A modified serial blood sampling technique and utility of dried-blood spot technique in estimation of blood concentration: application in mouse pharmacokinetics

摘要

Pharmacokinetic (PK) studies in mice usually require discrete and parallel blood sampling owing to a restriction on the volume of blood that can be withdrawn. This results in dosing large number of animals and generating composite PK profile. To reduce the number of animals and generate individual animal PK profiles, we developed a serial sampling technique via tail vein bleeding in mice, in which only 20–30 μL blood was withdrawn per time point. Due to the small blood volume, a dried-blood spot (DBS) technique was applied for sample processing. The utility of this technique was demonstrated using three test compounds (amodiaquine, chloroquine and chlorthalidone), with varying degrees of blood-to-plasma partition ratios. The PK studies were carried out in male Balb/c mouse weighing 25–30 g. The compounds were administered intravenously via the saphenous vein. Blood was collected by composite (retro-orbital plexus) or serial (tail vein bleeding) sampling techniques at different time points. Blood samples were processed as blood lysate or DBS. Blood or plasma samples were analyzed by sensitive and rapid UPLC-MS/MS methods. The blood concentrations (both from blood lysate and DBS) obtained from serial sampling matched with those from composite sampling. The ratio of blood AUC to plasma AUC correlated well with the in vitro blood-to-plasma partition ratio of the compounds. The systemic clearance and volume of distribution at steady state calculated from blood or plasma AUCs were in proportion to the respective AUCs. Our results indicated that the serial sampling technique would reduce the number of animals and also compound usage, as well as improve the quality of pharmacokinetic data. Also, the serial sampling technique does not require the use of anesthesia and allows estimation of inter-animal variability in PK. A small volume serial sampling is possible due to the availability of the DBS technique.