Eva Herzog

Reversal of dabigatran anticoagulation by prothrombin complex concentrate (Beriplex P/N) in a rabbit model.

Summary.  Background:  One limitation of the direct thrombin inhibitor dabigatran is the lack of specific antidotes that allow acute bleeding events to be managed or urgent interventional procedures performed. Prothrombin complex concentrates (PCCs) have served as a standard treatment for the reversal of coumarin anticoagulation.

Effective Reversal of Edoxaban-associated Bleeding with Four-factor Prothrombin Complex Concentrate in a Rabbit Model of Acute Hemorrhage

Background:Edoxaban is an oral, selective direct factor Xa inhibitor approved in Japan for venous thromboembolism prevention after orthopedic surgery. Data are lacking regarding reversal strategies for edoxaban; this study assessed whether four-factor prothrombin complex concentrate (Beriplex®/Kcentra®; CSL Behring GmbH, Marburg, Germany) can effectively reverse its effects on hemostasis using a previously described rabbit model. Methods:The study comprised assessments of thrombin generation in vitro, pharmacokinetic parameters, and edoxaban reversal in vivo. In a blinded in vivo stage, a standardized kidney incision was performed in animals (n = 11 per group) randomized to receive vehicle + saline, edoxaban (1,200 &mgr;g/kg) + saline, or edoxaban (1,200 &mgr;g/kg) + four-factor prothrombin complex concentrate (50 IU/kg). Animals were monitored for treatment impact on hemostasis and coagulation parameters. Data are median (range). Statistical tests were adjusted for multiple testing. Results:Edoxaban administration increased blood loss (30 [2 to 44] ml) and time to hemostasis (23 [8.5 to 30.0] min) compared with the control group (3 [1 to 8] ml and 3 [2.0 to 5.0] min, respectively). Biomarkers of coagulation (prothrombin time, activated partial thromboplastin time, whole blood clotting time) and thrombin generation parameters (e.g., peak thrombin, endogenous thrombin potential, lag time) were also affected by edoxaban. Administration of four-factor prothrombin complex concentrate significantly reduced time to hemostasis (to 8 [6.5 to 14.0] min, observed P < 0.0001) and total blood loss (to 9 [4 to 22] ml, observed P = 0.0050) compared with the edoxaban + saline group. Of the biomarkers tested, prothrombin time, whole blood clotting time, and endogenous thrombin potential correlated best with clinical parameters. Conclusion:In a rabbit model of hemostasis, four-factor prothrombin complex concentrate administration significantly decreased edoxaban-associated hemorrhage.

Correlation of Coagulation Markers and 4F-PCC-Mediated Reversal of Rivaroxaban in a Rabbit Model of Acute Bleeding

INTRODUCTION Rivaroxaban is an oral, selective direct factor Xa inhibitor approved for several indications in patients at risk of thrombotic events. One limitation of its clinical use is the lack of data pertaining to its reversal in situations where urgent response is critical (e.g. acute bleeding events or emergency surgery). MATERIALS AND METHODS This study assessed the effectiveness of a four-factor prothrombin complex concentrate (4F-PCC; Beriplex(®)/Kcentra(®)) for the reversal of rivaroxaban-associated bleeding in an in vivo rabbit model, and evaluated the correlations between in vitro coagulation parameters and haemostasis in vivo. RESULTS Administration of single intravenous doses of rivaroxaban (150-450 μg/kg) resulted in increased and prolonged bleeding following standardised kidney incision. Pre-incision treatment with 4F-PCC (25-100 IU/kg) resulted in a dose-dependent reversal of rivaroxaban (150 and 300 μg/kg)-associated increases in time to haemostasis and blood loss; no reversal was seen at the highest rivaroxaban dose (450 μg/kg). Of the in vitro biomarkers tested, thrombin generation and whole-blood clotting time correlated well with in vivo measures of 4F-PCC-mediated effects. Thrombin generation was highly reagent-dependent, with the assay initiated using the phospholipid-only reagent being the most predictive of effective haemostasis in vivo. CONCLUSIONS In summary, in a rabbit model of acute bleeding, treatment with 4F-PCC reduced bleeding to control levels following rivaroxaban 150 μg/kg and 300 μg/kg administration.

Biodistribution of the recombinant fusion protein linking coagulation factor IX with albumin (rIX-FP) in rats

INTRODUCTION The recombinant fusion protein linking coagulation factor IX with albumin (rIX-FP) is undergoing clinical trials for prophylaxis and on-demand treatment of haemophilia B patients. The aim of this study was to investigate the pharmacokinetics, whole-body and knee joint distribution of rIX-FP following intravenous administration to rats, compared with a marketed, non-fused rFIX and recombinant human albumin. MATERIAL AND METHODS [(3)H]-rIX-FP, [(3)H]-rFIX or [(3)H]-albumin were administered to rats followed by quantitative whole-body autoradiography over 24 or 240 hours, and the tissue distribution as well as elimination of radioactivity were measured. RESULTS Elimination of all radioactivity derived from the three proteins was shown to occur primarily via the urine. The tissue distribution of [(3)H]-rIX-FP and [(3)H]-rFIX (but not of [(3)H]-albumin) was comparable, both penetrating predominantly into bone, and well-perfused tissues, suggesting that the rIX moiety determines the distribution pattern of rIX-FP, while the albumin moity is responsible for the prolonged plasma and tissue retention. Detailed knee-joint analysis indicated rapid presence of [(3)H]-rIX-FP and [(3)H]-rFIX in synovial and mineralised bone tissue, mostly localised to the zone of calcified cartilage. Longest retention times were observed in the bone marrow and the endosteum of long bones. Intriguingly, [(3)H]-rIX-FP- and [(3)H]-albumin-derived radioactive signals were detectable up to 240 hours, while [(3)H]-rFIX-derived radioactivity rapidly declined after 1hour post-dosing correlating to the extended plasma half-life of [(3)H]-rIX-FP. CONCLUSION The prolonged plasma and tissue retention of rIX-FP achieved by albumin fusion may allow a reduction in dosing frequency leading to increased therapeutic compliance and convenience.

Thrombotic safety of prothrombin complex concentrate (Beriplex P/N) for dabigatran reversal in a rabbit model.

INTRODUCTION In vivo animal data have shown prothrombin complex concentrate (PCC) to be effective in preventing bleeding induced by excessive plasma levels of the direct thrombin inhibitor dabigatran. This animal model study was designed to determine the risk of thrombosis associated with administration of a PCC (Beriplex P/N) to reverse dabigatran-induced bleeding. MATERIALS AND METHODS Anesthetized rabbits were treated with initial 0, 75, 200 or 450 μg kg(-1) dabigatran boluses followed by continuous infusions to maintain elevated plasma dabigatran levels. At 15 min after the start of dabigatran administration, PCC doses of 0, 50 or 300 IU kg(-1) were administered. Thereafter, coagulation in an arteriovenous (AV) shunt was evaluated and histopathologic examination for thrombotic changes performed. Venous thrombosis was also assessed in a modified Wessler model. RESULTS At the suprapharmacologic dose of 300 IU kg(-1), PCC increased thrombus weight during AV shunting, but this effect could be prevented by dabigatran at all tested doses. AV shunt occlusion after PCC administration was delayed by 75 μg kg(-1) dabigatran and abolished by progressively higher dabigatran doses. High-dose treatment with 300 IU kg(-1) PCC resulted in histologically evident low-grade pulmonary thrombi; however, that effect could be blocked by dabigatran in a dose-dependent manner (p=0.034). In rabbits treated with high-dose PCC, dabigatran inhibited thrombus formation during venous stasis. PCC effectively reversed dabigatran-induced bleeding. CONCLUSIONS In this animal study, thrombosis after PCC administration could be prevented in the presence of dabigatran. PCC reversed dabigatran-induced excessive bleeding while retaining protective anticoagulatory activity of dabigatran.

Four-factor prothrombin complex concentrate reverses apixaban-associated bleeding in a rabbit model of acute hemorrhage.

Apixaban is a direct factor Xa inhibitor approved for the treatment and prevention of thromboembolic disease. There is a lack of data regarding its reversal in cases of acute bleeding or prior to emergency surgery that needs addressing.

Analysis of the safety and pharmacodynamics of human fibrinogen concentrate in animals.

Fibrinogen, a soluble 340kDa plasma glycoprotein, is critical in achieving and maintaining hemostasis. Reduced fibrinogen levels are associated with an increased risk of bleeding and recent research has investigated the efficacy of fibrinogen concentrate for controlling perioperative bleeding. European guidelines on the management of perioperative bleeding recommend the use of fibrinogen concentrate if significant bleeding is accompanied by plasma fibrinogen levels less than 1.5-2.0g/l. Plasma-derived human fibrinogen concentrate has been available for therapeutic use since 1956. The overall aim of the comprehensive series of non-clinical investigations presented was to evaluate i) the pharmacodynamic and pharmacokinetic characteristics and ii) the safety and tolerability profile of human fibrinogen concentrate Haemocomplettan P® (RiaSTAP®). Pharmacodynamic characteristics were assessed in rabbits, pharmacokinetic parameters were determined in rabbits and rats and a safety pharmacology study was performed in beagle dogs. Additional toxicology tests included: single-dose toxicity tests in mice and rats; local tolerance tests in rabbits; and neoantigenicity tests in rabbits and guinea pigs following the introduction of pasteurization in the manufacturing process. Human fibrinogen concentrate was shown to be pharmacodynamically active in rabbits and dogs and well tolerated, with no adverse events and no influence on circulation, respiration or hematological parameters in rabbits, mice, rats and dogs. In these non-clinical investigations, human fibrinogen concentrate showed a good safety profile. This data adds to the safety information available to date, strengthening the current body of knowledge regarding this hemostatic agent.

Reconstituted high-density lipoprotein can elevate plasma alanine aminotransferase by transient depletion of hepatic cholesterol: role of the phospholipid component.

Human apolipoprotein A‐I preparations reconstituted with phospholipids (reconstituted high‐density lipoprotein [HDL]) have been used in a large number of animal and human studies to investigate the physiological role of apolipoprotein A‐I. Several of these studies observed that intravenous infusion of reconstituted HDL might cause transient elevations in plasma levels of hepatic enzymes. Here we describe the mechanism of this enzyme release. Observations from several animal models and in vitro studies suggest that the extent of hepatic transaminase release (alanine aminotransferase [ALT]) correlates with the movement of hepatic cholesterol into the blood after infusion. Both the amount of ALT release and cholesterol movement were dependent on the amount and type of phospholipid present in the reconstituted HDL. As cholesterol is known to dissolve readily in phospholipid, an HDL preparation was loaded with cholesterol before infusion into rats to assess the role of diffusion of cholesterol out of the liver and into the reconstituted HDL. Cholesterol‐loaded HDL failed to withdraw cholesterol from tissues and subsequently failed to cause ALT release. To investigate further the role of cholesterol diffusion, we employed mice deficient in SR‐BI, a transporter that facilitates spontaneous movement of cholesterol between cell membranes and HDL. These mice showed substantially lower movement of cholesterol into the blood and markedly lower ALT release. We conclude that initial depletion of hepatic cholesterol initiates transient ALT release in response to infusion of reconstituted HDL. This effect may be controlled by appropriate choice of the type and amount of phospholipid in reconstituted HDL. Copyright

C1-esterase inhibitor treatment: preclinical safety aspects on the potential prothrombotic risk.

Human plasma-derived C1-esterase inhibitor (C1-INH) is an efficacious and safe treatment for hereditary angioedema. However, thrombotic events in subjects treated with C1-INH at recommended or off-label, high doses have been reported. In this study, we addressed the potential prothrombotic risk of C1-INH treatment in high doses using a non-clinical rabbit model. Following intravenous infusion of C1-INH to rabbits at doses up to 800 IU/kg, the exposure and the pharmacodynamic efficacy of C1-INH in rabbits were confirmed by activity measurements of C1-esterase, and coagulation factors XIa and XIIa, respectively. Potential prothrombotic effects were assessed following induction of venous and arterial thrombosis using in vivo models of venous and arterial stasis, complemented by various in vitro assays of coagulation markers. Administration of C1-INH at doses up to 800 IU/kg did not potentiate thrombus formation during venous stasis. In contrast, inhibition of arterial occlusion was observed upon C1-INH administration when compared with isotonic saline treatment, indicating antithrombotic rather than prothrombotic activity of high dose C1-INH treatment in vivo. This was further confirmed in vitro by decreased thrombin generation, increased activated partial thromboplastin time, clotting time and clot formation time, and inhibition of platelet aggregation. No relevant changes in fibrinolysis or in the levels of thrombin-antithrombin complexes, and prothrombin fragment 1+2 were observed upon high dose C1-INH treatment. The data suggest that treatment of healthy rabbits with high doses of C1-INH could potentially inhibit coagulation and thrombus formation rather than induce a prothrombotic risk.

Recombinant fusion protein linking factor VIIa with albumin (rVIIa-FP): Tissue distribution in rats.

INTRODUCTION A novel fusion protein linking coagulation factor VIIa with albumin (rVIIa-FP) is currently undergoing clinical investigations. OBJECTIVE This study was conducted to examine the biodistribution of rVIIa-FP in comparison to recombinant factor VIIa (rFVIIa). MATERIALS AND METHODS [(3)H]-rVIIa-FP (10mgkg(-1)) or [(3)H]-rFVIIa (1.6mgkg(-1)) were administered intravenously to rats, followed by quantitative whole-body and knee joint autoradiography for 24 ([(3)H]-rFVIIa) or 240 ([(3)H]-rVIIa-FP) hours post-dose. Pharmacokinetic and excretion balance analyses were performed. RESULTS In contrast to [(3)H]-albumin, the tissue distributions of [(3)H]-rVIIa-FP and [(3)H]-rFVIIa were similar. Within the knee, both were rapidly present within synovial and mineralized regions. Importantly, rVIIa-FP- and albumin-derived radioactivity were detectable up to 72-120hours, whereas [(3)H]-rFVIIa signals were already close to detection limits at 24hours. The longest rVIIa-FP retention times were observed in bone marrow and endosteum, in which the retention times were up to 5 times longer for rVIIa-FP compared with rFVIIa. Up to 8hours post-dose, 100% of radioactivity was assigned to unchanged [(3)H]-rVIIa-FP. Elimination of both proteins occurred primarily via the urine. CONCLUSIONS The data suggest that the FVIIa moiety is directing rVIIa-FPs tissue distribution while the albumin moiety is responsible for the prolonged tissue retention. Importantly, rVIIa-FP is highly concentrated and retained over a long period in the growth plate of the knee joint-a vulnerable site in haemophilia patients. Overall, these improved tissue distribution characteristics of rVIIa-FP may enhance compliance and allow a more convenient dosing frequency.