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

Thomas Harman; Michael Udoh; Dan L McElroy; Lyndsey L Anderson; Richard C Kevin; Samuel D Banister; Adam Ametovski; Jack Markham; Chris Bladen; Peter T Doohan; Quentin Greba; Robert B Laprairie; Terrance P Snutch; Iain S McGregor; John G Howland; Jonathon C Arnold

doi:10.3389/fphys.2023.1086243

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

Front Physiol. 2023 Apr 4:14:1086243. doi: 10.3389/fphys.2023.1086243. eCollection 2023.

Authors

Thomas Harman^{1

2}, Michael Udoh^{1

2}, Dan L McElroy³, Lyndsey L Anderson^{1

2}, Richard C Kevin^{1

2}, Samuel D Banister^{1

4}, Adam Ametovski^{1

4}, Jack Markham^{1

4}, Chris Bladen⁵, Peter T Doohan^{1

2}, Quentin Greba³, Robert B Laprairie⁶, Terrance P Snutch⁷, Iain S McGregor^{1

8}, John G Howland³, Jonathon C Arnold^{1

2}

Affiliations

¹ The Lambert Initiative for Cannabinoid Therapeutics, Brain and Mind Centre, The University of Sydney, Sydney, NSW, Australia.
² Discipline of Pharmacology, Sydney Pharmacy School, Faculty of Medicine and Health, The University of Sydney, Sydney, NSW, Australia.
³ Department of Anatomy, Physiology and Pharmacology, University of Saskatchewan, Saskatoon, SK, Canada.
⁴ School of Chemistry, Faculty of Science, The University of Sydney, Sydney, NSW, Australia.
⁵ Department of Biomedical Sciences, Macquarie University, Sydney, NSW, Australia.
⁶ College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon, SK, Canada.
⁷ Michael Smith Laboratories and Djavad Mowafaghian Centre for Brain Health, University of British Columbia, Vancouver, BC, Canada.
⁸ School of Psychology, Faculty of Science, The University of Sydney, Sydney, NSW, Australia.

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.

Grants and funding

This research was supported by the Lambert Initiative for Cannabinoid Therapeutics, a philanthropically funded centre for medicinal cannabis research at the University of Sydney, and the Australian National Health and Medical Research Council (NHMRC GNT1161571 to JA, SB, and IM). This work was completed in part with funding to RL from Canadian Institutes of Health Research (CIHR) Project Grant (201909) and GlaxoSmithKline-CIHR Partnership Grant (201704). The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication. All authors declare no other competing interests.