| Background and purpose: In recent years, recombinant hirudin(r-hirudin, rH), a polypeptide composed of 65 amino acids, which is a highly specific and the most potent thrombin inhibitor, has got a conspicuous development in its research and exploitation. Our research group has made study on pharmacodynamics and pharmacokinetics of domestic rH, in-depth demonstrating its potent anticoagulant and antithrombotic effects. This article is designed to further investigate the antithrombotic effect of domestic rH in experimental coronary artery and middle cerebral artery (MCA) thrombotic model of rats in order to provide preclinical pharmacological basis for its development as a new drug and its clinical uses. Methods:1. Preparation of thrombosis models: Thrombosis was induced by FeCl3 chemical damage to vessel endothelium. FeCl3 was locally applied on coronary artery and MCA of animals anesthetized for certain time (1h and 0.5h, respectivity) to cause thrombosis. Drugs were administrated before surgery. 2. Examination and determination of index and evaluation of pharmacological activity: (1) Coronary artery thrombosis: After 1h-application of FeCl3 on the artery, 1% Evans Blue was injected. The anti-artery thrombosis effect of rH was evaluated exactly after thrombosis by scores of infarcted area, Evans Blue content of left ventricle myocardium, change of ECG, superoxidant dismutase (SOD) activity and malondial dehyde (MDA ) content. Inhibitory rate of cardiac infarction was obtained according to the Evens Blue content. (2) MCA thrombosis: Neurological changes at 4h postoperation were scored and 24 hours after formation of thrombus, infarcted area of brain was measured and then, infarcted area inhibitory rate was calculated. Intra-vascular thrombosis was examined pathologically in both experiments. Results:1. Experiment of coronary artery thrombosis: In the research of time-effect relationship, after a single iv administration at a dose of 0.5mg/kg, rH exhibited a time-dependant attenuated anti-artery thrombosis effect. It was shown from score of myocardiac infarcted area, content of Evans Blue and change of J point and T wave as ECG index that with time going, the potency of rH began to weaken after its peak in 10 min postdose and lasted for about 1h. In the research of dose-effect relationship, rH iv 1.0, 0.5, 0.25 and 0.125mg/kg at time of effect peak resulted in a dose-dependent inhibitory effect on cardiac infarction with inhibitory rate of 80.84%, 65.15%, 40.77% and 21.20% respectively, yielding the 50% inhibitory dose (ID50) of 0.332mg/kg. Summation of infarced area score of model group and rH group treated with different dose was 39, 12, 16, 24 and 28 respectively. The change of J point and T wave, which served as myocardial infarction index, was measured in model group and rH group above, the average elevation being 0.31, 0.15, 0.16, 0.20, 0.26mv in J point and 0.22, 0.08, 0.15, 0.17, 0.15mv in T wave. Significantly improved SOD activity (54.64, 46.23, 44.41 and 41.75 Nu/mg prot) was abtained compared with 30.46 Nu/mg prot in model group. Correspondingly, MDA content was reduced from 2.31 nmol/mg prot in model group to 1.63, 1.74, 1.79 and 1.89 nmol/mg prot in rH group.2. Experiment of MCA thrombosis: rH iv 1.0, 0.5, 0.25mg/kg resulted in a marked dose-dependant inhibitory effect on infracted area, with the inhibitory rate being 79.24%, 61.82% and 46.29%, respectively and ID50 being 0.297 mg/kg. Neurological changes also displayed a good dose-effect relationship, which were scored as 1.8, 3.0 and 5.5 at descending order of doses while being 6.5 in model group. Pathology examination in both experiments demonstrated that with the dosage of rH increased, the intra-vascular formation of thrombi in rats receiving rH were reduced, or even disappeared.In addition, in the above two experiments heparin was used as positive control drug, moreover, Refludan was applied as potency comparison of same type of rH in experiment of coronary artery thrombosis formation.When the same dose was employed, domestic rH... |
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