© 1999 American Heart Association, Inc.
Thrombosis |
Intravenous and Oral Antithrombotic Efficacy of the Novel Platelet GPIIb/IIIa Antagonist Roxifiban (DMP754) and Its Free Acid Form, XV459
From DuPont Pharmaceuticals Company, Wilmington, Del (S.A.M., R.K., D.-X.M.).
Correspondence to Shaker A. Mousa, PhD, MBA, FACC, DuPont Pharmaceuticals Company, Experimental Station, E400/3470, 141 and Henry Clay Rd, Wilmington, DE 19880-0400. E-mail shaker.a.mousa{at}dupontpharma.com
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Abstract |
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Abstract—Currently used antiplatelet drugs, including aspirin, ticlopidine, and others, are effective against certain but not all of the many endogenous platelet activators. Because of their limited efficacy, a significant number of serious thromboembolic complications still occur, highlighting the need for a more effective therapy. DMP754 (roxifiban), a prodrug of XV459, is a recently discovered, potent antiplatelet agent with high affinity and specificity for platelet GPIIb/IIIa receptors that blocks platelet aggregate formation regardless of the agonist (IC50=0.030 to 0.05 µmol/L) or anticoagulant used for blood collection. DMP754 rapidly converts to its active free-acid form, XV459, which has a comparable high affinity for both resting and activated platelets (Kd=1 to 2 nmol/L) and a relatively slow rate of dissociation from resting platelets. The present study was undertaken to determine intravenous and oral antithrombotic efficacies of DMP754 and XV459 and to compare them with those of other antiplatelet and anticoagulant agents in canine models of arterial thrombosis. In these models, thrombosis was induced either electrolytically (200-µA anodal current) in the carotid artery or mechanically by external clamping of the femoral artery along with stenosis, which resulted in either total occlusive thrombus formation or cyclic flow reduction, respectively. DMP754 and XV459 were given either intravenously (0.1 mg/kg bolus) or orally (0.1 to 0.4 mg/kg). Additionally, the antithrombotic efficacies of DMP754, aspirin, heparin, and ticlopidine in the canine carotid artery electrolytic injury model were compared. DMP754 demonstrated oral bioavailability of 20.8% in dogs after administration at different doses and prevented cyclic flow reduction (ED90–100=<0.1 mg/kg IV or PO). Additionally, both DMP754 and XV459 (0.1 mg/kg IV or 0.3 to 0.4 mg/kg PO) demonstrated maximal antithrombotic efficacy in preventing electrically induced carotid and coronary artery thrombosis and significant antithrombotic efficacy (P<0.001) at relatively low doses in different settings of arterial thrombosis in the canine model. DMP754 resulted in a significant reduction in thrombus mass and sustained arterial blood flow with 100% prevention of occlusive and nonocclusive thrombosis. In contrast, administration of aspirin (10 mg/kg PO for 2 days), heparin (10 IU/kg IV bolus followed by 90 IU/kg IV infusion over 3 hours), or ticlopidine (300 mg/kg PO for 3 days) before initiation of arterial thrombosis did not reduce the incidence of electrolytic injury–induced occlusive arterial thrombosis. These studies demonstrated a distinct antithrombotic efficacy of DMP754 as compared with existing strategies and suggest potential intravenous and oral antithrombotic uses of DMP754 in the prevention and treatment of thromboembolic disorders.
Key Words: platelet GPIIb/IIIa • integrin • thrombosis • antiplatelet • anticoagulant • roxifiban
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Introduction |
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Intravascular thrombosis is one of the most frequent pathological events and a major cause of morbidity and mortality in western civilization. Critical steps in the development of acute coronary syndromes are the disruption, rupture, and erosion of atherosclerotic plaque with the formation of an either partially or completely occlusive thrombus.1 2 3 Factors that stimulate thrombosis include vascular damage, stimulation of platelets, and activation of the coagulation cascade. Platelet adhesion to exposed subendothelium surfaces of injured vessels with subsequent activation and the resulting aggregation have been shown to be associated with various pathological conditions, including cardiovascular and cerebrovascular thromboembolic disorders, such as unstable angina, myocardial infarction, transient ischemic attack, stroke, and atherosclerosis.3 4 5 6 7
The platelet glycoprotein IIb/IIIa complex (GPIIb/IIIa) has been identified as the final common pathway for all platelet agonists.8 9 The binding of adhesive proteins, such as fibrinogen, to GPIIb/IIIa causes platelets to aggregate.9 10 The binding of fibrinogen is mediated in part by the RGD recognition sequence, which is common to the adhesive proteins that bind to GPIIb/IIIa receptors.8 9 10 The first platelet GPIIb/IIIa antagonist developed was the chimeric 7E3 (ReoPro).11 ReoPro effectively inhibits platelet aggregation against all known platelet agonists in experimental animals and humans.12 13 However, the use of monoclonal antibodies as therapeutic agents might present certain limitations, such as immunogenicity, lack of oral bioavailability, and reversibility after intravenous administration. Thus, many groups have concentrated on developing small-molecule GPIIb/IIIa antagonists.14 15 16 17 18 19 The approach taken by the different groups has been to develop analogs of RGD or modifications of the RGD sequence to improve the pharmacodynamic (affinity and specificity for platelet GPIIb/IIIa integrin receptors) and the pharmacokinetic properties of the analogs.16 17 18 19
In contrast to the platelet GPIIb/IIIa antagonist approach, current antiplatelet drugs, including aspirin, ticlopidine, thromboxane A2 synthetase inhibitors, and hirudin, are mainly effective against 1 of the many platelet activators. Hence, the potential clinical benefits of an agent that inhibits platelet aggregation in response to all of these agonists should represent a more efficacious therapeutic approach than provided by current platelet inhibitors.4 5 6 18 Additionally, a higher incidence of coronary artery reocclusion after successful thrombolytic therapy or percutaneous coronary intervention is a persistent clinical problem despite the use of aspirin and/or heparin.6 7 19 Thus, prevention of reocclusion with an adjunctive pharmacological agent is is being actively pursued with different compounds, including anticoagulant and antiplatelet agents. Previous reports have described the clinical implications of different intravenous platelet GPIIb/IIIa antagonists, such as c7E3, Integrelin, tirofiban (Aggrastat), and RO44-9883.20 21 22 23 24 More recently, the antiplatelet efficacy of oral administration of platelet GPIIb/IIIa antagonists, such as xemilofiban, orbofiban, sibrafiban, lefradafiban, and others, has been demonstrated when these agents are given orally 2 or 3 times a day.25 26 27 28
In a recent report, XV459, the free-acid active form of DMP754, was demonstrated to have antiplatelet efficacy and specificity to platelet GPIIb/IIIa receptors.27 Roxifiban (DMP754), a methyl ester prodrug, has been shown to be 100% converted into XV459 on exposure to either blood or liver esterases.27 XV459 demonstrated a unique high-affinity binding to both resting and activated platelets, with a relatively slow platelet dissociation rate.29 The present study examined the potential antithrombotic efficacy of intravenous and oral administration of DMP754 and XV459 in various arterial thrombosis models in dogs.
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Methods |
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Reagents
Adenosine 5'-diphosphate (ADP), collagen, epinephrine, and other reagents used but not specifically mentioned were obtained from Sigma Chemical Co. Arachidonic acid was purchased from NuCheck Prep. Thrombin receptor agonist peptide was purchased from Peninsula Laboratories Inc. H-XV459 and DMP728 were custom-synthesized at DuPont NEN. DMP754 and XV459 (Figure 1
) were
synthesized at DuPont Merck Pharmaceutical Co. XV459 was used in all in
vitro studies described in this article.
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Antiplatelet Efficacy
Light Transmittance Aggregometry Assay
Venous blood was obtained from healthy human donors who had not
taken aspirin for at least 2 weeks before blood collection and from
dogs as previously described.29 Briefly, blood was
collected into either sodium citrate (3.2%) or heparin (14 IU/mL)
Vacutainer tubes. The blood was centrifuged for 10 minutes at
150g in a Sorvall RT6000 tabletop centrifuge with
H-1000 B rotor (DuPont) at room temperature, and platelet-rich
plasma (PRP) was removed. The remaining blood was centrifuged
for 10 minutes at 1500g at room temperature, and
platelet-poor plasma (PPP) was removed. Samples were assayed on a
PAP-4 platelet profiler using PPP as the blank (100%
transmittance). Two hundred microliters of PRP
(2x108 platelets/mL) was added to each test
tube, and transmittance was set at 0%. Twenty microliters of the
platelet agonist, ADP (final concentration, 100 µmol/L), was
added to each tube, and aggregation profiles were plotted (%
transmittance versus time). Maximal aggregation was obtained with ADP
at a final concentration of 100 µmol/L. Twenty microliters of
XV459 was added at different concentrations before the addition of ADP
(100 µmol/L). Results are expressed as the percentage of
inhibition of agonist-induced platelet aggregation or as the
IC50 (µmol/L).
Antiplatelet Efficacy in Anesthetized Dogs
Mongrel dogs of either sex were anesthetized by
administration of Nembutal sodium solution (30 mg/kg IV, Abbott
Laboratories). Animal studies were approved by the Animal Care and Use
Committee of DuPont Pharmaceuticals. DMP754 was administered orally
(single dose) in soft gelatin capsules at a dose of 0.1 or 0.3 mg/kg or
intravenously as a 0.1-mg/kg bolus. XV459 was administered
orally (single dose) in soft gelatin capsules at a dose of 0.4 mg/kg or
intravenously at a dose of 0.1 mg/kg. Blood samples were
placed on a platform rocker until assayed for platelet aggregation
within 2 hours as previously described. The antiplatelet efficacy
of DMP754 and XV459 was calculated by comparing the percentage of
aggregation in samples after administration of the agents to
percentages in baseline samples from the same animal.
Arterial Thrombosis Model in Dogs
In these models, different subsets of arterial
thrombosis were induced either (1) electrolytically (200-µA anodal
current) in the right or left carotid artery (CA) (occlusive
thrombosis) or (2) mechanically by external clamping of the right
or left femoral artery (FA) (4 to 5 times) along with stenosis
(cyclic flow reduction [CFR]). Each animal served as its own control
for the intravenous route of administration. To study oral
dosing, DMP754 or XV459 was administered 30 to 60 minutes before
anesthesia to minimize the effect of the anesthetic on oral
absorption of the test agent. DMP754 or XV459 was administered
intravenously or orally, and the same protocol of induction
of arterial thrombosis was repeated and extended for 3
hours. Arterial flow, ex vivo platelet aggregation and
template bleeding time, incidence of occlusion, time to occlusion,
incidence of CFR, and thrombus weight were monitored throughout the
procedure.
FA Thrombosis in Dogs (Folts' Model)
Mongrel dogs of either sex weighing 8 to 15 kg were
anesthetized and handled as previously described. The left and
right femoral artery (FA) was dissected and freed from fascia and
branches for a distance of 15 to 20 mm. A Doppler flow probe
was placed around the distal portion of the vessel segment, and flow
was monitored throughout the study. Animals were allowed to stabilize
for 20 minutes, and the hyperemic response of the dissected FA
was determined by 2 repeated, brief (20-second) total occlusions (3 to
5 minutes apart). After restoration of basal flow for 20 minutes, a
2.5-mm-long plastic cylinder was placed on the proximal portion of the
FA, creating a critical stenosis that reduced the lumen area of
the vessel by up to 80%, thereby preventing the hyperemic
response while minimally affecting basal flow. The clip was then moved
to one side, and a portion of the FA was mechanically damaged by gentle
clamping. The stenotic clip was then moved back onto the
damaged segment of the vessel. This resulted in repeated CFRs followed
by restoration of flow on dislodging or gentle shaking around the clip.
Baseline measurements of the frequency of CFRs were obtained, and then
either saline (control group) or DMP754 or XV459 (treated groups) was
administered intravenously as a single bolus (0.1 mg/kg) or
orally (0.1 and 0.3 mg/kg). CFRs were monitored for up to 3 hours after
treatment. Figure 2 is a
representative tracing showing the antithrombotic
efficacy (100% prevention of CFR) of DMP754 at 0.3 mg/kg PO.
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CA Thrombosis in Dogs (Electrolytic Injury Model)
The experimental procedure results in formation of a
platelet-rich intravascular thrombus at the site of the
electrolytically induced lesion. The response of the CA to the
electrolytic injury is similar to that observed in the canine
coronary artery, in which intimal wall injury secondary to
application of a direct anodal current leads to platelet adherence
with resultant occlusive thrombus formation. Animals were instrumented
as described earlier. A 20- to 30-mm segment of the left or right CA
was exposed and freed from fascia and branches were tied. Anodal
current was applied using an intravascular electrode composed of a
Teflon-insulated, silver-coated copper wire (28 gauge). Penetration of
the vessel wall by the electrode was facilitated by attaching the tip
of a 23-gauge hypodermic needle to the uninsulated part of the
electrode. Each intraarterial electrode was connected to
the positive pole (anodal) of a dual-channel stimulator (Ni-Cad
battery, 9 volts, connected to 250 000-Ohm potentiometer in series).
The cathode was connected to a distant subcutaneous site. The current
delivered to the arterial wall was monitored continuously
and maintained at 200 µA. Proper positioning of the electrode in the
CA was confirmed by visual inspection at the end of each experiment. In
all experiments, the anodal current was applied for a maximum of 3
hours. Flow was monitored throughout the experiment by use of a
Doppler flow probe (model 100, Triton Technology). Figure 3 is a representative
tracing showing the antithrombotic efficacy (100% prevention of
occlusive thrombus formation) of DMP754 at a dose of 0.3 mg/kg PO.
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Pharmacokinetics
Animals were fasted overnight and received standard certified
commercial dog food (400 g, Wayne Certified Dog Chow no. 8727) 6 hours
after administration of the dose over a 2-hour feeding period and were
given water ad libitum. Four dogs received 0.04 mg/kg XV459 in 5%
dextrose in water for injection as a single intravenous
dose via the jugular vein, and 4 dogs received 0.40 mg/kg DMP754 in
distilled water as a single oral gavage dose by means of intubation.
Blood samples (1.2 mL) were collected from the jugular vein by
venipuncture at predetermined time points into heparinized
Vacutainers (Becton-Dickinson) for plasma measurements of
XV459.
Liquid Chromatography/Mass Spectrometry/Mass
Spectrometry Assay for XV459
A specific and sensitive assay for the determination of XV459 in
dog plasma was developed using liquid
chromatography/mass spectrometry/mass spectrometry via
a turbo ion spray ionization source. The method involved solid-phase
extraction of XV459 and the internal standard (XU066) from plasma using
a C-8 end-capped column. The mobile phase consisted of acetonitrile,
water, and 98% formic acid (20:80:0.1, vol/vol/vol) running in an
isocratic mode. The analytes were separated on a 1-mmx150-mm YMC basic
S5 microbore column at a flow rate of 150 µL/min. The analytes
eluting from the column were ionized in the turbo ion spray ionization
source and sampled into the Sciex API III+ triple quadrupole mass
spectrometer. The mass spectrometer was operated in multiple-reaction
monitoring mode, in which parent ion (with a mass/charge ratio
[m/z] of 434 [XV459]), internal standard
(XU066) (with a m/z of 448), and their fragment
ions (with m/z values of 212 and 133,
respectively) were monitored.
Statistical Analysis
Data are expressed as mean±SEM. The design of the experimental
protocol in certain sections of the study allowed each animal to serve
as its own control for baseline values (% aggregation). Data were
analyzed by either paired or group analysis using
Student's t test or ANOVA when applicable; differences were
considered significant at P<0.05.
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Results |
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In Vitro Antiplatelet Efficacy
DMP754 administered after treatment with liver esterases and its free-acid form inhibited, in a concentration-dependent manner, agonist-induced (100 µmol/L ADP) platelet aggregation in platelets obtained from either dogs or humans (IC50=0.024 to 0.032 µmol/L) (Table 1
).
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Antiplatelet Efficacy in Dogs
Ex Vivo Platelet Aggregation
DMP754 demonstrated maximal antiplatelet efficacy when
administered at a dose of 0.1 mg/kg IV bolus or 0.3 mg/kg PO in
anesthetized dogs (Table 2). In
contrast, orally administered DMP754 at a dose of 0.1 mg/kg showed much
lesser antiplatelet efficacy (Table 2). Additionally,
maximal antiplatelet efficacy was demonstrated with XV459 at a dose
of 0.1 mg/kg IV (Table 2). Similarly, XV459 demonstrated maximal
antiplatelet efficacy at 0.4 mg/kg PO (Table 2). Neither
DMP754 nor XV459 affected platelet counts in whole blood (data not
shown).
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Bleeding Time
Both DMP754 and XV459 at a dose of 1.0 mg/kg IV significantly
(P<0.001) extended bleeding time to >30 minutes, as
compared with a baseline bleeding time of 3 to 4 minutes. Template
bleeding time increased to 6 to 8 minutes after administration of 0.1
mg/kg PO DMP754, extended to 12 to 15 minutes after administration of
0.4 mg/kg PO of XV459, and extended to 15 to 20 minutes after
administration of 0.3 mg/kg PO of DMP754.
Pharmacokinetic Parameters
The pharmacokinetics of XV459 after intravenous
administration and its bioavailability after oral administration of
DMP754 were investigated in dogs. After administration of single
intravenous bolus doses of 0.04, 0.4, and 1.0 mg/kg of
XV459 in dogs, plasma concentrations of XV459 declined
polyexponentially. The terminal half-life
(t1/2) was comparable for all 3 doses, with
mean values of 10.4, 11.8, and 12.2 hours, respectively. However, the
systemic plasma clearance (CL) and volume of distribution at steady
state (Vss) of XV459 increased with dose.
The mean CL values were 1.0, 4.1, and 6.3 mL/min per kg, whereas the
mean Vss values were 0.8, 3.4, and 4.4 L/kg
at doses of 0.04, 0.4, and 1.0 mg/kg, respectively.
The gastrointestinal absorption rate of DMP754 from normal saline was
moderate, with peak plasma concentrations of XV459 attained within 1.4
hours after oral administration of 0.4 mg/kg DMP754 in aqueous
solution. Plasma concentrations of XV459 had a mean
Cmax of 93.9 ng/mL. The apparent
bioavailability (F), defined as the percentage of XV459
formed after oral administration of DMP754, was 20.8% (Table 3). The postabsorptive plasma
concentrations of XV459 declined monoexponentially as a
function of time.
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Distribution of XV459 Between Platelet and Plasma
Compartments
DMP728, a potent platelet GPIIb/IIIa antagonist
with a relatively fast rate of dissociation from platelets, was
used in the present study to compare its platelet/plasma
distribution to that of XV459.15 16 The distribution of
3H-XV459 in human blood was compared with that of
3H-DMP728. Total blood XV459 concentration
(dissociated from platelets by EDTA plus free in plasma) was
markedly greater (10- to 12-fold) than corresponding free plasma
concentrations (Table 4). In contrast,
3H-DMP728 demonstrated equilibrium between PRP
and PPP (Table 4).
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Antithrombotic Efficacy in Dogs
Femoral Artery (FA) Thrombosis in Dogs (Folts' Model)
In this model, a platelet-dependent thrombus is produced in a
mechanically injured FA in the presence of a high degree of
arterial constriction. Under these conditions, FA blood
flow is occluded as a platelet-rich thrombus forms, which is
followed by restoration of flow on dislodging or gentle shaking around
the clip; this process results in thrombus formation and disappearance
in a cyclic manner (CFRs). This study examined the antithrombotic
efficacy of DMP754 and XV459 at doses of 0.1 mg/kg IV or PO and 0.3 to
0.4 mg/kg PO. DMP754 and XV459 both demonstrated maximal antithrombotic
efficacy at 0.1 mg/kg IV and at 0.3 to 0.4 mg/kg PO in
anesthetized dogs (Table 5). At
these doses, DMP754 and XV459 significantly prevented the incidence of
CFR during the 3 hours of the study (Table 5). In contrast, both
aspirin and ticlopidine were effective only in reducing, not
preventing, CFR in 30% to 40% of animals (data not shown).
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CA Thrombosis in Dogs (Electrolytic Injury Model)
The effect of DMP754 and XV459 on the incidence of occlusion, time
to occlusion, and thrombus weight of an electrolytically induced
thrombosis in a canine CA model was examined. DMP754 administered at
doses of 0.1 mg/kg IV and 0.3 to 0.4 mg/kg PO before the insult
resulted in 100% prevention of the incidence of occlusion (Table 5
). Additionally, both DMP754 and XV459 resulted in significant
prolongation of the time to occlusion along with a significant
reduction (P<0.01) in the weight of the thrombus formed
(Table 5). A significant prolongation of the time to occlusion
to >180 minutes (ie, 100% prevention of the incidence of occlusion
for up to the maximum period of the study) was demonstrated. In
contrast, aspirin (10 mg/kg PO for 2 days), heparin, and ticlopidine
(300 mg/kg PO for 3 days) administered before initiation of
arterial thrombosis did not reduce the incidence of
occlusive arterial thrombosis (Table 6).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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New targets for antiplatelet therapy have been identified as a result of a better understanding of the different processes involved in arterial thrombosis.18 30 Once endothelial damage occurs, platelet thrombus formation advances in 3 steps, including firstly, platelet adhesion and secondly, platelet activation by the various agonists or activators from damaged endothelium and from within activated platelets. The third and final step in platelet rich-thrombus formation is mediated exclusively by the GPIIb/IIIa receptor. Various large-scale, phase III clinical trials have illustrated the usefulness of ReoPro in the treatment of percutaneous coronary interventions.27 30 31 The first such study was the pivotal Evaluation of IIb/IIIa Platelet Receptor Antagonists 7E3 in Preventing Ischemic Complications (EPIC) trial, which enrolled >2000 high-risk patients scheduled to undergo percutaneous intervention. Although investigators in the EPIC trial found a relative 35% (13.1% versus 7.7%, P=0.008) reduction in the rate of composite end-point events in patients treated with ReoPro at 30 days, they also raised concern about excess and significant bleeding complications.13 In the Evaluation in PTCA to improve long-term outcome with Abciximab GpIIb/IIIa blockade EPILOG trial, a decrease in the incidence of bleeding was documented with lowering of the heparin dose. Additionally, a small-molecule, nonpeptide GPIIb/IIIa antagonist, tirofiban (Aggrastat) had significant clinical benefits in patients with unstable angina and non–Q-wave myocardial infarction (PRISM and PRISM-PLUS trials).3
Several other selective GPIIb/IIIa antagonists, including Integrelin, tirofiban (Aggrastat), and lamifiban, are in advanced stages of clinical development and are being developed primarily for intravenous use in the treatment and prevention of acute ischemic heart diseases. The Integrelin to Manage Platelet Aggregation to Prevent Coronary Thrombosis (IMPACT II) and Platelet glycoprotein IIb/IIa in Unstable angina: Receptor Supression Using Integrin Therapy (PURSUIT) trials with Integrelin and the Randomized Efficacy Study of Tirofiban for Outcomes and REstenosis (RESTORE) trial with tirofiban (Aggrastat), as well as other trials, have demonstrated significant clinical benefits in acute ischemic syndromes. Clinical studies with orally active GPIIb/IIIa antagonists, including xemilofiban (SC54684) and lefradafiban (BIBU104), demonstrated oral antiplatelet activity in humans with administration 2 to 3 times per day.25 26
The active free-acid form of Roxifiban has distinct platelet GPIIb/IIIa-binding characteristics along with a potent in vitro antiplatelet efficacy regardless of the activator or anticoagulant used for blood collection (citrate versus heparin).27 29 Those attributes result in the unique pharmacodynamic and pharmacokinetic properties of roxifiban shown in the present study. XV459 has been demonstrated to have high affinity for platelet GPIIb/IIIa and similar potency in inhibiting platelet aggregation regardless of the agonist or anticoagulant used.28 29 XV459 inhibited platelet aggregation and had comparable IC50 values regardless of the concentration of ADP used (10 or 100 µmol/L).29 XV459 has been shown to be a competitive inhibitor with high affinity for inhibiting fibrinogen binding to platelet GPIIb/IIIa receptors.29 A comparable high-affinity binding (Kd=0.0008 to 0.0025 µmol/L) of radiolabeled XV459 to either activated or unactivated platelets obtained from humans, baboons, or dogs was also demonstrated.27 29 XV459 demonstrated a high degree of selectivity toward the platelet GPIIb/IIIa receptors as compared with the closely related vitronectin receptors on endothelial cells or other adhesion receptors.28 Additionally, XV459 demonstrated high affinity for both activated and unactivated platelets along with relatively slow dissociation rates, suggesting a possible prolonged duration of in vivo antiplatelet effects.29 L-738,167, a nonpeptide GPIIb/IIIa antagonist, has been evaluated in various animal models and has been found to have an extended duration of antiplatelet efficacy and a slow rate of dissociation from platelets.32 33 This is in contrast to current intravenous platelet GPIIb/IIIa antagonists, such as Integrelin, tirofiban (Aggrastat), or lamifiban, and DMP728, which have a short duration of antiplatelet effects associated with their relatively fast rates of dissociation from human platelets.18 27 Because platelet glycoprotein GPIIb/IIIa receptor–specific antagonists, in addition to binding to plasma proteins, are also bound to blood platelets, an appreciable quantity of a compound in the platelet-rich buffy coat after conventional centrifugation of citrated blood might be lost. Therefore, drug concentrations in citrated PPP samples could be substantially lower than those in citrated PRP. EDTA, in addition to its anticoagulant property, may be used as a potential releaser of the drug bound to platelets by dissociating the calcium-dependent GPIIb/IIIa heterodimer. Hence, the type of biological matrix used for quantitation of drug concentrations at therapeutic levels could play a major role in the estimation of pharmacokinetic and pharmacodynamic parameters.34 Results demonstrated that XV459 exhibited nonlinear, dose-dependent pharmacokinetics in beagle dogs. The nonlinearity in pharmacokinetics suggested that prediction of plasma concentrations of XV459 after a certain dose and time will be difficult. The in vitro plasma protein-binding values of XV459 in fresh dog PRP at 5, 25, and 100 ng/mL were 85%, 85%, and 59%, respectively. The nonlinear clearance and distribution characteristics of XV459 appeared to be related to the saturable binding of XV459 to platelets.
Antiplatelet efficacy of DMP754 and XV459 was shown for the doses
used in the antithrombotic efficacy studies. DMP754 has minimal
antiplatelet efficacy when given at a dose of 0.1 mg/kg PO and
maximal antiplatelet efficacy at a dose of 0.3 mg/kg PO. This again
illustrates the steep dose-response relation of this class of
compounds. DMP754 at 0.3 mg/kg PO is as effective as 0.4 mg/kg PO of
XV459, suggesting improved oral antiplatelet efficacy of the
prodrug form (DMP754). This was further demonstrated by the improved
antithrombotic efficacy of DMP754 (0.3 mg/kg PO) as compared with XV459
(0.4 mg/kg PO) in limiting thrombus growth and improved CA blood flow.
A potent oral antithrombotic effect of DMP754 was shown at relatively
low unit doses (0.3 to 0.4 mg/kg PO) as compared with the relatively
higher unit dose required with other orally active GPIIb/IIIa
antagonists, such as SC-54684A (2.5 to 5.0 mg/kg PO BID in
dogs).25 In the CA thrombosis model in dogs, DMP754 (0.1
to 0.3 mg/kg IV or PO) exhibited antithrombotic efficacy in
electrolytically induced CA thrombosis. DMP754 at a dose of 0.3 mg/kg
PO is more effective than XV459 at a dose of 0.4 mg/kg PO in limiting
thrombus weight and blood flow in electrolytic injury–mediated
occlusive thrombosis in the CA (Table 3). In the case of
increased thrombogenic stimulus mediated by electrolytically induced
arterial thrombosis in the CA, DMP754 was capable of
limiting thrombus formation at the site of damage. However, it remains
to be determined in a clinical setting how long after
thrombolysis platelet inhibition must be sustained
to render the injured vessel and residual thrombus mass
nonthrombogenic. In contrast, aspirin (10 mg/kg PO for 2 days) and
ticlopidine (300 mg/kg PO for 3 days) administered before initiation of
arterial thrombosis were effective in reducing the
incidence of CFR but were ineffective in reducing the incidence of
electrolytic injury–induced occlusive arterial thrombosis.
Additionally, in the same models, hirudin, but not heparin, was shown
to have antithrombotic efficacy, which was reversed on rechallenge with
epinephrine (data not shown). The improved antithrombotic
efficacy of platelet GPIIb/IIIa antagonists as compared
with other agents might be due the universal effectiveness against all
known platelet activators.35 It is clear
that the efficacious doses vary according to the type of models used.
In the Folts' model, in which the thrombus is mainly
platelet-dependent, relatively lower oral doses of DMP754 (0.1
mg/kg) resulted in maximal antithrombotic efficacy. In contrast, in the
electrolytic injury model, in which the thrombus is associated with
mixed platelet, fibrin, monocyte, and red blood cells, relatively
higher doses of DMP754 (0.3 mg/kg PO) are required for maximal
antithrombotic efficacy. These data suggest that DMP754, a
low-molecular-weight GPIIb/IIIa receptor antagonist, may
have therapeutic potential as an effective intravenous and
oral antithrombotic agent in the prevention and treatment of
thromboembolic disorders.
Received December 16, 1998; accepted March 9, 1999.
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