Introduction

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Short-term blockade of the platelet fibrinogen receptor glycoprotein (GP)IIb/IIIa reduces ischemic complications in patients presenting with acute coronary syndromes or undergoing percutaneous coronary intervention (The EPIC Investigators, 1994; The CAPTURE Investigators, 1997; The EPILOG Investigators, 1997; Brener et al., 1998; The EPISTENT Investigators, 1998; The PRISM Study Investigators, 1998; The PRISM-PLUS Study Investigators, 1998). Several oral GPIIb/IIIa antagonists have been developed and have been studied in a number of large-scale clinical trials (Zablocki et al., 1995; Weller et al., 1996; Muller et al., 1997). However, xemilofiban, an oral GPIIb/IIIa antagonist, showed no long-term clinical efficacy in patients after coronary angioplasty (O'Neill et al., 2000). Two other compounds, sibrafiban and orbofiban, increased mortality and the risk of myocardial infarction in patients with acute coronary syndromes (Cannon et al., 2000; The SYMPHONY Investigators, 2000).

As with i.v. GPIIb/IIIa antagonists, oral GPIIb/IIIa antagonists inhibit fibrinogen binding and platelet aggregation (Cannon et al., 1998; Kereiakes et al., 1998) and are effective in preventing vascular occlusion in a number of animal models of acute thrombosis (Frederick et al., 1998). However, the long-term effects of receptor occupancy on platelet function and receptor number are unknown. It is possible that the clinical effect of long-term receptor occupancy would be limited by an alteration in GPIIb/IIIa receptor number or function, similar to that seen with G-protein-coupled receptors. Here, we examine the effect of chronic GPIIb/IIIa receptor blockade with xemilofiban on receptor number and function when administered over 6 months. GPIIb/IIIa receptor number and occupancy were quantified using two antibodies, mAb1 (LYP18) (Boukerche et al., 1989) and mAb2 (4F8). mAb1 binds to a site also recognized by abciximab but is not displaced by xemilofiban. mAb2 binds to a site on GPIIIa remote from the ligand binding site and its binding is inhibited by several GPIIb/IIIa antagonists. Inhibition of mAb2 binding is noncompetitive and is due to a conformational change in the receptor. Thus, mAb1 binding provides an estimate of GPIIb/IIIa receptor number, whereas mAb2 binding provides evidence of receptor occupancy (Quinn et al., 1999).

	Experimental Procedures

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Materials. The anti-GPIIIa monoclonal antibodies mAb1, clone LYP18; mAb2, clone 4F8; and GPIIb/IIIa receptor occupancy kits, which contain mAb1, mAb2, isotypic control antibody, sheep anti-mouse horseradish peroxidase-linked secondary antibodies, and calibration beads, were provided by Dr. M. Canton (Biocytex, Marseille, France). ADP was purchased from Sigma Chemical Co. (St. Louis, MO).

Effects of Xemilofiban In Vitro. Blood from healthy donors, who had not taken aspirin or any other antiplatelet agent in the previous 7 days, was collected into sodium citrate 3.8%. SC-54701 (0.01-320 ng/ml), the active metabolite of xemilofiban, was incubated with the blood for 30 min at room temperature. Aliquots of each dilution were incubated with mAb1 or mAb2 (10 µg/ml final concentration) at room temperature for 20 min. Antibody binding was determined using fluorescein isothiocyanate-labeled F(ab)₂ fragments of sheep anti-mouse IgG antibodies. The samples were fixed with 1 ml of 1% formaldehyde after a 10-min incubation and analyzed by flow cytometry (FACScan; Becton Dickinson, Oxford, UK) at 488-nm excitation. Platelet populations were gated according to their forward and side light scatter. Histograms were generated using 10,000 events and geometric mean fluorescence was calculated using the CELLQUEST software of the FACScan system (Becton Dickinson). The binding of an isotypic control antibody was taken as nonspecific binding and was subtracted from the observed geometric mean fluorescence. Calibration beads with a known amount of antibody per bead were used to convert the observed mean fluorescence intensity of the samples to the number of GPIIb/IIIa receptors per platelet as previously described (Quinn et al., 1999).

Effects of Xemilofiban In Vivo. This was a substudy of the EXCITE (evaluation of oral xemilofiban in controlling thrombotic events) trial, a multinational double blind, randomized, controlled trial of the efficacy and safety of xemilofiban in 7232 patients undergoing percutaneous coronary intervention. Males and females (nonpregnant, nonlactating) between the age of 21 and 80 years with clinically significant coronary artery disease (stable or unstable angina or previous myocardial infarction) suitable for coronary intervention were eligible for recruitment.

Study Protocol. Thirty to 90 min before the revascularization procedure, patients were randomized in a double blind fashion to 20 mg of xemilofiban or matching placebo and a maintenance dose of 10 or 20 mg of xemilofiban or matching placebo three times daily for 6 months. Aspirin was administered at a dose of 80 to 325 mg daily. Coronary angioplasty was performed in the usual manner. Heparin was administered before and during the procedure to maintain the activated clotting time between 250 and 350 s. Ticlopidine (250 mg) twice daily was administered to stented patients randomized to placebo and a matching ticlopidine placebo was administered to stented patients on xemilofiban for 2 to 4 weeks after the procedure.

Blood Samples. Blood samples were collected from a peripheral vein into 3.8% sodium citrate at baseline; at 1 to 2 h and 4 to 7 h after the initial dose of medication on day 1; and before and 1 h after drug administration on the morning of day 60 ± 7 and day 180 ± 7. Sampling was also performed at 24 and 48 h after the last dose of medication.

Platelet Aggregation. Platelet aggregation studies were performed within 2 h of blood sampling. PRP was prepared as described above and the remaining plasma was centrifuged at 2500g for 5 min to obtain platelet-poor plasma. Platelet aggregation was determined 3 min after the addition of ADP (20 µM) to PRP at 37°C by light transmission (Biodata PAP-4; Biodata Corporation). The platelet aggregation at the different time points was expressed as a percentage of baseline platelet aggregation, before administration of the drug.

GPIIb/IIIa Receptor Number and Occupancy. GPIIb/IIIa receptor number and occupancy were quantified using the GPIIb/IIIa receptor occupancy kit (Biocytex, Marseille, France), which contains the anti-GPIIIa monoclonal antibodies mAb1 (LYP18) and mAb2 (4F8) and calibration beads. Analyses were performed within 6 h of blood collection. The 1 in 4 dilution was performed in platelet-poor plasma from the corresponding time point to avoid dilution of xemilofiban. The samples were analyzed by flow cytometry as described above.

Plasma Xemilofiban. Analyses of plasma levels of xemilofiban were performed using a fluorescence polarization immunoassay, with a limit of detection of 3.5 ng/ml (Clinical Pharmacokinetics Laboratory, Buffalo, NY).

Statistical Analysis. Continuous data are presented as mean ± S.E. Analyses between xemilofiban and placebo-treated groups were performed by analysis of variance using DataDesk 6.0. Variables were assessed for systematic departure from normality using normal probability plots. All variables had correlations of >0.950 with the expected normal distribution. If there was a significant difference between the placebo and active treatment group, a three-category variable (placebo, 10 mg and 20 mg tds) was used to do Scheffé post hoc tests for the effect of dose. Plasma levels of SC-54701 were correlated with the percentage of baseline platelet aggregation using Pearson's correlation coefficient. Aggregation was log transformed to linearize the dose response. Values <1% and >99% were changed to 1 and 99% for the analysis.

	Results

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Effects of Xemilofiban In Vitro

The effect of SC-54701, the active metabolite of xemilofiban, on mAb1 and mAb2 binding in whole blood and PRP was studied in vitro by flow cytometry. mAb2 binding was inhibited by SC-54701 in a concentration-dependent manner with an IC₅₀ of 0.5 ± 0.1 × 10⁸ M in whole blood and 0.8 ± 0.2 × 10⁸ M in PRP, P > .05. mAb1 binding was unaffected by xemilofiban. The inhibition of mAb2 binding in PRP correlated with SC-54701 inhibition of ADP (20 µM)-induced platelet aggregation, with a correlation coefficient of 0.9, P < .0001 (Fig. 1). However, the dose response for inhibition of aggregation was steeper than for inhibition of mAb2 binding, with maximum inhibition of ADP-induced platelet aggregation seen at a 60% reduction in mAb2.

View larger version (11K): [in this window] [in a new window]   Fig. 1.   A, effect of SC-54701, the active metabolite of xemilofiban, on mAb1 () and mAb2 () binding to platelets in whole blood, in vitro. SC-54701 reduces mAb2 binding in a concentration-dependent manner with an IC50 of 2.7 ± 0.5 ng/ml, whereas mAb1 binding is unaffected (n = 5). B, correlation between SC-54701 inhibition of mAb2 binding in PRP, expressed as a percentage of control and inhibition of ADP (20 µM)-induced aggregation in PRP as a percentage of control.

Effects of Xemilofiban In Vivo

Patient Population. Thirty-two patients were enrolled in the study between January and April 1998 and their baseline characteristics are shown in Table 1. Twelve patients were randomized to placebo, 10 patients to xemilofiban (10 mg tds), and 10 patients to xemilofiban (20 mg tds). Four patients randomized to xemilofiban (10 mg tds) and one patient randomized to xemilofiban (20 mg tds) were withdrawn before day 60. The reasons were bleeding in two patients (one gastrointestinal bleed and one recurrent nosebleeds), myocardial infarction in one patient, withdrawal of consent in one patient, and failure to undergo a percutaneous coronary intervention in one patient. Day 60 follow-up data were not available for three patients randomized to placebo; one patient was withdrawn before day 60 with a femoral artery pseudoaneurysm and two patients failed to attend for their day 60 follow-up. Three patients were withdrawn before their 6-month visit; two patients on placebo (both with cardiac endpoints) and one patient on xemilofiban (10 mg tds) because open-labeled ticlopidine had been added to their treatment after coronary stent insertion. Two patients on xemilofiban (20 mg tds) did not return for their washout blood tests and one patient on placebo did not return for the 48-h blood test.

mAb1 and mAb2 Binding. In the study population, baseline GPIIb/IIIa receptor number determined by mAb1 binding was similar in the different treatment groups with 54,162 ± 2,455 receptors per platelet in patients randomized to placebo, 51,027 ± 2,886 in patients randomized to xemilofiban (20 mg tds), and 53,359 ± 2,602 in patients randomized to xemilofiban (10 mg tds) (P = .7). GPIIb/IIIa receptor number did not change during treatment or after drug withdrawal (Fig. 2A). Forty-eight hours after drug discontinuation, mAb1 identified 53,521 ± 3,957 receptors in patients on placebo; 45,249 ± 6,208 in patients on xemilofiban (10 mg tds); and 51,876 ± 5,031 in patients on 20 mg tds, P = .4 (Fig. 2). There was no difference in the number of sites recognized by mAb2 at baseline in the different treatment groups, with 43,501 ± 1,823 sites per platelet in patients on placebo; 38,913 ± 1,981 sites in patients randomized to xemilofiban (20 mg tds); and 39,064 ± 2,924 in patients randomized to 10 mg tds (P = .3) (Fig. 2B). Four to 7 h after the initial dose of 20 mg of xemilofiban, mAb2 binding was significantly reduced to 13,026 ± 1,722 in patients randomized to xemilofiban (20 mg tds) and 10,229 ± 1,097 in patients randomized to xemilofiban (10 mg tds) (P < .0001). mAb2 binding remained inhibited during the treatment period and had not recovered before drug administration on day 60 or 180 (P < .0001). mAb2 binding recovered slowly after the last dose on day 180 but was still significantly inhibited 48 h after drug withdrawal, P = .004 (Fig. 2B). mAb2 binding did not change in placebo-treated patients during the course of the study.

View larger version (46K): [in this window] [in a new window]   Fig. 2.   A, GPIIb/IIIa receptor number quantified by mAb1 binding, in patients randomized to placebo (), xemilofiban (10 mg tds) (), or xemilofiban (20 mg tds) (). B, GPIIb/IIIa receptor occupancy, quantified by mAb2 binding, in patients randomized to placebo (), xemilofiban (10 mg tds) (), or xemilofiban (20 mg tds) (). **P < .005, ***P < .001.

Inhibition of Platelet Aggregation. Maximum inhibition of ADP (20 µM)-induced platelet aggregation was observed at 4 to 7 h after the first dose of study medication, with aggregation at 86 ± 7% of baseline in patients on placebo, 4 ± 3% of baseline in patients on 10 mg tds, and 4 ± 2% in patients on 20 mg tds (P < .00001). Trough levels of ADP-induced platelet aggregation, before drug administration on day 60, were similar in the two treatment groups at 67 ± 14% of baseline in patients on 10 mg tds and 55 ± 14% of baseline in patients on 20 mg tds. However, trough levels on day 180 showed less inhibition at 76 ± 10 and 71 ± 12% of baseline, respectively (Fig. 3). One hour after drug administration on day 60, platelet aggregation was 55 ± 14 and 26 ± 10% of baseline on 10 and 20 mg tds xemilofiban, respectively. One hour after drug administration on day 80, platelet aggregation was 55 ± 13 and 42 ± 14% of baseline on 10 and 20 mg tds xemilofiban. Platelet aggregation had recovered within 24 h of drug discontinuation and although there was a trend toward increased platelet aggregation at 24 and 48 h after drug withdrawal in the active treatment groups, this did not reach statistical significance (P = .123).

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Ex vivo inhibition of platelet aggregation pre drug and 1 h post drug on day 1, 60, and 180, and 24 and 48 h after the last dose of medication in patients randomized to placebo (), xemilofiban (10 mg tds) (), or xemilofiban (20 mg tds) (). Results are expressed as a percentage of baseline aggregation. *P < .05, **P < .005.

Xemilofiban Plasma Levels. The plasma concentration of SC-54701, the active metabolite of xemilofiban, was assayed in 21 patients before and 1 h after drug administration on day 180. Twelve of the patients assayed were on active drug (eight patients randomized to 20 mg tds and four patients randomized to 10 mg tds) (Table 2). Plasma concentration of SC-54701 correlated with the corresponding platelet aggregation expressed as a percentage of baseline (r² = 0.77, P < .001) (Fig. 4), demonstrating that the metabolite SC-54701 was largely responsible for the platelet inhibitory effect of xemilofiban.

View larger version (11K): [in this window] [in a new window]   Fig. 4.   Correlation between platelet aggregation expressed as a percentage of baseline and plasma SC54701, the active metabolite of xemilofiban, measured pre and 1 h post the last dose of xemilofiban on day 180 (n = 12) and 24 and 48 h later (n = 9).

	Discussion

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Oral GPIIb/IIIa antagonists have failed to prevent coronary events in patients with acute coronary syndromes (Cannon et al., 2000; The SYMPHONY Investigators, 2000) and after coronary angioplasty (O'Neill et al., 2000), although short-term administration of i.v. agents has proved effective. One explanation is that the degree of receptor occupancy and inhibition of platelet aggregation achieved with oral dosing is inadequate. Although the goal with i.v. agents is to achieve a consistent >80% receptor occupancy and inhibition of platelet aggregation, this is not possible with oral therapy given the intermittent mode of administration. In this study, xemilofiban inhibited ADP (20 µM)-induced platelet aggregation by greater than 95% 4 to 7 h after the first dose of xemilofiban on day 1. However, with long-term therapy, trough levels were considerably lower at 30 to 45% inhibition, similar to previous reports (Kereiakes et al., 1998). The use of citrate anticoagulation may have overestimated the degree of platelet inhibition because low calcium concentrations increase the binding of certain GPIIb/IIIa antagonists (Phillips et al., 1997). However, the IC₅₀ for inhibition of platelet aggregation by xemilofiban is reduced by only 30% in citrated versus D-phenylalanyl-N-[4-[(aminoiminomethyl)amino]-1-(chloroacetyl)butyl]-L-prolinamide (PPACK)-treated platelet-rich plasma (IC₅₀ 4 × 10⁸ versus 6 × 10⁸ M, respectively; M. J. Quinn and D. J. Fitzgerald, unpublished data). This is a minor change compared with the change in IC₅₀ seen with eptifibatide (140-570 nM). Thus, the levels of platelet inhibition and receptor occupancy achieved in this study were well below the levels of inhibition (>80%) required to prevent complications at the time of coronary intervention (Coller, 1998). The low level of platelet inhibition may explain in part the lack of clinical benefit during long-term administration of xemilofiban.

A second issue with GPIIb/IIIa antagonists is that they may act as partial agonists (Cox et al., 2000) and so long-term administration may influence receptor number or limit the degree of platelet inhibition. GPIIb/IIIa antagonists are largely designed to mimic receptor recognition sites in adhesion proteins, particularly the arginine-glycine-aspartate sequence (Zablocki et al., 1995). Many antagonists induce an active conformation in the receptor, that is, one capable of binding fibrinogen (Peter et al., 1998). GPIIb/IIIa antagonists also induce the appearance of previously hidden regions, so called ligand-induced binding sites (Kouns et al., 1990; Honda et al., 1995). The functional relevance of these newly exposed regions is unclear. However, there is evidence that GPIIb/IIIa antagonists that induce ligand-induced binding sites provoke platelet activation, including Ca²⁺ transients and thromboxane formation and under some circumstances, platelet aggregation (Honda et al., 1998; Peter et al., 1998).

In addition to inducing the appearance of previously hidden epitopes, ligands may also induce the disappearance of an epitope, referred to as ligand-attenuated binding sites. Ligand attenuation of antibody binding has also been described for the fibronectin receptor (Mould et al., 1996). The mAb2 epitope on GPIIIa behaves as a ligand-attenuated binding site in that the mAb2 site is remote from the ligand binding site (Quinn et al., 1999). Recent experiments suggest that mAb2 binding reflects an active process. Thus, ligand-induced disappearance of the mAb2 epitope is not seen in purified GPIIb/IIIa or GPIIb/IIIa expressed in HEK cells and is prevented by the platelet inhibitor prostaglandin E₁ (M. J. Quinn and D. J. Fitzgerald, unpublished observations). Thus, in addition to reporting on receptor occupancy, the loss of the mAb2 epitope may signify partial agonist activity. Xemilofiban prevented mAb2 binding both in vitro and in vivo, suggesting that it possesses some partial agonist activity.

However, there was no evidence that this resulted in enhanced platelet aggregation, for example, during drug withdrawal when plasma levels fell. This contrasts with the findings of Peter et al. (1998) suggesting that at low levels of GPIIb/IIIa antagonism platelet aggregation is paradoxically enhanced. (It is worth noting that mAb2 detected levels of receptor occupancy below the threshold for inhibition of aggregation and was a far more sensitive measure of the presence of the drug.) There was also no change in GPIIb/IIIa receptor number, measured as mAb1 binding and no increase in receptor number during drug withdrawal.

The recent results of the EXCITE data suggest that oral GPIIb/IIIa antagonists are ineffective in preventing coronary events. Indeed, the OPUS trial (Cannon et al., 2000) and the unpublished SYMPHONY 2 have shown an increase in myocardial infarction and death, suggesting that the compounds act as partial agonists (Holmes et al., 2000). Our findings suggest that the degree of inhibition of platelet aggregation with xemilofiban in EXCITE was far less than achieved with i.v. agents and this may explain the lack of efficacy. Although xemilofiban induced a conformational change in the receptor, suggestive of a partial agonist effect, this did not translate into altered receptor number or enhanced platelet aggregation at low levels of receptor occupancy.