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: 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.

Description: Abstract 4440 It is known that some FVIII deficient patients who develop high titer FVIII inhibitors do not respond as expected to inhibitor bypassing agents. During an IRB approved study of laboratory monitoring of rFVIIa infusion in hemophilia patients, we had the opportunity to extensively study a patient who was known to respond poorly to standard dose (90 mg/kg) rFVIIa. We present here results from this patient included in this study and question whether it might be possible to predict poor response from in vitro measurements. Case history The patient is a 43 year old male with severe hemophilia A (FVIII6000 IU per infusion) to which he responded. He continues to bleed frequently with 7 documented bleeds requiring 21 infusions of FEIBA for treatment during the first six months of 2009. Methods This patient was one of 10 hemophiliacs participating in a clinical study of rFVIIa. Blood samples were drawn at baseline and at 0.5, 1, 2, 4 and 6 hours after a single dose of rFVIIa 90 mg/kg. Parameters measured included PT, PTT, fibrinogen level and whole blood assays (Hemodyne HAS, TEG®, and ROTEG®). Thrombin generation was measured in PPP and PRP by CAT. Plasma samples were analyzed for Prothrombin Fragment 1.2, FVII:C, FVII:Ag, FVIIa:ATIII and D-dimer. In addition, in this patient an in vitro spiking study of rFVIIa corresponding to doses of 90, 180 and 270 mg/kg was performed to determine the clotting parameters. Results At baseline, his PT was 9.6 seconds, PTT was 112 seconds, and fibrinogen was 238 mg/dl. Samples for TEG, HAS and ROTEG analysis all failed to clot when re-calcified and monitored for up to 60 minutes. Thirty minutes post infusion of rFVIIa, HAS parameters slightly improved (FOT=16 min, PCF 2.0 Kdyn) but quickly reverted to grossly abnormal at one hour. This is in marked contrast to the typical response of most patients in the study as demonstrated in (Fig.1). The R for TEG shortened to 14.4 min and CT for ROTEM decreased to 1094 sec after 30 minutes and remained measurable but grossly abnormal (30 min and 2000 sec) for the next six hours. MA (60 mm) and MCF (60 mm) normalized at 30 min and remained normal for the next six hours. Results of CAT were dependent on the sample type and clot triggering agent. Re-calcification in PRP resulted in shortening of T-lag to 21.5 minutes and a C-max of 15.8 nM both of which were grossly abnormal. T-lag for PRP clotted with 1pm TF was 9.9 min and shortened to 5 min post rFVIIa infusion. ETP when measurable was very low. For PPP clotted with 0.5 pM TF and 4mM phospholipid, the T-lag decreased from a baseline of 5 to