Pharmacokinetic Study With R-hirduidin In Rabbits By Means Of Bioassay

Aim:To develop a bioassay for determination of rabbit plasmaconcentration of r-hirudin (rH) newly developed in China, and therebyfurther study of Pharmacokinetics (PK) thereof. Methods:The rH plasma level was measured ex vivo by means ofantithrombin activity. The thrombin time (TT) prolongation was used asan antithrombin activity index. TT was measured in a coagulometer aftersuccessive addition of 50 μl pH 7.4 Tris-HCL buffer and 50 μl 5u / mlthrombin solution to 50 μl plasma. The concentration of rH in plasma wasread from the calibration curve relating Log TT prolongation rate to plasmaconcentration of rH. An appropriate dilution with plasma was inevitable ifrH level in plasma samples was beyond high limit of linear range.Thevalidation of the method including linearity,precision and recovery wasconducted at high ,middle and low concentrations by routineprocedures(n=5) as described in guiding principles of PK study issued bySFDA. The stability test was carried out by determing concentration ofstandard plasma sample of rH at different time post-preparation.25 rabbitswere randomly divided into 5 groups(each group containing 5animals).Group1,2,3 received iv administration of rH 4.0,2.0 and 1.0mg/krespectively;Group 4 was given sc rH 2.0mg/kg;Group 5 was subjected toligation of bilaterial renal arteries rendering animals to be of acute severefailaure of renal function and then received iv administration of rH2.0mg/kg.Blood samples were taken from ear maginal vein at pre-dosingand different times post-dosing. Results:The calibration curve constructed by logTT prolongation-rHconcentration plot had a good linearity over the range from 0.12 μg/ml to0.60 μg/ml (r= 0.999). The bioassay showed a good precision of <9.1 %(intra-day RSD) and <11.5%( inter-day RSD) as well as an high methodrecovery of 97.5 – 99.2 % at QC samples and dilution recovery of 92.4 ~94.8 % at 2~100- fold dilution The limit of quantification was 0.12 μg /ml.The PK behaviours of rH in rabbits after iv and sc administration could bedescribed by two-compartment model for iv administration andone-compartment model of extravascular administration,respectively,withthe first-order kinetics.The PK parameters obtained demonstrated a rapidelimination of rH from blood of rabbits with t1/2β being 58.4~59.0 min,anda non-wide distribution limited to extra-ceclular liquid with Vd of 0.09~0.12 L/kg.AUC was found to be proportional to rH doses, and t1/2,CL didnot change siganificantly with doses indicating the PK of rH in rabbits inthe range of doses utilized was in accordance with linear kinetics.Thebioavailability of rH after sc administration reached 94%.The rabbits withacute severe failure of renal functions presents a as much as 11 fold longert1/2 and 13-fold greater AUC than normal healthy rabbits(7084.4 min vs59.0 min,and 553.6 μg /ml×min vs 7084.4 μg /ml×min). Conclusion:The method developed by us is validated to fully meetsrequirements for rH PK study in rabbits. rH PK corresponds to thetwo-compartment model after iv dosing and the one-compartment modelafter sc dosing with the first-order kinetics.It eliminates from body rapidlywith t1/2β of near 1h ,and distributes only in extracellular space andabsorbed well from sc sites.Exeretion of rH by kidneys plays veryimportant role in the elimination of rH from the body.