Gil del Alcazar and coworkers have described radiosensitization of orthotopic GB by NVP-BEZ235 using the established glioma cell line U87 [
15]. The U87-driven tumors yet poorly mimic the infiltrative and neovascularization growth properties of clinical GB [
17]. Further, the U87 cell line was recently reported to represent a mix-up with another cell line [
18]. In order to investigate the radiosensitization properties of NVP-BEZ235 towards orthotopic GB driven by primary GIC that more faithfully mimic the clinical tumors growth, [
17] we preliminarily investigated the pharmacokinetics of NVP-BEZ235 using HPLC/MS. The relatively low dose of NVP-BEZ235 used in this study (25 mg/Kg i.p.) was chosen due to significant toxicity of higher doses towards our animals (data not shown). Consistently, in two independent animal studies utilizing nude rats and NOD/SCID mice in orthotopic xenograft models of GB, Netland and coworkers have found severe side effects of doses of NVP-BEZ235 higher than 25 mg/ml [
19]. The early termination of recent NVP-BEZ235 clinical trials due to elevated toxicity may confirm that safety dosing of NVP-BEZ235 should be thoroughly investigated prior to use [
20,
21]. Two hours after the i.p. administration of one single 25 mg/Kg body weight dose, NVP-BEZ235 could be detected in the brain of tumor-free mice at concentrations (111 × 10
− 9 M) significantly higher than the IC
50 for ATRi in cells (21 × 10
− 9 M)[
12] (Fig.
1e). Hence, In agreement with previous findings, [
15,
22] NVP-BEZ235 can diffuse to brain tissues at pharmacologically active levels after systemic administration, albeit the presence of the efflux ABC-transporters (in particular ABCG2) at the BBB may in part restrict its brain penetration [
22]. In a preliminary tumor radiosensitization study, we then developed primary adult COMI tumors in NOD SCID mice, whose immunodeficient phenotype is required for reproducible engraftment in virtually 100% of mice (Fig.
2a) [
23]. Unlike orthotopic tumors driven by established glioma cell lines such as U87, GIC-driven orthotopic tumors faithfully recapitulate the infiltrating and stem markers-expressing properties of the clinical tumors (Fig.
2a). In order to explore the radiosensitization properties of NVP-BEZ235, we adopted a low-dose protocol with a 25 mg/Kg NVP-BEZ235 i.p. administration followed four hours later by 0.5 Gy, this combination being repeated for three consecutive days. The low doses of IR (0.5 Gy) employed in the present study four hours after ATRi administration were chosen to limit additional toxic effects to the highly radiosensitive NOD SCID mice. Such low IR doses are actively investigated in both the preclinical and clinical settings for GB histotypes [
24‐
29] and their use may be particularly appropriate under radiosensitizing conditions where the killing effect is amplified. Under the above conditions, no elongation of median survival was observed in mice bearing GIC-driven orthotopic tumors treated with NVP-BEZ235 as compared to mice treated with vehicle. On the contrary, a trend towards reduced median survival (86 versus 99 days, respectively) was observed (Fig.
2b). This is consistent with the results by Netland and coworkers who found no survival benefit or inhibition of tumour growth in orthotopic xenograft models of GB developed in nude rats and NOD/SCID mice [
19]. Accordingly, the simultaneous treatment with NVP-BEZ235 and the MAPK inhibitor AZD6244 did not lead to synergistic radiosensitization of tumor cells, questioning a radiosensitizing effect of both inhibitors [
30]. Albeit the remarkable differences in the animal models and treatment protocols used may explain our failure to reproduce, even partially, the striking radiosensitization results obtained by Gil Del Alcazar and coworkers, our results support the conclusions by Netland and coworkers that the utility in vivo of NVP-BEZ235 is questionable due to toxicity and lack of efficacy towards GB [
19]. The early termination of recent NVP-BEZ235 clinical trials due to elevated toxicity and lack of clinical efficacy further support the conclusions of the preclinical studies [
20,
21].
AZD6738
We also describe for the first time effective brain penetration of AZD6738 using HPLC/MS. AZD6738 reached a concentration of 480 × 10
− 9 M (Fig.
1h) in brain tissues one hour after one single i.p. administration of 25 mg/Kg body weight, indicating that the concentration of AZD6738 achieved in the brain of our experimental animals exceeded the IC
50 for ATR kinase inhibition in cells (74 × 10
− 9 M) [
14]. Albeit AZD6738 at 25 mg/Kg body weight administered per os has been reported to enhance the therapeutic efficacy of cisplatin in xenograft NSCLC models, [
14] we could not observe any survival improvement of animals bearing orthotopic adult COMI GB treated for three consecutive days with i.p.-administered 25 mg/Kg AZD6738 followed four hours later by irradiation to the head with 0.5 Gy.
In conclusion, although the present results indicate favorable pharmacokinetics properties of ATR inhibitors NVP-BEZ235 and AZD6738, they do not at this stage lend support to a radiosensitizing effect towards orthotopic GIC-driven GB.