LC-MS/MS method for determination of r-RGD-hirudin in human plasma and its application in pharmacokinetic study.

OBJECTIVE: This study aimed to develop a simple, sensitive, and selective Liquid chromatography with a Mass spectroscopic method for simultaneous quantification of a recombinant bifunctional hirudin (r-RGD-Hirudin, Bifunctional Hirudin, BFH) in human plasma and verify its effectiveness. METHODS: The analytes and the internal standards from human plasma were extracted using the solid-phase extraction technique. The reconstituted samples were chromatographed on Waters C18 column (BEH 50 × 2.1 mm, 1.7 µm) using a mixture of 0.1% formic acid/acetonitrile (85%/15%, v/v) with gradient elution as the initial mobile phase at a flow rate of 0.3 mL/min. RESULTS: The effectiveness of the proposed method was verified over the concentration range of 10-2000 ng/mL for r-RGD-Hirudin. A linear calibration curve was obtained. The precision and accuracy of BFH in the intra- and inter-day runs fell within the range of ±15% at LQC, GMQC, MQC and HQC concentrations. The extraction recoveries and matrix effect at two quality control (QC) levels for BFH were confirmed to conform to the relevant requirement. CONCLUSION: The proposed method was successfully adapted to examine the pharmacokinetics of BFH in 40 Chinese healthy volunteers, respectively.

RGD-hirudin-based low molecular weight peptide prevents blood coagulation via subcutaneous injection.

Thromboembolic disease is a common cardio-cerebral vascular disease that threatens human life and health. Thrombin not only affects the exogenous coagulation pathway, but also the endogenous pathway. Thus, it becomes one of the most important targets of anticoagulant drugs. RGD-hirudin is an anticoagulant drug targeting thrombin, but it can only be administered intravenously. We designed a low molecular weight peptide based on RGD-hirudin that could prevent blood clots. We first used NMR to identify the key amino acid residues of RGD-hirudin that interacted with thrombin. Then, we designed a novel direct thrombin inhibitor peptide (DTIP) based on the structure and function of RGD-hirudin using homology modeling. Molecular docking showed that the targeting and binding of DTIP with thrombin were similar to those of RGD-hirudin, suggesting DTIP interacted directly with thrombin. The active amino acids of DTIP were identified by alanine scanning, and mutants were successfully constructed. In blood clotting time tests in vitro, we found that aPTT, PT, and TT in the rat plasma added with DTIP were greatly prolonged than in that added with the mutants. Subcutaneous injection of DTIP in rats also could significantly prolong the clotting time. Thrombelastography analysis revealed that DTIP significantly delayed blood coagulation. Bio-layer interferometry study showed that there were no significant differences between DTIP and the mutants in thrombin affinity constants, suggesting that it might bind to other sites of thrombin rather than to its active center. Our results demonstrate that DTIP with low molecular weight can prevent thrombosis via subcutaneous injection.

A novel hirudin derivative inhibiting thrombin without bleeding for subcutaneous injection.

Currently, anticoagulants would be used to prevent thrombosis. Thrombin is an effector enzyme for haemostasis and thrombosis. We designed a direct thrombin inhibitor peptide (DTIP) using molecular simulation and homology modelling and demonstrated that the C-terminus of DTIP interacts with exosite I, and N-terminus with the activity site of thrombin, respectively. DTIP interfered with thrombin-mediated coagulation in human, rat and mouse plasma (n=10 per group) and blocked clotting in human whole blood in vitro. When administered subcutaneously, DTIP showed potent and dose-dependent extension of aPTT, PT, TT and CT in rats (n=10 per group). The antithrombotic dose of DTIP induced significantly less bleeding than bivalirudin determined by transecting distal tail assay in rats. Furthermore, DTIP reached peak blood concentration in 0.5-1 hour and did not cause increased bleeding after five days of dosing compared to dabigatran etexilate. The antithrombotic effect of DTIP was evaluated in mice using lethal pulmonary thromboembolism model and FeCl3-induced mesenteric arteriole thrombus model. DTIP (1.0 mg/kg, sc) prevented deep venous thrombosis and increased the survival rate associated with pulmonary thromboembolism from 30 % to 80 %. Intravital microscopy showed that DTIP (1.0 mg/kg, sc) decelerated mesenteric arteriole thrombosis caused by FeCl3 injury. These data establish that DTIP is a novel antithrombotic agent that could be used to prevent thrombosis without conferring an increased bleeding risk.