Development of liposome-based drug delivery system to improve drug-like properties and anticancer activity of tyrosine kinase inhibitors with pyrazolo [3,4-d] pyrimidine structure.

Cancer is a multifactorial disease whose causes are still unknown. Frequently, mutations in several genes play an important role in cellular growth processes and, based on their specific roles, they are usually distinguished in proto-oncogenes and oncosuppressors. One of the most studied oncogenes is c-Src, encoding for a non-receptor tyrosine kinase protein that plays a multitude of roles in cell signalling. The activation of c-Src is involved in the control of many functions, including cell adhesion, growth, movement, and differentiation by a different set of cell surface receptors. c-Src is found to be over- expressed and mutationally activated in a wide variety of human cancers. Moreover, c- Src may have an influence on the development of the metastatic phenotype. Significant progresses in the understanding of cancer biology have prompted extensive research within novel classes of anticancer drugs. A number of tyrosine kinase inhibitors targeting the c-Src tyrosine kinase (as well as related tyrosine kinases) have been developed for therapeutic use. 4-aminopyrazolo [3,4-d] pyrimidines compounds were firstly used as c-Src family kinases (SFKs) inhibitors in 1996 but later demonstrated to inhibit all SFKs members with IC50 values in the low nanomolar range. These compounds have been extensively used for studying the biological pathways of SFKs and, even though they are a class of promising anti-tumor compounds because of their good activity against several cancer cell lines, these molecules showed a poor aqueous solubility, limiting them as clinical drug candidates. In the continuing effort to find new anticancer agents, our group conducted large studies on a series of new c-Src inhibitors with a pyrazolo [3,4-d] pyrimidine scaffold. The aim of this thesis was to improve the solubility profile and the pharmacokinetic properties of selected compounds, chosen on the base of their inhibitory activity on c- Src. To overcome the poor water solubility of these molecules, pyrazolo [3,4-d] pyrimidines compounds were encapsulated in liposomes formulations. Liposomes were prepared and characterized for size, ζ potential distribution and polydispersity index (PDI) by dynamic light scattering in order to obtain a homogeneous population of small unilamellar vesicles (SUV); subsequently, fluorescent liposomes were prepared to assess the ability of liposomes to interact with neuroblastoma (SH-SY5Y) and glioblastoma (U87) cells by confocal microscopy; finally, encapsulation efficiency and activity by cellular assays were studied to determine cytotoxicity, showing that liposomes loaded with pyrazolo [3,4-d] pyrimidine compounds determine a cytotoxic effect in SH-SY5Y (Neuroblastoma) or U87 (Glioblastoma) cell line equal to or higher than the free compound solubilised in DMSO. Moreover, biodistribution of liposomes loaded with pyrazolo [3,4-d] pyrimidine was evaluated in male Sprague Dawley rats after 24h of treatment. Altogether, the obtained data strongly indicate that the encapsulation of pyrazolo [3,4-d] pyrimidine compounds in liposomes represent an effective method to improve the biodistribution of pyrazolo [3,4-d] pyrimidines and attribute a therapeutic efficacy to this novel formulation. During my Ph.D. period, I also focused on the study of active targeting of liposomes against tumor areas that overexpress plasmin. The plasminogen/plasmin system plays a key role in tumor development and, in particular, in progression and metastasis. The aim of my project was to functionalize the surface of stealth liposomes encapsulating pyrazolo [3,4-d] pyrimidines with a peptide able to bind plasmin. Since plasmin is overexpressed in tumor areas, the bond between plasmin and the peptide in liposomes surface would lead to a destabilization of the liposomes and consequently to a greater drug release in the tumor site. The obtained liposomes were characterized after purification evaluating the size, the ζ potential distribution, the polydispersity index (PDI) and the encapsulation efficiency (EE). Furthermore, several tests to quantify the peptide bound to liposomes were performed, unfortunately with poor results. Moreover, peptide-bearing fluorescent liposomes were also prepared and tested on hepatocarcinoma cell line HepG2 to investigate any change in the cellular uptake. In addition, cytotoxicity assays on HepG2 cells were performed to test the efficacy of liposomes conjugated with the peptide with respect to non-conjugated liposomes, both encapsulating the selected compounds. Liposomes functionalized with the peptide resulted more stable and with better physicochemical properties than non-functionalized liposomes. Furthermore, confocal microscopy of fluorescent liposomes highlighted the ability of liposomes with the peptide to penetrate in hepatocarcinoma cells after 1h of treatment with respect to non-functionalized liposomes, not able to be internalized after the same time. Finally, cytotoxicity data showed that liposomes with the peptide have lower IC50 values than non-functionalized liposomes. In conclusion, data obtained showed that it was obtained a good functionalized liposomal formulation able to be recognized by hepatocarcinoma cells secreting plasmin. Further in vivo tests will be performed to study the ability of these liposomes to release the compound in tumor areas over-expressing plasmin.