ALBERT

Description: Abstract 1295 Poster Board I-317 Introduction Recent animal studies suggest that measurable amounts of factor VIIa and antithrombin (AT) complexes are formed and accumulate following rFVIIa administration. The in vivo rate of inhibition has been reported to be faster than the un-stimulated in vitro reaction between AT and free rFVIIa and of the same order of magnitude as the rate determined in the presence of tissue factor. To study the impact of AT inhibition on the elimination of rFVIIa in humans, we measured the pharmacokinetics (PK) of rFVIIa and rFVIIa-AT complex formation in 10 hemophilia A or B patients. Patients and Methods The PK of single-dose rFVIIa 90 μg/kg (Novo Nordisk A/S) was evaluated in 10 severe FVIII- or FIX-deficient patients in a non-bleeding state. The plasma concentrations of FVIIa activity (FVII:C), FVII antigen (FVII:Ag), FVIIa-AT, D-dimer and F1+2 fragment were determined immediately before, and at 0.5, 1, 2, 4 and 6 hours following rFVIIa dosing. Results Significant amounts of FVIIa–AT complex were formed in vivo after rFVIIa administration, and reached a maximum of 5.4 ± 0.8 nmol/L [mean ±SD] at 2 hours following rFVIIa administration and declined to 4.4 ± 0.9 nmol/L at 6 hours, as compared to 0.1 ± 0.05 nmol/L at baseline. While the FVII:C PK data in this study were consistent with previous data, there was greater total body clearance (Cltot), a larger volume of distribution (Vdss) and a shorter plasma half-life (T1/2) of FVII:C relative to FVII:Ag (Table). No change in D-dimer was observed after the administration of rFVIIa, while a slight increase in F1+2 fragment levels to 258 ± 73 pmol/L was measured 4 hours after rFVIIa dosing, as compared to 141 ± 45 pmol/L at baseline. Conclusion A significant divergence between the clearance of rFVIIa, as determined by either FVII:C or FVII:Ag measurements, can be accounted for by AT complex formation. Inhibition by AT appears thus to have a significant impact on the elimination of FVII:C activity from the circulation when rFVIIa is administered at a therapeutic dose. Similar to animal data, the formation of the FVIIa-AT complexes in vivo was faster than anticipated from in vitro studies, indicating that the exposure to the vessel wall stimulates the FVIIa inhibition by AT. Analyses of coagulation parameters did not indicate induction of systemic coagulation. Disclosures Ezban: NovoNordisk A/S: Employment. Pelzer:NovoNordisk: Employment. Agerso:NovoNordisk: Employment. Petersen:NovoNordisk: Employment. Hedner:NovoNordisk: Employment. Carr:NovoNordisk: Employment.

Description: Reports of the potential clinical utility of recombinant Factor VIIa (rFVIIa, NovoSeven®, NovoNordisk) in a variety of bleeding disorders have raised the possibility that this agent may have broad spectrum hemostatic properties. We have previously demonstrated that rFVIIa produces dose dependent correction of hemostatic status in patients with Factor VIII deficiency and/or Factor VIII inhibitor. In this study we measured the dose dependent effects of rFVIIa on Platelet Contractile Force (PCF), Clot Elastic Modulus (CEM) and Thrombin Generation Time (TGT) (Hemodyne® Analyzer, Hemodyne, Inc.) in patients with factor IX deficiency. Results were compared to those seen with recombinant factor IX (BeneFIX® [GI, Wyeth]) and factor nine concentrate (Mononine® [Aventis-Behring]). Blood from three patients with factor IX deficiency was collected via aseptic venipuncture into evacuated tubes containing 3.2% sodium citrate. The samples were spiked with increasing amounts of each hemostatic agent so as to yield levels ranging from 25 to 200% of the recommended dose. At baseline, each of the factor IX deficient patients had prolonged TGT, decreased PCF and decreased CEM. Despite the fact that each patient had less than 1% of normal factor IX activity, there was significant variability between the patients in their baseline values. TGT for the three patients varied from 587 to 1200 seconds. Baseline PCF ranged from 0.9 to 1.4 Kdynes, while CEM varied from 1.6 to 5.4 Kdynes/cm2. Normal TGT, PCF and CEM values for asymptomatic controls (mean ± SD, n=25) were 392±100 seconds, 7.8±1.3 Kdynes and 18.8±5.6 Kdynes/cm2 respectively. At the 100% of recommended dose, each of the factor IX agents produced shortening of the TGT, and increases in PCF and CEM. The degree of normalization of these parameters did not appear to depend on the baseline values. The patient with the worst baseline parameters completely normalized his values at 100% doses of BeneFIX® and Mononine®. The patient with the best baseline values also normalized his parameters with BeneFIX® but not with Mononine®. The patient with intermediate baseline abnormalities improved all parameters with both factor IX products but appeared to respond better to BeneFIX® at the 100% dose level. The response to NovoSeven®, at 100% of the dose recommended for treatment of factor VIII inhibitor patients, was detectable but clearly inferior to the response to the factor IX agents. These results indicate significant variability in hemostatic status between factor IX deficient patients both at baseline and in response to factor replacement. While rFVIIa has activity in these patients, the dose previously shown to correct TGT in factor VIII deficient patients may not be appropriate for factor IX deficient patients.

Description: P-selectin, a member of the selectin family, is a vascular cell adhesion molecule. It is expressed and stored in alpha granules of platelets and in Weibel-Palade bodies of endothelial cells, and upon activation, P-selectin is translocated/transferred to the membrane surface. A key function of the P-selectin is to mediate leukocyte, lymphocyte and platelet interactions in inflammation and possibly in the thrombus formation. A soluble variant, S-Psel, comprising the extracellular domain of P-selectin, has been identified in healthy individuals, but is markedly elevated in patient with vascular disorders. Recent work on S-Psel suggested that S-Psel may play a role in hemostasis/coagulation through the generation of procoagulant TF-bearing microparticles (MP), and therefore, has potential in treating patients with the bleeding disorders, like hemophilia. The aim of this study is to verify studies reporting that S-Psel exhibits in vitro and in vivo pro-coagulant activity. S-Psel and S-Psel-Fc (IgG) fusion were purchased commercially or prepared at Novo Nordisk Research US (NNRUS) and their biological activity was verified by P-selectin/Pselectin glycoprotein ligand-1(PSGL-1) interaction in vitro. The clotting times of human whole blood and plasma treated with S-Psel or S-Psel-Fc, or with an irrelevant human IgG control protein, were measured by thromboelastography and aggregometry respectively. After up to 8 hours of incubation with S-Psel and S-Psel-Fc at a concentration of 15ug/ml, we found no significant difference between samples treated with S-Psel, S-Psel-Fc and the IgG controls. The ability of S-Psel to generate TF-bearing microparticles in human whole blood was examined in a FXa substrate cleavage assay; however, no significant difference in cleavage was observed. Finally, we evaluated S-Psel in vivo. Hemophilia A mice were injected with recombinant mouse S-Psel-IgG or S-Psel-Fc (IgG) at the concentration of 1.2 mg/Kg body weight and human IgG was used as control. As suggested from published results, the effect of S-Psel was determined 6 h after the treatment. Contrary to previous reports, the results revealed no significant difference in bleed time and blood loss between the experimental and control group. In conclusion, we were unable to demonstrate the procoagulant activity of S-Psel in our laboratory either in vitro or in vivo.

Description: Abstract 4414 Introduction: A rFVIIa analogue (NN1731) with increased prothrombinase activity on the activated platelet surface relative to rFVIIa has been shown to result in a more rapid and less variable response in spiking experiments with hemophilia whole blood samples. In a recent pharmacokinetic study of rFVIIa in 10 non-bleeding hemophilia A and B patients who received a dose of 90 mg/kg rFVIIa, we noted that there were two divergent groups based on their laboratory response to rFVIIa. Study participants who achieved clot formation time determined by Hemodyne (FOT) or Rotational Thromboelastography (CT) 〈 15 min were noted to have a “rapid laboratory response”; conversely, those with a FOT and CT value ≥ 15 min were noted to have a “delayed laboratory response”. In order to determine whether the participants with a delayed laboratory response to rFVIIa 90 μ g/kg would have an improved response to higher doses of rFVIIa and to NN1731, additional blood samples were collected and spiked ex vivo. Patients and Methods: Blood samples from ten severe FVIII or FIX deficient patients were spiked with 1.28, 2.56, 3.84 μ g/mL rFVIIa (corresponding to 90, 180 and 270. Disclosures: Hedner: Novo Nordisk A/S: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Speakers Bureau. Ezban:Novo Nordisk A/S: Employment, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Description: Abstract 3485 Poster Board III-422 Introduction The clinical response of hemophilia patients with inhibitors to bypassing agent therapy can be unexpectedly poor. Indeed, there are reports of “poor responders” who either require alternative treatment or dose escalation. The lack of correlation between routine coagulation assays or factor antigen/activity levels with clinical efficacy contributes to the problem in these patients. Analysis of the clotting characteristics of “poor responders” is limited. In a recent study of rFVIIa PK in 10 non-bleeding hemophilia A and B patients, we noted that four of the ten had a remarkable attenuated response as seen in whole blood assays. We report here a comparison of clotting parameters in the “poor responders” versus robust responders to rFVIIa infusion, and attempt to define what might be the source of the altered response. Patients and Methods Ten severe FVIII or FIX deficient patients in a non-bleeding state were infused with a single-dose of rFVIIa (90 μg/kg). Platelet contractile force (PCF), clot structure (CEM,MA,MCF), and clot formation time (FOT,R,CT) were analyzed in whole blood by Hemodyne HAS, Thromboelastography, and Rotation Thromboelastography before, and at 0.5,1,2,4 and 6 hours following rFVIIa dosing. Thrombin generation parameters (Tlag, Cmax) were measured in PRP by the Calibrated Automated Thrombogram. Plasma concentrations of FVII:C and FVII:Ag were measured at each time point. Patients with a clot formation time (FOT, R, CT) ≥ 15 minutes following rFVIIa dosing were termed “Poor Responders”. Results There were few inter-group differences in baseline clotting characteristics. The values for all parameters are provided in Table 1. FVII PK were not different between the Responders and Poor-Responders. This can be appreciated from the PK parameters listed in the table as well as from relative lack of variability for the composite rFVIIa levels seen for the entire group of 10 patients (Figure 1, panels 1 and 2). Both groups had similar FVIIa Cmax and total body clearance values. However, the responders made significantly stronger clots (PCF, CEM) as can be appreciated in table 1 and even more dramatically in panels 3 and 4 of figure 1. In these panels, the responders and poor responders are plotted as separate groups. Even though the groups are small (n=6 vs 4) the minimal response (both in magnitude and duration) to rFVIIa in terms of platelet function (PCF) and clot structure (CEM) is grossly apparent. All three whole blood assays revealed significantly shorter time to clot formation (R, FOT, CT) in the responders. However, the MA (TEG) and MCF (ROTEG), and thrombin generation parameters (Tlag, max) failed to show significant inter-group differences following rFVIIa dosing. Conclusions These data suggest that the differences observed between Responders and Poor Responders are not due to PK influences, but may be related to differences in the effects of thrombin on platelet function. It is possible that whole blood assays may serve as a tool to monitor the clinical effects of rFVIIa. Changes in clot stiffness were better characterized by CEM compared to MA and MCF. There was good correlation between FOT, R and CT parameters to detect onset of clot formation. The thrombin parameters were highly dependent on sample type and triggering agent and did not significantly vary between the two groups. Further studies are needed to clarify the clinical significance of these findings. Disclosures: Ezban: NovoNordisk A/S: Employment. Hedner:NovoNordisk A/S: Employment.