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(Circulation. 1997;96:1130-1138.)
© 1997 American Heart Association, Inc.
Articles |
Profound and Sustained Inhibition of Platelet Aggregation by Fradafiban, a Nonpeptide Platelet Glycoprotein IIb/IIIa Antagonist, and Its Orally Active Prodrug, Lefradafiban, in Men
From the Departments of Biological Research (T.H.M., H.W.), Pharmacokinetics and Metabolism (R.B.), Chemistry (F.H.), and Project Management (J.K.) and the Center of Clinical Pharmacology (H.N.), Dr Karl Thomae GmbH, Biberach, Germany.
Correspondence to Dr Thomas H. Müller, Blood Transfusion Center, German Red Cross, Brandenburger Str. 21, D-26133 Oldenburg, Germany. E-mail ckmthm{at}nord.de
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Abstract |
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 Top Abstract IntroductionMethodsResultsDiscussionReferences |
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Background Clinical trials have demonstrated that platelet glycoprotein (GP) IIb/IIIa antagonists effectively prevent acute thrombotic events. Orally active GP IIb/IIIa antagonists are essential to evaluate the clinical benefit of long-term treatment. We therefore investigated platelet inhibition by the GP IIb/IIIa antagonist Fradafiban (BIBU 52; Fradafiban is the recommended INN of BIBU 52) and its orally administered prodrug, Lefradafiban (BIBU 104; Lefradafiban is the recommended INN of BIBU 104) in healthy subjects.
Methods and Results The activity and plasma levels of
Fradafiban and Lefradafiban were evaluated in double-blind,
placebo-controlled studies in 130 healthy male subjects. One to 15 mg
Fradafiban continuously infused over 30 minutes reversibly inhibited
platelet aggregation in platelet-rich plasma ex vivo in
response to 20 µmol/L ADP (5 mg, 100% inhibition at 27 minutes
after administration) and to both 1.0 (5 mg, 100%) and 10 µg/mL (15
mg, 97±3%) collagen. Single oral doses of Lefradafiban inhibited
ADP-induced aggregation by 59±14% (50 mg [mean±SD]; n=8), 90±12%
(100 mg), and 99±2% (150 mg) 8 hours after administration.
Correlations between activity and Fradafiban plasma levels were
identical after Fradafiban and Lefradafiban treatment. After day 1,
oral TID Lefradafiban treatment for 7 days inhibited aggregation by
31±9.6% (25 mg TID; n=8), 53±12% (50 mg; n=7), and 88±6.6% (75
mg; n=8) just before the next dose. A similar correlation between the
activity and Fradafiban plasma levels was observed at days 1, 2, and
7.
Conclusions Oral administration of Lefradafiban maintains the potent platelet GP IIb/IIIa antagonism of Fradafiban during treatment of healthy subjects for 1 week without signs of loss of the antiplatelet activity.
Key Words: platelet aggregation inhibitors • trials • pharmacology • glycoproteins • platelets
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Antiplatelet agents are useful in the prevention of thrombotic arterial occlusions leading to acute ischemic syndromes and their severe complications.1 2 The antithrombotic efficacy of the established platelet inhibitors is, however, limited. Substantial improvement in this therapeutic area is suggested by the results of the recently completed EPIC trial.3 4 The addition of c7E3 Fab, a humanized monoclonal antibody directed against the human platelet glycoprotein (GP) IIb/IIIa complex,5 6 7 for 12 hours to the standard antithrombotic regimen of aspirin combined with heparin reduced the incidence of ischemic complications in high-risk coronary angioplasty patients by 36% (30 days after percutaneous transluminal coronary angioplasty). Subgroup analysis has further revealed that in patients with unstable coronary artery disease and a relatively high risk of myocardial infarction, the therapeutic efficacy may be even higher.8 9
These clinical observations support the hypothesis that the binding of
fibrinogen and other multivalent ligands10 (eg, von
Willebrand factor) to the platelet GP IIb/IIIa complex
(
IIbß3 integrin) on the surface of
activated platelets cross-links adjacent platelets into
aggregates. Platelet-platelet interaction mediated by
conversion of the platelet GP IIb/IIIa to a receptor in the
presence of an excess of plasma proteins as ligands may
represent the final common step of platelet aggregation and
thus may be a prerequisite for platelet thrombus
formation.11 12
Specific blockade of these binding sites on platelets offers a novel therapeutic approach with potential advantages over established antiplatelet agents.13 Aspirin inhibits platelet aggregation by selective blockade of thromboxane A2–mediated platelet activation, whereas ticlopidine interferes with ADP-dependent platelet activation.14 These agents, therefore, affect platelet aggregation in ex vivo studies only in response to aggregatory agents dependent on these mediators. Platelet aggregation induced by relatively high concentrations of collagen (5 or 10 µg/mL) or thrombin is, if at all, only partially reduced even after the ingestion of maximal antiaggregatory doses of aspirin or ticlopidine. Aggregation in response to such potent proaggregatory stimuli thus could serve as a model to evaluate the platelet inhibitory profile of novel agents.
Oral activity is at present a prerequisite for long-term therapies. Monoclonal antibodies or peptidic GP IIb/IIIa antagonists usually have no or only very limited oral availability.15 16 In contrast, small synthetic molecules have been identified that result in substantial blockade of the GP IIb/IIIa receptor after oral administration.17 18 19 20 21 Fradafiban (BIBU 52; Fradafiban is the recommended INN of BIBU 52) is a nonpeptide mimetic of the arginine-glycine-aspartic acid recognition sequence22 :
Fradafiban, R1, R2=H; Lefradafiban (BIBU 104; Lefradafiban is the recommended INN of BIBU 104): R1=CO2CH3, R2=CH3.
Structure-activity relationships clearly indicate that the basic amidino group and the acidic carboxy group are decisive for the biological activity of Fradafiban. These functional groups are thought to correspond to the guanidino and ß-carboxy group of the arginine-glycine-aspartic acid recognition sequence, respectively. Fradafiban binds with high affinity and selectivity to the human platelet GP IIb/IIIa complex and potently inhibits human platelet aggregation in vitro.23 Fradafiban has only very limited oral activity probably due to its high polarity and thus poor absorption after oral ingestion. Esterification of the carboxyl group and acylation of the amidino group of Fradafiban led to a far less polar prodrug, Lefradafiban. Lefradafiban does not interact with the GP IIb/IIIa receptor and has to be converted metabolically to Fradafiban for platelet inhibition. Esterases, but not cytochrome P450–dependent enzymes, are involved in the conversion of Lefradafiban to Fradafiban in vivo.
The aim of the clinical pharmacology phase I studies described here was to investigate the antiaggregatory profiles of both Fradafiban and its orally active prodrug Lefradafiban in men. We measured the inhibition of platelet aggregation ex vivo in platelet-rich plasma (PRP) in response to both low and high concentrations of collagen and in response to ADP or a thrombin receptor agonist peptide. The inhibition of aggregation in whole blood induced by collagen (1 µg/mL) and ADP (5 and 20 µmol/L) was also determined. In addition, we monitored both the replacement of tritiated Fradafiban as a measure of the GP IIb/IIIa receptor occupancy and the Fradafiban plasma levels. These biochemical and pharmacological ex vivo measures demonstrate the potent platelet inhibition after treatment of healthy subjects with Fradafiban and its orally administered prodrug Lefradafiban.
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Methods |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Fradafiban {3-pyrrolidineacetic acid:5-[ [ [4'-(aminoimino-methyl)-[1,1'-biphenyl]4-yl]oxy]methyl]2-oxo-(3S-trans); molecular weight, 367.4 g/mol}, Lefradafiban {3-pyrrolidineacetic acid: 5-[ [ [4'(imino[(methoxycarbonyl)amino]methyl][1,1'-biphenyl]-4-yl]oxy]methyl]-2-oxo-methyl ester-(3S-trans); molecular weight, 439.5 g/mol}, and a thrombin receptor agonist peptide (serine-phenylalanine-leucine-leucine-arginine-asparagine-proline-asparagine-lysine-tyrosine-glutamic acid-proline-phenylalanine [TRAP])24 were prepared at Dr K. Thomae GmbH, Biberach, Germany.
Volunteer Studies
Three double-blind, placebo-controlled dose-escalation studies
were performed in healthy male subjects. All trials were approved by
the ethics committee Freiburger Ethikkomission. Written informed
consent was obtained from all participants. The participants were
enrolled after screening through history, physical examination, ECG,
urine analysis, and routine tests of hematology and clinical
chemistry to ensure good health. Aggregation in PRP induced by 1.5
µg/mL collagen was also tested, and no failure was observed.
Participants were instructed to refrain from taking any medication 14
days before and during the study. Subjects were treated in groups of
five (four subjects receiving Fradafiban or Lefradafiban and one
receiving placebo), and two groups were included per dose group. Blood
samples were always taken by separate venipuncture with a
18-gauge, 3/4-in needle at each time point.
Study 1
One, 3, 5, 10, and 15 mg Fradafiban was administered by
continuous intravenous infusion of an aqueous Fradafiban
solution (prepared from 2 mg/mL Fradafiban solutions by dilution
with a 5% glucose solution; a total volume of 25 mL was infused per
treatment) for 30 minutes. Blood samples were taken before treatment
and 27 as well as 150 minutes after the start of treatment. All
subjects completed the trial.
Study 2
A single oral dose of 10, 50, 75, 100, or 150 mg Lefradafiban
was administered at 8 AM. Blood samples were collected
before treatment and 1, 2, 4, 8, and 24 hours after the ingestion of
Lefradafiban (also 32 hours after administration in the 75- to 150-mg
dose groups and 48 hours after administration in the 100- and 150-mg
dose groups). All subjects completed the trial.
Study 3
Multiple oral doses of 25, 50, 75, and 100 mg TID Lefradafiban
were investigated. The treatment was started at 8 AM.
Subsequent doses were administered every 8 hours for 6 days followed by
a final dose on day 7 (8 AM). The minimal time interval
between meals and drug treatment was 2 hours. Blood samples were taken
before as well as 2, 4, and 8 hours after the first treatment (day 1),
just before as well as 2 and 8 hours after the fourth ingestion (day
2), and just before as well as 2, 4, and 8 hours after the last
treatment (day 7). One subject had to be excluded from the study just
before the start of treatment due to an injury (subject assigned to
placebo). Treatment was prematurely finished in two other subjects
after the first administration; one due to the failure to mention a
previous aspirin ingestion (assigned to 100 mg Lefradafiban). The other
subject (assigned to 50 mg Lefradafiban) was excluded after developing
fever most probably due to a viral infection. In a total number of four
subjects, slight signs of nose or gum bleeding were prolonged after 4
and 5 days, respectively, of treatment with 100 mg TID Lefradafiban
(Table 1
). The intensity of bleeding
signs remained light. Nevertheless, the treating physician decided to
withdraw medication in these subjects to avoid any possible risk. The
bleeding signs stopped within a few hours after discontinuation of the
treatment and without any additional therapy. Platelet count,
hemoglobin, and red blood cell count were not affected in any of the
subjects.
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Only three subjects in the 100-mg Lefradafiban dose group completed the
study as planned. This dose group could not be included in a meaningful
analysis of the effects (Table 2).
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3H-Fradafiban Binding to Platelets
PRP was prepared from blood anticoagulated with trisodium
citrate (13 mmol/L final concentration) through
centrifugation at 170g for 10 minutes. Then,
200 µL PRP was mixed with 10 µL 14C-sucrose (370 Bq)
and 10 µL 3H-Fradafiban (5 nmol/L final
concentration). After 20 minutes' incubation at room temperature, the
samples were centrifuged at 2000g for 5 minutes, the
supernatant was removed, 100 µL was counted for free ligand, and the
platelet pellet was dissolved in 200 µL of 0.2 mol/L NaOH.
An aliquot (180 µL) of this solution was mixed with 10 µL of 5
mol/L HCl and counted in a 2-mL scintillation cocktail. After
correction for spillover and extracellular space, the portion of
unoccupied binding sites for 3H-Fradafiban on the GP
IIb/IIIa receptor after treatment was calculated relative to the
individual pretreatment value.
Platelet Aggregation in Whole Blood
For logistic reasons, platelet aggregation in whole blood
could be investigated only in studies 2 and 3. Four aliquots of 2 mL
anticoagulated blood (trisodium citrate, 13 mmol/L) were
pipetted into polypropylene tubes and stirred (140 rpm) at 37°C for
10 minutes. To the control sample, 10 µL solvent was added. Then, 10
µL collagen (1.0 µg/mL) and 10 µL ADP (10 and 20
µmol/L), respectively, were added to the three aggregation
samples. Immediately before these additions and 1, 3, 6, and 10 minutes
later, 10 µL of blood was removed from the tubes and mixed with 10 mL
counting solution (44 mL formaldehyde [40%] + 1000 mL 0.9% saline).
Using the Ultra-Flo 100 Single Platelet Counter (Becton-Dickinson),
the number of single platelets (defined by the counting-window
preset of the manufacturer) in each sample was compared with the
corresponding control to calculate the extent of aggregation and
relative inhibition (in percent) of aggregation, respectively.
Platelet Aggregation in PRP
The aggregation tests were performed using six-channel
aggregometers for turbidometry (manufactured by H. Schwarz,
Feinmechanik, Dr K. Thomae GmbH). The photometric tracings were
recorded on six-channel recorders at a paper speed of 50
mm/min. Platelet-poor plasma prepared from each individual was used
to preadjust the photometric measurement to the minimal optical
density. All platelet aggregation tests were performed in
duplicate. PRP (297 µL) was pipetted into a cuvette and stirred with
a 3-mm-long bar (diameter, 1 mm) at 1250 rpm to preincubate at
37°C for 5 minutes in the aggregometer. Diluted collagen solutions (3
µL, prepared from the 1 mg/mL commercial batch; Hormon Chemie)
or ADP (Boehringer-Mannheim; 3 µL of a 2 mmol/L
solution) was added to induce platelet aggregation. In study 3, the
thrombin receptor-activating peptide (TRAP; 3 µL of a 2
mmol/L solution) was added to the PRP to stimulate platelet
aggregation.
After the addition of the stimulus, the optical density of the stirred PRP was recorded for 5 minutes. The area under this aggregation curve was determined. The inhibition (in percent) of collagen-, ADP-, or TRAP-induced platelet aggregation relative to the individual values observed before start of treatment was calculated.
The inhibition of platelet aggregation by Fradafiban in vitro did not differ with the use of PRP prepared from blood of identical donors anticoagulated either with citrate or the direct thrombin inhibitor hirudin (P=.6; paired t test). Platelet aggregation ex vivo was determined in citrate blood samples.
Fradafiban Plasma Levels
At each time point, 5 mL blood was drawn into tubes with EDTA.
The blood samples were stored in an ice-cooled water bath until
centrifugation. Plasma was prepared immediately through
centrifugation at 4°C, diluted with an equal volume
of 0.2 mol/L HCl, and stored at -20°C until
analysis.
Plasma concentrations of Fradafiban were measured using a validated high performance liquid chromatographic method with fluorescence detection. The lower limit of quantification was 1 ng/mL.
Statistical Analysis
The results are summarized as mean±SD. The association between
platelet inhibition and treatment dose was tested by fitting linear
models (least-squares method) for the platelet inhibition as a
function of dose levels (SAS/STAT release 6.11; SAS Institute).
Differences were considered statistically significant at a value of
P<.05. Concentration-response curves were fitted using the
sigmoid Emax model.
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Results |
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3H-Fradafiban Assay of the GP IIb/IIIa Receptor Occupancy
Fradafiban reversibly binds to the human platelet GP IIb/IIIa complex with a Kd value of 148 nmol/L. Kinetic experiments (data not shown) have demonstrated that both binding and dissociation of 3H-Fradafiban is very fast (<5 seconds) and reproducible for hours.
Binding of 3H-Fradafiban to platelets was measured in all three studies to quantify the GP IIb/IIIa receptor blockade in post-treatment blood samples. In PRP prepared from these samples, free and bound Fradafiban were in a steady state, and its overall concentration determined the extent of receptor occupancy. The minute amount of tritiated ligand added did not substantially increase the concentration of Fradafiban. Due to the rapid dynamic exchange of free Fradafiban molecules and Fradafiban bound to GP IIb/IIIa, the distribution of free versus bound 3H-Fradafiban reflected that of unlabeled Fradafiban molecules. 14C-Sucrose, which does not enter platelets, was added to determine the exact amount of plasma in the platelet pellet obtained after centrifugation and thus calculate the free 3H-Fradafiban in the platelet pellet without overestimation of platelet-bound radioactivity.
The number of binding sites per platelet determined in this assay were 65 601±11 675 (n=50) in study 1, 71 285±11 358 (n=39) in study 2, and 77 769±11 837 (n=50) in study 3. These results corroborate recent data of Wagner et al.25
Platelet Inhibition by Intravenous Infusion of
Fradafiban
Single doses of 1 to 15 mg Fradafiban were administered
intravenously via continuous 30-minute infusion. Blood
samples were taken both 27 and 150 minutes after the start of the
infusion to investigate the effects of the Fradafiban treatment on
platelet function ex vivo in relation to the individual
pretreatment values.
Three minutes before the end of the treatment (Fig 1: 27 minutes after start of treatment),
a dose of 1 mg Fradafiban half-maximally inhibited the binding of
3H-Fradafiban to the platelets in PRP
(P<.001 versus placebo-treated controls). This effect was
further increased in a dose-related manner. A mean inhibition of
96.0±1.1% (mean±SD, n=8; P<.001 versus control;
P<.001 versus 5 mg Fradafiban; P=.09 versus 10
mg Fradafiban) was observed in the highest dose group treated with 15
mg Fradafiban. The GP IIb/IIIa receptor antagonism also translated into
a dose-related inhibition of platelet aggregation in response to
different proaggregatory stimuli. The dose of 1 mg Fradafiban inhibited
1 µg/mL collagen-induced platelet aggregation in PRP by
70±16% (P<.001 versus control). In all subjects treated
with 3-mg doses of Fradafiban, platelet aggregation was
completely suppressed when induced by either 1 µg/mL collagen
or 20 µmol/L ADP ex vivo. Doses extrapolated to achieve a
half-maximal inhibition of platelet aggregation ex vivo were <1 mg
Fradafiban for 1 µg/mL collagen, 
2 mg Fradafiban for
20 µmol/L ADP, and 3 mg Fradafiban for 10
µg/mL collagen.
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Two hours after the end of the Fradafiban intravenous
infusion (Fig 1
: 2.5 hours after the start of treatment), the binding
of 3H-Fradafiban to the platelets was still inhibited
in a dose-related manner. Half-maximal inhibition required an
intravenous dose of 3 mg Fradafiban. Fifteen milligrams
of Fradafiban blocked the binding of labeled Fradafiban by 84.6±2.5%
at 2.5 hours after the start of treatment (P<.001 versus
control; P<.001 versus 5 mg Fradafiban; P=.07
versus 10 mg Fradafiban). Platelet aggregation in response to 1
µg/mL collagen was completely suppressed in all subjects
treated with 10 or 15 mg Fradafiban IV. The highest dose of 15 mg
Fradafiban also eliminated platelet aggregation in response to
20 µmol/L ADP at 2.5 hours after the start of treatment
(P<.001 versus control; P=.014 versus 10 mg
Fradafiban). Doses extrapolated to achieve a half-maximal inhibition of
platelet aggregation 2.5 hours after the start of treatment were
2 mg Fradafiban for 1 µg/mL collagen (PRP), 4 mg
Fradafiban for 20 µmol/L ADP (PRP), and 9 mg
Fradafiban for 10 µg/mL collagen.
Continuous infusion of total doses of 1 to 15 mg Fradafiban within 30
minutes corresponds to infusion rates of 0.44 to 6.7 µg/kg per
minute Fradafiban for a subject with a body weight of 75 kg. Our
findings indicate that these doses of Fradafiban both reproducibly and
reversibly inhibited platelet aggregation ex vivo in each dose
group of eight subjects. They were effective in a dose-related manner
not only in response to the conventional concentrations of collagen and
ADP but also in response to the very high concentration of 10
µg/mL collagen. The individual inhibition of platelet
aggregation ex vivo correlated well with the individual Fradafiban
plasma levels (see Fig 3). The concentration-response curve for
Fradafiban indicates that a plasma concentration of 54 ng/mL
Fradafiban half-maximally inhibits ADP-induced aggregation ex vivo
(r2=.99).
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Platelet Inhibition After Single Oral Doses of
Lefradafiban
Five different single doses of 10 to 150 mg Lefradafiban were
administered orally to evaluate the effects on various
parameters of platelet function ex vivo relative to the
individual pretreatment values. The effects were consistent in
the different test systems and their relative strengths agreed well
with the dose-response curves observed in the Fradafiban
intravenous study (Fig 1). The data for the inhibition of
20 µmol/L ADP-induced platelet aggregation (PRP) are
summarized in Fig 2 to represent
the time course of platelet inhibition after oral ingestion of
Lefradafiban.
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Ten milligrams of Lefradafiban did not affect ADP-induced platelet aggregation in comparison with the placebo treatment. The 50-mg Lefradafiban oral dose inhibited platelet aggregation by 90±5% at 2 hours (P<.001 versus control), by 59±14% at 8 hours (P<.001 versus control), and by <10% at 24 hours after administration (P=.04 versus control). Platelet aggregation was completely eliminated by 75 mg and all higher doses of Lefradafiban at 2 and 4 hours after administration. Twenty-four hours after administration, 75 mg Lefradafiban resulted in a mean inhibition of platelet aggregation of 22±19% (P<.001 versus control, P=.01 versus 50 mg Lefradafiban) and 150 mg Lefradafiban of 46±14% (P<.001 versus control, P=.005 versus 100 mg Lefradafiban). Platelet aggregation at 32 and 48 hours after administration did not differ from placebo treatment in any of the Lefradafiban dose groups and in response to any of the proaggregatory stimuli (data not shown).
Oral treatment with 50 to 150 mg Lefradafiban (ie, doses of 0.67 to 2 mg/kg for a person with a body weight of 75 kg) inhibited platelet aggregation ex vivo in a reproducible, reversible, and dose-dependent manner. The duration of the platelet inhibition was also related to the ingested dose of Lefradafiban.
Each individual value for the inhibition of 20 µmol/L
ADP-induced platelet aggregation (PRP) observed in the
Lefradafiban-treated subjects is represented in Fig 3 in relation to the individual
Fradafiban plasma levels measured in the same blood samples. The
(Fradafiban plasma) concentration-(platelet inhibition) response
curve fitted to all data from the Lefradafiban-treated subjects
demonstrates that a Fradafiban plasma concentration of 59 ng/mL
is required to half-maximally block ADP-induced aggregation ex vivo
(r2=.99). This concentration-response
curve is virtually identical to that fitted to the data from the single
intravenous doses of Fradafiban. Thus, we conclude that the
Fradafiban plasma levels generated in vivo after oral treatment with
Lefradafiban fully account for the extent of platelet inhibition
observed in Lefradafiban-treated subjects ex vivo.
The concentration-response curves for aggregation in response to 20 µmol/L ADP and 1.0 µg/mL collagen demonstrate that a 2.3- and a 2.5-fold higher, respectively, Fradafiban plasma level is required to half-maximally inhibit aggregation in whole blood compared with PRP (data not shown).
Platelet Inhibition After Multiple Oral Doses of
Lefradafiban
In the third trial, oral doses of 25, 50, 75, and 100 mg
Lefradafiban were administered every 8 hours for 6 days followed by the
last dose on day 7. Only three of the eight subjects assigned to the
highest dose completed the 7-day treatment (Table 2). Thus, the
inhibition of platelet aggregation (PRP) ex vivo in response to
either 20 µmol/L ADP (Fig 4) or 20 µmol/L of the
thrombin receptor activation peptide TRAP (Fig 5) is depicted for the placebo and the
25- to 75-mg Lefradafiban dose groups. The time course of day 1
represents the data observed after the first ingestion; day 2,
the data after the fourth administration; and day 7, the data after the
last ingestion. The treatment indicated by "0 hour" in these
figures was initiated at 8 AM on all 3 days. Platelet
aggregation was not affected in comparison with the individual
pretreatment controls in the placebo-treated subjects during the entire
7-day treatment course.
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In the 25-mg TID Lefradafiban dose group, the peak of the mean inhibition of ADP-induced platelet aggregation was 54±14% at 2 hours after administration on day 1 (P<.001 versus control), 69±14% at 2 hours on day 2 (P<.001 versus control), and 76±14% at 2 hours on day 7 (P<.001 versus control). Complete suppression of ADP-induced aggregation was observed in both the 50 mg TID and the 75 mg TID dose groups at 2 hours after administration on all days.
Platelet inhibition did not decline below 30% during the 7-day
treatment with 25 mg TID Lefradafiban. The trough of the inhibition was
50% before the fourth treatment with 50 mg Lefradafiban and 75%
thereafter. On days 2 and 7, the minimal effects of the 75 mg TID
regimen (average, 90%) exceeded those of the 50 mg TID treatment in
contrast to the first ingestion. Highly reproducible inhibition at a
slightly lower extent was also observed for platelet aggregation in
response to 20 µmol/L TRAP (Fig 5) during the entire
treatment course.
The consistent lack of a reduction in the antiaggregatory
activity in response to all the different proaggregatory stimuli
(including those not shown in Figs 4 and 5) after the 19th treatment on
day 7 in comparison with the first treatment on day 1 may indicate that
the platelets did not desensitize after prolonged treatment with
Lefradafiban.
The (Fradafiban plasma) concentration-(platelet inhibition)
response curves fitted to the data observed on days 1, 2, and 7 of the
oral Lefradafiban treatment demonstrate that ADP-induced aggregation
was half-maximally inhibited by 59 ng/mL
(r2=.99), 65 ng/mL
(r2=.99), and 71 ng/mL
(r2=.99) Fradafiban, respectively. The
close proximity of these curves (Fig 6),
furthermore, supports the conclusion that the antiaggregatory effects
of Fradafiban ex vivo were maintained for >6 days even after continued
in vivo exposure to therapeutic Fradafiban concentrations.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Three placebo-controlled studies were performed in healthy subjects to investigate the pharmacodynamics and pharmacokinetics of the nonpeptide GP IIb/IIIa antagonist Fradafiban (BIBU 52) and its orally active prodrug Lefradafiban (BIBU 104). This report summarizes the results of the analysis of platelet inhibition ex vivo.
The first study with Fradafiban in healthy volunteers confirmed the specific platelet-inhibitory profile of a GP IIb/IIIa antagonist observed both in vitro and in vivo in preclinical pharmacology. In men, continuous intravenous infusion of single doses of Fradafiban (1 to 15 mg IV within 30 minutes) inhibited platelet aggregation ex vivo not only in response to the conventional concentrations of collagen (1 to 2 µg/mL) and ADP (20 µmol/L) but also in response to high concentrations of collagen (5 and 10 µg/mL). The inhibition was reversible and related to the Fradafiban dose as well as to the strength of the proaggregatory stimulus.
Oral administration of single doses of 50 to 150 mg Lefradafiban
profoundly and reproducibly inhibited platelet aggregation ex vivo.
Platelet aggregation was impaired for 
24 hours after a single
dose and returned to the pretreatment values within 48 hours after
administration. Identical curves were obtained after the individual
Fradafiban plasma levels were correlated with the inhibition of
platelet aggregation ex vivo for both groups of subjects treated
with either Fradafiban IV or Lefradafiban PO.
In the third study, multiple oral doses of 25, 50, and 75 mg Lefradafiban TID for 6 days followed by a last dose on day 7 sustained the profound dose-related inhibition of platelet aggregation in response to collagen (1 to 10 µg/mL), ADP (20 µmol/L) as well as thrombin receptor-activating peptide (20 µmol/L TRAP) over the entire treatment course. Correlations between the individual Fradafiban plasma levels and the platelet inhibition measured on days 1, 2, and 7 did not reveal any sign of platelet desensitization to inhibition by Fradafiban, even after 6 days of sustained and profound GP IIb/IIIa antagonism.
These consistent results in >100 subjects confirm our hypothesis that Lefradafiban is an orally active agent that may be suitable for long-term interventions blocking the platelet GP IIb/IIIa receptor in humans.
As is to be expected from a potent and sustained inhibition of
platelet aggregation, minor bleeding events (Table 1) were observed
during prolonged treatment. The intensity of bleeding was always low.
The clinical relevance of the observed trend of increased bleeding with
higher doses must be confirmed in larger trials in patients. The
decision of the treating physician to prematurely stop 100 mg TID
Lefradfiban treatment in four of seven subjects also reflects the
present lack of an antidote. Dialysis should help to accelerate the
elimination of Fradafiban. Antibodies neutralizing synthetic GP
IIb/IIIa antagonists may also be useful.26
The need to generate the GP IIb/IIIa antagonist Fradafiban
from its prodrug Lefradafiban in vivo might be associated with
substantial variations of Fradafiban plasma levels and platelet
inhibition due to interindividual differences in the rates of
metabolic conversions. Our findings demonstrate, however,
highly reproducible levels of platelet inhibition in the subjects
treated with both 50 and 75 mg Lefradafiban (Figs 4 and 5). The TID
regimen sustained a reproducible and profound platelet inhibition
during the 6-day treatment. The peak and trough levels of
antiaggregatory activity observed after the fourth ingestion were
practically identical with those after the last ingestion on day 7. The
TID dose of 75 mg Lefradafiban PO maintained the mean inhibition of
platelet aggregation induced by 20 µmol/L ADP at
>90% (Fig 4) and the mean inhibition of platelet aggregation
induced by 20 µmol/L TRAP at >75% (Fig 5) after the
fourth ingestion.
Pharmacokinetic data from our studies with Lefradfiban, furthermore,
demonstrate an interindividual variation of <20% in the Fradafiban
plasma levels. This low variability suggests that both the absorption
of 25% after oral ingestion and the metabolic
conversion of the prodrug into Fradafiban were very reproducible in the
study population. Lefradafiban was administered 2 hours before or
after meals. Future studies should address the potential effects of the
food on the pharmacokinetics of Lefradafiban. The present data also
demonstrate that >90% of quantifiable drug in plasma is Fradafiban.
These data support the feasibility of this prodrug approach. Fradafiban
with its low protein binding shows a balanced (65% renal and
35% biliary) excretion pattern. Interestingly, the plasma levels
showed little dependence on the subject's body weight. Doubling the
weight from 50 to 100 kg decreased the plasma levels by only 20%.
The dominant half-life of 12 hours ensured low peak-to-trough
fluctuations in the Fradafiban plasma levels and may also limit
accumulation to the 1.8-fold of the initial peaks with TID dosing.
The mean accumulation factor of the plasma level peaks was 1.63 in the multiple Lefradafiban dose study. Neither these pharmacokinetic nor pharmacodynamic data provide evidence for excessive accumulation of drug levels and GP IIb/IIIa antagonism during the TID treatment with Lefradafiban for 6 days. The data furthermore demonstrate that after single as well as multiple oral administration of Lefradafiban, the blockade of platelet aggregation is reversible, whereas acetylsalicylic acid and ticlopidine apparently suppress platelet aggregation during the entire lifetime of the platelets. Moreover, we were not able to detect signs of a desensitization of the platelets in vivo to the profound inhibition by the GP IIb/IIIa antagonist Fradafiban generated in vivo even after prolonged treatment with Lefradafiban for 6 days. The findings of the present study corroborate and extend the ex vivo findings reported from treatment with GP IIb/IIIa antagonists for only a few hours.27 28 More than half of the platelet population exposed to Fradafiban after start of the Lefradafiban treatment may be replaced by new platelets at the end of the 1-week treatment. This finding of sustained antiaggregatory activity after several days of treatment with Lefradafiban must be supplemented by longer-term exposures to conclusively determine the effects of continued GP IIb/IIIa antagonism on thrombopoiesis and the function of newly formed platelets.
The pharmacological evaluation of novel platelet inhibitors in healthy subjects faces methodological difficulties. Platelet aggregation measured in stirred PRP or whole blood represents an in vitro model that at best reflects a few of the complex spatial and temporal patterns of interactions between the flowing blood and the injured vessel wall leading to mural platelet thrombus formation in patients with thrombosis. Diverse markers of platelet activation in patients in vivo have been developed to overcome the multiple limitations of such in vitro models. For studies in healthy subjects, however, these in vivo markers of thrombosis are not applicable. Potent inhibition of platelet aggregation ex vivo demonstrated by aggregometry does not necessarily translate into antithrombotic efficacy. Despite this precaution, the specific profile of a platelet inhibitor elaborated by aggregometry ex vivo in response to different stimuli (considering both the concentration and the nature of the stimulus) should be one of the pivotal determinants of the antithrombotic activity. Correlations between the inhibition of platelet function and plasma levels of the agent help also to identify essential features of the novel antiplatelet agent.
Profound inhibition of platelet aggregation independent of the specific nature of the proaggregatory stimulus is to be expected for GP IIb/IIIa antagonists. In addition to ADP and both conventional and high concentrations of collagen, we used TRAP to induce platelet aggregation. Thrombin is known to be an extremely potent activator of human platelet aggregation. Preclinical observations with direct thrombin inhibitors support the idea that thrombin may be a pivotal mediator of not only venous but also arterial thrombosis.29 30 31 The assessment of thrombin-induced platelet aggregation in PRP in ex vivo studies has been complicated by the problem of fibrin formation in the plasma due to the procoagulant activity of thrombin. Peptides that activate the thrombin receptor without converting fibrinogen to fibrin make it feasible to evaluate thrombin-mediated platelet aggregation directly in PRP. A substantially weaker inhibition of platelet aggregation ex vivo induced by 15 µmol/L TRAP (11–amino acid peptide) in comparison with 20 µmol/L ADP has recently been reported for patients treated with a bolus of c7E3.32
Our results with Fradafiban/Lefradafiban in men confirm the specific
advantage of GP IIb/IIIa antagonists to inhibit
platelet aggregation ex vivo independent of the nature of the
proaggregatory stimulus.5 Moreover, they demonstrate that
both the concentration and nature of the stimulus are essential
determinants for monitoring platelet inhibition ex vivo (Fig 1).
Several lines of evidence suggest that this reflects stimulus-dependent
mobilization of additional GP IIb/IIIa receptors from internal pools to
the platelet surface. Such differences in the strength of the
proaggregatory stimuli should be of value for the design of tests to
monitor platelet GP IIb/IIIa blockade ex vivo. They must also be
considered when different antiplatelet agents are compared ex vivo.
Aspirin selectively blocks thromboxane A2–mediated platelet activation. Ticlopidine specifically interferes with ADP-dependent platelet activation.14 The activation and aggregation of human platelets can be mediated by multiple signaling pathways.33 It is thus tempting to speculate that the limited antiaggregatory activity of the established antiplatelet agents accounts for their limited antithrombotic activity.11 12 Platelet aggregation in patients treated with these platelet inhibitors could be maintained by the unaffected pathways of platelet activation. As long as it is not feasible to identify or predict the temporal and spatial pattern of mediators involved in platelet activation and thrombotic complications in individual patients, the efficacy of these therapies should be less than optimal. Platelet GP IIb/IIIa antagonists are thus expected to offer a superior antiaggregatory profile by targeting the final step of platelet aggregation directly. The magnitude and optimal duration of the GP IIb/IIIa receptor blockade required for therapeutic activity in patients with thromboembolic disease both with and without concomitant aspirin treatment are not yet known. The highly reproducible and prolonged GP IIb/IIIa antagonism achieved with multiple doses of Lefradafiban should be adequate for the clinical evaluation of this pivotal issue of antithrombotic therapy. Treatment with such orally available GP IIb/IIIa antagonist for several weeks or even months should also help to address the importance of GP IIb/IIIa–dependent platelet/platelet interaction for the progression of various stages of vascular disease.
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Acknowledgments |
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The authors gratefully acknowledge the expert assistance of Monika Kink-Eiband, Günther Heinzel, Vera Koch, Karin Rühr, Johanna Schurer, Hans Schubert, Wolfgang Weiß, and the staff of the Centre of Clinical Pharmacology in Biberach.
Received November 20, 1996; revision received March 6, 1997; accepted March 11, 1997.
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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