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—i.e., concurrent temozolomide (TMZ) plus conformal
external beam radiotherapy (EBRT) followed by adjuvant TMZ—remains the cornerstone of glioma control for
all newly diagnosed patients, despite the fact that most tumors with active O6-methyguanine methyltransferase
(MGMT) gene promoters fail to respond to this protocol while those with silenced MGMT promoters do respond
initially but are prone to develop rapid resistance. One key issue with TMZ is the very minimal radiosensitization
activity shown by this drug in the MGMT-proficient setting. In a quest to find a TMZ derivative with better
radiosensitizer characteristics, we recently identified a novel chemical entity called NEO212—a derivatization of
TMZ generated by coupling TMZ to perillyl alcohol, a natural monoterpene. In a series of high-content/high-
throughput in vitro experiments, we generated highly quantitative data which demonstrate that, when compared
to TMZ at clinically relevant concentrations for GB, NEO212 exhibits a much-improved tumor cell killing efficacy
owing to its superior radiosensitization profile. Critically, the same outcome with NEO212 can be observed not
only with TMZ-sensitive GB cell lines, but also with isogenic variants of these that were further modified to
express high levels of MGMT. Moreover, we showed that the improved radiosensitization activity of NEO212 is
entirely depended on its alkylation capacity. Based on subsequent gas chromatography/mass spectrometry
(GC/MS) and in house-developed high-performance liquid chromatography (HPLC) analyses conducted on
isogenic pairs of GBM cell lines, we have also found that the tumor cell uptake of NEO212 is significantly better
than that of TMZ at equimolar concentrations. Importantly, because our data indicate that NEO212 behaves like
a prodrug of TMZ (i.e., once taken up by tumor cells, NEO212 releases intact TMZ), the enhanced
radiosensitization activity of NEO212 can ultimately be attributed to its superior tumor cell uptake resulting in
more extensive DNA alkylation. Based on these preliminary in vitro findings, we hypothesize that the superior
alkylation power of NEO212 at clinically relevant concentrations for GB will also allow for better synergisms with
EBRT thus leading to widespread in vivo double-strand breaks and irreparable DNA damage irrespective of
MGMT status. To further validate the activity of NEO212 in vivo, we propose the following Specific Aims. In Aim
1 we will conduct biodistribution, neuro-PK, and tissue alkylation studies with NEO212 administered by oral
gavage to glioma-bearing animals. In Aim 2 we will investigate the efficacy and neurotoxicity of NEO212 when
administered concurrently with EBRT in xenograft and syngeneic models of MGMT-expressing GB.Glioblastoma (GB), the most prevalent form of brain cancer in adults, is a devastating disease with very high
mortality rates. The current therapy for GB patients—brain surgery for tumor removal followed by a therapeutic
protocol called Stupp which combines chemotherapy and radiotherapy—offers only modest survival gains for
these patients. The main factor responsible for this outcome is the rapid development of resistance by GB tumors
to the chemotherapeutic component of the Stupp protocol. The studies proposed in this STTR Phase I are
focused on the development of a novel drug—called NEO212—which could potentially be used to circumvent
the mechanisms of drug resistance in GB when administered concurrently with radiotherapy.