Process for the synthesis of conjugates of hyaluronic acid and paclitaxel

Abstract

An industrial synthesis process of hyaluronan-paclitaxel conjugates obtained by introducing a 4-bromobutyric acid spacer / linker between hyaluronic acid and paclitaxel is described, said conjugates being characterized by high purity, related pharmaceutical compositions for the treatment of various forms of cancer are described.

Description

[0001] Purpose of the invention

[0002] This invention describes an industrial method for synthesizing hyaluronic acid-paclitaxel conjugates by introducing a 4-bromobutyric acid spacer / linker between hyaluronic acid and the aforementioned chemotherapeutic agent, according to a method of synthesis based on hyaluronic acid (HA) molecules and paclitaxel, and by using an original synthesis and purification method to ensure the purity of the conjugate in its final form. Invention Field

[0003] Paclitaxel It is an anticancer agent that exerts its antiproliferative effect by acting on the microtubule organization of the cytoskeleton system: by inhibiting the depolarization of the aforementioned microtubules, it prevents the normal dynamic reorganization that occurs during mitotic cell division (Manfredi JJ et al., J Cell Biol, 1982, 94: 688-696).

[0004] The main therapeutic indications for paclitaxel are for the treatment of advanced breast cancer, lung cancer, ovarian cancer, bladder cancer, prostate cancer, and endometrial cancer.

[0005] Paclitaxel is a water-insoluble compound; therefore, current pharmaceutical compositions utilizing it involve mixing it with Cremophor EL-ethanol (Pfeifer RW et al., Am J Hosp Pharm, 1993, 50:2520-2521) in a 1:1 ratio, and in this formulation, it is typically administered via continuous intravenous infusion. The presence of Cremophor EL represents a major cause of adverse reactions that usually occur during paclitaxel administration, namely simple onset of urticaria, dyspnea and bronchospasm, up to anaphylactic shock. For this reason, all patients treated with the paclitaxel-Cremophor EL pharmaceutical composition must adhere to a pre-dosing regimen consisting of dexamethasone (possibly related to antihistamines) before initiating treatment.

[0006] Therefore, we can state that the current clinical use... The formulation (and its route of administration) of these drugs limits their therapeutic efficacy. This is why current research focuses on both the synthesis of prodrug / water-soluble paclitaxel conjugates and novel formulations targeting the aforementioned anticancer drugs.

[0007] Paclitaxel is an alkaloid obtained through semi-synthesis from one of its precursors derived from the needles of the yew tree (Taxus bacon). Chemically, it consists of a 15-carbon taxane ring bonded to an oxyethane ring at C4 and C5, while an ester bond exists at C13, which is considered essential for its anticancer activity.

[0008] To overcome the aforementioned problems caused by the type of formulation, attempts have been made, for example, to encapsulate the anticancer agent in liposomes, nanocapsules, and microspheres with polymer walls (formed from biodegradable copolymers such as polylactic acid or non-biodegradable copolymers such as ethylene-vinyl acetate), or microspheres formed from polyphosphates loaded with paclitaxel, to create systems for prolonging drug release at the therapeutic site (Nuijen B et al., Investigational New Drugs, 2001, 19:143-153).

[0009] However, these new systems for encapsulating paclitaxel have issues with stability, preparation, and reproducibility.

[0010] In addition, various attempts have been made to solubilize the above-mentioned drugs with cyclodextrin, but the new formulations have not yielded the desired results (Nuijen B et al., Investigational New Drugs, 2001, 19:143-153).

[0011] Chemical research into new paclitaxel formulations that would make the drug more water-soluble while maintaining its efficacy as an anticancer agent has led to the synthesis of new analogs modified at the C2' and C7 positions, as well as the preparation of new conjugates defined as prodrugs.

[0012] For this reason, several attempts have been made to synthesize new prodrugs, which have led to the preparation of drugs such as acetyl-paclitaxel (Mellado W et al., Biochem Biophys Res Commun, 1984, 124(2):329-336) or the synthesis of new esters of the above drugs with succinic acid, glutaric acid and sulfonic acid at the C2' carbon. However, the new esters have been shown to be unstable in aqueous environments.

[0013] Furthermore, PEG (polyethylene glycol) is known to be used to derive the aforementioned chemotherapeutic agents by esterifying paclitaxel at the C2' position. Such novel molecules have been shown to be highly water-soluble, but also exhibit limited stability.

[0014] Finally, a new "delivery system" for the aforementioned drugs was developed, which is obtained by conjugating paclitaxel with serum albumin (HSA): the paclitaxel-HSA conjugate has been shown to be highly water-soluble, but there is no reliable efficacy data relative to paclitaxel itself (Nuijen B et al., Investigational New Drugs, 2001, 19:143-153).

[0015] Recently, a novel release system for paclitaxel, esterified with previously modified hyaluronic acid (HA) (i.e., derivatized with an amide hydrazine molecule bound to the carboxyl group of HA via an amide bond), has been synthesized (Luo Y et al., Biomacromolecules, 2000, 1(2):208-218) (US5,874,417). This yields a novel paclitaxel delivery system that allows the drug to directly reach the membrane surface of target tumor cells characterized by overexpression of the HA receptor CD44.

[0016] In an attempt to explore all the potential of associating paclitaxel with HA, a novel conjugate between HA and a chemotherapeutic agent was finally studied and synthesized, in which HA and the chemotherapeutic agent are covalently linked to each other indirectly through spacer / linker groups such as, for example, bromobutyric acid, which makes the final product more water-soluble and therefore easier to administer (EP2045270).

[0017] The scope of this invention is to identify a new method for preparation and purification that is particularly effective and overcomes the disadvantages of the prior art highlighted above. Invention Details

[0019] Therefore, the object of the present invention is a primitive method for preparing and purifying hyaluronic acid-paclitaxel (HA-paclitaxel) conjugates, comprising or consisting of the following steps:

[0020] a) In the presence of a catalyst (preferably DMAP (4-dimethylaminopyridine)), the carboxyl groups of the 4-bromobutyric acid spacer / linker are activated in a solution of paclitaxel in an organic solvent (preferably dichloromethane) containing 4-bromobutyric acid spacer / linker groups by an activator to obtain the intermediate of the HA-paclitaxel conjugate;

[0021] b) The intermediate obtained from the previous step a) is crystallized in an organic solvent (preferably n-heptane), then filtered and dried;

[0022] c) The intermediate obtained in step b) above is added to the TBA (tetrabutylammonium) salt of HA in DMSO;

[0023] d) The HA-paclitaxel conjugate thus obtained is purified by precipitation with an ethanol / water solution containing NaBr salt, followed by at least one subsequent washing in ethanol / water and further washing in ethanol, pre-drying at 40°C and passing through a saturated steam chamber at 40°C and atmospheric pressure, and finally drying under vacuum at 40°C. The conjugate has an ester bond between the carboxyl group of the HA polysaccharide and the spacer / linker group, which in turn is bonded to the hydroxyl group at carbon C2' of paclitaxel via its carboxyl group through the ester bond.

[0024] Another object of the present invention is to have HA-paclitaxel conjugates with a degree of derivatization or esterification in the range of 15% to 21% w / w, preferably in the range of 16% to 20% w / w, wherein the starting hyaluronic acid has a fermentation-derived origin and has a weight-average molecular weight of 140,000 Da to 250,000 Da, said conjugates being in the form of a fine and uniform powder having a purity of more than 98% by weight of the dried product and containing less than 1% by weight of dibromopaclitaxel.

[0025] Another object of the present invention is the preparation and purification of HA-paclitaxel conjugates as described, which are in the form of fine and uniform powders containing less than 1% by weight of dibromopaclitaxel, and are therefore extremely pure relative to the same conjugates obtained by the method according to EP2045270.

[0026] Another object of the present invention is a pharmaceutical composition consisting essentially of a conjugate prepared and purified by the methods described above, and a pharmacologically acceptable diluent / excipient, preferably formulated in sterile isotonic water containing 5% w / v glucose, for the treatment of advanced breast cancer, lung cancer, ovarian cancer, bladder cancer, prostate cancer, endometrial cancer, and mesothelioma.

[0027] The compositions according to the invention are used to treat all cancer forms in which paclitaxel, all known derivatives of the drug described above, and taxanes are generally used.

[0028] Therefore, the present invention describes and claims the preparation and purification of HA-paclitaxel conjugates according to an original synthetic method comprising the following steps: introducing a spacer / linker between hyaluronic acid and the above-mentioned chemotherapeutic agent by forming an ester bond between the carboxyl group and the spacer / linker of the polysaccharide, wherein the spacer / linker is further bonded to the hydroxyl group at carbon C2' of paclitaxel via its carboxyl group (always via an ester bond), wherein the spacer / linker used to synthesize the above conjugate is 4-bromobutyric acid.

[0029] This novel synthetic method for HA-paclitaxel conjugates differs from EP2045270 because it allows for:

[0030] • Formation of conjugates with higher purity;

[0031] • Eliminate the final dialysis;

[0032] • Reduce solvent use;

[0033] • Obtain the raw material (HA-paclitaxel conjugate) in a fine and uniform powder form, which is easy to sterilize by filtration once prepared in an aqueous solvent;

[0034] • Reduce synthesis time.

[0035] The HA-paclitaxel conjugate thus prepared and purified has a degree of derivatization in the range of 15% to 21% by weight (w / w), preferably in the range of 16% to 20% by weight (w / w).

[0036] The degree of derivatization or esterification of the above conjugates is defined as the weight percentage of paclitaxel relative to HA-paclitaxel. Therefore, 100 mg of the conjugate with a degree of derivatization of 15% to 21% w / w will contain 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, or 20 mg, or 21 mg of the chemotherapeutic agent paclitaxel, depending on the indicated degree of derivatization (for further illustration, a degree of derivatization of 20% w / w contains 20 mg paclitaxel / 100 mg conjugate). However, it will be apparent to those skilled in the art that small variations in the weight ratios between molecules can occur at the end of such industrial synthesis methods. Therefore, in describing and claiming protection for the 15% to 21% w / w derivatization range of the above conjugates, the applicant intends to claim protection for all reported percentage values ​​containing ±1%: as an example, a degree of 20% w / w is therefore intended to be 20% ± 1%.

[0037] The HA used to synthesize such HA-paclitaxel conjugates can be from any source, such as by extraction from rooster combs (e.g., according to EP0138572 or WO2018020458), or by a biotechnological route (e.g., according to EP2614088 or EP2614087), or preferably by a fermentation route (e.g., according to EP0716688), and has a strength of 400 Da to 3 × 10⁻⁶. 6 Da, especially 400 Da to 1×10 6 The weight-average molecular weight (MW) is 140,000 Da to 250,000 Da (weight-average molecular weight means molecular weight calculated using the intrinsic viscosity method (Terbojevich et al., Carbohydr Res, 1986, 363-377). The HA-paclitaxel described and claimed by the applicant has a degree of derivatization in the range of 15% to 21% w / w, preferably in the range of 16% to 20% w / w, and is preferably prepared from fermentation-derived HA having a weight-average molecular weight of 140,000 Da to 250,000 Da.

[0038] The HA-paclitaxel conjugates described above were prepared and purified according to the original synthetic method described above, which is an improvement over existing technologies, and are therefore detailed below:

[0039] - In the presence of a catalyst (preferably DMAP (4-dimethylaminopyridine)), the carboxyl group of 4-bromobutyric acid is activated in a solution of paclitaxel in an organic solvent (preferably dichloromethane) containing a spacer / linker group of 4-bromobutyric acid by an activator such as carbodiimide (preferably 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC)), thereby forming an ester between the hydroxyl functional group at the C2' carbon of paclitaxel and the carboxyl group of the spacer / linker group, for the formation of an intermediate of HA-paclitaxel conjugate, wherein the bond between the spacer / linker group and paclitaxel is an ester bond;

[0040] - By using organic solvents (preferably) (n-Heptane) Add to the above solution to allow the obtained intermediate to crystallize, then separate by filtration and dry; thus obtain Dry intermediate Then it is analyzed in order to determine by HPLC di-Br-paclitaxel To evaluate purity, due to the above crystallization steps, the dibromopaclitaxel was significantly reduced, confirmed to be less than 1% by weight, thus confirming that the purity of the product synthesized in this way is greater than 98% w / w;

[0041] Subsequently, by directly contacting the intermediate with the TBA (tetrabutylammonium) salt of HA in dimethyl sulfoxide (DMSO) (DMSO allows such intermediates to dissolve and optimizes the reaction for the synthesis compared to N-methyl-2-pyrrolidone (NMP) and dimethylformamide (DMF), nucleophilic substitution of the COO- of HA onto the carbon bound to the bromine of the spacer / linker group occurs. In this manner, an ester bond is formed between HA and the spacer / linker group previously bound to paclitaxel;

[0042] - The conjugate obtained therefrom is purified by the following steps: precipitate with an ethanol / water solution containing NaBr (T 40°C), then wash once or several times with ethanol / water, followed by washing with ethanol, then pre-dry at 40°C, place in a saturated water vapor chamber at atmospheric pressure at 40°C, and finally dry under vacuum at 40°C.

[0043] The applicant unexpectedly discovered that NaBr promotes the precipitation of the conjugate and thus its purification, while the NaBr is readily removed by subsequent washing of the conjugate with the above-mentioned aqueous alcohol solution.

[0044] The pre-drying step and the passage through a saturated water vapor chamber at atmospheric pressure are carried out at 40°C for at least 8 hours, while the drying step is carried out at 40°C under vacuum for at least 16 hours.

[0045] The purification step of this conjugate does not include dialysis.

[0046] The final product obtained is in the form of a fine and uniform powder, which is analyzed by HPLC to evaluate the degree of carboxyl derivatization or esterification and its filterability.

[0047] The HA-paclitaxel conjugates prepared and purified as reported above have thus been confirmed to be free of contaminants primarily represented by the following:

[0048] · Dibromopaclitaxel The esterification reaction of two 4-bromobutyric acid molecules with paclitaxel molecules at two different hydroxyl groups produces completely undesirable reaction byproducts. Since dibromopaclitaxel reacts with the two carboxyl functional groups of one or two HATBA molecules, and thus determines the formation of crosslinks completely lacking therapeutic efficacy, this impairs the sterilization of pharmaceutical compositions containing the target conjugate of the present invention by filtration through a 0.2 μm filter. Therefore, the reduction in the content of this minor product represents a significant improvement in the synthesis and purification methods of the conjugate.

[0049] • The crystallization of the intermediate in n-heptane, and the precipitation in ethanol followed by washing of the HA-paclitaxel final product in an aqueous alcohol solution, eliminate the need for further treatment. NaBr , solvent and Activator residue The crystallization process in n-heptane can actually occur and therefore only involves intermediates, thus yielding a pure product that is easily separated from the various residues present in the reaction medium.

[0050] Due to all the steps listed above, an extremely pure fine powder conjugate was obtained. No need for final dialysis process Therefore, it also determines that... Significant savings Solvent.

[0051] Therefore, the synthesis and purification method of the target HA-paclitaxel conjugate of the present invention differs from EP2045270 because it allows:

[0052] • Formation of conjugates with higher purity;

[0053] • Eliminate the final dialysis;

[0054] • Reduce solvent use;

[0055] • Obtain raw materials in the form of fine and uniform powder, which, once formulated in an aqueous solvent, are easily sterilized by filtration;

[0056] • Overall, reduce its synthesis time.

[0057] Examples of the preparation and purification of HA-paclitaxel conjugates are provided to better illustrate the scope and advantages of the invention; however, they do not in any way constitute a limitation on the scope of the claims.

[0058] Example 1: Preparation and purification of HA-paclitaxel conjugates with 18% w / w derivatization

[0059] HA (the HA used in such synthesis has a weight-average molecular weight of 160,000 Da to 230,000 Da) and paclitaxel Preparation of ester derivatives with a degree of derivatization (i.e., esterification of carboxyl groups) of 18% w / w.

[0060] 110.1 mg of 4-bromobutyric acid, 151.9 mg of EDC, and 19.5 mg of DMAP were added to 308.7 mg of paclitaxel dissolved in 15 mL of dichloromethane, and the mixture was stirred at 10 °C for at least 40 minutes. Water was then added to the resulting solution; after stirring for several minutes, the organic phase was allowed to separate. The organic phase was collected, while the aqueous phase (containing bromide residues and salts) was removed. 25 mL of n-heptane was added to the resulting organic phase at 10 °C to obtain crystals of the intermediate product, which were then separated by filtration and dried. In this manner, 301 mg of the dried intermediate product was obtained.

[0061] 300 mg of this intermediate was added to a solution of 1000 mg of HA-TBA (a salt of HA and tetrabutylammonium) dissolved in 44 mL of DMSO. After reacting at 40 °C for 1 day, 50 mL of DMSO was added to the solution. After 1 hour, a solution of ethanol / water in a 96 / 4 ratio containing 2% w / w NaBr was slowly added dropwise, thereby obtaining a precipitate of the resulting conjugate. This precipitate was washed several times in ethanol / water (8.5 / 1.5), and finally washed again with 100% ethanol, and then pre-dried at 40 °C for 8 hours. The pre-dried product was then placed at 40 °C under atmospheric pressure in a saturated steam chamber for another 8 hours, and finally dried at 40 °C under vacuum for at least 16 hours until completely dry.

[0062] The obtained HA-paclitaxel conjugate was then analyzed by HPLC (Rosato A., Urologic Oncology: Seminars and Original Investigations, 2006, 24: 207-215) to check the effective degree of derivatization, with a result of 18% w / w. The amount of dibromopaclitaxel was determined by HPLC analysis of the dried intermediate (Theodoridis G. et al., Application Note, August 1999: 40-44), with a result of less than 1% by weight (as shown below). The purity of the conjugate was greater than 98% by weight of the dried product.

[0063] The HA-paclitaxel conjugate obtained as described above is a white to pale yellow powder that is uniform and hygroscopic. When reconstituted into a pharmaceutical composition via an aqueous medium, it can be easily sterilized by filtration through a 0.2 μm filter.

[0064] Comparative Example 2: Preparation and purification of HA-paclitaxel conjugates according to WO2004035629 (EP2045270)

[0065] Preparation of ester derivatives of HA and paclitaxel with an esterification degree of 18% w / w carboxyl groups.

[0066] To demonstrate that the objective method of this invention is superior to the prior art represented by the method described in WO2004 / 035629, the applicant prepared an HA-paclitaxel conjugate according to Example 6 of WO2004 / 035629, changing the weight concentration of different components to prepare an HA-paclitaxel conjugate with an esterification degree of 18% w / w carboxyl groups, in order to compare the derivative with the HA-paclitaxel conjugate of Example 1 in terms of purity and filterability.

[0067] According to Example 1, the HA used has a weight-average molecular weight of 160,000 Da to 230,000 Da.

[0068] In short, 117.2 mg of 4-bromobutyric acid and 614.1 mg of EDC were added to 308.7 mg of paclitaxel dissolved in 15 mL of dichloromethane. Water was then added to the solution to remove all bromides and carbodiimide. Sodium sulfate was added to the resulting organic solution for dehydration, while the solvent was removed by rotary evaporation. Finally, 363 mg of the dried intermediate product was obtained.

[0069] 315 mg of the intermediate obtained was added to 1 g of HA-TBA dissolved in anhydrous NMP. The solution was stirred at room temperature for 7 days, then 20 mL of double-distilled water and 4 mL of saturated NaCl solution were added. The mixture was stirred for 1 hour to allow sodium to exchange with TBA ions. Subsequently, ethanol was added dropwise, and the resulting filamentous product was dissolved in water, dialyzed, and finally lyophilized.

[0070] The effective degree of derivatization was then examined by HPLC analysis of the HA-paclitaxel conjugate obtained according to Example 2, and the result was 18% w / w.

[0071] Therefore, the two conjugates from Example 1 and Example 2 were compared to identify and determine the presence of certain compounds in the dried intermediates from Examples 1 and 2. Dibromopaclitaxel The purity of the cross-linking reaction byproducts was verified by measuring their quantity (HPLC analysis of dried intermediates, Theodoridis G. et al., Application Note, August 1999: 40-44), and by passing them through a cellulose acetate filter. 0.2μ) verification Filterability This ensures that the products measured by technicians under a pressure of 2 atm are... sterility .

[0072] Filterability test :

[0073] For testing, a small steel canister (Sartorius Stedim, 0.22L volume) with a compressed air inlet (adjustable by a pressure gauge) at the top and an outlet at the bottom was used. 15 ml of a solution of HA-paclitaxel dissolved in 5% w / w glucose to a concentration of 12 mg / ml was filled into the canister. A 0.2 μm filter (Minisart, CE, sterile) was installed at the outlet at the bottom of the canister. The compressed air flow rate was adjusted to achieve a pressure of approximately 1.5 bar within the steel canister to force the solution through the sterile filter. A timer was started at the beginning of filtration, and a graduated cylinder was placed below the filter's outlet to measure the volume filtered over time.

[0074] result:

[0075] The HA-paclitaxel conjugate prepared according to Example 1:

[0076] Dibromopaclitaxel w / w (weight percentage relative to intermediate weight): 0.8%

[0077] Filterability (Solution of 12 mg / ml conjugate in 5% w / w glucose solution): 4.5 ml / min

[0078] The HA-paclitaxel conjugate prepared according to Example 2:

[0079] Dibromopaclitaxel w / w: 5.2%

[0080] Filterability : 0.04ml / minute.

[0081] In summary, the results obtained show that the HA-paclitaxel conjugate prepared according to the target method of the present invention is characterized by:

[0082] -Very high purity Dibromopaclitaxel The amount is completely negligible;

[0083] - As is known to those skilled in the art, the high filtration capacity of a 0.2 μ filter ensures the sterility of the filtrate (a filtration capacity of 0.04 ml / min indicates low product filtration / poor product filtration, therefore the product cannot be sterilized by a 0.2 μ filter, necessitating the selection of alternative sterilization methods such as heating or radiation sterilization; however, as is well known, these two methods determine the partial degradation of products undergoing such processes, especially ester derivatives).

[0084] Finally, the objective method of the present invention results in a significant reduction in the amount of solvent used, because in such a method, Purification dialysis of the conjugate was not performed. In contrast to methods known to those skilled in the art, the preparation time of the conjugates according to Examples 1-2 is significantly reduced.

[0085] Therefore, compared with the conjugates obtained by the prior art (WO2004035629), the resulting conjugates have the best properties, and in addition, considerable cost and time are saved.

Claims

1. A method for preparing and purifying hyaluronic acid-paclitaxel (HA-paclitaxel) conjugates, comprising or consisting of the following steps: a) Add 4-bromobutyric acid, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and catalyst 4-dimethylaminopyridine (DMAP) to paclitaxel dissolved in dichloromethane, and activate the carboxyl group of the above-mentioned 4-bromobutyric acid by EDC to obtain an intermediate of HA-paclitaxel conjugate; b) The intermediate obtained from the previous step a) is crystallized in the organic solvent n-heptane, then filtered and dried; c) The intermediate obtained in step b) above is added to the tetrabutylammonium (TBA) salt of HA in DMSO; d) Purification of the HA-paclitaxel conjugate thus obtained by the following steps: precipitation with an ethanol / water solution containing NaBr salt, followed by washing at least once in ethanol / water and further washing in ethanol, pre-drying at 40°C and passing through a saturated steam chamber at 40°C and atmospheric pressure, and finally drying under vacuum at 40°C, wherein the HA-paclitaxel conjugate has an ester bond between the carboxyl group of the HA polysaccharide and 4-bromobutyric acid, which in turn is bonded via its carboxyl group to the hydroxyl group at carbon C2' of paclitaxel through an ester bond. The starting hyaluronic acid has a weight-average molecular weight of 160,000 Da to 230,000 Da.

2. The method according to claim 1, wherein in purification step d), the pre-drying and passing through a saturated water vapor chamber at atmospheric pressure are carried out at 40°C for at least 8 hours, and the drying under vacuum at 40°C is carried out for at least 16 hours.

3. The method according to claim 1 or 2, wherein the starting hyaluronic acid has an extraction source, a biotechnological source, or a fermentation source derived from rooster combs.

4. An HA-paclitaxel conjugate having a degree of derivatization or esterification in the range of 15% to 21% w / w, wherein the starting hyaluronic acid is of fermentation origin and has a weight-average molecular weight of 160,000 Da to 230,000 Da, said conjugate being in the form of a fine and uniform powder having a purity of greater than 98% by weight of the dried product and containing less than 1% by weight of dibromopaclitaxel.

5. The HA-paclitaxel conjugate according to claim 4, having a degree of derivatization or esterification in the range of 16% to 20% w / w.

6. An HA-paclitaxel conjugate prepared and purified by any one of claims 1 to 3, which is in the form of a fine and uniform powder and contains less than 1% by weight of dibromopaclitaxel.

7. The conjugate according to claim 6, wherein the degree of derivatization or esterification is in the range of 15% to 21% w / w.

8. The conjugate according to claim 7, wherein the degree of derivatization or esterification is in the range of 16% to 20% w / w.

9. A pharmaceutical composition comprising the conjugate according to any one of claims 4 to 8 and a pharmacologically acceptable diluent / excipient for the treatment of advanced breast cancer, lung cancer, ovarian cancer, bladder cancer, prostate cancer, endometrial cancer, and mesothelioma.

10. The pharmaceutical composition according to claim 9, wherein it is formulated in sterile isotonic water containing 5% w / v glucose.