Developing a Clinically Relevant Radiosensitizer for Temozolomide-resistant Gliomas - Despite important advances in surgical techniques, imagistic modalities and computer-assisted stereotactic delivery of radiation therapy, the prognosis for patients with glioblastoma (GB) remains grim and has not significantly changed in decades. The Stupp protocol—concurrent temozolomide (TMZ) plus cranial external beam radiotherapy (EBRT) followed by adjuvant TMZ—remains the cornerstone of glioma control for all newly diagnosed patients. This is despite the fact that most GB patients who harbor tumors that express O6- methyguanine methyltransferase (MGMT) or are deficient in mismatch repair (dMMR) fail to respond to this protocol. TMZ has minimal radiosensitization activity in these tumors, which are therefore considered to be TMZ- resistant from the time of diagnosis. In a quest to find a TMZ derivative with better radiosensitization properties, we recently identified a novel chemical entity called NEO212—which is a derivatization of TMZ generated by coupling TMZ to perillyl alcohol, a natural monoterpene. In a series of in vitro experiments conducted with TMZ- resistant GB cell lines, we generated highly quantitative data which demonstrate that NEO212 chemoradiation exhibits superior tumor cell killing efficacy compared to TMZ chemoradiation. Moreover, these outcomes were further confirmed in multiple animal models of TMZ-resistant GB in which the animals received clinically relevant doses of NEO212 chemoradiation or TMZ chemoradiation according to a treatment schedule designed to mimic the Stupp protocol. Critically, the observed gains in therapeutic efficacy after NEO212 chemoradiation in these animal models did not happen at the expense of additional systemic toxicities (especially in the bone marrow). Based on these encouraging preclinical findings, we hypothesize that the superior on-target alkylation power of NEO212, at equivalent dosages with TMZ, allows for the generation of a type of DNA lesion (i.e., N-methylpurine adducts) that is more efficiently converted by radiotherapy into lethal, much harder to repair double-strand breaks (DSB). This could explain why the classical mechanisms of TMZ-resistance in GB are so efficiently circumvented by the NEO212 chemoradiation regimen. To confirm this mechanism of action for NEO212 and to support further the preclinical advancement of NEO212 toward an IND for a phase 2a clinical trial for newly diagnosed GB patients with unmethylated MGMT promoter tumors, we are proposing in this application the following Specific Aims. In Aim 1 we will conduct comparative tumor and organ biodistribution, neuroinflammation, and tissue alkylation studies with clinically relevant doses of NEO212 and TMZ that will be gavaged as pills to tumor-free and tumor-bearing rats. Additional studies will be conducted in vitro in which base-excision repair (BER) intermediates will be carefully quantified in TMZ-resistant GB cell lines incubated with clinically relevant concentrations of either NEO212 or TMZ. In Aim 2 we will complete a multi-dose drug escalation toxicology study with NEO212 chemoradiation in a large animal species (dogs) at Charles River Laboratories.
