The Pilot And Preclinical Study Of RGD-SAK, A Novel Mutant Of Staphlokinase

Acute myocardial infarction is among the most prominent causes of death in the world. Staphylokinase (SAK) is a plasminogen activator and a promising blood clot-dissolving agent with clinical potency that is at least as good as tPA. However, it is far from ideal and the adverse effects, such as incomplete recanalization, delayed reperfusion time, reocclusion, bleeding and allergic reaction, restrict the wide application of SAK. It is well known that platelets play a pivotal role in arterial thrombosis and the binding of surface glycoprotein GPâ…¡b/â…¢a to fibrinogen mediates platelet aggregation. GPâ…¡b/â…¢a receptor antagonist, such as Arg-Gly-Asp (RGD) peptide had been proved to have obvious antiplatelet activity in thrombus formation experiments. Based on these theories, the previous work in our lab had substituted K35 with Arg to constitute a RGD motif, resulting in a novel SAK variant, designated as RGD-SAK, which was supposed to be recognized by the activated GPâ…¡b/â…¢a on the surface of platelet membrane. The preliminary experiments proved that RGD-SAK possess the bifunction to target platelet-rich clots and to block platelets aggregation.In order to provide an empirical basis for the establishment of standard operating procedure (SOP) for further large scale production and characterize the functional properties of RGD-SAK for the further clinical research, we carried out the pilot study of RGD-SAK and the preclinical study including pharmadynamics, pharmacokinetics and safety evaluation.RGD-SAK was expressed, purified and characterized in pilot scale. By fermentation and temperature induction in a 300 L fermentator, RGD-SAK was high effectively expressed. SDS-PAGE showed that the target protein was expressed after 0.5 h of induction and reached peak after 2.5 h. The protein was isolated by homogenization and purified by sequential chromatography steps through SP-Sepharose, Sephadex G-50, and Q-Sepharose and high purity protein was obtained with a purity＞98% by RP-HPLC analysis. Molecular mass spectrometric determination by LC-MS showed that RGD-SAK had a molecular weight of 15,478 D. The yield of purified RGD-SAK was estimated to be 300 mg per liter culture and the recovery of protein was more than 60% during purification. The results of quality control indicated that the product at pilot scale was qualified for clinical use. Biochemical analysis indicated that RGD-SAK maintained the similar structure and the fibrinolytic activity of SAK. Comparing with the standard SAK, the fibrinolytic activity of RGD-SAK was 120,000 IU/mg, which was at least equal to or slightly higher than SAK. Measurement ofplatelet binding activity in vitro demonstrated that RGD-SAK had a much higher affinity with platelets than SAK (P＜0.05). In vitro platelet-rich clot lysis assay demonstrated that the engineered mutant outperformed the non-manipulated SAK. The time required for 50% platelet-rich clot lysis was reduced significantly across different concentrations of RGD-SAK comparing with SAK (P＜0.05). Meanwhile, RGD-SAK was found to inhibit ADP-induced platelet aggregation in a concentration-dependent manner while SAK had negligible effect on platelet aggregation (P＜0.05). These results indicated that RGD-SAK possessed the bifunction to target platelet-rich clots and to block platelets aggregation, and thus may serve as a more potential thrombolytic agent with platelet-targeted fibrinolytic and antiplatelet aggregation activities in compared with SAK.Thrombolytic and anti-reocclusion efficacy of RGD-SAK observed by coronary artery angiography was compared with SAK in the porcine coronary balloon injury model. The coronary angiograph was detected by DSA (digital substrate angiography) to observe the vessel patency before thrombosis, thrombus formation, 90 rain, 24 h and 30 days after thrombolytic therapy. Blood samples were collected for analysis of D-dimer, cTnT (cardiac troponin T), activated partial thromboplastin time (aPTT), prothrombin time (PT), thrombin time (TT), fibrinogen (Fg) and platelet aggregation. The results revealed that RGD-SAK 50,000 IU/kg had more thrombolytic efficacy than that of equivalent dose SAK in 90 min after administration (P＜0.05). Meanwhile, the angiographically documented coronary artery recanalization rate also indicated that the intermediate-dose RGD-SAK had at least equivalent efficacy compared to that of SAK. The rethrombosis rate after 30 days following thrombolysis in different groups was compared and the results indicated four of six swine had rethrombosis in SAK 50,000 IU/kg group while there was no rethrombosis both in intermediate and high-dose RGD-SAK group (P＜0.05), which revealed that RGD-SAK had excellent efficacy in preventing arterial rethrombosis after thrombolysis. The concentration of D-dimer and cTnT in RGD-SAK 50,000 IU/kg group were higher than that of SAK 50,000 IU/kg group in 90 min and 24 h after administration (P＜0.05), which correlated with the corresponding thrombolytic efficacy. Meanwhile, RGD-SAK could inhibit ADP-induced platelet aggregation in a dose dependent manner both in vivo, while the SAK had almost no effect on platelet aggregation, which partially account for the anti-reocclusion efficacy of RGD-SAK after recanalization. There was no significant difference in thrombin time, prothrombin time, activated partial thromboplastin time and fibrinogen levels in plasma between any treatment group and the control group (P＞0.05), which indicated different regimens in the study had no significant effect on the hemostatic function and did not induced an overt systemic lytic state.To observe the effect of RGD-SAK on prevention of restenosis after coronary angioplasty in a swine model, the injured coronary arteries were dissected and examined histopathologically and the intima area ration was measured. To explore the pharmacological mechanism involved in the anti-restenosis, the PDGF-BB concentration, superoxide (.O^2-), NADPH Oxidase activity and NADPH Oxidase subunits (p22^phox and gp91^phox) expression were determined. The results showed that the injured arteries revealed marked eccentric neointima formation with cell proliferation of vascular smooth muscle cells. RGD-SAK 50,000 IU/kg can inhibited the restenosis significantly compared with the group treated wth equal regimen SAK in 30 days after balloon injury (P＜0.05). The blood analysis indicated that RGD-SAK 50,000 IU/kg can significantly decrease the platelet-derived-growth-factor (PDGF) relase in comparison with the same dose of SAK (P＜0.05). The determination of·O^2-. and NADPH oxidase in the coronary segments indicated that RGD-SAK 50,000 IU/kg can reduce the DPI-dependent·O^2-. production (P＜0.05), which was correlated to the downregulated expression of NADPH oxidase in the same site. Westem blot analysis results showed that the expression of two subunits of NADPH oxidase p22^phox, gp91^phox was significantly suppressed by RGD-SAK 50,000 IU/kg (P＜0.05), while SAK 50,000 IU/kg could not exhibit this effect. These results suggested that RGD-SAK can prevent the formation of restenosis after balloon injury and the mechanisms may be involved the anti-PDGF-release and the sequential downregulation of NADPH oxidase p22^phox, gp91^phox, which can affect the new intima formation by regulating the production of·O^2-.To study the pharmacokinetics of RGD-SAK in rats, different dose of ¹²⁵I-RGD-SAK was injected via caudal vein and the pharmacokinetic parameters, organ distribution and average accumulative samples discharged in different organ were assessed. The results showed that the pharmacokinetics of RGD-SAK was fitted with 3-compartment model, and there were no significant differences among the pharmacokinetic parameters of 3 doses. RGD-SAK was mainly distributed in kidney and discharged in the organ.Safety evaluation was assessed in a series of experiments. Acute toxicity experiment revealed that the maximum tolerance dose (MTD) of RGD-SAK was 2.5g/kg. All the physiological indexes of the treated mouse change little by 14d feeding experiment. The long-term toxicity of RGD-SAK was assessed in Rhesus monkeys, and the results showed that there were no effects on body weight, absolute or relative organ weights, ophthalmology, or electrocardiogram during the research dose range. Some adverse effects were observed in the high-dose (15mg/kg/d) group including the proteinuria, mild hematuria, significant prolongation of aPTT, PT and TT as well as the hemorrhage in the major organs (P＜0.05). These results indicated that RGD-SAK is well tolerated at doses up to 15mg/kg/d. Hemolysis test verified the RGD-SAK had not haemolytic adverse effect. To study the teratogenicity and mutagenicity of RGD-SAK, the Ames test and chromosome aberration of mammals cells were investigated to observe whether RGD-SAK would cause mutation of gene or chromosome mutagenesis. The study shows that RGD-SAK has no teratogenicity and mutagenicity.In conclusion, the present study established the sophisticated pilot method, which provided an empirical basis for the further large scale production. The function analysis in vitro and in vivo indicated that RGD-SAK was a more potent clot-dissolving agent for thrombolytic therapy and prevention of rethrombosis in comparison with SAK in the porcine coronary thrombus model. Meanwhile, RGD-SAK was exploited to anti-restenosis after balloon injury by preventing the release of PDGF, which can downregulate the expression of NADPH oxidase p22^phox, gp91^phox and decrease the production of·O^2-. Pharmacokinetics and safety evaluation of RGD-SAK testify the potential drug is effective and safe for the further clinical investigation.