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  • Title: Pharmacokinetics study of recombinant hirudin in the plasma of rats using chromogenic substrate, ELISA, and radioisotope assays.
    Author: Jiang SY, Jiao J, Zhang TT, Xu YP.
    Journal: PLoS One; 2013; 8(6):e64336. PubMed ID: 23785400.
    Abstract:
    AIM: To compare the analytical methods used to study the pharmacokinetics of recombinant hirudin in the plasma of rats that had been injected with (125)I-recombinant hirudin. METHODS: 2.0 mg/kg (125)I-recombinant hirudin were injected intravenously into rats. The recombinant hirudins in the plasma was analyzed by chromogenic substrate assay, enzyme-linked immunosorbent assay (ELISA), total radioisotope assay (RA) and trichloroacetic acid pre-treated total radioisotope assay (TCA-RA). RESULTS: The chromogenic substrate assay standard curve was linear over the concentration range from 3.12 to 40.00 ng/ml for the recombinant hirudin in plasma. The relative standard deviations (RSD) for the intra- and inter-day variation were 5.0 to 6.3% and 11.9 to 12.6%, respectively. The recoveries of recombinant hirudin was 89.8% to 100.7%. The limit of quantification (LOQ) was 3.12 ng/ml. The concentration-time curve of the recombinant hirudin in the plasma could be explained as a two-compartment model. Pharmacokinetic parameters, including the half-life of distribution phase (t1/2 α), the half-life of elimination phase (t1/2 β), volume of apparent distribution (Vd), and area under the concentration-time curve from zero to infinite time (AUC0-t) were 7.59 min, 46.99 min, 0.17 L/kg, and 204.5 mg/L/min, respectively, as determined by chromogenic substrate assay; 6.41 min, 47.28 min, 1.24 L/kg, and 575.18 mg/L/min, respectively, as determined by ELISA; 3.69 min, 701.90 min, 0.04 L/kg, and 4189 mg/L/min, respectively as determined by RA; and 4.57 min, 724.9 min, 0.09 L/kg, and 2329 mg/L/min, respectively, as determined by TCA-RA. CONCLUSIONS: The chromogenic substrate assay on the concentration dynamics of the recombinant hirudin in the plasma is a specific, sensitive, and accurate analytical method for pharmacokinetic studies. Moreover, the pharmacokinetic parameters determined by the chromogenic substrate assay and ELISA are congruent except for AUC.
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