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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 11 (June 1), 1998:
pp. 4197-4205
Biochemical and Pharmacological Properties of SANORG 34006, a
Potent and Long-Acting Synthetic Pentasaccharide
By
J.M. Herbert,
J.P. Hérault,
A. Bernat,
R.G.M. van Amsterdam,
J.C. Lormeau,
M. Petitou,
C. van Boeckel,
P. Hoffmann, and
D.G. Meuleman
From the Haemobiology Research Department, Sanofi Recherche,
Toulouse, France; and NV Organon, Oss, The Netherlands.
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ABSTRACT |
SANORG 34006 is a new sulfated pentasaccharide obtained by chemical
synthesis. It is an analog of the "synthetic pentasaccharide" (SR
90107/ ORG 31540) which represents the antithrombin (AT) binding site
of heparin. SANORG 34006 showed a higher affinity to human AT than SR
90107/ORG 31540 (kd = 1.4 ± 0.3 v 48 ± 11 nmol/L), and it is a potent and selective catalyst of the inhibitory
effect of AT on factor Xa (1,240 ± 15 anti-factor Xa U/mg v
850 ± 27 anti-factor Xa U/mg for SR 90107/ORG 31540). In vitro,
SANORG 34006 inhibited thrombin generation occurring via both the
extrinsic and intrinsic pathway. After intravenous (IV) or subcutaneous (SC) administration to rabbits, SANORG 34006 displayed a
long-lasting anti-factor Xa activity and inhibition of thrombin
generation (TG) ex vivo. SANORG 34006 was slowly eliminated after IV or
SC administration to rats, rabbits, and baboons, showed exceptionally long half-lives (between 9.2 hours in rats and 61.9 hours in baboons), and revealed an SC bioavailability near 100%. SANORG 34006 displayed antithrombotic activity by virtue of its potentiation of the
anti-factor Xa activity of AT. It strongly inhibited thrombus
formation in experimental models of thromboplastin/stasis-induced
venous thrombosis in rats (IV) and rabbits (SC) (ED50
values = 40.0 ± 3.4 and 105.0 ± 9.4 nmol/kg, respectively). The
duration of its antithrombotic effects closely paralleled the ex vivo
anti-factor Xa activity. SANORG 34006 enhanced
rt-PA-induced thrombolysis and inhibited accretion of
125I-fibrinogen onto a preformed thrombus in the rabbit
jugular vein suggesting that concomitant use of SANORG 34006 during
rt-PA therapy might be helpful in facilitating thrombolysis and
preventing fibrin accretion onto the thrombus under lysis. Contrary to
standard heparin, SANORG 34006 did not enhance bleeding in a rabbit ear incision model at a dose that equals 10 times the antithrombotic ED50 in this species and, therefore, exhibited a favorable
therapeutic index. We suggest that SANORG 34006 is a promising compound
in the treatment and prevention of various thrombotic diseases.
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INTRODUCTION |
IN THE PAST SEVERAL YEARS, considerable
progress has been made in developing ideal
antithrombotics.1,2 Recently, SR 90107/ORG 31540, a new
pentasaccharide obtained by total chemical synthesis,3 which represents the minimal sequence of the heparin chain that interacts with antithrombin (AT), has been shown to display
antithrombotic activity by virtue of its potentiation of the
anti-factor Xa activity of AT.3,4 This compound, which
accelerates the inhibition by AT of factor Xa but not thrombin,
possesses antithrombotic efficacy in various thrombosis models without
deleterious effects on hemostasis.5-9 When pharmacodynamics
of the anti-factor Xa effect and tolerance of SR 90107A/ORG 31540 were
investigated in healthy volunteers10 the drug was well
tolerated, suggesting that accelerating the inhibition of factor Xa is
a promising approach for the treatment of thrombosis. The complex
structure of SR 90107/ORG 31540 requires numerous synthetic steps, and,
to simplify the synthesis, analogs of SR 90107/ORG 31540 have been
synthesized.3 The biochemical and pharmacological
activities of one of these compounds, SANORG 32701, a newly developed
analog of SR 90107/ORG 31540, have been described
recently.11
In an attempt to find new pentasaccharides with longer half-lives, we
have designed SANORG 34006, an O-methylated, O-sulfated pentasaccharide. It has been postulated that the specific binding of
pentasaccharides to AT in the circulation governs their pharmacokinetic properties.12 In particular, time courses of plasma
anti-factor Xa activities for different pentasaccharides could be
predicted from their affinities (kd) for AT, the elimination half-life
of AT itself, and the plasma AT concentration.12 In the
present study we describe the in vitro and in vivo activities of SANORG 34006.
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MATERIALS AND METHODS |
Drugs
Recombinant human tissue factor (Inovin) was obtained from Dade (Baxter
Diagnostics, Deerfield, IL). Tissue thromboplastin and standard heparin
(162 IU/mg, sodium salt from pig intestinal mucosa, mean molecular
weight 15,000) were purchased from Sigma Chemical Co (L'isle d'Abeau,
France). rt-PA (Actilyse) was obtained from Boehringer Ingelheim
(Ingelheim, Germany). Human  -thrombin (3,000 IU/mg) was
from Centre de Transfusion Sanguine (Strasbourg, France).
125I-human fibrinogen (4.1 MBq/mg) was purchased from
Amersham (Amersham, UK). Human factor Xa, human AT, human
heparin-cofactor II (HC-II), chromogenic substrates
Bz-Ile-Glu-(Piperidyl)-Gly-Arg-pNA (S-2222), and D-Phe-Pip-Arg-pNA
(S-2238) were from Kabivitrum (Stockholm, Sweden). SR 90107/ORG 31540, a pentasaccharide that represents the minimal sequence on the heparin
chains, interacting with AT and SANORG 34006 (Fig 1) were from Sanofi Recherche
(Toulouse, France). The pharmaceutical research and development of SR
90107/ORG 31540 and SANORG 34006 is pursued by a partnership agreement
between Organon (Oss, The Netherlands) and Sanofi (Gentilly, France). All compounds were solubilized in saline and administered as indicated. All other chemicals and solvents were of reagent grade from Prolabo (Paris, France).
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| Fig 1.
Comparative structures of SR 90107/ORG 31540 and
SANORG 34006. SR 90107/ORG 31540 is the -methyl glycoside
of the pentasaccharide which represents the unique sequence in heparin
that binds to AT. SANORG 34006 belongs to the so-called
"non-glycosaminoglycan" series of analogs of SR 90107/ORG
31540.
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In Vitro Studies
Inhibitory activity of SANORG 34006 on factor Xa and thrombin.
Anti-factor Xa activities were determined by a modification of the of
Teien and Lie procedure13 as recently
described.11 Human factor Xa (2.4 nkat/mL) was
incubated for 2 minutes with human AT (0.17 U/mL) at 37°C in the
presence of various concentrations of the oligosaccharides in
Tris/maleate 20 mmol/L buffer pH 7.4, NaCl 150 mmol/L. To measure the
residual factor Xa activity, S-2222 (dissolved in 50 mmol/L Tris/HCl
buffer, pH 8.4, NaCl 175 mmol/L, EDTA 27.5 mmol/L, polybrene 1 mg/mL)
was added (0.25 mmol/L final). The reaction was stopped 2 minutes later
by the addition of a 50% aqueous acetic acid solution and the
absorbency at 405 nm was read on a spectrophotometer. The percentage of
inhibition was then calculated as [% Inhibition = 100 × (Optical Density [OD] Blank  OD Sample)/OD
Blank]. The activity per milligram of the compounds was
determined by comparison with a calibrated standard (SR 90107/ORG
31540) using Excel 4.0 Software (Microsoft, Redmond, CA).
Affinity for human AT.
Affinity of SANORG 34006 for human AT was determined by fluorescence as
described by Atha et al16 using a Perkin Elmer LS-50 type
spectrofluorimeter (Perkin Elmer, Norwalk, CT) at a  excitation = 280 nm and a emission = 338 nm at 37°C, under
continuous stirring. Oligosaccharides were added into 2 mL of Tris-HCl
buffer 0.01 mmol/L pH 7.0 containing NaCl 0.15 mol/L and 5 to 60 nmol/L
AT. The ratio and the concentrations of AT-oligosaccharide complex were
calculated and dissociation constants (kd) were determined from
Scatchard plots using RS/1 software.
Anticoagulant activity.
The activated partial thromboplastin time (APTT) was measured with
Actin FS (Dade-Baxter, Dülingen, Switzerland) in a Amelung-Baxter KC10 coagulometer. APTT was expressed in units per
milligram, taking unfractionated heparin (180 U/mg) as the standard.
The thrombin generation (TG) methodology was adapted from Hemker et al,14 as previously reported.15 TG was
triggered either by kaolin (5 µg/mL final concentration) (intrinsic
TG) or by rabbit brain thromboplastin (extrinsic TG) in defibrinated
human platelet-poor plasma supplemented with 2 µmol/L of cephalin.
Aliquots were taken every 15 seconds and the concentration of thrombin
was measured using the chromogenic substrate S-2238. The total
amount of thrombin generated was quantified by computing the area under
the TG curve (AUC).
In Vivo Studies
Ex vivo anti Xa activities in different species.
Pentobarbital anesthetized (30 mg/kg, intraperitoneal
[IP]) male Sprague-Dawley rats (275 to 300 g;
Iffa-Credo, L'arbresle, France), pentobarbital
anesthetized (30 mg/kg, intravenous [IV]) male New Zealand rabbits
(2.7 to 3.0 kg; Lago, Vonnas, France), and male conscious
baboons (Papio Ursinus, 7 to 9 kg provided by CAPE, Capetown, South
Africa) were treated by subcutaneous (SC) or IV injections of SANORG
34006 or SR 90107/ORG 31540. Arterial blood samples (2 mL) were
withdrawn in a 3.8% trisodium citrate solution (1/9 vol/vol). Each
blood sample was centrifuged (1,800g, 10 minutes) and the
platelet-poor plasma was stored at 20°C. The anti-factor Xa
activity of SANORG 34006 was determined as described above by measuring
the residual activity of factor Xa added to diluted plasma supplemented
with human AT.
Stasis-induced venous thrombosis in the rabbit.
Venous thrombosis was induced in pentobarbital anesthetized (30 mg/kg
IV) male New Zealand rabbits according to Buchanan et al,17
with slight modifications.11 Each jugular vein was isolated and two loose sutures were placed 2 cm apart. Recombinant human tissue
factor (1 ng/kg) was injected 5 minutes before the induction of stasis.
Both jugular-vein segments were occluded by the distal and proximal
sutures and stasis was maintained for 15 minutes. The veins were opened
longitudinally, and the thrombus, if apparent, was removed, blotted on
filter paper, and weighed. Wet weights of thrombi were averaged for
left and right jugular veins. Test compounds or vehicle were
administered SC at indicated time points before the IV injection of
tissue factor.
Stasis-induced venous thrombosis in the rat.
Thrombus formation by a combination of stasis and hypercoagulability
was induced as originally proposed by Vogel et al.5,11 The
abdomen of pentobarbital-anesthetized (30 mg/kg, IP) male Sprague-Dawley rats (250 to 300 g) was opened and the vena cava exposed. SANORG 34006 or SR 90107/ORG 31540 were administered IV 5 minutes before thrombosis induction. Two loose sutures were prepared
0.7 cm apart on the inferior vena cava and all collateral veins were
ligated. Human tissue factor (1 ng/kg, IV) was injected into the dorsal
vein of the penis. Ten seconds after the end of the injection, stasis
was established by tightening the two sutures, the proximal and then
the distal. The abdominal cavity was provisionally closed and stasis
was maintained for 10 minutes. The cavity was then reopened, the
ligated segment was opened longitudinally, and the formed thrombus was
removed, rinsed, blotted on filter paper, dried overnight at 60°C,
and weighed.
125I-fibrinogen accretion on a preformed thrombus in the
jugular vein of the rabbit.
The antithrombotic effect of SANORG 34006 was assessed by measuring its
ability to inhibit the accretion of 125I-fibrinogen onto
autologous nonradioactive venous thrombi preformed in the jugular veins
of rabbits as recently described.11,18 Male New Zealand
rabbits (2.5 to 3 kg) were anesthetized with sodium pentobarbital (30 mg/kg, IV). Both jugular veins were exposed and a 2-cm segment was
isolated on either side. Each jugular vein segment was emptied of
blood, and blood flow was temporarily interrupted by proximal and
distal clamps. One milliliter of blood was collected from a carotid
canula and mixed with 50 µL of human -thrombin (20 U/mL), and 150 µL of clotting blood was immediately injected into the isolated
segment. A 10-cm length silk thread was passed longitudinally through
the forming thrombus and the vessel wall to keep the thrombus in place.
Blood flow was restored 2 minues later. Fifteen minutes after the
thrombus was formed, each animal was injected with 20 µCi of
125I-fibrinogen. Five minutes later, indicated doses of
SANORG 34006, SR 90107/ORG 31540, or heparin were infused over a 4-hour
period with 10% of the dose as an IV bolus. Control animals were
infused with the same volume of saline. At the end of the 4-hour
infusion period, the venous segments containing the thrombi were tied
off, slit open longitudinally, and the remaining thrombi were removed. The specific activity of the whole-blood fibrinogen was estimated from
the mean of blood samples taken at hourly intervals throughout the
infusion. The ratio of radioactivity of the thrombus to circulating radioactivity was used as an estimate of thrombus size.
Lysis of a jugular vein thrombus in the rabbit.
The thrombolytic effect of rt-PA in association with SANORG 34006, SR
90107/ORG 31540, or heparin was evaluated by the lysis of a
standard-sized, preformed 125I-fibrinogen-labeled thrombus
produced in the external jugular vein of rabbits.11,19 The
jugular vein of pentobarbital-anesthetized male New Zealand rabbits (30 mg/kg, IV) was uncovered, and all tributaries at a distance of 4 cm
from the main bifurcation of the external jugular and facial veins were
ligated. A silk thread was then inserted through the vessel to anchor
the thrombus and avoid embolization. After ligation of the vein with
two vessel clamps, the enclosed blood was removed and exchanged with
200 µL of citrated rabbit blood mixed with 0.5 µCi
125I-labeled human fibrinogen and 10 IU human thrombin.
After clot formation (30 minutes), the surgical clamps were removed and
the blood flow was restored. Thrombolysis was performed by infusion of
rt-PA via the contralateral marginal ear vein. SANORG 34006 or saline
were administered as IV bolus injections at the start of the infusion.
At the end of the rt-PA infusion period (4 hours), the thrombus was
carefully removed from the vein and weighed. The amount of
radioactivity remaining in the clot was determined with a gamma counter
(1261 Multigamma counter; Wallac, Turku, Finland). The
extent of 125I-fibrino(geno)lysis was calculated at the end
of the infusion as the difference between the radioactivity originally
incorporated in the thrombus and that remaining in the residual
thrombus. Percent decrease of the thrombus-associated
radioactivity was calculated with reference to the initial
125I-fibrinogen added into the initial clot.
Bleeding test in the rabbit.
Male New Zealand rabbits were anesthetized with sodium pentobarbital
(30 mg/kg, IV). SANORG 34006, SR 90107/ORG 31540, or standard heparin
were administered IV. Five minutes later, five standardized incisions
were made through the ear with a scalpel blade (no. 21; Swann-Norton,
Sheffield, UK) as reported.11 Care was taken
to avoid any macroscopically visible vessel. The ear was immersed in a
500 mL saline bath at 37°C under continuous stirring. Blood loss
was determined 10 minutes later by measurement of the hemoglobin
content of the water bath after the addition of hemolizing reagent
(Zapoglobin, Coultronics, France).
Statistical Analysis
The results shown are arithmetic means ± SEM. IC50
values were calculated using the 4-parameter logistic model and shown
with a 95% confidence interval. The adjustment was obtained by
nonlinear regression using the Levenberg-Marquard algorithm in RS/1
software (BBN, Cambridge, MA). ED50 values were calculated
from the dose-response curves by fitting the logistic equation to the
data by means of nonlinear regression. Data were statistically analyzed
using the Mann-Whitney test with the Holm-Bonferroni adjustment. A
significant difference was accepted at the P < .05 level.
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RESULTS |
In Vitro Studies
Anti-factor Xa activity of SANORG 34006.
SANORG 34006 inhibited the activity of human factor Xa in the presence
of human AT in a dose-dependent manner with an IC50 value
of 2.9 ± 0.2 nmol/L (n = 6). Under the same experimental conditions, SR 90107/ORG 31540 showed a slightly lower inhibitory potency (IC50 = 4.1 ± 0.7 nmol/L, n = 6). SANORG 34006 exhibited a specific activity of 1,240 ± 15 anti-factor Xa U/mg
versus 850 ± 27 anti-factor Xa U/mg for SR 90107/ORG 31540 and 180 ± 11 anti-factor Xa U/mg for heparin
(Table 1). The affinity constant (kd) of SANORG 34006 for human AT was 34-fold higher than that measured for SR
90107/ORG 31540 (Table 1). Neither SANORG 34006 nor SR 90107/ORG 31540 exhibited AT activity in the presence of AT or heparin cofactor II (not
shown).
Effect of SANORG 34006 on thrombin generation and APTT.
SANORG 34006 inhibited TG in human plasma in a dose-dependent manner
(Fig 2). As previously shown for SR
90107/ORG 31540,15 SANORG 34006 impaired TG via the
extrinsic pathway more efficiently than via the intrinsic one showing
IC50 values of 0.5 (0.3 to 1.1) µmol/L and 1.8 (1.1 to
3.7) µmol/L (means + confidence intervals at 95%), respectively. The
potency of SANORG 34006 to inhibit TG was similar to that observed for
SR 90107/ORG 31540 and heparin (Table 1). Contrary to heparin, SANORG
34006 and SR 90107/ORG 31540 did not prolong APTT as previously
described for this class of compounds11 (Table 1).
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| Fig 2.
Inhibition of TG in human plasma by SANORG 34006. The
effects of SANORG 34006 on intrinsic (A) and extrinsic TG (B) are
shown. Results shown are the means of three different experiments
performed in triplicate. The corresponding AUCs shown in the legend are in nmol/L · min 1. SD bars are not shown
for clarity of graph reading.
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Ex Vivo Studies
Anti-factor Xa and anticoagulant activities of SANORG 34006.
The ex vivo anti-factor Xa and anticoagulant activities of SANORG
34006 after SC injections of 30, 100, and 300 nmol/kg to rabbits were
compared in Fig 3. Ex vivo anti-factor Xa
activity in plasma increased as a function of the injected dose (Fig
3A). SANORG 34006 also inhibited TG ex vivo in a dose-dependent manner (Fig 3B). The kinetics of the ex vivo TG inhibition paralleled the time
course of the ex vivo anti-factor Xa activity in plasma (Fig 3).
After administration of the lowest dose (30 nmol/kg SC), TG was
significantly inhibited for at least 24 hours. At 100 and 300 nmol/kg
SANORG 34006 SC, TG was significantly inhibited during the whole
investigation period (56 hours). Comparative pharmacodynamics of the
anti-factor Xa effect of SANORG 34006 after IV and SC administration of 100 nmol/kg to rats, rabbits, and baboons are shown in
Table 2. After IV administration of 100 nmol/kg SANORG 34006, cmax was reached immediately in the
three species being slightly lower in baboons compared with rats and
rabbits. Half-life and AUC increased with increasing size of the
species. Estimated half-lives were 9.2 hours in rats, 22.8 hours in
rabbits, and 61.9 hours in baboons (Table 2). Half-lives and AUC were
inversely correlated with the measured clearance values in the three
species (Table 2). After SC administration of 100 nmol/kg SANORG 34006, cmax was measured after 2.1 hours in rats, 3.4 hours in
rabbits, and 1 hour in baboons. Half-life, AUC, and clearance showed
the same species dependency as observed for IV SANORG 34006 administration (Table 2). Estimated SC bioavailability was 100% for
rats and rabbits and 72% for baboons. After SC administration of
SANORG 34006 to rabbits, cmax and AUC showed a quasi-linear
dose-dependency, whereas half-life and clearance did not vary as a
function of dose. Table 2 also summarizes pharmacodynamic parameters
associated with IV and SC administration of SR 90107/ORG 31540 to rats,
rabbits, and baboons. The main difference compared with SANORG 34006 is the markedly increased clearance which results in a distinct decrease in half-life and AUC.
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| Fig 3.
Pharmacodynamics of the anti-factor Xa effect of SANORG
34006 in the rabbit. SANORG 34006 was administered SC ( , 30;  ,
100;  , 300 nmol/kg) to rabbits and anti-factor Xa activity (A) or intrinsic TG inhibition (AUC measurement) (B) were determined at
indicated time points after administration. Results are expressed as
means ± SD (n = 6).
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Table 2.
Pharmacokinetic Parameters Measured for SANORG 34006 and
SR 90107 After IV or SC Administration to Rats, Rabbits, and
Baboons
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In Vivo Studies
Stasis-induced thrombosis after injection of tissue factor in the
rabbit.
SANORG 34006 was tested for its ability to inhibit thrombus formation
in a venous thrombosis model in the rabbit using a combination of a
thrombogenic challenge (1.0 ng/kg of tissue factor) and stasis. Thrombus weight under control conditions in this model was 155.9 ± 10.3 mg (n = 10). SANORG 34006 administered sc 2 hours before thrombosis induction displayed a dose-dependent antithrombotic effect.
Maximum inhibition of thrombosis (58%) was observed at a dose of 100 nmol/kg (Fig 4A). The ED50
value was 105 ± 9.4 nmol/kg (n = 10). In this experimental model,
SR 90107/ORG 31540 and standard heparin were as potent as SANORG 34006 showing ED50 values of 74 ± 6.1 and 83 ± 10.2 nmol/kg, respectively, after SC administration (n = 10). However,
standard heparin totally inhibited thrombus formation at a dose of 200 nmol/kg SC, whereas SR 90107/ORG 31540 and SANORG 34006 showed lower
efficacy (maximum inhibition between 50% and 60% at 100 to 300 nmol/kg SC). The antithrombotic effect of 100 nmol/kg SANORG 34006 SC
plateaued at 53% to 58% during 12 hours after administration (Fig
4B). This correlates with the pharmacokinetic data that were obtained
after SC administration of 100 nmol/kg to rabbits (Table 2). In this
rabbit venous thrombosis model, SANORG 34006 exhibited a much longer
antithrombotic activity (t1/2 = 16.4 hours) than SR
90107/ORG 31540 and heparin (t1/2 = 1.8 and 2.9 hours,
respectively) at doses of 100 nmol/kg SC (Fig 4B).
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| Fig 4.
Effect of SANORG 34006 on stasis-induced venous
thrombosis in the rabbit. (A) Dose-response relationships: SANORG 34006 (), SR 90107/ORG 31540 (), or standard heparin () were
administered SC 2 hours before stasis and administration of tissue
factor as described in Materials and Methods. (B) Effect kinetics:
SANORG 34006 (), SR 90107/ORG 31540 (), or standard heparin ()
were administered SC at the dose of 100 nmol/kg. Their antithrombotic activities were determined at indicated time points after
administration. Each point represents the mean ± SEM of 10 animals.
Statistical analysis was performed using the Mann-Whitney test:
*P < .05 versus controls.
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Stasis-induced thrombosis after injection of tissue factor in the
rat.
Stasis after thrombogenic challenge induced a thrombus with an average
weight of 6.1 ± 0.2 mg (n = 20) in this rat model under control
conditions. IV injection of SANORG 34006 inhibited thrombus formation
in a dose-dependent manner (Fig 5). The
ED50 value was 40.0 ± 3.4 nmol/kg. SR 90107/ORG 31540 showed a comparable antithrombotic activity with an IV ED50
value of 68.6 ± 8.4 nmol/kg (Fig 5). Standard heparin was more
potent than the two pentasaccharides in this rat venous thrombosis
model (ED50 = 8.8 ± 2.1 nmol/kg IV). As already
observed in the rabbit venous thrombosis model (Fig 4), SANORG 34006 exhibited prolonged antithrombotic activity compared with SR 90107/ORG
31540 and standard heparin (Fig 5B). The half-lives in this rat model
after administration of 100 nmol/kg IV were 3.7, 0.7, and 1.25 hours
for SANORG 34006, SR 90107/ORG 31540, and standard heparin,
respectively.
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| Fig 5.
Effect of SANORG 34006 on stasis-induced venous
thrombosis in the rat. (A) Dose-response relationships: SANORG 34006 (), SR 90107/ORG 31540 (), or standard heparin ()
were administered IV 5 minutes before stasis and administration of
tissue factor as described in Materials and Methods. (B) Effect
kinetics: SANORG 34006 (), SR 90107/ORG 31540 (), or standard
heparin () were administered IV at the dose of 100 nmol/kg. Their
antithrombotic activities were determined at indicated time points
after administration. Each point represents the mean ± SEM of 10 animals. Statistical analysis was performed using the Mann-Whitney
test: *P < .05 versus controls.
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125I-fibrinogen accretion onto a preformed thrombus in
the rabbit.
The effects of a 4-hour infusion of SANORG 34006, SR 90107/ORG 31540, and heparin on 125I-fibrinogen accretion onto preformed
thrombi are shown in Fig 6. In
saline-treated animals, 5.5 ± 0.8 µg (n = 9) of
125I-fibrinogen were accreted onto the preformed thrombi
during the 4-hour period. SANORG 34006 was much more potent
in inhibiting 125I-fibrinogen accretion (ID50
value = 14 ± 4 nmol/kg IV) than standard heparin (ID50
value = 141 ± 21 nmol/kg IV) and SR 90107/ORG 31540 (ID50 value = 285 ± 12 nmol/kg IV) (Fig 6).
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| Fig 6.
Effect of SANORG 34006 on fibrinogen accretion onto a
preformed thrombus in the rabbit jugular vein. A thrombus was formed in
the jugular vein of rabbits and 125I-fibrinogen (20 µCi)
was administered IV 15 minutes later. The indicated doses of SANORG
34006 (), SR 90107/ORG 31540 (), standard heparin (), or
saline were then administered as a 4-hour infusion. Radioactivity of
the thrombus was determined at the end of the 4-hour infusion period.
Results are expressed as percent inhibition of
125I-fibrinogen accretion compared with saline-treated
control animals. Each point represents the mean ± SEM of 10 animals.
Statistical analysis was performed using the Mann-Whitney test:
*P < .05 versus controls.
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Effect of SANORG 34006 on rt-PA-induced thrombolysis.
The effects of IV bolus injections of SANORG 34006, SR 90107/ORG 31540, and heparin on rt-PA thrombolysis expressed as isotope recovery balance
are shown in Fig 7A. In the control group,
infused with saline instead of rt-PA for 4 hours, the average degree of thrombolysis was 18.9% ± 2.0% (n = 8), either as a result of
spontaneous lysis and/or washout of unclotted radiolabeled
material. This effect was similar to that observed in the absence of
rt-PA after single-bolus injections of SANORG 34006, SR 90107/ORG
31540, or heparin (200 or 300 nmol/kg IV). Infusion of rt-PA (0.5 mg/kg) resulted in a 43.1% ± 9.5% (n = 8)
125I-fibrino(geno)lysis, which was significantly enhanced
(P < .05) by a bolus IV injection of SANORG 34006 (300 nmol/kg) (Fig 7A). A similar efficacy of SANORG 34006 was observed when
thrombolysis was expressed as decrease of the thrombus weight (Fig 7B).
Thrombus weight decreased from a control value of 134.8 ± 9.1 mg to
61.4 ± 8.6 mg (n = 8) in the rt-PA-treated group. Concomitant
treatment with SANORG 34006 further reduced thrombus weights in a
dose-dependent manner. SR 90107/ORG 31540 and heparin demonstrated
comparable effects in this rt-PA thrombolysis model (Fig 7A and B).
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| Fig 7.
Effect of SANORG 34006 on rt-PA-induced thrombolysis in
the rabbit. A 125I-fibrinogen-labeled thrombus was formed
in the jugular vein of rabbits. After thrombus formation, blood flow
was restored and a 4-hour rt-PA infusion (0.5 mg/kg) was administered.
SANORG 34006, SR 90107/ORG 31540, or heparin were administered at
indicated doses as a bolus IV injection at the beginning of the rt-PA
infusion. At the end of the infusion period, the amount of
radioactivity remaining in the clot was determined with a gamma counter
(A) and the thrombus was weighed (B). The extent of
125I-fibrino(geno)lysis was calculated as the difference
between the radioactivity originally incorporated in the thrombus and the remaining radioactivity in the residual thrombus. Values are means ± SEM (n = 8). Statistical analysis was performed using the
Mann-Whitney test: *P < .05 versus rt-PA-treated group.
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Nonhemorrhagic effect SANORG 34006 in the rabbit.
The hemorrhagic risk associated to a treatment with SANORG 34006 was
evaluated using a rabbit ear incision model. This model has been shown
to be highly sensitive to anticoagulants and to glycoprotein IIb-IIIa
inhibitors but not to aspirin and clopidogrel.20 IV
administration of standard heparin at doses  83 nmol/kg significantly increased blood loss, whereas Fraxiparin, a low-molecular-weight (LMW) heparin, showed a significantly lower efficacy.
SANORG 34006 or SR 90107/ORG 31540 did not affect bleeding when
administered at a dose of 1,000 nmol/kg IV
(Fig 8).
View larger version (30K):
[in this window]
[in a new window]
| Fig 8.
Nonhemorrhagic effect SANORG 34006 in the rabbit.
SANORG 34006, SR 90107/ORG 31540, LMW heparin (Fraxiparin),
or heparin were administered IV at indicated doses 5 minutes before ear
incision. Blood loss were determined during 10 minutes as described in
Materials and Methods. Results are expressed as mean volumes ± SEM (n
= 10). Statistical analysis was performed using the Mann-Whitney test: *P < .05 versus controls.
|
|
| |
DISCUSSION |
It is now well admitted that the inhibition of factor Xa represents an
attractive approach for clinical intervention in various thrombotic
disorders.21 Results obtained in this study confirm and
extend these observations and clearly show that synthetic analogs of
the unique AT binding sequence present on the heparin chains4-9 represent a new class of antithrombotic agents.
One of these compounds, SANORG 34006, interacts with human AT with a
dissociation constant in the nanomolar range. This affinity for human
AT is more than 10 times higher than that observed for the
"natural" representative of this class of compounds: SR 90107/ORG 31540.4,11,15 Binding of the pentasaccharides to AT induces a conformational change which results in an accelerated, selective inactivation of factor Xa.22 In that respect, SANORG 34006 is a potent and selective AT-dependent inhibitor of human factor Xa
showing an IC50 value which compared favorably with that of SR
90107/ORG 31540. Nevertheless, despite its much higher affinity for AT
and a higher anti-factor Xa activity, the resulting overall anticoagulant effect elicited by SANORG 34006 as demonstrated by its
effect on thrombin generation appeared to be only slightly enhanced
compared with that of SR 90107/ORG 31540. In that respect, SANORG 34006 efficiently impaired thrombin generation in human plasma, with
IC50 values in the microgram per milliliter range, the
extrinsic pathway being more sensitive to SANORG 34006 than the
intrinsic one. Similar results which have been already observed with SR
90107/ORG 3154015 and other pentasaccharides11
can be explained by the indirect impairment of factor VIIa formation by
such factor Xa-inactivating compounds.23 Alternatively,
the recently described AT-mediated inhibition of the factor
VIIa-tissue factor complex by the pentasaccharides24 might
also account for the observed greater susceptibility of the coagulation
extrinsic pathway to inhibition by AT-binding pentasaccharides such as
SANORG 34006.
Because these data show that SANORG 34006, in the presence of AT, is a
potent inhibitor of factor Xa, we then evaluated the pharmacodynamics
of the anti-factor Xa effect of an SC or IV administration of SANORG
34006 by means of measurement of its anti-factor Xa activity in
plasma. SANORG 34006 exhibited a strong, dose-dependent anti-factor Xa
activity after SC and IV administration to rabbits, rats, and baboons.
This anti-factor Xa effect almost completely paralleled the inhibition
of thrombin generation measured ex vivo. In contrast to unfractionated
and LMW heparin, these pentasaccharides represent single molecular
entities with well-defined pharmacological targets. Thus, circulating
anti-factor Xa activity may be considered as reflecting the true
plasma concentration of this class of compounds. In the range of doses
investigated, the elimination half-life of SANORG 34006 (t1/2) was independent of the dose as already shown for
other pentasaccharides.12,25 Interestingly, SANORG 34006 was cleared more slowly than SR 90107/ORG 31540 in these three species.
Some investigators suggested that this difference could result from the
very high affinity of such compounds for AT, accounting for a half-life
that tended to be close to that of AT itself.25 Similarly,
other investigators suggested that the elimination half-life of
structural analogs of the AT-binding pentasaccharide might be
correlated to their affinity for AT because they could show a relation
between the kd and half-life of elimination in rats.12
Based on allometric scaling,26 which gave predictions for
SR 90107/ORG 31540 in agreement with the experimental data obtained in
a recently published clinical trial in humans,10 the
determination of the pharmacodynamics of the anti-factor Xa effect
parameters of SANORG 34006 suggested that in humans the terminal
half-life for this compound would be around 80 hours. This value,
markedly different from the value observed for SR 90107/ORG 31540, will
allow a once-a-week administration of the compound. Provided that this
extrapolated data will be confirmed in an appropriate clinical study in
humans, this compound might therefore be used in several clinical
settings where high anti-factor Xa activity is needed with a necessary
long-lasting anticoagulation.
The antithrombotic potency of this compound was then evaluated in
comparison with that of SR 90107/ORG 31540 and standard heparin. The
role of factor Xa bound to fibrin and/or platelets and its
subsequent effect on prothrombin activation has been emphasized, showing that these complex phenomena ensure the progressive growth of
the thrombus over several hours.27 SANORG 34006 displayed marked dose-dependent antithrombotic activity in different experimental models of thrombosis in rats and rabbits such as stasis-induced venous
thrombosis in rats and rabbits where thrombus formation occurs because
of a combination of high-grade stenosis and thrombogenic challenge,
fibrinogen accretion to a preformed thrombus in the rabbit jugular
vein, and potentiation of t-PA-induced thrombolysis of a thrombus
implanted in the rabbit jugular vein. Moreover, the curves describing
the relationship between the doses, the subsequent plasma
concentrations, and the resultant antithrombotic effect were parallel,
suggesting that only one mechanism is involved in the antithrombotic
property of SANORG 34006, namely its pure anti-factor Xa effects. One
important observation was that, compared with SR 90107/ORG 31540, SANORG 34006 exhibited an extended duration of antithrombotic activity
as already demonstrated for its anti-factor Xa activity in plasma.
Therefore, these studies clearly show that SANORG 34006 is an
efficacious antithrombotic drug after IV or SC administration. This
compound, which inhibited several types of experimental venous thrombosis without affecting bleeding, exhibited an extremely long
duration of action compared with any other compound of this kind. In
contrast with other anticoagulants such as thrombin inhibitors, SANORG
34006, which did not affect thrombin- or adenosine diphosphate (ADP)-induced platelet aggregation (2% and 5% inhibition at 10 µmol/L, respectively), would not compromise the hemostatic response of platelets, thereby indicating the possibility of using it alone or
as an adjunctive agent with other antithrombotic drugs without the risk
of abnormal bleeding.
Thus, this compound appears a promising antithrombotic agent with
reduced bleeding tendency and an extended half-life.
| |
FOOTNOTES |
Submitted August 12, 1997;
accepted January 26, 1998.
Address reprint requests to J.M. Herbert, PhD, Sanofi
Recherche, Haemobiology Research Department, 195 Route d'Espagne,
31036 Toulouse, France.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We thank F. Roye, A. Hubert, M. Sainte-Marie, C. Gaich, and C. Avril
for their technical assistance.
| |
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Abstract
Introduction
Abstract