PRECLINICAL CHARACTERIZATION OF SFK INHIBITORS, PYRAZOLO[3,4-d]PYRIMIDINE SCAFFOLD-BASED DERIVATIVES, FOR CANCER TREATMENT
Creators: name: RANGO, ENRICO
Publisher: name: Universit� degli Studi di Siena
Year: 2021

description: The first part of this thesis essentially focuses on the preclinical characterization of Si306, a pyrazolo[3,4-d]pyrimidine derivative, identified as a very promising anticancer agent. This compound has shown a favorable in vitro and in vivo activity profile against neuroblastoma (NB) and glioblastoma (GBM) models by acting as a competitive inhibitor of c-Src tyrosine kinase. Nevertheless, the good antitumor activity of Si306 is associated with sub-optimal aqueous solubility, which might hinder its further development. In this context, drug delivery systems were developed to overcome the poor aqueous solubility obtaining suitable formulations for their in vivo use in the treatment of NB. Si306 was encapsulated in liposomal nanoparticles: i. Stealth liposomes and ii. Immunoliposomes decorated with AntiGD2 monoclonal antibody, which specifically binds GD2 antigen expressed by NB cells. Both liposomal suspensions resulted stable and showed excellent morphological and physio-chemical properties. The liposomal suspensions exhibited increased cytotoxic activity against different NB cell lines; in particular, AntiGD2-decorated liposomes, due to the interaction with the antigen, showed the ability to bind and be internalized in different NB cells and the cellular association increases proportionally with increasing GD2 antigen expression. The pharmacokinetic (PK) and tissue biodistribution (BD) profiles were evaluated by treating healthy male mice intravenously at two dosages of 5 and 25 mg of Si306/kg of body weight. Higher plasma exposure of Si306 was observed when it is delivered by liposomes compared to Si306 administered in free form. Moreover, an immediate distribution of Si306 followed by a concentration decrease in a time-dependent manner was observed in all organs analyzed. An increased concentration of Si306 was observed in the liver, spleen, and lungs when it is delivered by liposomes. A preliminary PK and BD study on NB orthotopic mouse model demonstrated increased tumor uptake of Si306 when it is encapsulated in liposomes compared to drug-free. Finally, in vivo efficacy and survival studies conducted on orthotopic NB mice models revealed the ability of Si306-loaded immunoliposomes to reduce tumor growth and to significantly increase the survival rate.
In the second section, gold nanoparticles (AuNPs) conjugated to Si306 were developed to improve its solubility, but also to increase its ability to cross the blood-brain barrier and to have a therapeutic action against (GBM). This section describes the design, the preparation, and the characterization of AuNPs conjugated with Si306. AuNPs-Si306 showed a good loading efficacy (65%), optimal stability in polar media and human plasma, and a suitable morphological profile. Antitumoral activity of AuNPs-Si306 was evaluated in in vitro GBM model, also in combination with radiotherapy (RT). Results demonstrated that AuNPs had a basal radio-sensitization ability and that AuNPs-Si306, when used in combination with RT, was more effective in inhibiting tumor cell growth with respect AuNPs and free Si306. 
In the third section, Si409, a pyrazolo[3,4-d]pyrimidine derivative, showed the ability to inhibit key members of SFK involved in Diffuse Large B-cell Lymphoma (DLBCL) and to reduce the proliferation of several B-cell tumor cell lines. Also, low cytotoxic activity of Si409 was measured on healthy human peripheral blood mononuclear cells (PBMCs). Si409 showed to be safe by exhibiting a reduction in hERG current but only at concentrations at least 10-fold higher than the median IC50 value recorded in tumor cell lines. PK studies showed high phase II metabolism caused by glucuronide conjugation, which reduces plasma levels and increases the clearance of Si409. Despite the low plasma levels, the treatment with Si409 in ABC-DLBCL xenograft mouse model revealed a considerable volumetric reduction of the tumor mass suggesting an interesting in vivo pharmacological potency of this compound.