Antitumoral bioconjugates of hyaluronic acid or its derivatives obtained by indirect chemical conjugation

[0001] Tumor development, growth and progression to primary and secondary metastases are very complex biological processes that require the sequential organization of coordinated (organoselective) cell processes.

[0002] The spread of cancer cells that leads to the formation of metastases occurs as a result of their detachment from the main growth site, with their penetration in the capillary bed and / or lymphatic system.

[0003] Over the past few years, the progressive knowledge of life processes that cause the onset, development, spread and implantation of a tumor and its metastasis, has offered scientists not only the opportunity to study, synthesize and / or experiment with new chemical molecules as new agents with anti-cancer effects, but also facilitated research and improvement of new therapeutic therapies that overcome problems related to the toxicity of anti-cancer drugs, and above all, understanding chemical-biological mechanisms that cause resistance to the above drug.

[0004] One of the main problems associated with cancer treatment actually relates to the possible tumor "resistance" to drug treatment after an initial positive response.

[0005] These "resistances" are associated with biological / biochemical changes in the functioning of the cancer cell, such as, for example:

• changes in cellular drug transport;

• changes in affinity with respect to that from a possible metabolism inhibitor;

• a significant increase in the ability of cells to self-neutralize the medicine.

[0006] Recently published scientific experiments (Misra et al., The Journal of Biological Chemistry, 2003, 278 (28): 25285-25288) have shown how initial / simultaneous in vitro treatment of cancer cells resistant to some chemotherapy drugs, hyaluronic acid oligomers with very low molecular weight, restored the initial sensitivity of cells to the drug. However, the experimental data obtained so far have not completely explained how / why chemotherapy sensitivity has been restored, even if it has been observed that these oligomers can interfere with various intracellular molecular processes responsible for the acquisition of drug resistance and thus tumor growth and diffusion.

[0007] The pharmacological action of the above oligomer becomes possible because, since it binds to the CD-44 receptor (specific for hyaluronic acid), it manages to negatively affect the binding of the native HA receptor, which interaction is responsible for the coordination of numerous cell function and, above all, a cancer cell.

[0008] By its binding (and subsequent internalization) to its receptor present in the cell membrane, HA is in fact involved in the activation of many processes that are essential for cell life, such as regulation of adhesion / growth processes and cell migration, enters the chemotactic mechanism during inflammatory processes, plays a major role in scarring processes and, as mentioned above, in the migration of cancer cells when metastases.

[0009] Many solid tumors have in fact exhibited large amounts of HA, which may consequently facilitate the invasion of cancer cells by other tissues and organs.

[0010] Forms of cancer, such as, for example, cancers, melanomas, lymphomas, breast cancers, colorectal cancers, and lung cancers, overexpress the transmembrane CD-44 receptor: in these cell lines, experiments with anti-receptor antibodies (which, as a result, "block" the receptor, preventing its binding to native HA) have demonstrated an effective ability to inhibit growth and metastasis of tumors, this shows how to "disrupt" HA binding to its receptor causes a disturbance of numerous events of fundamental importance for cell life, and therefore shows the actual involvement of HA in the development of tumor mass.

[0011] It is known that some anti-cancer drugs that have been used for years in the field of oncology with satisfactory clinical results have been chemically modified to:

• overcome the problem of their specific toxicity in order to make a new treatment strategy consisting of directing the cancer drug directly to the cancer cell by binding it to HA as fully described above, in many cancer phenotypes HA specific CD-44 receptor is overexpressed on their cell surface (this is the mechanism of active management, which increases the cellular effectiveness of the drug by reducing its systemic toxicity). The binding and internalization of the polymer also lead the drug to the cancer cell, increasing its effectiveness;

• increase their solubility (binding of fat-soluble drugs to highly hydrophilic molecules such as HA has been shown to significantly increase the solubility of the drug itself in the circulatory system).

[0012] The solubility of capillary bed chemotherapy drugs actually represents an important condition for their pharmacological efficacy, however, some drugs that have been shown to be extremely active for various types of cancers, such as, for example, camptothecin and their irinotecan and topotecan derivatives, paclitaxel and derivatives Vinca alkaloids as a result of their high insolubility they have problems related to intravenous administration (and in the case of hormones and anti-hormones also intramuscularly), which may limit and limit their clinical use.

[0013] For the reasons mentioned above (solubility and toxicity), new chemotherapy drugs have been synthesized that are created as a result of chemical binding (direct or indirect through a spacer consisting of amino acids or peptides with a short chain of amino acids) or a simple combination of some anti-cancer drugs containing a lactone ring (such as, for example, doxorubicin, paclitaxel, vincristine, vinblastine and camptothecin derivatives) with hyaluronic acid (HA) (US Patent 6,291,671).

[0014] Other conjugates include anti-cancer drugs such as paclitaxel and camptothecin bound to a polymer consisting of polyglutamic acid, optionally bound to HA (US Patent No. 5,977,163).

[0015] Other new types of chemotherapy drugs are also known, represented by anti-cancer doxorubicin covalently bound to both HA (chemically modified dihydrazide) and a carrier such as polymeric hydroxypropylmetaacrylamide (International Patent Application WO 02/090390).

[0016] New drug carriers are also known, consisting of chemically conjugated polysaccharides with amino acid chains in turn covalently attached to anti-cancer drugs such as doxorubicin (US Patent No. 5,688,931). In addition, other release systems have been improved for the same reason, e.g. encapsulation of doxorubicin in liposomes containing lipid HA derivatives (Peer D. et al., Neoplasia, 2004, 6 (4): 343-353; Eliaz RE et al., Cancer Research, 2001, 61: 2592-2601).

[0017] It is known, for example, that to overcome the problems associated with camptothecin derivatives, to change their pharmacokinetic profile and reduce their toxicity by increasing their therapeutic efficacy, irinotecan has been coupled to the carboxy methyl dextran polymer / carrier using a spacer represented by triglycin peptide (Satoshi Okuno et al., Cancer Research, 2000, 60: 2988-2995; US Patent 5,892,043).

[0018] The resulting prodrug has proved to be active in its therapeutic efficacy as it remains in circulation for a prolonged period of time increasing its accumulation in tumor mass, while reducing its systemic toxicity; however, for many of the conjugates previously described, final experimental data are not yet available that document its efficacy with respect to the unconjugated drug. In addition, an article by Ouchi T. et al. "Design of polysaccharide-5-fluorouracil conjugates exhibiting antitumor activities" (ACS Symposium Series, American Chemical Society / Oxford University Press, US, vol. 469, August 15, 1991, pages 71-83) discloses hyaluronic acid conjugates to 5-fluorouracil by a molecular spacer that forms an amide bond with the HA carboxyl group. Also EP0506976 discloses conjugates of hyaluronic acid to 5-fluorouracil or cytosine arabinoside via an amide bond with the HA carboxyl group. WO2004 / 035629 discloses conjugates of hyaluronic acid and / or its derivatives to taxol via a molecular spacer that forms an ester or amide bond with the HA carboxyl group and / or its derivative.

[0019] Also known is a derivative of paclitaxel, covalently bound to HA by a previously derivatized hydrazide (US Patent No. 5,874,417), or bound directly to HA or indirectly through a spacer of different nature capable of forming various types of bonds chemicals that increase the solubility and, consequently, the effectiveness of the drug (patent application EP 1560854).

[0020] The present invention describes and claims new HA conjugates obtained from the intermediate binding between polysaccharide and fat-soluble anti-cancer drugs, such as, for example, analogous pyrimidine products, to overcome problems related to their solubility (if present), their toxicity, and primarily to restore and increase the effectiveness of the drug in cancer cells that have acquired pharmacological resistance to the drug itself. The state of the art, represented by the previously described derivatives, is therefore exceeded because the applicant is able to demonstrate the pharmacological superiority of the new conjugates, objects of the present invention, due to the extremely high cytotoxic activity of these derivatives relative to tumor cells.

[0021] This new pharmacological efficacy enables the use in clinical pharmacology of innovative chemotherapy drug therapies for the treatment of primary and / or secondary tumors that no longer respond to any treatment after the formation of Multi Drug Resistance (MDR), which generally threatens the possibility of effective treatment of the patient, and therefore, in the final analysis, drastically shortens his life expectancy.

[0022] By resolving / overcoming MDR, the new derivatives, object of the present invention, change the patient's final prognosis, thereby enabling the solution / reduction of cancer pathology.

DETAILED DESCRIPTION OF THE INVENTION [0023] The present invention describes and claims a new group of conjugates / derivatives and methods for their preparation, consisting of hyaluronic acid (HA) (and / or its derivatives) and anti-cancer drugs indirectly coupled via a molecular bridge called "element spacer ", consisting of an aliphatic, araliphatic, alicyclic or heterocyclic, linear or branched chain, with or without heteroatoms.

[0024] In particular, the present invention relates to chemical-pharmaceutical conjugates of hyaluronic acid and / or its derivatives, obtained by an indirect bond between a polysaccharide and an anti-cancer drug, through a molecular spacer that forms an ester bond with the carboxyl group of HA and / or its derivative , provided that the spacer is not a hydrazide or polypeptide, wherein the drug is selected from antimetabolites, the degree of substitution at the carboxyl group of hyaluronic acid and / or one of its derivatives ranges from 1 to 20%; wherein said hyaluronic acid derivatives are selected from the following

- HA salt-forming with organic and / or inorganic bases with a molecular weight of 50-730KDa or a high molecular weight of 750-1230 KDa;

- HA esters with alcohols from the series of aliphatic, araliphatic, cycloaliphatic, aromatic, cyclic and heterocyclic alcohols, with the percentage of esterification, which may vary depending on the type and length of alcohol used, from 1 to 75%;

- HA amides with amines from the series of aliphatic, araliphatic, cycloaliphatic, aromatic, cyclic and heterocyclic amines with an amidation percentage ranging from 1 to 10%;

- O-sulphated HA derivatives up to the 4th degree of sulphation;

- internal HA esters with a percentage of internal esterification in the range of 0.5 to 10%, preferably 5%;

- deacetylated HA derivatives derived from deacetylation of the N-acetylglucosamine fraction, with a deacetylation percentage preferably ranging from 0.1 and 30%;

- percarboxylated HA derivatives obtained from the oxidation of primary hydroxyl groups of the N-acetylglucosamine fraction with a percarboxylation degree in the range of 0.1 to 100%.

[0025] HA (and / or one of its derivatives) and the drug are therefore indirectly coupled via one or more ester-type covalent bonds that partly or fully include polysaccharide carboxyl groups and chemical functions (e.g. hydroxyl, carboxy, amino , etc.) belonging to a spacer that in turn binds to a cancer drug selected as described in detail below.

[0026] The derivatives that can be prepared according to the present invention have various physico-chemical properties that can be modulated by choosing the type of binding and degree of substitution to improve the initial characteristics of the chemotherapy drug, such as:

• solubility, • mechanical and rheological properties, • resistance to hydrolytic degradation, making the new conjugate more effective in its cytotoxic activity, the derivative of which will have a new mechanism of action, thus overcoming the pharmacological resistance to the drug itself, acquired by the tumor cell ( as described above).

[0027] As is known, many anti-cancer drugs used in chemotherapy have limited, if any, solubility in water or saline solutions; this means that for their administration, it is necessary to refer to organic solvents and oils, which, although introducing the drug into the solution, have a specific toxicity with side effects that require medical intervention before administering the product.

[0028] In some cases, for the chemotherapy drug, Irinotecan, the active form (SN38) is even chemically modified (prodrug) causing it to dissolve and to promote the release of its metabolite, which is active after intravenous administration. However, this is the reason for the low availability of the SN38 metabolite at the target site, and therefore high cytotoxic doses are required, consequently increasing undesirable side effects.

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[0029] International literature (Mathijssen RH et al., Clin Cancer Res, 2001, 7: 21822194) indicates that the anti-tumor activity of SN38 is 100 to 1000 times higher compared to its commercial prodrug; therefore, the possibility of coupling SN38 with hyaluronic acid or one of its derivatives allows obtaining compounds with increased efficiency, and thanks to the necessity of administering smaller doses, with less side effects associated with the dispersion of the drug in areas that are not attacked by cancer. The combination of anti-cancer drugs used in chemotherapy with HA also enables the active substances to be "targeted" to their target site, and thus towards the tumor tissue. Therefore, emphasis is placed on an active mechanism of targeting between the conjugate and the tumor cell, which increases the local concentration of the drug near the tumor area, and consequently the efficacy. In this way, by further reducing the distribution of the derivative to healthy tissues, greater product tolerance with respect to the free drug is guaranteed.

[0030] The second main advantage of the present invention is the possibility, mainly due to the presence of chemically modified HA, to transform the conjugate into a three-dimensional biomaterial (for topical application) processed into various forms, such as, for example, hydrogel, nano- or microspheres or spun fibers rail as fabric or non-woven products; in this case, the chemically modified polysaccharide matrix is ​​in close contact with the tumor mass, acts as a controlled drug release system at the application site, and therefore promotes greater efficacy on the part of the drug itself. After exerting an antitumor effect, the derivative degrades naturally and safely to the body, completely releasing the anticancer active substance and hyaluronic acid. The products generated by the invention, whether in the form of classic pharmaceutical compositions or degradable biomaterials, are therefore characterized by greater tolerance to the unmodified active substance and higher pharmacological activity, in some cases up to several orders of magnitude in relation to that exhibited by the active substance forming it ; both effects can be attributed to the specific affinity of hyaluronic acid for receptors such as CD44 present in cancer cells. These effects are enhanced when the conjugate drug is administered in the form of three-dimensional materials, in direct contact with the cancer. The combination of these features is that the derivatives / conjugates of the present invention clearly outweigh what is available in the art when it comes to the local or systemic treatment of various types of cancers and different origins that have also become resistant to traditional chemotherapy.

[0031] The molecular weight of hyaluronic acid used in the present invention varies from 400 to 3000000 Da, preferably in the range from 5000 to 1000000 Da, and even more preferably from 30,000 to 500,000 Da, it can be of extractive, fermentative or biosynthetic origin. The covalent bond with the spacer involves the carboxyl group of the D-glucuronic acid of the repeating polymer unit, in percent ranging from 1 to 100% (degree of substitution), which is an ester bond with the functional group of the molecular spacer selected, which therefore acts as connection between hyaluronic acid and a chemotherapy drug.

[0032] The spacer consists of an aliphatic, araliphatic, alicyclic or heterocyclic chain, linear or branched containing or not containing heteroatoms, which may include hydroxyl, carboxyl, carbonyl, amino (excluding hydrazides and polypeptides), epoxy groups, acid chlorides, thiols, nitriles, halogens, anhydrides, isocyanates and isothiocyanates; carboxylic acid bromides, iodides and chlorides with a C2 to C10 aliphatic chain are preferred, and in particular bromides such as bromopropionic acid or bromobutyric acid. The degree of substitution is in the range of 1 to 20%.

[0033] HA derivatives that can be used in the new conjugates, an object of the present invention, are listed below:

1. HA salt-forming salts with organic and / or inorganic bases with a molecular weight of 50-730KDa (EP0138572 B1) or a high molecular weight of 7501230 KDa (EP 535200 B1);

2. Hyaff®: HA esters with alcohols from the series of aliphatic, araliphatic, cycloaliphatic, aromatic, cyclic and heterocyclic alcohols, with the percentage of esterification, which may vary depending on the type and length of alcohol used, from 1 to 75%, preferably from 30 to 50 % (EP 216453 B1);

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3. Hyadd^™: HA amides with amines from the series of aliphatic, araliphatic, cycloaliphatic, aromatic, cyclic and heterocyclic amines, with an amidation percentage ranging from 1 to 10%, preferably 4% (EP 1095064 B1);

4. O-sulphated HA derivatives, up to the 4th degree of sulphation (EP 0702699 B1);

5. ACP®: HA internal esters with a percentage of internal esterification ranging from 0.5 to 10%, preferably 5% (EP 0341745 B1);

6. Deacetylated HA derivatives: derived from deacetylation of the N-acetylglucosamine fraction, with a deacetylation percentage preferably ranging from 0.1 and 30%, while all HA carboxyl groups can be converted into a salt with organic and / or inorganic bases (EP 1313772 B1 );

7. Hyoxx ™: percarboxylated HA derivatives, obtained from the oxidation of primary hydroxyl groups of the N-acetylglucosamine fraction with a percarboxylation degree in the range from 0.1 to 100%, preferably from 25 and 75%. All HA carboxyl groups can be salified with organic and / or inorganic bases (patent application EP 1339753).

[0034] Drugs used in the coupling reaction with HA fall into the following category:

• anti-metabolites: such as, for example, analogous folic acid products (including methotrexate), analogous pyrimidine products (including fluorouracil and Ι-β-D-arabinofuranosylcytosine: Ara-C).

[0035] An analogous pyrimidine product such as fluorouracil and Ara-C is particularly suitable for the purposes of the present invention.

[0036] Identified drugs and hyaluronic acid (and / or one of its derivatives) are indirectly bonded via a spacer by forming ester bonds by the following procedures:

1. a functional group of a suitably selected spacer (such as, for example, a carboxyl group, amino group, halide, etc.), also containing a second group (named "leaving group") capable of reacting with the carboxyl functional group of HA (e.g. halide: bromine, iodine or chlorine) reacts with a functional group belonging to the anti-cancer molecule represented, for example, by a hydroxyl, amino, carboxyl or mercaptan group. The reaction may optionally require activation of one of the functional groups involved with an activating agent (e.g. activation of the carboxyl group by means of carbodiimides). In the second stage, by direct contact with a tetraalkylammonium (preferably tetrabutylammonium) HA salt in an anhydrous environment, the compound consisting of the modified drug reacts leading to nucleophilic substitution of the leaving group (e.g. bromide) at the HA carboxyl group, causing the formation of an ester bond between HA and a spacer;

2. the carboxyl group of hyaluronic acid or one of its derivatives is bonded via a nucleophilic bond to a suitable spacer, which is then bonded to the functional group of the anti-cancer molecule (in all ways known to experts in the field);

3. the carboxyl group of HA or one of its derivatives is activated by means of an activating agent, for example carbodiimide, and reacts with the hydroxyl function of the appropriately selected spacer, previously or later bound to the drug (in all ways known to experts in the field);

[0037] Oncological applications relating to the use of conjugates consisting of hyaluronic acid (and / or one of its derivatives) and the anti-cancer active substance are closely related to the tumor response to the conjugate drug. According to the intended applications, bioconjugates can therefore be administered orally, intravenously, intraarterially, intrathecally, intramuscularly, subcutaneously, intraperitoneally, intraarticularly, topically, transdermally, locoregally or in combination (therefore, the procedure of both local and systemic administration has been reserved). Tumors that can be treated include, for example (without limitation) cancers of the pancreas, breast, large intestine and rectum, lungs and respiratory system in toto (whole), head and neck, liver, stomach, testicles, ovary, endometrium, prostate, bladder, brain, leukemia, lymphomas, melanoma, Kaposi's sarcoma, osteosarcoma, neuroblastoma and skin cancer.

[0038] The following are some examples of the preparation of bioconjugates between hyaluronic acid and / or its derivatives and chemotherapy drugs with anti-tumor activity, for illustrative purposes.

Example 1: Preparation of a hyaluronic acid ester derivative with a MW of 440 kDa and 5-fluorouracil with a degree of substitution at the carboxyl group of about 15%.

[0039] 680 mg ethylene carbonate and about 10 mg NaOH were added to 510 mg fluorouracil dissolved in 15 ml DMF. The whole mixture was heated and the reaction left for a further 1 hour at reflux. The product recovered by precipitation was dissolved in anhydrous DMSO / pyridine 50/50 mixture with 1.00 g of ptoluenesulfonyl chloride. After about 15 hours, the product was recovered by precipitation and added to the HATBA solution dissolved in DMSO (3.60 g in 180 ml DMSO). The solution was kept stirring at 38 ° C for about 3 days and finally 20 ml milliQ water and 7 ml saturated NaCl solution were added. The whole mixture was allowed to stir for 1 hour to allow sodium exchange with TBA. Then, ethanol was added dropwise, and the obtained filamentous product was dissolved in water, dialyzed and lyophilized.

Example 2: Preparation of an hyaluronic acid ester derivative with a MW of

200 kDa and Ι-β-D-arabinofuranosylcytosine (Ara-C) with a degree of substitution at the carboxyl group of about 18%.

[0040] 100 mg Ara-C, 80 mg EDC and 69 mg 4-bromobutyric acid were dissolved in 10 ml water. The whole mixture was reacted for about 1 hour, and finally the solvent was stripped off by evaporation under reduced pressure on a rotary evaporator. The product was purified by column chromatography. The intermediate thus obtained was dissolved in a solution of 20 mg / ml 1.10 g HATBA in DMSO and allowed to react for 7 days at room temperature. 5 ml of saturated NaCl solution was added to recover the product, thereby allowing the carboxyl groups of hyaluronic acid to form the sodium salt. The polymer was precipitated by the addition of ethyl alcohol dropwise, and after filtration and redissolution in water, it was dialyzed to eliminate solvent and salt residues, and finally freeze-dried.