mm3 (10 mm in diameter) or when the tumors in rats developed at 2400 mm3 (mean diameter 17 mm). A single dose of PG-paclitaxel in saline or paclitaxel in Cremophor EL vehicle was given in doses ranging from 40 to 160 mg equiv Paclitaxel / kg body weight. In control experiments, saline, Cremophor vehicle [50/53 Cremophor / ethanol diluted with saline (1: 4)], solution of PG (PM 38K) saline and a paclitaxel / PG mixture was used. Tumor growth was determined daily (FIGURE 7A, 7B, 7C, 7D and 7E) by measuring three orthogonal tumor diameters. The volume of the tumor was calculated according to the formula (A x B x C) / 2. Absolute growth retardation (RCA) in mice was defined as the time in days for tumors treated with several drugs to develop from 500 to 2,000 mm3 in mice minus the time in days for tumors treated with saline control to grow from 500 to 2,000 mm3. Table 1 summarizes the toxicity of PG paclitaxel in rats compared to paclitaxel / Cremophor. Table 2 summarizes the data referring to the effect of PG-paclitaxel against tumors of MCa-4, FSa-ll and HCa-l in mice. The data is also summarized in FIGURE 7A-FIGURE 7E.

-Paclitaxel in Rats Fischer * P * Drugs were administered intravenously in Fischer rats having 13762F tumor (female, 130 g) in a single injection. * J ^ F a The PG-paclitaxel solution was prepared by dissolving the conjugate in saline (8 mg equiv Paclitaxel / ml). The volume injected at 60 mg / kg was 0.975 ml per rat. b The Paclitaxel Cremophor solution was prepared by dissolving paclitaxel in a 1: 1 mixture of ethyl alcohol and Cremophor (30 mg / ml). This stock solution was further diluted with saline (1: 4) before the injection. The final concentration of paclitaxel in the solution was 6 mg / ml). The volume injected at 60 mg / kg was 1.3 ml per rat. forgetting the polymer in saline (22 mg / ml). The injected dose was 0.3 g / kg (1.8 ml per rat) which was equivalent to the paclitaxel dose of 60 mg / kg. d The Cremophor vehicle was prepared by diluting a mixture of diethyl alcohol and Cremophor (1: 1) with saline (1: 4). Table 2: The Antitumor Effect of PG-Paclitaxel Against Different Types of Murine Tumors in vivo * were treated with several doses of PG-paclitaxei (40-120 mg equiv Paclitaxel / kg) in saline or paclitaxel in Cremophor vehicle in a single iv injection Control animals were treated with saline (0.6 ml), vehicle Cremophor (0.5 ml) solution of PG in saline, or PG g / kg) plus paclitaxel (80 mg / kg). b The tumor growth was determined by the daily measurement of three orthogonal diameters with calibrators and the volume was calculated as (a x b x c) / 2. In square brackets sample the number of mice used in each group. The time in days to grow from 500 mm ° to 2000 mm3 is presented as mean + normal deviation, c Absolute growth delay (RCA) defined as the time in days for tumors with several drugs to grow from 500 to 2000 tumors treated with control of saline solution to grow from 500 to 2000 mm3. d The time in days to grow from 500 to 2000 mm3 was compared for the treatment of groups and the saline group using 5 Student's t test. The P values have two sides and were taken as significant when they were less than or equal to 0.05. Example 3 Polyethylene glycol-Paclitaxel Synthesis of polyethylene-paclitaxel glycol (PEG-paclitaxel) Synthesis was achieved in two steps. First 2'-succinyl-paclitaxel was prepared according to a reported procedure (Deutsch et al., 1989). Paclitaxel (200 mg, 0.23 mmol) and succinic anhydride (228 mg, 2.22 mmol) were allowed to react in anhydrous pyridine (6 ml) at room temperature for 3 hours. The The pyridine was then evaporated and the residue was treated with water, stirred for 20 minutes and filtered. The precipitate was dissolved in acetone, water was added slowly and the fine crystals were recovered to give 180 mg of 2'-succinyl-paclitaxel. PEG-paclitaxel was synthesized by a coupling reaction mediated by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). To a solution of 2'-succinyl-paclitaxel (150 mg, 0.18 mmol) and methoxypolyoxyethylene amine (PEG-NH2, MW 5000, 900 mg, 0.18 mmol) in methylene chloride was added EEDQ (180 mg, 0.72 mmol). The reaction mixture was stirred at room temperature for 4 hours. The raw product is chromatographed on silica gel with ethyl acetate followed by chloroform-methanol (10: 1). This gave 350 mg of product. 1 H NMR (CDCl 3) d 2.76 (m, succinic acid, COCH 2 CH 2 CO 2), d 3.63 (PEG, OCH 2 CH 2 O), d 4.42 (C7-H) and d 5.51 (C2'-H). The maximum UV absorption was 288 nm, which was also characteristic for paclitaxel. Binding to PEG improved the aqueous solubility of paclitaxel (> 20 mg paclitaxel equivalent / ml of water). PEG-Paclitaxel hydrolytic stability PEG-paclitaxel was dissolved in phosphate pH buffer (0.01 M) at various pH at a concentration of 0.4 mM and the solutions were allowed to incubate at 37 ° C with moderate agitation. At selected time intervals, aliquots (200 μl) were removed and lyophilized. The resulting dry powders were redissolved in methylene chloride for gel permeation chromatography (CPG analysis). The CPG system consisted of a Perkin-Elmer PL mixed gel column, a Perkin-Elmer Socratic CL pump, a 900 PE Nelson series interface, a UV / Vis Spectra-Physics detector and a data. The eluent (methylene chloride) was run at 1.0 ml / min with a UV detector set at 228 nm. The retention times of PEG-paclitaxel and paclitaxel were 6.1 and 8.2 min, respectively. The peak areas were quantified and the percentage of PEG-paclitaxel remaining and the percentage of paciitaxel related were calculated. The mean life of PEG-paclitaxel determined by linear least squares at pH 7.4 was 54 minutes. The half-life at pH 9.0 was from paciitaxel eduction of PEG-paclitaxel at pH 7.4 shown in FIGURE 8. Cytotoxicity studies of PEG-paclitaxel using melanoma cells from B16 mice in vitro. Following the procedure described in the DTPA-paclitaxel cytotoxicity studies, melanoma cells were plated in 24-well plates at a concentration of 2.5 x 10 4 cells / ml and a minimum essential medium of Dublecco 50:50 (DME) was developed and F12 medium containing 10% calf serum from bovine at 37 ° C for 24 hours at 97% humidified atmosphere of 5.5% CO2. The medium was then replaced with fresh medium containing paclitaxel and its derivatives in concentrations ranging from 5 x 10"9 M to 75 x 10" 9. After 40 hours, the cells were released by trypsination and counted in a Coulter counter. The final concentrations of DMSO (used to dissolve paclitaxel) and 0.05 M sodium bicarbonate solution (used to dissolve PEG-paclitaxel) in the cell medium was less than 0.01%. This amount of solvent has no effect on the growth of the cells determined by the control studies. In addition, the PEG in the concentration scale used to generate a concentration of 5 x 10"9 M to 75 x 10" 9 M did not have any defects on the proliferation either. Antitumor effect of PEG-paclitaxel against MCa-4 tumor in mice To evaluate the anti-tumor efficacy of PEG-paclitaxel against solid breast tumors, MCa-4 cells were injected (5 × 10 5 right of C3Hf / Kam female mice) As described in Example 1 with DTPA-paclitaxel, when the tumors grew to 8 mm (about 2 weeks) ), a single dose of paclitaxel or PEG-paclitaxel was given at 10, 5, 20 and 40 mg paclitaxel equivalent / kg of body weight Paclitaxel was initially dissolved in absolute ethanol with a volume equal to Cremophor. (1: 4 by volume) and? With a sterile physiological solution within 15 minutes of ^ injection. PEG-paclitaxel was dissolved in saline (6 mg equiv.

Paclitaxel / ml) and filtered through a sterile filter (Millipore, 4.5 μm). Saline, paclitaxel vehicle, absolute alcohol: Cremophor (1: 1) diluted with saline (1: 4) and PEG solution in saline (600 mg / kg body weight) were used in control experiments. The tumor growth was determined 15 daily, measuring three orthogonal diameters of the tumor. When the tumor size reached 12 mm in diameter, the tumor growth retardation was calculated. The tumor growth curve is shown in FIGURE 9. At a dose of 40 mg / kg, either PEG-paclitaxel or paclitaxel 20 effectively retarded tumor growth. Paclitaxel was more effective than PEG-paclitaxel, although the difference is not statistically significant. Tumors treated with paclitaxel required 9.4 days to reach 12 mm in diameter while tumors treated with PEG-paclitaxel required 8.5 days. 25 Statistically, these values were significant (p> 0.05) compared to their corresponding controls, which were 6, 7 days for the paclitaxel vehicle and 6.5 days for the PEG saline solution (FIGURE 4). While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that variations may be applied to the compositions, methods and in the steps or sequence of steps of the methods described. in the present without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are both physically and physiologically related can be substituted for agents described herein, while the same or similar results could be achieved. All similar substitutes and apparent modifications for those skilled in the art are considered within the spirit, scope and concept of the invention defined by the appended claims.

References The following references, to the extent that provide details of illustrative procedures or other supplementary details for those exhibited herein, are specifically incorporated herein by reference. Bartoni and Boitard, Yin vitro and in vivo antitumor activity of free, and encapsulated taxol, "J. Microencapsulation, 7: 191-197, 1990. Cortes, J.E. and Pazdur, R.," Docetaxel ", Journal of Clinical Oncology, 13: 2643-2655, 1995. Deutsch et al, "Synthesis of congeners and prodrugs 3. Water-soluble produgs of taxol with potent antitumor activity," J. Med. Chem., 32: 788-792, 1989. Eiseman and others, "Plasma pharmacokinetics and tissue distribution of paclitaxel in CD2F1 mice," Cancer Chemother. Pharmacol., 34: 465-471, 1994. Fidler, et al., "The biology of cancer invasion and metastasis," Adv. Cancer Res., 28: 149-250, 1987. Goldspiel, "Taxol pharmaceutical issues: preparation, administration, stability, and compatibility with other medications," Ann. Pharmacotherapy, 28: S23-26, 1994. Greenwaid et al., "Highly soluble water Taxol derivatives, 7-polyethylene glycol esters as potential products," J. Org. Chem., 60: 331-336, 1995.

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Claims (4)

1. # CLAIMS 1. A composition comprising an anti-tumor drug conjugated with a water-soluble polymer or metal chelator and wherein the anti-tumor drug is paclitaxel, docetaxel, etopside, teniposide, camptothecin or epothilone.
2. 2. The composition of claim 1, wherein the anti-tumor drug is paclitaxel.
3. 3. The composition of claim 1, wherein the anti-tumor drug is docetaxel.
4. 4. The composition of claim 1, wherein the anti-tumor drug is conjugated to a water-soluble metal chelator. The composition of claim 4, wherein it further comprises a chelated metal ion. 6. The composition of claim 5, wherein the ion The metallic chelate is selected from the group consisting of aluminum, boron, calcium, chromium, cobalt, copper, dysprosium, erbium, europium, gadolinium, gallium, germanium, holmium, indium, iridium, iron, magnesium, manganese, nickel, platinum, rhenium, rubidium, ruthenium, samarium, sodium, technetium, thallium, tin, yttrium and zinc. 7. The composition of claim 5, wherein the chelated metal ion is a radionuclide. The composition of claim 7, wherein the radionuclide is selected from the group consisting of 67Ga, 68Ga. 111ln, 99mTc, 90Y, 114mln and 193mPt. 9. A composition comprising 111ln-DTPA-paclitaxel dication 4, wherein the water-soluble chelator is selected from the group consisting of (DTPA), ethylenediaminetetraacetic acid (EDTA), 1,4,7,10-tetraazacyclododecane-N , N ', N ", N'" - tetraacetate (DOTA), acid 5-tetraazacyclotetradecane-N, N ', N ", N'" - tetracetic (TETA), hydroxyethylidene diphosphate (HEDP), dimercaptosuccinic acid (DMSA), diethylenetriaminetetramethylenephosphonic acid (DTTP), DPDP and 1- (p-aminobenzyl) -DTPA . The composition of claim 4, wherein the chelator 10 is diethylenetriaminepentaacetic acid (DTPA). The composition of claim 1, wherein the water-soluble polymer is selected from the group consisting of poly-d-glutamic acid, poly-1-glutamic acid, poly-d-glutamic acid, poly-d-aspartic acid , poly-1-aspartic acid, poly-d1-aspartic acid, 15 polyethylene glycol, polyacrylic acid, poly (2-hydroxyethyl-1-glutamine), carboxymethyl dextran, hyaluronic acid, human serum albumin, alginic acid and a combination of the mimes. The composition of claim 12, wherein the polymer is further defined as a copolymer with polycaprolactone, Polyglycolic acid, polylactic acid, polyacrylic acid poly-2-hydroxyethyl-1-glutamine), carboxymethyl dextran, hyaluronic acid, human serum albumin, polyalgénico acid or combination thereof. Claim 12, wherein the polymer has a molecular weight of from about 5,000 to about 100,000. 15. The composition of claim 12, wherein the polymer has a molecular weight of from about 20,000 to about 80,000. 16. The composition of claim 12, wherein the polymer has a molecular weight of from about 30,000 to about 60,000. 17. The composition of claim 12, wherein the water soluble polymer is conjugated to the 2'- and / or 7-hydroxyl of paclitaxel or docetaxel. 18. The composition of claim 12, wherein the water soluble polymer is polyethylene glycol. 19. The composition of claim 12, wherein the water soluble polymer is poly-1-glutamic acid. The composition of claim 12, wherein the water soluble polymer is poly-1-aspartic acid. 21. The composition of claim 1, dispersed in a pharmaceutically acceptable vehicle solution. 22. A method for determining an antitumor drug uptake by tumor tissue comprising the steps of: a) obtaining a composition comprising a conjugate of paclitaxel or docetaxel and a metal chelator and a chelated metal ion; b) contacting the tumor tissue with the composition; and c) detecting the presence of the chelated metallic element in said tumor tissue; wherein the presence of the chelated metal ion in said tumor tissue indicates paclitaxel by the tumor tissue. 23. A method of claim 22, wherein the anti-tumor drug is paclitaxel. 24. A method of claim 22, wherein the metal ion # chelate is a radionuclide and such detection is the representation 10 scintillation chart. 25. The method of claim 22, wherein the tumor tissue is a subject and said composition is administered to said subject. 26. A method of treating cancer in a subject comprising the steps of: a) obtaining a composition comprising paclitaxel or docetaxel conjugated with a water soluble or chelating polymer and dispersed in a pharmaceutically acceptable solution; and b) administering said solution to said subject in an amount effective to treat the cancer. 27. The method of claim 26, wherein the composition comprises paclitaxel. The method of claim 26, further comprising determining the absorption of paclitaxel or docetaxel in said tumor 25 prior to administration, wherein the determination is administered with a conjugated chelate-metal ion-paclitaxel or docetaxel to said subject and detected the presence of the metal ion in said tumor. 29. The method of claim 26, wherein the cancer is breast cancer, ovarian cancer, malignant melanoma, lung cancer, gastric cancer, colon cancer, head and neck cancer or leukemia. 30. The method of claim 26, wherein the cancer is breast cancer. 31. The method of claim 26, wherein the cancer is ovarian cancer. A method for decreasing at least one symptom of an autoimmune disease comprising administering to a subject having a systemic autoimmune disease an effective amount of a composition comprising paclitaxel or docetaxel conjugated with poly-1-glutamic acid or poly-1 acid -aspartic. 33. The method of claim 32, wherein the composition comprises paclitaxel. 34. The method of claim 32, wherein the composition comprises poly-1-glutamic acid. 35. The method of claim 32, wherein the disease of the autoimmune system is rheumatoid arthritis. 36. A method for inhibiting arterial restenosis or arterial occlusion after vascular trauma comprising administering to a subject in need thereof, as a composition comprising poly-1-glutamic acid or poly-1-aspartic acid. 37. The method of claim 36, wherein the composition comprises paclitaxel. 38. The method of claim 36, wherein the composition comprises poly-1-glutamic acid. 39. The method of claim 36, wherein the subject is a patient with coronary bypass, vascular surgery, organ transplantation or coronary or arterial angioplasty. 40. The method of claim 36, wherein the composition is coated on a stent mass and said stent mass is implanted at the site of vascular trauma. 41. A pharmaceutical composition comprising paclitaxel or docetaxel conjugated to a water soluble or chelating polymer. 42. The composition of claim 41, wherein the water soluble polymer is poly-1-aspartic acid or poly-1-glutamic acid. 43. The composition of claim 41, wherein the chelator is DTPA. 44. The composition of claim 43, further comprising a therapeutic amount of a chelated radionuclide. 45. A method for obtaining an image of a subject comprising: a) administering to said subject an effective amount of agent 25 of claim 6; and b) measuring the signals of the scintillation graphic representation to obtain an image. 46. An implantable median device, wherein the device is coated with a composition comprising paclitaxel or docetaxel conjugated with a polyglutamic acid or polyaspartic acid in an amount effective to inhibit the proliferation of muscle cells. 47. The implantable medical device of claim 46, further defined as a stent mass coated with said composition. 48. The implantable medical device of claim 47, wherein said Stent mass is adapted to be used after balloon angioplasty and the composition is effective to inhibit restenosis. 49. A composition comprising polyglutamic acid conjugated to the 2 'or 7-hydroxyl of paclitaxel. 50. A composition comprising polyaspartic acid conjugated to the 2 'or 7-hydroxyl of paclitaxel. 51. The composition of claim 49, where the polyglutamic acid is conjugated with the 2 'and the 7-hydroxyl of paclitaxel. 52. The composition of claim 40, wherein the polyaspartic acid is conjugated with the 2 'and the 7-hydroxyl of paclitaxel. 53. A composition comprising an anti-tumor drug conjugated with d-glutamic acid, poly-1-glutamic acid, poly-d-glutamic acid, poly-d-aspartic acid, poly-1-aspartic acid, polyhydric acid, d1-aspartic acid, polyacrylic acid, poly-2-hydroxyethyl-1-glutamine. carboxymethyl dextran, hyaluronic acid, human serum albumin, alginic acid or combination thereof, and wherein said anti-tumor drug is paclitaxel, docetaxel, etopside, teniposide, camptothecin or epothilone. 54. The composition of claim 53, wherein the composition is a pharmaceutical composition comprising paclitaxel or docetaxel conjugated to a water soluble polymer, wherein said water soluble polymer is selected from the group consisting of -d-glutamic acid, poly-1-glutamic acid, poly-d-1-glutamic acid, poly-d-aspartic acid, poly-1-aspartic acid, poly-d-1-aspartic acid, polyacrylic acid, poly-2-h hydroxyethyl-1-glutamine, carboxymethyl dextran, hyaluronic acid, human serum albumin, alginic acid or combination thereof. 55. The composition of claim 54, wherein the polymer has a molecular weight of from about 5,000 to about 100,000. 56. The composition of claim 55, wherein the polymer has a molecular weight of from about 20,000 to about 80,000. 57. The composition of claim 56, wherein the polymer has a molecular weight of about 30,000 to about 60,000. 58. The composition of claim 54, wherein the composition comprises paclitaxel or docetaxel conjugated to a water soluble polymer, wherein the water soluble polymer is selected from the group consisting of poly-d-glutamic acid, poly-1 acid -glutamic, or poly-d1-glutamic acid or a combination thereof.