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Aim To produce poly (lactic-co-glycolic acid) (PLGA) microspheres, containing a staphylokinase variant (K35R, DGR) with reduced immunogenecity and antiplatclet aggregation activities,which allowed the preservation of protein stability during both particle processing and drug release.Methods DGR-loaded microspheres were fabricated using a double emulsion-solvent evaporation technique. The effects of preparative parameters, such as stirring rate, polymer concentration, and the excipients of both internal and external aqueous phase (W2 ), on DGR encapsulation efficiency and microsphere characteristics were investigated. In vitro and in vivo release of DGR were conducted and the cause for instability of DGR during release was also investigated. Results Moderate ultrasonic treatment of aqueous DGR/dichloromethane mixtures caused approximately. Eighty four per cent DGR denaturation.However, the activity recovery of DGR almost amounted to 100% when 2% polyvinyl alcohol (PVA) was added into the aqueous phase. It was found that NaCl in the external water phase significantly increased DGR encapsulation efficiency. Furthermore, NaCl in the external water phase played a role in determining size and surface morphology of microsphere. In vitro release test showed a burst release of DGR from microspheres, followed by sustained release of 50% total activity over 15 days. In vivo experiments showed that DGR released from microspheres sustained 5 days. Denaturation of DGR within microspheres might be resulted from acidic microclimate. Conclusion The stability of DGR was effectively protected during microencapsulation and a relatively high encapsulation efficiency of DGR was obtained. PLGA microspheres could be an effective carrier for DGR.
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The recombinant fusion protein staphylokinase-hirudin(rSFH) was purified from the high density-fermented engineered E.coli by means of ion-exchange chromatography (IEC) and gel filtration (GF). The purity of rSFH reached to more than 98% determined by RP-HPLC and SDS-PAGE, and the yield was up to 0.7g per liter of fermentation broth. The analysis of homologous dimmer of rSFH appeared during the purification and calculation of the surface hydrophobic area had been carried out by means of hydrophobic chromatography and MALD-TOF. The influence of sodium chloride and temperature on the behavior of rSFH reversible dimerization was analyzed by high performance sized- exclusive chromatography(HPSEC). It is concluded that the hydrophobic interaction played an important role in the reversible dimerization of rSFH.
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Objective To study on the immunogenicity of SAK and its mutant using PAb.Methods 4 New Zealand white rabbits subcutaneously injected with corresponding antigens were grouped into group SAK involving 2 rabbits and group SAK2 involving 2 rabbits.The antiserums were collected 1 week after third injection.The rabbit IgG fraction was precipitated with saturated(NH4)2SO4 and purified by DEAE-Sepharose column chromatography,and then the antibody titer was determined by ELISA.The immunoreactivity of antigen to polyclonal antibody against SAK and SAK2 was tested using ELISA.Results The immunoreactivity of SAK2 to polyclonal antibody against SAK was sharply reduced to a very low level determined by ELISA,which indicated that some epitopes of SAK had been deleted.Conclusions The immunoreactivity of SAK to polyclonal antibody against SAK2 did not get a dramatically change compared with SAK2,which indicated that the reconstruction of epitope did not create new epitope.
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Objective To observe the effects of recombinant staphylokinase (r-SAK) on platelet activation parameters in patients with acute myocardial infarction(AMI) by intravenous thrombolysis in order to investigate the clinical thrombolytic efficacy of r-SAK therapy in AMI comparing with recombinant tissue-type plasminogen activator(rt-PA) therapy.Methods Thirty-three patients with AMI within 12 h after the onset were selected and divided randomly into the r-SAK therapy group(n=17) and rt-PA therapy group(n=16).Coronary artery angiography(CAG) was performed 90 min after thrombolytic therapy in patients.Thrombin-antithrombin complex(TAT) and alpha granule membrane protein(GMP-140) were measured by similar commercial enzyme-linked immunosorbent assay(ELISA).Results In r-SAK group and rt-PA group,the plasma contents of GMP-140 2 h after thrombolytic therapy were significantly higher than before therapy(P0.05).In rt-PA group,the plasma content of TAT 2 h after thrombolytic therapy increased significantly(P0.05).) Conclusion r-SAK has similar effect with rt-PA and it will become available for highly fibrin-selective thrombolytic therapy of AMI.Thrombolytic treatment with r-SAK can improve the injury of myocardial microperfusion.
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OBJECTIVE:To study the pharmacokinetics of recombinant staphylokinase(r-SAK)in a thrombosis model of femoral artery in rabbits.METHODS:Large glass plates were used for modified agar-well diffusion assay to measure r-SAK concentration in plasma of rabbits which had been administered different doses drug by different means respective?ly.RERULTS:The pharmacokinetics of r-SAK infusion in thrombosis model of the femoral artery in rabbits fits two-com?partment model,the plasma levels and the diameter of lytic circles showed a good linear correlation under the scope of20～2500IU/ml(r=0.9960),and the averaged recovery rate was(96.05?9.20)%.The peak concentrations of low,medial and high dose drop group and single iv group are(2.28?1.06),(3.54?0.32),(6.12?1.61)and(5.16?1.02)?g/ml.CONCLUSION:The biological assays is a simple,reliable method to determine the plasma level.