Increased Accumulation and Retention of rhFVIIa (eptacog beta) in Knee Joints of Hemophilia A Mice Compared to Wild-Type Mice.

Our earlier studies showed that recombinant human factor VIIa (rhFVIIa) administered intravascularly in mice disappeared rapidly from the circulation. However, a small fraction of rhFVIIa that entered extravascular remained functionally active for an extended period. The present study aims to investigate the dose-dependency of rhFVIIa accumulation and retention in mouse knee joints and test whether the hemophilic condition affects rhFVIIa sequestration in joints. Wild-type and FVIII-/- mice were injected with three doses of rhFVIIa (eptacog beta, 90, 250, and 500 µg/kg) via the tail vein. At varying times following rhFVIIa administration, blood and knee joints were collected to measure FVIIa activity and antigen levels in plasma and joint tissues. Joint tissue sections were analyzed by immunohistochemistry for the presence of rhFVIIa. Vascular permeability was assessed by either Evans Blue dye or fluorescein dextran extravasation. The study showed that rhFVIIa accumulated in knee joints of wild-type and FVIII-/- mice in a dose-dependent manner. rhFVIIa antigen and FVIIa activity could be detectable in joints for at least 7 days. Significantly higher levels of rhFVIIa accumulation were observed in knee joints of FVIII-/- mice compared with that of wild-type mice. Immunohistochemical analyses confirmed higher levels of rhFVIIa retention in FVIII-/- mice compared with wild-type mice. Additional studies showed that FVIII-/- mice were more permissible to vascular leakage. In conclusion, the present data demonstrate a dose-dependent accumulation of rhFVIIa in knee joints, and the hemophilic condition enhances the entry of rhFVIIa from circulation to the extravascular. The present data will be useful in improving rhFVIIa prophylaxis.

Administration of recombinant FVIIa (rFVIIa) to concizumab-dosed monkeys is safe, and concizumab does not affect the potency of rFVIIa in hemophilic rabbits.

Essentials Hemophilia patients on concizumab prophylaxis may need rFVIIa to treat breakthrough bleeds. Effect and safety of concizumab + rFVIIa were tested in vitro and in vivo. Concizumab + rFVIIa had no additive effects on bleeding in hemophilic rabbits. High steady-state levels of concizumab did not affect the safety of rFVIIa in cynomolgus monkeys. SUMMARY: Background Concizumab is a monoclonal antibody (mAb) against tissue factor pathway inhibitor (TFPI), currently in clinical development as a subcutaneous prophylactic therapy for hemophilia A/B with and without inhibitors. In patients with inhibitors, the treatment choice for breakthrough bleeding will comprise bypassing agents, e.g. activated recombinant FVIIa (rFVIIa) or activated prothrombin complex concentrates. Objectives To explore the effect and safety of concizumab and rFVIIa when they are simultaneously present. Methods Human blood made hemophilic with a FVIII antibody was spiked with increasing concentrations of concizumab, rFVIIa, or concizumab and rFVIIa in combination, and this was followed by thrombin generation test or thromboelastography. Blood loss in hemophilic rabbits was measured when concizumab, rFVIIa or concizumab + rFVIIa was administered either before or during cuticle bleeding. In a safety study, cynomolgus monkeys were exposed to high steady-state concizumab concentrations and given three doses of rFVIIa, and then subjected to full necropsy and histopathological examination. Results In human blood, concizumab + rFVIIa had more pronounced procoagulant effects under hemophilic conditions than the sum of individual responses. In contrast, concizumab + rFVIIa had no additional effects on blood loss in hemophilic rabbits as compared with rFVIIa or concizumab alone. In cynomolgus monkeys, the macroscopic and microscopic pathological examinations revealed no thrombi or other signs of excessive coagulation activation. Both rFVIIa and concizumab caused increases in thrombin-antithrombin and D-dimer concentrations; this effect tended to be additive with concomitant administration. Conclusions Concizumab did not affect the potency or safety of rFVIIa in vivo. These results support a clinical evaluation of rFVIIa at standard dose (90 µg kg-1 ) to treat breakthrough bleeds in concizumab clinical trials.

Phase 1, single-dose escalating study of marzeptacog alfa (activated), a recombinant factor VIIa variant, in patients with severe hemophilia.

Essentials Marzeptacog alfa (activated) [MarzAA] is a novel variant of activated human factor VII. A phase 1 dose escalation trial of MarzAA was conducted in subjects with severe hemophilia. MarzAA was safe and tolerated at intravenous doses up to 30 µg kg-1 Data observed support further trials for hemophilia patients with inhibitors to factors VIII/IX. SUMMARY: Background Marzeptacog alfa (activated) (MarzAA), a new recombinant activated human factor VII (rFVIIa) variant with four amino acid substitutions, was developed to provide increased procoagulant activity and a longer duration of action in people with hemophilia. Objectives To investigate the safety, tolerability, immunogenicity, pharmacokinetics (PK) and pharmacodynamics (PD) of single ascending intravenous bolus doses of MarzAA in non-bleeding patients with congenital hemophilia A or B with or without inhibitors. Methods This international, phase 1, open-label study (NCT01439971) enrolled males aged 18-64 years with severe hemophilia A or B, with or without FVIII or FIX inhibitors. Subjects were assigned to single-dose MarzAA cohorts (0.5, 4.5, 9, 18 or 30 µg kg-1 ). Blood sampling was performed predose and postdose, and subjects were monitored for 60 days postdose. Safety endpoints included adverse events, vital sign changes, electrocardiograms, laboratory abnormalities, and immunogenicity; secondary endpoints included evaluation of PK and PD. Results Overall, in 25 patients, MarzAA was well tolerated at all dose levels tested, and was not associated with dose-limiting toxicity. No treatment-emergent severe or serious adverse events occurred. MarzAA showed linear dose-response PK across the 4.5-30 µg kg-1 dose range, with a terminal half-life of ⁓ 3.5 h. Dose-dependent shortening of the activated partial thromboplastin time and prothrombin time, and evidence of an increase in peak thrombin as determined with a thrombin generation assay, were observed at all doses. Conclusions MarzAA was tolerated at doses up to 30 µg kg-1 . The safety profile and pharmacological effects observed support further clinical trials for the treatment of hemophilic patients with inhibitors.

Prediction of human pharmacokinetics of activated recombinant factor VII and B-domain truncated factor VIII from animal population pharmacokinetic models of haemophilia.

Various experimental animal models are used in haemophilia research, however, little is known about how well the different species predict pharmacokinetic (PK) profiles in haemophilia patients. The aim of the current study was to describe the plasma concentration-time profile of recombinant activated factor VII (rFVIIa) and recombinant factor VIII (rFVIII) in several experimental animal models using population PK modelling, and apply a simulation-based approach to evaluate how well the developed animal population PK models predict human PK. PK models were developed for rFVIIa and rFVIII in mice, rats, monkeys, and dogs using nonlinear mixed-effects modelling, accounting for inter-individual variability, nonlinear kinetics and covariate effects. Three scaling principles were applied to predict human PK: proportional scaling to body weight from single species, scaling with fixed theory-based allometric exponents from single species, and allometric interspecies scaling with estimated allometric coefficients and exponents. The plasma concentration-time profile of rFVIIa and rFVIII in mice, rats, monkeys and dogs were accurately described by the developed species-specific PK models, accounting for nonlinear kinetics and gender-specific difference in clearance for rFVIII. The predictive performance of the animal population PK models of rFVIIa and rFVIII revealed significant species-variation. The developed PK models of rFVIIa and rFVIII in monkeys and dogs along with allometric interspecies scaling revealed high predictive performance for human PK, and may promote rational decision-making in future first-in-human trials for rFVIIa and rFVIII variants.

Safety and dose-dependency of eptacog beta (activated) in a dose escalation study of non-bleeding congenital haemophilia A or B patients, with or without inhibitors.

INTRODUCTION: Varying initial doses of activated eptacog beta (recombinant human FVIIa, rhFVIIa) may provide therapeutic options when treating bleeding in patients with congenital haemophilia who have developed inhibitory antibodies to factor VIII (FVIII) or factor IX (FIX). This study evaluated escalated doses of a new rhFVIIa product as a prelude to selecting the doses for clinical efficacy evaluation in haemophilia patients. AIM: To assess the safety, pharmacokinetics, and laboratory pharmacodynamics of 3 doses of rhFVIIa in non-bleeding patients with congenital haemophilia A or B with or without inhibitors. METHODS: Adult male patients (18-75 years old) with congenital haemophilia A or B (with or without inhibitors) received infusions of rhFVIIa at doses of 25, 75 or 225 µg/kg body weight. Ten patients were treated at each dose level, and each patient received 2 different dose levels. Descriptive methods were used to analyse the data. RESULTS: Administration of rhFVIIa at all doses was well tolerated. Pharmacokinetic analyses showed that peak FVIIa plasma levels (Cmax ) were approximately proportional to dose and correlated well with peak thrombin generation. Total AUC0-inf also was approximately dose proportional. Clot formation and duration correlated with FVIIa activity. Repeat doses did not produce an immunological response. CONCLUSION: In the first dose-escalation study of rhFVIIa to support product registration, eptacog beta at doses of 25, 75, and 225 µg/kg was pharmacodynamically active and well tolerated in non-bleeding patients with congenital haemophilia A or B.

The pharmacokinetics and pharmacodynamics of single-dose and multiple-dose recombinant activated factor VII in patients with haemophilia A or B.

Monitoring recombinant activated factor VII (rFVIIa) treatment outcomes remains challenging. Thromboelastography (TEG) and the thrombin generation assay (TGA), measure coagulation dynamics over time and are being assessed as potential methods for evaluating and monitoring haemophilia treatment. Lack of standardized TEG/TGA methods makes it difficult to compare results and to establish a correlation with clinical outcomes. AIMS: To compare the pharmacokinetics (PK) of rFVIIa after 3×90 µg kg-1 doses vs a single dose (270 µg kg-1 ) in haemophilia patients and to evaluate TEG/TGA results postdosing to determine how these assays relate to PK findings. METHODS: Patients in this open-label, single-centre, randomized, crossover trial received one injection of 270 µg kg-1 rFVIIa crossed over with three injections of 90 µg kg-1 rFVIIa in a non-bleeding state. For TEG, kaolin and tissue factor were used as activators; TGA was performed on frozen platelet-rich and platelet-poor plasma, with and without corn trypsin inhibitor. FVIIa activity was evaluated using in vivo samples. RESULTS: TGA showed a dose-dependent effect of rFVIIa on thrombin generation; TEG revealed lower dose-dependent effects. Both showed some differences between single-/multiple-dose rFVIIa; both supported the PK findings. CONCLUSION: While TEG and TGA are not yet clinically predictive, both supported the PK results. Data suggest that, while a single dose of 270 µg kg-1 rFVIIa provides slightly higher haemostatic potential than the multiple-dose regimen of 3×90 µg kg-1 , the latter results in prolonged activity levels compared with a higher single dose.

Anticoagulant Reversal and Anesthetic Considerations.

Bleeding complications are a common concern with the use of anticoagulant agents. In many situations, reversing of neutralizing their effects may be warranted. Prothrombin complex concentrate replaces coagulation factors lowered by warfarin, as does fresh frozen plasma, but in a more concentrated form. Protamine negates the effect of heparin and combines chemically with heparin molecules to form an inactive salt. It also partially reverses the effects of low-molecular-weight heparin. Recombinant activated factor VII is a nonspecific procoagulant that activates the extrinsic clotting pathway, resulting in thrombin generation, but does not directly neutralize the activity of any of the new oral anticoagulants.

Modificar la especificación técnica del producto farmacéutico Factor VIIA o EPTACOG ALFA (Activado) 2 punto 4 mg/vial + sdiluyente - AM incluido en el Petitorio Farmacológico de ESSALUD / MModify the technical specification of the pharmaceutical product Factor VIIA or EPTACOG ALFA (Activated) 2 score 4 mg / vial + solvent-AM included in the Pharmacological Request of ESSALUD

La Carta Número 304-DETS-IETSI-ESSALUD-2016 y el Informe Número 135-SDEPFyOT-DETS-IETSI-ESALUD-2016 que sustenta la modificación del producto farmacéutico FACTOR VIIA o EPTACOG ALFA (Activado) 2.4 mg/vial + diluyente - AM en el Petitorio Farmacológico de ESSALUD. SE RESUELVE: MODIFICAR: La especificación técnica del producto farmacéutico FACTOR VIIA o EPTACOG ALFA (Activado) 2.4 mg/vial + diluyente - AM en el Petitorio Farmacológico de ESSALU según consta en tabla de la Resolución. DISPONER: que la Dirección de Evaluación de Tecnologías Sanitarias del Instituto de Evaluación de Tecnologías en Salud e Investigación haga de conocimiento a todos los órganos desconcentrados, órganos prestadores nacionales, establecimientos de salud y demás órganos que correspondan, la presente Resolución. DISPONER: que la Dirección de Evaluación de Tecnologías Sanitarias del Instituto de Evaluación de Tecnologías en Salud e Investigación coordine con la Gerencia Central de Tecnologías de Información y Comunicaciones, la publicación de la inclusión de los referidos medicamentos en el Petitorio Farmacológico en la pagina WEB de ESSALUD.

Entering new areas in known fields: recombinant fusion protein linking recombinant factor VIIa with recombinant albumin (rVIIa-FP) - advancing the journey.

The novel fusion protein linking recombinant factor VIIa with recombinant albumin (rVIIa-FP) is designed to extend the half-life of recombinant factor VIIa (rFVIIa) and improve the care of hemophilia A or B patients with inhibitors. Preclinical studies in various animal models have demonstrated markedly improved pharmacokinetic and pharmacodynamic properties, as well as prolonged retention in the joint tissues, of rVIIa-FP compared with a commercially available rFVIIa (NovoSeven®). A phase I study in healthy volunteers - the first study in the PROLONG-7FP program - confirmed that rVIIa-FP has a good tolerability profile in doses of up to 1,000µg/kg and has demonstrated enhanced pharmacodynamic activity relative to rFVIIa. The half-life of rVIIa-FP at the highest dose investigated in the study was 8.5hours, which represents a 3- to 4-fold half-life extension compared with rFVIIa. Encouraging results from preclinical and phase I studies have led to the initiation of clinical studies of rVIIa-FP in patients with congenital hemophilia A or B and inhibitors, and in patients with confirmed factor VII deficiency. The results from these studies are awaited with interest by clinicians and patients alike.

New Insights Into the Treatment of Glanzmann Thrombasthenia.

Glanzmann thrombasthenia (GT) is a rare inherited autosomal recessive bleeding disorder of platelet function caused by a quantitative or qualitative defect of platelet membrane glycoprotein IIb/IIIa (integrin αIIbß3), a fibrinogen receptor required for platelet aggregation. Bleeds in GT are variable and may be severe and unpredictable. Bleeding not responsive to local and adjunctive measures, as well as surgical procedures, is treated with platelets, recombinant activated factor VII (rFVIIa), or antifibrinolytics, alone or in combination. Although platelets are the standard treatment for GT, their use is associated with the risk of blood-borne infection transmission and may also cause the development of platelet antibodies (to human leukocyte antigens and/or αIIbß3), potentially resulting in platelet refractoriness. Currently, where rFVIIa is approved for use in GT, this is mostly for patients with platelet antibodies and/or a history of platelet refractoriness. However, data from the prospective Glanzmann's Thrombasthenia Registry (829 bleeds and 206 procedures in 218 GT patients) show that rFVIIa was frequently used in nonsurgical and surgical bleeds, with high efficacy rates, irrespective of platelet antibodies/refractoriness status. The mechanisms underpinning rFVIIa effectiveness in GT have been studied. At therapeutic concentrations, rFVIIa binds to activated platelets and directly activates FX to FXa, resulting in a burst of thrombin generation. Thrombin converts fibrinogen to fibrin and also enhances GT platelet adhesion and aggregation mediated by the newly converted (polymeric) fibrin, leading to primary hemostasis at the wound site. In addition, thrombin improves the final clot structure and activates thrombin-activatable fibrinolysis inhibitor to decrease clot lysis.

Rapid rFVIIa enhanced on-demand dosing in haemophilia inhibitor patients.

Recombinant factor VII activated (rFVIIa) is a bypassing agent widely used in haemophilia A and B patients with antibodies against coagulation factors VIII or IX. When used according to the correct doses, rFVIIa may control bleeding, subclinical bleeding and rebleeding, avoiding the effect of neutralising inhibitors. Because of the fast action of the rFVIIa, haemostasis occurs promptly and enables a fast bleeding control with on-demand treatment in home or in surgical setting. Rapidity is also a distinguishing feature in preparation and injection of rFVIIa to cope the restraining times of busy patients and parents. The effective haemostatic activity of rFVIIa enables a sustained bleeding control, which is implemented with every other day (eod) administration and suited for enhanced on-demand therapy and short-term repeated infusions use of rFVIIa to prevent microhaemorrhages or rebleeding. Comprehensive appreciation of these pharmacological and pharmacodynamic' characteristics will likely be a further stimulus to the wider enhanced on-demand use of rFVIIa.

Changes in the amino acid sequence of the recombinant human factor VIIa analog, vatreptacog alfa, are associated with clinical immunogenicity.

BACKGROUND: Vatreptacog alfa, a recombinant human factor VIIa (rFVIIa) analog developed to improve the treatment of bleeds in hemophilia patients with inhibitors, differs from native FVIIa by three amino acid substitutions. In a randomized, double-blind, crossover, confirmatory phase III trial (adept(™) 2), 8/72 (11%) hemophilia A or B patients with inhibitors treated for acute bleeds developed anti-drug antibodies (ADAs) to vatreptacog alfa. OBJECTIVES: To characterize the formation of anti-vatreptacog alfa ADAs in hemophilia patients with inhibitors. METHODS/PATIENTS: This was a post hoc analysis of adept(™) 2. Immunoglobulin isotype determination, specificity analysis of rFVIIa cross-reactive antibodies, epitope mapping of rFVIIa single mutant analogs and pharmacokinetic (PK) profiling were performed to characterize the ADAs. RESULTS: Immunoglobulin isotyping indicated that the ADAs were of the immunoglobulin G subtype. In epitope mapping, none of the rFVIIa single mutant analogs (V158D, E296V or M298Q) contained the complete antibody epitope, confirming that the antibodies were specific for vatreptacog alfa. In two patients, for whom PK profiling was performed both before and after the development of ADAs, vatreptacog alfa showed a prolonged elimination phase following ADA development. During the follow-up evaluation, the rFVIIa cross-reactivity disappeared after the last vatreptacog alfa exposure, despite continued exposure to rFVIIa as part of standard care. CONCLUSIONS: Results from the vatreptacog alfa phase III trial demonstrate that the specific changes made, albeit relatively small, to the FVIIa molecule alter its clinical immunogenicity.

Enhanced Pharmacokinetics of Factor VIIa as a Monomeric Fc Fusion.

Recombinant Factor VIIa (rFVIIa) is utilized for on-demand treatment of bleeding episodes in hemophilia patients with neutralizing antibodies (inhibitors) against Factor VIII or Factor IX, but a short half-life in the circulation (~2.5hrs) limits its use in a prophylactic setting. Recombinant FVIIa variants with improved pharmacokinetic properties may enable improved treatment and prevention of bleeding episodes in the inhibitor population. In this study we describe recombinant FVIIaFc (rFVIIaFc), a recombinant Fc-fusion protein generated to utilize the neonatal Fc receptor (FcRn)-mediated recycling pathway that protects immunoglobulin G from catabolism. On the basis of activity, rFVIIaFc exhibited a 5.5-fold extension in terminal half-life in hemophilia A mice compared to rFVIIa. The potency of rFVIIaFc was comparable to that of rFVIIa in thrombin generation assay and ROTEM. In agreement with these data, rFVIIaFc and rFVIIa showed similar acute efficacy at comparable molar doses in the tail clip bleeding model in hemophilia A mice. Taken together, these studies demonstrate enhanced pharmacokinetics and similar hemostatic properties for rFVIIaFc compared to rFVIIa.

A comparison between recombinant activated factor VII (Aryoseven) and Novoseven in patients with congenital factor VII deficiency.

In order to establish the efficacy and biosimilar nature of AryoSeven to NovoSeven in the treatment of congenital factor VII (FVII) deficiency, patients received either agent at 30 µg/kg, intravenously per week for 4 weeks, in a randomized fashion. The primary aim was to compare FVII:coagulation activity (FVII:C), 20 minutes after recombinant activated FVII (rFVIIa) injection, in the 2 groups. A secondary measure was self-reported bleeding. The median interquartile baseline range of the plasma level of activated FVII (FVIIa) activity in the 2 groups was 1.6 (1.1-14.0) IU/dL and 5.0 (1.1-25.5) IU/dL. All patients achieved levels of FVIIa (FVII:C) >30 IU/dL, 20 minutes after the injection of rFVIIa. Bleeding was similar between the 2 groups, with a comparable decrease in severity and frequency compared to the last month prior to treatment. AryoSeven is similar to NovoSeven in increasing postinjection FVIIa activity as well as in clinical safety and efficacy.

NovoSeven (recombinant factor VIIa) for the treatment of bleeding episodes and perioperative management in patients with Glanzmann's thrombasthenia.

Glanzmann's thrombasthenia is a rare inherited autosomal recessive bleeding disorder caused by qualitative or quantitative defects of the platelet membrane glycoprotein IIb/IIIa. The ensuing lack of platelet aggregation is frequently associated with mucocutaneous bleeding that may be variable in both frequency and intensity, ranging from minimal bruising to severe and life-threatening hemorrhages. A number of treatment modalities have been proposed to manage the bleeding episodes, which include local measures, antifibrinolytic agents, platelet transfusions and recombinant activated factor VII. The role of this bypassing hemostatic agent for treatment or prevention of bleeding episodes in Glanzmann's thrombasthenia patients is critically analyzed in this review.

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-FP's 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.

Predicting dosing advantages of factor VIIa variants with altered tissue factor-dependent and lipid-dependent activities.

BACKGROUND: Recombinant factor VIIa (rFVIIa) is an FX-cleaving coagulation enzyme licensed for the treatment of bleeding episodes in hemophiliacs with inhibitory antibodies. Even though the optimal dosing and comparative dose efficacy of rFVIIa remain poorly understood, genetic or chemical modifications of rFVIIa have been proposed, with the goal of achieving faster and longer hemostatic action. No ongoing trial is currently comparing rFVIIa variants with each other. OBJECTIVES AND METHODS: We used mathematical modeling to compare the pharmacokinetics, dose-response (pharmacodynamics) and dose-effect duration (pharmacokinetics/pharmacodynamics) of rFVIIa variants to predict their optimal doses. The pharmacodynamic (PD) model of FXa generation by FVIIa in complexes with tissue factor (TF) and procoagulant lipids (PLs) was validated against published ex vivo and in vitro thrombin generation (TG) experiments. To compare variants' safety profiles, the highest non-thrombogenic doses were estimated from the clinical evidence reported for the licensed rFVIIa product. RESULTS: The PD model correctly described the biphasic TF-dependent and PL-dependent dose response observed in TG experiments in vitro. The pharmacokinetic/PD simulations agreed with published ex vivo TG data for rFVIIa and the BAY 86-6150 variant, and explained the similar efficacies of a single dose of 270 µg kg(-1) (as reported in the literature) and repeated doses of 90 µg kg(-1) of unmodified rFVIIa. The duration of the simulated hemostatic effect after a single optimal dose was prolonged for rFVIIa variants with increased TF affinity or extended half-lives, but not for those with modulated PL activity. CONCLUSIONS: Some modifications of the rFVIIa molecule may not translate into a prolonged hemostatic effect.

The kidneys play an important role in the clearance of rFVIIa in rats.

INTRODUCTION: Previous distribution and histological studies have indicated that the kidneys and renal proximal tubular cells play a role in clearance of rFVIIa. However, the relative importance of the kidneys in clearance of rFVIIa has not previously been addressed. The objective of the present study was to evaluate the importance of the kidneys in the clearance process of rFVIIa after iv administration to rats using a nephrectomy model. MATERIALS AND METHODS: A nephrectomized rat model was established and validated using inulin, a compound primarily cleared by the kidneys, as a test substance and several physiological parameters were monitored to ensure viability and robustness of the model. The model was then used for pharmacokinetic evaluation of renal clearance of rFVIIa. The pharmacokinetic parameters for rFVIIa were evaluated both by use of standard non-compartmental methods and by use of mixed effects methods, where a pharmacokinetic model was used to simultaneously model all data from healthy, sham operated, and nephrectomized rats. RESULTS: Nephrectomized animals showed stable rectal temperature, SpO2 and pulse and as expected, clearance of inulin was essentially abolished compared to control animals (p<0.001). For rFVIIa, nephrectomy resulted in a clearance and terminal half-life of 34mL/h/kg and 2.8h compared to 68mL/h/kg and1.9h in rats exposed to sham surgery (p<0.0001 for both parameters). CONCLUSION: The present data show that about 50% of the total clearance of rFVIIa from circulation in rats under isoflurane anaesthesia is due to renal clearance.

Sustained pro-haemostatic activity of rFVIIa in plasma and platelets in non-bleeding pigs may explain the efficacy of a once-daily prophylaxis in humans.

Recombinant factor VIIa (rFVIIa) is registered for treatment of inhibitor-complicated haemophilia, and a once-daily prophylactic administration of rFVIIa is successful in reducing the number of bleeding events. This suggests that a single rFVIIa dose has a pro-haemostatic effect up to 24 hours (h), which is difficult to explain given its half-life of 2 h. In this study, six pigs received a 90 µg/kg rFVIIa bolus. Plasma was collected and platelets were isolated at various time points up to 48 h, and analysed for FVIIa levels and associated haemostatic activity. Elevated plasma FVIIa levels were detected up to 24 h post-administration (36 (32-56) mU/ml [median (interquartile range [IQR]), 24 h] vs 2 (2-14) mU/ml [baseline]). Corresponding prothrombin time (PT) values remained shortened compared to baseline until 24 h post-administration (9.4 (9.3-9.9) seconds (s) [24 h] vs 10.5 (10.2-11.0) s [baseline], p ≤0.01). The lag time in thrombin generation testing as well as clotting times in plasma-based assays were shortened up to 12 or 24 h post-administration, respectively (lag times 1.8 (1.7-2.1) minutes (min) [12 h] vs 2.3 (2.3-2.6) min [baseline], p ≤0.01 and clotting times 3.8 (3.2-3.9) min [24 h] vs 5.2 (4.6-5.5) min [baseline], p ≤0.001). Platelet FVIIa levels were elevated up to 48 h (7.7 (3.4-9.0) ng VIIa/mg actin [48 h] vs 2.5 (0.7-4.8) ng VIIa/mg actin [baseline]). In conclusion, elevated and haemostatically active plasma and platelet FVIIa levels are detectable up to 24-48 h following rFVIIa administration in pigs. This prolonged pro-haemostatic effect of FVIIa may explain the prophylactic efficacy of a once-daily rFVIIa treatment.