Improvement of pyrazolo[3,4-d]pyrimidines pharmacokinetic properties in cancer therapy: nanosystem and prodrugs approaches

The protein kinases overexpression and iperactivation are presents in several kind of tumor. Pyrazolo[3,4-d]pyrimidines represent a promising class of compounds capable of inhibiting several oncogenic tyrosine kinases, which represent an attractive target for the development of new therapeutic agents against cancer. The anticancer activity of pyrazolo[3,4-d]pyrimidines was confirmed in several cancer cell lines, both from solid tumors (i.e. osteosarcoma SaOS-2, prostate PC3, neuroblastoma SH-SY5Y, glioblastoma U87, rabdomyosarcoma8, mesothelioma, medulloblastoma, medullary thyroid carcinoma and leukemia (i.e. 32D-p210 and 32D-T315I) and Burkitt lymphomas. In vivo data, generated in subcutaneous xenograft mouse models further supported the antitumor activities of several pyrazolo[3,4-d]pyrimidines against leukemia (32D-p210 and 32D-T315I),12 neuroblastoma (SH-SY5Y),4 and glioblastoma (U87). To date, the library of pyrazolo[3,4-d]pyrimidines synthesized by our research group consists of more than 400 members. Despite the promising anticancer activity, these molecules showed a poor aqueous solubility. This issue could threat the future development of pyrazolo[3,4-d]pyrimidines as clinical drug candidates. In this work, with the aim of improving their solubility profile and consequently their pharmacokinetic properties, three different approaches were evaluated. As a first proposal, four pyrazolo[3,4-d]pyrimidines were chosen on the basis of their anti-neuroblastoma activity and were encapsulated in albumin nanoparticles and liposomes. Albumin nanoparticles and liposomes were prepared and characterized regarding size and ζ-potential distribution, polidispersity index, entrapment efficiency and activity against SH-SY5Y human neuroblastoma cell line. The most promising nanosystem was chosen to perform further studies: confocal microscopy, stability and drug release in physiological conditions, and biodistribution. In the second approach the prodrugs strategy was applied. To synthesize the full set of pyrazolo[3,4-d]pyrimidines prodrugs, a versatile one-pot two-step procedure was set up. Next, the plasma half-life, the ability to overcome biological membranes, metabolic stability as well as water solubility were analysed. Then the most interesting prodrugs were tested in a cellular assay with human GBM U87 cell line. As in vivo proof of concept, the pharmacokinetics of the most promising pair of drug and prodrug were studied in an orthotopic mouse model of GBM. Finally, selectively activated prodrugs by a tumor-associated enzymes were developed. This approach allows to generate active parent drug at the target site, as well as increase the solubility of our molecules. Tumor-associated enzymes, overexpressed in the tumoral environment, can be used as a trigger for specific prodrugs activation. Therefore we synthesized a plasmin-activated pyrazolo[3,4-d]pyrimidines prodrugs by the inclusion of a small peptide, selectively recognized by this tumor-associated enzymes. SI113 was the selected compound to develop this approach. Indeed, SI113 showed an interesting activity profile against hepatocellular carcinoma (HCC), where the enzyme plasmin is overexpressed. Also in this case the plasma half-life, the ability to overcome biological membranes, metabolic stability as well as water solubility were analysed. In addition the hydrolysis ability of prodrugs in the presence of the enzyme plasmin was evaluated. Afterwards the synthesized compounds were tested in a cellular assay with hepatocellular carcinoma HepG2 cell line. In vitro cytotoxicity was also determinated by pre-incubation of the cell with the plasmin. Based on obtained results, in vivo experiments in xenograft mouse models of hepatocellular carcinoma and in vivo studies of pharmacokinetics are ongoing.