| Objective: To develop a bioassay for determination of r-hirudin (rH) in urine and bile of rabbits and thereby study urinary and biliary excretion and pharmacokinetics (PK) of rH in rabbits so as to provide experimental data for its development and clinical application.Methods: rH urine and bile concentrations were measured by thrombin time (TT) method based on ex vivo antithrombin activity of rH. The validation of developed method including linearity, precision and recovery was carried out at high, middle and low concentration by routine procedures (n=5). Method recovery was obtained by found concentration divided by added concentration. Dilution recovery (%) was calculated by the formula: determined concentration (Cd) of diluted samples×diluted ratio / added concentration×100.In order to inspect the stability of samples, the urine and bile standard solutions were prepared at high and low concentrations and stored at room temperature and–20℃and examined at different time with respect to their concentrations. Fifteen male rabbits, which were randomly divided into high, middle and low dose groups, were anesthetized with iv urethane (25%-4ml/kg bw) and then were subjected to the surgical opening of abdominal cavity and cannulation of bilateral ureters and bile common duct with plastic tube for collection of samples. Physiological saline was infused into ear marginal vein at a constant rate of 0.5ml/min to maintain stable urinary flow. rH (1.0mg/ml) was iv given to 3 groups of rabbits via ear marginal vein at doses of 2.0,1.0 and 0.5 mg/kg, respectively. Samples of urine and bile were collected from respective catheter prior to dosing and different time (10, 20, 30, 40, 60, 90,120,180,240,360min) postdosing. Meanwhile, the citrated blood was taken from ear marginal vein prior to dosing and 5, 15, 25, 35, 50, 75, 105, 150, 210,300 min ( middle point time of urine collection intervals) and then centrifuged at 3000r/min for 15 min to yield plasma samples. Sigma-minus method was used for urinary pharmacokinetic analysis. rH plasma concentration was determined according to the bioassay reported before by us. Creatinine concentration in urine and plasma were assayed with reference to Jaffe's picrate method and used to calculated CLCR and thereby estimate GFR.Results: Log prolongation rate was found to be linearly related to rH concentration in urine and bile over the range of 0.49～2.50μg/ml (r>0.995). The validation of methodology showed high precision and recovery. rH was undetectable in bile sample. The cumulative excretion of rH in urine accounted for about 60% of dose. The main pharmacokinetic parameters were t1/2 58.08～63.97min, MRT 66.59～75.37min, CLR 1.67～1.78ml/min/kg (urinary PK study) and t1/2 59.76～62.71min, MRT 70.41～71.39min, CLt 2.80～2.98ml/min/kg (plasma PK study), CLCR 3.18～4.52ml/min/kg, as well as renal reabsorption rate (Ra) 47.20～58.61%.Conclusion: The urinary excretion is the main elimination route of rH in rabbits with no rH biliary excretion being found. The t1/2,MRT determined using urinary data are in good agreement with those using plasma data, and all indicate a rapid elimination of rH in rabbits. Both plasma and urine data demonstrate that rH follows linear pharmacokinetics. It is estimated that approximately 40% of rH dosed is cleared through non-renal and non-biliary routes. Since CLR is smaller than CLCR, it is reasoned that rH excretes into urine by both renal glomerular filtration and tubular reabsorption. |
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