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Vol. 295, Issue 1, 212-218, October 2000
Cardiovascular Diseases Research (P.C.W., E.J.C., R.M.K., R.P.M., C.A.W., A.M.S.), Chemical and Physical Sciences (M.L.Q., R.R.W.), Drug Metabolism and Pharmacokinetics (M.R.W.), DuPont Pharmaceuticals Company, Wilmington, Delaware
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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SK549 (mol. wt. 546 Da) is a synthetic, selective inhibitor of human coagulation factor Xa (fXa) (Ki = 0.52 nM). This study compared the antithrombotic effects of SK549 and a series of benzamidine isoxazoline fXa inhibitors with aspirin, DuP 714 (a direct thrombin inhibitor), recombinant tick anticoagulant peptide, or heparin in a rabbit model of electrically induced carotid arterial thrombosis. Compounds were infused i.v. continuously from 60 min before electrical stimulation to the end of the experiment. Values of ED50 (dose that increases the carotid blood flow to 50% of the control) were 0.12 µmol/kg/h for SK549, 0.56 µmol/kg/h for aspirin, 0.14 µmol/kg/h for DuP 714, 0.06 µmol/kg/h for recombinant tick anticoagulant peptide, and >100 U/kg/h for heparin. The EC50 (plasma concentration that increased blood flow to 50% of the control) for SK549 was 97 nM. Unlike aspirin and heparin, SK549 was efficacious and, at 1.5 µmol/kg/h i.v. (n = 9), maintained carotid blood flow at 87 ± 6% of control level for greater than 90 min. Unlike heparin, SK549 inhibited ex vivo fXa activity but not ex vivo thrombin activity. There was a highly significant correlation between Ki (fXa) and ED50 of a series of fXa inhibitors (r = 0.85, P < .001). Therefore, these results suggest that SK549 is a novel, potent, and effective antithrombotic agent in a rabbit model of arterial thrombosis. It is likely that SK549 exerts its antithrombotic effect through selective inhibition of fXa. Furthermore, SK549 may be clinically useful for the prevention of arterial thrombosis.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The
clinical usefulness of anticoagulants such as warfarin (Coumadin) and
the successful development of low-molecular-weight heparin for the
treatment and prevention of thromboembolic diseases have generated
great interest in designing new inhibitors of blood coagulation
(Turpie, 1998
). The most promising new inhibitors of blood coagulation
are inhibitors of thrombin or factor Xa (fXa) (Fevig and Wexler, 1999;
Hauptmann and Stürzebecher, 1999).
Both naturally occurring and synthetic thrombin inhibitors have been
well studied for the past two decades (Hauptmann and Stürzebecher, 1999). However, it is still not clear whether the desired antithrombotic effects of these inhibitors can be achieved without undesirable bleeding complications (Turpie, 1998). Thus, there
is an increasing interest in developing synthetic and selective fXa
inhibitors (Fevig and Wexler, 1999; Sinha, 1999).
Similar to thrombin inhibitors, both naturally occurring and synthetic
fXa inhibitors are available and have been shown to be potent
antithrombotic agents in animal models of thrombosis (Wong et al.,
1996; Kaiser, 1998; Hauptmann and Stürzebecher, 1999). However,
in contrast to thrombin inhibitors, it is believed that inhibition of
fXa may reduce the production of thrombin by either the extrinsic or
intrinsic pathways without interfering with a basal level of thrombin
activity necessary for normal hemostasis (Harker et al., 1997).
Recently, DuPont Pharmaceuticals has discovered an interesting novel
series of potent and selective nonpeptide fXa inhibitors, exemplified
by (
)-5-isoxazolecarboxamide,
3-[3-(aminoiminomethyl)phenyl]-N-5-[2'-(aminosulfonyl)-[1,1'-biphenyl]-4-yl]-4,5-dihydro-5-(1H-tetrazol-1-ylmethyl)-trifluoroacetic acid salt (SK549) (Fig. 1) (Quan et al.,
1999a,b; Wong et al., 2000). SK549 is a potent and selective fXa
inhibitor (Ki: fXa, 0.52 nM; thrombin, 400 nM; trypsin, 45 nM; tissue plasminogen activator, >33,000 nM; plasmin,
890 nM) (Wong et al., 2000). It has a low plasma clearance of 0.3 l/h/kg in rabbits and 0.7 l/h/kg in dogs (Quan et al., 1999a). Given
i.v. or intraduodenally, SK549 is a potent antithrombotic agent in a
rabbit model of arteriovenous shunt thrombosis (Wong et al., 2000).
Although the model of arteriovenous shunt thrombosis is useful in
predicting antithrombotic efficacy of fXa inhibitors, it may not be a
very physiologically relevant model. Furthermore, the effectiveness of
SK549 for the prevention of arterial thrombosis has not been studied.
Therefore, we evaluated in this study the antithrombotic effect of
SK549 and its chemical analogs in a rabbit model of arterial
thrombosis. Thrombosis in this model was produced by electrically
induced injury of the carotid artery. Because arterial thrombosis in
humans usually occurs in areas of medium-to-high blood flow and shear
stress with a triggering factor of vascular injury (Badimon, 1997), the model of electrical current-induced arterial thrombosis (ECAT) in
rabbits may have some pathophysiological relevance to the human disease. In this study, we also included heparin, aspirin, DuP 714 (a
direct thrombin inhibitor) (Kettner et al., 1990; Knabb et al., 1992),
and recombinant tick anticoagulant peptide (rTAP, a potent and
selective fXa inhibitor) (Neeper et al., 1990) for comparison.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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All experiments were conducted in accordance with the regulations of the Animal Care and Use Committee of the DuPont Pharmaceuticals Company.
Reagents.
The following drugs and chemicals were used in
this study: chromogenic substrates S-2222 and S-2238 (Chromogenix AB
products distributed by DiaPharma Group, Inc., West Chester, OH); human 
-thrombin and fXa (Enzyme Research Laboratories, Inc., South Bend,
IN); protease inhibitor cocktail (Complete; Boehringer Mannheim GmbH,
Indianapolis, IN); human 
-thrombin (ICN Biomedicals, Inc., Costa
Mesa, CA); activated partial thromboplastin time (APTT) reagent, ADP,
aspirin, and thromboplastin with calcium (Sigma Chemical Co., St.
Louis, MO); and heparin (Upjohn, Kalamazoo, MI). Nonpeptide fXa
inhibitors were synthesized at DuPont Pharmaceuticals Company. Purified
rTAP was prepared from culture medium of Saccharomyces cerevisiae as described by Neeper et al. (1990) with modifications.
Electrically Induced Arterial Thrombosis Model in Rabbits.
Experiments, using a modification of the methods of Hladovec (1971) and
Guarini (1996), were conducted on male New Zealand White rabbits
(2.7-3.1 kg). The rabbits were anesthetized with ketamine (50 mg/kg + 50 mg/kg/h i.m.) and xylazine (10 mg/kg + 10 mg/kg/h i.m.). The left
femoral vein and artery were isolated and catheterized. Both common
carotid arteries were carefully isolated. Carotid blood flow was
measured with a calibrated flow probe (3.5-mm circumference) that was
linked to an electromagnetic flowmeter (FM501D; Carolina Medical
Electronics, Inc., King, NC). A stainless steel bipolar hook electrode
was placed on the carotid artery and positioned caudally in
relationship to the flow probe. A piece of Parafilm (7 × 30 mm)
was placed under the electrode to protect the surrounding tissue.
Thrombosis was induced by applying a direct electrical current of 4 mA
for 3 min to the external arterial surface, using a constant current
unit and a d.c. stimulator (S88D; Grass Instruments Co., Quincy, MA).
We chose to stimulate the carotid artery at 4 mA because we found that
in a preliminary study, electrical stimulation at 1 mA for 3 min did
not produce occlusive thrombus within 40 min. However, increasing the
current to 4 mA produced a more reproducible occlusion of the injured carotid artery within 40 min. In this study, carotid blood flow was
monitored continuously before and after electrical stimulation. The
left carotid artery served as a control artery. If the left carotid
artery did not occlude within 5 to 40 min after electrical stimulation,
these rabbits were not included in the study. Rabbits that were
excluded from the study represented about 5% of rabbits used.
). The
ED50 (dose that increased blood flow to 50% of
the control) value for each compound was estimated by a nonlinear least
square regression program (DeltaGraph; Delta Point, Monterey, CA) using
the Hill sigmoid Emax equation.
Antithrombotic Studies in Arterial Thrombosis. Rabbits were dosed i.v. with saline vehicle (6 ml/kg/h), heparin (64 and 100 U/kg/h), aspirin (2, 6, 17, and 56 µmol/kg/h), DuP 714 (0.06, 0.2, and 0.6 µmol/kg/h), rTAP (0.03 and 0.05 µmol/kg/h), and SK549 (0.04, 0.09, 0.15, 0.45, and 1.5 µmol/kg/h). In addition, rabbits were dosed i.v. with each of a series of nonpeptide fXa inhibitors listed in Fig. 1. Average carotid blood flow over 90 min was determined for each treatment group and the ED50 was estimated as described above.
In some rabbits treated with SK549, concentrations of SK549 in plasma samples, taken during electrically induced arterial thrombosis, were determined by liquid chromatography-tandem mass spectrometry method (Wong et al., 2000). The EC50 (plasma
concentration that increased blood flow to 50% of the control) value
for SK549 was estimated by a nonlinear least square regression program
as described above.
Mean blood pressure and heart rate were determined in some rabbits
treated with the saline vehicle and SK549 at 1.5 µmol/kg/h. Mean
blood pressure was measured by connecting a femoral arterial catheter
to a pressure transducer (Gould Inc., Oxnard, CA) coupled to a
polygraph (Grass Instruments Co.). Heart rate was recorded by the polygraph.
Scanning Electron Microscopy of Rabbit Carotid Artery. Segments of carotid artery, collected from both the sham and electrically induced injury rabbits, were fixed in a solution of 4% paraformaldehyde with 1% glutaraldehyde overnight at 4°C. Subsequent postfixation was in 1% osmium tetroxide containing 1.5% potassium ferricyanide for 1 h at room temperature. After several buffer washes, they were dehydrated in a graded ethanol series. Final drying was accomplished by passing the samples through several changes of hexamethyldisilazane followed by air drying. Samples were mounted and coated with gold/palladium before examination in a JEOL JSM 840 scanning electron microscope.
Coagulation Assays. Arterial blood samples for the determination of ex vivo APTT, prothrombin time (PT), thrombin time (TT), anti-fXa, and antithrombin activity were collected in tubes containing one-tenth the volume of 0.109 M sodium citrate before and at the end of the test.
APTT, PT, and TT were measured with a fibrometer (BBL Fibrosystem; Becton Dickinson, Cockeysville, MD) (Kettner et al., 1990). APTT was
measured by incubating 100 µl of platelet-poor plasma with 100 µl
of APTT reagent for 3 min, followed by addition of 100 µl of 25 mM
CaCl2. PT was measured by incubating 100 µl of plasma for 2 min at 37°C, followed by addition of 200 µl of
prewarmed thromboplastin with calcium. TT was measured by incubating
200 µl of plasma and 50 µl of fibrometer buffer for 2 min at
37°C, followed by addition of 50 µl of thrombin (24 U/ml). Data
points were the mean of duplicate measurements and were expressed as a
ratio of treated versus baseline control.
Ex vivo anti-fXa and antithrombin activities were measured using a
modification of the method of Sato et al. (1998). Anti-fXa and
antithrombin activities were determined using the chromogenic substrates S-2222 and S-2238, respectively. Assays were performed in a
Spectramax Plus spectrophotometer (Molecular Devices, Sunnyvale, CA).
All assays were performed in 96-well plates (no. 3604; Corning Inc.,
Corning, NY). Thirty microliters of S-2222 (500 µM) or S-2238 (500 µM) was added to a mixture of 30 µl of buffer (0.1 M Tris, pH 7.5;
0.2 M NaCl), 30 µl of platelet-poor plasma, and 30 µl of human fXa
(0.625 µg/ml) or human thrombin (0.2 U/ml). This resulted in a final
volume of 120 µl. The incubation period for the anti-fXa assay was 15 min at 37°C, and for the antithrombin assay was 45 min at 37°C. At
the end of the incubation period, the enzyme reaction was quenched with
the addition of 12 µl of 12× Complete protease inhibitor cocktail.
The hydrolysis of the chromogenic substrates was assayed by measuring
absorbance at 405 nm at 37°C. Anti-fXa or antithrombin activity was
calculated by comparing the values of optical density from samples
taken in the post-treatment period to those taken in the pretreatment period.
Ex Vivo Platelet Aggregation.
In some experiments, arterial
blood samples, for the determination of ex vivo platelet aggregation,
were collected before and after SK549 at 1.5 µmol/kg/h i.v. Platelet
aggregation was measured with a platelet aggregometer (Model PAP-4D;
BioData, Horsham, PA). Two hundred microliters of platelet-rich plasma was incubated for 3 min at 37°C. Percentages of platelet aggregation were determined 4 min after the addition of 20 µl of the agonist (ADP
at 10 µM and -thrombin at 35 nM, final concentration).
Statistical Analysis.
Statistical analyses used were
correlation, linear regression, ANOVA, and Duncan's new multiple range
test (Cody and Smith, 1991). A value of P < .05 was
considered statistically significant. All data are means ± S.E.
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Results |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Scanning Electron Microscopy of Rabbit Carotid Artery.
Longitudinal sections of normal and electrically injured rabbit carotid
arteries were examined by scanning electron microscopy. The luminal
surface of the normal artery was covered with intact endothelium with
some scattered platelets (Fig. 2). In the
injured carotid artery, the endothelial surface was disrupted and
numerous platelets and networks of fibrin were attached to the injured site (Fig. 2).
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Antithrombotic Effect of Heparin.
Average control carotid
blood flow before electrical stimulation was about 17 ml/min. After
electrical stimulation of the carotid artery, thrombus formation was
induced and blood flow was gradually decreased to less than 5% in 35 to 40 min in vehicle-treated animals (Fig.
3). Heparin was given as an i.v. infusion
starting 60 min before electrical stimulation. Heparin at 100 U/kg/h,
but not at 64 U/kg/h, slightly improved the patency of the injured artery. At 40 min after electrical stimulation, animals treated with
heparin at 100 U/kg/h i.v. had an average carotid blood flow of 26 ± 14% of the control level (Fig. 3).
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Antithrombotic Effect of Aspirin, DuP 714, and rTAP.
Figure
4 shows effects of aspirin (2 to 56 µmol/kg/h i.v.), DuP 714 (0.06 to 0.6 µmol/kg/h i.v.), and rTAP
(0.03 and 0.05 µmol/kg/h i.v.) on the average carotid blood flow over
90 min. Control carotid blood flow in these groups averaged 23 ml/min. Average carotid blood flow over 90 min was reduced to less than 20% in
vehicle-treated animals after electrical stimulation (Fig. 4). Aspirin,
DuP 714, and rTAP caused a dose-dependent increase in average carotid
blood flow over 90 min with ED50 values of 56, 0.14, and 0.06 µmol/kg/h, respectively (Fig. 4). Values of time to
occlusion (in min) for the vehicle and aspirin at 2, 6, 17, and 56 µmol/kg/h were 23 ± 2, 26 ± 4, 28 ± 5, 69 ±
9, and 88 ± 3, respectively; for the vehicle and DuP 714 at 0.06, 0.2, and 0.6 µmol/kg/h were 25 ± 3, 38 ± 8, 90 ± 0, and 90 ± 0, respectively; and for the vehicle and rTAP at 0.03 and 0.05 µmol/kg/h were 25 ± 3, 40 ± 6, and 75 ± 11, respectively. Significant increases in time to occlusion were
observed for aspirin at 17 and 56 µmol/kg/h, for DuP 714 at 0.06 to
0.6 µmol/kg/h, and for rTAP at 0.05 µmol/kg/h (P < .05, compared with vehicle).
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Blood Pressure and Heart Rate Effects of SK549. Effects of the saline vehicle and SK549 on blood pressure and heart rate were evaluated in a separate group of animals. Compared with the vehicle (n = 4), SK549 at 1.5 µmol/kg/h i.v. (n = 4) did not change blood pressure significantly (76 ± 3 mm Hg for SK549 and 86 ± 7 mm Hg for vehicle) and heart rate significantly (182 ± 13 bpm for SK549 and 173 ± 9 bpm for vehicle).
Ex Vivo Effects of SK549 on Platelet Aggregation and Coagulation
Parameters.
At 1.5 µmol/kg/h i.v. (n = 4), SK549
did not alter the ex vivo platelet aggregation induced by either ADP or
-thrombin (ADP, 51 ± 3% for the control and 54 ± 4%
for SK549; or -thrombin, 67 ± 4% for the control and 68 ± 6% for SK549).
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Relationship between Ki (fXa) and In
Vivo Potency ED50.
A series of benzamidine isoxazoline
fXa inhibitors and rTAP were evaluated against purified human fXa for
their inhibitory effects on fXa activity and in a rabbit model of ECAT
for their antithrombotic activities, expressed as
Ki and ED50,
respectively (Fig. 1). We observed a highly significant correlation
between Ki and ED50
(r = 0.85, P < .001) as shown in Fig.
10.
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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SK549 is a potent and selective fXa inhibitor with a
Ki of 0.52 nM against human fXa (Wong et
al., 2000). Compared with other well characterized small-molecule fXa
inhibitors, SK549 is 58, 2.5, 13.5, and 2.5 times more potent in terms
of Ki than DX-9065a (Ki = 30 nM), YM-60828
(Ki = 1.3 nM, reported by Taniuchi et al., 1998), RPR120844 (Ki = 7 nM, reported by
Leadley et al., 1999), and RPR208566 (Ki = 1.31 nM, reported by Heran et al., 2000), respectively. In addition,
given intraduodenally and i.v. to rabbits, SK549 effectively prevented
thrombus formation in a model of arteriovenous shunt thrombosis (Wong
et al., 2000). In this study, we demonstrated clearly that SK549 given
i.v. to rabbits is also a potent antithrombotic agent in a model of
arterial thrombosis.
Our study is the first comparative evaluation of the dose-dependent
antithrombotic effects of small-molecule fXa inhibitors such as SK549,
the peptide fXa inhibitor rTAP, standard heparin, and the direct
thrombin inhibitor DuP 714 in a rabbit model of arterial thrombosis.
Although the fXa inhibitor YM-60828 has been evaluated in the rat ECAT
model (Kawasaki et al., 1998), the antithrombotic effect of a
small-molecule fXa inhibitor has not been previously reported in a
similar ECAT model in rabbits. Furthermore, we believe that the rabbit
is a better choice of animal model than the rat for evaluating the
antithrombotic effect of small-molecule fXa inhibitors. Studies have
reported that rabbit fXa, but not rat fXa, and human fXa have similar
binding affinity to enzyme substrate and small-molecule inhibitors of
fXa (Hara et al., 1995; Taniuchi et al., 1998).
We reasoned that the ECAT model, which mimics clinical arterial
thrombosis, may be physiologically more relevant than the arteriovenous
shunt thrombosis model. The former but not the latter model involves
additional factors that are important for the mechanism of thrombus
formation, such as high shear rate and endothelial injury (Badimon,
1997). In this study, we used external electrical stimulation to induce
endothelial injury. Scanning electron microscopy confirmed endothelial
injury at the site where the electrical stimulation was applied. The
injured vessel was covered with platelets and fibrin. The platelet
deposition is consistent with the findings of Badimon (1997) showing
that the de-endothelialized vessel wall, which is exposed to blood at
high shear rate, would induce platelet deposition on the exposed
vessel. In addition, the tissue factor in the subendothelial
extracellular matrix of the denuded vessel would activate blood
coagulation cascade and contribute significantly to thrombin formation
and fibrin deposition (Pawashe et al., 1994).
We observed that heparin is a weak antithrombotic agent in the ECAT
rabbit model. Previously, we showed that heparin at 64 U/kg/h
completely inhibited the thrombus formation in the rabbit arteriovenous
shunt model (Wong et al., 2000). However, in this study heparin at a
higher dose of 100 U/kg/h, which increased APTT by greater than 6-fold,
did not prevent arterial thrombosis in rabbits. Our finding is
consistent with previous reports showing that heparin is a weak
antithrombotic agent for the treatment of arterial thrombosis in humans
and animals (Schumacher et al., 1993; Kawasaki et al., 1998; Lockyer
and Kambayashi, 1999; Heran et al., 2000; Hirsh and Bates, 2000). This
may be related to the ineffective inhibition of clot-bound fXa and
thrombin by the complex of antithrombin III with heparin (Teitel and
Rosenberg, 1983; Weitz et al., 1990). Unlike heparin, the potencies of
nonpeptide fXa inhibitors tested in the ECAT and arteriovenous shunt
model were very similar in both models. A possible explanation is that nonpeptide fXa inhibitors, because of their small size, may penetrate and inhibit the clot-bound fXa better than the complex of antithrombin III with heparin (Hérault et al., 1997).
Our study also shows that aspirin is not a very effective
antithrombotic agent in the ECAT rabbit model, which is consistent with
other reports showing that aspirin is a poor antithrombotic agent in
similar ECAT models in rats (Bernat et al., 1993; Schumacher et al.,
1993). It is believed that aspirin at the high dose we studied is
effective in blocking the formation of platelet-aggregating prostanoids
such as thromboxane A2. However, at the same time aspirin also blocks the production of platelet-inhibitory prostanoids such as prostacyclin, which may account for its poor antithrombotic effect in arterial thrombosis models (Bernat et al., 1993; Schumacher et al., 1993; Lockyer and Kambayashi, 1999). The weak
antithrombotic potency of aspirin may also be related to its lack of
effects on blood coagulation with thrombin and fibrin formation, which play a role in arterial thrombosis.
We observed that DuP 714 is a potent antithrombotic agent in the ECAT
rabbit model. A similar antithrombotic efficacy of DuP 714 has also
been reported in a rabbit model of arteriovenous shunt thrombosis
(Knabb et al., 1992). In the ECAT rabbit model, the dose of DuP 714 to
achieve maximal antithrombotic effect (reflected by an increase in
blood flow) resulted in a 4.8-fold prolongation in APTT. In contrast,
the maximal antithrombotic dose of SK549 only prolonged APTT by
2.4-fold. It is not known how much systemic anticoagulation can be
tolerated without bleeding complications during antithrombotic therapy.
However, bleeding complications occurred in clinical trials with
hirudin (a naturally occurring direct thrombin inhibitor) for the
treatment of myocardial infarction (for references, see Kaiser,
1998). Whether a lower level of anticoagulation induced by fXa
inhibitors may account for a reduced incidence of bleeding
complications in patients remains to be determined.
SK549 is a potent antithrombotic agent in the ECAT model with an
EC50 of 97 nM, which is very close to the potency
of 62 nM observed in the arteriovenous shunt thrombosis model (Wong et al., 2000). Although SK549 was as effective as rTAP in the
arteriovenous shunt thrombosis rabbits (Wong et al., 2000), it appears
that SK549 was more effective than rTAP in the ECAT rabbits. For
instance, rTAP at 0.05 µmol/kg/h, which exerted an antithrombotic
effect of 91% in the arteriovenous shunt thrombosis rabbits (Wong et al., 2000), produced an antithrombotic effect of 46% (reflected by an
increase in blood flow) in the ECAT rabbits. On the other hand, SK549
at 1.5 µmol/kg/h, which exerted an antithrombotic effect of 94% in
the arteriovenous shunt rabbits (Wong et al., 2000), produced an
antithrombotic effect of 87%, respectively, in the ECAT rabbits. The
mechanism responsible for the increased effectiveness of SK549 in the
ECAT rabbits compared with rTAP is not clear, but could be related to
the slow binding kinetics of rTAP to fXa (Eisenberg et al., 1992).
To substantiate that the antithrombotic effect of benzamidine isoxazoline fXa inhibitors is due to the inhibition of fXa, the correlation between the inhibitory constants for the fXa, Ki, and the in vivo antithrombotic potencies, ED50, of a series of benzamidine isoxazoline fXa inhibitors was determined. Our study shows a good correlation between Ki and ED50, supporting that inhibition of fXa is the primary mechanism of the antithrombotic effect of these nonpeptide fXa inhibitors. This is further substantiated by the finding that SK549 at antithrombotic doses selectively inhibited ex vivo fXa but not thrombin activity.
It should be noted that SK549 at 1.5 µmol/kg/h does not alter blood
pressure and heart rate, suggesting that the involvement of hemodynamic
effect in its antithrombotic effect is not likely. In addition, the
antithrombotic effect of SK549 may not be due to inhibition of platelet
aggregation because SK549 at the maximal antithrombotic dose of 1.5 µmol/kg/h did not inhibit the ex vivo platelet aggregation induced by
ADP or -thrombin.
Some studies have used the time to occlusion as an index of
antithrombotic effect (Kawasaki et al., 1998; Heran et al., 2000), which, we believe, may overestimate the antithrombotic efficacies of
the compounds. For instance, this study shows that aspirin and rTAP
could produce high values of time to occlusion, but low-to-moderate levels of blood perfusion (as reflected by the blood flow). Thus, the
time to occlusion has important limitations as an index of antihrombotic effect, which must be taken into account in the interpretation of antithrombotic effect of a test agent.
APTT is universally used to monitor the therapeutic level of
heparin-induced anticoagulation (Kher et al., 1997; Bajaj and Joist,
1999). The dose of heparin that doubles the APTT is often taken as a
measure of adequate heparin administration. In this study, although
heparin at 100 U/kg/h i.v. increased APTT by greater than 6-fold, it
had only a minimum antithrombotic effect in the ECAT rabbit model.
Heparin at this dose also increased TT by greater than 7-fold and did
not change PT. In contrast, the maximal antithrombotic effect of SK549
was associated with a 2.4-fold increase in APTT and less than 2-fold
increase in PT. SK549 did not change TT, supporting that the
antithrombotic effect of SK549 is not related to thrombin inhibition.
Although APTT and PT are very useful for monitoring heparin and
warfarin therapy, respectively, in the clinic (Kher et al., 1997; Bajaj
and Joist, 1999), our study shows that these tests are not sensitive
enough to monitor the antithrombotic effect of the fXa inhibitor SK549.
It appears that measuring anti-fXa activity is a sensitive method for
assessing fXa inhibitors ex vivo.
In summary, our study shows that SK549 is a novel, potent, and effective antithrombotic agent in a rabbit model of arterial thrombosis. It is likely that SK549 exerts its antithrombotic effect through selective inhibition of fXa. Thus, SK549 may be clinically useful for the prevention of arterial thrombosis.
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Acknowledgments |
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We thank Dr. M. Thoolen for helpful comments; Danielle M Timby for plasma level determinations; A. Liauw, C. Ellis, and J. Luettgen for technical assistance; and Drs. J. Duke and S. Rosenfeld for providing rTAP.
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Footnotes |
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Accepted for publication June 30, 2000.
Received for publication April 12, 2000.
1 Presented in part at the 72nd Scientific Sessions of the American Heart Association, November 7-10, 1999, Atlanta, Georgia (Abstr. 2483).
2 Current address: Tularik, Inc., South San Francisco, CA.
Send reprint requests to: Dr. Pancras C. Wong, DuPont Pharmaceuticals Company, P.O. Box 80400, Wilmington, DE 19880-0400. E-mail: pancras.c.wong{at}dupontpharma.com
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Abbreviations |
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fXa, factor Xa;
SK549, ()-5-isoxazolecarboxamide,
3-[3-(aminoiminomethyl)phenyl]-N-5-[2'-(aminosulfonyl)-[1,1'-biphenyl]-4-yl]-4,5-dihydro-5-(1H-tetrazol-1-ylmethyl)-trifluoroacetic acid salt;
ECAT, electrical current-induced arterial thrombosis;
rTAP, recombinant tick anticoagulant peptide;
APTT, activated partial
thromboplastin time;
PT, prothrombin time;
TT, thrombin time.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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, n = 11), and
heparin at 64 (
, n = 6) and 100 U/kg/h i.v. (
,
n = 6) on carotid blood flow (expressed as percentage
of control carotid flow) after thrombus induction in ECAT rabbits.
Significant difference was observed among the groups (ANOVA:
F2,10 = 17.44, P < .0005).
Effect of heparin at 100 U/kg/h i.v. on carotid blood flow was
significantly different from the vehicle and heparin at 64 U/kg/h i.v.
(P < .05, Duncan's multiple range test). Mean ± S.E.
, n = 10), 0.45 (
,
n = 10), and 1.5 (
, n = 9)
µmol/kg/h i.v. on carotid blood flow (expressed as percentage of
control carotid flow) after thrombus induction in ECAT rabbits.
Means ± S.E.
