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PURPOSE Despite significant advances in multimodal approaches for the treatment of glioblastoma (GBM), the outcome for the patients remains dismal. Previous research from our lab has shown that aromatase (CYP19A1), an enzyme that catalyzes in situ production of estrogens, is markedly elevated in GBM tissues. Additionally, letrozole (LTZ), an aromatase inhibitor with extensive record of efficacious use in breast cancer, exhibited significant activity against GBM in preclinical studies. The purpose of this study was to investigate the mechanism(s) of LTZ activity in GBM. METHODS Patient-derived GBM cells with varying aromatase expression and other genotypic/phenotypic characteristics were employed including aromatase expressing G43, G75, G76, BT-142-gfp-luc and aromatase-null G80 cells. The effects of LTZ on cell viability, neurosphere growth, DNA damage (assessed by measuring phosphorylated histone product ỴH2A.X using flow cytometry) and induction of apoptosis (caspase3/7 activity) were assessed. The effects of exogenous addition of estradiol (E2) was also examined. RESULTS LTZ treatment (72 hours) caused a marked reduction in viability and growth of all GBM cells (IC50 ranging from 0.08 to 0.7 µM) except in G80 cells where LTZ had no effects. LTZ treatment also resulted in inhibition of E2 synthesis in a time and dose-dependent fashion. Furthermore, in aromatase expressing cells LTZ treatment induced apoptosis as measured by increase in Caspase-3/7 activity and triggered significant DNA double strand break as determined by ỴH2A.X formation. The addition of exogenous E2 (250 pg/ml) abrogated the effects of LTZ on cell viability, DNA damage and induction of apoptosis. CONCLUSIONS Our study suggests that the cytotoxic effects of LTZ against GBM are mediated, at least partially, by the inhibition of aromatase activity which results in estrogen deprivation. These mechanistic insights are important to facilitate repurposing LTZ as a novel anti-GBM drug.

PURPOSE AMLAL-101 is a novel agent which preferentially targets α3, α5 subtypes of ɣ-amino butyric acid receptors and shows anti-tumor activity against disparate cancer types. AMLAL-101 is being advanced as an ‘add-on’ to potentiate treatment of primary and metastatic brain cancers. However, AMLAL-101 must penetrate the blood-brain barrier (BBB) and show sufficient brain retention. The primary purpose of this study was to determine the plasma pharmacokinetics (PK) and quantitative estimate of the BBB permeability of AMLAL-101. METHODS We performed intracranial microdialysis, employing jugular vein cannulated Sprague-Dawley rats which facilitated simultaneous serial blood and brain extracellular fluid (ECF) sampling. AMLAL-101 was injected i.p. at 5 mg/kg and serial blood and brain ECF samples collected up to 10 h post-dosing. Plasma and ECF samples were analyzed by LC/MS-MS and plasma and ECF concentration vs time PK profiles determined. In vivo recovery analysis was performed using retrodialysis and rapid equilibrium dialysis employed to determine the extent of protein binding. RESULTS AMLAL-101 plasma protein binding was 85% and in vivo recovery from ECF was 25%. AMLAL-101 peak concentration (Cmax) in plasma and brain ECF were 15 µM and 13.8 µM, respectively. The plasma and brain ECF area under the concentration (AUC0-10) were 27.5 h.µg/mL and 24.10 h.µg/mL, respectively. The brain partitioning of unbound AMLAL-101 (Kp,uu; determined either as a ratio of brain ECF Cmax:unbound plasma Cmax or brain ECF AUC: unbound plasma AUC), were 6.13 and 4.13, respectively. The elimination half-life of AMLAL-101 was 3 h for both brain ECF and plasma. CONCLUSIONS These results suggest that AMLAL-101 has the requisite BBB permeability required for brain cancer therapeutics. AMLAL-101 shows significant brain retention when compared to a chemically similar agent that does not show anti-cancer activity, which may contribute to efficacy of AMLAL-101 as an anti-tumor agent for treatment of brain cancers.