The use of combination tyrosine kinase inhibitors for the treatment of glioblastoma multiforme
Glioblastoma is the most common primary brain tumor which has a median survival of only 14.6 months. The central nervous system localization, heterogeneity of the cells and resistance to treatment constitute a significant challenge to establishing an efficient and durable therapy. Glioblastoma lesions contain a dense network of defective blood vessels which are permeable to high molecular weight constructs such as nanocarriers. Furthermore, glioblastoma lesions are known to depend upon several tyrosine kinases in order to facilitate their development, progression, invasion and treatment resistance. As a consequence, we assessed the cytotoxicity of multiple tyrosine kinase inhibitors (TKIs), singularly or in combination, against a panel of glioblastoma cell lines in vitro. The most promising combination of crizotinib, an inhibitor of Met and ROS1, and dasatinib, an inhibitor of SRC and focal adhesion kinase (FAK), were pursued as the lead compounds. In addition, these TKIs were encapsulated into polystyrene co-maleic acid micelles to improve their pharmacokinetic profiles in vivo by selective accumulation in tissues with fenestrated vasculature. The micelles were characterized based on their loading, charge, size, solubility and release rate. Both formulations were assessed for their ability to alter protein expression profiles, induce cell death and reduce proliferation. The ability of the treatments to prevent migration, invasion and angiogenesis in a panel of glioblastoma cell lines was also assessed. The micelles had favorable physicochemical characteristics, with a size sufficient to promote a prolonged plasma half-life, a near neutral charge to prevent opsonization and a steady release rate. The expression and phosphorylation of the target kinases Met, ROS1, SRC, and FAK were consistently reduced by the combination treatment. The combination treatment also reduced the expression of EGFR despite neither TKI targeting this receptor. The inhibition of these targets and subsequent suppression of downstream AKT activity induced apoptotic cell death in glioblastoma cells. The cell death following removal of the drug after a 72 h treatment period, defined as washout, was assessed to determine the ability of the cells to escape from treatment-induced cell death. Following washout, the apoptosis was maintained for 72 h following the removal of the drugs in the free but not the micellar formulations. The induction of cell death correlated with the induction of polyploidy in the free but not micellar formulations. This polyploidy was not causative of cell death but was associated with genetic instability. Both free and micellar formulations showed effective prevention of invasion, migration and angiogenesis in vitro. This study demonstrated the efficacy of the combination of crizotinib and dasatinib in a panel of glioblastoma cell lines. This combination strongly induced apoptotic cell death. Furthermore, the combination also effectively combated several treatment resistance pathways such as invasion and vascular mimicry, potentially augmenting the efficacy of the current therapies. The micellar formulations show comparable efficacy to the free formulations but also provide the potential for the improvement of in vivo pharmacokinetic parameters. In conclusion, the combination of crizotinib and dasatinib shows significant promise in vitro and warrants further investigation in vivo for the treatment of glioblastoma.
Advisor: Greish, Khaled; Rosengren, Rhonda; Taurin, Sebastien
Degree Name: Doctor of Philosophy
Degree Discipline: Pharmacology and Toxicology
Publisher: University of Otago
Keywords: Glioblastoma; Dasatinib; Crizotinib; Micelles; combination; tyrosine kinase inhibitors
Research Type: Thesis