© 2004 Schattauer GmbH, Stuttgart
Theme Issue Letter to the Editor
Combined coagulation phase-directed factor Xa inhibition with
heparin compounds and DX-9065a – A direct and selective antagonist
Dear Sir,
The development of novel anticoagulants for use in patients
with acute coronary syndromes (ACS) has evolved steadily,
emphasizing the importance of cellular surface biochemistry
and the highly integrated contribution of platelets, leukocytes
and endothelial cells in the thrombotic process. Accordingly,
tissue factor and its primary complexing coagulation proteases,
factor VIIa and Xa, represent attractive targets for pharmacologic inhibition of thrombin generation (1).
Although combined or “multi-site directed” pharmacologic
therapy is an appealing construct for the attenuation of vascular
thrombosis in ACS, pharmacokinetic and pharmacodynamic
relationships that may potentially influence safety, efficacy and
dosing must be considered carefully.
DX-9065a is the first in a class of synthetic, small-molecule,
direct factor Xa inhibitors. Its antithrombotic efficacy has been
demonstrated in animal models of venous thrombosis (2), arteriovenous shunt thrombosis (3, 4), vein graft thrombosis (5) and
disseminated intravascular coagulation (6). In a double-blind
trial of 73 patients with stable coronary artery disease (7),
DX-9065a given as a 72-hour infusion was well tolerated and
plasma drug concentrations correlated strongly with anti-factor
Xa activity (r=0.97).
Because unfractionated heparin (UFH) and low molecular
weight heparin (LMWH) represent the current standard of care
for anticoagulant therapy in ACS and both inhibit factor Xa
(as well as other coagulation proteases) indirectly through an
antithrombin III-dependent mechanism, we conducted a series
of in vitro and ex vivo experiments to determine their pharmacodynamic interactions with DX-9065a.
Blood samples were obtained by standard venipuncture
from healthy donors (n=6). The initial 2 ml of blood was
discarded and the remainder placed in tubes containing 3.2%
sodium citrate. Following gentle mixing, samples were transCorrespondence to:
Richard C. Becker, M.D.
Cardiovascular Thrombosis Center
Duke University Medical Center
Durham, NC 27715, USA
Tel.: + 1 919 668-8926
E-mail: [email protected]
ferred to tubes containing either DX-9065a (Daiichi Pharmaceutical, Co. Ltd., Tokyo, Japan) (0, 0.05, 0.1, 0.2, 0.4, 0.8 ug/ml),
UFH (Elkin-Simm) (0, 0.5, 1.0, 2.5, 5.0 U/mL), enoxaparin
(Aventis Pharmaceuticals) (0, 0.5, 1.0, 2.5, 5.0 U/mL),
DX-9065a plus UFH or DX-9065a plus enoxaparin. Lower
concentrations of UFH (maximum 1.0 U/mL) and enoxaparin
(1.0 U/mL) were used for the combination experiments.
Several mLs of blood were transferred to tubes containing
CaCl2 (final concentration 12.5 nM) and incubated at 37°C for a
total of four minutes. Immediately following incubation, 50 ul
of blood was placed on a coagulation test cartridge (whole blood
microcoagulation system, Hemochron Jr®, International Technidyne Corporation, Princeton, NJ). Prothrombin time (PT),
(upper limit 100 seconds) and Activated Clotting Time (ACT)
(upper limit 400 seconds) measurements were performed
according to the manufacturers specifications. Anti-Xa measurements were made using a chromogenic assay (Rotachrome;
Diagnostica Stago Inc.).
Patients scheduled to undergo elective percutaneous coronary intervention (PCI) were approached for study participation.
Blood samples were obtained either before (control) or 15 minutes after intravenous administration of UFH (average bolus
dose 3000 U). Some patients received a platelet glycoprotein
(GP) IIb/IIIa receptor antagonist and all had been given aspirin
(325 mg) within the prior 6 hours.
0.45 mL of blood was added to tubes containing DX-9065a
(0, 0.05, 0.1, 0.2, 0.4, 0.8 µg/ml). After gentle mixing, 50 ul of
blood was placed on coagulation test cartridges for measurement of PT, INR and ACT (Microcoagulation System
Hemochron Jr®, International Technidyne Corporation, Princeton, NJ). All coagulation testing was performed at the “point of
care” using a separate coagulation monitor for each DX-9065a
concentration (to minimize the effect of measurement delay on
test results)
To examine the sensitivity of each coagulation measurement
to DX-9065a alone and in combination with either UFH or
enoxaparin, the PT was mathematically transformed using the
mean of the highest ACT value as a reference. The transformed
PT was defined as
X PT (8). The slope, Y value,
of the regression line represents a reproducible and comparable
dose response parameter (9).
Received April 21, 2004
Accepted after resubmission October 5, 2004
Thromb Haemost 2004; 92: 1229–31
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Letter to the Editor
A)
Figure 1: Anti-Xa
response to UFH,
Enoxaparin,
DX-9065a and
their combination.
A). Anti-Xa measurements for DX-9065a
alone and in combination with unfractionated heparin.The anticoagulant effect was
additive and particularly robust with DX9065a concentrations
≥ 0.20 µg/mL. B). AntiXa measurements for
DX-9065a alone and
in combination with
enoxaparin. The anticoagulant effect, although overall less robust than with UFH,
was additive.
B)
Unfractionated heparin at concentrations ranging from 0.5
to 5.0 U/mL prolonged the whole blood PT (r=0.87, Y=35.1 +
13.3) and ACT (r= 0.92, Y=134.0 + 6.4). Similar observations
were made for enoxaparin; however, the slopes were lower for
each coagulation measurement.
DX-9065a in concentrations up to 0.8 µg/mL prolonged the
whole blood PT (r=0.94, Y=114.8 + 4.53) and ACT (r=0.93,
Y=143.6 + 9.7). Transforming the PT yielded a Y value of 280 +
11.1, suggesting that the whole blood PT is a more sensitive
coagulation measure than ACT for determining the anticoagulant effects of DX-9065a. For each concentration of DX-9065a
there was a corresponding whole blood PT and ACT value within the devices range of reportable results.
UFH in relatively low concentrations (0.5 U/ml) added to
DX-9065a prolonged the whole blood PT(Y=322.1 + 27.9) and
ACT (Y=355.1 + 32.9). The transformed PT slope value was
786.0 + 65.8. Concentrations of DX-9065a above 0.2 µg/mL
prolonged both the PT and ACT measures beyond reportable
limits. UFH, in moderate concentrations (1.0 U/mL), added to
DX-9065a caused marked prolongation of the PT (Y=537.0 +
49.8) and ACT (Y=654.3 + 60.5), surpassing the upper limits of
reportable results at concentrations of DX-9065 approaching
0.2 µg/mL. The coagulation measurement responses for enoxaparin in combination with DX-9065a were similar to those
observed with UFH; however, the slopes were lower.
Anti-Xa measurements using a chromogenic assay in
response to DX-9065a, UFH, enoxaparin and their combination
are shown in Figure 1. There was a concentration-dependent
increase in anti-Xa activity with DX-9065a. Approximately
0.2 µg/mL DX-9065a produced a similar degree of factor Xa
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Letter to the Editor
inhibition (based on a chromogenic assay) to UFH and enoxaparin at 0.5 U/mL. The anticoagulant effects of DX-9065a
(<0.2 µg/mL) combined with either UFH or enoxaparin
(0.5 U/mL) were additive. At higher concentrations (DX-9065a
> 0.2 µg/mL and UFH or enoxaparin 1.0 U/mL) the response
was more robust. Similar observations were made in our ex vivo
experiments.
The development of new anticoagulants for thrombotic disorders of the cardiovascular system must carefully consider
their mechanism of action, pharmacodynamic profile and full
spectrum of coagulation protease inhibiting properties when
used alone or in combination with other drugs. DX-9065a, a
direct and selective factor Xa inhibitor prolongs whole blood
coagulation measures and, in combination with either UFH or
the LMWH preparation, enoxaparin (antithrombin III-dependent, indirect inhibitors), provokes additive anticoagulant effects.
The work of Rezaie and colleagues (10) suggests that 7 of the
11 basic residues of the heparin-binding exosite of thrombin are
conserved at similar three dimensional locations in fXa; they
are Arg93, Lys96, Arg125, Arg165, Lys169, Lys236, and Arg240. In
addition, their findings support overlapping binding sites on
fXa for fVa and heparin, providing insights regarding the relative contribution of heparin and the heparin-antithrombin III
complex neutralization of free fXa and fXa existing within the
prothrombinase complex (which is not accessible for binding to
AT). In contrast, DX-9065a inhibits fXa in both the plasma
phase and prothrombinase complex in the presence or absence
of prothrombin (11).
The unique binding characteristics for heparin and
DX-9065a to fXa provide distinct targets for maximal pharmacologic inhibition and a mechanistic explanation for our findings both in vitro and ex vivo (12, 13).
DX-9065a in combination with either UFH or enoxaparin
provoked a rapid anticoagulant response, as reflected in whole
blood coagulation measures and anti-Xa activity. This effect has
not been reported previously. Although the overall clinical
significance of these observations is unknown, our in vitro and
ex vivo experiments suggest that a systemic state of moderateto-high intensity anticoagulation should be anticipated in clinical settings where heparin concentrations in excess of 0.5 U/ml
coexist with DX-9065a levels of 0.2 µg/ml or greater. The common practice of combined pharmacotherapy in acute coronary
syndromes with use of aspirin, clopidogrel and GPIIb/IIIa receptor antagonists could potentially “shift” the balance of safety
even further with lower concentrations of either or both anticoagulants particularly in the setting of invasive procedures.
The potential influence of combined and rapidly sequenced
anticoagulant pharmacotherapy on clinical outcome should not
be underestimated and may be highly relevant for drug development, clinical trial design, and patient care.
Richard C. Becker1, 2, John Alexander1, 2,You Fu Li3,
Edwin Bovill4, Frederick A. Spencer3,Thomas L. Robertson5,
Satoshi Kunitada5, Christopher K. Dyke1, 2,
Robert A. Harrington1, 2
1
Cardiovascular Thrombosis Center, Duke University
Medical Center, Durham, North Carolina, USA
2Duke Clinical Research Institute
Durham, North Carolina, USA
3Laboratory for Vascular Biology Research, UMASS Memorial Medical Center,Worcester, Massachusetts, USA
4Department of Pathology, University of Vermont, Burlington,Vermont, USA
5Daiichi Pharmaceuticals, Inc.,Tokyo, Japan
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