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
.