Visualizing the spatial distribution of Alprazolam in the mouse brain following nasal administration using desorption electrospray ionization mass spectrometry imaging

Alprazolam (Alp), a triazolobenzodiazepine, is widely prescribed for the treatment of sleep disorders, anxiety, and panic disorder. While oral administration remains the standard route, its slow onset of action has prompted interest in intranasal delivery as an alternative, offers the potential for direct drug delivery to the brain. This study aims to develop a fast-acting intranasal formulation of Alp (Alp-nd). Safety evaluations were conducted using mucociliary toxicity tests in bullfrogs and ciliary toxicity tests in rats, demonstrating that Alp-nd was safe and did not cause damage to the nasal mucosa. The righting reflex test was employed to assess the dose-response effect of Alp-nd on sleep in mice, revealing a 95 % effective dose (ED₉₅) of 1.81 mg/kg, which is lower than that observed with intragastric administration (Alp-ig). A comparison of the pharmacodynamics, plasma pharmacokinetics, and brain distribution between intranasal and intragastric administration was performed. Sleep phase analysis revealed that Alp-nd significantly shortened sleep latency and prolonged sleep duration compared to Alp-ig. LC-MS/MS analysis indicated that Alp plasma concentrations were significantly higher at 5 and 10 min post-dosing with Alp-nd compared to Alp-ig. Furthermore, desorption electrospray ionization-time of flight mass spectrometry imaging (DESI-MSI) demonstrated that Alp-nd significantly increased Alp content in the olfactory bulb within 2-5 min, and in the cortex, thalamus and hypothalamus within 2-30 min, and in the pineal gland within 5-30 min. These results suggest that the Alp-nd shortens sleep latency via the nose-to-brain pathway and extends sleep duration by increasing Alp concentrations in sleep-related brain regions.