Ingo Pragst

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.

A factor XIIa inhibitory antibody provides thromboprotection in extracorporeal circulation without increasing bleeding risk.

Blocking the enzyme that initiates the intrinsic coagulation pathway protects against thrombosis in bypass systems and does not cause excess bleeding in vivo. When Life-Saving Is Life-Threatening We all need a vacation sometimes. For the heart and lungs, that time can come during surgery (such as cardiopulmonary bypass procedures), in instances of organ failure (for example, in septic patients), or while awaiting a replacement organ for transplantation. When the heart and lungs take time off, oxygenation of the blood needs to occur outside of the body by circulation through a cardiopulmonary bypass system (also called a heart-lung machine). In order to prevent blood clot formation in the extracorporeal circuit, an anticoagulant is added to the system. Anticoagulants block thrombus formation that would occlude the circulation; however, the drugs also interfere with the body’s ability to stop bleeding at the site of injury. Thus, an ideal anticoagulant would only block blood clotting in thrombosis without causing excess bleeding. Now, Larsson et al. describe a new antibody that prevents thrombosis and facilitates blood flow in a specific heart-lung machine without causing bleeding in large animal models. The anticoagulant heparin is used most often during extracorporeal oxygenation and targets multiple components of the blood coagulation cascade that are necessary formation of fibrin—a clotting protein essential for stemming injury-related blood loss. The authors used phage display to identify an antibody that binds to and inhibits the protease activity of factor XIIa (FXIIa), a protein that controls fibrin formation in vitro but does not appear to be required for cessation of bleeding from injury sites. A fully humanized version of the antibody, called 3F7, protected against pathological thrombosis in the extracorporeal bypass system without increasing bleeding from injuries in rabbits. 3F7 had the added benefits of a broad therapeutic range and easy monitoring at the point of care. And because 3F7 doesn’t cause bleeding, it should not require neutralization after surgery and can simply be cleared from the patient’s circulation naturally. Even with optimal heparin treatment, bleeding remains the most common complication of anticoagulation therapy. Additional mechanistic and clinical studies will show whether 3F7—or an optimized version—will be able to give heparin a vacation from facilitating extracorporeal circulation and possibly other scenarios that require safe anticoagulation. Currently used anticoagulants prevent thrombosis but increase bleeding. We show an anticoagulation therapy without bleeding risk based on a plasma protease factor XII function-neutralizing antibody. We screened for antibodies against activated factor XII (FXIIa) using phage display and demonstrated that recombinant fully human antibody 3F7 binds into the FXIIa enzymatic pocket. 3F7 interfered with FXIIa-mediated coagulation, abolished thrombus formation under flow, and blocked experimental thrombosis in mice and rabbits. We adapted an extracorporeal membrane oxygenation (ECMO) cardiopulmonary bypass system used for infant therapy to analyze clinical applicability of 3F7 in rabbits. 3F7 provided thromboprotection as efficiently as heparin, and both drugs prevented fibrin deposition and thrombosis within the extracorporeal circuit. Unlike heparin, 3F7 treatment did not impair the hemostatic capacity and did not increase bleeding from wounds. These data establish that targeting of FXIIa is a safe mode of thromboprotection in bypass systems, and provide a clinically relevant anticoagulation strategy that is not complicated by excess bleeding.

Animal model and clinical evidence indicating low thrombogenic potential of fibrinogen concentrate (Haemocomplettan P).

The objective of this study was to characterize the thrombogenicity of one pasteurized fibrinogen concentrate (Haemocomplettan P) in clinical use for over 20 years. Thrombus formation during venous stasis was assessed in rabbits receiving either 100 or 250 mg/kg Haemocomplettan P. Thrombotic adverse events possibly related to Haemocomplettan P were documented in a long-standing pharmacosurveillance program. A systematic review of thrombotic events in Haemocomplettan P clinical studies was also conducted. There was no evidence of thrombus formation during venous stasis in any Haemocomplettan P-treated animal. The pharmacosurveillance program spanned 22 years, during which a Haemocomplettan P quantity equivalent to more than 250 000 doses of 4 g each was distributed in 21 countries. Nine spontaneous reports of thrombotic adverse events possibly related to Haemocomplettan P were compiled, corresponding to an incidence rate of 3.48 events per 105 treatment episodes (95% confidence interval, 1.59–6.61 events per 105 treatment episodes). In 10 clinical studies involving 298 patients, one patient developed nonfatal venous thrombosis and pulmonary embolism to which fibrinogen concentrate may have contributed. Additionally, four other patients experienced arterial ischemic events that were likely attributable to massive hemorrhage and hypotension rather than fibrinogen replacement. Evidence from an animal model of venous stasis, a comprehensive pharmacosurveillance program, and a systematic review of clinical studies indicates that the thrombogenic potential of fibrinogen concentrate is low.

Novel Formulation of a Reconstituted High-Density Lipoprotein (CSL112) Dramatically Enhances ABCA1-Dependent Cholesterol Efflux

Objective—The ability of high-density lipoprotein (HDL) to remove cholesterol from atherosclerotic plaque is thought to underlie its inverse correlation with cardiovascular risk. Our objective was to produce and characterize a human apolipoprotein AI (apoA-I) product optimized to treat clinical atherosclerotic disease. Approach and Results—A new formulation of full length, plasma-derived human apoA-I termed CSL112 was designed to maximize the cholesterol efflux from cells and exhibit favorable pharmacological properties. CSL112 is a disc-shaped particle that strongly elevates cholesterol esterification and shows good pharmacokinetics in rabbits. Infusion of CSL112 into rabbits caused a strong and immediate increase in the ATP binding cassette transporter A1 (ABCA1)-dependent efflux capacity of plasma, an increase in plasma unesterified cholesterol and rapid subsequent cholesterol esterification. In the presence of human plasma, CSL112 was significantly more potent than native HDL at enhancing cholesterol efflux from macrophages, and the efflux elevation was predominantly via the ABCA1 transporter. Consistent with this observation, addition of CSL112 to plasma led to generation of high levels of HDL-VS, a favorable substrate for ABCA1. The lipid profile of plasma did not affect these behaviors. In studies with whole human blood, CSL112 reduced expression of intercellular adhesion molecule 1 and cytokine secretion, and as with cholesterol efflux, these activities were substantially greater than those of native HDL assayed in parallel. Conclusions—CSL112 has favorable pharmacological properties and strongly elevates the ability of plasma to withdraw cholesterol from cells. Preferential elevation of ABCA1-dependent efflux may target atherosclerotic plaque for cholesterol removal and this property makes CSL112 a promising candidate therapy for acute coronary syndrome.

Preclinical efficacy and safety of rVIII-SingleChain (CSL627), a novel recombinant single-chain factor VIII

INTRODUCTION The preclinical efficacy and safety of rVIII-SingleChain (CSL627), a novel recombinant single-chain factor VIII, was assessed in a series of animal studies. MATERIALS AND METHODS In the tail-clip bleeding model, hemophilia A mice were injected with escalating doses (1-150 IU/kg) of rVIII-SingleChain, B-domain deleted (BDD) rFVIII (ReFacto AF(®)), or full-length rFVIII products (Advate(®), Helixate(®)). Total blood loss and the percentage of animals in which hemostasis occurred were assessed in this observer-blinded, randomized study. In a second non-randomized study in hemophilia A mice, thromboelastographic analysis, thrombin generation, and activated partial thromboplastin time assays were performed. General safety and toxicity were assessed in three animal species, including determination of the prothrombotic potential of rVIII-SingleChain in a rabbit venous thrombosis model. RESULTS Under acute bleeding conditions, the effect of rVIII-SingleChain on total blood loss and hemostasis was indistinguishable from BDD and full-length rFVIII. rVIII-SingleChain and full-length rFVIII (both 20 IU/kg) corrected thromboelastographic parameters, activated partial thromboplastin time, and thrombin generation to a similar degree in hemophilia A mice. In a thrombosis model, the effect of rVIII-SingleChain on thrombus incidence was non-significant and comparable to BDD rFVIII at doses up to 500 IU/kg. Treatment with rVIII-SingleChain did not cause anaphylactic reaction or local intolerance in safety and toxicity studies, and demonstrated an excellent overall safety profile. CONCLUSIONS rVIII-SingleChain showed convincing hemostatic efficacy and excellent tolerability in animal studies, warranting continued investigation in human Phase I/III trials (AFFINITY).

Prevention of hereditary angioedema attacks with a subcutaneous C1 inhibitor

BACKGROUND Hereditary angioedema is a disabling, potentially fatal condition caused by deficiency (type I) or dysfunction (type II) of the C1 inhibitor protein. In a phase 2 trial, the use of CSL830, a nanofiltered C1 inhibitor preparation that is suitable for subcutaneous injection, resulted in functional levels of C1 inhibitor activity that would be expected to provide effective prophylaxis of attacks. METHODS We conducted an international, prospective, multicenter, randomized, double‐blind, placebo‐controlled, dose‐ranging, phase 3 trial to evaluate the efficacy and safety of self‐administered subcutaneous CSL830 in patients with type I or type II hereditary angioedema who had had four or more attacks in a consecutive 2‐month period within 3 months before screening. We randomly assigned the patients to one of four treatment sequences in a crossover design, each involving two 16‐week treatment periods: either 40 IU or 60 IU of CSL830 per kilogram of body weight twice weekly followed by placebo, or vice versa. The primary efficacy end point was the number of attacks of angioedema. Secondary efficacy end points were the proportion of patients who had a response (≥50% reduction in the number of attacks with CSL830 as compared with placebo) and the number of times that rescue medication was used. RESULTS Of the 90 patients who underwent randomization, 79 completed the trial. Both doses of CSL830, as compared with placebo, reduced the rate of attacks of hereditary angioedema (mean difference with 40 IU, –2.42 attacks per month; 95% confidence interval [CI], –3.38 to –1.46; and mean difference with 60 IU, –3.51 attacks per month; 95% CI, –4.21 to –2.81; P<0.001 for both comparisons). Response rates were 76% (95% CI, 62 to 87) in the 40‐IU group and 90% (95% CI, 77 to 96) in the 60‐IU group. The need for rescue medication was reduced from 5.55 uses per month in the placebo group to 1.13 uses per month in the 40‐IU group and from 3.89 uses in the placebo group to 0.32 uses per month in the 60‐IU group. Adverse events (most commonly mild and transient local site reactions) occurred in similar proportions of patients who received CSL830 and those who received placebo. CONCLUSIONS In patients with hereditary angioedema, the prophylactic use of a subcutaneous C1 inhibitor twice weekly significantly reduced the frequency of acute attacks. (Funded by CSL Behring; COMPACT EudraCT number, 2013‐000916‐10, and ClinicalTrials.gov number, NCT01912456.)

Pharmacological characteristics of a novel, recombinant fusion protein linking coagulation factor VIIa with albumin (rVIIa‐FP)

Recombinant factor VIIa (rFVIIa) is approved for use in controlling bleeding episodes in people with hemophilia who have developed inhibitors to replacement therapy. Due to its short half‐life (t½), frequent injections are required, limiting its use as a prophylactic treatment. A novel, recombinant fusion protein linking coagulation factor VIIa with albumin (rVIIa‐FP) has been developed to extend the t½ of rFVIIa.

Improved kinetics of rIX‐FP, a recombinant fusion protein linking factor IX with albumin, in cynomolgus monkeys and hemophilia B dogs

Summary.  Background:  Prophylaxis of hemophilia B, at present, requires multiple infusions of human factor (F)IX concentrates per week. A FIX molecule with a prolonged half‐life has the potential to greatly improve the convenience of, and adherence to, prophylaxis.

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.

Non-clinical pharmacokinetics and pharmacodynamics of rVIII-SingleChain, a novel recombinant single-chain factor VIII

INTRODUCTION rVIII-SingleChain (CSL627), a novel recombinant coagulation factor VIII (FVIII), is under investigation in a phase I/III clinical programme (AFFINITY) for the treatment of haemophilia A. Non-clinical studies were conducted to investigate the pharmacokinetic/pharmacodynamic profile of rVIII-SingleChain in comparison with full-length recombinant FVIII. MATERIALS AND METHODS Binding affinity of rVIII-SingleChain for von Willebrand factor was investigated by surface plasmon resonance analysis. The pharmacokinetic profile of rVIII-SingleChain was compared with a marketed full-length recombinant FVIII concentrate (Advate(®)) in haemophilia A mice, von Willebrand factor knock-out mice, Crl:CD (SD) rats, rabbits and cynomolgus monkeys. Systemic FVIII activity or antigen levels were recorded. Procoagulant activity was measured in an FeCl3-induced arterial occlusion model and by recording thrombin generation activity (ex vivo) after administration of 200-250 IU/kg rVIII-SingleChain or full-length FVIII to haemophilia A mice. RESULTS rVIII-SingleChain displayed a high affinity for von Willebrand factor (KD=44 pM vs. 139 pM for full-length recombinant FVIII). In all animal species tested, rVIII-SingleChain had more favourable pharmacokinetic properties than full-length recombinant FVIII: clearance was decreased and area under the curve and terminal half-life were enhanced vs. full-length recombinant FVIII, while in vivo recovery and volume of distribution were equivalent. rVIII-SingleChain showed a prolonged thrombin generation potential and prolonged procoagulant activity vs. full-length recombinant FVIII in an FeCl3-induced arterial occlusion model. CONCLUSIONS rVIII-SingleChain had a higher affinity for von Willebrand factor than full-length recombinant FVIII and displayed favourable pharmacokinetic/pharmacodynamic properties in non-clinical models.