| With rapid development of gene engineering techniques, the recombinant polypeptide drugs have been widely used in clinical therapeutic practices. The R&D and clinical rational application of this kind of drugs need first to elucidate their pharmacokinetics (PK) properties. However, the PK study of polypeptide drugs continues to be a great difficult problem, with no ideal method available to date, the combination of different methods are usually required. In this paper, PK of the recombinant hirudin (rH), an highly specific thrombin inhibitor, has been studied in order to develop a routine methodology of polypeptide drug PK study and an individual character method suitable for rH PK study, and to develop a routine methodology for study of metabolic products in animals by means of analysis of urinary metabolites of rH in rats, and thereby to build up a basis for R&D and clinical application of newly made domestic rH.(N-Ile’-Thr-63-desulfato-r-hirudin).Methods1. A double antibody sandwich ELISA method was established for determination of the rH in rat plasma and tissue homogenate samples, and thereby to study the rH PK and tissue distribution after intravenous(iv) of rH.2. Based on antithrombin activity of rH, a chromogenic substrate assay (CSA) was established for determination of the rH in rat plasma and urine samples, and thereby to study the rH plasma PK and urinary excretion after iv of rH.3. rH was labeled with125I, the resultant125I-rH was employed to study the rH PK and metabolic process after iv of125I-rH.4. The125I-rH radioactivity in rat plasma and urine samples was measured by means of the gel chromatography and the gamma-counter so as to study the metabolic processes in rat plasma and metabolic products in rat urine. Urinary metabolic products were isolated and purified by the gel chromatography and the reversed-phase HPLC, and subsequently subjected to MW measurement using TOF-MS and identification of chemical structure as well as amino acid sequence by the amino acid analyzer.Results1. The rH concentration/content in body fluids and tissues of rats was measured by ELISA assay with the limit of detection of0.25ng·mL-1and good linearity ranged from0.25ng·mL-1to3.0ng·mL-1(r=0.999). The rH plasma concentration over time after iv injection at doses of0.5,1.0and2.0mg·kg-1was characterized by a rapid distribution phase followed by the relatively slow elimination phase and could be described by the bi-exponential equation for two-compartment open model and followed the first-order kinetics; the main PK parameters t1/2α=5-6min, t1/2β=45-47min, AUC (μg·mL-1·min) were 127.8±27.8,330.8±115.9,575.1±39.4proportional to doses; demonstrating that the PK behaviour of the tested drug complies with the linear kinetics. After iv of1.0mg·kg-1, parent rH were hardly detected in urine, feces and bile samples; Tissue distribution experiment showed that the rH distributed rapidly into tissue after iv dosing at1.0mg·kg-1and the rH contents in tissues reached a peak at15min postdose and afterwards decreased gradually, the rH content was found to be highest in lung (78.88±32.3ng·g-1) at15minutes, in kidney (24.39±9.71ng·g-1) at1hour and in liver (7.55±11.33ng·g-1) at3hours.2. The established CSA showed good linearity in the range from3.125ng·mL-1to40ng·mL-1(r=0.995) for plasma and from6.25ng·mL-1to75ng·mL-1(r=0.997) for urine. The main plasma kinetic parameters based on the CSA at dose of2.0mg·kg-1were similar to that based on the ELISA except for AUC. After iv rH at0.5,1.0and2.0mg·kg-1the percent urinary cumulative excretion in relation to dose between0-12h was found22.3%,23.2%and23.3%, respectively. This dose-independent excretion manner indicated that rH urinary excretion followed the first-order i.e. linear kinetics.3. The PK of rH in rats was also studied by the125I-rH isotope tracer technology. The parameter t1/20was701.8±198.7min on a total radio activity (RA) basis, and724.8±81.2on a TCA precipitation-radio activity (TCA-RA) basis. The percent cumulative excretion of rH in urine was62.1%between0-12h based on the RA detection.4. The rH was shown to be metabolized in plasma, followed by being excreted in urine as metabolites. One of these metabolites showed potent anti-thrombin activity and was found to be composed of51-52amino acids with molecular weight being5162Da,.Conclusion1. By validation of methodology it has been shown that the established ELISA and the CSA are highly sensitive and specific for rH bio-analysis in rats and, therefore, completely meet requirements for rH PK study. The rH PK in the rat can be described by the open two-compartment model and follows the first-order kinetics with rapid elimination from body (t1/246min or so).2. The PK parameters obtained by the RA and TCA-RA are quite different from that by the ELISA assay and by the chromogenic substrate method.3. The rH undergoes extensive metabolic degradation in rats, with only extremely minute amount of unchanged rH recovered in urine of rats. Some metabolic products excreted into urine have anti-thrombin activity. Among them, the strongest in activity is the metabolite having molecular weight of5162Da and composed of51-52amino acids. rH has been found for the first time by us to be metabolized mainly in plasma but not in liver and kidneys as described in foreign literature. |
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