(Circulation. 1995;91:403-410.)
© 1995 American Heart Association, Inc.
Articles |
SC-54684A: An Orally Active Inhibitor of Platelet Aggregation
From the Departments of Cardiovascular Diseases Research (N.S.N., S.G.P.-K., A.K.S., B.B.T., J.A.S., N.F.H., L.W.K., L.P.F.) and Chemical Research (J.A.Z.), Searle, Skokie, Ill; the Departments of Cardiovascular Diseases (B.T.K.) and Monsanto Corporate Research (K.B.), St Louis, Mo; Monsanto Corporate Research (W.E.), St Louis, Mo; the Department of Pharmacokinetics and Drug Metabolism (M.H.), Lilly Mont-Saint-Guibert Development Center, Mont St Guibert, Belgium; and Janssen Research Foundation (P.J.), Beerse, Belgium.
Correspondence to Nancy Nicholson, Searle, 4901 Searle Parkway, Skokie, IL 60077.
 |
Abstract |
|---|
 Top Abstract IntroductionMethods Results Discussion References |
|---|
Background Intravenous therapy has been shown to be beneficial in the prevention of acute platelet-associated thrombotic events. However, orally active agents would be advantageous for chronic therapy. Fibrinogen receptor antagonists block the fibrinogen/platelet interaction and thus inhibit a step required for thrombus formation. To date, no orally active fibrinogen binding inhibitors have been characterized. SC-54684A, now in clinical trial, is the orally active prodrug of a potent and specific fibrinogen binding antagonist.
Methods and Results We measured inhibition of
125I-fibrinogen binding to activated platelets and
inhibition of aggregation in platelet-rich plasma to selected agonists
and showed IC50s of 1.0x10-8 and 3 to
7x10-8 mol/L, respectively. Specificity of the active
moiety was determined by studying its effect on the binding of (1)
neutrophils to interleukin (IL)-1ß–stimulated endothelial cells, (2)
endothelial cells to fibronectin, and (3) vitronectin to isolated
vitronectin and fibrinogen receptors. No effect was observed on the
binding neutrophils to IL-stimulated endothelial cells or endothelial
cell binding to fibronectin. There was a fivefold separation between
binding to isolated receptors of vitronectin and fibrinogen.
Collagen-induced aggregation was inhibited by 80%, and bleeding time
was increased 
2.5-fold when the active moiety was infused to steady
state at 0.2 µg/kg per minute in dogs. When the ester prodrug was
given orally and the active moiety was given intravenously, the oral
systemic activity was 20%. Pharmacokinetic analysis after
intravenous infusion of the prodrug or active moiety showed that the
prodrug was rapidly converted to the active moiety; the active moiety
had a t
of 6.5 hours. When the prodrug
was
administered both orally and intravenously, the systemic availability
of the active moiety was 62%.
Conclusions SC-54684A, an orally active antiplatelet drug now in clinical trial, is shown to be a potent, specific fibrinogen binding inhibitor that blocks platelet aggregation to a wide variety of known stimuli and has good bioavailability in animals.
Key Words: platelets • platelet aggregation inhibitors • thrombosis
|
Introduction |
|---|
|
TopAbstractIntroductionMethods Results Discussion References |
|---|
Several important thrombotic diseases such as unstable angina and myocardial infarction have been shown to be related to platelet aggregation leading to thrombus formation. Although intravenous drugs may be useful in acute situations, long-term administration for prevention of secondary events may require the use of orally active agents. Currently available oral antiplatelet agents, such as aspirin and ticlopidine, have been shown to be beneficial in reducing myocardial reinfarction and mortality after coronary occlusions.1 The protection afforded by these agents is remarkable because aspirin and ticlopidine have only limited platelet inhibitory activity. Aspirin inhibits only the cyclooxygenase pathway leading to thromboxane production,2 and ticlopidine interferes with the ability of adenosine diphosphate (ADP) to stimulate platelets.3 4 In each case, these agents do not completely inhibit such important platelet agonists as thrombin, collagen (which is exposed in injured vessels), or shear forces (which occur in injured or atherosclerotic vessels5 ). Nevertheless, these agents exert protective effects, suggesting that substances that are effective against a wider variety of agonists could yield even greater protection.
Inhibitors of fibrinogen (fgn) binding to its receptor glycoprotein
(GP) IIb/IIIa on activated platelets represent a class of
compounds that inhibit platelet aggregation to all known stimuli.
Several reports in the literature have described molecules that are
active in inhibiting platelet aggregation when administered
intravenously (7E3, Integrelin, DPM 728,
SC-49992).6 7 8 9 10 11 12
In
this report, we describe the pharmacological properties of the ester
prodrug SC-54684A (SCp, Fig 1
, a hydrochloride salt in
which the carboxylic acid of the aspartate is converted to an ethyl
ester) and the active moiety, SC-54701A (SCa), in which the carboxylate
is not esterfied. SC-54684A is currently in clinical trial as an
antithrombotic agent for chronic use.
|
We show that SCa is a potent inhibitor of fgn binding to platelets, resulting in inhibition of platelet aggregation. The prodrug ethyl ester (SCp) is readily absorbed after oral administration and is rapidly metabolized to SCa. Furthermore, we show that SCa is specific for GP IIb/IIIa compared with other integrins sharing the same ß3-subunit. We believe that SCp has greater bioavailability than other orally active agents thus far reported and that SCp has a sufficiently long half-life to make it a viable candidate for clinical development as an oral agent for treatment of thrombotic diseases resulting from platelet aggregation.
|
Methods |
|---|
|
TopAbstractIntroductionMethods Results Discussion References |
|---|
SCa and SCp were prepared at Searle. SCp, ethyl 3-(S)-[[4-(aminoiminomethyl)phenyl]amino]-1,4-dioxobutyl]amino]-4-pentynoate, was synthesized by reacting commercially available 4-aminobenzamidine hydrochloride with succinic anhydride followed by a mixed anhydride coupling with ethyl 3(S)-amino-4-pentynoate.13 The (S) ethyl 3-amino-4-pentynoate was prepared through the addition of 1-lithio-2-trimethylsilylacetylene to 4-benzoyloxyazetidinone followed by isolation and treatment of the product with anhydrous HCl in ethanol, which afforded the racemic material that was separated using classic techniques and subsequently deprotected. SCa was prepared by cleavage of SCp, using pig liver esterase (Sigma) in phosphate buffer (pH 7.4, 0.1 mol/L, Sigma).
Semipurified unlabeled fgn (Kabi, Division of Helena Laboratories) was prepared as previously described.14 Platelet agonists included ADP (BioData) and collagen (equine tendon, Chronolog).
Dogs (beagles; weight, 7 to 10 kg) were obtained from White Eagle Laboratories, Doylestown, Pa. Platelet-rich plasma (PRP) was prepared from blood obtained from dogs and from normal human volunteers who reported that they had not taken nonsteroidal anti-inflammatory agents for at least 14 days.
Human vitronectin receptors (
vß3) were
purified from placenta and human fibrinogen receptors
(IIbß3) were purified from outdated
platelets as previously described (References 15 and 16, respectively).
Human vitronectin was purified from fresh frozen plasma.17
Biotinylated human vitronectin was prepared by coupling NHS-biotin from
Pierce Chemical Co to purified vitronectin.18 Assay
buffer, OPD substrate tablets, and radioimmunoassay grade bovine serum
albumin (BSA) were obtained from Sigma. Antibiotin antibody was
obtained from Calbiochem. Linbro microtiter plates were obtained from
Flow Labs. Soybean trypsin inhibitor and all cell culture and buffer
reagents were purchased from Sigma. Cell culture plasticware was
purchased from Costar, except the 96-well nontissue culture grade
microtiter plates were purchased from Dynatech. The CellTiter 96 Assay
was purchased from Promega. Human umbilical vein endothelial cells
(HUVEC, for the endothelial cell adhesion assay) at passage 1 were
purchased from Cell Systems.
Fibrinogen Binding
Fgn binding was assessed in a manner
similar to that of
Marguerie et al19 and Plow et al,20 as
modified by Nicholson et al.14 Responses at each
concentration were averaged, and the standard error was calculated. The
IC50 was estimated from each individual dose-response
curve. The IC50s were averaged, and standard error was
calculated.
In Vitro Platelet Aggregation: Inhibition of Aggregation of Human
and Dog PRP
PRP was prepared as previously described,14
and
platelet aggregation was measured as an increase in light transmission.
Agonists included ADP and collagen: ADP, 20 µmol/L; collagen in
human, 4 µg/mL; and collagen in dogs, 33 µg/mL. All agonists were
titrated to concentrations greater than that required for maximal
aggregation. Responses at each concentration were averaged, and the
standard error was calculated. The IC50s were estimated
from the dose-response curves, and averaged and standard error was
calculated.
Specificity Studies
Neutrophil–Endothelial Cell
Adhesion Assay
Isolation and fluorescent labeling of human
neutrophils. Isolation and labeling of neutrophils with Dil
(1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyamine
perchlorate,
Molecular Probes) were as described by Look et al.21
Cell adhesion assay. Monolayers of HUVECs (Clonetics Corp) were grown on fibronectin-coated, 96-well tissue culture plates and activated with interleukin (IL)-1ß for either 4 or 20 hours in complete endothelial growth medium (Clonetics Corp) containing 5% fetal bovine serum. Subsequently, the monolayers were washed three times with 300 µL per well Hanks' balanced salt solution (HBSS) containing Ca2+/Mg2+ and 0.1% BSA to ensure complete removal of the cytokine. One hundred microliters of SCa (in HBSS/BSA) then was added to the appropriate wells and the plates incubated at 37°C for 30 minutes, after which a microscopic analysis was carried out to confirm that there was no obvious effect on endothelial cell morphology. Dil-labeled human neutrophils were added to each well (3x105/50 µL) and the plate returned to the 37°C incubator for 20 minutes to allow the neutrophils to settle and bind to the HUVEC. Unbound neutrophils were removed by three buffer washes (300 µL per well). During the third wash, a clear adhesive plate sealer was placed over the buffer-filled wells, and the plate was inverted and centrifuged at 200g for 3 minutes. After removing the sealer (maintaining the plate in an inverted position) and decanting the buffer by shaking, the wells were washed an additional time, decanted, and the plate centrifuged in an inverted position to remove all residual liquid from the wells.
The
number of neutrophils bound was determined by solubilizing each
well in 100 µL of 0.5% Triton-X100 for 45 minutes (37°C) and
transferring 80-µL samples to a fluoricon assay plate (Baxter
Healthcare Corp) for quantitation of fluorescence in a Pandex
fluorescence concentration analyzer plate reader (
ex=545 nm,
em=575 nm). Cell number was established by comparison to a
standard
curve of the Dil-labeled neutrophils prepared by a 12-step serial
dilution on a separate 96-well plate.
Endothelial Cell
Adhesion Assay
HUVECs were grown to passage 4 in MCDB131, 4% fetal
bovine
serum, 1% human serum, 1 µg/mL gentamycin sulfate. Tissue culture
plates (Costar, 24-well) were fibronectin coated for 2 hours at room
temperature with 20 µg/mL human fibronectin in Dulbecco's
Ca2+/Mg2+-free phosphate-buffered saline. The
solution was aspirated and cells plated into the wells at a 1:4 split
and cultured for 3 days in 5% CO2 at 37°C. Subconfluent
cell cultures (3 days after seeding at a 1:4 split) were removed from
the incubator, medium aspirated, and replaced with 0.6 mL of complete
medium containing 250 µmol/L GRGDSP or 50 µmol/L SCa. (Note:
Previous experiments with bovine pulmonary artery cells [data not
shown] indicated that cells sensitive to 100 µmol/L GRGDSP detached
more rapidly when sparse rather than confluent cultures were used.
Human cells were unaffected by GRGDSP.) The plate was returned to the
incubator for 1 hour and 50 minutes. The plate then was removed and
medium aspirated from each well just before taking a photomicrograph
using a x20 phase objective on a Nikon diaphot phase microscope
equipped with a Nikon 8008 camera set for automatic exposure with TMAX
100 film (Kodak). Photography was completed within 10 minutes.
Solid-Phase Receptor Assays
The purified human
vitronectin receptor
(vß3) and purified human fgn receptor
(IIbß3) assays were conducted as
previously described.22 23
Bioassay
The concentration of the active agent in canine
plasma was
estimated using plasma from treated dogs to inhibit PRP from donor
dogs. Blood was collected in citrate (9:1) from nontreated dogs and
centrifuged (500g, 3 minutes, x2) to yield PRP. The
remaining blood sample was centrifuged at 2000g for 10
minutes to obtain platelet-poor plasma (PPP), used to set baseline in
an aggregometer and for dilution of samples. Plasma (225 µL) from
treated dogs was mixed with 225 µL of PRP from donor dogs, and
aggregation was determined.14 Percent inhibition was
calculated, and the concentration of active compound in plasma from
treated dogs was estimated by comparing the inhibition observed with a
standard curve prepared using plasma that had been spiked with known
amounts of SCa. If the plasma from the treated animals produced 80% to
100% inhibition, the plasma was diluted with PPP to produce results
that were within the 20% to 80% range of the standard curve.
Ex
Vivo Platelet Aggregation and Determination of Oral Systemic
Activity
Dogs received either SCp administered orally (single dose) by
gelatin capsule or SCa (1 mg/kg in 4 mL of saline/water [50:50])
injected intravenously into the cephalic vein over 1 minute. Before
dosing, two control blood samples (2x2 mL) were drawn by venipuncture
from the cephalic vein. Blood was centrifuged at 266g for 6
minutes to prepare PRP. Percent aggregation before administration of
compound (baseline) was established. Blood samples were drawn at
selected time intervals after administration of compound for the first
14 hours and again at 24 hours after dosing for determination of
platelet aggregation. The remaining blood was centrifuged for 2 minutes
at 12 000g to prepare PPP for determination of the
concentration of compound in the plasma samples by bioassay (see
above). Inhibition of collagen-induced platelet aggregation was
determined by comparing aggregation responses in the samples containing
compound with the responses of samples before administration of
compound (baseline).
Oral Systemic Activity
Concentration of active compound in
plasma samples was
determined by bioassay from both the oral study and the intravenous
study. These concentrations were plotted versus the time the sample was
taken and the area under the curve (AUC) was calculated. AUCs for
intravenous (IV) and oral (PO) treatments were corrected for the
respective doses, and oral systemic activity (OSA) was expressed in
percentage as (PO AUC÷IV AUC)x100.
Bleeding Time
Bleeding time was assessed in the upper lip of
anesthetized
beagles. An 18-gaugex2-in. catheter (Becton Dickinson) was placed in
the antecubital vein on the forelimb for the infusion of drug or 0.9%
NaCl. The infusion was continued for 5 hours (0.15 mL/min, Harvard
programmable infusion pump model 22). A catheter was placed in the
jugular vein to obtain blood samples for the determination of platelet
aggregation. Pharmacodynamic steady state (inhibition of platelet
aggregation) was achieved at approximately 2 hours. When steady state
was achieved, the bleeding time measurement was determined according to
the methods described by Jergens et al24 using a
spring-loaded device (Simplate II, Organon Teknika Corp) to make the
incisions (maximum time of observation, 30 minutes). The bleeding times
from the two cuts were averaged and used as the bleeding time value for
that particular test. Statistical analysis of bleeding times was
carried out by the nonparametric (ranks) maximum t test
method.25 Significance was assumed when P<.05
was found.
Pharmacokinetics
In one experiment, 14C-labeled
ester
([14C]-SCp) and acid ([14C]-SCa)
were each
administered intravenously to one female beagle dog at a dose of 2.5
mg/kg. The same compounds were also administered intragastrically as a
solution to two female beagle dogs at the same dose. Blood was
collected at selected times between 0 and 24 hours after
administration. Urine and feces were collected each day for 4 days
after administration. The total 14C radioactivity was
counted in blood, plasma, urine, and some fecal samples. The plasma
from each of the dogs (intravenous or oral, acid or ester) was
prepared, and corresponding samples were pooled and profiled by
high-pressure liquid radiochromatography (HPLRC). The ester and acid
concentrations were estimated using the HPLRC profiles. Samples of 24-
and 48-hour urine and feces were also analyzed by HPLRC.
|
Results |
|---|
|
TopAbstractIntroductionMethods Results Discussion References |
|---|
In Vitro Platelet Effects
SCa inhibited fgn binding with an IC50 of 1.0±0.2x10-8 mol/L, as shown in the dose-response curve (Fig 2
). With respect to platelet aggregation, the
dose-response curves in Fig 3 illustrate that the
IC50 for SCa in human PRP was
7.0±0.4x10-8
mol/L when collagen was used as the agonist and
3.5±0.3x10-8 mol/L with ADP as the agonist. This
figure
also illustrates that inhibition in dog PRP was similar to that
observed in human PRP, with IC50s of
6.1±0.8x10-8 mol/L and
6.7±0.5x10-8 mol/L
for ADP and collagen, respectively. In addition, Fig 3 presents
data on the comparison of the activity of SCa with SCp. SCp had an
IC50 of 4.6x10-6 mol/L in
collagen-stimulated
dog PRP. (Note: This amount of activity can be accounted for by the
known 1% contamination of SCp with the free acid SCa.) The relative
lack of activity in response to SCp confirms that the ester acts as a
prodrug, which requires in vivo metabolism for conversion to the active
moiety.
|
|
Specificity
We have examined the specificity of SCa for GP
IIb/IIIa in a
variety of models. The effect of SCa was determined on the binding of
neutrophils to HUVEC stimulated with IL-1. In response to IL-1, the
HUVECs express endothelial leukocyte adhesion molecule-1 (ELAM-1) and
intercellular adhesion molecule-1 (ICAM-1) in a time-dependent
fashion.26 ELAM-1 binds to a carbohydrate containing
moiety (sialyl Lewisx structure) on neutrophils, while
ICAM-1 binds to the heterodimeric B2 integrins (CD11a/CD18
and CD11b/CD18). SCa at concentrations up to 10 µmol/L had no effect
on the binding of neutrophils to endothelial cells expressing either
ELAM or ICAM (data not shown).
SCa competed with vitronectin for
binding to isolated vitronectin
receptors (vß3) with an IC50
of 1.6±0.1x10-5 mol/L compared with its
inhibition of
binding to isolated fibrinogen receptors
(IIbß3) with an IC50 of
4.7±0.7x10-10 mol/L (Fig 4).
Thus, SCa is about 34 000 times
more specific for IIbß3 than for
vß3.
|
SCa did not affect spreading or attachment of subconfluent HUVECs when compared with cells treated with vehicle (phosphate-buffered saline). After 2 hours of incubation with SCa, no differences were observed between the experimental and control chambers (comparisons not shown). The endothelial cells remained well spread, with distinct ruffling edges. The peptide GRGDSP also did not disrupt HUVEC attachment or spreading (data not shown), which suggests that non-RGD integrins may be involved in human endothelial cell–substratum interactions.
Bleeding Time
Control bleeding time was 2.66±0.52
minutes (Fig 5). Bleeding time was significantly increased
(approximately 2 times control) at 0.10 µg/kg per minute, which
resulted in approximately 35% inhibition of platelet aggregation
induced by collagen. At a dose of 0.2 µg/kg per minute when
collagen-induced aggregation was inhibited by 80%, bleeding time was
increased about 2.5-fold. At 0.3 µg/kg per minute, when aggregation
was inhibited by >85%, bleeding times were 5 times control (15
minutes). No animals had bleeding times over the 20 minutes.
|
In Vivo Oral Systemic Activity
When administered orally, SCp
completely inhibited platelet
aggregation for more than 24 hours after a single dose of 7.5 mg/kg
(data not shown). Likewise, when administered intravenously at 1 mg/kg,
platelet activity was completely inhibited for more than 8 hours, and
inhibition was still detectable 24 hours later (data not shown). AUCs
were determined from plasma concentrations determined by bioassay
plotted against time (Fig 6). A comparison of the AUC
for SCp administered orally and the AUC for SCa administered
intravenously indicated an OSA of 18.7%.
|
Pharmacokinetics
We administered 14C-labeled
compound (both SCa and
SCp) (Table) to dogs both by the intragastric and
intravenous routes and measured the amount of radiolabeled compound in
the plasma as a function of time after each route of administration.
(Note: The study was repeated using unlabeled compound. Data from this
experiment [mean±SD] are in parentheses after the
14C-labeled data.) Half-life data are presented in Fig
7. Shown in Fig 8 are the amounts of
SCp or SCa detected in the plasma after intragastric or intravenous
administration of either SCp or SCa.
|
|
|
After intravenous administration
of SCa, the half-life of the ß-phase
for elimination of SCa (two-compartment open model) was 6.5 hours
(4.7±1.0 hours) (Fig 7
and Table), the
total plasma clearance was 0.3
L/h per kilogram (0.42±0.02 L/h per kilogram) and the volume of
distribution (Vd/area) was 2.8 L/kg (2.9±0.7 L/kg). The unchanged
compound was the only radioactive constituent in every plasma sample
and in the 0- to 48-hour urine sample. Seventy-five percent of the
administered radioactive dose was recovered in the urine after 4 days.
In contrast, after intravenous administration of SCp, the apparent
elimination half-life (two-compartment open model) was 1.6 hours
(1.4±0.8 hours) (Fig 7 and Table), with a
plasma clearance of 2.05 L/h
per kilogram (2.02±0.32 L/h per kilogram) and a volume of distribution
(Vd/area) of 4.6 L/kg (4.3±3.5 L/kg). Most of the dose was rapidly
transformed into the free acid. Only a small amount of the ester was
eliminated in the urine, accounting for 11.6% of the radioactivity
recovered in 0- to 24-hour urine. The acid was the major radioactive
compound in urine, accounting for 73.8% of the radioactivity excreted
in 0- to 24-hour urine.
In contrast to intravenously administered SCa, the radioactivity eliminated in the urine when SCp was administered intravenously was only 40.5% of the dose, while 52.2% of the dose was excreted in the feces, suggesting that a significant amount of SCp was eliminated by biliary excretion. In 0- to 24-hour feces, the radioactivity was almost exclusively recovered as the radiolabeled free acid (92.9%). Only 4.3% was detected as the ester. These data may suggest that after excretion of SCp in the bile, the compound may be metabolized into SCa in the lumen of the intestine.
After oral administration of SCp, the mean percentage of the dose excreted in the urine was 28.6±14.8%. In the 0- to 48-hour urine, the acid and the ester accounted for 23.8±15.5% and 0.9±0.3% of the dose, respectively, showing that most of the radioactivity was excreted in the urine as radiolabeled free acid.
When the acid [14C]-SCa was given orally, the mean [14C]-free acid maximum plasma concentration (Cmax) was 0.205 µg/mL. This value was close to the Cmax observed after administration of the ester (0.172 µg/mL); however, the time to reach the maximum plasma concentration was shorter (1.5 hours).
In each animal, when the total radioactivity
plasma concentration was
100 ng/mL, the concentration of total radioactivity in blood was
approximately equal to or greater than the plasma concentration. This
indicates a considerable distribution of the total radioactivity in red
blood cells and/or platelets at concentrations 100 ng/mL.
Bioavailability
After oral administration, the absolute
bioavailability of
ester prodrug [14C]-SCp expressed in terms of the ester
in the plasma was low (11.8%, Fig 8a). Compound was, however,
present
in greater quantity as the free acid form (Figs 8b and
8c). The
systemic availability of the active metabolite [14C]-SCa
after oral administration of the prodrug [14C]-SCp was
21.3% (systemic availability=AUC1/AUC2 where
AUC1=AUC0-24h of SCa when SCp was administered
IG and AUC2=AUC0-24h of SCa when the active
molecule SCa was administered intravenously) (Fig 8b). In
contrast,
when we administered SCp intragastrically and intravenously and
measured SCa after both routes of administration, the AUC ratio was
61.5% (Fig 8c). The time to reach the maximum plasma
concentration of
the acid (tmax) was 3 hours, and the mean maximum plasma
concentration of the SCa (Cmax) was 0.172 µg/mL. A
plateau was observed from 3 to 5 hours.
The absolute bioavailability of
[14C]-SCa given
intragastrically was only 8.5% (Fig 8d), considerably lower
than when
SCp was given intragastrically, indicating that the ester prodrug is
needed to increase absorption after oral administration.
|
Discussion |
|---|
|
TopAbstractIntroductionMethods Results Discussion References |
|---|
Intravenous agents for prevention of platelet aggregation are useful for the treatment of acute thrombotic events such as reocclusion after thrombolytic therapy or emergency angioplasty in an acute-care setting. However, intravenous therapy is not appropriate for chronic treatment, ie, prevention of thrombotic disorders including recurrence of myocardial infarction or unstable angina. For chronic therapy, an orally active agent is desirable because of ease of administration and greater patient compliance. SC-54684A is currently in clinical trial for chronic therapy in the prevention of platelet-associated thrombus formation.
SCp is a prodrug with excellent oral availability that has been shown to inhibit platelet aggregation in human and animal platelets in vitro (as the active moiety) as well as in intact dogs. The compound is potent and effectively inhibits ex vivo platelet aggregation in dogs at reasonable doses. (In a separate study,27 we have determined that 2 mg/kg BID at steady state leads to a minimum of 50% platelet inhibition during a 24-hour period.) In this study, we have reported that a single oral dose of 7.5 mg/kg resulted in complete inhibition of platelet aggregation for more than 24 hours. Likewise, platelet aggregation was completely inhibited for >8 hours after a single dose of 2.5 mg/kg. SCp leads to an active metabolite that inhibits platelet aggregation by inhibiting fibrinogen binding and should, therefore, be effective in response to all known platelet agonists. Agents that act through this mechanism should provide greater protection compared with other compounds, such as aspirin, which only inhibit platelet aggregation in response to specific stimuli.28
Recently, reports have appeared in which peptide mimetics29 30 were described as being orally available. However, this is the first report in which a detailed analysis of bioavailability is reported, and the level of absorption is higher than reported for other agents.29 Our data suggest that >50% of the administered prodrug SCp is absorbed after oral administration and that about half that amount is converted to the active agent SCa.
Low bioavailability is an undesirable characteristic because it can lead to variation in absorption between individuals or even in a single individual as a function of external factors, such as diet. The oral systemic activity of the active compound after administration of the prodrug exceeded 20%. On the other hand, for purposes of safety, it is desirable that a large fraction of the administered compound be absorbed. After oral administration of the ester, the systemic availability was measured to be >60% ([SCp IG/SCp IV] measuring plasma concentrations of SCa), suggesting that even though not all of the prodrug was converted to biologically active material, >60% of the dose was absorbed, limiting the potential for increased levels of absorption due to dietary effects or individual variations.
Since an agent such as SCa has the potential of being used chronically
for an extended period of time, it was expected that a high degree of
specificity would lead to fewer side effects. We have shown that SCa is
highly specific for the platelet IIbß3
integrin relative to vß3, which may be an
indication of increased safety. In contrast, the antibody 7E3 in
development is not specific to IIbß3
relative for vß3.31 32
Furthermore, after conversion of SCp to its active form, there is
little further metabolism, suggesting that there is little opportunity
for the formation of toxic metabolites.
Bleeding time has been advanced as an important measurement, even
though there is considerable controversy as to the correlation of
bleeding time measurements with clinical blood loss.33
Some studies have reported inhibition of aggregation with no increase
in bleeding time,34 while still others report greater
levels of bleeding associated with platelet inhibition.11
In an animal model, the correlation between the level of inhibition
determined in ex vivo platelet preparations and the bleeding time found
at the time of blood sampling may be influenced by the type of agonist
and concentrations used in the aggregation studies. For example, the
lack of effects on bleeding time at 100% inhibition of ex vivo
platelet aggregation reported for TP9201 was measured using ADP at
concentrations titrated to give 80% of maximal aggregation (5
µmol/L).34 When results were reported for DMP728,
increased bleeding time was reported at levels that inhibited ex vivo
aggregation in response to 10 µmol/L ADP.11 In the
present studies, we used a supramaximal dose of collagen (33
µg/mL) to produce ex vivo platelet aggregation. We found a small
increase in bleeding time at doses that inhibited this aggregation
response 80%. However, it is difficult to relate these results to each
other because of the differences in methodology. Nevertheless, it is
apparent that some compounds appear to produce beneficial effects in a
variety of animal models without substantially increasing template
bleeding times.
In addition, the efficacious dose at which the bleeding time measurements are made could be influenced by the choice of animal model. TP9201 was reported to be effective in preventing clot formation in an inverted artery model at doses that did not increase bleeding time.34 This model has been described as being a very platelet-rich model.35 In contrast, the canine electrical damage model is associated with a less homogenous thrombus that is formed by a fibrin/platelet clot.36 The effectiveness of a compound in preventing thrombus formation may be influenced by the nature of the injury, which leads to different forms of thrombus. For example, compounds directed solely against platelet aggregation, such as inhibitors of fibrinogen binding, may appear more potent in models that are highly platelet dependent. While, for example, DMP728 led to increased bleeding time at efficacious doses, the increase may be a reflection of a higher concentration of compound required to prevent thrombus formation in a model that is not exclusively platelet dependent. We believe that the present data suggest that SC-54684A represents a highly bioavailable agent that only minimally increases bleeding times at doses that lead to significant inhibition of platelet aggregation. Whether these results will be translated into less bleeding in a clinical situation is not known, however, particularly because the clinical relevance between increases in template bleeding time and clinical events remains controversial.
Summary
SCp is a prodrug of a potent, specific, and
relatively stable
metabolite, SCa. After oral administration, a significant amount of the
compound is absorbed, leading to inhibition of platelet aggregation.
Clinical trials with SCp have shown it to be potent and safe in
humans.
Received May 27, 1994; accepted August 19, 1994.
|
References |
|---|
|
TopAbstractIntroductionMethods Results Discussion References |
|---|
1. Theroux P. Antiplatelet and antithrombotic therapy in unstable angina. Am J Cardiol. 1991;68:92B-98B. [Medline] [Order article via Infotrieve]
2. Smith JB, Willis AL. Aspirin selectively inhibits prostaglandin production in human platelets. Nature: New Biol. 1971;231:235-237. [Medline] [Order article via Infotrieve]
3. Thebault JJ, Blatri CE, Blanchard JF, Panak EA. Effects of ticlopidine, a new platelet aggregation inhibitor, in man. Clin Pharmacol Ther. 1975;18:485-491. [Medline] [Order article via Infotrieve]
4. Pengo V, Boschello M, Marzari A, Baca M, Schivazappa L, Dalla-Volta S. Adenosine diphosphate (ADP)-induced alpha-granule release from platelets of native whole blood is reduced by ticlopidine but not by aspirin or dipyridamole. Thromb Haemost. 1986;56:147-150. [Medline] [Order article via Infotrieve]
5. Ruggeri ZM. Von Willebrand factor as a target for antithrombotic intervention. Circulation. 1992;86(suppl III):III-26-III-29.
6. Tcheng JE, Ellis SG, Kleiman NS, Harrington RA, Mick MJ, Navetta FI, Worley S, Smith JE, Kereiakes DJ, Kitt MM, et al. Outcome of patients treated with the GPIIb/IIIa inhibitor integrelin during coronary angioplasty: results of the IMPACT Study. Circulation. 1993;88(suppl I):I-595. Abstract.
7. Schulman SP, Goldschmidt-Clermont PJ, Navetta FI, Chandra NC, Guerci AD, Califf RM, Ferguson JJ, Willerson JT, Wolfe CL, Bahr R, et al. Integrelin in unstable angina: a double-blind randomized trial. Circulation. 1993:88(suppl I):I-608. Abstract.
8. Simoons MI, van den Brand M, Hoorntije JCA, de Feytor P, Zijlstra F, Heyndricks G, van den Wieken R, De Bono D, Rutsch W, Weisman HF, et al. Chimeric 7E3 antiplatelet antibody Fab for treatment of refractory unstable angina: a placebo-controlled pilot study. J Am Coll Cardiol. 1993;21:269A. Abstract.
9. Kleiman NS, Ohman EM, Ellis SG, Aguirre FV, Weisman HP, Schaible T. Infarct vessel patency is enhanced by profound platelet inhibition with fibrinogen receptor blockade with 7E3 in patients receiving thrombolysis for acute myocardial infarction. Circulation. 1992;86(suppl I):I-260. Abstract.
10.
Simoons MI, DeBoer MJ, Marcel JBM, van den Brand M, von
Miltenberg AJM, Hoorntje JCA, Heyndrickx GR, van den Wieken LR, De Bono
D, Rutsch W, et al. Randomized trial of a GPIIb/IIIa platelet receptor
blocker in refractory unstable angina. Circulation. 1994;89:596-603.
11.
Mousa SA, Bozarth JM, Forsythe MS, Jackson SM, Leamy A, Diemer
MM, Kapil RP, Knabb RM, Mayo MC, Pierce SK, et al. Antiplatelet and
antithrombotic efficacy of DMP 728, a novel platelet GP IIb/IIIa
receptor antagonist. Circulation. 1994;89:3-12.
12. Nicholson NS, Panzer-Knodle SG, King LW, Taite BB, Keller BT, Tjoeng FS, Engleman VW, Giorgio TD, Feigen LP. SC-49992: a potent and specific inhibitor of platelet aggregation. Thromb Res. 1994;74:523-535. [Medline] [Order article via Infotrieve]
13. Zablocki JA, Miyano M, Garland B, Pireh D, Schretzman L, Bovy P, Rico J, Lindmark RJ, Tjoeng FS, McMackins DE, et al. Fibrinogen receptor antagonists: SAR of a new orally active series. 205th ACS Meeting, Denver, Medi-3, March 29, 1993.
14. Nicholson NS, Panzer-Knodle SG, Salyers AK, Taite BB, King LW, Miyano M, Gorczynski RJ, Williams MH, Zupec ME, Tjoeng FS, et al. In vitro and in vivo effects of a peptide mimetic (SC-47643) of RGD as an antiplatelet and antithrombotic agent. Thromb Res. 1991;62:567-578. [Medline] [Order article via Infotrieve]
15.
Lam SC, Plow EF, Ginsberg MH. Platelet membrane glycoprotein
IIb heavy chain forms a complex with glycoprotein IIIa that binds
Arg-Gly-Asp peptides. Blood. 1989;73:1513-1518.
16. Pytela R, Pierschbacher MD, Argraves S, Suzuki S, Rouslahti E. Arginine-glycine-aspartic acid adhesion receptors. Methods Enzymol. 1987;144:475-489. [Medline] [Order article via Infotrieve]
17. Yatohgo T, Izumi M, Kashiwagi H, Hayashi M. Novel purification of vitronectin from human plasma by heparin affinity chromatography. Cell Struct Funct. 1988;13:281-292. [Medline] [Order article via Infotrieve]
18.
Charo IF, Nannizzi L, Phillips DR, Hsu MA, Scarborough RM.
Inhibition of fibrinogen binding to GP IIb/IIIa by a GP IIIa peptide.
J Biol Chem. 1991;266:1415-1421.
19.
Marguerie GA, Plow EF, Edgington TS. Human platelets possess
an inducible and saturable receptor specific for fibrinogen. J
Biol Chem. 1979;254:5357-5363.
20.
Plow EF, Pierschbacher MD, Ruoslahti E, Marguerie GA, Ginsberg
MH. The effect of Arg-Gly-Asp-containing peptides on fibrinogen and von
Willebrand factor binding to platelets. Proc Natl Acad Sci
U S A. 1985;82:8057-8061.
21. Look DC, Rapp SR, Keller BT, Holtzman MJ. Selective induction of intercellular adhesion molecule-1 by interferon in human airway epithelial cells. Am J Physiol. 1992;263(Lung Cell Mol Physiol 7):179-187.
22.
Orlando RA, Cherish DA. Arginine-glycine-aspartic acid binding
leading to molecular stabilization between integrin
vß3 and its ligand. J Biol
Chem. 1991;266:19543-19550.
23.
Niiya K, Hodson E, Bader R, Byers-Ward V, Kozial JA, Plow EF,
Ruggeri ZM. Increased surface expression of the membrane glycoprotein
IIb/IIIa complex induced by platelet activation: relationships to the
binding of fibrinogen and platelet aggregation. Blood. 1987;70:475-483.
24. Jergens AE, Turrentine ME, Kraus KH, Johnson GS. Buccal mucosa bleeding of healthy dogs and of dogs in various pathologic states, including thrombocytopenia, uremia, and von Willebrand's disease. Am J Vet Res. 1987;48:1337-1342. [Medline] [Order article via Infotrieve]
25. Robertson T, Wright FT, Dykstra RL. Order Restricted Statistical Inference. New York: John Wiley & Sons, Ltd; 1988:180.
26. Vadas MA, Gamble JR, Smith WB. In: Harlan JM, Liu DY, eds. Adhesion: Its Role in Inflammatory Disease. New York: WH Freeman and Co; 1992:65-81.
27.
Szalony JA, Haas NF, Salyers AK, Taite BB, Mehrotra DV,
Feigen LP, Nicholson NS. Extended inhibition of platelet aggregation
with the orally active platelet inhibitor SC-54684A.
Circulation. 1995;91:411-416.
28.
Pytele R, Pierschbacher MS, Ginsberg MH, Plow EF, Ruoslahti E.
Platelet membrane glyoprotein IIb/IIIa: a member of a family of RGD
specific adhesion receptors. Science. 1986;231:1559-1562.
29. Gould RJ, Varret JS, Ellis JD, Holahan MA, Stranieri MT, Theoharides AD, Lynch JJ, Friedman RA, Duggan ME, Ihle NC, et al. Characterization of L-703,014, a novel fibrinogen receptor antagonist, following oral administration to dogs. Thromb Haemost. 1993;69:539. Abstract. [Medline] [Order article via Infotrieve]
30. Pike NB, Foster MR, Hornby EJ, Lumley P. Effect of the fibrinogen receptor antagonist GR144053 upon platelet aggregation ex vivo following intravenous and oral administration to the Marmoset and Cynomolgus monkey. Thromb Haemost. 1993;69:1071. Abstract.
31.
Conforti C, Dominguez-Jimonoz D, Zanetti A, Gimbrone MA,
Cramona O, Marchisio PC, Dejana E. Human endothelial cells express
integrin receptors on the luminal aspect of their membrane.
Blood. 1992;80:437-446.
32. Coller BS, Scudder LE, Beer J, Gold HK, Folts JD, Cavagnaro J, Jordan R, Wagner C, Iuliucci J, Knight D, et al. Monoclonal antibodies to platelet GPIIb/IIIa as antithrombotic agents. Ann N Y Acad Sci. 1991;614:193-213. [Medline] [Order article via Infotrieve]
33.
Lind SE. The bleeding time does not predict surgical bleeding.
Blood. 1991;77:2547-2552.
34. Collen D, Lu JR, Stassen J-M, Vreys I, Yasuda T, Bunting S, Gold HK. Antithrombotic effects and bleeding time for prolongation with synthetic platelet GPIIb/IIIa inhibitors in animal models of platelet-mediated thrombosis. Thromb Haemost. 1994;71:95-102. [Medline] [Order article via Infotrieve]
35.
Jang I-K, Gold HK, Ziskind AA, Fallon JT, Holt RE, Leinbach
RC, May JW, Collen D. Differential sensitivity of erythrocyte-rich and
platelet-rich arterial thrombi to lysis with recombinant tissue-type
plasminogen activator. Circulation. 1989;79:920-928.
36. Romson JL, Haack DW, Lucchesi BR. Electrical induction of coronary artery thrombosis in the ambulatory canine: a model for in vivo evaluation of anti-thrombotic agents. Thromb Res. 1980;17:841-853.[Medline] [Order article via Infotrieve]
This article has been cited by other articles:
 |
|
 |
|
  G. Thibault, P. Tardif, and G. Lapalme Comparative Specificity of Platelet {alpha}IIb{beta}3 Integrin Antagonists J. Pharmacol. Exp. Ther., March 1, 2001; 296(3): 690 - 696. [Abstract] [Full Text]
|
|
|
|
 |
|
 M. Quinn, A. Deering, M. Stewart, D. Cox, B. Foley, and D. Fitzgerald Quantifying GPIIb/IIIa Receptor Binding Using 2 Monoclonal Antibodies : Discriminating Abciximab and Small Molecular Weight Antagonists Circulation, May 4, 1999; 99(17): 2231 - 2238. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. G. Frederick, O. D. Suleymanov, J. A. Szalony, B. B. Taite, A. K. Salyers, L. W. King, L. P. Feigen, and N. S. Nicholson Protective Effect of Oral Xemilofiban in Arterial Thrombosis in Dogs : Increased Activity in Combination With Aspirin Circulation, August 25, 1998; 98(8): 813 - 820. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. H. Muller, H. Weisenberger, R. Brickl, H. Narjes, F. Himmelsbach, and J. Krause Profound and Sustained Inhibition of Platelet Aggregation by Fradafiban, a Nonpeptide Platelet Glycoprotein IIb/IIIa Antagonist, and Its Orally Active Prodrug, Lefradafiban, in Men Circulation, August 19, 1997; 96(4): 1130 - 1138. [Abstract] [Full Text]
|
|
|
|
 |
|
 T. Prueksaritanont, L. M. Gorham, M. J. Breslin, J. H. Hutchinson, G. D. Hartman, K. P. Vyas, and T. A. Baillie In Vitro and In Vivo Evaluations of the Metabolism, Pharmacokinetics, and Bioavailability of Ester Prodrugs of L-767,679, A Potent Fibrinogen Receptor Antagonist. An Approach for the Selection of a Prodrug Candidate Drug Metab. Dispos., August 1, 1997; 25(8): 978 - 984. [Abstract] [Full Text]
|
|
|
|
|
|
C. Simpfendorfer, K. Kottke-Marchant, M. Lowrie, R. J. Anders, D. M. Burns, D. P. Miller, C. S. Cove, A. C. DeFranco, S. G. Ellis, D. J. Moliterno, et al. First Chronic Platelet Glycoprotein IIb/IIIa Integrin Blockade : A Randomized, Placebo-Controlled Pilot Study of Xemilofiban in Unstable Angina With Percutaneous Coronary Interventions Circulation, July 1, 1997; 96(1): 76 - 81. [Abstract] [Full Text]
|
|
|
|
|
|
J. J. Cook, G. R. Sitko, M. A. Holahan, M. T. Stranieri, J. D. Glass, B. C. Askew, C. J. McIntyre, D. A. Claremon, J. J. Baldwin, G. D. Hartman, et al. Nonpeptide Glycoprotein IIb/IIIa Inhibitors. 15. Antithrombotic Efficacy of L-738,167, a Long-Acting GPIIb/IIIa Antagonist, Correlates with Inhibition of Adenosine Diphosphate-Induced Platelet Aggregation but not with Bleeding Time Prolongation J. Pharmacol. Exp. Ther., May 1, 1997; 281(2): 677 - 689. [Abstract] [Full Text]
|
|
|
|
|
|
S. A. Mousa, W. F. DeGrado, D.-X. Mu, R. P. Kapil, B. R. Lucchesi, and T. M. Reilly Oral Antiplatelet, Antithrombotic Efficacy of DMP 728, a Novel Platelet GPIIb/IIIa Antagonist Circulation, February 1, 1996; 93(3): 537 - 543. [Abstract] [Full Text]
|
|
|
|
|
|
L. G. Frederick, O. D. Suleymanov, L. W. King, A. K. Salyers, N. S. Nicholson, and L. P. Feigen The Protective Dose of the Potent GPIIb/IIIa Antagonist SC-54701A Is Reduced When Used in Combination With Aspirin and Heparin in a Canine Model of Coronary Artery Thrombosis Circulation, January 1, 1996; 93(1): 129 - 134. [Abstract] [Full Text]
|
|
|
|
|
|
J. A. Szalony, N. F. Haas, A. K. Salyers, B. B. Taite, N. S. Nicholson, D. V. Mehrotra, and L. P. Feigen Extended Inhibition of Platelet Aggregation With the Orally Active Platelet Inhibitor SC-54684A Circulation, January 15, 1995; 91(2): 411 - 416. [Abstract] [Full Text]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||