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OBJECTIVE@#To investigate the antithrombotic effects of recombinant hirudin and its mechanism.@*METHODS@#Sixty male Kunming mice were randomly divided into 6 group (=10):control group, model group, aspirin (25 mg/kg) group, recombinant hirudinlow, middle and high dose (0.05, 0.1, 0.2 mg/kg) groups.Except mice in control group, 2.5 mg/kg carrageenan was injected intraperitoneallyto mice in the other groups to produce thrombosis on the mice tail. The mice in aspirin group were administrated intraperitoneally 25 mg/kg aspirin, the mice in recombinant hirudinlow, middle and high dose groups were administrated intraperitoneally 0.05, 0.1, 0.2 mg/kg combinanthirudin, the mice in control group and model group were administrated intraperitoneallynormal saline at the same volume respectively at 24 h, 0.5 h before injecting carrageenan and 24 h after injecting carrageenan. The black tail length of mice and the incidence of black tail were observed at 48h after injection of carrageenan; prothrombin time (PT), activated partial thromboplastin time (APTT), tissue plasminogen activator (t-PA), type-1 plasminogen activator inhibitor (PAI-1), 6-keto-PGF1α, and thromboxane B2 (TXB2) level in mice plasma were determined.@*RESULTS@#As compared with control group, the mice in model group presented tail thrombosis; PT level in plasma was significantly shortened (<0.01), PAI-1 and TXB2levels in plasma were significantly increased (<0.01), while the t-PA and 6-keto-PGF1α levels in plasma in model group were significantly decreased (<0.01). As compared with model group, the thrombus length in the tail was significantly shortened (<0.05, <0.01), PT level was obviously prolonged (<0.01), and the plasma levels of PAI-1 and TXB2 were significantly decreased (<0.01), while the plasma levels of t-PA and 6-keto-PGF1α were significantly increased (<0.01)in the mice of recombinant hirudin low dose, middle dose, high dose groups and aspirin group. As compared with aspirin group, the thrombus length in the tail was significantly increased (<0.05), PT level was obviously shortened (<0.01), and the plasma levels of PAI-1 and TXB2 were significantly increased (<0.01)in the mice of recombinant hirudin low dose group; the plasma level of 6-keto-PGF1α was significantly decreased (<0.01, <0.05) in the mice of recombinant hirudin low dose and middle dose groups; the plasma levels of PAI-1 and TXB2 were significantly increased (<0.01, <0.05)in the mice of recombinant hirudin middle dose group.@*CONCLUSIONS@#The recombinant hirudin can fight against thrombosis, its antithrombotic mechanisms may be related to its influence on the exogenous coagulation system and the promotion of fibrinolysis function.
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Animals , Male , Mice , Blood Coagulation , Fibrinolytic Agents , Hirudins , Pharmacology , Recombinant Proteins , Thromboxane B2 , Tissue Plasminogen ActivatorABSTRACT
OBJECTIVE:To study the pharmacokinetic characteristics of Recombinant hirudin enteric-coated capsule by single and multiple administration in Beagle dogs. METHODS:12 Beagle dogs were divided into single ig group and single iv group by random control method,6 in each group. Recombinant hirudin 0.2 mg/kg was intragastrically administrated or intravenously inject-ed,blood sample was collected;after 2 weeks of cleaning,12 dogs were intragastrically administrated recombinant hirudin 0.2 mg/kg,for 7 d. Sample blood was collected,referred to multiple ig group. Recombinant hirudin concentration in plasma was deter-mined by enzyme-linked immunosorbent assay,and pharmacokinetic parameters were calculated by DAS 2.0 software. RESULTS:The results showed that pharmacokinetics by ig and iv recombinant hirudin in Beagle dogs fitted to two-compartment model,abso-lute bioavailability of ig Recombinant hirudin enteric-coated capsule was(14.908±1.868)%;the pharmacokinetic parameters in sin-gle ig group and multiple ig group were tpeak of(2.105±0.243),(3.000±0.000)h,t1/2β of(8.660±2.965),(14.870±2.710)h, cmax of(10.700±0.872),(12.05±1.587)ng/mL,AUC0-1440 min of(55.250±4.386),(58.978±6.002)ng·h/mL,without statistical significances in two groups(P>0.05). CONCLUSIONS:The ig Recombinant hirudin enteric-coated capsule can be absorbed into the blood to a certain extent. There is no accumulation for ig Recombinant hirudin enteric-coated capsule for several days,and it dose not change the pharmacokinetic characteristics.
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Objective To investigate the protective effect and mechanism of recombinant hirudin on avulsed flap and to explore the application of recombinant hirudin in clinical treating avulsed flap.Methods 108 Sprague-Dawley rats were randomly divided into three groups:local injecting recombinant hirudin (group A),local injecting low molecular heparin (group B),and local injecting physiological saline (group C).The far ends were sutured after forming dorsal avulsed flap with local freeing.The levels of P-selectin and VEGF were detected,and the inflammatory cell aggregation and formation of microthrombus were observed in pathological section.On 14th postsurgery day the survival rate of flap was calculated.Results The content of P-selectin in group A was lower than that of groups B and C,with a statistical difference (P＜0.05).The content of VEFG in group A was higher than that of groups B and C,with a statistical difference (P＜0.05).On 14th postsurgery day,group A was higher than that in groups B (P＜0.05)and C (P＜0.01) in the survival rate of flap.Conclusions Locally injected recombinant hirudin can actively suppress the production of P-selectin,reduce accumulation of inflammatory cells,prevent information of microthrombus,improve ischemia and anoxia of avulsed flaps,decrease injuries of endotheliocyte,enhance the expression of VEGF,and stimulate hyperplasia of neogenesis capillaries that elevate survival rate of avulsed flaps.
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Background Recombinant hirudin variant Ⅲ(rHV3) can effectively prevent galactose-induced human lens epithelial cells LECs injury,but little is known about the molecular mechanism of its action.Objective The present study was to investigate the effects of rHV3 on the expression of apoptosis-related genes in damaged LECs induced by galactose.Methods The rHV3 was extracted by our research group,and the biological activity of rHV3 was identified by titration of thrombase according to Markwardt's method.Human LECs (SRA01/04) were cultured using 125×10-3 mol/L D-galactose+10% FBS+D/F12 medium to establish the damaged human LECs model.rHV3 was added into the medium of the damaged human LECs model.Human LECs were cultured in D/F12 medium containing 10% FBS as normal control.The expression of apoptosis-related genes,such as aldose reductase (AR),bax,bcl2 and p53,in LECs at the mRNA level was detected using RT-PCR.The abundance ratio of target genes was presented with the absorbance (A) of gene mRNA/GAPDH mRNA.Results Compared to the normal control group,the A values of AR mRNA/GAPDH mRNA,bax mRNA/GAPDH mRNA and p53 mRNA/GAPDH mRNA were significantly elevated in model group (t=3.90E-06,t=8.44E-04,t=5.15E-08,P＜0.01).However,in the rHV3-treated group,the A values of AR mRNA/GAPDH mRNA,bax mRNA/GAPDH mRNA and p53 mRNA/GAPDH mRNA were lower than those of model group (t=5.90E-06,t=1.51E-04,t=3.42E-06,P＜0.01).The bcl2 mRNA/GAPDH mRNA was markedly downregulated in the model group when compared with the normal control group (t=1.86E-05,P＜0.01);while after rHV3 addition,bcl2 mRNA/GAPDH mRNA increased in comparison with the model group (t=8.56E-05,P＜0.01).Conclusion 125×10-3mol/L D-galactose induces the damage and apoptosis of human LECs.rHV3 likely plays a protective function on D-galactose-induced damage of human LECs by inhibiting the polyol pathway and mitochondria-mediated pathway.
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OBJECTIVE:To prepare polyclonal antibody for recombinant hirudin Ⅲ (rHV3). METHODS: The antigens were prepared through two typical methods: one was conjugated with rHV3 itself, another was coupled with bovine serum albumin (BSA). Conjugation results were detected through electrophoresis. Then the rabbits, guinea pigs and rats were immunized and with serum sample taken for determination of antibody titer by double immunodiffusion test. RESULTS: Antibodies were produced in rabbits and DHP guinea pigs after immunization using rHV3-BSA as antigen, with antibody titer of 1∶16. CONCLUSION: After being immunized with rHV3-BSA, rabbits and DHP guinea pigs can produce polyclonal antibody for rHV3.
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brain and fatty. The accumulation excretion ratios within 96 h were 70.16% in urine and only 1.35% in feces after iv administration in rats, while within 24 h was 3.46% in bile. Conclusion The results of pharmacokinetics of novel r-hirudin in rats given by iv determined by RA are different from those determined by TCA-RA, but the trends indicated by the two methods are the same; urine excretion is the major excretion pathway.
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AIM: To investigate the anti-DIC effect of the domestic recombinant hirudin (rH) in order to provide preclinical pharmacological basis for its new drug application. METHODS: DIC was induced by constant iv infusion of thrombin (100 U?kg -1 ?h -1 ?1 h) plus PAMBA (50 mg?kg -1 ?h -1 ?1 h). Platelet count (PLT), PT and fibrinogen (FIB) determination as well as plasmaprotamine paracoagulation (3P) test served as diagnostic indexes of DIC, while pathlogical examination of microthrombosis of lungs and kidneys was followed. RESULTS: rH 125,62.5,31.25 and 15.625 ?g?kg -1 iv. gtt demonstrated a dose-dependant inhibitory effect on DIC. The inhibitory effects on DIC of rH(%) were 89.38%, 52.60%, 49.71% and 25.58% for PT, and 100.01% , 61.86%, 58.40% and 32.68% for PLT, and 85.22%, 73.01%, 33.60% and 14.86% for FIB respectively at the descending order of rH doses. CONCLUSION: Domestic rH being tested has powerful anti-DIC effect which is comparable with refludan—an r-hirudin product approved by European Community for marketing.