Abstracts

Rationale: Given the diverse and complex nature of the epilepsies, the search for new pharmaco-therapeutic options is an urgent necessity. Our goal was to evaluate the effects of a single-dose of HUF-101, a synthetic cannabidiol analogue, on behavioral, electroencephalographic and histochemical parameters in experimental models of epilepsy.  Methods: (Protocol 1) Adult male Wistar Audiogenic Rats (WAR) were submitted to a chronic protocol of acoustic stimulation (2 per day/10 days; Audiogenic Kindling - AuK). All rats developed tonic-clonic (brainstem) and limbic seizures. The day after AuK, thirty minutes before the test stimulus, animals which showed severe and moderate seizures were treated with HUF-101 (10 mg/kg; i.p; N= 6) or vehicle (N= 5), respectively. (Protocol 2) Adult male Wistar-Old rats were treated with either vehicle (n= 6) or HUF-101 (n= 6; 10 mg/kg; i.p), 30 minutes before intra-hippocampal microinjection of pilocarpine (PILO) (1.2 μg/1μL) to induce Status Epilepticus (SE). SE was abolished 90 minutes after its EEGraphic onset with diazepam (5 mg/kg; i.p.). Animals were sacrificed 24 h after the SE and brains were processed for morphological analysis of necrotic neurodegeneration, using Fluorojade C (FJC) histochemistry. (Protocol 3) Adult male Wistar-Old rats were submitted to amygdala rapid kindling (ARK), which consisted in 10 daily electrical stimuli, during 2 consecutive days. In the third day rats were injected with vehicle (N= 7) or HUF-101 (10 mg/kg; i.p, N= 7). Thirty minutes after the treatment animals were exposed to the test stimulus. Behavioral responses were classified according to the Racine/Pinel & Rovner scales and behavioral sequences were analyzed second-to-second (neuroethology analysis). EEGraphic data were acquired from Piriform Cortex, Hippocampal Formation and Amygdaloid Complex (CEUA: 27/2019).  Results: A single dose of HUF-101(10 mg/kg ip) after AuK in WARs had no significant effects on latencies for wild running/tonic-clonic seizures, as well as on seizures severity. In the PILO model, however, HUF-101 promoted neuroprotection indicated by qualitative reduction in FJC+ cells in CA3 and CA1 hippocampal neurons, but not in the hilus in animals treated with HUF-101, compared to vehicle-treated rats. Lastly, ARK animals treated with HUF-101 had a reduction of the limbic seizures severity at the test stimulus compared to vehicle-treated rats (p<0.05, Mann Whitney test). Additionally, neuroethological analysis confirmed the suppressive effect of HUF-101 in limbic seizures, given the reduced expression of limbic behaviors. Spectral EEG analysis confirmed the HUF-101 effect on limbic seizures, with reduction of epileptogenic activity, demonstrated by comparisons of amplitudes of EEG signal, signal duration, over the distribution of the frequencies (with presence of lower frequencies) at the last ARK stimulus, when compared to the same period of the test stimulus.  Conclusions: Our data, with combined behavioral, EEGraphic and histochemical analyses, indicate promising HUF-101 anticonvulsant and neuroprotective effects experimental models of epilepsy.  Funding: This study was supported by FAPESP (2014/50891-1), CNPq/FAPESP INCT Translational Medicine, CAPES: PROEX-Physiology and PROEX-Neurology, FAEPA.