Scale up and pharmacokinetic study of a novel mutated chimeric tissue plasminogen activator (mt-PA) in rats (original) (raw)

Because of high mortality caused by cardiovascular diseases, various fibrinolytic agents with diverse pharmacokinetic and pharmacodynamic properties have been developed. A novel mutated chimeric tissue plasminogen activator (mt-PA) was developed by the removal of first three domains of t-PA, insertion of GHRP sequence and mutation towards resistance to plasminogen activator inhibitor-1 (PAI-1). Mt-PA protein was expressed in Expi293F cells. The expression level of mt-PA was found to be 5000 IU/ mL. Following purification, the pharmacokinetic properties of mt-PA were evaluated in three doses in rats. Data related to mt-PA were best fitted to two compartment model. With the increase in dose, the Area Under the plasma concentration-time Curve (AUC 0→∞) increased. The elimination half-life (t 1/2) of mt-PA was in the range of 19.1-26.1 min in three doses while that of Alteplase was 8.3 min. The plasma clearance (CLp) of mt-PA ranged from 3.8 to 5.9 mL/min in three doses, which was several times lower than that of Alteplase (142.6 mL/min). The mean residence time (MRT) of mt-PA ranged from 23.3-31.8 min in three doses, which was 4-5 times greater than that of Alteplase (6 min). Mt-PA showed extended half-life and mean residence time and is a good candidate for further clinical studies. Cardiovascular diseases, caused by disorders of heart and blood vessels, account for 17.3 million deaths per year that is expected to grow to more than 23.6 million by 2030 1,2. In 2011, the estimated annual costs of cardiovascular diseases and stroke amounted to a total of more than $320.1 billion 1. Thrombolytic drugs particularly plasminogen activators (PAs) play an essential role in this respect and PAs can clear circulatory occlusions due to fibrin clot or thrombus. PAs convert plasminogen to the active serine protease plasmin which, in turn, dissolves fibrin, the insoluble matrix of clots 3. Tissue-type plasminogen activator (t-PA) is one of the fibrin-specific serine proteases that plays a crucial part in the fibrinolytic system 4,5. T-PA is composed of a single chain polypeptide of 527 amino acids and includes 17 disulfide bridges 6. The mature form of t-PA comprises five distinct domains: a finger domain (F) involved in the high-affinity binding of t-PA to fibrin and hepatic clearance of t-PA 7 , an epidermal growth factor-like domain (EGF) which contributes to the hepatic clearance of t-PA 8 , a kringle 1 domain (K1) which is important in the uptake of t-PA by mannose receptors on liver cells 9 , a K2 domain involved in the high-affinity binding to fibrin and activation of plasminogen, and a serine protease domain (S) where the catalytic activity of t-PA takes place 10. The main inhibitor of t-PA is PAI-1, a member of the serpin family (serine-protease inhibitor), which plays its role as a pseudo-substrate for target serine proteases 11. PAI-1 is synthesized by endothelial cells and hepatocytes, and partially by the α-granules of platelets 12. Similarly, plasmin is inhibited mainly by α 2-antiplasmin, yet plasmin-bounded fibrin is never inhibited 6. Because of the short plasma half-life (4-6 min) of Alteplase 13 , a large dose is required to obtain therapeutic blood levels, which in turn may lead to higher bleeding and re-occlusion risks due to a decreased plasma