Water-soluble promedicines of paklitaxel

The present invention relates to a pharmaceutical composition containing an anti-cancer drug, its uses for the preparation of drugs, and for the coating of medical devices intended to be implanted into the body. Generally, the invention relates to the field of pharmaceutical compositions for the treatment of cancer, autoimmune diseases and restenosis. The invention also relates to the field of the pharmaceutical preparation of anti-cancer agents such as paclitaxel (paclitaxel) (Taxol) and docetaxel (docetaxel) (Taxotere), in particular the preparation of water-soluble paclitaxel by conjugating the drug with water-soluble groups.

Paclitaxel, an anti-microtubule agent extracted from Pacific yew tree needles and bark, Taxus brevifolia, showed remarkably anti-cancer activity in human cancer in phase I and early phase II and III trials (Horwitz et al., 1993). Initially, this was reported in cases of advanced ovarian and breast cancer. Considerable activity has been documented in small cell and non-small cell lung cancer, in head and neck cancers, and in metastatic melanoma. However, a major obstacle to the development of clinical trials with paclitaxel was its lack of water solubility.

Docetaxel is semisynthetically made from 10-deacetyl baccatin III (10-deacetyl baccatin III), a non-cytotoxic precursor extracted from common yew needles (Taxus baccata) and esterified with a chemically synthesized side chain (Cortes and Pazdur, 1995). Various cancer cell lines, including those of the breast, lung, ovary, colon and rectum, and melanomas, were sensitive to docetaxel. Docetaxel has been used in clinical trials to obtain complete or partial responses in breast, ovarian, head and neck cancer and malignant melanoma.

Paclitaxel is typically prepared as a concentrated solution containing 6 mg of paclitaxel per milliliter of Cremophor EL (polyoxyethylated castor oil) and dehydrated alcohol (50% v / v) and must then be diluted prior to administration (Goldspiel, 1994). The amount of Cremophor EL required to deliver the desired doses of paclitaxel is much higher than any other drug suspended in Cremophor. Cremophor has been associated with certain toxic effects including vasodilatation, dyspnoea and hypotension. This medium has also been shown to cause severe hypersensitivity in laboratory animals and humans (Weiss et al., 1990). In fact, the maximum dose of paclitaxel that can be administered by bolus intravenous injection to mice is determined by the acute lethal toxicity of the vehicle - Cremophor (Eiseman et al., 1994). Furthermore, Cremophor EL, a surfactant, is known to leach plastifi189 698 phthalate minerals such as di (2-ethylhexyl) phthalate (DEHP) from polyvinylchloride bags and cannula. DEPH is known to be hepatotoxic to animals and carcinogenic to rodents. It has also been shown that this formulation produces solid particles over time and therefore requires filtration during administration (Goldspiel, 1994). Thus, in order to ensure the safe delivery of the drug to the patients, special precautions for the formulation and administration of the paclitaxel solutions are necessary, leading inevitably to increased costs.

Earlier attempts to prepare water-soluble paclitaxel have involved the preparation of a paclitaxel prodrug by placing solubilizing groups such as succinate and amino acids at the 2'-hydroxyl group or the 7-hydroxyl position (Deutsch et al., 1989; Mathew et al., 1992). However, these prodrugs did not provide sufficient chemical stability for the further development of the trials. For example, Deutsche et al. (1989) reported the 2'-succinate derivative of paclitaxel, but the water solubility of its sodium salt is only about 0.1%, and the triethanolamine and N-methylglucamine salts were only soluble in about 1⁰%. Moreover, it was reported that the amino acid esters were unstable. Similar results were reported by Mathew et al. (1992). Greenwald et al. reported the synthesis of the highly water-soluble 2'- and 7'-esters of taxol polyethylene glycol (Greenwald et al., 1994), but no data on the tumor activity of these compounds in vivo (Greenwald et al., 1995) were presented.

Other attempts to solve these problems have involved the entrapment of paclitaxel in both liposomes and nanospheres (Bartoni and Boitard, 1990). The liposome form was reported to be as effective as free paclitaxel, however only the liposome form containing less than 2% paclitaxel was physically stable (Sharma and Straubinger, 1994). Unfortunately, the form in the nanospheres turned out to be toxic. Thus, there is still a need for a water-soluble form of paclitaxel that can provide effective amounts of paclitaxel and docetaxel without the drawbacks of drug insolubility.

Another obstacle to the widespread use of paclitaxel is the limited sources from which paclitaxel is produced, which makes paclitaxel therapy expensive. For example, a course of treatment can cost several thousand dollars. An additional disadvantage is that not all tumors respond to paclitaxel therapy, which may be because paclitaxel does not penetrate the tumor.

U.S. Patent No. 4,960,790 teaches that various single amino acid residues and derivatives, referred to as "alkylated amino acids," can be conjugated to taxol (col. 2, in 19-57). The compounds disclosed therein are 2 'and / or 7' esters of taxol and amino acids or alkylated amino acids.

There remains an urgent need for effective forms of paclitaxel and related drugs that are water-soluble and have long serum half-lives for the treatment of tumors, autoimmune diseases such as rheumatoid arthritis, as well as the prevention of vasoconstriction associated with trauma such as like plastic surgery and arthroplasty.

It is an object of the present invention to overcome these and other disadvantages inherent in the prior art by providing compositions comprising a chemotherapeutic and anti-angiogenic drug such as paclitaxel or docetaxel coupled with, for example, water-soluble polymers such as polyglutamic acid or polyaspartic acid, or a water-soluble chelator. metal. These compositions have been shown herein to be surprisingly effective as anti-tumor agents against exemplary tumor models and are expected to be at least as effective as paclitaxel or docetaxel in diseases or conditions for which the effectiveness of taxanes or taxoids is known. The compositions of the invention deliver water-soluble taxoids overcoming the insolubility inconvenience of these drugs and also provide the benefit of controlled drug release, so it has been shown in animal models here that tumors eradicated after a single intravenous administration.

The present invention relates to a pharmaceutical composition comprising an anti-cancer drug as an active ingredient and a pharmaceutically acceptable carrier, characterized in that the anti-cancer drug is conjugated to a water-soluble polymer selected from the group consisting of poly (d-glutamic acid), poly (acid

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1-glutamic acid), poly (dl-glutamic acid), poly (d-aspartic acid), poly (acid

1-aspartic acid), poly (dl-aspartic acid), polyacrylic acid, poly (2-hydroxyethyl-1-glutamine), carboxymethyldextran, hyaluronic acid, human serum albumin, alginic acid and a combination thereof or a metal chelator, where the anti-cancer drug is paclitaxel, docetaxel, etopside, teniposide, camptothecin, or epothilone.

Preferably the anti-cancer drug is paclitaxel, docetaxel or camptothecin, more preferably paclitaxel or docetaxel, in particular paclitaxel or docetaxel.

The conjugate in the composition of the invention preferably comprises up to 35%, more preferably 15-25% of an anti-cancer drug. The molecular weight of the conjugate in the composition of the invention is in the range 45,000-55,000.

As the water-soluble polymer, a copolymer of the above-defined polyamino acids with polycaprolactone, polyglycolic acid, polylactic acid, polyacrylic acid, poly (2-hydroxyethyl-1-glutamine), carboxymethyldextran, hyaluronic acid, human serum albumin or a combination thereof may also be used.

Preferably the polymer has a molecular weight of from about 5,000 to about 100,000, more preferably from about 20,000 to about 80,000, and most preferably from about 30,000 to about 60,000. Preferably, the water-soluble polymer is conjugated to the 2 'and / or 7-hydroxyl group of paclitaxel in the composition of the invention. or docetaxel.

As the water-soluble polymer, the composition preferably comprises poly (1-aspartic acid) or poly (1-glutamic acid). In one embodiment, the composition comprises poly (1-glutamic acid), and in another embodiment, poly (1-aspartic acid).

Preferably, poly (glutamic acid) is conjugated to the 2'- or 7-hydroxyl group of paclitaxel, similarly poly (aspartic acid) is conjugated to the 2'- or 7-hydroxyl group of paclitaxel.

When functional groups such as the above-mentioned C2'-hydroxyl group of paclitaxel are used for drug conjugation, an unstable bond, in this case an ester, was used to ensure release of the active drug form from the polymeric carrier.

Preferably the composition is dispersed in a pharmaceutically acceptable carrier solution. Such solution should be sterile or aseptic, and may include water, buffers, isotonic agents, or other ingredients known to those skilled in the art that, when administered to animals or humans, should not cause allergic or other serious reactions.

Preferably the composition of the invention comprises paclitaxel or docetaxel conjugated to a water-soluble polymer selected from the group consisting of poly (d-glutamic acid), poly (1-glutamic acid), or poly (dl-glutamic acid) or a combination thereof.

In another embodiment, the composition of the invention comprises an anti-cancer drug conjugated to a water-soluble metal chelator.

Preferably the chelator is diethylenetriaminepentaacetic acid (DTPA).

The composition according to the invention may additionally contain a therapeutic amount of a chelated metal ion, especially 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. The chelated metal ion may be a radionuclide, i.e. a radioactive isotope of one of the mentioned metals, in particular selected from the group consisting of⁶⁷Ga, ⁶⁸Ga, ^1HIn, ^99mTc, ⁹⁰Y, ^I14mIn and ^193mFri.

Preferably the composition of the invention comprises a water-soluble chelator selected from the group consisting of diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), -N, N ', N, N'-1,4,7,10-tetraazacyclododecane-tetraacetate (DOTA), tetraazacyclotetradecane-N, N ', N' ', N'-tetraacetic acid (TETA), hydroxyethylidene diphosphonate (HEDP), dimercaptosuccinic acid (DMSA), diethylenetriaminetetramethylenephosphonic acid (DTTP), 1,6-diaminohexane-N, N, N, N ', N'-tetraacetic, ethylenebis (oxyethylenenitrile) tetraacetic acid, DPDP and 1- (p-aminobenzyl) -DTPA, especially diethylenetriaminepentaacetic acid (DTPA).

In a specific embodiment, the composition of the invention comprises ^H1In-DTPA-paclitaxel.

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The invention furthermore relates to the use of a composition comprising paclitaxel or docetaxel conjugated with a water-soluble polymer as defined above or a chelator and dispersed in a pharmaceutically acceptable solution for the manufacture of a medicament for the treatment of cancer, especially breast cancer, ovarian cancer, malignant melanoma, lung cancer, gastric cancer, cancer. colon cancer, prostate cancer, head and neck cancer, Kaposi's sarcoma or leukemia, and in particular breast cancer or ovarian cancer.

In a preferred embodiment, the composition comprises paclitaxel.

In another aspect, the invention relates to the use of a composition comprising paclitaxel or docetaxel conjugated to poly (1-glutamic acid) or poly (1-aspartic acid) in the manufacture of a medicament for the treatment and / or alleviation of at least one symptom of a systemic autoimmune disease.

Paclitaxel and poly (1-glutamic acid) are preferably used. Such a composition is preferably used for the treatment of rheumatoid arthritis, which is known to respond in some cases to taxol administered in a standard Cremophor-based formulation (US Patent 5,583,153). It is believed that in treating tumors, the effectiveness of the water-soluble taxoids of the invention is not diminished by conjugation with the water-soluble groups, and that the water-soluble prodrugs may act as a controlled drug release agent that releases the active drug over a given period of time. Therefore, the compositions of the invention are expected to be as effective in rheumatoid arthritis as, for example, Taxol, but will have a controlled release advantage. It is also understood that the compositions of the present invention may be used in conjunction with other drugs such as an angiogenesis inhibitor (AGM-1470) (Oliver et al., 1994) or methotrexate.

In a further aspect the invention relates to the use of a composition comprising paclitaxel or docetaxel conjugated to poly (1-glutamic acid) or poly (1-aspartic acid) in the manufacture of a medicament for inhibiting restenosis or occlusion of an artery following vascular injury.

Paclitaxel and poly (1-glutamic acid) are preferably used.

A composition of this type is preferably used to treat a patient with coronary bypass surgery, post-vascular surgery, organ transplant, or coronary or arterial plasticization.

The finding that paclitaxel also inhibits vasoconstriction after balloon plasticization indicates that the water-soluble paclitaxels and docetaxels disclosed in the present invention will find various uses besides direct parenteral administration (WO 9625176).

It is contemplated that in these embodiments, the water-soluble paclitaxel may be combined with an implantable medical device, or alternatively the water-soluble paclitaxel may be passively adsorbed onto the surface of the device to be implanted. For example, vascular prostheses can be coated with polymer-drug conjugates by dipping into or spraying their conjugate solution. Suitable materials for implanting devices should be biocompatible and non-toxic and may be selected from metals such as nickel titanium alloys, steel, or biocompatible polymers such as hydrogels, polyurethanes, polyethylenes, ethylene vinyl acetate copolymers, etc.

In a preferred embodiment, water-soluble paclitaxel, in particular a PG-paclitaxel conjugate, covers a vascular prosthesis intended for insertion into an artery or vein after balloon angioplasty. Thus, the invention may be determined, in some broader aspects, to be useful for inhibiting arterial restenosis or arterial occlusion following a vascular injury. In patients with coronary bypass surgery, after vascular surgery, organ transplant, or coronary or arterial plastic surgery, the composition according to the invention may be administered directly, intravenously, or it may even poke a vascular prosthesis which is implanted immediately after a vascular injury.

A further object of the invention is therefore the use of a composition containing paclitaxel or docetaxel conjugated with water-soluble polyamino acids.

189 698 in an amount effective in inhibiting the proliferation of smooth muscle cells, to coat medical devices intended for implantation into the body.

Preference is given to using polyamino acids selected from the group consisting of polyglutamic acids and poly aspartic acids.

The medical device is in particular a vascular prosthesis, a vascular fistula, a catheter, an artificial implant, a needle electrode, a pacemaker, and in particular a vascular prosthesis coated with the said composition.

The vascular prosthesis is adapted for use after balloon angioplasty, and said composition is effective in inhibiting restenosis.

The disclosure herein could also be used to prepare water-soluble polymeric conjugates of other therapeutic agents, contrast agents, and drugs, including ethopside, teniposide, fludarabine, doxorubicin, daunomycin, emodin, 5-fluorouracil, FUDR, estradiol, camptothecin, retinic acid. , verapamil, epothilones and cyclosporine. In particular, agents with a free hydroxyl group could be coupled to the polymers via similar chemical reactions as described for paclitaxel herein. Such conjugation would be within the scope of the daily chemistry practitioner and as such would fall within the scope of the claimed invention. These factors could include, but are not limited to, ethopside, teniposide, camptothecin, and epothilones. The term "conjugated water-soluble polymer" as used herein means the covalent association of a drug with a polymer or chelator.

It is also understood that the water-soluble conjugates of the present invention may be administered in conjunction with other drugs including other anti-cancer or anti-cancer drugs. Such combinations are known. The water-soluble paclitaxel or docetaxel of the invention may be combined with platinum in certain types of treatment, antibiotics such as, for example, doxorubicin or daunorubicin, or other drugs that are used in combination with Taxol.

The conjugation of chemotherapeutic drugs with polymers is an interesting way to reduce systemic toxicity and improve the therapeutic index. Polymers with a molecular weight greater than 30 kDa do not readily diffuse through the normal capillaries and glomerular endothelium, thereby protecting normal tissue from undesirable drug-induced toxicity (Maeda and Matsumura, 1989; Reynolds, 1995). On the other hand, it has been shown that malignant tumors often have damaged capillary endothelium and a more permeable vascular system than normal tissues (Maeda and Matsumura, 1989; Fidler et al., 1987). Thus, a drug-polymer conjugate that normally remains in the vasculature can selectively pass from blood vessels to tumors, causing the therapeutically active drug to accumulate in the tumor. In addition, drug-polymer conjugates can act as a drug-controlling depot, causing tumor cells to be exposed to the drug for longer. After all, water-soluble polymers can be used to stabilize drugs as well as to solubilize otherwise insoluble compounds. Currently, a wide variety of synthetic and natural polymers have been tested for their ability to enhance tumor specific drug delivery (Kopecek, 1990, Maeda & Matsumura, 1989). However, only a few are currently undergoing clinical trial, among them SMANCS in Japan and HPMA-Dox in the UK (Maeda, 1991; Kopecek and Kopeckova, 1993).

In the present disclosure, the term "taxoid" means those compounds that include paclitaxel and docetaxel, and chemicals having a "taxon skeleton" (Cortes and Pazdur, 1995) and may be isolated from natural sources such as yew or cell culture, or may be chemically synthesized molecules, and is preferably a chemical compound of the general formula C47H51NO14, including ester 6.12b, bis (acetyloxy) -12- (benzoyloxy) -2a, 3,4,4a, 5,6,9,10,11, 12.12a, 12b-dodecahydro-4,11-dihydroxy-4a, 8,13,13-tetramethyl-5-oxo-7,1 1-methane-1H-cyclodeca [3,4] -benz- [1,2-b] oxet [2aR- [2aa, 4β, 4aP, 6β, 9a (aR * pS *), 11a, 12a, 12aa, 12ba,]] - P- (benzoylamino) -a-hydroxybenzenopropionic acid -9-yl. It is understood that for certain types of tumors, paclitaxel and docetaxel differ in effectiveness, and that in the practice of this invention, those tumors that are more sensitive to a given taxoid should be treated with a water-soluble conjugate of that taxoid.

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By preparing the drug and metal chelator conjugate with a chelated metal ion, and then contacting the tumor tissue with the composition, the presence of chelated metal ion in the tumor tissue can be detected. This presence is indicative of the uptake of the chelated metal ion by the tumor tissue. The chelated metal ion may be a radionuclide and may be detected scintigraphically. The composition should be administered to animals or humans with evidence of tumor tissue.

In certain embodiments, the compositions of the present invention can be used in the treatment of a patient afflicted with cancer.

The method of treating a tumor may include some evaluation of paclitaxel or docetaxel uptake by the tumor prior to administration of a therapeutic amount of the drug or prodrug. The method may include certain imaging methods, discussed below, in which the paclitaxel-chelator-metal chelate is administered to a patient and then detected in the tumor. This step provides a cost-effective way to conclude that in cases where drug does not penetrate the tumor, tumor response to DTpA-paclitaxel treatment is not to be expected. By using imaging to predict responses to paclitaxel and identify patients who are unlikely to respond to treatment, it will be possible to save the latter from high costs and a waste of valuable time. It is assumed that unless a rational amount of a chemotherapeutic agent is deposited in the tumor, the likelihood of the tumor responding to this agent is relatively low.

Compositions of the invention can be used in a method of obtaining an image of a patient's body. A body image is obtained by administering to the patient an effective amount of a radioactive metal ion chelated with a paclitaxel chelator conjugate and measuring scintigraphic radioactive metal signals to obtain an image.

The terms "polyglutamic acid" or "polyglutamic acid" as used herein mean poly (glutamic acid), poly (d-glutamic acid), poly (dl-glutamic acid), and the terms "poly aspartic acid" or "poly aspartic acid" mean poly (1-aspartic acid), poly (d-aspartic acid) and poly (dl-aspartic acid).

Unless defined inac / ei, all technical and scientific terms used have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although certain methods and materials similar to or equivalent to those described herein may be eastos / waee in the practice or research of the present invention, the preferred methods and materials will now be described.

Figure 1A. Chemical structure of paclitaxel, PEG-paclitaxel and DTPA-paclitaxel.

Figure IB. 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 B & lt; 1 & gt; cesarean cells.

Figure 3. Preventive / oral DTPA-paclitaxel effect on MCa-4 breast tumors.

Figure 4. Mean time (days) of tumor diameter growth to 12 mm after treatment with Pain, DTPA-paclitaxel, and PEG-PAC.

Figure 5. Gamma-scintigraphy of mice affected by MCa-4 tumors after intravenous injection ^11,Ie-DTPA-paclitakshlu i ^ulIn-DTPA.

The arrow points to the tumor.

Figure 6. Distribution of hndr / lithium PG-paclitaxel determined in PBS at pH 7.4 at 37 ° C. represents the percentage of paclitaxel that remained bound to soluble PG, --Δ-represents the percentage of wntw / re / eeg / metabolites, - Δ-represents the percentage of wntw / re / eeg / metabolites.

Figure 7A. Antimicrobial action of PG-paclitaxel in rats affected by mouse mammary tumor (13762F). - □ - represents the response to a single intravenous dose of PG (0.3 g / kg); --A— represents the response to paclitaxel (40 mg / kg), --O-- represents the response to PG-paclitaxel (equivalent to 60 mg paclitaxel / kg).

Figure 7B. Preventive effect of PG-paclitaxel and paclitaxel in mice affected by OCa-1, pi ^^ ssa ^ ao / 0 ^ e / ^ Ti ^ idi <per p (/ edinium dose of PG dosing (0.8 (0.8 ( gkg);

□ A-- represents the response to paclitaxel (80 mg / kg),

- □ - represents the response to PG-paclitaxel (equivalent to 80 mg paclitaxel / kg),

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--O - represents the response to PG-paclitaxel (equivalent to 160 mg paclitaxel / kg).

Figure 7C. Antitumor activity of PG-paclitaxel in mice affected by MCa-4 breast carcinomas. - □ - represents the response to a single intravenous dose of saline solution, "□ - represents the response to a single intravenous dose of PG (0.6 g / kg); shows the response to PG-paclitaxel (40 mg / kg), -0- shows the response to PG-paclitaxel (equivalent to 60 mg paclitaxel / kg), -O- shows the response to PG-paclitaxel (120 mg / kg).

Figure 7D. Antitumor effect of PG-paclitaxel on mouse soft tissue sarcomas (FSa-II). represents the response to a single intravenous dose of saline, --O- represents the response to a single intravenous dose of PG (0.8 g / kg); —O-- represents the response to paclitaxel (80 mg / kg), --Δ— represents the response to PG-paclitaxel (equivalent to 160 mg paclitaxel / kg).

Figure 7E. Antitumor effect of PG-paclitaxel on syngeneic liver cancer in mice (HCa-I). - shows the response to a single intravenous dose of saline solution, "A" shows the response to a single intravenous dose of PG (0.8 g / kg); --O— represents the response to PG-paclitaxel (80 mg / kg), —A— represents the response to PG-paclitaxel (equivalent to 160 mg paclitaxel / kg).

Figure 8. Release profile of paclitaxel from PEG-paclitaxel in phosphate buffer (pH 7.4). Paclitaxel, -X-; PEG-paclitaxel, -O-.

Figure 9. Antitumor effect of PEG-paclitaxel on MCa-4 breast tumors. represents the response to a single intravenous injection of PEG in saline (60 mg / ml), - - represents the response to Cremophor / alcohol, --O-- represents a single dose of 40 mg paclitaxel / kg body weight, - shows PEG-paclitaxel at 40 mg paclitaxel equivalent / kg body weight.

The present invention stems from the development of new water-soluble formulations of paclitaxel and docetaxel and the finding of the surprising efficacy of these formulations against tumor cells in vivo. Paclitaxel (PG-paclitaxel) conjugated poly (1-glutamic acid) when administered to mice afflicted with ovarian cancer (OCa-I) caused a significant delay in tumor growth compared to the same dose of paclitaxel without PG. paclitaxel and PG showed a tumor growth delay initially, but after ten days the tumors re-enlarged to a size comparable to the untreated control group. In addition, at the maximum tolerated dose (MTD) of the PG-paclitaxel conjugate (equivalent to 160 mg paclitaxel / kg), tumor growth was completely inhibited, tumors disappeared, and mice observed for two months post-treatment did not have tumor recurrence (MTD: defined as the maximum dose that was caused 15% or less weight loss over two weeks after a single intravenous injection). In parallel, studies were conducted on the anti-tumor activity of PG-paclitaxel in rats with rat mammary adenocarcinoma (13762F). Again, complete tumor eradication was observed with a dose of PG-paclitaxel equivalent to 40-60 mg paclitaxel / kg. These surprising results show that the polymer-drug conjugate PG-paclitaxel is effective in eradicating advanced solid tumors in both mice and rats after a single intravenous injection. Furthermore, PG-paclitaxel with a 40-day half-life at pH 7.4 is one of the most stable water-soluble paclitaxel derivatives known (Deutch et al., 1989; Mathew et al., 1992; Zhao and Kingston, 1991).

It has also been shown here that DTPA-paclitaxel is as effective as paclitaxel in an in vitro antitumor assay using the B16 melanoma cell line. DTPA-paclitaxel as compared to paclitaxel did not show any significant differences in antitumor activity against MCa-4 breast tumor at a dose of 40 mg / kg body weight in a single injection. In addition, it was shown that DTPA-paclitaxel was labeled^H.1ndem accumulated, as shown by gamma-scintigraphy, in MCa-4 tumor, confirming that the anti-cancer drug conjugated chelator of the present invention is useful and effective in tumor imaging.

The novel compositions and uses of the invention provide significant advantages over previously known compositions and uses and it is anticipated that water-soluble paclitaxels will improve the efficacy of paclitaxel based anti-cancer therapy by providing paclitaxel-derived compositions with controlled release paclitaxel and soluble in the water. These compositions eliminate the need for solvents with which there are side effects seen with previously known paclitaxel compositions. In addition, radiolabelled paclitaxel, while retaining anti-tumor activity, will also be useful in tumor imaging. Thus, the present invention makes it possible to determine, by means of scintigraphy, single photon emission computed tomography (SPECT) or positron emission tomography (PET), whether paclitaxel will be taken up by a given tumor. This term can then be used to decide on the effectiveness of an anti-cancer treatment. This information may be helpful for the clinician in selecting patients for paclitaxel therapy.

Paclitaxel can be made soluble in two ways: by conjugating paclitaxel with water-soluble polymers that serve as drug carriers and by derivatizing the anti-cancer drug with water-soluble chelators. The second method also provides the possibility of labeling with radionuclides (e.g.^1HIn, ⁹⁰Y, ^I66Ho, ⁶⁸Ga, ^99mTc) for nuclear imaging and / or radiotherapy research. The structure of paclitaxel, polyethylene glycol-paclitaxel (PEG-paclitaxel), polyglutamic acid-paclitaxel (PG-paclitaxel) conjugate and diethylenetriaminepentaacetic acid-paclitaxel (DTPA-paclitaxel) are shown in Figure 1.

In some embodiments of the present invention, DTPA-paclitaxel or other paclitaxel-chelator conjugates such as, for example, EDTA-paclitaxel, DTTP-paclitaxel or DOTA-paclitaxel, can be formulated as water-soluble salts (sodium salt, potassium salt, tetrabutylammonium salt, calcium salt, iron salt, etc.). These salts will be useful as therapeutic agents in the treatment of a tumor. Second, DTPA-paclitaxel or other paclitaxel-chelator conjugates will be useful as diagnostic agents which, when labeled with a radionuclide such as ^mIn or 9 ^mTc can be used as radioactive tracers in combination with nuclear imaging methods to detect certain tumors. It is understood that other taxane derivatives, in addition to paclitaxel (taxol) and docetaxel (taxotere), may be used in the compositions and uses of the present invention, and all such compositions and uses are included in the appended claims.

DTPA-paclitaxel toxicity, pharmacokinetics and tissue distribution studies showed that for mice, the LD50 (50% lethal dose) of DTPA-paclitaxel, observed with a single intravenous injection, is approximately 110 mg / kg body weight. A direct comparison with paclitaxel is difficult to make due to intravenous dose volume limitations imposed by the limited solubility of paclitaxel and the toxicity of the vehicle. However, in view of the present disclosure, those skilled in the art of chemotherapy could determine effective and maximum tolerated doses for human use in clinical trials.

In some embodiments of the invention, vascular prostheses coated with polymer-paclitaxel conjugates can be used to prevent restenosis, occlusion of arteries after balloon angioplasty. Recent results of clinical trials using balloon-dilated vascular prostheses in coronary artery plastic surgery show a significant improvement in patency and a reduction in the number of restenoses compared to standard balloon angioplasty (Serruys et al., 1994). According to the "response to injury" hypothesis, new vessel intimal formation is associated with cell proliferation. It is now widely believed that smooth muscle cell proliferation (SMC) is a critical process leading to vascular damage in spontaneous and accelerated atherosclerosis (Phillips-Hughes and Kandarpa, 1996). Since the phenotypic proliferation of SMCs following arterial trauma has been found to mimic tumor cells, it is possible that the use of anti-cancer drugs may be useful in preventing SMC accumulation in the newly formed intima. Vascular prostheses coated with antiproliferative factors linked to a polymer in such a way as to allow the release of these factors over an extended period of time in sufficient concentration will therefore prevent intimal and muscular growth into the lumen of the vessel, thereby reducing restenosis.

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Since paclitaxel was able to suppress collagen, which induces arthritis in a mouse model (Oliver et al., 1994), the use of the embodiments of the present invention in the treatment of autoimmune and / or inflammatory diseases such as rheumatoid arthritis is also contemplated. Binding of paclitaxel to tubulin shifts the equilibrium towards stable microtubule polymers, making this drug a potent inhibitor of eukaryotic cell replication by blocking cells in late G2 mitosis. Several mechanisms may be associated with paclitaxel-induced arthritis suppression. For example, paclitaxel's specific cytotoxic phase can affect rapidly proliferating inflammatory cells, and in addition, paclitaxel inhibits cell mitosis, migration, chemotaxis, intracellular transport, and H production.₂ABOUT₂ by neutrophils. In addition, paclitaxel may exhibit anti-angiogenic activity by blocking the coordinated migration of endothelial cells (Oliver et al., 1994). Therefore, the polymer conjugated prodrugs of the present invention are considered to be as useful as free paclitaxel in the treatment of rheumatoid arthritis. The polymer conjugate form disclosed herein should also have the advantage of delayed and extended release and greater solubility of the drug. There is also this aspect of arthritis treatment in which preparations can be injected or implanted directly into the affected joints.

Pharmaceutical preparations of paclitaxel or docetaxel suitable for injectable use include sterile aqueous solutions or suspensions and sterile powders for the extemporaneous preparation of sterile injectable solutions or suspensions. In all cases, the injectable form must be sterile and liquid. It must be stable under the conditions of manufacture and storage and must be preserved against the contamination with microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium and includes, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. The action of microorganisms can be prevented by using various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride.

Injectable sterile solutions are prepared by adding the active compounds in the required amount with various of the other ingredients enumerated above to the appropriate solvent, followed by filtered sterilization as necessary. Generally, suspensions are prepared by adding the various sterilized active ingredients to a sterile vehicle that contains the dispersion medium and the desired 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 methods which provide, from a previously sterile filtered solution, a powder containing the active agent plus an additional desired ingredient.

As used herein, the phrase "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coating agents, antibacterial and antifungal agents, and isotonic agents, and the like. The use of such media and agents for pharmaceutically active substances is well known. Any conventional media and agents are contemplated for use in the therapeutic compositions, except those which are to some extent incompatible with the active ingredient. Supplementary active ingredients can also be incorporated into the compositions.

The phrase "pharmaceutically acceptable" also refers to molecular entities and compositions that do not cause allergic or similar adverse reactions when administered to animals or humans.

For parenteral administration, for example in the form of aqueous solutions, the solution, if necessary, should be suitably buffered and the liquid solvent previously brought to the desired isotonicity with an appropriate saline or glucose solution. These individual aqueous solutions are particularly suitable for intravenous or intraperitoneal administration. In this view, sterile aqueous media that can be employed will be known to those skilled in the art in light of the present disclosure.

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The following examples are included to demonstrate the preferred embodiments of the invention. It should be apparent to those skilled in the art that the techniques disclosed in the following examples represent techniques selected by the inventor to function well in the practice of the present invention, and it is obvious that they are the preferred method for their practical application. However, in light of the present disclosure, those skilled in the art should recognize that numerous changes may be made to the particular embodiments of the invention, which are disclosed below, to achieve the same or similar results without departing from the spirit and scope of the invention.

Example 1

DTPA-paclitaxel

DTPA-paclitaxel synthesis:

Diethylenetriaminepentaacetic acid anhydride (DTPA A) (210 mg, 0.585 mmol) was added to a solution of paclitaxel (100 mg, 0.117 mmol) in dry DMF (2.2 ml) at 0 ° C. The reaction mixture was stirred overnight at 4 ° C. The slurry was filtered (0.2 µm Millipore filter) to remove unreacted DTPA anhydride. The filtrate was washed with distilled water, stirred for 20 min. at 4 ° C and the precipitate was collected. The crude product was purified by conducting preparative TLC on C8 silica gel plates developed with acetonitrile / water (1: 1). The Rf value for paclitaxel is 0.34. The band above the corresponding paclitaxel for which the Rf ranged from 0.65 to 0.75 was removed by scraping, eluted with acetonitrile / water (1: 1), and after removal of the solvent, 15 mg of DTPA-paclitaxel was obtained (yield 10.4%). ): m.p. > 226 ° C with decomposition. The UV spectrum (sodium salt in water) shows an absorption maximum at a wavelength of 228 nm, which is also characteristic of paclitaxel. Mass spectrum: (FAB) m / e 1229 [M + H] +, 1251 [M + Na], 1267 [M + K]. In the 1 H NMR spectrum (DMSO-d₆) NCH groups resonance₂CH₂N and CH₂COOH DTPA manifests itself with appropriately complex series of signals at δ 2.71 - 2.96 ppm and a multiplet at 6 3.42.The shift of the signal corresponding to the C7-H group resonance from 4.10 ppm for paclitaxel to 5.51 ppm indicates the estrification of carbon at position 7. The rest of the spectrum corresponds to the structure of paclitaxel.

DTPA-paclitaxel sodium was also prepared by adding a solution of DTPA-paclitaxel in ethanol to an equivalent amount of 0.05 M NaHCO 3 followed by lyophilization to obtain a water-soluble solid powder (solubility> equivalent to 20 mg paclitaxel / ml).

Hydrolysis resistance of DTPA-paclitaxel:

The hydrolysis resistance of DTPA-paclitaxel was tested under conditions of continuous motion. Briefly, 1 mg of DTPA-paclitaxel was dissolved in 1 ml of 0.5 M NaHCO3 aqueous solution (pH 9.3) and analyzed by HPLC. The HPLC system consisted of a Waters 150 x 3.9 mm (ID) Nova-Pak column coated with 4 µm C18 silica gel and a Perkin-Elmer isocratic LC pump, Nelson 900 series PE connectors, Spectra-Physics UV / Vis detector and a teleddating station. The eluate (acetonitrile / methanol / 0.02 M ammonium acetate = 4: 1: 5) was passed at a rate of 1 ml / min when detected by UV light with a wavelength of 228 nm. The retention times of DTPA-paclitaxel and paclitaxel were 1.38 and 8.83 minutes, respectively. Peak areas were calculated and compared to the standard curve to determine the DTPA-paclitaxel and paclitaxel concentrations.

Estimated half-life

DTPA-paclitaxel in 0.5 M NaHCO3 solution at room temperature was approximately 16 days.

Effect of DTPA-paclitaxel on the in vitro growth of murine Bn melanoma cells,

Cells were placed in a 24-well plate at a concentration of 2.times.10⁴ cells / ml and allowed to grow at 37 ° C for 24 hours in a 97% humidity and 5.5% CO2 atmosphere on a 50:50 modified Dulbecco essential minimum (Dulbecco's medium) (DEM) medium and Fj2 medium containing 10% of calf serum. The medium was then replaced with fresh medium containing DTPA-paclitaxel at a concentration of 14

189 698 in the range from 5x10 '⁹ up to 75x10 '⁹ M. After 40 hours, cells were split by trypsinization and counted on a Coulter counter. The final concentration of DMSO (used to dissolve paclitaxel) and 0.05 M sodium bicarbonate solution (used to dissolve DTPA-paclitaxel) in cell medium was less than 0.01%. This amount of solvent had no effect on cell growth, which was confirmed by control tests.

The effect of DTPA-paclitaxel on the growth of Bi6 melanoma cells is shown in Figure 2. After incubation for 40 hours at various concentrations, DTPA-paclitaxel and paclitaxel were compared for cytotoxicity. The IC50 for paclitaxel and DTPA-paclitaxel are 15 nM and 7.5 nM, respectively.

Antitumor activity in a breast cancer model (MCa-4):

Female C3Hf7Kam mice were implanted with breast cancer cells (MCa-4) in the muscles of the right thigh (5 x 10 ⁵ cells / mouse). When the tumors had grown to 8 mm (approximately 2 mg / kg each), a single dose of either polyol or DTPA-paclitaxel was administered at the equivalent of 10, 20 and 40 mg paclitaxel / kg body weight. body. Saline solution and a 50/50 mixture of absolute alcohol / Cremophor dissolved in saline (1: 4) were used in the control tests. Tumor growth was determined daily by measuring three orthogonal tumor diameters. When the tumor size reached 12 mm in diameter, tumor growth retardation was assessed. Mice were anesthetized when the cancer was approximately 15 mm in size.

The tumor growth curve is shown in Figure 3. Compared to the control group, both palkitaxel and DTPA-paclitaxel showed antitumor activity at the dose of 40 mg / kg. Data was also analyzed to determine the average number of days a tumor reaches 12 mm in diameter. Statistical analyzes showed that DTPA-paclitaxel at a dose of 40 mg / kg (p <0.01) significantly delayed tumor growth compared to the saline control group. The mean time it takes for a tumor to reach 12 mm in diameter for DTPA-paclitaxel was 12.1 days compared to 9.4 days for paclitaxel (Figure 4).

Radiolabelling of DTPA-paclitaxel ¹¹¹Ic.

40 µl of 0.6 M sodium acetate buffer (pH 5.3), 40 µl of 0.06 M sodium citrate buffer (pH 5.5), 20 µl of DTPA-paclitaxel solution in ethanol (2 % w / v) and 20 µl of solution ⁿ InCh (1.0 mCi) in sodium acetate buffer (pH 5.5). After an incubation period of about 30 minutes at room temperature. marked¹l¹The Ic-DTPA-pakliaaxel was purified by passing the mixture through a C18 Sęp-Pac cartridge using saline and then ethanol as the mobile phase. Free 'Ic-DTPA (<3%) was removed with saline, while 11¹The Ic-DTPA-paclitaxel was collected in the ethanol wash. The ethanol was evaporated under nitrogen and the labeled product was placed back into the saline solution. Radiochemical yield: 84%.

Analysis ^πiIc-DTPA-paclitaxel:

For analysis of the reaction mixture and purity ¹l¹In-DTPA-plkditaxel was used by HPLC. The system consisted of an LDC binary pump, a Waters 100 x 8.0 mm (ID) column coated with 5 µm ODS silica gel. The column was eluted at a flow rate of 1 ml / min with a gradient mixture of water and methanol (gradient from 0% to 85% methanol over 15 minutes). The gradient system was monitored with a Nal crystal detector and a Spectra-Physizs UV / Vis detector. According to HPLC analysis, purification on the Sep-Pac cartridge removed most of it^luIn-DTPA which has a retention time of 2.7 minutes. ^1hThe Ic-DTPA probably came from the trace DTPA impurities contained in DTPA-paclitaxel. The radiochromatogram of the 'iln-DTPA-paclitaxel in combination with the UV chromatogram indicates that the peak at 12.3 minutes is in fact due to the target compound. Under the same chromatographic conditions, paclitaxel had a retention time of 17.1 minutes. Chromatographic analysis showed that the final radiochemical purity of the preparation was 90%.

Comprehensive scintigraphy:

Female C3Hf / Kam mice were implanted with breast cancer cells (Mca-4) in the muscles of the right thigh (5 x 10⁵ cells). When the tumors grew to a diameter of 12 mm, the mice were divided into two groups. In group I, mice were generally anesthetized by an intraperitoneal injection of sodium pentobarbitol and then dosed with DTPA-paclitaxel (100-200 mCi) via the tail vein. A y-camera equipped with a collimator was placed above the mice (3 mice per group). A series of 5 minute logs were collected at 5, 30, 60, 120, 240 minutes and 24 hours post injection. In group II, the same procedures were performed except that the mice were fed for control ^noIn-DTPA. Figure 5 shows gamma-scintigraphy of animals after injection^noIn-DTPA and ⁱⁿln-DTPA-paclitaxel. ^H1In-DTPA was characterized by rapid disappearance from plasma, rapid and high urinary excretion with minimal renal retention, and negligible retention in tumor, liver, intestine and other organs and parts of the body. For comparison,^{11 T.}In-DTPA-paclitaxel shows a pharmacological profile similar to that of paclitaxel (Eiseman et al., 1994). The radioactivity in the brain was negligible. Liver and kidney have the highest tissue: plasma ratio. Biliary excretion of DTPA-radiolabeled paclitaxel or its metabolites through the biliary was one of the major routes of clearance from the blood. Unlike paclitaxel, significant amounts¹¹¹ Ir ^^ lDT ^ yA ^ f ^^ akl ^^ axel were excreted by the kidneys, which played only a secondary role in clearing the body of paclitaxel. The tumor showed significant uptakeⁱⁿIn-DTPA-paclitaxel. These results show that¹¹¹ In-DTPA-paclitaxel is able to detect certain tumors and allows quantification of the uptake ¹1¹ In-DTPA-paclitaxel by neoplasms, which may consequently aid in selecting patients for paclitaxel treatment.

Example 2

Polyglutamic acid-paclitaxel

This example demonstrates the conjugation of paclitaxel with a water-soluble polymer, poly (glutamic acid) (PG). The potential of water-soluble polymers used as drug carriers has already been established (Kopecek, 1990; Maeda and Matsmura, 1989). In addition to their ability to dissolve otherwise insoluble drugs, polymer conjugates act as slow release stores that release drugs in a controlled manner.

Synthesis of PG-paclitaxel

PG was chosen as the carrier for paclitaxel because it can be readily degraded by lysosomal enzymes, is stable in plasma, and contains a functional group suitable for drug attachment. A number of anti-cancer drugs, including adriamycin (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.

PG sodium salt (C.P. 34K, Sigma, 0.34 g) was dissolved in water. The pH of the aqueous solution was adjusted to 2 using 0.2 M HCl. The precipitate was collected, dialyzed with distilled water, and lyophilized to give 0.29 g of PG.

To a solution of PG (75 mg, M repeat unit 170, 0.44 mmol) in dry DMF (1.5 ml) was added 20 mg of paclitaxel (0.023 mmol, PG / paclitaxel molar ratio = 19), 15 mg of dicyclohexylcarboimide ( DCC) (0.073 mmol) and a trace of dimethylaminopyridine (DMAP). The reaction was run for four hours at room temperature. Thin layer chromatography (TLC, silica) showed the complete conversion of paclitaxel (Rf = 0.55) to the polymer conjugate (Rf = 0, Clftyl / MeOH mobile phase = 10: 1). The reaction mixture was poured into chloroform. The resulting precipitate was collected and dried in vacuo to yield 65 mg of the polymer-drug conjugate. It is possible to synthesize polymer conjugates with various paclitaxel content by changing the weight ratio of paclitaxel to PG

The sodium salt of the PG-paclitaxel conjugate was obtained by dissolving the product in 0.5 M NaHCO3. The aqueous PG-paclitaxel solution was dialyzed with distilled water (MWCO 1,000) to remove low molecular weight contaminants and excess NaHCO3 salt. After lyophilization of the dialysate, 88.6 mg of a white powder was obtained. The paclitaxel contained in this polymeric conjugate was determined using the UV technique (described below) at 21% (w / w). Yield (conversion to paclitaxel related polymer, UV): 93%. PG-paclitaxel with a higher paclitaxel content (greater than 35%) can be synthesized in the same way by simply increasing the ratio of paclitaxel to PG.

1 H-NMR (GE spectrometer model GN 500, 500 MHz, in D.₂O): δ = 7.75 to 7.36 ppm (aromatic components of paclitaxel); δ = 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 (C5-H), 4.39 ppm (C7-H), 4.10 ppm

189 698 (C'20-H), 1.97 ppm (OCOCH3), and 1.18-1.20 ppm (C-CH3) were assigned to the aliphatic component of paclitaxel. The remaining resonance components of paclitaxel are obscured by the resonance components of PG. The resonance components of PG at 4.27 ppm (H-α), 2.21 ppm (Hy) and 2.04 ppm (Η-β) are consistent with the pure PG spectrum. The couplings of the paclitaxel-conjugated polymer are too poorly cleaved to be recorded at the available resolution. The water solubility is> 20 mg paclitaxel / ml.

Characterization of PG-paclitaxel

The ultraviolet (UV) spectrum was obtained using a Beckman DU-640 spectrophotometer (Fullerton, CA). The amount of PG conjugated paclitaxel was estimated from the UV spectrum based on standard curves plotted for known paclitaxel concentrations in methanol (X = 228 nm), assuming that the polymer conjugate in water and the free drug in methanol had the same molar extinction coefficients and that both obey the Lambert-Beer law. As shown in the UY spectrum, PG-paclitaxel shows an absorption characteristic of paclitaxel with a shift of the X wavelength from 228 to 230 nm. The paclitaxel concentration in PG-paclitaxel was estimated from the standard curves plotted for the known paclitaxel concentrations in methanol for the absorption band at 228 nm assuming that the conjugated polymer in water at 230 nm and the free drug in methanol at 228 nm had the same molar extinctions and that both obey the Beer-Lambert law.

Study of PG-paclitaxel using gel permeation chromatography

The relative molecular weight of PG-paclitaxel was determined using Gel Permeation Chromatography (GPC). The GPC system consisted of two model III LDC pumps connected to the LDC gradient master, the PL GPC gel column, and the Waters 990 array photodiode array detector, the eluate (DMF) was passed at 1.0 ml / min, di ^^ on UV detection at 270 nm. As by GPC analysis, the attachment of paclitaxel to PG leads to an increase in the molecular weight of PG-paclitaxel, as indicated by a retention time shift from 6.4 minutes for PG to 5.0 minutes for the PG-paclitaxel conjugate. The calculated molecular weight for PG-paclitaxel containing 15-25% paclitaxel (w / w) is in the range 45-55 kDa. The crude product contained low molecular weight impurities (retention time 8.0 to 10.0 minutes and 11.3 minutes) that could 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 phosphate buffered solutions (PBS, 0.01 M) at pH 6.0, pH 7.4 and pH 9.6 at a paclitaxel equivalent concentration of 0.4 mM. The solutions were incubated at 37 ° C with gentle shaking. At selected time intervals, samples of the solution (100 µl) were removed, mixed with an equal volume of methanol and analyzed by high performance liquid chromatography (HPLC). The HPLC system consisted of a reverse phase silica column (Nova-Pac, Waters, CA), a methanol-water mobile phase (2: 1, v / v) passed at a flow rate of 1.0 mL / min, a detector photodiode. The concentration of PG-bound paclitaxel, free paclitaxel and other degradation products in each sample was calculated by comparing the peak areas with the separately obtained standard curves prepared for paclitaxel, assuming that the molar extinction coefficient of each peak at 228 nm is the same as that of paclitaxel. The conjugate half-life was estimated at 132, 40 and 4 days at pH 6.0, pH 7.4 and 9.6, respectively, determined by linear least squares method. HPLC analysis showed that incubation of PG-paclitaxel in PBS solutions yielded paclitaxel and several other substances including one more hydrophobic than paclitaxel itself (metabolite-1). In fact, the amount of metabolite-1, which is most likely 7-epipaclitaxel, formed in PBS at pH 7.4 after 100 hours of incubation exceeds that of paclitaxel (Fig. 6).

In vitro research

The samples obtained from the PBS solution at pH 7.4 were analyzed using the tubulin polymerization test. The tubulin fusion reaction was performed at 32 ° C in PEM buffer (pH 6.9) with a tubulin concentration (bovine brain, Cytoskeleton Inc., Boulder, CO) of 1 mg / ml (10 pM) in the presence of experimental samples (equiv. 1). , (0 pM Paclitaxel) and 1.0 mM GTP.

189 698

Tubulin polymerization was carried out by measuring the absorbance of the solution at 340 nm during its duration. After 15 minutes, calcium chloride (125 mM) was added to measure CaCl2-induced depolymerization of the microtubules. While PG-paclitaxel freshly dissolved in PBS was inactive in the microtubule formation process, samples of PG-paclitaxel incubated for three days participated in tubulin polymerization. The formed microtubules were resistant to the CaCF-induced depolymerization process.

The effect of PG-paclitaxel on cell growth was also investigated by determination of the tetrazole salt (MTT) (Mosmann, 1983). MCF-7 cells or 13762F cells were seeded in a 96-well microtiter plate at a concentration of 2 x 10⁴ cells / ml and 24 hours later they were treated with various concentrations of PG-paclitaxel, paclitaxel and PG and incubated for a further 72 hours. Then, MTT solution (20 µl, 5 mg / ml) was added to each well and incubated for 4 hours. The supernatant was aspirated and the MTT formazan produced by the metabolically convertible cells was measured in a microplate with a fluorescence reader at a wavelength of 590 nm. After a period of three days, PG-paclitaxel inhibited tumor cell proliferation to an extent similar to that of free paclitaxel. For the human breast cancer cell line MCF-7, the IC50 values ​​obtained were 0.59 pM for paclitaxel and 0.82 pM for PG-paclitaxel (measured in the appropriate number of paclitaxel units). Regarding the 13762F cell line, the efficacy of PG-paclitaxel (IC50 = 1.86 pM) was comparable to that of paclitaxel (IC50 = 6.79 pM). For both cell lines, the IC50 of PG alone was greater than 100 pM.

In vivo anti-tumor activity

All animal studies were conducted using the MD Anderson Cancer Center in accordance with the institute's guidelines. C3H / Kam mice were bred and housed in pathogen-free facilities of the Department of Experimental Radiation Oncology. Single tumors were generated in the right thigh muscle of female C3H / Kam mice (25-30 g) by injection of 5 x 105_my_S.and_ch ovarian cancer cells (OCa-I), breast cancer (MCa-4), liver cancer (HCa-I), or fibrosarcoma (Fsa-II). In a parallel study, female Fischer 344 rats (125-150 g) were inoculated with 1.0 x 10 5 viable 13762F cancer cells in 0.1 M PBS. Treatment was started when tumors in the mice had reached a volume of 500 mm³ (10 mm in diameter), or when the tumors in rats reached a volume of 2400 mm 3 (corresponding to a diameter of 17 mm). A single dose of PG-paclitaxel in saline or paclitaxel on the Cremophor EL vehicle was administered at doses ranging between 40 and 160 mg eq. paclitaxel / kg body weight. Saline solution, Cremophor carrier (50/50 Cremophor / ethanol diluted with saline solution (1: 4)), PG salt solution (M.C. 38K) and Paclitaxel / PG mixture Tumor growth was determined daily (Figures 7A, 7B, 7C, 7D and 7E) by measuring three orthogonal tumor diameters. Tumor volume was calculated according to the formula (A x B x C) / 2. Absolute growth delay (AGD) in mice was defined as the time difference in days required for a treated tumor to grow in volume from 500 to 2000 mm3 with different drugs and the time in days required for a tumor control treated with saline solution to expand in volume. 500 to 2000 mm³. Table 1 summarizes the acute toxicity of PG-paclitaxel in rats compared to the paclitaxel / Cremophor mixture. Table 2 summarizes the data on the effects of paclitaxel against MCa-4, Fsa-II and HCa-I tumors in mice. Data is also collected in Figs. 7A-7E.

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Table 1

Acute toxicity of PG-paclitaxel in Fischer rats *

<td>Group</td><td>Dose (mg / kg)</td><td># Deaths induced by toxicity</td><td>Weight loss in%</td><td>Time of greatest danger (days)</td><td>Time of complete recovery (days)</td>

<td>PG-paclitaxel "</td><td> 60</td><td> 1/4</td><td> 15,7</td><td> 7</td><td> 14</td>

<td>PG-paclitaxel^and</td><td> 40</td><td> 0/4</td><td> 11,1</td><td> 6</td><td> 11</td>

<td>Paclitaxelb</td><td> 60</td><td> 1/4</td><td> 16,7</td><td> 6</td><td> 15</td>

<td>Paclitaxelb</td><td> 40</td><td> 0/3</td><td> 17,9</td><td> 6</td><td> 16</td>

<td>Salt solution</td><td>1.0 ml</td><td> 0/2</td><td> 5,2</td><td> 1</td><td> 7</td>

<td>PG^C.</td><td>0.3 g / kg</td><td> 0/2</td><td> 4,3</td><td> 2</td><td> 8</td>

<td>Carrier Cremophord</td><td>2.0 ml</td><td> 0/2</td><td> 6,9</td><td> 1</td><td> 9</td>

* Drugs were administered intravenously to Fischer rats affected by the 13762F tumor (female, 130g) as a single injection.

a. PG-paclitaxel solution was prepared by dissolving the conjugate in saline (equiv. 8 mg paclitaxel / ml). The injection volume corresponding to a dose of 60 mg / kg was 0.975 ml per rat.

b. The paclitaxel solution in Cremophor was prepared by dissolving paclitaxel in a 1: 1 mixture of ethyl alcohol and Cremophor (30 mg / ml). This stock solution was then diluted with saline (1: 4) prior to injection. The final concentration of paclitaxel in the solution was 6 mg / ml. The injection volume corresponding to a dose of 60 mg / kg was 1.3 ml per rat.

c. The PG solution was prepared by dissolving the polymer in saline (22 mg / ml). The injection dose was 0.3 g / kg (1.8 ml per rat) corresponding to a paclitaxel dose of 60 mg / kg.

d. The Cremophor vehicle was prepared by diluting a mixture of ethyl alcohol and Cremophor (1: 1) with saline (1: 4).

Table 2

In vivo anti-tumor activity of PG-paclitaxel against various types of murine tumors

<td>Bump</td><td>The drug</td><td>Time of growth⁰⁰500-2000 mm3</td><td>Household appliances °</td><td>t-testd</td>

<td></td><td>Salt solution</td><td> 4,8±0,8 (5)</td><td></td><td> -</td>

<td></td><td>PG (0.6 g / kg)</td><td> 9,3±1,1 (4)</td><td> 4,5</td><td> 0,0114</td>

<td></td><td>Cremophor carrier</td><td> 6,1±0,7 (5)</td><td> 1,3</td><td> 0,265</td>

<td>. AND</td><td>PG-Pakl (40 mg / kg)</td><td> 8,6±1,2 (4)</td><td> 3,8</td><td> 0,026</td>

<td>ΐνίνύ-'τ</td><td>PG-pakl (60 mg / kg)</td><td> 14,2±1,5 (5)</td><td> 9,4</td><td> 0,0001</td>

<td></td><td>PG-pakl (120 mg / kg)</td><td> 44,4±2,9 (5)</td><td> 39,6</td><td> &lt;0,0001</td>

<td></td><td>Paclitaxel (40 mg / kg)</td><td> 9,0±0,6 (4)</td><td> 4,2</td><td> 0,0044</td>

<td></td><td>Paclitaxel (60 mg / kg)</td><td> 9,3±0,3 (5)</td><td> 4,5</td><td> 0,0006</td>

<td></td><td>Salt solution</td><td> 1,9±0,1 (5)</td><td></td><td> -</td>

<td></td><td>PG (0.8 g / kg)</td><td> 2,8±0,2 (6)</td><td> 0,9</td><td> 0,0043</td>

<td></td><td>Cremophor carrier</td><td> 2,2±0,2 (6)</td><td> 0,3</td><td> 0,122</td>

<td>Fsa-II</td><td>PG-pakl (80 mg / kg)</td><td> 3,8±0,4 (6)</td><td> 1,9</td><td> 0,0016</td>

<td></td><td>PG-pakl (160 mg / kg)</td><td> 5,1±0,3 (13)</td><td> 3,2</td><td> &lt;0,0001</td>

<td></td><td>Paclitaxel (80 mg / kg)</td><td> 4,2±0,3 (6)</td><td> 2,3</td><td> 0,0002</td>

<td></td><td>PG + paclitaxel</td><td> 3,0±0,2 (6)</td><td> 1,1</td><td> 0,0008</td>

<td></td><td>Salt solution</td><td> 7,3±0,3 (5)</td><td> -</td><td> -</td>

<td></td><td>PG (0.8 g / kg)</td><td> 7,7±0,4 (4)</td><td> 0,4</td><td> 0,417</td>

<td></td><td>Cremophor carrier</td><td> 6,8±0,8 (5)</td><td> -0,5</td><td> 0,539</td>

<td></td><td>PG-Pakl (40 mg / kg)</td><td> 8,2±0,7 (5)</td><td> 0,9</td><td> 0,218</td>

<td>nta'1</td><td>PG-pakl (80 mg / kg)</td><td> 8,6±0,2 (5)</td><td> 1,3</td><td> 0,0053</td>

<td></td><td>PG-pakl (160 mg / kg)</td><td> 11,0±0,8 (4)</td><td> 3,7</td><td> 0,0023</td>

<td></td><td>Paclitaxel (80 mg / kg)</td><td> 6,4±0,5 (5)</td><td> -0,9</td><td> 0,138</td>

<td></td><td>PG + paclitaxel</td><td> 6,7±0,4 (5)</td><td> -0,6</td><td> 0,294</td>

a. Mice affected by tumors in their right limb with a size of 500 mm³ were treated with various doses of PG-paclitaxel (40-120 mg paclitaxel / kg equivalent) in saline or with paclitaxel in Cremophor vehicle administered as a single intravenous injection. Control animals were treated with saline (0.6 ml), Cremophor vehicle (0.5 ml), PG solution in saline or PG plus paclitaxel (80 mg / kg).

189 698

b. The growth of e / anchor was determined on the basis of the measurements of three (x-tog) meshed diameters with the use of a caliper, and the volume (bliceae) was determined with the formula (A x B x C) / 2. The number of mnsen in each group is given in parentheses. Time in days corresponding to an increase from 500 to 2000 mm³ shown as value ± standard deviation.

c. Δbs / lutez absolute growth delay (AGD) ezfieiowae / as the difference in time measured in days necessary for lzceoezg / tumor growth from 500 to 2000 mm³ with the use of various drugs and the time of malignancy / in days necessary for tumor growth for the group treated with k / etrolysis r / etw / r salt from 500 to 2000 mm3,

d. The rise time from 500 to 2000 mm3 Ι ^^ ι in days was compared for the litter group (s) and the saline control group using the Student's Test. The P values ​​are ambiguous, and are considered significant when they are smaller

Example 3

P / polyethylene glycol-paclitakszl

Synthesis of polyethylene glycol-paclitaxel (PEG-paclitakschlu)

This synthesis was carried out in two stages. First, 2'-succinyl-paclitaxel was prepared according to the reported procedure (Deutsch et al., 1989). Paclitaxel (200 mg, 0.23 mmol) and succinic anhydride (288 mg, 2.22 mmol) were allowed to react for 3 hours at room temperature in anhydrous pyridine (6 ml). Then, pyridine was evaporated, the residue was taken up in water, stirred for 20 minutes and then filtered. The precipitate was dissolved in acetone, then water was slowly added, and fine crystals were collected to give 180 mg of 2'-succinyl-paclitakschl. PEG-paclitaxel was synthesized by a coupling reaction involving N-ethoxyncarb / ethyl / -2-ethoxn-1,2-dihndrochieolin (EEDQ). EEDQ (180 mg, 0.72) was added to a solution of 2'-succinenyl-paclitaxel (160 mg, 0.18 mmol) and methoxy-polyoxyethylene amine (PEG-NH2, MW 5000, 900 mg, 0.18 mmol) in methylene chloride. mmoles). The reaction mixture was stirred at room temperature for 4 hours. The crude product was subjected to the chromatograph! on kszem gel (onkojvym in οΙυΖοπκ acetate^and followed by chloroform-methanol (10: 1). 350 mg of product were obtained.

1 H NMR (CDCl3) δ 2.76 (m, succinic acid, COCH2CH2CO2), δ 3.63 (PEG, OCH2CH2O), δ 4.42 (C7-H) and δ 5.51 (C2'-H). The maximum abs / rbcyjee in the UV spectrum occurred at 228 nm, which is also characteristic of paclitaxel. PEG attachment significantly improved the solubility of Pakel in water (> 20 mg paclitaxel equivalent / ml in water).

PEG-paclitaxel resistance to hydrolysis.

PEG-paclitaxel was dissolved in different pH phosphate buffer (0.01 M) to give a concentration of 0.4 mM, and the solutions were allowed to incubate at 37 ° C with gentle shaking. At selected time intervals, samples (200 µl) were removed and lyophilized. The thus obtained dry powders were redissolved in methylene chloride for gel / permeation chromatography (GPC analysis). The GPC system consisted of a Perkie-Elmer PL mixed gel column, Pzrkin-Elmer isocratic LC pump, Nelson 900 series PE connectors, Spzktaln / -physical detector UV / Vis and a teledation station. The eluate (methylene chloride) was passed through at a flow rate of 1.0 ml / min and UV detection was made at 228 nm. The retention times of PEG-paclitaxel and paclitaxel were 6.1 and 8.2 minutes, respectively. The peak areas were calculated and the percentage of paclitaxel remaining as PEG-paclitaxel and the percentage of paclitaxel released were calculated. The half-life of PEG-paclitaxel based on least squares at pH 7.4 was 54 minutes. The half-life at pH 9.0 was

7.6 minutes The release profiles of pacli-paclitaxel from PEG-paclitaxel at pH 7.4 are shown in Figure 8.

In vitro cytotoxicity study of PEG-paclitaxel using murine B 16 melanoma cells

P / d / beie, as in the procedure described in the cnSotoxinosis study □ TPA-pa ^ ak ^ lu, melanoma cells were plated in a 24-well plate at a concentration of 2.5 x 10⁴ cells / ml ip / were salted for 24 g / day at 37 ° C to grow on 50:50 Dulbecco Necessary Minimum Medium (DME) and F12 medium containing 10% calf serum in an atmosphere of 97% humidity and 5, 5% CO2. The medium was then replaced with fresh medium containing pacliSakszl or its p / ch / dee in concentrations ranging from 5 × 10 '⁹ up to 75xl0 '⁹ M. After 40 hours, cells were dissociated by trypsinization and run on a C counter. The final concentration of DMSO (used

189 698 for dissolving paclitaxel) and 0.05 M sodium bicarbonate solution (used to dissolve PEG-paclitaxel) in cell medium was less than 0.01%. This amount of solvent had no effect on cell growth as confirmed by control studies. Moreover, the use of PEG in such a concentration as to ensure the appropriate concentration of paclitaxel from 5x10 '⁹ M up to 75x10 '⁹ M also had no effect on cell proliferation.

Antitumor activity of PEG-paclitaxel against MCa-4 tumor in mice.

To determine the anti-tumor efficacy of PEG-paclitaxel against solid breast tumors, female C3Hf / Kam mice were implanted with Mca-4 cells in the right thigh muscles (5 x 10⁵ cells). As described in Example 1 for DTPA-paclitaxel, when tumors had grown to 8 mm (approximately 2 weeks), paclitaxel or PEG-paclitaxel was administered in a single dose equivalent to 10, 20 and 40 mg paclitaxel / kg body weight. First, paclitaxel with an equal volume of Cremophor was dissolved in absolute ethanol. Then, 15 minutes before injection, this stock solution was diluted (1: 4 by volume) with sterile physiological solution. PEG-paclitaxel was dissolved in saline (equiv. 6 mg paclitaxel / ml) and filtered through a sterile filter (Millipore, 4.5 µm). Saline solution, paclitaxel vehicle, absolute alcohol: Cremophor mixture (1: 1) diluted with saline (1: 4) and PEG solution in saline solution (600 mg / kg body weight) were used in the control studies. Tumor growth was determined daily by measuring three orthonormal tumor diameters. When the tumor diameter reached 12 mm, the tumor growth delay was calculated.

Tumor growth curves are shown in Figure 9. At the 40 mg / kg dose, both PEG-paclitaxel and paclitaxel were effective in inhibiting tumor growth. Paclitaxel was more effective than PEG-paclitaxel, but the differences were not statistically significant. Paclitaxel treated tumors take 9.4 days to reach 12 mm in diameter, while PEG-paclitaxel treated tumors take 8.5 days. Statistically, these values ​​are significant (p> 0.05) compared to the corresponding control results of 6.7 days for paclitaxel vehicle and 6.5 for PEG in saline (Figure 4).

While the composition and use of the present invention have been described in accordance with the preferred embodiments of the invention, it will be apparent to all those skilled in the art that any modifications to the compositions, methods and steps or the order of steps described herein may be used without departing from the purpose and spirit of the present invention. applications. More specifically, it is evident that certain agents that are both chemically and physiologically related can be used interchangeably with the agents described herein when the same or similar results are obtained. All such substitutions and modifications known to those skilled in the art are considered to be consistent with the form, object and purpose of the present invention as defined in the claims.

189 698

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