Study On Development Of Methods For Determination Of R-RGD-Hirudin In Vivo And Applications In Clinical Trial

Hirudin is an anticoagulative product from salivary glands of medicinal leech. It is a powerful and specific thrombin inhibitor. As Direct thrombin inhibitors (DTIs), hirudin is a new class of therapeutics possessing theoretic advantages over unfractionated heparin (UFH). In contrast to UFH, DTIs do not activate platelets, have no circulating inhibitors, and bind to both free and clot-bound thrombin. These theoretical advantages have spurred clinical trials investigating DTIs in a variety of cardiovascular indications. Unlike heparin, which has to bind to anti-thrombin III to exert its actions, hirudin binds directly to thrombin, inhibits fibrin-bound as well as fluid-phase thrombin. It also inhibits fibrinogen clotting and thrombin-catalyzed haemostatic reactions.A novel bi-functional hirudin molecule, r-RGD-Hirudin, were engineered by inserting a RGD tripeptide to a given domain of the wt-Hirudin molecular. In theory, r-RGD-Hirudin has been able to inhibit both thrombin and platelet aggregation. It is anticipated that r-RGD-Hirudin replaces heparin or wt-Hirudin with higher efficacy and safety because of its bi-functional action and its low effective dosage.A reliable and validated LC-MS method was established for determination of r-RGD-Hirudin in human serum. Instead of liquid-liquid extraction or solid phase extraction, ultrafiltration was used for water solubility drug r-RGD-Hirudin extraction. After that, freeze drying was used for concentration. The experiment conditions including pre-processing procedure and LC-MS have been investigated and optimized. Comparing with reported assays, the method showed significant improvement in specificity, linearity, precision and sensitivity(LLOQ=25ng/mL), and it has been successfully applied in clinical research of r-RGD-Hirudin.Also a simple and rapid CE-MS methods was developed for quantification of r-RGD-Hirudin in human urine. The assay using a capillary online concentration technique achieved adequate sensitivity of 250ng/mL while required no preprocessing procedure of urine sample. Successfully applied to clinical trial, the method appeared to be suitable for TDM and PK/PD study of r-RGD-Hirudin.