Abstract
Background: Chronic insomnia is associated with elevated cardiovascular disease risk, and current therapeutic options for this condition remain inadequate. Melatonin (MT) combined with cannabidiol (CBD) may exert synergistic effects on improving sleep; the underlying pharmacological drug-drug interactions (DDI) and interspecies differences in their combined actions remain unknown. Purpose: This study aimed to evaluate the pharmacokinetic characteristics of combined drug formulations by utilizing DDI-based approaches so as to underpin the efficacy and safety of the formulation. Methods: Overexpressing hPEPT1 in MDCK cells, multiple species liver microsomes, equilibrium dialysis, and a static DDI model were employed to assess CBD's effects on MT's cellular uptake, inhibitory effect, enzymatic phenotype, protein binding, and human AUC changes. Results: CBD significantly increased MT exposure in dogs but caused dose-dependent biphasic changes in rats. MT negligibly affected CBD PK. In vitro, CBD inhibited MT metabolism with species differences: potent competitive inhibition in dogs (IC(50) = 3.42 ± 1.30 μM), weaker inhibition in rats/humans (IC(50) = 13.54 ± 1.15/16.47 ± 4.23 μM). CBD also demonstrated mechanism-based inhibition (K(I) = 25.63 μM, K(inact) = 0.063 min(-1)) against human CYP1A2-mediated MT metabolism. Acidic conditions revealed that CBD inhibited PEPT1-mediated MT uptake. CBD exhibits high and MT moderate protein binding. Static model predictions aligned with in vivo dog/rat data project a worst-case human MT AUC increase up to 12-fold. Conclusions: This study identifies the critical role of PEPT1 in MT absorption and elucidates the dual mechanisms of CBD; namely, absorption inhibition and metabolic delay in regulating MT pharmacokinetics, which exhibits interspecies differences.