The first-line cluster headache medication verapamil alters the circadian period and elicits sex-specific sleep changes in mice

Verapamil is the first-line preventive medication for cluster headache, an excruciating disorder with strong circadian features. Whereas second- and third-line preventives include known circadian modulators, such as melatonin, corticosteroids, and lithium, the circadian effects of verapamil are poorly understood. Here, we characterize the circadian features of verapamil using both in vitro and in vivo models. In Per2::LucSV reporter fibroblasts, treatment with verapamil (0.03–10 µM) showed a dose-dependent period shortening of the reporter rhythm which reached a nadir at 1 µM, and altered core clock gene expression at 10 µM. Mouse wheel-running activity with verapamil (1 mg/mL added to the drinking water) also resulted in significant period shortening and activity reduction in both male and female free-running wild-type C57BL6/J mice. The temporal patterns of activity reduction, however, differ between the two sexes. Importantly, piezo sleep recording revealed sexual dimorphism in the effects of verapamil on sleep timing and bout duration, with more pronounced adverse effects in female mice. We also found altered circadian clock gene expression in the cerebellum, hypothalamus, and trigeminal ganglion of verapamil-treated mice. Verapamil did not affect reporter rhythms in ex vivo suprachiasmatic nucleus (SCN) slices from Per2:Luc reporter mice, perhaps due to the exceptionally tight coupling in the SCN. Thus, verapamil affects both peripheral (trigeminal ganglion) and central (hypothalamus and cerebellum) nervous system structures involved in cluster headache pathophysiology, possibly with network effects instead of isolated SCN effects. These studies suggest that verapamil is a circadian modulator in laboratory models at both molecular and behavioral levels, and sex is an important biological variable for cluster headache medications. These observations highlight the circadian system as a potential convergent target for cluster headache medications with different primary mechanisms of action.

维拉帕米是丛集性头痛（cluster headache）的一线预防用药，丛集性头痛是一种伴有显著昼夜节律特征的剧痛性神经系统疾病。二、三线预防药物多为已知的昼夜节律调节剂，如褪黑素（melatonin）、糖皮质激素（corticosteroids）与锂盐（lithium），但目前学界对维拉帕米的昼夜节律调控效应仍知之甚少。本研究通过体外（in vitro）与体内（in vivo）模型，系统表征了维拉帕米的昼夜节律特性。在Per2::LucSV报告基因成纤维细胞中，0.03~10 µM浓度的维拉帕米处理可使报告基因节律呈现剂量依赖性的周期缩短，该效应在1 µM浓度时达到最低点；10 µM浓度的维拉帕米还可改变核心钟基因（core clock gene）的表达水平。对野生型C57BL6/J小鼠给予饮水给药（1 mg/mL维拉帕米加入饮用水中）的转轮活动实验显示，无论雌雄小鼠，其自由活动节律的周期均显著缩短，活动量亦出现下降。但活动量降低的时间模式存在性别差异。尤为关键的是，压电睡眠记录（piezo sleep recording）结果揭示，维拉帕米对睡眠时相与睡眠片段持续时间的影响存在性别二态性，雌性小鼠的不良反应更为显著。本研究还发现，经维拉帕米处理的小鼠，其小脑（cerebellum）、下丘脑（hypothalamus）与三叉神经节（trigeminal ganglion）内的核心钟基因表达均出现异常改变。维拉帕米对离体视交叉上核（suprachiasmatic nucleus, SCN）脑片的Per2:Luc报告基因节律无明显影响，这可能源于视交叉上核内部极强的神经元耦合特性。综上，维拉帕米可作用于参与丛集性头痛病理生理过程的外周（三叉神经节）与中枢（下丘脑、小脑）神经系统结构，其调控效应可能依赖于神经网络层面的整体作用，而非仅针对视交叉上核的单一效应。本研究表明，在实验模型中，维拉帕米在分子与行为层面均具备昼夜节律调节活性，且性别是影响丛集性头痛治疗药物疗效的重要生物学变量。上述发现提示，昼夜节律系统可作为具有不同核心作用机制的丛集性头痛治疗药物的潜在趋同靶点。