In vivo pharmacokinetics in mice
A bolus of 30 mg/kg of test compounds were administered via oral gavage to isoflurane-anesthetized control mice. Tissue samples, including muscle, brain, and serum, were collected at 2-, 4-, 6-, and 18-h post-administration, from three mice at each time point. Collected tissues were promptly preserved at -80ºC until further processing. Frozen muscle and brain samples were pulverized in liquid nitrogen using a steel mortar immersed in dry ice. Approximately 100 mg of pulverized tissue and 100 µL of serum were then incubated with a 1% formic acid-acetonitrile solution, with equal volumes of tissue mass for muscle and brain, and three times for serum. Following a 90-s ultrasound bath for muscle and a 10-s bath for serum and brain samples, the extracts underwent centrifugation at 9600 g and 4ºC for 5 minutes. The resulting supernatants were collected and stored at -80ºC until further quantification. Quantitative determination of compounds in the biological samples was carried out using liquid chromatography-tandem mass spectrometry (LC-MS/MS) at the Research General Services SGIker of UPV/EHU (Vitoria-Gasteiz). The pharmacokinetic parameters following oral administration were calculated using the PKSolver add-in program . Noncompartmental analysis was employed to compute the pharmacokinetic parameters of the parent compound. The terminal slope was automatically estimated using regression with the largest adjusted R2. The parameters inferred included the terminal half-life (t1/2), maximum concentration (Cmax), and the time taken to reach the maximum concentration (Tmax). Subsequently, the brain-to-serum (Cb:Cs) was calculated based on the obtained pharmacokinetic parameters .