MX2008001439A - Antitumoral bioconjugates of hyaluronic acid or its derivatives obtained by indirect chemical conjugation - Google Patents

Abstract

The present invention describes a new group of bioconjugates which can be obtained by means of indirect synthesis, via a molecular spacer, between hyaluronic acid and/or its derivatives and drugs with an antitumoral activity belonging to different groups, their preparation process and use in the oncological field. The new derivatives, in relation to the type of bond and Substitution degree, have different physico-chemical properties which improve their tolerability and efficiency and allow a more accurate modulation of the dosage, exploiting an active targeting mechanism.

Description

BIOCONJUGADOS ANTITUMORALES OF HYALURONIC ACID OR ITS DERIVATIVES OBTAINED BY CHEMICAL CONJUGATION HINT The development of a tumor, its growth and progression. towards primary and secondary metastasis are highly complex biological processes that require a sequential organization of coordinated (organ-selective) cellular events. The spread of tumor cells that leads to the formation of a metastasis occurs as a result of their separation from the primary growth site followed by their penetration into the circulatory bed and / or into the lymphatic system. In recent years, the progressive knowledge of vital processes that cause the initiation, development, dissemination and implantation of a tumor and its metastasis has not only offered researchers the possibility of studying, synthesizing and / or experimenting with new chemical molecules as new agents antitumor drugs but also has facilitated the study and improvement of new therapies of treatment that overcome the problems related to the toxicity of antineoplastic drugs and, above all, an understanding of the chemical-biological mechanisms that cause resistance to the previous drug. One of the main problems related to the treatment of tumors, in fact, refers to the possible "resistance" of the tumor to pharmacological treatment after an initial positive response. These "resistances" are associated with biological / biochemical variations in the functioning of the tumor cell such as, for example: • alterations in the cellular transport of the drug; • affinity changes with respect to this in the part of a possible metabolic inhibitor; • Substantial increase in the ability of the cell itself to activate the drug. The recently published scientific experiments (Misra et al., The Journal of Biological Chemistry, 2003, 278 (28): 25285-25288) have been shown as the pre / co-treatment in vitro of tumor cells resistant to some chemotherapy drugs with hyaluronic acid oligomers having a very low molecular weight. -established the initial sensitivity of the cell to the drug. The experimental data thus far obtained, however, have not fully clarified the how / why the sensitivity to chemotherapy is re-established, even if it has been observed that these oligomers are capable of interfering with several molecular events within the cell responsible for the acquisition of resistance to the drug and therefore the growth and diffusion of the tumor. The pharmacological action of the above oligomer becomes possible because, as it binds itself to the CD-44 receptor (specifically hyaluronic acid), it manages to interfere negatively with the native HA-receptor bond, an interaction that is responsible of the coordination of numerous cellular functions and, above all, of the tumor cell.

Through this link (and subsequent internalization) with its receptor present in the cell membrane, HA in fact participates in the activation of many events that are of fundamental importance for cellular life such as, for example, the regulation of cellular processes. adhesion / growth and cell migration, enters the chemtic mechanism during inflammatory processes, plays a major role in healing processes and, as mentioned above, in the migration of tumor cells for the formation of metastasis. Many solid tumors, in fact, have shown high amounts of HA that can consequently facilitate the invasion of other tissues and organs in the part of tumor cells. Tumor forms such as, for example, carcinomas, melanomas, lymphomas, breast tumors, colon-rectal and lung tumors, overexpress the transmembrane receptor CD-44: in these cell lines, the experiments carried out with anti- receptors (which consequently "block" the receptor by preventing its binding to native HA) have shown the effective ability to inhibit growth and tumor metastasis, thus demonstrating how the "interference" of the HA link with its receptor causes a disturbance of numerous events of fundamental importance for cellular life and showing, consequently, the real participation of HA in the development of the tumor mass. It is known that some anticancer drugs that have been used for years in the oncology field with satisfactory clinical results have been chemically modified to: »overcome the problem of their intrinsic toxicity with the aim of carrying out a new treatment strategy consisting of guiding the antineoplastic drug directly to the tumor cell by binding it to HA where, as described above, many tumor phenotypes overexpress the CD-44 receptor specific for HA on its cell surface (i.e. active target that increases the cellular efficacy of the drug by reducing its systemic toxicity). The binding and internalization of the polymer also carry the drug inside the tumor cell increasing its ericacy • increasing its solubility (it has been shown that the binding of fat-soluble drugs with strongly hydrophilic molecules such as, for example, HA, considerably increases the solubility of the drug by itself in the circulatory system). The solubility of chemotherapy drugs in the circulatory bed, in fact, represents the essential condition of their pharmacological efficacy, some drugs, however, that have proven to be extremely active in various types of tumors such as, for example, captothekines and their derivatives of irinotecan and topotecan, paclitaxel and alkaloids, Vinca derivatives, as a result of their high insolubility have problems related to intravenous administration (and, for hormones and anti-hormones also intramuscular) that can limit and restrict its clinical application. For the reasons cited above (solubility and toxicity) new chemotherapy drugs have been synthesized, which are created from the chemical bond (direct or indirect by means of a separator consisting of amino acids or peptides with a chain of amino acids letter) or simple association of some antineoplastic drugs containing a ring lactone (such as, for example, doxorubicin, paclitaxel, vincristine, vinblastine and camptothecins derivatives) with hyaluronic acid (HA) (US patent 6, 291, 671). Other conjugates comprise antineoplastic drugs such as paclitaxel and camptothecins linked to a polymer consisting of polyglutamic acid possibly associated with HA (U.S. Patent 5,977,163). Other new types of chemotherapy drugs are also known, represented by antitumor doxorubicin linked covalently to both HA (chemically modified with dihydrazide) and to a carrier such as the hydroxy-propyl-methacrylamide polymer (International Patent Application WO 02/090390). New vehicle drugs are also known, consisting of chemically conjugated polysaccharides with amino acid chains in turn covalently linked to antineoplastic drugs such as doxorubicin (U.S. patent 5,688,931). In addition, for the same reason, other delivery systems have been perfected, consisting, for example, in the encapsulation of doxorubicin in liposomes containing lipid derivatives of HA (Peer D. et al., Neoplasia, 2004, 6 (4): 343 -353; Eliaz RE et al., Cancer Research, 2001, 61: 2592-2601). It is known, for example, that in order to overcome the problems of camptothecin derivatives, to alternate their pharmacokinetic profile and reduce their toxicity by increasing their therapeutic efficacy, irinotecan has been conjugated with the carboxy-methyl-dextran polymer / vehicle by medium of a separator represented by a triglycine peptide (Satoshi Okuno et al., Cancer Research, 2000, 60: 2988-2995, U.S. patent 5, 892, 043). The resulting prodrug has proven to be active in its therapeutic efficacy since it remains in circulation for a prolonged period of time increasing its accumulation in the tumor mass, reducing its systemic toxicity contemporaneously; for many of the previously described conjugates, however, the definitive experimental data are not yet available, which document their efficacy with respect to the unconjugated drug. The paclitaxel derivative is also known, covalently linked to HA previously derived with hydrazide (patent of E. U. 5, 874, 17), or directly linked to HA, or indirectly by means of a separator of a variable nature capable of forming different types of chemical bonds that increase the solubility and consequently the efficacy of the drug (patent application EP 1560854). The present invention describes and claims new HA conjugates obtained from the indirect link between the polysaccharide and fat-soluble antineoplastic drugs such as, for example, irinotecan and Vinca alkaloids, or with soluble or partially soluble chemotherapy drugs such as doxorubicin and pyrimidine analog products, to overcome the related problems with its solubility (if present), its toxicity and, above all, to re-establish and increase the therapeutic efficacy of the drug in tumor cells that have acquired pharmacological resistance to the drug by themselves. The state of the art represented by the previously described derivatives is consequently exceeded in the present since the Applicant is able to demonstrate the pharmacological superiority of the new conjugates, object of the present invention, thanks to the extremely high cytotoxic capacity of these derivatives towards Neoplastic cells. This new pharmacological efficacy allows the application in clinical pharmacology of innovative chemotherapeutic therapies, for the treatment of primary and / or secondary tumors that do not respond more to any medical treatment following the formation of Multiple Drug Resistance (MDR) that generally endangers the possibility of an effective treatment of the patient and consequently, in the last analysis, drastically reduces your life expectancy. When resolving / overcoming MDR, the new derivatives, object of the present invention, change the final prognosis of the patient, consequently allowing the solution / reduction of the tumoral pathology.

DETAILED DESCRIPTION OF THE INVENTION The present invention describes and claims a new group of conjugates / derivatives and their preparation process, consisting of hyaluronic acid (HA) (and / or its derivatives) and antitumor drugs, indirectly conjugated by means of a molecular bridge called "separator" consisting of an aliphatic, araliphatic, alicyclic or heterocyclic, linear or branched chain with or without heteroatoms. In particular, the subject of the present invention are chemical-pharmaceutical conjugates of hyaluronic acid and / or its derivatives obtained through an indirect bridge between the polysaccharide and a drug with an antitumor action, through a molecular separator that forms a bond of amide or ester with the carboxylic group of HA and / or its derivative, with the proviso that said separator is not a hydrazide or a polypeptide. The HA (and / or one of its derivatives) and the drug, therefore, are conjugated in an indirect manner by means of one or more covalent bonds of the amide or ester type that partially or totally include the carboxylic groups of the polysaccharide and a functional chemical (eg, a hydroxyl, carboxyl, amine, etc. group) belonging to the separator, which, in turn, binds to the selected antitumor drug, as described in detail below. The derivatives that can be obtained according to the present invention have different physical-chemical properties that can be modulated through the selection of the type of link and degree of substitution, to improve the characteristics of the initial chemotherapy drug, such as: • solubility, • mechanical and rheological characteristics, • resistance to hydrolytic degradation, making the new conjugate more efficient in its cytotoxic action, a derivative that will have a new mechanism of action in this way will overcome the pharmacological resistance to the drug itself acquired by the tumor cell (as described above). As is known, many anti-tumor chemotherapy drugs have limited, if not nonexistent, solubility in water or saline solutions; This means that for its administration, it is necessary to resort to oils and organic solvents that, although they bring the drug in solution, have an intrinsic toxicity with side effects that require medication interventions before the administration of the product. In some cases, for the drug of Irinotecan chemotherapy, the active form (SN38) is still modified chemically (prodrug) to make it soluble and to promote the release of its metabolite that is active after intravenous administration. This however causes a low availability of the metabolite SN38 at the target site, thus requiring the administration of high cytotoxic dosages with a consequent amplification of undesired side effects.

The international literature (Mathijssen RH et al., Clin Cancer Res, 2001, 7: 2182-2194) indicates that the anti-tumor activity of SN38 is 100 to 1000 times higher with respect to its commercial prodrug; consequently the possibility of conjugating SN38 with hyaluronic acid or one of its derivatives according to the present invention, allows that the compounds with an increased efficacy are obtained and, thanks to the need to lower the dosages of administration, with fewer side effects related to the dispersion of the drug in areas not reached by neoplasia. The conjugation of anti-tumor chemotherapy drugs with HA also allows the active principle to be "directed" towards its target, and consequently towards the neoplastic tissue. Therefore, emphasis must be given to an active target mechanism between the conjugate and the neoplastic cell that increases the local concentration of drug close to the neoplastic area and consequently the efficacy. In this way, in addition, by reducing the distribution of the derivative to healthy tissues, a greater tolerance of the product with respect to the free drug is guaranteed. A second fundamental advantage deriving from the present invention is the possibility, mainly thanks to the presence of chemically modified HA, of technologically transforming the conjugate into a three-dimensional biomaterial (to be applied locally) processed in various forms such as, for example, hydrogel, nano or microspheres or of fibers in turn spun as woven or nonwoven products; in this case the chemically modified polysaccharide matrix is in close contact with the tumor mass, it acts as a controlled release system of the drug at the application site and therefore favors greater efficiency in the drug part by itself. Once the antineoplastic action has been exercised, the derivative degrades naturally and safely to the organism, completely releasing the antitumor active ingredient and hyaluronic acid. The products generated by the invention, whether in the form of classical pharmaceutical compositions or degradable biomaterials, are therefore characterized by a greater tolerance with respect to the active ingredient not modified and a higher pharmacological activity, in some cases even by several orders of magnitude with respect to that expressed by the active principle that forms them; both effects can be attributed to the specific affinity of hyaluronic acid towards receptors such as CD44 present in tumor cells. These effects are highlighted when the conjugated drug is administered in the form of a three-dimensional material, in direct contact with the neoplasm. The combination of these characteristics is such that the derivatives / conjugates of the present invention surpass in a different manner what is available in the state of matter in the local or systemic therapy of various kinds of neoplasia and of different origins, which have also been become resistant to traditional chemotherapy therapy. The hyaluronic acid used in the present invention has a molecular weight ranging from 400 to 3,000,000 Da, preferably ranging from 5,000 to 1,000,000 Da, and even more preferably from 30,000 to 500,000 Da, it may be an extractor, fermentor or intimate biosource origin. . The covalent bond with the separator includes the carboxyl group of D-glucuronic acid of the repeating unit of the polymer, in a percentage that varies from 1 to 100% (degree of substitution), which forms an ester or amide bond with the functional group of the selected molecular separator, which consequently acts as a connection between the hyaluronic acid and the chemotherapeutic drug. The separating agent consists of an aliphatic, araliphatic, alicyclic or heterocyclic, linear or branched chain containing or not containing heteroatoms, which may comprise hydroxyl, carboxyl, carbonyl, amine groups (with the exclusion of hydrazides and polypeptides), epoxy groups, chlorides of acids, thiols, nitriles, halogens, anhydrides, isocyanates and isothiocyanates; bromides, iodides and chlorides of carboxylic acids with an aliphatic C 2 to Cι chain are preferred, and in particular bromides such as bromopropionic acid or bromobutyric acid. The degree of substitution preferably ranges from 1 to 50% and even more preferably from 1 to 20%; for conjugation with doxorubicin a substitution of 3 to 15% is preferable while for conjugation with SN38 a substitution of 1 to 10% is preferred. The HA derivatives that can be used in the new conjugates, object of the present invention, are listed below: 1. HA salified with organic and / or inorganic bases having a molecular weight of 50-730KDa (EP0138572B1) or a high molecular weight 750 -1230 KDa, (EP 535200 Bl); 2. Hyaff®: HA esters with alcohols of the aliphatic, araliphatic, cyclo-aliphatic, aromatic, cyclic and heterocyclic series, with a percentage of esterification that can vary depending on the type and length of alcohol used, from 1 to 75%, preferably 30 to 50% (EP 216453 Bl); 3. Hyadd ™: HA amides with amines of the aliphatic, araliphatic, cyclo-aliphatic, aromatic, cyclic and heterocyclic series, with a percentage of amidation ranging from 1 to 10%, preferably 4% (EP 1095064 Bl); O-sulfated derivatives of HA up to the 4th degree of sulfation (EP 0702699 Bl); ACP®: internal HA esters with an internal esterification percentage varying from 0.5 to 10% and preferably 5% (EP 0341745 Bl); Deacetylated HA: derivatives of the deacetylation of the fraction of N-acetyl-1-glucosamine with a percentage of deacetylation preferably varying from 0.1 to -30%, while all carboxyl groups of HA can be salified with organic and / or inorganic bases (EP 1313772 Bl); Hyoxx TM, percarboxylated derivatives of HA obtained from the oxidation of the primary hydroxyl of the N-acetyl-glucosamine fraction with a degree of percarboxylation ranging from 0.1 to 100%, preferably from 25 to 75%. All the groups HA carboxylic acids can be salified with organic and / or inorganic bases (patent application EP 1339753). The drugs used in the conjugation reaction with HA preferably belong to the following categories: • nitrosurients, • antimetabolites: such as, for example, folic acid analogues (among which are methotrexate), pyrimidine analogs (among which are they are fluorouracil and ina-1-ß-D-Arabino-furanosilcitos ina: Ara-C), • alkaloids: such as, for example, vincristina and vinblastina (Vinca alkaloids) and the active metabolite of irinotecan, SN38, • antibiotics and analogous products: such as, for example, doxorubicin and epirubicin, • biological response modifiers, • diterpenoids, • synthetic hormones and antihormones: such as, for example, extradiol. Doxoru icinaa and the metabolite of irinotecan, SN38, are particularly suitable for the purposes of the present invention. The identified drugs and hyaluronic acid (and / or one of its derivatives) are indirectly linked by means of a separator through the formation of ester bonds with the following procedures: 1. a properly selected functional group of the separator (such as example, a carboxyl group, an amine group, a halide, etc.), also containing a second group (called "leaving group") capable of reacting with the HA carboxyl function (for example, a halide: bromine, iodine or chlorine ) reacts with a functional group belonging to the antitumor molecule represented, for example, by a hydroxyl, an amine, a carboxyl or an ercaptan.

The reaction may possibly require the activation of one of the included functions by means of an activation agent (for example the activation of a carboxyl group by means of carbodiimides). In a second phase, by contact directly with a tetraalkylammonium (preferably tetrabutylammonium) salt of HA in an anhydrous environment, the compound consisting of the modified drug reacts giving rise to a nucleophilic substitution of the leaving group (for example bromide) in the carboxyl of the HA, causing the formation of an ester link between HA and separator; 2. the carboxyl group of hyaluronic acid or one of its derivatives is linked by nucleophilic linkage to a suitable separator which is subsequently linked to a function of the antitumor molecule (in all ways known to experts in the field); 3. the carboxyl group of HA or one of its derivatives is activated with an activating agent, for example a carbodiimide, and is reacted with a hydroxyl function of the separator suitably selected, previously or subsequently bound to the drug (in all known ways by experts in the field). The identified drugs and hyaluronic acid (and / or one of their derivatives) are indirectly linked by means of a separator through the formation of amide bonds with the following methods: 1. the carboxyl group of hyaluronic acid or one of its derivatives is activated with an activating agent such as, for example, a carbodiimide, and is reacted with an amine function of the separator appropriately selected, previously or subsequently bound to the selected drug (in all ways known to experts in the field). The oncological applications relating to the use of conjugates consisting of hyaluronic acid (and / or one of its derivatives) and antitumor active are closely related to the response of the neoplasm to the conjugated drug. According to the contemplated uses, the bioconjugates can therefore be administered orally, intravenously, intra-arterially, intrathecally, intra-muscularly, subcutaneously, intraperitoneally, intra-articularly, topically, transdermally, loco-regionally, or in a combination thereof (therefore both a local and systemic administration procedure is claimed). Neoplasms included in the treatment, for example, can be (without limits) tumors of the pancreas, breast, colon-rectum, lung and respiratory system intoto, head-neck, liver, stomach, testes, ovary, endometrium, prostate, bladder, brain, leukemia, lymphomas , melanoma, Kaposi's sarcoma, osteosarcoma, neuroblast and skin cancer. Some examples of preparation of bioconjugates between hyaluronic acid and / or its derivatives and chemotherapy drugs with an anti-tumor activity are provided herein for purely illustrative and non-limiting purposes. Example 1: preparation of an ester derivative of hyaluronic acid having a MW of 200 kDa and SN-38 with a degree of substitution of about 15% (Figure 1) 199 mg of SN-38 are dissolved in 50 ml of Acetonitrile and 383 mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide (EDC), 258 mg of 4-bromobutyl acid and 40 mg of DMAP are added to the solution. The development of the solution is followed by TLC chromatography (silica stationary phase with fluorescence indicator and eluent of chloroform-acetonitrile 60:40). He The product is recovered by precipitation and purified by chromatography on a silica column using chloroform-methanol 99: 1 as eluent. The intermediate compound thus obtained is dried at room temperature under high vacuum. 0.84 g of tetrabutylammonium salt of hyaluronic acid (HATBA) are dissolved in 43 ml of N-methyl-2-pyrrolidone (NMP) at room temperature. The intermediate compound is added to the solution and the entire mixture is allowed to react at room temperature. After 7 days of reaction the solution is diluted with 5 ml of water and 5 ml of saturated sodium chloride. The whole mixture is left under stirring for 1 hour to allow the exchange of the sodium with the TBA ion. Ethanol is subsequently added dropwise and the obtained filamentous product is dissolved in water, dialyzed and, finally, lyophilized. Example 2: preparation of an ester derivative of hyaluronic acid (MW 31 kDa) and SN-38 with a degree of substitution at the carboxyl of about 10% 200 mg of SN-38 are dissolved in 50 ml of DMSO and 1.00 g of Ethylene carbonate are added to the solution. The solution is heated to 50 ° C and the Development of the solution is followed by means of TLC chromatography on silica plates. At the end of the reaction, the product is recovered by precipitation and dried at room temperature under a high vacuum. 175 mg of intermediate compound thus obtained are dissolved in an anhydrous mixture of 90:10 DMSO / pyridine with 85 mg of p-toluenesulfonyl chloride. When the intermediate compound has been converted to the corresponding toxylate, it is recovered by precipitation and dissolved in a solution of HATBA in NMP (0.68 g of polymer in 34 ml of NMP). The entire mixture is allowed to react for 7 days at room temperature. 4 ml of a saturated solution of NaCl is added to the solution and the mixture is left under stirring for 1 hour to allow the exchange of the sodium with the TBA ion. Ethanol is subsequently added dropwise and the obtained filamentous product is dissolved in water, dialyzed and, finally, lyophilized. Example 3: preparation of an ester derivative of hyaluronic acid with a MW of 55 kDa with vinblastine with a degree of substitution at the carboxyl of about 10% 308 mg of vinblastine are dissolved in 30 ml.

My chloroform and 120 mg of 4-bromobutyric acid and 150 mg of EDC are then added. After a while, water is added to the solution for the removal of the bromide and carbodiimide. The organic solution is anhydrous by means of sodium sulfate and the solvent is removed in a rotary evaporator. 300 mg of intermediate compound thus obtained are added to 1.70 g of HATBA dissolved in an anhydrous NMP and the solution is kept under stirring at room temperature for seven days. At the end, the complete mixture is left under stirring for 1 hour with 6 ml of saturated NaCl solution to allow the exchange of the sodium with the TBA ion. Ethanol is subsequently added dropwise and the obtained filamentous product is dissolved in water, dialyzed and, finally, lyophilized. Example 4: Preparation of an ester derivative of hyaluronic acid with a MW of 440 kDa and 5-fluorouracil with a degree of substitution at the carboxyl of about 15% 680 mg of ethylene carbonate and about 10 mg of NaOH are added to 510 mg of fluorouracil dissolved in 15 ml of DMF. The whole mixture is heated and the reaction is allowed to continue for 1 hour at reflux temperature. He The product recovered by precipitation is dissolved in an anhydrous mixture of DMSO / pyridine 50/50 with 1.00 g of p-toluenesulfonyl chloride. After approximately 15 hours, the product is recovered by precipitation and added to a solution of HATBA dissolved in PMSO (3.60 g in 180 ml of PMSO). The solution is kept under agitation at 38 ° C for about 3 days and at the end 20 ml of illiQ water and 7 ml of a saturated solution of NaCl are added. The whole mixture is left under stirring for 1 hour to allow the exchange of the sodium with the TBA ion. Ethanol is subsequently added dropwise and the obtained filamentous product is dissolved in water, dialyzed and lyophilized. Example 5: preparation of an ester derivative of hyaluronic acid with a MW of 200 kPa and 1-β-P-Arabino-furanosylcytosine (Ara-C) with a degree of substitution at the carboxyl of approximately 18% 100 mg of Ara- C, 80 mg of EPC and 69 mg of 4-bromobutyric acid are dissolved in 10 ml of water. The entire mixture is reacted for about 1 hour and finally the solvent is removed by evaporation under reduced pressure in a rotary evaporator. The product is purified by means of column chromatographic separation. The intermediate compound thus obtained is dissolved in a solution at 20 mg / ml of 1.10 g of HATBA in PMSO and reacted for 7 days at room temperature. 5 ml of a saturated solution of NaCl are added to recover the product, thus allowing salification with sodium of the hyaluronic acid carboxyls. The polymer is precipitated by adding ethanol dropwise, and after filtering and redissolving it in water, it is dialyzed to remove solvent and salt residues and finally lyophilized. Example 6: Preparation of an ester derivative of hyaluronic acid with a MW of 120 kDa and 17β-extradiol with a degree of substitution at the carboxyl of about 20% 140 mg of 17 β-extradiol are dissolved in 50 ml of DMSO and 380 mg of l- (3-dimethylaminopropyl) -3-ethylcarbodiimide (EDC), 262 mg of 4-bromobutyl acid are added to the solution. The product is recovered by precipitation and purified by chromatography on a silica column. The intermediate compound thus obtained is dried at room temperature under a high vacuum. 0.80 g of tetrabutylammonium salt of Hyaluronic acid (HATBA) are dissolved in 40 ml of N-methyl-2-pyrrolidone (NMP) at room temperature. The intermediate compound is added to the solution and the entire mixture is allowed to react at room temperature. After 7 days of reaction the solution is diluted with 5 ml of water and 5 ml of a saturated solution of sodium chloride. The whole mixture is left under stirring for 1 hour to allow the exchange of the sodium with the TBA ion. Ethanol is then added dropwise and the filamentous product obtained is dissolved in water, dialyzed and, in the end, lyophilizes. Example 7: preparation of the partial ester between 200 kDa hyaluronic acid and SN38 and self-degradation of HA derivative 200 mg of SN38 are dissolved in 50 ml of DMSO and 375 mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide (EDC ), 330 mg of 4-bromobutyl acid are added to the solution. The development of the solution is monitored by means of TLC chromatography (silica stationary phase with fluorescence indicator and eluent of chloroform-acetonium trill 60:40). The product is recovered by precipitation and purified by chromatography on a silica column using chloroform methanol 99: 1 as eluent. The intermediate compound thus obtained is dried at room temperature under high vacuum. 0.84 g of tetrabutylammonsalt of hyaluronic acid (HATBA) are dissolved in 43 ml of N-methyl-2-pyrrolidone (NMP) at room temperature. After removing the solution to react for 7 days, 34 mg of triethylamine is added to the reaction solution and the whole mixture is stirred for 30 '. A solution of 87 mg of 2-chloro-1-methyl-pyridine iodide in 10 ml of PMSO is slowly added dropwise over a period of 45 'and the mixture is maintained at 30 ° for 15 h. A solution consisting of 15 ml of water and 0.5 g of sodchloride is then added and the resulting mixture is poured slowly into 300 ml of acetone with continued stirring. A precipitate is formed that is filtered and rinsed three times with 25 ml of acetone-water 5: 1 and three times with acetone (50 ml). The product is dried under a high vacuum at 38 ° C. Example 8: Preparation of a 220 kDa hyaluronic acid amide derivative with doxorubicin with a degree of substitution at the carboxyl of about 5% (Figure 2) 2. 00 g of HATBA are dissolved in 100 ml of DMSO with a low water content. The solution is insufflated with gaseous hydrochloric acid until an aliquot of the solution is removed and diluted with 1:10 water, the pH test is between 4.5 and 5. Carbonyl diimidazole (55 mg) is subsequently added to the solution and the complete mixture is left under stirring at room temperature for 1 h. At the end, 1.4 g of doxorubicin are added to the solution and the mixture is allowed to react for 24 hours at room temperature. 5 ml of a saturated solution of NaCl are added to recover the product, thus allowing salification with sodof the hyaluronic acid carboxyls. The polymer is precipitated by adding ethanol drop by drop and after filtering and redissolving it in water, it is dialyzed to eliminate solvent and salt residues and finally it is lyophilized. Example 9: preparation of the partial ester between hyaluronic acid and doxorubicin and self-degradation with condensation Ugi 500 mg of polymer obtained according to example 8 are dissolved in 5 ml of distilled water. The pH of the solution is decreased to about 4 by the addition of concentrated hydrochloric acid. 15 mg of ethyl ester di-hydrochloride from usina, 250 μl of aqueous formaldehyde solution at 40% and 250 μl of cyclohexyl isocyanide are added to the solution. After 15 minutes of reaction the gel is placed on dialysis in a basic solution of sodcarbonate for approximately 24 h and finally dialyzed against water until a solution conductivity of less than 40 μs. The polymer is recovered by lyophilization. Example 10: preparation of an ester derivative of hyaluronic acid with a MW of 200 kPa and Poxorubicin with a degree of substitution at the carboxyl of approximately 10% 325 mg of Poxorubicin Hydrochloride are dissolved in 50 ml of NMP, after adding 0.3 ml of Et3N, 420 mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide ( EPC) and 280 mg of 4-bromobutyric acid are subsequently added to the solution. The development of the solution is monitored by means of TLC chromatography (silica stationary phase with fluorescence indicator and eluent of dichloromethane-methanol 80:20). The product is purified by chromatography in a column using chloroform: methanol 99: 1 as eluent. The intermediate compound thus obtained is dried at room temperature under high vacuum. 0.75 g of tetrabutylammonsalt of hyaluronic acid (HATBA) are dissolved in 40 ml of N-methyl-2-pyrrolidone (NMP) at room temperature. The intermediate compound is added to the solution and the entire mixture is allowed to react at room temperature. After 7 days of re-action the solution is diluted with 5 ml of water and 5 ml of saturated sodchloride. The whole mixture is left under stirring for 1 hour to allow the exchange of the sodwith the TBA ion. Ethanol is subsequently added dropwise and the obtained filamentous product is dissolved in water, dialyzed and, finally, lyophilized. In Vitro Experimentation: Evaluation of the antiproliferative activity of the HA / SN38 ester conjugate having a degree of substitution equal to 10% and 15%, in the HT29 colic adenocarcinoma cell line. The aliquots of the derivatives obtained from examples 1 and 2 are characterized by a cytotoxicity test in vi tro in a line of colonic tumor cells called HT29. The comparison is affected with SN38 dissolved in PMSO. The HA derivatives are solubilized in a 5% glucose solution at a concentration of 5 mg / ml. The test is carried out by depositing in a plate with 96 cavities, 3000 cells per cavity; After 24 hours of incubation at 37 ° C, the cells are placed in contact with the solutions and after an additional 48 hours the cell vitality is determined by means of a MTT colorimetric assay (Pezinot F. et al., J Immunol Methods, 1986, 22 (89): 271-277). The proliferation curves referring to the two ester conjugates of HA are shown in the graphs (on the left the cytotoxic activity of the derivative at 15%, on the right that at 10%), see figure 4. When comparing EC50 data With SN38 in dimethylsulphoxide (PMSO) the following results are obtained: The results confirm that the new derivatives of HA / SN38 show the same cytotoxic activity of the active metabolite SN38 which, as described above, has 100 to 1,000 times the activity of its commercial prodrug, Irinotecan. The experimentation carried out therefore affirms a much higher efficacy of the new derivative with respect to the reference drug currently used in clinical practice. In order to demonstrate what is established above with respect to the efficacy of the new conjugates (object of the present invention) as antineoplastic drugs able to overcome the pharmacological resistance acquired by neoplastic cells no longer sensitive to the drug per se, the following experiments are carried out. in vi tro: Cytotoxicity test in PHP / K12 cells of the chemotherapy drug doxorubicin compared to its amide conjugate with HA The cell line uses rat derivatives of the BPIX strain treated with 1,2-dimethylhydrazine. These cells do indeed express the same tumor antigens as human colon-rectum adenocarcinoma and, for this reason, are used as a preclinical study model for the same tumor time.

The cell line above has also acquired resistance to chemotherapy treatment (this is called "Multiple Drug Resistance": MPR). To assess the degree of cellular vitality the VIVA / DEAD Cell Vitality Test (Molecular Probes) is used, which allows metabolically active cells to be distinguished from dead cells; the latter emit green fluorescence at a nuclear level while living cells emit red fluorescence located in the cell membrane and in the cytoplasm. After coloring, the cells are analyzed using a confocal microscope and the percentage of live / dead cells is evaluated by counting at least 500 live or dead cells per sample. The amide conjugate of doxorubicin with HA (Hydox) is used for experimentation, having a degree of substitution of 5% obtained according to example 8, compared to the drug as such, at different concentrations. Figure 3: after 48 hours of treatment, the results of the VIVAS / DEAD test represented in figure 3, clearly indicate how the Hydox conjugate is able to exert, in a dose-dependent manner, a cytotoxic effect on cells that have acquired a certain resistance to chemotherapy, much higher than the corresponding non-conjugated doxorubicin, used under the same concentrations. Hydox, in fact, also proves to be active at low concentrations such as 0.25 μM, while the reference drug in this dosage has no cytotoxic effect. By doubling the concentration, the conjugate in question has 35% more cytotoxicity, thus allowing the use of lower dosages of drug with minor side effects in cells that do not respond more to classical chemotherapeutic therapies due to acquired resistance to the drugs antineoplastic from above. The description of the invention being such, it is evident that these methods can be modified in various ways. These modifications should not be considered as diverging from the spirit and perspectives of the invention and all the modifications that will appear evident to the experts in the field they include in the scope of the following claims.

Claims (30)

1. CLAIMS 1. Chemical-pharmaceutical conjugates of hyaluronic acid and / or its derivatives obtained through an indirect link between the polysaccharide and a drug with an antitumor action, through a molecular separator that forms an ester or amide bond with the group HA carboxylic acid and / or its derivative, with the proviso that said separator is not a hydrazide or a polypeptide.
2. 2. Chemical-pharmaceutical conjugates according to claim 1, wherein the drug is selected from the following groups: nitrosoureas, antimicrobials, alkaloids, antibiotics and analogous products, biological response modifiers, diterpenoids, synthetic hormones and antihormones.
3. 3. Chemical-pharmaceutical conjugates according to claim 2, wherein the drug is an antimetabolite consisting of a pyrimidine analog product such as fluorouracil and Ara-C.
4. 4. Chemical-pharmaceutical conjugates according to claim 2, wherein the drug is an alkaloid such as vincristine, vinblastine and the active metabolite of irinotecan SN38.
5. 5. Chemical-pharmaceutical conjugates according to claim 2, wherein the drug is an antibiotic such as doxorubicin.
6. 6. Chemical-pharmaceutical conjugates according to claim 2, wherein the drug is a hormone such as extradiol.
7. 7. Chemical-pharmaceutical conjugates according to claim 1, wherein the degree of substitution in the carboxyl of hyaluronic acid and / or one of its derivatives varies from 1 to 100%.
8. 8. Chemical-pharmaceutical conjugates according to claim 7, wherein the drug is doxorubicin and the degree of substitution in the carboxyl of hyaluronic acid of the doxorubicin-linked separator ranges from 1 to 20%.
9. 9. Chemical-pharmaceutical conjugates according to claim 7, wherein the drug is SN38 and the degree of substitution in the carboxyl of hyaluronic acid of the separator bound to SN38 ranges from 3 to 15%.
10. 10. Chemical-pharmaceutical conjugates according to claim 1, wherein the hyaluronic acid and / or one of its derivatives has a weight molecular that varies from 400 to 3xl06 Da.
11. 11. Chemical-pharmaceutical compounds according to claim 10, wherein the hyaluronic acid preferably has a molecular weight ranging from 5,000 to lxlO6 Da.
12. 12. Chemical-pharmaceutical compounds according to claim 11, wherein the hyaluronic acid preferably has a molecular weight ranging from 30,000 to 0.5x106 Pa.
13. 13. Chemical-pharmaceutical conjugates according to claim 1, wherein the hyaluronic acid derivative is a salt with organic and / or inorganic bases.
14. 14. Chemical-pharmaceutical conjugates according to claim 1, wherein the hyaluronic acid derivative is an ester of hyaluronic acid with alcohols of the aliphatic, araliphatic, cycloaliphatic, aromatic, cyclic and heterocyclic series with a percentage of non-esterification. higher than 75%
15. 15. Chemical-pharmaceutical conjugates according to claim 1, wherein the hyaluronic acid derivative is an amide of hyaluronic acid with amines of the aliphatic, araliphatic, cycloaliphatic series, aromatic, cyclic and heterocyclic with an amidation percentage that varies from 1 to 10%.
16. 16. Chemical-pharmaceutical conjugates according to claim 1, wherein the hyaluronic acid derivative is a 0-sulphated derivative of hyaluronic acid up to the 4th degree of sulfation.
17. 17. Chemical-pharmaceutical conjugates according to claim 1, wherein the hyaluronic acid derivative is an internal ester with an esterification percentage ranging from 0.5 to 10%.
18. 18. Chemical-pharmaceutical conjugates according to claim 1, wherein the hyaluronic acid derivative is obtained by deacetylation of the N-acetylglucosamine fraction, with a percentage of deacetylation ranging from 0.1 to 30%.
19. 19. Chemical-pharmaceutical conjugates according to claim 1, wherein the hyaluronic acid derivative is a percarboxylated derivative obtained by oxidation of the primary hydroxyl of the N-acetylglucosamine fraction, with a degree of percarboxylation ranging from 0.1 to 100% .
20. 20. Chemical-pharmaceutical conjugates according to claim 1, wherein the separator consists of an aliphatic, araliphatic, alicyclic, heterocyclic, linear or branched chain optionally containing heteroatoms, having hydroxyl, carboxyl, carbonyl, amine, epoxy, acid chlorides groups , thiols, nitriles, halogens, anhydrides, isocyanates, and isothiocyanates.
21. 21. Chemical-pharmaceutical conjugates according to claim 20, wherein the separator is represented by carboxylic acids with a number of carbon atoms ranging from 2 to 10.
22. 22. Chemical-pharmaceutical conjugates according to claim 21, in where the separator is bromopropionic acid.
23. 23. Chemical-pharmaceutical conjugates according to claim 21, wherein the separator is bromobutyric acid.
24. 24. Pharmaceutical compositions having one or more of the chemical-pharmaceutical conjugates according to the previous claims, as an active ingredient.
25. 25. Pharmaceutical compositions of according to claim 24 for oral, intravenous, intra-arterial, intrathecal, intramuscular, subcutaneous, intraperitoneal, intra-articular, topical, transdermal administration or for direct administration at the site of the neoplasia.
26. 26. Three-dimensional biomaterials according to claim 1 processed in the form of hydrogels, nano- and microspheres, spun or non-woven fibers.
27. 27. Use of the chemical-pharmaceutical conjugates according to claims 1-23 for the preparation of pharmaceutical compositions to be used in the oncological field.
28. 28. Use according to claim 26 for the systemic or local treatment of tumors of the pancreas, breast, colon-rectum, lung and respiratory system intoto, head-neck, liver, stomach, testes, ovary, endometrium, prostate, bladder, brain, leukemia, lymphomas, melanoma, Kaposi's sarcoma, osteosarcoma, neuroblastoma and skin cancer.
29. 29. Process for the preparation of chemical-pharmaceutical conjugates according to claims 1-23 by indirect conjugation of hyaluronic acid or one of its derivatives and a drug having an anti-tumor activity by means of a separator forming an ester bond with the carboxyl group of hyaluronic acid according to the following alternative methods a), b) or c): la) a functional group of the suitably selected separator also containing a second leaving group capable of reacting with the carboxyl function of HA, reacts with a functional group belonging to the selected antitumor molecule; lia) the reaction may possibly require the activation of one of the included functions by means of an activating agent such as carbodiimides; Illa) in a second phase, by direct contact with a salt of tetra-alkylammonium (preferably tetrabutyl ammonium) of HA in an anhydrous environment, the compound consisting of the modified drug reacts giving rise to a nucleophilic substitution of the leaving group in the carboxyl of the HA, causing the formation of an ester bond between HA and separator; Ib) the carboxyl group of hyaluronic acid is linked by nucleophilic linkage to a suitable separator which is subsequently linked to a function of the molecule antitumor; le) the carboxyl group of HA is activated with an activating agent and reacted with a hydroxyl function of the separator suitably selected, previously or subsequently linked to the drug. Process for the preparation of the chemical-pharmaceutical conjugates according to claims 1-23 by the indirect conjugation of hyaluronic acid or one of its derivatives and a drug having an antitumor activity by means of a separator that forms an amide bond with the carboxyl group of hyaluronic acid according to the following procedure: the carboxyl group of hyaluronic acid is activated with an activating agent and reacted with an amine function of the separator suitably selected, previously or subsequently bound to the drug.