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.

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.

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.

A Phase II clinical trial of a mixture of plasma-derived factor VIIa and factor X (MC710) in haemophilia patients with inhibitors: haemostatic efficacy, safety and pharmacokinetics/pharmacodynamics.

MC710, a mixture of plasma-derived activated factor VII and factor X at a protein weight ratio of 1:10, is a novel bypassing agent for haemostasis in haemophilia patients with inhibitors. In a Phase II trial, we evaluated the haemostatic efficacy and safety of single doses of MC710, and investigated pharmacokinetic and pharmacodynamic parameters in nine joint bleeding episodes in six male haemophilia patients with inhibitors. This trial was a multi-centre, open-label, non-randomized study of two doses (60 and 120 µg kg(-1) as FVIIa dose), allowing the re-administration of different MC710 dosages to the same subjects. Haemostatic efficacy was assessed by evaluating reduction in pain and swelling, as well as increase in range of motion in a bleeding joint. The results of the study showed that in nine bleeding episodes, seven treatments were rated as 'excellent' or 'effective' according to investigator's rating system of efficacy at 8 h after administration. No serious or severe adverse events were observed after administration; furthermore, measurement of several diagnostic markers revealed no signs or symptoms of disseminated intravascular coagulation (DIC). The haemostatic potential of MC710 was confirmed at doses of 60 and 120 µg kg(-1) in this trial. MC710 is thus expected to be a safe and efficacious novel bypassing agent for controlling bleeding in haemophilia patients with inhibitors.

Comparative pharmacokinetics of factor VIII and recombinant factor IX: for which coagulation factors should half-life change with age?

The half-life of factor VIII (FVIII) increases with the age of the patient, while studies on recombinant factor IX (rFIX) and factor VIIa (rFVIIa) have not demonstrated corresponding age-related changes. The purpose of this analysis was to relate the changes in FVIII and rFIX pharmacokinetics (PK) with age to developmental changes in body size and fluid volumes and explain why the elimination half-life of FVIII, but not of rFIX, would change with age, and to consider how the findings could be applied prospectively to other coagulation factors. Published PK data for FVIII from 186 patients aged 1-74 years and for rFIX from 56 patients aged 4-56 years were used. The relationships of FVIII and rFIX clearance (CL) with body weight could be described by allometric expressions. Relative changes in CL with age or weight were similar for FVIII and rFIX. The age-related change in volume of distribution at steady state (V(ss)) of rFIX was parallel to the change in CL in the children while for FVIII the change was much less pronounced. Elimination half-life was clearly age-dependent for FVIII while only a very weak trend could be seen for rFIX. Limited data suggest that rFVIIa in this respect resembles rFIX, with parallel changes in CL and V(ss) producing insignificant change in half-life. To what extent the elimination half-life of a coagulation factor would show a correlation with age can in principle be predicted from the characteristics of its CL and distribution.

Pharmacokinetic properties of two different recombinant activated factor VII formulations.

Recombinant factor VIIa is indicated for treatment of bleeding episodes in patients with haemophilia A or B with inhibitors; in FVII deficiency and in Glanzmann's thrombasthenia. The aim of the study reported here was to compare the pharmacokinetic profiles between two formulations of rFVIIa that are produced in two different cell lines and media: Chinese hamster ovary cells cultured in a serum-free medium (CHO-rFVIIa) and baby hamster kidney cells cultured in a non-human serum-based medium (BHK-rFVIIa). Two clinical trials were performed; one in healthy subjects and the other in patients with congenital haemophilia A or B, with or without inhibitors. Subjects were recruited into a two-way crossover trial and were randomized to receive a dose of CHO-rFVIIa and BHK-rFVIIa. Healthy subjects received one dose of 90 µg CHO-rFVIIa kg(-1) bodyweight (bw) in the newly developed room-temperature stable rFVIIa formulation and one dose of 90 µg BHK-rFVIIa kg(-1) bw, in the original rFVIIa formulation. Patients with haemophilia received one dose of 270 µg CHO-rFVIIa kg(-1) and one dose of 270 µg BHK-rFVIIa kg(-1), both in the room-temperature stable formulation. The trials showed higher FVII activity levels [higher area under the plasma concentration-time curve (AUC)] following administration of CHO-rFVIIa than after BHK-rFVIIa. Therefore, bioequivalence could not be established. The difference in FVII activity levels is believed to be a result of different glycosylation patterns between the two products. Neither the use of CHO-rFVIIa nor the use of one single dose of 270 µg kg(-1) of the newly developed room-temperature stable rFVIIa raised any safety concerns.

Clinical pharmacological study of a plasma-derived factor VIIa and factor X mixture (MC710) in haemophilia patients with inhibitors--phase I trial.

MC710, a combined product of plasma-derived activated factor VII (FVIIa) and factor X (FX) at a protein weight ratio of 1:10, is a novel bypassing agent for haemostasis in haemophilia patients with inhibitors. In this study, pharmacokinetic (PK), pharmacodynamic (PD) parameters and safety of single doses of MC710 were investigated in 11 male haemophilia patients with inhibitors in a non-bleeding state. This was a multi-centre, open-labelled, non-randomized, active controlled crossover, dose-escalation study of five doses (20-120 µg kg(-1) of FVIIa) with re-administration of different MC710 dosages to the same subjects. The active controls were NovoSeven (120 µg kg(-1)) and/or FEIBA (50 and 75 U kg(-1)) which were used to compare PD parameters. The area under the curve (AUC) and maximum plasma concentration (C(max)) of MC710 active ingredients increased dose-dependently within the range of 20 and 120 µg kg(-1). After administration of MC710, activated partial thromboplastin time (APTT) was dose-dependently improved and prothrombin time (PT) was shortened to approximately 6 s at 10 min, and APTT improvement and PT shortening effects were maintained until 12 h after administration of MC710 at all doses. No serious or severe adverse event was observed after administration of MC710; furthermore, several diagnostic marker values and those changes did not indicate any signs of disseminated intravascular coagulation (DIC). These results suggest that MC710 would have haemostatic potential equal to or greater than NovoSeven and FEIBA and was be tolerable when given at doses up to 120 µg kg(-1).

Pharmacokinetics and pharmacodynamics of rFVIIa and new improved bypassing agents for the treatment of haemophilia.

Animal models have played a critical role in developing our understanding of haemophilia and its treatment. For example, studies in mice and dogs have provided insights into the pharmacokinetics and pharmacodynamics of recombinant activated factor VII (rFVIIa). Such studies have shown that antithrombin has a significant impact on clearance of rFVIIa, which explains discrepancies between the antigen and activity half-lives of rFVIIa. Animal studies have also shown that the major clearance organs for rFVIIa are the liver and the kidneys, whereas distribution studies suggest that FVII and rFVIIa leave the circulation and enter the tissues, before returning to the circulation through the lymph. One agent that has benefited greatly from the use of animal models in its development is vatreptacog alfa, a new analogue of rFVIIa. Promising in vitro results, including increased generation of FXa, shortened clotting times and increased clot stability, were subsequently confirmed in animal models. In a severe tail-bleed model in FVIII knock-out mice, reduction in maximal blood loss was substantially greater with vatreptacog alfa than with rFVIIa, FVIII or plasma-derived activated prothrombin complex concentrate. In a mouse model of joint bleeding, rFVIIa and vatreptacog alfa significantly reduced bleeding compared with vehicle-treated haemophilic controls. More recently, a model of endothelial injury based on mouse cremaster muscle has been developed. Overall, animal models are a valuable tool in elucidating the haemostatic process and the effects of therapeutic agents, although direct extrapolation to the clinical setting should be done with caution.

Pharmacokinetics, pharmacodynamics and safety of recombinant canine FVIIa in a study dosing one haemophilia A and one haemostatically normal dog.

Recombinant human FVIIa (rhFVIIa) corrects the coagulopathy in hemophilia A and B as well as FVII deficiency. This is also the case in dogs until canine anti-human FVIIa antibodies develop (~2 weeks). Recombinant canine factor VIIa (rcFVIIa), successfully over-expressed by gene transfer in haemophilia dogs, has provided long-term haemostasis (>2 years). However, pharmacokinetics (PK), pharmacodynamics (PD) and safety of rcFVIIa after pharmacological administration have not been reported. We therefore wanted to explore the safety, PK and PD of rcFVIIa in dogs. A pilot study was set up to evaluate the safety as well as PK and PD of rcFVIIa after a single intravenous dose of 270 µg kg(-1) to one HA and one haemostatically normal dog and to directly compare rcFVIIa with rhFVIIa in these two dogs. Single doses of rcFVIIa and rhFVIIa were well tolerated. No adverse events were observed. Pharmacokinetic characteristics including half-life (FVIIa activity: 1.2-1.8 h; FVIIa antigen 2.8-3.7 h) and clearance were comparable for rcFVIIa and rhFVIIa. Kaolin-activated thromboelastography approached normal in the HA dog with the improvement being most pronounced after rcFVIIa. This study provided the first evidence that administering rcFVIIa intravenously is feasible, safe, well tolerated and efficacious in correcting the haemophilic coagulopathy in canine HA and that rcFVIIa exhibits pharmacokinetic characteristics comparable to rhFVIIa in haemophilic and haemostatically competent dogs. This strengthens the hypothesis that rcFVIIa can be administered to dogs to mimic the administration of rhFVIIa to humans.

Pharmacokinetics and safety of a 270 mcg kg-1 dose of room temperature stable recombinant activated factor VII in patients with haemophilia.

Recombinant factor VIIa (rFVIIa), a haemostatic bypassing agent, has been shown to be effective and well-tolerated in patients with haemophilia at standard doses of 90 and 270 mcg kg(-1). A new room temperature stable formulation of rFVIIa was recently developed that was shown to be bioequivalent to and maintain the safety and efficacy profiles of the original formulation at a dose of 90 mcg kg(-1). The aim of this study was to examine the pharmacokinetics and safety of rFVIIa-RT at a 270 mcg kg(-1) dose. The pharmacokinetics and safety of a 270 mcg kg(-1) dose of the newly formulated room-temperature stable recombinant activated factor VII (BHK-rFVIIa-RT) was evaluated in 23 subjects with congenital haemophilia A or B. The pharmacokinetic profile for the 270 mcg kg(-1) dose of BHK-rFVIIa-RT was in line with what was previously observed for the 90 mcg kg(-1) dose. The AUC(last) and C(max) of BHK-rFVIIa-RT at 270 mcg kg(-1) (346.65 h IU mL(-1) and 146.12 IU mL(-1) respectively) were proportionally higher than those observed at the lower 90 mcg kg(-1) dose of BHK-rFVIIa-RT (113.26 h IU mL(-1) and 52.83 IU mL(-1)) demonstrating the dose-dependent nature of BHK-rFVIIa-RT activity. There were no thromboembolic events or related serious adverse events reported with the increased dose of BHK-rFVIIa-RT, and no patients withdrew because of adverse events. This indicates that BHK-rFVIIa-RT was well tolerated at a higher dosage and maintains the favourable safety profile established by rFVIIa. Therefore, the 270 mcg kg(-1) dose of BHK-rFVIIa-RT shows dose-dependent pharmacokinetic effects that do not appear to increase the risk of serious adverse events.

Monitoring rFVIIa 90 µg kg⁻¹ dosing in haemophiliacs: comparing laboratory response using various whole blood assays over 6 h.

Recombinant FVIIa is a haemostatic agent administered to patients with severe FVIII or FIX deficiency with inhibitors. Although rFVIIa is effective at stopping bleeding, a reliable assay to monitor its effect is lacking. To characterize the pharmacokinetics and global coagulation effects of rFVIIa for 6 h following a IV dose of 90 µg kg⁻¹. Ten non-bleeding subjects with severe FVIII or FIX deficiency were infused with a single-dose of rFVIIa 90 µg k⁻¹ body weight and blood was collected before and at 0.5, 1, 2, 4 and 6 h postdose. Global haemostasis was characterized throughout the study utilizing whole blood analyses (Hemodyne HAS, TEG, ROTEM). The clearance and half-life of factor FVII:C was estimated as 39.0 ± 8.8 mL h⁻¹ kg⁻¹ and 2.1 ± 0.2 h respectively. There was good inter-assay agreement with respect to clot initiation parameters (R, CT and FOT) and these parameters all fell to a mean of approximately 9 min following rFVIIa dosing. The platelet contractile force (PCF) and clot elastic modulus (CEM) were positively correlated to FVII:C (P < 0.0001), and these parameters were dynamic throughout the 6-h period. The MA and MCF did not correlate to FVII:C nor did they significantly change during the study. Prothrombin F1 + 2 significantly increased following rFVIIa dosing (P < 0.001), but remained steady throughout the study. There was no change in D-dimer concentrations over time. The FOT, R and CT characterized clot initiation following rFVIIa dosing. The PCF and CEM were correlated to FVII:C and characterized the dynamics of platelet function and clot strength over the rFVIIa dosing interval. The clinical significance of these findings needs additional study.

Variability of thromboelastographic responses following the administration of rFVIIa to haemophilia A dogs supports the individualization of therapy with a global test of haemostasis.

The efficacy of recombinant factor VIIa (rFVIIa) therapy in haemophilia A is challenged by the lack of a reliable monitoring tool for treatment response. This is further complicated by the significant inter-patient variability associated with this response. Thromboelastography (TEG), a real time global haemostatic test has shown superiority over conventional tests of haemostasis and has proven efficiency in the monitoring of bypass agents such as rFVIIa and FEIBA™. However, this evaluation has been limited to a few case studies or very small patient series. In this study, six severe haemophilia A dogs were treated with a clinically relevant single dose of rFVIIa, and therapy was monitored by thromboelastography predrug and at 15, 30 and 60 min postdrug administration using citrated whole blood samples activated with tissue factor and compared with non-tissue factor-activated samples. Despite the homogeneity of the tested dogs, a clear inter-individual variation was observed in the pre-and post-rFVIIa Thromboelastography analyzes. The improvement of global haemostatic parameters was seen as early as 15 min following drug administration, with a peak for factor VIIa activity in plasma at the same time. There is a significant correlation between plasma FVIIa and TEG parameters 15 min postinjection, and the baseline TEG profile influences the individual postdrug administration outcome. Together, these data support the value of TEG not only as an effective monitoring haemostatic test, but also as a tool for individualization of therapy to achieve the best haemostatic and cost effectiveness of rFVIIa therapy.

Safety, pharmacokinetics and efficacy of factor VIIa formulated with PEGylated liposomes in haemophilia A patients with inhibitors to factor VIII--an open label, exploratory, cross-over, phase I/II study.

Recombinant activated factor VIIa (FVIIa) is a bypassing agent used to treat bleeding episodes in haemophilia patients with inhibitors to factor VIII (FVIII) and factor IX. The pharmacological effect of FVIIa is short-lived and therefore with the recommended dose of 90 µg kg(-1), a bleeding episode is treated with multiple injections. A long-acting form of FVIIa that can ensure adequate haemostasis with a single infusion, without increasing the thrombotic risk, would therefore be beneficial. PEGylated liposomes (PEGLip) have been shown to bind FVIIa and to improve haemostatic efficacy in preclinical experiments. In the present phase I/II clinical trial, we assessed the safety and efficacy of PEGLip-formulated FVIIa in severe haemophilia A patients (FVIII≤1%) with inhibitors to FVIII. Each patient received one prophylactic infusion of standard FVIIa and one prophylactic infusion of PEGLip-formulated FVIIa. The order of the infusions was randomized and the two infusions were separated by a ten-day washout period. Efficacy assessed by thromboelastography revealed that PEGLip-FVIIa induced significantly shorter clotting times and produced higher clot firmnesses than standard FVIIa. Thrombin generation assays showed that PEGLip-FVIIa induced faster thrombin generation and higher peak levels of thrombin than standard FVIIa. These effects lasted up to 5 h postinfusion. Measurements of D-dimer, prothrombin fragment 1+2 and fibrinogen showed no significant differences between the PEGLip-FVIIa and standard FVIIa treatments. PEGLip-FVIIa therefore showed improved haemostatic efficacy without increased risk of thrombosis and may be further developed for the treatment for bleeding episodes in haemophilia patients with inhibitors.

Plasma elimination kinetics for factor VII are independent of its activation to factor VIIa and complex formation with plasma inhibitors.

The mechanism for the elimination of factor VII (FVII) from the circulation is unknown, just as it is unclear how activation of FVII to FVIIa and subsequent complex formation with antithrombin III (AT) or alpha2-macroglobulin (alpha2M) affects clearance. The possibility that the clearance mechanism involves activation and inhibitor complex formation as obligatory intermediate reactions is examined in this study. Human and murine sera were spiked with human FVIIa in the absence and presence of heparin and analysed for complex formation. Complex formation in vivo was studied after intravenous injection of (125)I-VIIa in mice; and the pharmacokinetics (PK) of human and murine FVIIa was studied in normal mice. Furthermore, comparative PK studies were performed with FVII, FVIIa, active site blocked FVIIa and a preformed FVIIa-AT complex in normal and alpha2M-deficient mice. The data demonstrated that FVIIa-AT complexes and to a much lesser extent FVIIa-alpha2M-complexes accumulated in vivo after FVIIa administration. FVIIa-AT accounted for about 50% of total FVIIa antigen left in the circulation after 3 hours. All FVII derivatives studied including FVII, FVIIa and FVIIa-AT were cleared with similar rates suggesting an elimination kinetics which is unaffected by FVII activation and subsequent inactivation by plasma inhibitors.

Evaluation of intraosseous delivery of factor VIIa during hemorraghic shock in the pig.

The purpose of this investigation is to determine if intraosseous infusion (IO) is a suitable method for the delivery of recombinant human factor VIIa (rFVIIa) during hemorrhagic shock. The measures that were used to evaluate IO delivery suitability included: (1) determination of clinically significant local or systemic toxicity and (2) demonstration that systemic blood levels of rFVIIa increased rapidly following administration. Our results indicate that there was no evidence of significant local or systemic toxicity following infusion and that the systemic blood concentration of rFVIIa peaks immediately after the end of infusion. This result suggests that the systemic blood level profiles of rFVIIa delivered by IO infusion are similar to those that could be produced by intravenous (IV) administration. Furthermore, in all 25 test animals, access to the systemic circulation during shock was achieved as evidenced by rapid increase in a marker dye (flourescein) or rFVIIa in the blood. We conclude that administration of rFVIIa via IO infusion is a reasonable safe method and is likely to produce blood levels required for improved hemostasis during shock.

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.

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.

Prolonged in-vivo half-life of factor VIIa by fusion to albumin.

For the treatment of haemophilia patients with inhibitors, recombinant factor VIIa (rFVIIa) is available as a therapeutic option to control bleeding episodes with a good balance of safety and efficacy. However, the short in-vivo half-life of approximately 2.5 hours makes multiple injections necessary, which is inconvenient for both physicians and patients. Here we describe the generation of a recombinant FVIIa molecule with an extended half-life based on genetic fusion to human albumin. The recombinant FVII albumin fusion protein (rVII-FP) was expressed in mammalian cells and upon activation displayed a FVII activity close to that of wild type FVIIa. Pharmacokinetic studies in rats demonstrated that the half-life of the activated recombinant FVII albumin fusion protein (rVIIa-FP) was extended six- to seven-fold compared with wild type rFVIIa. The in-vitro and in-vivo efficacy was evaluated and was found to be comparable to a commercially available rFVIIa (NovoSeven((R))). The results of this study demonstrate that it is feasible to develop a half-life extended FVIIa molecule with haemostatic properties very similar to the wild-type factor.

Use of albumin fusion technology to prolong the half-life of recombinant factor VIIa.

A significant proportion of patients with haemophilia A develop inhibitors to administered factor VIII (FVIII) and require therapy with bypassing agents such as activated factor VII (FVIIa) or activated prothrombin complex concentrates. NovoSeven is a commercially available recombinant FVIIa (rFVIIa) with a very short half-life of approximately 2.4 hours. As a result, patients generally require multiple, frequent infusions for the management of bleeding episodes. Thus, there is growing interest in extending the circulating half-life of coagulation factors through the use of innovative drug delivery and formulation technologies. One such approach uses albumin fusion technology in which human albumin is genetically fused to the C-terminus of rFVIIa via a flexible glycine serine linker. The properties of this rFVIIa fusion protein (rVIIa-FP) have recently been examined in pre-clinical studies. Results from these investigations demonstrate the feasibility of this approach, which successfully extended the half-life and biological activity of rFVIIa without compromising haemostatic efficacy. These data suggest that rVIIa-FP may be a promising therapy for the treatment of haemophilia patients with inhibitors and warrants further investigation in clinical trials.

Overview of the human pharmacokinetics of recombinant activated factor VII.

AIMS: To review the pharmacokinetics of rFVIIa in various patient populations, and to discuss the differences observed between groups. METHODS: Based on a registry of Novo Nordisk studies, 14 studies evaluating rFVIIa pharmacokinetics following single and multiple bolus administration in healthy volunteers, adult and paediatric patients with congenital haemophilia and inhibitors, patients undergoing liver surgery and in patients with cirrhosis, inherited FVII deficiency, upper gastrointestinal bleeding or severe trauma were identified. Data on rFVIIa PK, analyzed with noncompartmental and population pharmacokinetic methods, were extracted. RESULTS: Plasma clearance was a more robust parameter than half-life for comparing rFVIIa pharmacokinetics between groups. In healthy volunteers and patients with no or low-level bleeding (e.g. adults with haemophilia, nonbleeding patients with cirrhosis), plasma clearance was relatively low (30-40 ml kg(-1) h(-1)). In children with haemophilia and adults with high-level bleeding (e.g. cirrhotic patients undergoing orthotopic liver transplantation or resection) and patients with congenital FVII deficiency, plasma clearance was relatively higher (60-90 ml kg(-1) h(-1)). CONCLUSIONS: Comparison of plasma clearance rates in different patient populations suggested that subjects fall into two distinct groups. These differences may have clinical implications in terms of how to adapt the rFVIIa dosing regimen, depending on the expected bleeding rate/blood loss and underlying disease.