Accuracy of repeated blood sampling in rats: a new technique applied in pharmacokinetic/pharmacodynamic studies of the interaction between warfarin and co-enzyme Q10.

In the past, combined pharmacokinetic/pharmacodynamic (PK/PD) modeling studies of oral anticoagulants in rats have been hampered by the technical problems of blood sampling. In the present study, a semi-micromethod of preparing serial plasma samples for accurate assessment of the prothrombin times (PT) and clotting factor VII activity (CFA) in rats is proposed. The method consists of orbital bleeding, blood sample weighing, gravity calculation and buffer volume adjustment. No significant differences of CFA (percentage normal) were found between citrate-diluted and undiluted plasma. This technique was employed to examine the possibility of PK/PD interaction between warfarin and Co-enzyme Q10 (CoQ10). Rats were given a single oral dose (1.5 mg/kg) of warfarin either alone or on day 4 of an 8-day oral dosing regimen of 10 mg/kg CoQ10 daily. Serial plasma and serum samples, which were subjected to respectively measurements of the anticoagulant effects and concentrations of warfarin and its main metabolites, were obtained over a 96-h period following warfarin administration. All rats survived the whole experiment and maintained a stable condition except for a marked hematocrit decrease. CoQ10 significantly augmented warfarin metabolism but showed little effect on the absorption of warfarin. CoQ10 alone had no apparent effect on either the PT or CFA. The concomitant administration of CoQ10 and warfarin does not significantly affect the anticoagulant effect of warfarin. In conclusion, the proposed serial orbital bleeding technique in rats to prepare an accurate citrate-diluted plasma for PT and CFA measurement is rapid and reliable.