gms | German Medical Science

Objective: The activity of single-agent targeted molecular therapies in glioblastoma multiforme (GBM) has to date been limited. Dysregulation of the PI3K/AKT/mTor pathway has been implicated in GBM and agents targeting this pathway, including the tyrosine kinase inhibitor axitinib (AXI) and the rapalog everolimus (EVE), have shown promising results in vitro and in rodent models. Still, it is unclear how these drugs differentially affect tumor growth and whether they act synergistically. This study explored the in vivo effectiveness of these drugs alone and in combination in an orthotopic mouse model of GBM and undertook in vitro characterization to elucidate drug synergy.

Methods: Human glioma LN229 cells were stereotactically injected into female NSG mice brains (n=41) and tumor growth was assessed with 3T MRI. After two weeks, mice were randomized into one of the following treatment arms: AXI (25mg/kg/d), EVE (5mg/kg/d), AXI+EVE, DMSO, ethanol (EtOH) or DMSO+EtOH with ongoing behavioural scoring for 2 weeks, at which point tumor growth was assessed by MRI. Mice were then sacrificed and tumors histopathologically analysed. To examine agent synergy in vitro, MTT viability assays were performed on immortalized LN229 cells treated with a range of drug and vehicle concentrations (alone or in combination, 0-1600μg/ml) at 24h and 48h time-points. Hill equation dose-effect curves were plotted and combination indices (CI) calculated based on Loewe additivity.

Results: AXI and EVE mono-therapy did not significantly reduce tumor growth in inoculated mice in comparison with vehicle-treated controls. Moreover, the treatment combination of AXI+EVE was no more effective in reducing tumor volume than the control combination DMSO+EtOH. However, AXI and EVE monotherapies reduced evidence of neurological dysfunction vs. control. In vitro CI analysis demonstrated that AXI+EVE acted synergistically at both 24h and 48h time-points (CI<1). Although the DMSO+EtOH combination showed antagonism at 24h (CI>1), by 48h strong synergistic activity was demonstrated (CI<1).

Conclusion: The suboptimal in vivo effectiveness of AXI and EVE mono- and dual-therapies led us to perform an in vitro confirmatory study that demonstrated strong synergistic activity of both combination AXI+EVE and control DMSO+EtOH therapy at the extended 48h timepoint. The ability of solvents to modulate drug effectiveness and the potential of drug and control synergy to change over a longer time-course must be borne in mind when designing in vitro studies.