The mean values and variation in the measurements for BD and BV were higher for the second physician performing the biopsies. Subjects in the 5 and 10 μg/kg rFVIIa treatment groups were biopsied only by the first physician. The treatment groups had smaller standard deviations for BD (3.6 and 6.6 minutes) and BV (8.7 and 5.6 mL). The subjects in the placebo and 20 μg/kg rFVIIa groups were biopsied either by the first or second physician and had larger standard deviations for BD (16.8 and 19.7 minutes) and BV (12.0 and 30.4 mL).
In an attempt to account for the influence of the variability between physicians, analyses of rFVIIa effects on BD and BV incorporated adjustment for physician. In addition, the statistical analysis model used for BD and BV performs satisfactorily even for large changes in standard deviation if the CV is approximately constant across treatment groups. In these analyses, monotonicity was observed for the effect of rFVIIa dose on BD and BV, although this was only significantly different from placebo for blood loss in the 10 μg/kg (P = 0.007) and 20 μg/kg (P = 0.001) dose groups (Table 3). However, a separate analysis for physician 1, who was completely responsible for the 5 and 10 μg/kg dose groups, can be useful. The effects of rFVIIa on BV in subjects biopsied by physician 1 were significant for the 10 and 20 μg/kg dose groups for physician 1 (P = 0.006 and P = 0.013, respectively) (Table 4). The effect of rFVIIa on BD was significant only in the 20 μg/kg dose group for physician 1 (P = 0.048) (Table 4).
Effect of rFVIIa on Clot Dynamics
Clot dynamics were assessed using TEG®. A longitudinal approach was performed using a mixed-effect model. Treatment with rFVIIa significantly decreased time to clot onset (R) and increased the clot angle (A), compared with placebo (P < 0.005 for all dose groups) (Fig. 3). The effects of rFVIIa on any parameter of clot dynamics were not observed 3 hours after Bx2 or 1 hour after Bx3. No significant differences were observed in other TEG® parameters across treatment groups.
The adverse events reported in this study were burning sensation (placebo, n = 3), suture-related complications (placebo, n = 2), eczema (40 μg/kg rFVIIa, n = 2), catheter site hemorrhage (20 μg/kg rFVIIa, n = 1), vessel puncture at site of hematoma (20 μg/kg rFVIIa, n = 1), postprocedural complication (10 μg/kg rFVIIa, n = 1), dizziness (20 μg/kg rFVIIa, n = 1), headache (placebo, n = 1), agitation (20 μg/kg rFVIIa, n = 1), ecchymosis (placebo, n = 1), erythema (40 μg/kg rFVIIa, n = 1), and rash (placebo, n = 1). All adverse events were graded as mild or moderate in severity and were related to the biopsy procedure. No thromboembolic complications were reported.
Because platelet response to clopidogrel (i.e., inhibition of platelet aggregation by clopidogrel) is highly variable,22–24 with poor response rates occurring in as many as 40% to 60% of patients,25–27 it was critical to include reliable PI screening to exclude subjects nonresponsive to clopidogrel. In the current study, 56% of subjects failed to demonstrate an antiplatelet effect measured as PI. Previous studies report that up to 60% of clopidogrel-treated subjects exhibit a poor clopidogrel response, a poor response being arbitrarily defined as less than 30% to 40% inhibition of platelet aggregation.25–27 Furthermore, the variability (20.3% SD) in PI in those subjects who met the cutoff levels was similar to the variability reported from secondary post hoc analyses of clopidogrel-treated subjects by Serebruany et al.28 (20.8% SD). Factors such as age (older than 55 years),27 increased body weight,26,29 and conditions that increase platelet reactivity, including diabetes,27,30 acute coronary syndrome, and acute stroke31 may affect response to clopidogrel. It is more likely that other factors affected subject response to clopidogrel because most subjects in this study did not have any of these characteristics that affect response to clopidogrel. Factors that influence the absorption and biotransformation of clopidogrel and/or variability in the P2Y12 adenosine diphosphate receptor affecting platelet activation and aggregation32 may explain the clopidogrel response pattern. Furthermore, there are several other mechanisms and pathways for platelet aggregation (e.g., activation of platelet aggregation by thromboxane A2).33,34 Therefore, targeting activation of the P2Y12 adenosine diphosphate–receptor pathway alone may not sufficiently attenuate the entire aggregation process.
The punch biopsy model has advantages over the original Ivy and Simplate bleeding tests. The Ivy method has limited use in the clinic because of its poor correlation with actual patient bleeding.35 When healthy volunteers were treated with acetylsalicylic acid, bleeding times did not significantly increase using the Simplate bleeding test (P > 0.05).36 The punch biopsy model produced significantly longer BD and BV in healthy volunteers after anticoagulant therapy.15 For these reasons, the punch biopsy model was deemed a suitable model to investigate the ability of rFVIIa to mitigate the effects of clopidogrel on BD and BV. The reasons for the limited ability of the punch biopsy model to demonstrate an effect of rFVIIa on mitigating drug-induced (warfarin15 or clopidogrel) coagulopathies measured by BD remain unclear. Other than the inter-investigator variability that was specific to this study, the nature of the injury (capillary effects), the interaction of the local anesthetic with rFVIIa at the area of injury, or simply the limited effects of rFVIIa in such a small vessel bleeding model are possible explanations.
The effects of rFVIIa on BV, unlike BD, were consistent with the ex vivo time to clot onset (TEG®-R) and clot angle (TEG®-A) results (Fig. 3). These results are similar to the previously reported findings in which rFVIIa corrected the effect of warfarin on all ex vivo TEG® parameters,15 and suggest that time to clot formation affects BV but not BD.
Limitations of the Study
Dual therapy with aspirin and clopidogrel is the common practice for inhibition of platelet aggregation for preventing cardiovascular events.34 This study might have benefited from the use of dual antiplatelet drugs and/or increased loading/maintenance dose26,37,38 to allow a larger proportion of subjects to achieve the PI cutoff levels and possibly a more uniform clopidogrel response. However, the percentage of subjects who did not respond to clopidogrel treatment (56%) was similar to that reported in a previous study that used dual therapy with aspirin.18
A signal on BV for the 10 and 20 μg/kg rFVIIa dose groups and for BD in the 20 μg/kg rFVIIa dose group, for the initial physician, clearly underscores the impact of technical skill in this bleeding model. The premature termination of the study restricted enrollment of subjects in the 40 μg/kg dose group and did not allow for the investigation of the 80 μg/kg dose group, a dose that is within the trial product label. Even though a signal showed an rFVIIa effect for BV in the 10 and 20 μg/kg rFVIIa dose groups as compared with placebo, the results for BD at these doses were not convincing.
Despite these noted limitations of the punch biopsy model, rFVIIa mitigated the effects of clopidogrel-induced bleeding on BV. It is possible that the inhibition of platelet aggregation was compensated by rFVIIa-mediated enhancement of thrombin generation and thrombin-mediated activation of platelet aggregation,33,39 which was reflected more in the BV measurements than in BD. As such, the results of this study cannot be extrapolated to clinical hemorrhages because the effectiveness of rFVIIa to mitigate clopidogrel-associated bleeding has not been tested.
This exploratory study was designed to investigate the effect of escalating doses of rFVIIa in clopidogrel-mediated bleeding in a punch biopsy model. Despite study limitations, rFVIIa (10 and 20 μg/kg) significantly mitigated clopidogrel-induced effects on BV. Furthermore, in subgroup analyses of subjects, 20 μg/kg rFVIIa showed a significant reduction of clopidogrel-induced BD as well as on BV. The clinical enhancement of coagulation by rFVIIa was also reflected in ex vivo clotting parameters (TEG®-R and TEG®-A).
Brett E. Skolnick, PhD, is currently affiliated with Novo Nordisk Inc, Princeton, NJ; Magdy Shenouda, MD, is currently affiliated with Iberia Clinical Research, Eatontown, NJ; Anthony E. Pusateri, PhD, is currently affiliated with US Army Medical Research and Materiel Command, Fort Detrick, MD; and Marcus E. Carr, MD, PhD, FACP, is currently affiliated with Pfizer Inc., Collegeville, PA.
Name: Brett E. Skolnick, PhD.
Contribution: This author helped design the study, conduct the study, analyze the data, and write the manuscript.
Attestation: Brett E. Skolnick designed the study, has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.
Conflicts of Interest: Brett E. Skolnick is an employee of Novo Nordisk.
Name: Magdy Shenouda, MD.
Contribution: This author helped conduct the study and write the manuscript.
Attestation: Magdy Shenouda has seen the original study data and approved the final manuscript.
Conflicts: Magdy Shenouda received financial compensation for his role as investigator of the study.
Name: Naum M. Khutoryansky, PhD.
Contribution: This author helped design the study, analyze the data, perform statistical analysis, and write the manuscript.
Attestation: Naum M. Khutoryansky has seen the original study data, supervised the analysis and review of the data, and approved the final manuscript.
Conflicts of Interest: Naum M. Khutoryansky is an employee of Novo Nordisk.
Name: Anthony E. Pusateri, PhD.
Contribution: This author helped conduct the study, write the manuscript, and contributed to acquisition of laboratory data.
Attestation: Anthony E. Pusateri has seen the original study data and approved the final manuscript.
Conflicts of Interest: Anthony E. Pusateri was an employee of Novo Nordisk at the time of study.
Name: Don Gabriel, MD, PhD.
Contribution: This author helped conduct the study, write the manuscript, and served as an external independent Safety Officer.
Attestation: Don Gabriel has seen the original study data and approved the final manuscript.
Conflicts of Interest: Don Gabriel received financial compensation for his role as Safety Officer of the study.
Name: Marcus E. Carr, MD, PhD, FACP.
Contribution: This author helped write the manuscript.
Attestation: Marcus E. Carr has seen the original study data and approved the final manuscript.
Conflicts of Interest: Marcus E. Carr was an employee of Novo Nordisk at the time of study.
We thank all the study subjects for their participation. We also thank Dr. Sandra Connolly and other study staff at MDS Pharma Services, Neptune, NJ, and the facility staff at Novo Nordisk Research Facility US (NNRUS), New Brunswick, NJ, including Jun D. Guzman, and Petula Fraser-Davies at Novo Nordisk, Princeton, NJ, for acquisition of data, study supervision, and administrative, technical, and/or material support. Dr. Alvin Estilo, Novo Nordisk's Safety Officer, assembled a safety group (including internal Novo Nordisk members not involved in the study conduct and an external independent Safety Officer, Dr. Don Gabriel) to review safety findings at the conclusion of each dose tier. We thank Abha Chandra, PhD, Neal Okarter, PhD, and Charlotte Yap, MSc, of Novo Nordisk, for providing writing and editorial assistance with the manuscript. We also thank Hongli Wang, MSc, and Thomas Henschel, employees of Novo Nordisk, for performing the statistical analyses.
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© 2011 International Anesthesia Research Society
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