Introduction: The incredible progress of cardiosurgery and thromboprophylaxis requires predictable anticoagulants therapy. All anticoagulants, especially parenteral, may cause uncontrolled bleeding, need fast neutralization in case of emergency. Protamine, the only clinically approved unfractionated heparin (UFH) binder, reverses partially enoxaparin; does not reverse fondaparinux. Protamine[1],[2] and some other alternative antidotes[3],[4] under development exert significant adverse effects. Previously, we showed various polymers able to bind UFH in vitro and in vivo[5],[6]. Currently, we aimed to develop an effective and safe fully synthetic copolymer-PEG41-PMAPTAC53, as a new antidote of UFH, enoxaparin and fondaparinux.
Materials and Methods: To investigate effectiveness of PEG41-PMAPTAC53 in vitro, its complexes with UFH were assessed with a colorimetric method using Azure A. The neutralization of UFH, enoxaparin and fondaparinux was evaluated by measuring of activated partial thromboplastin time (aPTT) and anti-factor Xa activity (anti-Xa). Three procedures were performed to investigate effectiveness in vivo. In the first, thrombosis was induced by electrical stimulation in the carotid artery of the rat. The bleeding time and blood cell count were measured after removal of thrombus. The blood samples were collected from the heart for measurement of aPTT and anti-Xa. In the second procedure, enoxaparin or fondaparinux were injected before the bleeding time measurement alone or followed by intravenous infusion of PEG41-PMAPTAC53. After anticoagulant administration, blood samples were taken from the heart for measurement of anti-Xa. In the third procedure, rats treated with enoxaparin alone or followed by infusion of PEG41-PMAPTAC53. Activated clotting time (ACT) was measured in blood samples collected from tail at several time points after enoxaparin administration. To investigate the safety, blood pressure, heart rate, body temperature, oxygen saturation, perfusion, peak CO2, respiratory rate were monitored after antidote administration. Finally, biochemical parameters in serum were measured in rats and major organs were collected for histological examination. Additionally, the body distribution was analyzed in NMRI-Foxn1nu/Foxn1nu mice receiving UFH followed by PEG41-PMAPTAC53 conjugated with AlexaFluor®750 dye using In-vivo MS FX PRO system.
Results: PEG41-PMAPTAC53 completely neutralized the activities of UFH, enoxaparin, and fondaparinux at in vitro conditions. In the in vivo study, UFH decreased thrombus weight, prolonged aPTT, bleeding time, and increased anti-Xa activity. PEG41-PMAPTAC53 reversed UFH effects without changing blood count. Enoxaparin and fondaparinux prolonged bleeding time, increased anti-Xa and induced a significant rise in the ACT. Infusion of PEG41-PMAPTAC53 restored all parameters and rapidly shortened ACT value to normal levels. After infusion of polymer in efficient neutralizing dose, there were no significant changes in cardiopulmonary and biochemical parameters. The fluorescence of PMAPTAC53 was detected in the bladder. PEG41-PMAPTAC53 showed no toxic effects on tissues.
Discussion: This study presents fully synthetic copolymer-PEG41-PMAPTAC53, for reversal parenteral anticoagulants. PEG41-PMAPTAC53 successfully neutralized UFH, enoxaparin, and fondaparinux in vitro. We confirmed efficacy and good safety profile of PEG41-PMAPTAC53 in various in vivo experimental setups.
Conclusion: PEG41-PMAPTAC53 has very promising properties and may help patients taking parenteral anticoagulants with uncontrolled bleeding.
Grant no. DEC-2011/03/B/NZ7/00755 from National Science Centre in Poland.; Grant no. UMO-2013/09/D/ST5/03864 from National Science Centre in Poland.
References:
[1] Kimmel SE, Sekeres M, Berlin JA, Ellison N. Mortality and adverse events after protamine administration in patients undergoing cardiopulmonary bypass. Anesth Analg. 2002; 94:1402-1408.
[2] Levy JH, Schwieger IM, Zaidan JR, Faraj BA, Weintraub WS. Evaluation of patients at risk for protamine reactions. J Thorac Cardiovasc Surg. 1989; 98:200–204.
[3] Povsic TJ, Vavalle JP, Aberle LH, Kasprzak JD, Cohen MG, Mehran R, Bode C, Buller CE, Montalescot G, Cornel JH, Rynkiewicz A, Ring ME, Zeymer U, Natarajan M, Delarche N, Zelenkofske SL, Becker RC, Alexander JH; RADAR Investigators. A Phase 2, randomized, partially blinded, active-controlled study assessing the efficacy and safety of variable anticoagulation reversal using the REG1 system in patients with acute coronary syndromes: results of the RADAR trial. Eur Heart J. 2013; 34:2481-9.
[4] Mahan CE. A 1-year drug utilization evaluation of protamine in hospitalized patients to identify possible future roles of heparin and low molecular weight heparin reversal agents. Thromb Thrombolysis 2014; 271-278.
[5] Kaminski K, Plonka M, Ciejka J, Szczubialka K, Nowakowska M, Lorkowska B, Korbut R, Lach R. Cationic derivatives of dextran and hydroxypropylcellulose as novel potential heparin antagonists. J Med Chem. 2011; 54:6586-6596.
[6] Kalaska B, Kaminski K, Sokolowska E, Czaplicki D, Kujdowicz M, Stalinska K, Bereta J, Szczubialka K, Pawlak D, Nowakowska M, Mogielnicki A. Nonclinical evaluation of novel cationically modified polysaccharide antidotes for unfractionated heparin. PLOS ONE 2015; 17:e0119486.