MEPIRAPIM-derived synthetic cannabinoids inhibit T-type calcium channels with divergent effects on seizures in rodent models of epilepsy

Abstract

Background: T-type Ca²⁺ channels (Ca_v3) represent emerging therapeutic targets for a range of neurological disorders, including epilepsy and pain. To aid the development and optimisation of new therapeutics, there is a need to identify novel chemical entities which act at these ion channels. A number of synthetic cannabinoid receptor agonists (SCRAs) have been found to exhibit activity at T-type channels, suggesting that cannabinoids may provide convenient chemical scaffolds on which to design novel Ca_v3 inhibitors. However, activity at cannabinoid type 1 (CB₁) receptors can be problematic because of central and peripheral toxicities associated with potent SCRAs. The putative SCRA MEPIRAPIM and its analogues were recently identified as Ca_v3 inhibitors with only minimal activity at CB₁ receptors, opening the possibility that this scaffold may be exploited to develop novel, selective Ca_v3 inhibitors. Here we present the pharmacological characterisation of SB2193 and SB2193F, two novel Ca_v3 inhibitors derived from MEPIRAPIM. Methods: The potency of SB2193 and SB2193F was evaluated in vitro using a fluorometric Ca²⁺ flux assay and confirmed using whole-cell patch-clamp electrophysiology. In silico docking to the cryo-EM structure of Ca_v3.1 was also performed to elucidate structural insights into T-type channel inhibition. Next, in vivo pharmacokinetic parameters in mouse brain and plasma were determined using liquid chromatography-mass spectroscopy. Finally, anticonvulsant activity was assayed in established genetic and electrically-induced rodent seizure models. Results: Both MEPIRAPIM derivatives produced potent inhibition of Ca_v3 channels and were brain penetrant, with SB2193 exhibiting a brain/plasma ratio of 2.7. SB2193 was further examined in mouse seizure models where it acutely protected against 6 Hz-induced seizures. However, SB2193 did not reduce spontaneous seizures in the Scn1a ^+/- mouse model of Dravet syndrome, nor absence seizures in the Genetic Absence Epilepsy Rat from Strasbourg (GAERS). Surprisingly, SB2193 appeared to increase the incidence and duration of spike-and-wave discharges in GAERS animals over a 4 h recording period. Conclusion: These results show that MEPIRAPIM analogues provide novel chemical scaffolds to advance Ca_v3 inhibitors against certain seizure types.

Keywords: 6 Hz; Cav3 channels; Dravet syndrome; GAERS; cannabinoid; epilepsy; mepirapim.

FIGURE 1

Chemical structures of MEPIRAPIM and its analogues.

FIGURE 2

MEPIRAPIM analogues are potent inhibitors of Ca_v3 channels. Concentration-response curves of SB2193 (A), SB2193F (B), and NNC 55-036 (C) at Ca_v3 channel subtypes (n = 5–8). Calcium response was measured in the presence of varying concentrations of each compound as measured using a fluorometric imaging plate-reader (FLIPR) calcium flux assay and expressed as a percentage of response to vehicle. Data are expressed as mean ± SEM and curves represent fit to a four-parameter log function.

FIGURE 3

SB2193 inhibited Ca_v3.1 in whole-cell patch-clamp electrophysiology experiments (n = 7–8). Data are expressed as mean ± SEM and curves represent fit to a four-parameter log function.

FIGURE 4

Calculated binding mode of SB2193 (orange) and SB2193F (green) at Ca_v3.1 (PDB: 6KZP) (Zhao et al., 2019) using GlideXP Docking (Friesner et al., 2006) (top). 2D Ligand Interaction Diagram depicting the summarizing the interactions between SB2193F and the Ca_v3.1 binding site (bottom).

FIGURE 5

Pharmacokinetic analysis of MEPIRAPIM analogues in mouse plasma and brain samples. Concentration-time curves for SB2193 in mouse plasma (A) and brain (B) and SB2193F in mouse plasma (C) and brain (D) following 10 mg/kg i.p. injections. Data are expressed as mean ± SEM with n = 4 per timepoint.

FIGURE 6

SB2193 treatment in Scn1a ^+/− mice. Spontaneous generalised tonic-clonic seizure (GTCS) frequency of individual untreated and SB2193-treated Scn1a ^+/− mice (A). Treatment was administered orally through supplementation in chow (5,000 mg/kg chow) from postnatal day 18 and spontaneous GTCSs were quantified over 48 h from postnatal day 23–24. Sub-chronic SB2193 treatment had no effect on spontaneous seizure frequency (p = 0.67, Mann-Whitney U test, n = 17–19). Survival curves comparing untreated and SB2193-treated Scn1a ^+/− mice (B). SB2193 treatment had no effect on survival of Scn1a ^+/− mice to P30 (p = 0.09, Mantel-Cox log rank).

FIGURE 7

SB2193 treatment in Swiss mice administered 6 Hz corneal stimulation. Percentage of Swiss mice protected from seizure in the 6 Hz seizure model following acute treatment with SB2193 or valproic acid (n = 10 per group). Drugs were administered via intraperitoneal injection. SB2193 (100 mg/kg) increased the percentage of mice protected (χ² = 6.33, p = 0.01) as did valproic acid (300 mg/kg) (χ² = 15.54, p < 0.001) (*p < 0.05, ****p ≤ 0.0001).

FIGURE 8

SB2193 increased spike and wave discharge (SWD) incidence and duration in GAERS (males: n = 9; females: n = 5). SB2193 treatments (Veh, 10, 30, 100 mg/kg) were administered (i.p.) ∼20 min prior to EEG recordings (4 h). SWDs are reported as the normalized percentage change from baseline (A, C, E, G) and raw data sampled over the 4 h recording window (B, D, F, H). Representative EEG traces show 30 min samples from a single rat following the four treatments; insets depict zoomed-in 2 min traces (I). SWD event durations were assessed semi-autonomously using Matlab (script = “EEG Seizure Analyzer”) and confirmed by an experimenter based on pre-established criteria (7–12 Hz; burst activity > 3x baseline for at least >0.5 m). (J) Panels show a Matlab-produced SWD event (top left) alongside an interictal event (top right) and events are highlighted within an expanded 2 min sample trace (bottom). Compared to vehicle, 30 and 100 mg/kg SB2193 increased SWD incidence (A). SB2193 dose-dependently increased SWD total duration (C) and 100 mg/kg SB2193 increased average duration (E). No significant effect of treatment on SWD average frequency was observed (G). Similarly, raw data analyses revealed an increase in SWD incidence at 30 mg/kg, total duration at 30 and 100 mg/kg (D) and average duration at 100 mg/kg (F). Values are reported as mean ± SEM and significance is denoted as p = 0.05*, 0.01** or 0.001***. Males and females were pooled for these analyses; however, sex has been differentiated in the figures for clarity (circles, males; squares, females). BL: baseline; Veh: vehicle; Avg: average. See Results for details of statistical analyses.