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 mu M) showed a dose-dependent period shortening of the reporter rhythm which reached a nadir at 1 mu M, and altered core clock gene expression at 10 mu M. 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.