Pharmacological modulation of circadian rhythms in brain microvasculature

The interface between the blood and brain is vital for the protection of neurological processes. A key regulatory mechanism of the blood-brain border (BBB) is the circadian rhythm, an intrinsic timekeeping system that maintains 24-h rhythms. At the BBB, the endothelial cell clock has been implicated in time-of-day differences in removal of drugs from the brain; however, pharmacologic modulation of the BBB clock remains unexplored. To investigate circadian dynamics in the BBB, we isolated murine brain microvessels (MVs) and used a Color-Switch PER2::iLuc system to longitudinally monitor rhythms. Isolated MVs consisting of endothelial cells and pericytes preserved vascular morphology for 4 days and circadian oscillations were maintained ex vivo under forskolin synchronization. MVs were treated with known small-molecule modulators of the circadian clock including flavonoids; nobiletin, tangeretin, sinensetin; a CLOCK inhibitor CLK8; CRY stabilizer KL001; and REV-ERBα agonist SR9009. Of these, sinensetin increased rhythms and KL001 decreased rhythms consistently in both MVs and a human microvascular cell line. KL001 and sinensetin administration to mice altered circadian gene expression and BBB circadian rhythms of efflux. These findings establish that BBB circadian rhythms are pharmacologically tractable and highlight small-molecule modulators as promising tools to optimize CNS drug delivery using chronopharmacology.