Inhibition of endocannabinoid hydrolases MAGL, FAAH and ABHD6 by AKU-005 reduces ex vivo cortical spreading depression.

BACKGROUND: Migraine is a common neurovascular disorder that remains currently untreated in half of the patients. One third of migraine patients experience aura, which is associated with the development of cortical spreading depolarization (CSD), a wave of depolarization involving neurons and glial cells. Cannabinoids have proven to be a promising class of compounds for the treatment of migraine pain. In this study, we are proposing a new strategy to counteract development of CSD and downstream events via multicomponent enhancement of the endocannabinoid system (ECS) by using a AKU-005, to simultaneously target several key endocannabinoids hydrolases. To this end, we profiled the activity of selective endocannabinoid hydrolases and their inhibition by AKU-005 and analyzed the effect of AKU-005 on the development of CSD in an ex vivo cortical slice model.

METHODS: The inhibitory profile of AKU-005 was evaluated by a glycerol assay of lysates from HEK293 cells expressing mouse and human MAGL and ABHD6. After ex vivo treatment of cortex slices of Wistar rats and C57 BL/6 J-OlaHsd mice, endocannabinoids were quantified by mass spectrometry (LC-MS/MS), and activity of the hydrolases MAGL, FAAH, and ABHD6 were measured by activity-based protein profiling (ABPP). The effect of AKU-005 on ex vivo CSD wave in cortical slices was studied by live calcium imaging.

RESULTS: Ex vivo, AKU-005 inhibited MAGL, FAAH, and ABHD6, increasing 2-arachidonoylglycerol (2-AG) and anandamide (AEA) levels in rat cortex under both basal and CSD conditions. In mice, AKU-005 showed a milder effect, inhibiting MAGL only under CSD conditions and increasing 2-AG levels in both basal and CSD states. In vitro analyses confirmed the ex vivo findings for rats and revealed basal MAGL inhibition in mice cortex. AKU-005, previously reported as a double MAGL/FAAH-inhibitor, also inhibited overexpressed mouse and human ABHD6, a little studied 2-AG-hydrolyzing enzyme in brain. In line with these results, AKU-005 reduced CSD events in cortical slices from both rodent species, with higher efficacy in rats.

CONCLUSIONS: Given the distinct profile of endocannabinoids hydrolases activities between rats and mice in the brain areas associated with migraine, AKU-005 may target multiple endocannabinoid hydrolases to serve as an efficient treatment option for migraine with aura.