Abstracts

TSC is a rare genetic disorder caused by mutations in either the TSC1 or TSC2 genes, leading to the loss of function and increase activity of the mechanistic target of rapamycin (mTOR). Approximately 85% of those affected with TSC exhibit epilepsy, which is either not treatable or only partially treated by current anti-epileptic drugs. To address this, preclinical mouse models of TSC-specific epilepsy have been developed to further translational drug development. In the Tsc1^flox/flox; GFAP-Cre⁺ conditional knockout (Tsc1 CKO), Tsc1 is knocked out in glial fibrillary acidic protein (GFAP)-expressing cells, including astrocytes and some neurons. The standard comparator is rapamycin at 3 mg/kg as the positive control. In this study, we sought to define the dose relationship of two clinical mTOR inhibitors (rapamycin and everolimus) on seizure reduction and mortality. A better understanding of this relationship will facilitate the interpretation of the effects of novel compounds in this model.

Tsc1 CKO mice were bred and genotyped at PsychoGenics (Paramus, NJ) using breeding pairs derived from the Wong Laboratory (Washington University, St. Louis). Tsc1 CKO mice were separated into seven treatment groups [rapamycin (R-5000, LC Labs) and everolimus (RAD001, MCE) at 0.03, 0.3, and 1 mg/kg, and a vehicle control (1.6% ETOH, 5% DMSO, 5% PS80, 88.4% H2O)]. All groups were balanced across sex. Mice were implanted between P20-21, and QD dosing began at P21. Ten days of EEG (P45-P54) were analyzed for generalized tonic-clonic seizure frequency and duration. Seizures were detected manually by scorers blinded to treatment condition. Weekly body weights and survival data were collected to the end of study and tissue was collected at P57 for bioanalyses. Whole brains from n=3-4 per treatment group were used to determine protein levels of p70S6K and S6 as well as other relevant biomarkers. Drug levels were examined by LC/MS in plasma and brain two hours post final dose.

Both test articles significantly reduced seizure frequency compared to the vehicle group (rapamycin and everolimus at 0.3 and 1 mg/kg). A dose-related decrease in mortality was also found for each test article. Decreased seizure duration was associated with decreased seizure frequency, but changes in duration were hard to statistically assess at the high doses due to the general lack of seizures. Brain and plasma drug concentrations were proportionately dose-related across compartments. Total protein levels of S6 and p70S6K remained unchanged across treatment groups, indicating that the effects were due to phosphorylation inhibition. Total 4E-BP1 levels varied in response to treatment, suggesting that prolonged mTOR inhibition may influence 4E-BP1 expression and stability.