Novel potent doxorubicin derivatives able to self nanoassembly

Doxorubicin is an essential component of first-line chemotherapy in the treatment of a variety of solid and hematopoietic tumors. It plays a central role in the treatment of breast cancers, sarcomas, lymphomas, and leukemias. The therapy-limiting toxicity for this drug is cardiomyopathy, which may lead to congestive heart failure and death. Chemical conjugation of doxorubicin with squalene –a natural acyclic isoprenoid- and nanoparticles preparation could be an innovative formulation that could determine a different biodistribution of the nanosystem, supposing to target the drug to the tumor site by EPR effect, and so limiting cardiotoxicity and toxic effects in other tissues. The novel approach of squalenoyl derivatives active prodrugs, able to nanoassembly in structures, have been demonstrated both for hydrophilic compounds, such as gemcitabine, and also for hydrophobic compounds, such as paclitaxel. In this context two derivatives were synthesized, covalently linking the drug directly with 1,1’,2-tris-nor-squalenoic acid, forming an ester linkage, and by an hydrazone linkage with 1,1’,2-tris-nor-squalenhydrazide. The compounds were then fully characterized and then formulated as nanoparticles, by simple solvent displacement method and without any surfactant addition. Nanoparticles suspensions were characterized showing a mean diameter of (221.9±11) nm (polydispersity index 0.164) and a positive Z potential (+35.51) due to the protonated amino group on the sugar moiety. CAC for SQDOXO HCl was found to be around 10-4 M, corresponding to 0.96 mg/L. Furthermore the stability of both the suspension and the prodrugs were evaluated in different media (basic and acidic conditions and in serum) monitoring the size of nanoparticles and the doxorubicin release by HPLC analysis. After physico-chemical evaluations was selected, for in vivo studies, only the more promising compound composed by a direct ester between hydroxyl group of doxorubicin and 1,1′,2-tris-nor-squalenoic acid. Several in vitro tests were performed on pancreatic ca. cell lines ((MIA PaCa 2, PANC 1, BxPc3) to evaluate IC50 toxicity, cell migration and cell apoptosis. In vitro assays revealed relevant anticancer activity of SQDOXO HCl in comparison with free doxorubicin but more important data emerged from the in vivo evaluations. These studies were performed on M109 (murine lung ca), MiaPaca2 and Panc1 (human pancreatic adenocarcinomas) in CD2F1 bearing mice, after evaluation of the Maximum total tolerated dose. After 20 days from implant of M109, MiaPaca2 (30 days) and Panc1 (50 days), by comparison with free doxorubicin, Tumor growth inhibition (%) at maximum total tolerated dose on mice CD2F1 was as follows. Compound MiaPaca2 Panc1 M109 DOXO 29 4 3 SQDOXO HCl 95 82 90 Moreover, the cardiotoxicity studies (Troponin T levels in serum) showed that SQDOXO NAs didn’t show serious cardiotoxic effects in mice, in contrast to free doxorubicin. In conclusion we have demonstrated that squalenoylation process of doxorubicin offers several pharmacological benefits and, hence, is expected to provide a positive therapeutic advantage in cancer therapy.