Brugia Flavia, Ivanov Konstantin, Aroviita Auni, Giniatullina Raisa, Lehtonen Marko, Malm Tarja, Savinainen Juha, Giniatullin Rashid, Della Pietra Adriana
A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, Finland.
Institute of Biomedicine, University of Eastern Finland, Kuopio, Finland.
J Headache Pain. 2025 Apr 23;26(1):85. doi: 10.1186/s10194-025-02030-2.
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.
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.
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.
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.
偏头痛是一种常见的神经血管疾病，目前仍有半数患者未得到治疗。三分之一的偏头痛患者会经历先兆，这与皮层扩散性抑制（CSD）的发生有关，CSD是一种涉及神经元和神经胶质细胞的去极化波。大麻素已被证明是一类有前景的治疗偏头痛疼痛的化合物。在本研究中，我们提出了一种新策略，即通过使用AKU - 005多组分增强内源性大麻素系统（ECS），以对抗CSD及其下游事件的发展，同时靶向几种关键的内源性大麻素水解酶。为此，我们分析了选择性内源性大麻素水解酶的活性及其被AKU - 005抑制的情况，并在离体皮层切片模型中分析了AKU - 005对CSD发展的影响。
通过对表达小鼠和人类单酰甘油脂肪酶（MAGL）和α/β-水解酶结构域蛋白6（ABHD6）的HEK293细胞裂解物进行甘油测定，评估AKU - 005的抑制谱。对Wistar大鼠和C57 BL/6 J - OlaHsd小鼠的皮层切片进行离体处理后，通过质谱（LC - MS/MS）对内源性大麻素进行定量，并通过基于活性的蛋白质谱分析（ABPP）测量水解酶MAGL、脂肪酸酰胺水解酶（FAAH）和ABHD6的活性。通过实时钙成像研究AKU - 005对皮层切片中离体CSD波的影响。
在离体实验中，AKU - 005抑制了MAGL、FAAH和ABHD6，在基础状态和CSD条件下均增加了大鼠皮层中2 - 花生四烯酸甘油酯（2 - AG）和花生四烯乙醇胺（AEA）的水平。在小鼠中，AKU - 005的作用较温和，仅在CSD条件下抑制MAGL，并在基础状态和CSD状态下均增加2 - AG水平。体外分析证实了大鼠的离体实验结果，并揭示了对小鼠皮层中基础MAGL的抑制作用。AKU - 005先前被报道为双MAGL/FAAH抑制剂，它也抑制过表达的小鼠和人类ABHD6，ABHD6是一种在脑中研究较少的2 - AG水解酶。与这些结果一致，AKU - 005减少了两种啮齿动物物种皮层切片中的CSD事件，在大鼠中效果更高。
鉴于在与偏头痛相关的脑区中，大鼠和小鼠的内源性大麻素水解酶活性存在明显差异，AKU - 005可能靶向多种内源性大麻素水解酶，作为伴有先兆偏头痛的一种有效治疗选择。
