WO2009074678A2

WO2009074678A2 - Anticancer conjugates of camptothecin to hyaluronic acid

Info

Publication number: WO2009074678A2
Application number: PCT/EP2008/067433
Authority: WO
Inventors: Stefano Norbedo; Francesca Dinon; Susanna Bosi; Massimo Bergamin; Erminio Murano
Original assignee: Eurand Pharmaceuticals Limited
Priority date: 2007-12-12
Filing date: 2008-12-12
Publication date: 2009-06-18
Also published as: WO2009074678A3; IE20070900A1

Abstract

New conjugates are described, containing camptothecin connected to hyaluronic acid via a linker, where the linker is bound to the hyaluronic acid by means of an ester bond. The ester bond involves on one side, a hydroxyl group of hyaluronic acid and, on the other side, a carboxyl group present on the linker; the linker is covalently bonded to camptothecin. Pharmaceutical compositions thereof, their use in the treatment of pathologies responsive to camptothecin, and a process to prepare said conjugates are also described.

Description

NEW ANTICANCER CONJUGATES Field of the invention

The present invention relates to novel conjugates of hyaluronic acid with camptothecin. Prior art

Amongst the problems encountered in different types of therapies, such as cancer therapy are: (a) small molecule (anticancer drugs) are mainly hydrophobic in nature hence have poor water solubility and consequently their biological properties are impaired and (b) insufficient selectivity for specific tissues or cells. Camptothecin is a water insoluble, optically active pentacyclic alkaloid obtained from Camptotheca acuminata tree. 20(S)-Camptothecin and its derivatives are cytotoxic agents that are thought to act by stabilising a topoisomerase l-induced single strand in the phosphodiester backbone of DNA, thereby preventing ri- legation. This leads to the production of a double strand DNA break during replication, which results in apoptosis, if not repaired. 20(S)-Camptothecins exhibit excellent antitumour activity against human cancer cell lines and in vivo animal xenografts. In addition to its water insolubility, its pharmacologically important lactone ring is unstable in human plasma where it is present mainly as its open hydroxy-acid form, which is captured by albumin, thus inactivating the drug. The primary shortcomings of camptothecins are the formation of a labile drug target complex and instability of the lactone ring. On the basis of the reversibility of the ternary complex and formation of lethal lesions during DNA replication, optimal cytotoxic effects are expected with prolonged exposure to the drug. Several camptothecin derivatives have been synthetised and undergone clinical development but despite their promising clinical role, the over all therapeutic impact of available camptothecin derivatives has been modest; many approaches to optimise their therapeutic indices are being evaluated. Researchers tried to solve the problems by preparing new compounds. Topotecan and irinotecan are synthetic derivatives designed to facilitate parenteral administration of the active lactone form of the compound by introducing functional groups to enhance solubility. They are now well-established components in the chemotherapeutic management of several neoplasms. Topotecan has good activity in patients treated previously with ovarian and small cell lung cancer and is currently approved for use in the United States as second-line therapy in these diseases, lrinotecan is a prodrug that undergoes enzymatic conversion to the biologically active metabolite 7-ethyl-10-hydroxy-camptothecin (SN38). It is presently the treatment of choice when used in combination with fluoropyrimidines as first-line therapy for patients with advanced colorectal cancer or as a single agent after failure of 5-fluorouracil-based chemotherapy. Several additional camptothecin derivatives are in various stages of clinical development, they however present some disadvantages such as they are not suitable for targeting to specific tissues or cell receptors.

In the attempt to optimize drug efficacy, possible strategies may involve chemical modifications of molecular structure. One strategy is to covalently bind the drug to a water soluble polymeric material. A number of polymers have been used to conjugate hydrophobic drug molecules, including camptothecins, such as polyethylene glycol (PEG), poly(L-glutamic acid)-camptothecins, poly(N-(2- hydroxypropyl)methylacrylamide), carboxymethyldextran, beta- cyclodextrin. Such strategies however do not allow to have a targeting to the desired sites; in some cases the polymer does not recognise any targets at all either because of its very chemical nature or because it has lost its original/native targeting capability, due to the chemical modification. In fact, the functional and biological properties of the native polymer may be easily lost when the chemical modification involves groups that are essential for their maintenance.

These polymers (poly(L-glutamic acid), polyethylenglycol (PEG) and carboxymethyldextran (CMD) lack in bioactivity and targeting capabilities. Differently, native hyaluronic acid has shown advantages over the other polymers because of its capability to target the drug to the diseased site. Anti-cancer polymeric drugs can traverse through the cancer site either by enhanced permeability and retention (EPR) module, a passive mechanism, or by active targeting using specific interactions between receptors on the cell surface. Hyaluronic acid can not only operate through the EPR module, but also have a number of recognised cell receptors in the body and it may interact with other structures such as in particular proteoglycans; among the different receptors, the CD44 may be quoted, which is over expressed in many tumor types. Conjugates of hyaluronic with various drugs have been disclosed in WO2006/122954, WO2007/085629, PCT/EP2007/057772. Although improvements in drug efficacy have been reported for these conjugates, none of them has shown optimal characteristics with respect to targeting capability, drug stability, reproducible release rate. In particular, little progress has so far been made as to stabilising the specific camptothecin molecule, to obtain an optimal receptor targeting and a timely and reproducible release of the active molecule. Summary of the invention New conjugates have now been found, wherein camptothecin is connected to hyaluronic acid via a linker, said linker being bound to the hyaluronic acid by means of an ester bond. The ester bond involves on one side, any of the hydroxyl groups of hyaluronic acid and, on the other side, a carboxyl group present on the linker; the linker is covalently bonded to camptothecin. The presence of these linkages provides peculiar characteristic allowing a precise control over the site and rate of drug release. High-efficacy camptothecin-containing medicaments are thus obtained, allowing a more precise targeting of the patient's diaseased sites, and an optimised duration of activity after administration. Detailed description of the invention In the present text, hyaluronic acid is also referred as "HA" or "hyaluronan".

One essential feature of the present invention is the presence of an ester bond connecting the camptothecin-carrying linker and hyaluronic acid. These esters involve any of the hydroxyl groups of hyaluronic acid and a carboxyl group present on the linker. By "any of the hydroxyl groups" are meant both primary and secondary hydroxyl groups of HA, irrespective of their positions on the HA molecule: however, no other groups of HA (such as carboxyl groups of glucuronic acid or amine of deacetylated N-acetylglucosamine residue) are involved in any linkages with the linker and or the drug. The degree of substitution (DS) indicates the percent of hydroxyl groups of hyaluronic acid involved in the ester with the drug-carrying linker; in the present conjugates the DS can be varied depending on reaction conditions (i.e. the amount of drug /linker, temperature, reaction time, solvent). The modulation of the DS results in conjugates having different levels of drug loading, useful for different therapeutic purposes. Preferred DS range between 1 and 50%, more preferably between 5 and 30% mol/mol.

The term hyaluronic acid (or HA or hyaluronan) means a polysaccharide composed of a disaccharidic repeating unit, consisting of D-glucuronic acid and 2- acetamido-2-deoxy-D-glucose (N-acetyl-D-glucosamine) bound by β(1 → 3) glycosidic linkage; the D-glucuronic acid residue may either be in the acid form or in the form of a salt. Each repeating unit is bound to the next one by a β(1 →4) glycosidic linkage that forms a linear polymer. The HA used for the preparation of the instant conjugates has preferably a weight average molecular weight (MW) comprised between 5,000 and 1 million, and more preferably from 5,000 to 500,000. The term hyaluronic acid (or HA or hyaluronan) encompasses both the free acid and its salified form with e.g. alkaline metals (preferably Na or K), earth- alkaline metals (preferably Ca or Mg), transition metals (preferably Cu, Zn, Ag, Au, Co, Ag). The term hyaluronic acid (or HA) also includes derivatives thereof, e.g. wherein some hydroxyl groups are derivatised with groups selected from: -NH₂, - NHCOCH₃, OR, -OCOR, -SO₂H, -OPO₃H₂, -O-CO-(CH₂)_n-COOH, -O-(CH₂)_n- OCOR, wherein n is 1 -4 and R is C₁-C₁₀ alkyl. The term camptothecin (or CPT, or the drug) comprises camptothecin, having formula (I):

(I)

and derivatives thereof; examples of said derivatives are structurally modified camptothecins containing further groups, such as for example hydroxyl, amino, alkylamino, fluoro groups. Examples of structurally modified camptothecins are: 7,10-modified-CPT (such as irinotecan), 9,10-modified-CPT (such as topotecan), 7,9,10,1 1 -modified-CPT (such as exatecan), 10,1 1 -modified-CPT, 9-amino-CPT derivatives, 20-amino-CPT derivatives, their prodrugs.

The linker (meant in its free state, i.e. before engaging in the present conjugates) always contains at least one carboxyl group, necessary for esterifying the HA; it also contains at least one other group useful for covalently linking the drug, e.g. further carboxyl groups, hydroxyl groups, amino groups.

Suitable linkers are linear or branched, aliphatic, aromatic or araliphatic C₂-C₂O dicarboxylic acids, aminoacids, peptides, linear or branched, aliphatic, aromatic or araliphatic C₂-C₂₀ dicarboxylic acid linked to aminoacids or to peptides. The role of the linker consists in creating a spacer between the hyaluronic acid and the drug. The linker engages, on one side, the hydroxyl groups of HA via the ester linkage and, on the other side, the drug via a covalent bond. When the linker is a dicarboxylic acid linked to aminoacid or to peptide, the carboxylic group forming the ester bond with the HA may be the free acid group of the dicarboxylic acid or that of the aminoacid or that of the peptide.

Preferred linkers are: succinic acid, succinic acid linked to aminoacids, succinic acid linked to peptides.

Preferred aminoacids according to the invention are selected from the group consisting of alanine, valine, leucine, isoleucine, methionine, glycine, serine, cysteine, asparagine, lysine, glutamine, aspartic acid, glutamic acid, proline, histidine, phenylalanine, triptophane and tyrosine. Preferred peptides according to the invention are peptides consisting of different combinations of the above aminoacids, they are preferably di-, tri- or tetra-peptides. The present conjugates are stable compounds, they are free of undesired reaction by-products and impurities that can be harmful to their practical pharmaceutical use. After administration to the body, by the effect of hydrolysis, they release the drug and the HA in form of native HA, which is a biocompatible and bioabsorbable and of no toxicity for the body. These conjugates allow to obtain pharmaceutical compounds retaining the pharmacological efficacy of the drug over an extended period of time. Therefore, they can be successfully used in the treatment/prevention of all diseases responsive to CPT. At the same time they can show some properties not observed in the therapeutic agent alone, for examples higher affinities for some cells or tissues, different bioavailability profiles.

Accordingly, a further aspect of the invention is the use of the above conjugates in the manufacture of a medicament for the treatment/prevention of cancer and all diseases responsive to CPT, i.e. diseases on which CPT is effective. The invention thus includes said conjugates for use in the treatment/prevention of all diseases responsive to CPT. Further provided is a method of treating/preventing said diseases, characterised by administering an effective amount of the above described conjugates to a patient in need thereof. Among the diseases responsive to CPT, cancer is especially worth mentioning, in particular: colorectal, gastroinstestinal, lung, central nervous system, head and neck, ovarian, cervical, breast, pancreatic, lymphoma, melanoma cancers. Further aspect of the invention is a pharmaceutical composition containing the above conjugates in admixture with pharmaceutically acceptable excipients and/or diluents. The pharmaceutical composition may be either in the liquid or solid form; it may be administered through oral, parenteral, topical, intraarticular route, etc. Systemic administration may occur by intravenous, intraperitoneal, intramuscular, subcutaneous route. Particularly interesting are the injectable pharmaceutical compositions. A further aspect of the invention is a process for the preparation of the above described conjugates. The process presents the advantage that it can be easily performed directly on the HA in its native form, preferably in its salified form; HA needs not to be chemically modified (covalent modification) before reacting with the linker. Furthermore, this process is applicable to HA within the entire range of molecular weights characteristic of the present invention, i.e. from 5,000 to 1 ,000,000.

The said process includes the step of linking the drug to the linker, followed by the step of forming the ester linkage between hydroxyl groups of HA and a carboxyl group present on linker. The type of bond between the linker and the drug is not critical and can be obtained by any suitable chemical reaction forming a covalent bond: esters, ethers, carbonates, carbammates, are examples of such bonds. There are some derivatives of camptothecin that contain reacting groups other than hydroxyl, that are useful for the formation of different linkages; e.g., when an amino group is present, the resulting covalent bond may be an amine, carbammate, amide, ureide bond. Preferably, since the camptothecin and its derivatives contain a hydroxyl group and the linker may contain a carboxyl group (in addition to the one required for the esterification with HA), the drug-linker bond is conveniently of ester type. Thus the drug is treated with the linker (e.g. properly protected succinic acid like its t-butyl monoester, or hemisuccinates wherein succinic acid forms an amide with an aminoacid or peptide), in a chlorinated organic solvent (e.g. methylene chloride, chloroform, 1 ,2-dichloroethane) in the presence of a catalyst (e.g. p- dimethylamino-pyridine, 4-pyrrolidinopyridine, 4-tetramethylguanidinopyridine) and a coupling agent (e.g. diisopropylcarbodiimide, dicyclohexylcarbodiimide, 1 -(3- dimethylaminopropyl)-3-ethylcarbodiimide) for a period of time ranging from 4 to 24 hours, preferably 12 hours, thus obtaining a drug-linker monoester; the resulting monoester is isolated from the reaction mixture according to any known techniques. COOH-protecting groups can be removed by treatment with a strong acid, e.g. tetrafluoroacteic acid. The thus obtained drug-linker compound is dissolved in a polar aprotic solvent (e.g. dimethylsulfoxide, Λ/,Λ/-dimethylformamide, Λ/,Λ/-dimethylacetamide, N- methylpyrrolidone) and reacted with an activating agent (e.g. N-hydroxysuccinimide (NHS), 1 -hydroxybenzotriazole) and in the presence of a coupling agent (e.g. diisopropylcarbodiimide, dicyclohexylcarbodiimide, 1 -(3- dimethylaminopropyl)-3-ethylcarbodiimide). The activated drug-linker compound is then reacted with hyaluronic acid, preferably in form of a salt, e.g. quaternary ammonium salts like tetrabutylammonium or other tetra aryl/alkyl ammonium salts; the reaction is carried out at temperature ranging from 20 °C to 60 °C for 16-120 hours. The final conjugate is isolated from the reaction mixture according to known procedures. Salts of HA are obtainable by standard procedures, e.g. by treatment with the relevant base, or by ion exchange resins.

When the linker molecule includes an aminoacid or a peptide, the hydroxyl group of camptothecin may be linked to the carboxyl function of said aminoacid/peptide by treatment of the drug with the corresponding N-protected aminoacid/peptide (such as Cbz-glycine-OH), yielding the corresponding ester derivative by regeneration of the amino group. Then, the resulting product having the free NH₂ group is treated with a monoester of C₂-C_2O dicarboxylic acid to give the corresponding monoamide. The thus obtained drug-linker compound is then reacted with HA affording the final conjugate. Alternatively, the reaction sequence involves: reacting the drug with C₂-C₂₀ dicarboxylic acid (e.g. succinic acid) to give the corresponding monoester (e.g. hemisuccinate) of the camptothecin hydroxyl group; the monoester is then treated with an aminoacid (e.g. glycine) or a peptide, in which the carboxyl group is protected; after removal of the protecting group and treatment with HA in the above referred conditions, the final conjugate is obtained. The invention is now illustrated by the following non limiting examples. EXPERIMENTAL PART Materials and methods HA: hyaluronic acid. TBA: tetrabutylammonium. DMF: Λ/,Λ/-dimethylformamide. DMSO: dimethylsulfoxide. DIEA: N,N-diisopropylethylamine. DMAP: 4- dimethylaminopyridine. DCM: dichloromethane. DIPC: diisopropylcarbodiimide. TFA: trifluoroacetic acid. THF: tetrahydrofuran. MeOH: methanol. EtOH: ethanol. CPT: campthotecin (formula I) EDC: N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide. Cbz: benzyloxycarbonyl. Boc: ter/-butoxycarbonyl. HOBt: 1 -hydroxybenzotriazole. NHS: N- hydroxysuccinimide. EtOAc: ethyl acetate.

The structures of the intermediates CPT-linker and the final conjugates were supported by NMR. ¹H NMR, ¹H DOSY, ¹³C NMR, HSQC spectra confirmed the covalent linkage of the drug on the linker and confirmed the ester linkage of the linker on the hydroxyl groups of hyaluronic acid.

NMR spectra were taken on a Varian Inova 500 spectrometer, equipped with a linear gradient along the z axis and on a Varian Mercury 200 spectrometer, in D₂O for HA derivatives and as specified for other intermediates. The determination of the CPT content in conjugates with HA by NMR was achieved by integrating the peaks of CPT versus the peaks of the hyaluronan backbone in the proton NMR spectrum. Hyaluronic acid of MW 20,000 was used as starting material, unless otherwise noted.

The TBA salt of hyaluronic acid was prepared by ion exchange. Briefly, Amberlite IRA-120 resin was treated with excess 20% tetrabutylammonium hydroxide solution for 24h, then it was washed with water. A solution of HA in water (5%) was then gently mixed with the resin for 24h. Filtration, concentration and freeze- drying afforded HA TBA salt with stoichiometric TBA content, as confirmed by proton NMR. Example 1. CPT-20-O-hemisuccinate. To a solution of 2.98g (17.1 mmol) of succinic acid mono-tert-butyl ester and 1.40g (1 1.5mmol) of p-dimethylaminopyridine in 200ml of dichloromethane were added, while stirring at room temperature, 2.68ml (17.3mmol) of diisopropylcarbodiimide and 3.0Og (8.62mmol) of CPT. After stirring overnight, the resulting suspension was diluted with 80ml of dichloromethane to obtain a solution which was washed with 0.1 N HCI solution and dried over anhydrous sodium sulfate. Then it was filtered and evaporated to dryness in a rotary evaporator. The residue was crystallized with 100ml of MeOH, filtered and washed with MeOH. After drying on the filter, the solid was treated with 50ml of a 40% v/v solution of trifluoroacetic acid in dichloromethane and, after 1 h standing at room temperature, the resulting greenish solution was evaporated to dryness in a rotary evaporator. The residue was crystallized with 100ml of MeOH, filtered and washed with MeOH and diethyl ether. After drying in vacuo, 3.67g (8.19mmol, 95%) of a pale yellow solid were obtained. Example 2. HA-O-CO-(CH?)?-CO-20-O-CPT. To a solution of 800mg (1.8mmol) of CPT hemisuccinate from Example 1 in 20ml of DMSO were added, stirring at room temperature under nitrogen, 299mg (2.6mmol) of NHS and 278μl_ (1.8mmol) of DIPC. After 7h, 558mg (0.9mmol) of HA TBA salt were added and the resulting solution was heated at 50°C for 16h. 2ml of saturated NaCI solution were then added and the mixture was stirred for 30min. Then 100ml of EtOH were added under stirring and the mixture was filtered. The solids were washed with DMF and EtOH, then dissolved in 20ml of water and dialysed against water. Freeze-drying afforded 390mg of a white solid. DS in CPT by proton NMR: 13% mol/mol. Example 3. HA-O-CO-(CHp)p-CO-20-O-CPT.

The procedure of Example 2 was repeated, with the exception of the different temperature for the conjugation step, which was room temperature. 5ml were worked up after 24h, affording 90mg of a white solid. DS in CPT by proton NMR: 2% mol/mol.

5ml were worked up after 48h, affording 72mg of a white solid. DS in CPT by proton NMR: 3.5% mol/mol. The remainder of the reaction mixture was worked up after 5 days, affording 57mg of a white solid. DS in CPT by proton NMR: 7% mol/mol.

Example 4. HA-O-CO-(CHp)p-CO-20-O-CPT.

To a solution of 896mg (2.0mmol) of CPT hemisuccinate from Example 1 in 20ml of DMF were added, stirring at room temperature under nitrogen, 345mg (3.0mmol) of NHS and 309μl_ (2.0mmol) of DIPC. After 7h, 620mg (LOmmol) of HA TBA salt were added and the resulting solution was heated at 50°C for 16h. 2ml of saturated NaCI solution were then added and the mixture was stirred for 30min. Then 100ml of EtOH were added under stirring and the mixture was filtered. The solids were washed with DMF and EtOH, then dissolved in 20ml of water and dialysed against water. Freeze-drying afforded 340mg of a white solid. DS in CPT by proton NMR: 18% mol/mol. Example 5. CPT-20-O-CO-CHp-NHBoc.

To a solution of 3.0Og (17.1 mmol) of Boc-Gly-OH and 1.4Og (1 1.4mmol) of 4-dimethylaminopyridine in 100ml of dichloromethane were added 2.68ml (17.1 mmol) of diisopropylcarbodiimide and 2.0Og (5.75mmol) of CPT. After stirring at room temperature overnight, the resulting suspension was diluted with 50ml of dichloromethane, washed with 0.1 N HCI solution, dried over anhydrous sodium sulfate, filtered and evaporated. The residue was crystallized with the minimal amount of methanol, filtered, washed with methanol and dried to give 2.32g (81 %) of a white solid. Example 6. CPT-20-O-CO-CHp-NHp-TFA

1.5Og (2.97mmol) of CPT-20-O-CO-CH₂-NHBoc from Example 5 were dissolved in 100ml of a 40% solution of trifluoroacetic acid in dichloromethane. After 1 h solvents were removed in a rotary evaporator and the residue was crystallized with diethyl ether, filtered, washed with diethyl ether and dried to give 1.5Og (0.289mmol, 97%) of a pale yellow solid. Example 7. CPT-20-O-CO-CHp-NHCO-(CHp)p-COOH.

To a solution of 174mg (17.3mmol) of succinic anhydride, 0.50ml (2.9mmol) of DIEA and 4mg (0.03mmol) of DMAP in 50ml of dichloromethane were added, at room temperature under nitrogen, 750mg (1.44mmol) of CPT-20-O-CO-CH₂- NH₂-TFA from Example 6. After 18h, the solution was diluted with 25ml of dichloromethane, washed with 0.1 N HCI solution, washed with saturated NaHCO₃ solution, dried over anhydrous sodium sulfate, filtered and evaporated. The residue was crystallized with the minimal amount of methanol, filtered, washed with methanol and dried to give 650mg (1.29mmol, 89%) of a white solid. Example 8. HA-OCO-(CHp)p-CONH-Glv-20-O-CPT.

To a solution of 250mg (0.495mmol) of CPT-20-O-CO-CH₂-NHCO-(CH₂)₂-COOH from Example 7 and 86mg (0.75mmol) of Λ/-hydroxysuccinimide in 10ml of dimethylsulfoxide were added, with stirring under nitrogen at room temperature, 77μl_ (0.50mmol) of diisopropylcarbodiimide. After 16h, 310mg (0.50mmol) of HA TBA salt were added, and the resulting solution was heated at 50 °C for 24h. After cooling to room temperature, 1.0ml of saturated NaCI solution were added and stirring was continued for 30min. The mixture was poured into 40ml of EtOH while stirring, the resulting slurry was stirred for 10min and then filtered and washed with EtOH. The solid was suspended in 30ml of dimethylformamide, slurried for 30min, filtered and washed once with dimethylformamide and twice with MeOH. After drying on the filter, the solid was dissolved in 40ml of water and dialysed against water. Then the solution was filtered through a 0.22μ pore size membrane and freeze-dried to give 23Og of a white solid. DS in CPT by proton NMR: 10% mol/mol. Example 9. CPT-20-O-CO-(CHp)p-CONH-Glv-OH.

To a solution of 800mg (1.79mmol) of CPT-20-O-hemisuccinate from Example 1 , 549μl_ (3.94mmol) of triethylamine and 343mg (1.79mmol) of EDC hydrochloride in 80ml of dichloromethane were added 330mg (1.96mmol) of Gly-O-tert-Bu hydrochloride. After stirring at room temperature overnight, the solution was washed with 0.1 N HCI solution, dried over anhydrous sodium sulfate, filtered and evaporated in a rotary evaporator. The residue was crystallized with the minimal amount of methanol, filtered, washed with methanol and dried to give a white solid. This solid was dissolved in 25ml of TFA containing 5% v/v of water. After 1.5h the solvents were removed in a rotary evaporator and the residue was crystallyzed with methanol. Filtration, washing with methanol and drying gave 620mg (1.23mmol, 68%) of an off-white solid. Example 10. CPT-20-O-CO-(CHp)p-CONH-Glv-O-HA To a solution of 250mg (0.495mmol) of CPT-20-O-CO-(CH₂)₂-CONH-Gly-OH from Example 9 and 1 14mg (0.99mmol) of Λ/-hydroxysuccinimide in 7ml of dimethylsulfoxide were added, with stirring under nitrogen at room temperature, 77μl_ (0.50mmol) of diisopropylcarbodiimide. After 16h, 250mg (0.403mmol) of HA TBA salt were added, and the resulting solution was heted at 50 °C for 24h. After cooling to room temperature, 0.7ml of saturated NaCI solution were added and stirring was continued for 30min. The mixture was poured into 30ml of EtOH while stirring, the resulting slurry was stirred for 10min and then filtered and washed with EtOH. The solid was suspended in 25ml of dimethylformamide, slurried for 30min, filtered and washed once with dimethylformamide and twice with MeOH. After drying on the filter, the solid was dissolved in 30ml of water and dialysed against water. Then the solution was filtered through a 0.22μ pore size membrane and freeze-dried to give 145mg of a white solid. DS in CPT by proton NMR: 9% mol/mol.

Example 11. TFA-HpN-Phe-Leu-Glv-20-O-CPT. To a suspension of 348mg (LOOmmol) of CPT in 20ml of DCM were added, stirring at room temperature under nitrogen, 653mg (1.5mmol) of Boc-HN-Phe- Leu-Gly-OH, 158mg (1.3mmol) of DMAP and 309μl_ (2.0mmol) of DIPC. After 16h the resulting solution was washed with 0.1 N HCI solution and with saturated NaHCO₃ solution, then it was dried over anhydrous sodium sulfate, filtered and evaporated to dryness. The solid residue was recrystallized from MeOH/diethyl ether to obtain a solid. This was dissolved in 5ml of a 40% solution of TFA in DCM and stirred for 2h at room temperature. The solvents were removed under reduced pressure and traces of TFA were removed by co-evaporating with small portions of diethyl ether obtaining 501 mg of a solid residue. Example 12. HOOC-(CHp)_?CO-Phe-Leu-Glv-20-O-CPT.

To a solution of 490mg (0.63mmol) of TFA-H₂N-Phe-Leu-Gly-20-O-CPT from Example 1 1 in 10ml of DMF were added, stirring at 0°C under nitrogen, 109mg (0.63mmol) of mono tert-butyl succinate, 85mg (0.63mmol) of HOBt, 216μl_ (1.26mmol) of DIEA and 146mg (0.76mmol) of EDC hydrochloride. The reaction mixture was then allowed to reach room temperature overnight. The solvent was removed under reduced pressure and the residue partitioned between EtOAc and water. The aqueous phase was extracted with EtOAc. The combined organic phases were washed with 10% citric acid, saturated NaHCO₃ solution and brine, then they were dried over anhydrous sodium sulfate, filtered and evaporated to dryness. The residue was dissolved in 5ml of a 40% solution of TFA in DCM. After 2h solvents were removed under reduced pressure and traces of TFA were removed by co-evaporating with small portions of diethyl ether obtaining 489mg of a solid.

Example 13. HA-OCO-(CH?)?-CO-HN-Phe-Leu-Glv-20-O-CPT. To a solution of 489mg (0.63mmol) of HOOC-(CH₂)₂CO-Phe-Leu-Gly-20-O-CPT from Example 12 in 20ml of DMSO were added, stirring at room temperature under nitrogen, 108mg (0.90mmol) of NHS, 154μl_ (0.90mmol) of DIEA and 132mg (0.70mmol) of EDC hydrochloride. After 16h, 390mg (0.63mmol) of HA TBA salt were added and the resulting solution was heated at 50 °C for 24h. After cooling to room temperature, 2ml of saturated NaCI solution were added and the mixture was stirred for 30min. Then 80ml of EtOH were added under stirring and the mixture was filtered. The solids were washed with DMF and EtOH, then dissolved in 15ml of water and dialysed against water. Freeze-drying afforded 220mg of a white solid. DS in CPT by proton NMR: 7% mol/mol. Example 14. HA-O-CO-(CH?)?-CO-20-O-CPT Several conjugates were prepared by scaling-up a process as substantially described in Example 2; in this new set of experiments the conjugation step was carried out at room temperature at different reaction times ranging from about 13 hours to 150 hours thus affording final conjugates having different DS values. The DS values increased with increasing the reaction time. All the reactions were carried out starting from 1 g HA-TBA and they afforded conjugates in amount of about 500 mg (HA-CPT sodium salt). The DS of isolated freeze dried conjugates was: 6%, 8%, 19%, 26% mol/mol (measured by NMR) Example 15. Determination of number of cells positive to the CD44 receptor Standard CD44 hyaluronic acid receptor expression is determined by flow cytometry, using a monoclonal Ab (mouse anti-human) conjugated with fluorescein isotiocyanate (FITC). Cells were recovered from the flasks with trypsin- EDTA solution, washed with PBS and centrifuged at 400 x g for 5 min. Samples (0.5x10⁶ cells each) were loaded and cells were stained with CD44-FITC (2 mg, at a diluition of 1 :10, for 60 min, 4°C in the dark, and washed twice with PBS-NaN₃- BSA). The cells were then fixed with 1 % Paraformaldehyde, for 15 min, washed twice with PBS. Aliquots treated with an irrelevant isotype matched and FITC MoAb, run in parallel, were used to set the gates in the monoparametric histogram to include less than 2% aspecific fluorescence events. All flow cytometric analyses were performed with the a FACScan flow cytometer (Becton Dickinson, San Jose, CA) equipped with an argon laser at 488 nm. At least 10000 events were acquired for each sample. Histograms were analysed with the WinMDI software (Dr. J. Trotter, Scripps Research Institute, La JoIIa, CA, USA).

The table clearly shows that H-460 and HT-29 have the highest cell density of CD44 receptors and that H-460/M2 cell line shows the highest number of CD44 positive cells. Example 16. Antiproliferative activity

Antiproliferative activity of the conjugates of the invention was determined on three lines of carcinoma (HT29: colon rectal carcinoma, H460: lung carcinoma, H460M2: lung metastatic carcinoma) which are sensitive to camptothecin and irinothecan. The original sample of the HT29 and H460 tumour lines was from ATCC and was regularly reconstituted and stored in liquid nitrogen after the second in vitro propagation. The original sample of the H460M2 tumour was from lstituto dei Tumori of Milan, and it was regularly reconstituted and stored in liquid nitrogen after the second in vitro passage. Tumor cells were incubated in 96-well plates for 5 days in complete RPMH 640 Medium (Sigma Chemical Co.) supplemented with 10% FBS fetal bovine serum, 1 % L-glutamine 100x (200 mM) and 1 % penicillin G-streptomycin (Euroclone) at 37°, and in controlled atmosphere (5% CO₂), with irinotecan and DDSs of example 14; the DDS were tested at doses equimolar with those of CPT, in the range of 1 nM and 100 μM. Irinothecan was used as standard controls. Cytotoxicity was determined by the MTT test, by measuring cell viability as the cell metabolic capacity to transform the tetrazolium salt of MTT in the blue formazan, by mitochondrial dehydrogenases; the blue colour was read at 570 nm with a spectrophotometer (Automated Microplate Reader EL31 1 , BIO-TEK Instruments, Vermont, USA).

On day 0, in 96-well plates, 1000cells/well/100μl complete medium were plated. On day 1 , medium was replaced with 100μl/well/complete medium containing the conjugate at the concentrations of 10^"9M; 3x10^"8M; 10^"8M; 3x10^"7M; 10^"7 M; 3x10^"6 M; 10^"6 M; 3x10^"5 M; 10^"5 M; 3x10^"4 M; 10^"4 M. Each concentration was studied in triplicate. Cell viability, as determined by the MTT test was done on day 6, after 5 days treatment. Each well was added with 10μl/1 OOμl complete medium containing MTT (prepared previously by dissolving 5 mg/ml MTT in PBS, sterilized with filters at a cut-off of 0,22nm and stored at 4⁰C). Plates were put in an incubator at 37°C for 4hrs, then medium was eliminated and each well was added with 100μl IGEPAL (HCI 0,01 N + 10% Igepal, Sigma Chemical Co., USA). Plates were read with a spectrophotometer at 570 nm wavelength. IC50, which is the minimal pharmacological concentration to inhibit cell growth by 50% as compared to the untreated controls was calculated.

Antitumour effect of the conjugates of the invention on various tumour cells is reported in the Table 1 ; the effect has been compared to that of Irinothecan. This reference is the CPT analogous compound that is currently used in therapy; CPT is insoluble in aqueous solution and therefore it cannot be used in therapy and for both reasons it is not a suitable reference.

Table 1 shows the values of the concentration (IC50, nM) of the DDS and of Irinothecan necessary to reduce the cell growth of various tumours lines to 50% of the growth of the control. Table 1

These data show that the present conjugate have a cell cytotoxicity that is higher than that of Irinothecan.

Claims

1. Conjugate of camptothecin or its derivatives, with hyaluronic acid, wherein camptothecin is connected to hyaluronic acid through a linker, the latter being connected to any of hydroxyl groups of hyaluronic acid by means of an ester bond.

2. Conjugate according to claim 1 , where the linker is chosen among linear or branched, aliphatic, aromatic or araliphatic C₂-C₂O dicarboxylic acid, aminoacid, peptide, linear or branched, aliphatic, aromatic or araliphatic C₂-C₂₀ dicarboxylic acid linked to aminoacid or to peptide.

3. Conjugate according to anyone of claims 1 -2, where the linker is chosen among succinic acid, succinic acid linked to aminoacid, or succinic acid linked to peptide.

4. Conjugate according to anyone of claims 2-3, wherein the aminoacid is chosen from alanine, valine, leucine, isoleucine, methionine, glycine, serine, cysteine, asparagine, lysine, glutamine, aspartic acid, glutamic acid, proline, histidine, phenylalanine, triptophane and tyrosine.

5. Conjugate according to anyone of claims 2-4, wherein the peptide is a di-, tri- or tetra-peptide, including one or more among alanine, valine, leucine, isoleucine, methionine, glycine, serine, cysteine, asparagine, lysine, glutamine, aspartic acid, glutamic acid, proline, histidine, phenylalanine, triptophane and tyrosine.

6. Conjugate according to anyone of claims 1 -5, wherein the percentage of the hydroxyl groups of hyaluronic acid connected by means of the ester bond with the linker is between 1 % and 50%.

7. Pharmaceutical composition comprising the conjugate of any one of claims 1 - 6, in admixture with pharmaceutically acceptable excipients and/or diluents.

8. Pharmaceutical composition according to claim 7, adapted for oral, parenteral, topical, intraarticular, intravenous, intraperitoneal, intramuscular, or subcutaneous administration.

9. Conjugate according to anyone of claims 1 -6 or the pharmaceutical composition according to anyone of claims 7-8 for use in the treatment or prevention of diseases responsive to camptothecin therapy.

10. Conjugate or composition according to claim 9, wherein the disease is cancer.

1 1. Conjugate according to claim 10, wherein the disease is selected from the group consisting of colorectal cancer, gastroinstestinal cancer, lung cancer, central nervous system cancer, head and neck cancer, ovarian cancer, cervical cancer, breast cancer, pancreatic cancer, lymphoma and melanoma.

12. Process for the preparation of the conjugate of anyone of claims 1 -6, comprising the following steps:

(i) forming a covalent bond between camptothecin or its derivatives and the linker;

(ii) forming an ester bond between the product of (i) and hyaluronic acid, said ester bond involving any of hydroxyl groups of HA and a carboxyl group of the linker.

13. Process according to claim 12, wherein in step (i) the bond between camptothecin and the linker is chosen among ester, ether, carbonate, amine, carbammate, amide, ureide bond.

14. Process according to claim 13, wherein camptothecin is reacted with the linker in a chlorinated solvent, in presence of a catalyst and a coupling agent.

15. Process according to claim 14, wherein the chlorinated solvent is chosen from the group consisting of methylene chloride, chloroform, 1 ,2-dichloroethane, the catalyst is chosen from p-dimethylaminopyridine, 4-pyrrolidinopyridine and 4- tetramethylguanidinopyridine, the coupling agent is chosen from the group consisting of diisopropylcarbodiimide, dicyclohexylcarbodiimide and 1 -(3- dimethylamino-propyl)-3-ethylcarbodiimide, and the reaction is performed in a period of time ranging from 4 to 24 hours, thus obtaining a drug-linker monoester.

16. Process according to anyone of claims 12-15, wherein step (ii) comprises reacting the product of step (i) with an activating agent, in a polar aprotic solvent, in the presence of a coupling agent, and reacting the thus obtained product with hyaluronic acid.

17. Process according to claim 16, wherein the polar aprotic solvent is selected from the group consisting of dimethylsulfoxide, Λ/,Λ/-dimethylformamide, N, N- dimethylacetamide and Λ/-methylpyrrolidone, the activating agent is selected from the group consisting of N-hydroxysuccinimide and 1 -hydroxybenzotriazole; the coupling agent is selected from the group consisting of diisopropylcarbodiimide, dicyclohexylcarbodiimide and 1 -(3-dimethylaminopropyl)-3-ethylcarbodiimide, and the hyaluronic acid is used in the form of tetrabutylammonium or other tetra aryl/alkyl ammonium salts.

18. Process according to anyone of claims 16-17, wherein the reaction with the quaternary ammonium salt of hyaluronic acid is performed at a temperature comprised between 20 and 60°C for a period of time in the range from16 to 120 hours.