Overall, our study failed to demonstrate that brain interstitial temozolomide concentrations were increased by use of standard dose regadenoson; which we pre-specified as an increase in temozolomide brain concentration by ≥ 50%. Importantly, regadenoson did not alter temozolomide plasma concentrations which could result in changes in temozolomide related efficacy or toxicity. Although these results are consistent with our previous negative imaging study , they are at odds with the preclinical data that demonstrated increased drug delivery with one small dose of regadenoson [16, 19, 24, 36]. This difference in effect has raised further questions regarding BBB differences between mice and humans relating to expression and function of CNS adenosine A2A receptors. Alternatively, activation of A2A receptors and subsequent BBB disruption in the brains of glioblastoma patients may differ from the activation of A2A receptors in the normal brain vasculature. Previous preclinical studies demonstrated regadenoson’s ability to decrease cell–cell adhesion integrity while potentially modifying efflux transporter expression within 0.5–2 h after administration [19, 36, 38]. While we anticipated that regadenoson might increase drug entry across the BBB, drug exit from the CNS could also be facilitated resulting in decreased temozolomide concentrations in brain interstitium. The effect on transport is further compounded by the studies by that demonstrated temozolomide’s ability to bind to the multi-drug resistance protein, P-glycoprotein; which likely plays a significant role in glioblastoma temozolomide resistance . Interestingly, regadenoson has been shown to downregulate P-glycoprotein expression in brain endothelial cells thus increasing CNS drug delivery in in vitro BBB models and non-tumor bearing rodents . Thus, these combined findings add to the plausibility of temozolomide efflux by P-glycoprotein along with inadequate P-glycoprotein inhibition within brain/brain tumor parenchyma by regadenoson, thereby not causing a significant increase in brain interstitial temozolomide concentrations.