Subcutaneous Marzeptacog Alfa (Activated) for On-Demand Treatment of Bleeding Events in Subjects With Hemophilia A or B With Inhibitors.

Marzeptacog alfa (MarzAA) is under development for subcutaneous treatment of episodic bleeds in patients with hemophilia A/B and was studied in a phase III trial evaluating MarzAA compared with standard-of-care (SoC) for on-demand use. The work presented here aimed to evaluate MarzAA and SoC treatment of bleeding events on a standardized four-point efficacy scale (poor, fair, good, and excellent). Two continuous-time Markov modeling approaches were explored; a four-state model analyzing all four categories of bleeding improvement and a two-state model analyzing a binarized outcome (treatment failure (poor/fair), and treatment success (good/excellent)). Different covariates impacting improvement of bleeding episodes as well as a putative relationship between MarzAA exposure and improvement of bleeding episodes were evaluated. In the final four-state model, higher baseline diastolic blood pressure and higher age (> 33 years of age) were found to negatively and positively impact improvement of bleeding condition, respectively. Bleeding events occurring in knees and ankles were found to improve faster than bleeding events at other locations. The covariate effects had most impact on early treatment success (≤ 3 hours) whereas at later timepoints (> 12 hours), treatment success was similar for all patients indicating that these covariates might be clinically relevant for early treatment response. A statistically significant relationship between MarzAA zero-order absorption and improvement of bleedings (P < 0.05) were identified albeit with low precision. No statistically significant difference in treatment response between MarzAA and intravenous SoC was identified, indicating the potential of MarzAA for treatment of episodic bleeding events with a favorable subcutaneous administration route.

Model-Informed Support of Dose Selection for Prophylactic Treatment with Dalcinonacog Alfa in Adult and Paediatric Hemophilia B Patients.

INTRODUCTION: Dalcinonacog alfa (DalcA), a novel subcutaneously administered recombinant human factor IX (FIX) variant is being developed for adult and paediatric patients with hemophilia B (HB). DalcA has been shown to raise FIX to clinically meaningful levels in adults with HB. This work aimed to support dosing regimen selection in adults and perform first-in-paediatric dose extrapolations using a model-based pharmacokinetic (PK) approach. METHODS: A population PK model was built using adult data from two clinical trials (NCT03186677, NCT03995784). With allometry in the model, clinical trial simulations were performed to study alternative dosing regimens in adults and children. Steady-state trough levels and the time-to-reach target were derived to inform dose selection. RESULTS: Almost 90% of the adults were predicted to achieve desirable FIX levels, i.e. 10% FIX activity, following daily 100 IU/kg dosing, with 90% of the subjects reaching target within 1.6-7.1 days. No every-other-day regimen met the target. A dose of 125 IU/kg resulted in adequate FIX levels down to 6 years, whereas a 150 IU/kg dose was needed below 6 down to 2 years of age. For subjects down to 6 years that did not reach target with 125 IU/kg, a dose escalation to 150 IU/kg was appropriate. The children below 6 to 2 years were shown to need a dose escalation to 200 IU/kg if 150 IU/kg given daily was insufficient. CONCLUSION: This study supported the adult dose selection for DalcA in the presence of sparse data and enabled first-in-paediatric dose selection to achieve FIX levels that reduce risk of spontaneous bleeds.

Model-informed pediatric dose selection of marzeptacog alfa (activated): An exposure matching strategy.

Marzeptacog alfa (activated) (MarzAA) is an activated recombinant human rFVII variant intended for subcutaneous (s.c.) administration to treat or prevent bleeding in individuals with hemophilia A (HA) or B (HB) with inhibitors, and other rare bleeding disorders. The s.c. administration provides benefits over i.v. injections. The objective of the study was to support the first-in-pediatric dose selection for s.c. MarzAA to treat episodic bleeding episodes in children up through 11 years in a registrational phase III trial. Assuming the same exposure-response relationship as in adults, an exposure matching strategy was used with a population pharmacokinetics model. A sensitivity analysis evaluating the impact of doubling in absorption rate and age-dependent allometric exponents on dose selection was performed. Subsequently, the probability of trial success, defined as the number of successful trials for a given pediatric dose divided by the number of simulated trials (n = 1000) was studied. A successful trial was defined as outcome where four, three, or two out of 24 pediatric subjects per trial were allowed to fall outside the adult exposures after s.c. administration of 60 µg/kg. A dose of 60 µg/kg in children with HA/HB was supported by the clinical trial simulations to match exposures in adults. The sensitivity analyses further supported selection of the 60 µg/kg dose level in all age groups. Moreover, the probability of trial success evaluations given a plausible design confirmed the potential of a 60 µg/kg dose level. Taken together, this work demonstrates the utility of model-informed drug development and could be helpful for other pediatric development programs for rare diseases.

Phase III dose selection of marzeptacog alfa (activated) informed by population pharmacokinetic modeling: A novel hemostatic drug.

Marzeptacog alfa (activated) (MarzAA) is an activated recombinant human FVII (rFVIIa) variant developed as subcutaneous (s.c.) administration for the treatment or prevention of bleeding episodes in patients with hemophilia A (HA) or hemophilia B (HB) with inhibitors and other rare bleeding disorders. Population pharmacokinetic (PK) modeling was applied for dose selection for a pivotal phase III clinical trial evaluating s.c. MarzAA for episodic treatment of spontaneous or traumatic bleeding episodes. The population PK model used MarzAA intravenous and s.c. data from previously completed clinical trials in patients with HA/HB with or without inhibitors. Based on the model, clinical trial simulations were performed to predict MarzAA exposure after different dosing regimens. The exposure target was identified using an exposure-matching strategy with a wild-type rFVIIa but adjusting for the difference in potency between the two compounds. Simulations demonstrated a sufficient absorption rate and prolonged exposure following a single 60 µg/kg dose leading to 51% and 70% of the population reaching levels above the target after 3 and 6 h, respectively. According to the phase III protocol, if a second dose was required after 3 h because of a lack of efficacy, 90% of the population was observed to be above target 6 h after the initial dose. The model-informed drug development approach integrated information from several trials and guided dose selection in the pivotal phase III clinical trial for episodic treatment of an acute bleeding event in individuals with HA or HB with inhibitors without the execution of a phase II trial for that indication.

Tail Vein Transection Bleeding Model in Fully Anesthetized Hemophilia A Mice.

Tail bleeding models are important tools in hemophilia research, specifically for the assessment of procoagulant effects. The tail vein transection (TVT) survival model has been preferred in many settings due to sensitivity to clinically relevant doses of FVIII, whereas other established models, such as the tail clip model, require higher levels of procoagulant compounds. To avoid using survival as an endpoint, we developed a TVT model establishing blood loss and bleeding time as endpoints and full anesthesia during the entire experiment. Briefly, anesthetized mice are positioned with the tail submerged in temperate saline (37°C) and dosed with the test compound in the right lateral tail vein. After 5 min, the left lateral tail vein is transected using a template guide, the tail is returned to the saline, and all bleeding episodes are monitored and recorded for 40 min while collecting the blood. If no bleeding occurs at 10 min, 20 min, or 30 min post-injury, the clot is challenged gently by wiping the cut twice with a wet gauze swab. After 40 min, blood loss is quantified by the amount of hemoglobin bled into the saline. This fast and relatively simple procedure results in consistent and reproducible bleeds. Compared to the TVT survival model, it uses a more humane procedure without compromising sensitivity to pharmacological intervention. Furthermore, it is possible to use both genders, reducing the total number of animals that need to be bred, in adherence with the principles of 3R's. A potential limitation in bleeding models is the stochastic nature of hemostasis, which can reduce the reproducibility of the model. To counter this, manual clot disruption ensures that the clot is challenged during monitoring, preventing primary (platelet) hemostasis from stopping bleeding. This addition to the catalog of bleeding injury models provides an option to characterize procoagulant effects in a standardized and humane manner.

Prophylactic administration of glycoPEGylated factor IX provides protection and joint outcome superior to recombinant factor IX after induced joint bleeding.

BACKGROUND: Following induced joint hemorrhage, hemophilia B results in the abnormal persistence of iron deposition, inflammation, and neovascularity of the synovial tissue, as well as deterioration of the bone articular surface and strength. Previously, we demonstrated that a factor IX (FIX) replacement protein with extended circulating FIX activity, glycoPEGylated FIX nonacog beta pegol (N9-GP), could improve synovial and osteochondral parameters in F9 knockout mice when administered after joint injury. OBJECTIVE: We explored the use of N9-GP prior to unilateral joint hemorrhage and compared to unmodified recombinant FIX (rFIX). METHODS: Pharmacodynamics, histology, and microcomputed tomography were used to assess the effects of prophylactic administration of glycoPEGylated FIX. RESULTS: In comparison to rFIX, N9-GP significantly improved soft tissue histological parameters, as well as bone outcome at 2 weeks post injury, while performing equally in reduction of blood present in the joint space assessed 1 day after injury. CONCLUSIONS: These results indicate that, in comparison to rFIX, the prophylactic use of extended half-life FIX provides superior protection from bleeding-induced joint damage, manifested by improved correction of histologic parameters.

Absence of functional compensation between coagulation factor VIII and plasminogen in double-knockout mice.

Plasminogen deficiency is associated with severely compromised fibrinolysis and extravascular deposition of fibrin. In contrast, coagulation factor VIII (FVIII) deficiency leads to prolonged and excessive bleeding. Based on opposing biological functions of plasminogen and FVIII deficiencies, we hypothesized that genetic elimination of FVIII would alleviate the systemic formation of fibrin deposits associated with plasminogen deficiency and, in turn, elimination of plasminogen would limit bleeding symptoms associated with FVIII deficiency. Mice with single and combined deficiencies of FVIII (F8-/-) and plasminogen (Plg-/-) were evaluated for phenotypic characteristics of plasminogen deficiency, including wasting disease, shortened lifespan, rectal prolapse, and multiorgan fibrin deposition. Conversely, to specifically examine the role of plasmin-mediated fibrinolysis on bleeding caused by FVIII deficiency, F8-/- and F8-/-/Plg-/- mice were subjected to a bleeding challenge. Mice with a combined deficiency in FVIII and plasminogen displayed no phenotypic differences relative to mice with single FVIII or plasminogen deficiency. Plg-/- and F8-/-/Plg-/- mice exhibited the same penetrance and severity of wasting disease, rectal prolapse, extravascular fibrin deposits, and reduced viability. Furthermore, following a tail vein-bleeding challenge, no significant differences in bleeding times or total blood loss could be detected between F8-/- and F8-/-/Plg-/- mice. Moreover, F8-/- and F8-/-/Plg-/- mice responded similarly to recombinant FVIII (rFVIII) therapy. In summary, the pathological phenotype of Plg-/- mice developed independently of FVIII-dependent coagulation, and elimination of plasmin-driven fibrinolysis did not play a significant role in a nonmucosal bleeding model in hemophilia A mice.

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.

Mouse embryonic stem cell adherent cell differentiation and cytotoxicity assay.

There are thousands of environmental chemicals for which there is limited toxicological information, motivating the development and application of in vitro systems to profile the biological effects of xenobiotic exposure and predict their potential developmental hazard. An adherent cell differentiation and cytotoxicity (ACDC) assay was developed using pluripotent mouse embryonic stem cells (mESCs) to evaluate chemical-induced effects on both stem cell viability and differentiation. This assay uses an In-Cell Western technique after a 9-day culture. DRAQ5/Sapphire700 cell/DNA stains are used to quantify cell number and myosin heavy chain (MHC) protein is used as a marker of cardiomyocyte differentiation. MHC is corrected for cell number, thereby separating cytotoxicity and effects on differentiation. The ACDC assay can be used to evaluate the effects of xenobiotics on mESC differentiation and cell number in the same sample.

Development of a flow cytometric assay for detection of coated platelets in dogs and evaluation of binding of coated platelets to recombinant human coagulation factor VIIa.

OBJECTIVE: To develop an antibody-based flow cytometric assay to detect coated platelets in dogs and to characterize the interaction of recombinant human coagulation factor VIIa with activated platelets from dogs with hemophilia A. SAMPLE: Platelets from 4 dogs with hemophilia A, 4 dogs with hemophilia B, 4 dogs with von Willebrand disease, and 6 hemostatically normal dogs. PROCEDURES: Freshly isolated platelets were activated with thrombin, convulxin, or a thrombin-convulxin combination. Resulting platelet phenotypes were resolved on the basis of P-selectin and fibrinogen expression, and binding of recombinant human coagulation factor VIIa to these distinct platelet subpopulations was measured by use of a flow cytometric assay. RESULTS: Coated platelets were identified on the basis of expression of α-granule fibrinogen and were generated in response to stimulation with the thrombin-convulxin combination but not to stimulation with either agonist alone. Approximately 70% of the platelets from dogs with hemophilia A, hemophilia B, and von Willebrand disease and from the control dogs had the coated platelet phenotype. Recombinant human coagulation factor VIIa bound preferentially to coated platelets with a mean ± SD binding equilibrium constant of 2.6 ± 0.5µM. CONCLUSIONS AND CLINICAL RELEVANCE: Formation of coated platelets in dogs was similar to that in humans. Recombinant human coagulation factor VIIa bound preferentially to coated platelets from dogs. IMPACT FOR HUMAN MEDICINE: A similar mechanism of action for recombinant human coagulation factor VIIa may exist in dogs and humans. The potential for use of dogs in the study of bleeding disorders in humans was strengthened.

Tissue factor and factor VIIa cross-species compatibility.

Knowledge about species compatibility is crucial for proper interpretation of data from in vivo experiments with human proteins in pharmacological models and of data from cross-species in vitro experiments. Information about the cross-species compatibility of tissue factor (TF) and coagulation factor (F) VII (FVII) has accumulated since the early history of coagulation research. Many observations were connected to the introduction and development of the prothrombin time (PT) assay where fibrin clot formation was observed when tissue extracts of different origins were added to recalcified human or non-human plasmas. Studies on cross-species TF-FVIIa compatibility entered into a new area with the cloning and recombinant expression of TF and FVII from a number of species as well as with the possibility of specific amino acid substitution. TF and/or FVIIa from cattle, dog, rabbit, mouse, rat and zebrafish have been purified and characterized in varying detail. In addition to adding knowledge about the species-specific TF-FVIIa interactions, cross-species studies often reveal information which adds to the general view of the structural and functional properties of the human TF-FVIIa complex. This review briefly outlines the features of human TF and FVIIa, their intermolecular interactions, and the biological effects of TF-FVIIa complex formation and compares this information to findings obtained in studies addressing TF or FVIIa of non-human origin. By examples we point to difficulties which may arise from the transcendence across species borders and how some cross-species data have advanced our understanding of the structure and function of the human TF-FVIIa complex.

Canine specific ELISA for coagulation factor VII.

Canine coagulation factor VII (FVII) deficiency can be hereditary or acquired and may cause life threatening bleeding episodes if untreated. FVII procoagulant activity can be measured by FVII activity (FVII:C), but assays for measurement of canine specific FVII antigen (FVII:Ag) have not been available to date. In this study, a canine specific ELISA for measurement of FVII:Ag in plasma was developed and validated. The FVII:Ag ELISA correctly diagnosed homozygous and heterozygous hereditary FVII deficiency. Together with activity based assays, such as FVII:C, the FVII:Ag ELISA should be valuable in the diagnosis of hereditary canine FVII deficiency.