Phyarmacokinetic Studies Of A Plasminogen Activator From Gloydius Brevicaudus Venom

In 1993,A novel plasminogen activator form Trimeresurus stejnegeri venom(TSV—PA) has been identified and purified by Yun Zhang for the first time.It offers the new subject for developing new medicine from venom. The laboratory of Snake Venom has purified novel plasminogen activator from Gloydius brevicaudus venom(GBV)and analyse its characterization and biological activities by animal experiment, and the results shown that the GBV-PA can treated and protected the thrombus in animal. In order to analyse its metabolic process in vivo, this study was designed to isolate and purify GBV-PA and study its pharmacokinetic, as a basic data for its further clinical study.1. Purification and characterization of the plasminogen activator from Gloydius brevicaudus Venom.1.1 Purification of GBV-PAAffinity chromatography of Gloydius brevicaudus Venom in Benzamidine Sepharose 6B resulted in the separation of two protein peaks. The fractionⅡwas collected and purify though gel filtration of Superdex G-75, which resulted in three protein peaks. The descending branch of fractionⅡcan specifically activate plasminogen through an enzymatic reaction estimated by the fibrin plate method. GBV-PA was purified from the fractionⅡby Lichrospher C18 4.6/250 reverse chromatography.1.2 The characterization of GBV-PAIt was homogeneous as a single band in both reducing and non-reducing showed on SDS-polyacrylmide gel electrophoresis (SDS-PAGE,Tris system) with molecular weight 40KDa as calculated by Image Master VDS system.2. Purification and characterization of the special antibody of GBV-PA.2.1 Purification of special antibody of GBV-PA Affinity chromatography of special antibody in CNBr-activated Sepharose 4B resulted in two protein peaks. Collected the fractionⅡ.2.2 Characterization of antibodyBy using the double immunodiffusion assay to determined the characteristic of this antibody, there was a clearly immunoprecipitate line between antibody and GBV-PA and affinity chromatography fractionⅡ.3. Determination of the GBV-PA in plasma by inhibiting BA-ELISA3.1 The assay of inhibiting BA-ELISAThe GBV-PA was diluted with coating buffer. To each well of a 96-well microtitre plate (Costa Corning Incorporated, USA) was added 0.1ml solutions. After overnight incubation at 4℃, the sealing buffer was added and the plates were incubated for 1hr at 37℃. After washing the plates with PBST, the samples were added to the wells. After an incubation of 90min at 37℃, the plates were washed again with the washing buffer and then incubated with biotin labeled anti-horse IgG for 90min at 37℃. Anther wash and added avidin labeled HRP in each well for 30min at 37℃. Then peroxidase substrate solution (ortho-phenylenediamine, hydrogen peroxide in pH 5.0 citrate-phosphate buffer) was added. The reaction was kept at 37℃for 30min in the dark, and then stopped by the addition of 2mol/L sulphuric acid. The absorbance at 492nm was measured.3.2 Determination of the best concentration of coated antigenThe GBV-PA was diluted with coating buffer in succession, but fixed the concentration of special antibody,biotin labeled IgG and avidin labeled HRP, and then carried out ELISA protocol. Made the absorbance of 492nm in 1.0 as the best choice in negative (contain antibody but no antigen), and the absorbance of 492nm in minimal as the best choice in blank (contain nothing but Beagle plasma). The best concentration of coated antigen was 0.5μg/ml.3.3 Determination of the best concentration of antibodyAs the method described before, fixed the concentration of coated antigen, biotin labeled IgG and avidin labeled HRP. The best concentration of antibody was 0.6×10-3U/ml3.4 Determination of the stander curve and the sensibility of inhibiting BA-ELISABeagles'blood without GBV-PA was diluted 1:20 in PBS immediately for serial dilution. The standard curve was established by the linear regression of△A492nm and the logarithmic does of GBV-PA under the condition of setting the best concentration of coated antigen and antibody. The stander curve was y=0.3173x+0.3491 (r=0.9927) when the antigen was between 1ng/ml～32ng/ml. The sensibility reached to 1ng/ml.4. Collection the blood, bile and urine samples4.1 Collection and examination of blood samples in BeaglesThirty-six Beagles were divided into six groups, received i.v. 300μg/kg, i.v.75μg/kg ,a.v.75μg/kg ,a.v.37.5μg/kg, a.v. 18.75μg/kg of GBV-PA. Heparin (3125U/kg) was used to prevent thrombosis by i.v. injection. Following drug administration, blood samples were collected and plasma drug concentrations were determined at 1, 5, 10, 20, 30, 60, 120, 240, 480, 720, and 1440min respectively. The intra-arterial injection groups was collected the sample right after the drug was administrated after 10min. all of the samples was diluted in PBS and centrifuged in 3000rpm for 10min.The intravenous injection 300μg/kg and 75μg/kg of GBV-PA shown a two compartment open model. t1/2αwas 3 and 7min respectively, t1/2βwas 5861 and 5107 respectively, the apparent volume of distribution was 0.25 and 0.3L/kg respectively, the formula was C(t)=1815e-0.2181t+107e-0.0046t and C(t)=4897e-0.1862t+39e-0.0056t respectively. The intra-arterial injection 75μg/kg,37.5μg/k and 18.75μg/kg of GBV-PA shown that, Cmax was 6472ng/ml,1413ng/ml and 1442ng/ml respectively, tpeak was 15min.4.2 Collection and examination of bile and urine samples in ratsSix rats were used, anesthetized by ethylcarbamate (0.16mg/kg). Before administration, isotonic Na chloride was administrated (10ml/min) by intervenous drop infusion in left femoral GBV-PA (300μg/kg) was intravenous injection, the bile and urine samples were collected at different time point, after diluted in PBS, all of the samples were examined by inhibiting BA-ELISA. GBV-PA was excreted mainly from kidney. After one hour, there was 7.06% of GBV-PA excreted in urine, but in bile, there contained little, just 0.005% compared to 12.78% in urine.ConclusionsThe sensitivity of inhibiting BA-ELISA is reached 1ng/ml. The tpeak is 15min during the group of intra-arterial injection. Intra-arterial injection of GBV-PA (75μg/kg) has a higher peak plasma concentration compare to intravenous injection. Intra-arterial injection is a effected method of administration.