Abstract 2201: Preclinical development of novel pyrazolo[3,4-d]pyrimidines structure-based TKIs for the treatment of glioblastoma
Authors: Mancini Arianna , Fallacara Anna Lucia , Zamperini Claudio , Iovenitti Giulia , Molinari Alessio , Rango Enrico , Angelucci Adriano , Schenone Silvia , Botta Maurizio 
Published: August 2019

Abstract
The aim of this study was the preclinical development of a set of pyrazolo[3,4-d]pyrimidine structure-based small molecules ATP-competitive SRC inhibitors for the therapy of glioblastoma (GBM), a rare but extremely aggressive and infiltrative glial tumor with a very limited response to therapies. Ongoing preclinical and clinical studies of tyrosine kinase inhibitors (TKIs) for the therapy of GBM identified SRC family inhibitors as a significant and effective therapeutic strategy (i.e. dasatinib, bosutinib, PP2).
In vitro Screening: From the wide in-house synthesized pyrazolo[3,4-d]pyrimidines’ library, nine compounds have been selected to be formulated and tested in in vitro and in vivo GBM pharmacological models. Among all the examined formulations, the prodrug approach has been applied to the candidate drugs, introducing the N-methylpiperazine moiety by a carbamate-based linker (in vivo enzymatically hydrolysable). Drugs and corresponding prodrugs have been screened on the basis of their SRC Ki and in vitro ADME properties. GBM U87 cell assays showed IC50 in the sub-micromolar range (1.5 - 15.0 µM) for almost all compounds and improved cellular uptake of the prodrugs in respect to the corresponding drugs.
PK and Toxicity: After that, the most promising compound has been selected for further in vitro assays and in vivo tests. In vivo pharmacokinetics and pharmacodynamics have been evaluated for the compound by various formulations, routes of administration and dosages. In all cases investigated, the compound has been proved to reach the brain. As expected, prodrug plasmatic MRT and AUC confirmed the realization of an extended release formulation that allows a higher overall exposure to the drug. In addition, considering an estimated oral bioavailability of 40.4% and a 11-fold higher Cmax in respect to i.v. and i.p. administration, the oral route can be identified as the most advantageous for the compound for GBM treatment. Moreover, an acute toxicity study revealed that the LD50 for i.v. toxicity is more than 100 mg/kg with a good tolerability.
In vivo Efficacy Tests: The selected compound and its prodrug were orally administered in GBM orthotopic mouse model and results indicated a median survival rate 10% greater for prodrug-treated mice in respect to drug-treated mice and 35% greater in respect to control group. The combination effect of the compound and radiotherapy was also investigated in subcutaneous U87 xenografted mice model and resulted in a reduction of the tumor growth close to 80% in respect to the control growth.
Conclusions: Taking into account all the data collected regarding their pharmacological properties, pyrazolo[3,4-d]pyrimidines SRC inhibitors class of compounds deserve further investigation to show its potential to become a new therapy for GBM.
Citation Format: Arianna Mancini, Anna Lucia Fallacara, Claudio Zamperini, Giulia Iovenitti, Alessio Molinari, Enrico Rango, Adriano Angelucci, Silvia Schenone, Maurizio Botta. Preclinical development of novel pyrazolo[3,4-d]pyrimidines structure-based TKIs for the treatment of glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2201.