Jun 1, 2015
Bioxodes to present preclinical data for its main product, Ir-CPI, at ISTH 2015 annual congress.
Efficacy of a novel contact pathway inhibitor, Ir-CPI, in an extracorporeal membrane oxygenator model
Combe S1,2, Fromes Y3, Krezel C2, Gueret P4,5, Amiral J6, Guyaux M2 and Godfroid E2
1Hopitaux Universitaires Paris Centre, Paris, France; 2Bioxodes,Marche-en-Famenne, Belgium; 3Universite Pierre et Marie Curie, Paris; 4GETBO EA3878, Brest; 5Hemostasis Unit Hematology Laboratory, University Hospital, Rennes; 6Hyphen Biomed, Neuville sur Oise, France
Background: Ir-CPI, a protein derived from the tick Ixodes ricinus salivary, is a serine protease inhibitor of both FXIIa and FXIa. Heparins remain suboptimal in managing bleeding complications when extracorporeal life support is required.
Aims: The aim of this study was to evaluate whether inhibition of the contact phase of the coagulation cascade might confer antithrombotic activity.
Methods: Twelve Beagle dogs were included. A pediatric ECMO system was connected between the carotid artery and the jugular vein, and a cardiopulmonary bypass was maintained for 90 min at the level of full-flow systemic perfusion. Anticoagulation was performed by UFH (300 IU kg_1) with protamine reversal at the end of the procedure (N = 3). Three investigative groups (3 x N = 3) received various doses of Ir-CPI (bolus and infusion) total dose 4.5 to 24 mg kg_1. After disconnection, circuits and cannulae were examined for clot and protein deposits. All animals were followed-up and sampled for determination of Ir-CPI plasma concentration (Mass Spectrometry), aPTT, FXI and FXII residual activities, before necropsy on Day 7.
Results: All 12 animals completed procedure. First Ir-CPI group showed fibrin deposits, increased pressure gradient in the circuit, and subsequent decreased gas exchanges. In further animals perfusion of Ir-CPI was initiated earlier in order to decrease the observed clot initiation and to improve gas exchanges. High dose achieved close to optimal control. Low dose was less satisfactory but perfusion remained possible. A dose-dependent and related increase in Ir-CPI plasma concentration, increase in aPTT ratio and inhibition of factors XI and XII (20% and >70% respectively) were measured over the course of the procedure. No side effects were observed and necropsies confirm the absence of thrombosis or hemorrhage.
Conclusion: Ir-CPI can be used as a single agent to inhibit the intrinsic coagulation pathway and achieve anticoagulation. Efficiency can be improved without increasing bleeding risk.
Disclosure of Interest: S. Combe Shareholder of: the company developing agent, Consultant for: the company developing agent, Y. Fromes Consultant for: the company developing agent, C. Krezel Consultant for: the company developing agent, P. Gueret Grant/Research Support from: the company developing agent, J. Amiral: None declared, M. Guyaux Shareholder of: the company developing agent, Consultant for: the company developing agent, E. Godfroid Shareholder of: the company developing agent, Employee of: the company developing agent.
Antithrombotic effects of Ir-CPI in an arterio-venous shunt model in the rabbit
Guyaux M1, Gueret P2,3, Becher F4, Amiral J5, Simon S4 and Godfroid E1
1Bioxodes, Marche-en-Famenne, Belgium; 2Hemostasis Unit Hematology Laboratory, University Hospital, Rennes; 3GETBO EA3878, Brest; 4Laboratoire Etudes et Recherches Immunoanalyse, CEA Saclay, Gif sur Yvette; 5Hyphen Biomed, Neuville sur Oise, France
Background: Ir-CPI is a 67 aa protein derived from the salivary glands of the tick Ixodes ricinus. It is a contact phase inhibitor targeting specifically factors XIa and XIIa.
Aims: The aim of the study was to evaluate the antithrombotic potential and the pharmacokinetic-pharmacodynamic relationships of Ir-CPI.
Methods: The experiment was performed on New Zealand rabbits (n = 34) anesthetized with ketamine-xylazine. An arterio-venous (AV) shunt device containing a silk thread was connected between the femoral artery and vein. Ir-CPI was administered intravenously at variable doses as a bolus alone or followed by a continuous infusion starting 5 min before the opening of the AV-shunt. Thrombus weight was measured at the end of a 40 min shunt period. Blood samples were taken 10 min before and 45 min after administration. Plasma was prepared to monitor the activated partial thromboplastin time (aPTT), FXI and FXII activities and Ir-CPI concentration. The aPTT was measured using actin FS as reagent. Factors XI and XII activities were measured using FXI or FXII deficient plasmas and aPTT (Cephen) method. Ir-CPI plasma concentration was determined by mass spectrometry and enzyme-linked immunosorbent assay.
Results: Ir-CPI reduced thrombus weight by 36.8%, 62.1% and 97.2% at 1, 3 and 5 mg kg_1, iv and by 90.4% at 3 mg kg_1 followed by 2.3 mg kg_1 h infusion. Ir-CPI plasma concentrations increased proportionally with the dose. Antithrombotic activity (>90%) corresponded to a plasma Ir-CPI level of 2500 ng mL_1, a prolongation of the aPTT of 50%, an inhibition of FXI and FXII activities of 35– 40%. The in vivo effects on aPTT, FXI and FXII and Ir-CPI exposure level were consistent with the data obtained in vitro using rabbit plasma spiked with Ir-CPI.
Conclusion: These results demonstrate the antithrombotic potential of Ir-CPI in a model of AV-shunt in the rabbit whilst establishing the relationships between the antithrombotic efficacy, the Ir-CPI circulating concentration, the aPTT prolongation and the inhibition of FXI and FXII.
Disclosure of Interest: M. Guyaux Shareholder of: company developing agent, Consultant for: company developing agent, P. Gueret Grant/Research Support from: the company developing agent, F. Becher Grant/Research Support from: company developing agent, J. Amiral: None declared, S. Simon Grant/Research Support from: company developing agent, E. Godfroid Shareholder of: the company developing agent, Employee of: the company developing agent.
Evaluation of the effects of a novel contact pathway inhibitor, IR-CPI, on in vitro platelet function and coagulation
Jennings LK1,2, Kotha J2, Cardenas J2, Herr M2, Bhal V2, Dixon M2, White MM2, Combe S3 and Godfroid E3
1Vascular Biology/Medicine, University of Tennessee Health Science Center; 2CirQuest Labs, Memphis, USA; 3Bioxodes SA, Marche-en-Famenne, Belgium
Background: Ir-CPI, the tick Ixodes ricinus salivary protein, is a serine protease inhibitor under development as a novel anticoagulant. Ir-CPI inhibits Factors (F) XIIa, XIa, and kallikrein generation, prolongs the activated partial thromboplastin time, and confers antithrombotic activity in preclinical models.
Aims: The study aim was to test Ir-CPI effects on platelet function using light transmission aggregometry (LTA), clot retraction, and P-selectin expression and to characterize Ir-CPI inhibition of the contact activated coagulation pathway by assessing FXa and FIXa activation time and activity.
Methods: Ir-CPI effects on LTA in response to 20 µM ADP and 1.6 mM arachidonic acid (AA) were studied using consented blood donors (n = 5). Platelet P-selectin expression was evaluated using flow cytometry (n = 4) following activation by contact pathway initiation. Ir-CPI effects on PRP clot retraction was measured (n = 5), and FXa and FIXa generation were determined by quantitative chromogenic assays (n = 3).
Results: LTA response as well as clot retraction were unaffected by Ir-CPI pretreatment. However, the percent of P-selectin positive platelets was decreased following Ir-CPI treatment (0.5, 1, and 2 µM) by 5%, 9%, and 18%, respectively, vs. control. FXa activation lag times were prolonged to 704, 752, 826, 1013 s with increasing concentrations of Ir-CPI (1, 2, 4, 8 µM, respectively) vs. control (611 s). Similarly, FIXa activation lag times were 790, 834, 876, and 902 s, respectively, vs. 581 s (control). Also, the mean reaction rates (OD min_1) of FXa and FIXa with added Ir-CPI were decreased ~2.3-fold.
Conclusion: We demonstrated that Ir-CPI does not affect in vitro platelet aggregation response or clot retraction. However, Ir-CPI dose dependently blocked contact pathway mediated platelet P-selectin expression and the rate of FXa and FIXa activation and activity. This study confirms specific effects of Ir-CPI on the contact pathway of coagulation and provides a basis for monitoring Ir-CPI.
Disclosure of Interest: L. Jennings Grant/Research Support from: Bioxodes, J. Kotha: None declared, J. Cardenas: None declared, M. Herr: None declared, V. Bhal: None declared, M. Dixon: None declared, M. White: None declared, S. Combe Consultant for: Bioxodes, E. Godfroid Shareholder of: Bioxodes, Employee of: Bioxodes.Return