Journal of the American College of Cardiology

Volume 49, Issue 25, June 2007DOI: 10.1016/j.jacc.2007.02.065

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Recombinant Nematode Anticoagulant

Protein c2 in Patients With Non–ST-
Segment Elevation Acute Coronary
Syndrome
The ANTHEM–TIMI-32 Trial
Robert P. Giugliano, Stephen D. Wiviott, Peter H. Stone, Daniel I. Simon, Marc J.
Schweiger, Alain Bouchard, Massoud A. Leesar, Michael A. Goulder, Steven R.
Deitcher, Carolyn H. McCabe, Eugene Braunwald and ANTHEM–TIMI-32 Investigators
Author + information
Recombinant Nematode Anticoagulant Protein c2 in Patients With Non–ST-Segment Elevation
Acute Coronary Syndrome: The ANTHEM–TIMI-32 Trial

Robert P. Giugliano, Stephen D. Wiviott, Peter H. Stone, Daniel I. Simon, Marc J. Schweig, Alain
Bouchard, Massoud A. Leesar, Michael A. Goulder, Steven R. Deitcher, Carolyn H. McCabe,
Eugene Braunwald, for the ANTHEM-TIMI-32 Investigators

We evaluated ascending doses of recombinant nematode anticoagulant protein c2 (rNAPc2), a

tissue factor/activated factor VII inhibitor in 255 patients with non–ST-segment elevation acute
coronary syndrome (nSTE-ACS) and found: 1) a dose-related increase in international
normalized ratio and suppression of F1.2; 2) no difference in significant bleeding; and 3) >50%
reduction in ischemia by continuous 3-channel electrocardiography with higher-dose rNAPc2
(≥7.5 μg/kg). Procedure-related thrombosis occurred in 5 of 26 patients receiving open-label
rNAPc2 without unfractionated heparin, but did not occur if heparin was administered. In patients
with nSTE-ACS managed with standard antithrombotics and an early invasive approach,
additional proximal inhibition of the coagulation cascade with rNAPc2 is promising, although
some heparin may be necessary to avoid procedure-related thrombus formation.

Abstract
Objectives We sought to evaluate the safety and efficacy of recombinant nematode
anticoagulant protein c2 (rNAPc2) in patients with non–ST-segment elevation acute coronary
syndrome (nSTE-ACS).

Background Recombinant NAPc2 is a potent inhibitor of the tissue factor/factor VIIa complex
that has the potential to reduce ischemic complications mediated by thrombin generation.

Methods A total of 203 patients were randomized 4:1 to double-blinded intravenous rNAPc2 or
placebo every 48 h for a total of 1 to 3 doses in 8 ascending panels (1.5 to 10 μg/kg). All patients
received aspirin, unfractionated heparin (UFH), or enoxaparin and early catheterization;
clopidogrel and glycoprotein IIb/IIIa blockers were encouraged. Two subsequent open-label
panels evaluated 10 μg/kg rNAPc2 with half-dose UFH (n = 26) and no UFH (n = 26). The
primary end point was the rate of major plus minor bleeding. Pharmacokinetics,
pharmacodynamics, continuous electrocardiography, and clinical events were assessed.

Results Recombinant NAPc2 did not significantly increase major plus minor bleeding (3.7% vs.
2.5%; p = NS) despite increasing the international normalized ratio in a dose-related fashion
(trend p ≤ 0.0001). Higher-dose rNAPc2 (≥7.5 μg/kg) suppressed prothrombin fragment F1.2
generation compared with placebo and reduced ischemia by >50% compared to placebo and
lower-dose rNAPc2. Thrombotic bailout requiring open-label anticoagulant occurred in 5 of 26
patients treated without UFH, but none in the half-dose UFH group (19% vs. 0%; p = 0.051).

Conclusions In patients with nSTE-ACS managed with standard antithrombotics and an early
invasive approach, additional proximal inhibition of the coagulation cascade with rNAPc2 was
well tolerated. rNAPc2 doses ≥7.5 μg/kg suppressed F1.2 and reduced ischemia, though some
heparin may be necessary to avoid procedure-related thrombus formation. (Anticoagulation With
rNAPc2 to Eliminate MACE/TIMI
32; http://www.clinicaltrial.gov/ct/show/NCT00116012?order=1; NCT00116012)

Despite intensive therapy with antiplatelet agents, anticoagulants, anti-ischemic therapy, and an
early invasive strategy, 10% to 15% of high-risk patients with non–ST-segment elevation acute
coronary syndrome (nSTE-ACS) have recurrent myocardial infarction or die in the next 30 days
(1). Plaque rupture, whether spontaneous or following balloon inflation, exposes subendothelial
tissue factor (TF) and releases microparticles containing TF, a highly procoagulant material
found in elevated concentration in the necrotic atherosclerotic core. This results in the rapid
initiation of platelet activation and aggregation and thrombus formation (2). Current standard
antithrombotic therapies (heparins, aspirin, clopidogrel, glycoprotein [GP] IIb/IIIa blockers) do not
suppress thrombin generation (3) and percutaneous coronary intervention (PCI) enhances it (4).
Because proximal inhibitors of the coagulation cascade are potent inhibitors of new thrombin
generation, several agents (5) have been developed to exploit this potential advantage over
drugs that act more downstream in the cascade, such as heparins.

Recombinant nematode anticoagulant protein c2 (rNAPc2) is an 85-amino acid serine protease

inhibitor fashioned after the original protein isolated from the hematophagous hookworm
parasite Ancylostoma caninum(6). The anticoagulant activity results from inhibition of the
catalytic complex of TF/tissue factor/activated factor VII (fVIIa) by a unique mechanism that
requires the initial binding of rNAPc2 to zymogen or activated factor X before the formation of the
final ternary inhibitory complex with TF/fVIIa (7) (Fig. 1).The elimination half-life in man is 50 to
60 h and is independent of dose and route of administration (intravenous or subcutaneous) (8).
Earlier studies of rNAPc2 have demonstrated that it safely and effectively inhibits thrombin
generation in patients undergoing elective PCI (9) and reduces the incidence of deep venous
thrombosis when administered prophylactically in patients undergoing elective total knee
arthroplasty (10).

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Figure 1
Mechanism of Action of rNAPc2

Tissue factor/activated factor VII (TF/fVIIa) inhibition by recombinant nematode

anticoagulant protein c2 (rNAPc2) involves the following steps: 1) rNAPc2 binds tightly to
zymogen factor X; 2) docking of the rNAPc2-fX complex to TF/fVIIa assembled on a
procoagulant phospholipid surface; and 3) presentation of the reactive loop of rNAPc2 to
the active site of fVIIa, resulting in a tightly bound quarternary inhibitory complex. c2 =
rNAPc2; TF = tissue factor; VIIa = fVIIa; X = factor X.

The primary goal of the present phase 2 trial was to evaluate the safety and efficacy of a range of
doses of rNAPc2 in patients with nSTE-ACS managed with standard therapies, including an early
invasive strategy. We hypothesized that proximal inhibition of the coagulation cascade with
rNAPc2 would reduce new thrombin generation and recurrent ischemia following nSTE-ACS
without substantially increasing bleeding. The heparin de-escalation phase of this study was an
open-label pilot experience to explore the feasibility of the stepwise withdrawal of heparin in
patients treated with the highest studied dose of rNAPc2 with the goal of optimizing treatment
simplicity, safety, and efficacy.

Methods
Patient population
The ANTHEM–TIMI-32 (Anticoagulation With rNAPc2 to Help Eliminate Major Adverse Cardiac
Events–Thrombolysis In Myocardial Infarction-32) trial was a multicenter trial consisting of 2
parts: a randomized, double-blind, placebo-controlled, ascending rNAPc2 dose-ranging phase
and a single-arm, open-label, stepwise, unfractionated heparin (UFH) de-escalation pilot study of
10 μg/kg rNAPc2. In the dose-ranging phase, approximately 25 patients in each of 8 dose panels
were randomized to receive treatment with rNAPc2 or placebo in a 4:1 ratio. In the UFH de-
escalation phase, 2 open-label panels of 26 patients each with half-dose or no UFH were
studied.

Eligibility criteria included: age 18 to 75 years, hospitalization with moderate to high-risk nSTE-
ACS with ischemic discomfort at rest lasting ≥5 min consistent with ACS within 48 h of
randomization, and management with an early invasive strategy (≤72 h). In panels 1 to 3,
moderate to high risk was defined as a TIMI risk score for nSTE-ACS (11) of ≥3. Subsequently, a
protocol amendment also permitted patients with nSTE-ACS and either ST-segment deviation
≥0.5 mm (depression or transient elevation) or an elevated cardiac marker (troponin or creatinine
kinase-myocardial band) to be enrolled regardless of the TIMI risk score.

Exclusion criteria were ST-segment elevation myocardial infarction, ACS due to a distinct
nonatherosclerotic mechanism, increased bleeding risk, fibrinolytics within 24 h, planned
coronary artery bypass graft (CABG) surgery within 7 days, creatinine >4-mg/dl, anemia,
aminotransferase >3 times normal, allergy to aspirin, history of heparin-induced
thrombocytopenia, pregnancy, or other conditions placing the patient at increased risk. In the
heparin de-escalation phase, low-molecular-weight heparin within 12 h before randomization was
not permitted.

The study was conducted according to the International Conference on Harmonization Good
Clinical Practice Guidelines, approved by the institutional review boards/ethics committees of
each center, and registered with the National Institutes of Health (NCT00116012). Written
informed consent was obtained from all patients.
Study protocol
The study design is shown in Figure 2.A total of 255 patients (203 randomized in a 4:1 fashion to
rNAPc2 or placebo in the dose-finding phase, 52 patients enrolled in the single-arm, open-label
heparin de-escalation phase) participated from 28 centers in the U.S. and Canada between
November 2002 and May 2006. A central randomization system was used that involved a
permuted-block design in which assignment was blocked by site (block size of 5).

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Figure 2
ANTHEM–TIMI-32 Design

The study had 2 phases: (A)a double-blind, randomized, placebo-controlled, ascending

dose-ranging phase and (B)a single-arm, unblinded heparin de-escalation phase. In the
dose-ranging phase (A), 203 patients with non-ST-segment elevation acute coronary
syndrome (nSTE-ACS) managed with an early catheterization were randomized 4:1 to
recombinant nematode anticoagulant protein c2 (rNAPc2) or matching placebo
administered intravenously (IV) every 48 h for 1 to 3 doses in 8 panels of ∼25 patients
each. In the heparin de-escalation phase (B), 26 patients received 10 μg/kg rNAPc2 and
half-dose unfractionated heparin (UFH), followed by a second panel of 26 patients treated
with 10 μg/kg rNAPc2 and no heparin. *Encouraged per current practice guidelines. †10
μg/kg dose panel repeated. ASA = aspirin; ECG = electrocardiogram; Enox = enoxaparin;
GP = glycoprotein.

In the dose-ranging portion of the trial, we evaluated rNAPc2 doses (μg/kg) of 1.5, 2.0, 3.0, 4.0,
5.0, 7.5, and 10.0 (2 panels) in a double-blind fashion compared with placebo. In the heparin de-
escalation phase we studied 10.0 μg/kg rNAPc2 (open label) without a concomitant control
group. An independent data safety monitoring board, guided by an algorithm designed to
terminate study of an unsafe dose (see Statistical analysis), reviewed data at the completion of
each panel and approved the testing of the next dose. Patients received intravenous study drug
(single injection over 60 s) followed by coronary angiography and revascularization (if
appropriate). Additional doses of study drug every 48 h were permitted during hospitalization,
provided CABG was not required and the patient did not experience clinically significant bleeding
or other adverse event.

All patients received daily oral aspirin (75 to 325 mg). The use and timing of clopidogrel, GP
IIb/IIIa blockers, and other standard ACS therapies were encouraged per current practice
guidelines (12) but left to the investigator’s discretion.
In dose finding, either UFH (60 U/kg bolus [maximum 5,000 U], 12 U/kg/h initial infusion
[maximum 1,000 U/h] titrated to an activated partial thromboplastin time [aPTT] 1.5 to 2 times
control) or enoxaparin (1.0 mg/kg subcutaneously every 12 h [every 24 h if the calculated
creatinine clearance was <30 ml/min]) was required. In the first 3 dose panels, enoxaparin was
the only anticoagulant permitted between randomization and 48 h after the last dose of study
drug. However, a protocol amendment subsequently allowed the use of UFH after new external
data (1) demonstrated similar outcomes between these agents. During PCI, dosing algorithms
were used (see Appendix) to ensure adequate and uniform anticoagulation.

In the first panel of the heparin de-escalation phase, half-dose UFH (30 U/kg bolus [maximum
2,500 U]; 6 U/kg/h infusion [maximum 500 U/h] with no subsequent adjustment) was
administered before and during coronary angiography/intervention. In the second panel, no
additional anticoagulant was permitted between randomization and 48 h after the last dose of
study drug (including during PCI), unless required for management of a thrombotic complication
or other strong clinical indication (e.g., CABG).

Blood collection and assays

Blood specimens to assess the complete blood count (CBC), plasma drug concentration, and
pharmacodynamic effect (international normalized ratio [INR], prothrombin fragment 1.2 [F1.2])
were obtained at baseline, 2 to 6 h, 24 h (CBC only), 48 h (if not discharged), and 7 days after
last study drug, and 42 days after randomization. The INR and F1.2 (assessed by enzyme-linked
immunoabsorbent assay [ELISA]) were measured by Icon Laboratories (Farmingdale, New York)
using a Stago Sta Analyzer and ELX800 Biotek instrument, respectively. The F1.2 concentration
during dose ranging was measured using polyclonal antibodies (Enzygnost F1+2 assay,
reference range 0.40 to 1.10 nmol/l, sensitivity 0.04 nmol/l), but this methodology was replaced
by a more specific assay using monoclonal antibodies during the heparin de-escalation phase
(Enzygnost F1+2 [monoclonal] assay, reference range 69 to 229 pmol/l, sensitivity 20 pmol/l) by
the manufacturer (Dade Behring, Deerfield, Illinois). Plasma drug concentrations were analyzed
by ELISA (panels 1 to 3 by Corvas International, San Diego, California; all other panels by Alta
Bioanalytical Laboratories, San Diego, California).

Blood samples were obtained at baseline and on day 42 to assess for the development of
antibodies to rNAPc2 using rabbit polyclonal antibodies to NAPc2. If antibodies were present at
day 42, then patients were retested again at 3 and 6 months, and antibodies were assessed for
neutralizing activity.

Local measurement of INR during dose finding was not permitted between randomization and 7
days after study drug to maintain the study blind, because rNAPc2 increases the INR in a dose-
related fashion (8). The aPTT (which is only minimally affected by rNAPc2 [13]) was measured
daily and 6 h after any change in the UFH dose. The activated clotting time was used to guide
UFH dosing during PCI (see Appendix). Serial assessment of cardiac markers of necrosis were
performed in the first 24 h and after recurrent ischemia or revascularization.

3-lead electrocardiographic monitoring

All patients underwent continuous ambulatory 3-lead electrocardiographic (ECG) monitoring
(Lifecard CF ambulatory ECG recorder; Reynolds Medical, Braintree, Massachusetts) for
ischemia from randomization through 7 days after the last dose of study drug. Monitoring
continued during and after PCI but was terminated just before CABG surgery. Continuous ECGs
were assessed by a central core laboratory blinded to treatment assignment. An ischemic
episode was defined as ≥1.0 mm horizontal or downsloping ST-segment depression or >1.0 mm
ST-segment elevation lasting at least 1 min and separated from other episodes by at least 5 min,
occurring in any of the 3 leads. Ischemic events occurring during catheterization or PCI were
censored.
Clinical follow-up
Return visits to the clinic were made 7 days after the last dose of study drug and 42 days after
randomization. Follow-up phone calls to assess for bleeding and intercurrent events were made
at 3 and 14 days after the last dose and at 3 and 6 months after randomization to assess for
death and ischemic complications.

Clinical end points and definitions

The prespecified primary end point of the study was the combined rate of adjudicated major plus
minor hemorrhage using the standard TIMI criteria (14) (hereafter termed “significant”
hemorrhage) from randomization through 7 days after the last dose of study drug. A TIMI major
hemorrhage was defined as an intracranial hemorrhage or overt bleeding associated with a >5
g/dl fall in hemoglobin. A TIMI minor hemorrhage was defined as a nonintracranial overt bleed
associated with a 3 to 5 g/dl decrease in hemoglobin. Additional clinical safety end points
included the rates of transfusions, serious adverse events, and antibody formation to rNAPc2.

Prespecified efficacy end points included pharmacokinetic (plasma drug concentration) and
pharmacodynamic (INR, F1.2) measures and the percentage of patients with ischemia in the first
48 h on 3-lead continuous ECG. Additional prespecified ECG end points of interest included the
average number of ischemic events per patient and the ST-product, defined as the product of the
maximal extent of ST-segment deviation multiplied by the number of minutes of deviation.

Reinfarction, clinical recurrent ischemia, and stroke were defined with the standard TIMI
definitions (15). Thrombotic bailout (TBO) was defined as an episode of refractory ischemia
occurring after administration of the first dose through 7 days after the last dose of study drug
that required the use of open-label anticoagulation to manage any one of the following
complications due to thrombosis: 1) ischemia lasting ≥20 min despite management with standard
therapies other than anticoagulants; 2) clinical instability (e.g., hypotension); 3) a decrement in
TIMI flow grade; 4) a coronary artery dissection with decreased flow; 5) distal embolization; 6)
side branch closure; or 7) abrupt closure of the culprit artery. All of the clinical end points
described were adjudicated by an independent blinded clinical event committee. In suspected
cases of TBO during the heparin de-escalation phase, angiographic results assessed by a
blinded central angiographic core laboratory were provided to the event committee for
consideration.

Biomarkers were obtained at baseline, 2 to 6 h, 48 h, and 7 days after last dose, and 42 days
after randomization in the dose-ranging phase. These included cardiac troponin I, high-sensitivity
C-reactive protein, brain natriuretic peptide, and matrix metalloproteinase 9, measured in the
TIMI Biomarker Core Laboratory using established methods (Beckman Coulter AccuTnI and
Hitachi 917 Analyzer; Roche Diagnostics, Indianapolis, Indiana; and Beckman Coulter Access 2
BNP; ELISA assay, R&D Systems, Minneapolis, Minnesota; respectively).

Statistical analysis
The selection of 25 patients per panel was guided by the desire to gain experience with the use
of rNAPc2 in combination with aspirin, heparins, clopidogrel, GP IIb/IIIa inhibitors, and an early
invasive strategy. Safety boundaries were set to exclude rates of significant hemorrhage that
were relatively 20% greater than the predicted historical control rate of 6% (i.e., absolute rate
>7.2%) based on the observations from the National Investigators Collaboration with Enoxaparin
3 (NICE-3) registry (16), using a 1-sided 95% confidence interval. In the heparin de-escalation
phase, a second safety boundary was set to exclude a rate of TBO that was relatively 20%
greater than the historical control rate of TBO (6.2%) observed in the REPLACE-2 (Randomized
Evaluation of PCI Linking Angiomax to Reduced Clinical Events) trial (17) (i.e., absolute rate
>7.44%) using a 1-sided 95% confidence interval, because the risk of thrombosis with reduced-
dose heparin is unknown.
The prespecified primary safety and efficacy end points were based on the intention-to-treat
principle among patients who received any dose of study drug. For the prespecified continuous
ECG analysis, patients were required to have at least 48 h of interpretable data. Analyses were
performed using 2-sided tests at the 0.05 level of significance. No adjustments were made for
multiple comparisons.

Baseline characteristics and safety and efficacy end points were summarized using descriptive
statistics, including mean values ± SD, median values with interquartile ranges, and event rates,
as appropriate, using SAS version 9.1 (SAS Institute, Cary, North Carolina). Differences between
rNAPc2 and placebo were analyzed via Fisher exact test (binary outcomes), ttests (continuous
normally distributed data), and the Wilcoxon rank sum test (continuous non-normally distributed
data). Comparisons involving more than one rNAPc2 dose were performed using the chi-square
test (binary outcomes), 1-way analysis of variance (continuous normally distributed data), and
Kruskal-Wallis test (continuous non-normally distributed data). The Pearson correlation test was
used to assess the correlation of log concentration rNAPc2 with INR.

The trial was designed as a collaborative effort between the TIMI Study Group, the Nottingham
Clinical Research Limited (NCRL), and the sponsor. The data were collected by NCRL. The
NCRL and the TIMI Study Group performed the prespecified and exploratory analyses
independently of the sponsor.

Results
Patients
Baseline characteristics are shown in Table 1and concomitant treatments in Table
2(see Appendixfor complete data in each dose panel). No significant differences were found
between patients who received rNAPc2 or placebo.

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Table 1
Patient Baseline Characteristics

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Table 2
In-Hospital Therapies and Procedures

Pharmacokinetics and pharmacodynamics

Dose-related increases in plasma rNAPc2 concentration and INR were observed at 2 to 6 and 48
h (Fig. 3).The log concentration was correlated with INR at 2 to 6 and 48 h (ρ = 0.51 and ρ =
0.49, respectively; p < 0.0001 for both). Patients who received increasing doses of rNAPc2 had
progressively less thrombin generation (lower F1.2) at 2 to 6 h (trend p = 0.001) and 48 h (trend
p = 0.002), with statistically significant reductions observed with doses ≥7.5 μg/kg compared with
placebo (Fig. 4).

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Figure 3
Pharmacokinetics and Pharmacodynamics

(A)Increasing doses of recombinant nematode anticoagulant protein c2 (rNAPc2) were

associated with increased plasma drug concentrations at 2 to 6 and 48 h. (B)rNAPc2
increased the international normalized ratio (INR) in a dose-related fashion at 2 to 6 and 48
h.

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Figure 4
rNAPc2 Suppresses New Thrombin Generation

Prothrombin fragment 1.2 (F1.2), a marker of new thrombin generation, was reduced
signficantly at 2 to 6 and 48 h with ≥7.5 μg/kg recombinant nematode anticoagulant
protein c2 (rNAPc2) compared with placebo. Lower-dose rNAPc2 (1 to 5 μg/kg) blunted the
increase in F1.2 seen at 48 h in the placebo group.

Continuous ECG
The rate of ischemia by continuous ECG through day 7 was similar in patients receiving rNAPc2
and placebo (18% vs. 21%; p = NS). At doses of rNAPc2 that suppressed F1.2 through 48 h
(≥7.5 μg/kg), ischemia was reduced by >50% using a variety of continuous ECG measures
compared with low-dose rNAPc2 and placebo (Table 3).
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Table 3
Continuous 3-Lead Electrocardiographic Results

Biomarkers
There were no differences (absolute concentrations or change from baseline) in cardiac troponin
I, high-sensitivity C-reactive protein, brain natriuretic peptide, or matrix metalloproteinase-9
through 42 days across treatment groups in the dose-ranging phase or when comparing all
patients who received rNAPc2 versus placebo (data not shown).

Safety
There were 9 significant hemorrhages (3.5%), including 4 major and 5 minor, in the entire trial; all
were associated with a procedure. In dose ranging, there was no difference in the incidence of
significant hemorrhage between rNAPc2 and placebo (4.3% for the pooled doses of 1.5 to 10
μg/kg rNAPc2 vs. 2.5% for placebo; p = NS). No statistical trend was observed across ascending
doses of rNAPc2 individually (Fig. 5)or when grouped as placebo, low-dose (1.5 to 5.0 μg/kg),
and high-dose (≥7.5 μg/kg; 2.5% vs. 2.9% vs. 4.5%, respectively; p = 0.77). All 4 major
hemorrhages occurred at the highest dose (10 μg/kg); 3 were patients with excessive bleeding
requiring transfusions after CABG. A shorter interval between the last dose of rNAPc2 and CABG
surgery was associated with a higher risk of bleeding (40% vs. 10% vs. 0% for surgery
performed <48 h vs. 48 to 96 h vs. >96 h, respectively, after the last dose of study drug; trend p =
0.077). Other safety outcomes, including rates of other (neither major nor minor) bleeding (9.8%
vs. 10.0%), frequency (6.5% vs. 2.5%) and average number (2.2 vs. 2.0) of units of red blood cell
transfusions, and serious adverse events (33% vs. 30%) were similar between rNAPc2 and
placebo, respectively (all p = NS). No patients required treatment with fVIIa concentrate to
reverse rNAPc2 anticoagulation (18). Three patients receiving rNAPc2 developed mild
thrombocytopenia (93,000, 98,000, and 99,000 cells/mm3) with no clinical sequelae.

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Figure 5
Hemorrhagic Events With rNAPc2

There was no difference between the rates of major plus minor hemorrhage among
patients receiving recombinant nematode anticoagulant protein c2 (rNAPc2) versus
placebo (3.7% vs. 2.5%, respectively), nor was there a trend across ascending doses (p =
NS). All major and minor bleeding events were related to procedures; none were
spontaneous. *3/4 major hemorrhages were CABG-related, 2 to 3 days after surgery.
CABG = coronary artery bypass graft; Hgb = hemoglobin; ICH = intracranial hemorrhage;
TIMI = Thrombolysis In Myocardial Infarction; UFH = unfractionated heparin.

Anti-rNAPc2 antibody formation occurred in 13% of patients receiving rNAPc2. No patients with
positive antibodies experienced symptoms (e.g., allergic reaction, anaphylaxis) attritibutable to
the antibody, nor were there differences in INR or clinical outcomes among those with or without
antibodies.

Clinical events
Clinical event rates were low and there were no differences in the rates of death, reinfarction,
clinical recurrent ischemia, stroke, and late revascularization either individually or in composite
between the treatment groups (Table 4).The composite event rate was 24% among patients
receiving higher dose (≥7.5 μg/kg) rNAPc2 versus 38% for placebo (p = 0.15). Thrombotic bailout
with an open-label antiocoagulant occurred in 5 patients during heparin de-escalation, all whom
received rNAPc2 10 μg/kg and no heparin (19% vs. 0% for no-heparin vs. half-dose heparin
treatment groups, respectively; p = 0.051). Of the 14 patients who had PCI in the no-heparin
group, 4 had TBO (new intrastent or coronary artery thrombus formation). The fifth TBO event
was an episode of clinical unstable angina without new ischemic ECG changes that required an
increased dose of intravenous nitroglycerin and open-label anticoagulation before PCI.

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Table 4
Clinical Events Through 42 Days

Discussion
This is the first randomized trial of a proximal inhibitor of the coagulation cascade that targets TF
in patients with ACS. We found that intravenous rNAPc2 in doses of 1.5 to 10 μg/kg was well
tolerated in patients with nSTE-ACS managed with multiple antithrombotic agents and an early
invasive strategy. However, all 4 observed major hemorrhages occurred with the highest dose.
The INR was increased and new thrombin generation (measured by F1.2) was suppressed in a
dose-dependent fashion that correlated with plasma drug concentration. We observed 3
important issues with regard to the bleeding risk of rNAPc2. First, similar to other antithrombotic
agents, the risk of bleeding is highest around the time of procedures. Indeed, all significant
hemorrhages were related to the femoral access site or CABG surgery. Second, there was no
statistical increase in bleeding rates with rNAPc2 (all doses pooled) compared with placebo, a
result that is consistent with in vitro modeling of rNAPc2 and various antithrombotic agents that
demonstrated no exaggerated effect on clotting and platelet function (13). However, in the
subgroup of patients undergoing CABG, we observed a tendency toward increased bleeding
rates with surgery performed <48 hours of rNAPc2 administration, particularly following the
highest dose studied (10 μg/kg).

Suppression of ischemia on continuous ECG has been associated with a reduction in clinical
ischemic events in studies with enoxaparin (19) and GP IIb/IIIa blockers (20), including patients
who underwent PCI (21). A novel finding in the ANTHEM–TIMI-32 trial was that the doses of
rNAPc2 that suppressed F1.2 through 48 h were also associated with a ≥50% reduction in
ischemia on continuous ECG. This provides a link between the mechanism of action of this drug
and the hypothesized benefit in patients with plaque rupture and vessel wall injury. A larger trial
would be required to test the hypothesis that rNAPc2 reduces clinical events, because the
current trial was not powered for such an assessment. Because rNAPc2 has a half-life of 50 to
60 h and can be administered subcutaneously, prolonged inhibition of new thrombin generation
with rNAPc2 for several weeks has the potential to improve the recommended discharge regimen
after ACS, which currently does not include routine use of an anticoagulant.

Although several small studies have explored the use of reduced-dose UFH (22) or no heparin
(23) during PCI, the risk of intraprocedural thrombosis remains a concern (24). In the ANTHEM–
TIMI-32 trial, we observed thrombotic complications in 5 of 26 patients (19%) who were treated
with 10 μg/kg rNAPc2 without UFH. However, no thrombotic complications occurred when half-
dose UFH was administered. In 4 out of 5 cases of TBO, new intracoronary thrombus formed
during PCI. Catheter-related thrombosis has been reported more frequently with the parenteral
factor Xa inhibitor fondaparinux than UFH despite flushing catheters with heparin (25). Similarly,
thrombus formation on the guiding catheter and wire has been reported with enoxaparin despite
concomitant therapy with aspirin, clopidogrel, and GP IIb/IIIa blockers (26).

These observations, although small in number, raise the possibility that inhibitors of the terminal
portion of the coagulation cascade (i.e., “distal protection”) may be necessary during
intravascular procedures to inhibit thrombin. Distally acting agents may be more effective in
inhibiting the contact pathway that is activated when catheters are introduced into the
bloodstream (27). Although proximal inhibitors of the clotting cascade may be more effective in
reducing thrombin generation, even with high doses of a proximal inhibitor, thrombus formation
can still occur when foreign bodies are present and heparin is withheld. The present data are
consistent with emerging clinical (25,26) and experimental (27) data which suggest that
concomitant inhibitors of the contact pathway that act on formed thrombus may be necessary
during intracoronary procedures. Therefore, we believe that reduced-dose distal-acting
antithrombins should be tested in combination with proximal inhibitors currently in development.

Study limitations
This phase 2 trial was not designed with sufficient power to exclude small differences in safety or
clinical efficacy. The trial enrolled moderate- to high-risk patients in whom an early invasive
strategy was planned and CABG was not intended; therefore, the observed results may not apply
to other patients. We did not control for prior therapies and concurrent treatments (other than
antithrombotics), which may have affected the biomarkers and outcomes. Too few patients (on
average 3 per panel) received more than 1 dose of rNAPc2 to draw any meaningful conclusions
from the administration of multiple doses. The heparin de-escalation phase was open label
without concurrent controls.

Conclusions
In patients with nSTE-ACS managed with standard therapies including an early invasive
approach, addition of rNAPc2, a potent inhibitor of the TF/fVIIa complex, at doses up to 10 μg/kg,
was well tolerated. The observation that doses ≥7.5 μg/kg suppressed F1.2 (a marker of
thrombin generation) may explain the observed >50% reduction in ischemia detected by
continuous ECG. Some heparin may be necessary to prevent catheter-based thrombosis.
Coronary artery bypass graft surgery should be avoided within 48 hours of dosing. Future trials of
rNAPc2 in nSTE-ACS are needed to assess the potential clinical role of this novel agent.

Appendix
 For supplementary tables and a detailed list of investigators, study committees, and core
laboratories, please see the online version of this article.

Appendix
Recombinant Nematode Anticoagulant Protein c2 in Patients With Non-ST-Segment Elevation
Acute Coronary Syndrome: The ANTHEM-TIMI-32 Trial

[S0735109707012028_mmc1.doc]

Footnotes
 ↵1 Dr. Deitcher is an employee of Nuvelo, Inc.
 ↵2 Dr. Giugliano serves as a consultant to Schering-Plough and GlaxoSmithKline and has
received honoraria for continuing medical education activity from Sanofi-Aventis, Bristol-Myers
Squibb, Schering-Plough, GlaxoSmithKline, and Genentech.
 ↵3 Dr. Wiviott serves as a consultant to Biogen-Idec, Forrest Laboratories, and Transform and
has received honoraria from Eli Lilly, Daiichi Sankyo, and Accumetrics.
 ↵4 Dr. Braunwald has participated in symposia/advisory board meetings/consultancies for
AstraZeneca, Bayer, Daiichi Sankyo, Eli Lilly, Momenta, Pfizer, Sanofi-Aventis, Schering-Plough,
and Scios/Johnson & Johnson.
 See the Appendixfor a complete list of the ANTHEM–TIMI-32 Investigators.
 Supported by Nuvelo, Inc., San Carlos, California.
 The TIMI Study Group receives research grant support from Sanofi-Aventis, Schering-Plough,
Novartis, Johnson & Johnson, and Sunol for studies of anticoagulants.

Abbreviations and Acronyms

CABG
coronary artery bypass graft
F1.2
prothrombin fragment 1.2
GP
glycoprotein
INR
international normalized ratio
nSTE-ACS
non–ST-segment elevation acute coronary syndrome
PCI
percutaneous coronary intervention
rNAPc2
recombinant nematode anticoagulant protein c2
TF/fVIIa
tissue factor/activated factor VII
TIMI
Thrombolysis In Myocardial Infarction
UFH
unfractionated heparin

 Received January 3, 2007.

 Revision received January 31, 2007.
 Accepted February 12, 2007.
  American College of Cardiology Foundation

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