Abstracts

Rationale: Cannabidiol (CBD) oral solution (Epidiolex®) is approved in the US and other countries for the treatment of seizures associated with tuberous sclerosis complex (TSC), but the mechanism of drug action remains to be fully elucidated. The aim of this research was to better understand the drug action of CBD in TSC model systems.

Methods: The potential anti-seizure and anti-tumor efficacy of purified CBD (Jazz Pharma. Research UK Ltd) was assessed in two in vivo models of TSC; (1) the Tsc1^GFAPCKO model of epilepsy, where mice were dosed i.p. for 46 days with CBD 50 or 100 mg/kg or rapamycin 3 mg/kg to assess the effects on spontaneous seizure via electroencephalogram (EEG) and (2) female BALB/c nude mice injected with Tsc2^-/- cystadenoma 105K cells, where mice were dosed i.p. for 28 days with CBD 5, 50 or 100 mg/kg, rapamycin 0.03, 0.3 or 3 mg/kg, or their combination to assess the effects on tumor volume using calipers.
In vitro, 1-10 µM CBD, 50 nM rapamycin (an mTORC1 inhibitor), CBD+rapamycin and 30 µM C188-9 (a signal transducer and activator of transcription 3 [STAT3] inhibitor), were tested in TSC cell line models (Tsc2^-/- mouse embryonic fibroblasts and TSC2^-/- patient derived angiomyolipomas 621-101 cells, with their respective wild-type controls). To examine drug action, assays assessed colony formation, cell migration/invasion, western blotting of target proteins, vascular mimicry, gene expression (RNA sequencing), and transcription of hypoxia inducible factor 1a (HIF-1a) and STAT3.


Results: CBD reduced spontaneous seizures in the TSC murine model. CBD, rapamycin and their combination repressed tumor growth in vitro and in vivo. In vitro, treatment with CBD, rapamycin or their combination repressed wound healing and cell migration/invasion. Under hypoxia, treatment with CBD, rapamycin or their combination reduced HIF-1a protein levels and activity. In several assays CBD had an inhibitory effect when rapamycin had none. For instance, transcription activity and phosphorylation of STAT3 were markedly reduced with CBD but were unaltered with rapamycin. Under hypoxic conditions, CBD potently blocked angiogenesis in a manner consistent with that of a STAT3 inhibitor, while rapamycin had no effect. In CBD-treated TSC2-deficient 621-101 cells, significant changes were observed in the gene interaction networks associated with cell cycle control, inflammation, and mitochondrial activity.

Conclusions: This study reveals that CBD treatment inhibits spontaneous seizures, tumor growth and modulates STAT3/HIF-1a signaling in an mTORC1-independent manner in TSC model systems, showing differentiation from the mechanisms through which rapamycin works. Evidence that CBD can effectively block STAT3 and HIF-1a in TSC2-deficient cells warrants further research, as both STAT3 and HIF-1a are drug targets in neuroinflammation and cancer.

Funding: Jazz Pharmaceuticals Research UK Ltd.