Tirofiban (Aggrastat )

Transcription

1 Cardiovascular Drug Reviews Vol. 17, No. 3, pp Neva Press, Branford, Connecticut Tirofiban (Aggrastat ) Jacquelynn J. Cook, Bohumil Bednar, Joseph J. Lynch, Jr., Robert J. Gould, Melissa S. Egbertson, Wasyl Halczenko, Mark E. Duggan, George D. Hartman, Man-Wai Lo, Gail M. Murphy, Lawrence I. Deckelbaum, Frederick L. Sax, and Eliav Barr Merck Research Laboratories, West Point, PA, USA Key Words: Antiplatelet Coronary artery syndromes Non-Q-wave myocardial infarction Platelet glycoprotein (GP) IIb/IIIa antagonist Unstable angina INTRODUCTION An evolving understanding of the biochemical mechanisms involved in platelet activation and aggregation has led to an appreciation that the binding of the platelet surface glycoprotein (GP) IIb/IIIa receptor ( IIb 3 integrin) to fibrinogen is a prerequisite, pivotal event in thrombus formation regardless of aggregatory stimulus (12,20,48,58). This has led to the design, synthesis, and clinical assessment of GPIIb/IIIa antagonists, which bind to the platelet GPIIb/IIIa receptor and thereby prevent its binding to fibrinogen, as potential antithrombotic interventions (1,35). Tirofiban (Aggrastat, MK-0383; L-700,462) is a parenteral, highly specific, and potent GPIIb/IIIa antagonist that was approved for patient use in 1998 for the treatment of unstable angina and non-q-wave myocardial infarction, with clinical studies continuing to explore expanded therapeutic indications. CHEMISTRY The medicinal design of tirofiban was based on the observation that the Arg Gly Asp (RGD) triad constitutes a specific recognition element in the binding of fibrinogen to GPIIb/IIIa receptors (12,48,58). Therefore, we sought a potent, small molecule mimic of the RGD sequence. Recognizing that the separation distance between the positively charged argininyl and the negatively charged aspartyl centers of RGD was a key determinant of potency, we carried out a directed search of the Merck Sample Collection. Using defined distance and atom requirements and eliminating peptide structures, we uncovered a tyrosine analog (Fig. 1, Lead Structure 1) as a structurally novel, moderately potent lead structure (IC mol for inhibition of in vitro platelet aggregation). Optimization at the N-terminus revealed that the 4-(4-piperidinyl)butyl linkage could Address correspondence and reprint requests to Jacquelynn J. Cook, WP46-300, Merck Research Laboratories, West Point, PA, USA FAX: (215) ; jacquelynn-cook@merck.com. 199

2 200 J. J. COOK ET AL. FIG. 1. Chemical structures of Lead Structure 1 and tirofiban. replace that of Lead Structure 1 with simplification of molecular structure via removal of a chiral center and only modest increase in molecular weight. At the C-terminus, the (S)-butylsulfonylamino group was the optimal replacement affording both potency and appropriate physical properties (29). Overall, optimization at both termini resulted in a 3000-fold increase in potency for inhibition of platelet aggregation with maintenance of selectivity for GPIIb/IIIa over closely related integrins (18). The dramatic increase in potency associated with the (S)-butylsulfonylamino substituent was without precedent in the peptide literature and suggested a novel exosite interaction at the receptor. IN VITRO PHARMACOLOGY Historically, inhibition of platelet aggregation has been used to assess potency in the design of GPIIb/IIIa antagonists. However, inhibition of platelet aggregation does not reflect the affinity of GPIIb/IIIa antagonists for receptors on resting platelets. Further, inhibition of platelet aggregation does not provide a true measure of the affinity for highly potent compounds, since the observed IC 50 can never be less than 50% of the concentration of receptors used in the assay (8). To measure the affinity of GPIIb/IIIa antagonists for resting and activated forms of the receptor, we isolated both forms of the receptor from human platelets and developed several assays using these receptors (8). Displacement by tirofiban of the fluorescent GPIIb/IIIa antagonist L-736,622 (17) from resting and activated forms of the receptor solubilized in Triton-X-100 micelles provided a sixfold higher K d value for the resting receptor than for the activated form (Table 1). Equilibrium binding measurements with radiolabeled tirofiban and competition measurements with the fluorescent GPIIb/IIIa antagonist L-762,745 (7,17) on resting human platelets confirmed that affinity measurements using purified, micelle-solubilized receptors provided correct values of the affinity for the receptors on human platelets (Table 1). Competition measurements between unlabeled and radiolabeled tirofiban (Fig. 2) provided the dissociation rate constant from the receptors on resting platelets (Table 1). Using the measured equilibrium dissociation constant, K d, of 15 nmol and dissociation rate constant, k off, of s 1,we calculated an association rate constant, k on,of (k on k off /K d ). The value of k off indicates that the dissociation of tirofiban from receptors is a relatively fast process with t 1/2 11 s, while the binding is near diffusion controlled (see k on in Table 1). The determination of binding constants on activated platelets was impaired by the transient character of the activation process that prevents the achievement of equilibrium under the same conditions. Since the RGD sequence is utilized by several members of the integrin family to modulate cellular adhesion and migration, the determination of the specificity of tirofiban

3 TIROFIBAN 201 TABLE 1. Binding parameters of tirofiban in binding to purified human GPIIb/IIIa and receptors on human platelets Purified GPIIb/IIIa receptors Activated form Resting form K d (nmol) 1.9 ± ± 4 Resting human platelets (method) [ 3 H]-L-700,462 a L-762,745 K d (nmol) 15 ± 2 b 15 ± 3 k off (s 1 ) (t 1/2 11 s) k on (nmol 1 s 1 ) c a L-700,462, tirofiban. b Data are mean ± S.D. c Calculated using K d of 15 nmol and k off of s 1 (k on k off /K d ). in binding to GPIIb/IIIa was of importance. An interaction of tirofiban with other members of the integrin family, determined by measuring the IC 50 for inhibition of attachment of human umbilical vein endothelial cells (HUVEC) to fibrinogen, vitronectin, and fibronectin, provided IC 50 values >100 mol, indicating a high selectivity of tirofiban for GPIIb/IIIa (18). The binding of ligands to activated and unactivated GPIIb/IIIa ( IIb 3 integrin) induces conformational changes in the extracellular domains of the receptor that can be detected by antibodies. The epitopes recognized by these antibodies have been termed ligandinduced binding sites (LIBS) and are present on both the IIb and 3 subunits (21,22,32, 36). Although the physiological significance of LIBS expression is not known, neoepitopes of IIb 3 have been speculated to play a role in platelet aggregation (32), clot retraction (21), cellular adhesion (21,36), and drug-induced thrombocytopenia (6,15). The induction of LIBS on IIb 3 can be detected with anti-libs antibodies that bind preferentially to occupied forms of the receptors. We developed three monoclonal antibodies, mab3-758, mab10-758, and mab15-758, that recognize epitopes on the 3 subunit of the integrin IIb 3. Two of these antibodies, mab and mab15-758, are anti-libs antibodies that preferentially bind to neoepitopes exposed on IIb 3 upon ligand binding, while the binding of mab3-758 to the GPIIb/IIIa is not affected by RGD ligands (39). As depicted in Fig. 3, tirofiban induced such epitopes upon binding to GPIIb/IIIa receptors on platelets as indicated by an increase in the relative fluorescence for antibodies mab and mab15-758, while the binding of the control antibody mab3-758 was not affected. It is important to stress that all GPIIb/IIIa antagonists tested thus far, including integrilin, orbofiban, and lamifiban, induce LIBS recognized by mab and mab15-758, although the affinity of mab for induced epitopes varies for different antagonists, indicating subtle differences in the structure of induced epitopes. The effects of tirofiban on platelet aggregation versus secretion have been assessed in vitro in human platelet-rich plasma (PRP) stimulated with ADP or thrombin receptoractivating peptide (TRAP) (45). Tirofiban inhibited platelet aggregation and ATP secretion from dense granules in response to both agonists. However, the differential magnitude

4 202 J. J. COOK ET AL. FIG. 2. Dissociation rate constant for tirofiban from resting human gel-filtered platelets (GFP). GFP ( platelets/ml) were incubated with 6 nmol of radiolabeled tirofiban ([ 3 H]L-700,462) and allowed to equilibrate. At zero time, unlabeled tirofiban (1 mmol) was added, and at the indicated times platelets were centrifuged for 10 s at 12,000 rpm. An aliquot of the supernatant was immediately removed and the radioactivity was counted. The Free CPM as a function of time were fit to a single exponential to determine the dissociation rate constant s 1. of effect on aggregation versus ATP release varied with the agonist tested, suggesting that the extent of inhibition of secretion is dependent in part on the platelet stimulus (45). Recently Pedicord et al. (46) reported inhibition of platelet procoagulant activity by the cyclic peptide GPIIb/IIIa antagonist DMP728 and confirmed a similar effect for the anti-gpiib/iiia antibody c7e3 (abciximab, ReoPro ) reported initially by Reverter et al. (50). Using an assay for the measurement of thrombin generation in human PRP in vitro, we found, upon activation by tissue factor (similar to the assay used by Reverter et al. (50)), that tirofiban inhibited thrombin generation in a concentration-dependent manner, reaching a maximum inhibition of 35% to 40% when all GPIIb/IIIa receptors were occupied by antagonist. Similarly, inhibition of thrombin generation has been reported ex vivo in PRP obtained from patients receiving tirofiban (34). Therefore, the inhibition of thrombin generation may contribute to the overall antithrombotic effects of tirofiban. The effect of tirofiban on neutrophil-platelet adhesion also has been assessed ex vivo in citrated whole blood obtained from patients receiving tirofiban (61). Tirofiban reduced the number of platelets bound per neutrophil both in the basal state and following stimulation with the agonists ADP, TRAP, and ADP plus epinephrine. Such an inhibition of plateletneutrophil binding may prevent mass accumulation of platelets and/or neutrophils on injured endothelium, thereby contributing to the antithrombotic effect of tirofiban (61).

5 TIROFIBAN 203 FIG. 3. The induction of neoepitopes on resting human gel-filtered platelets (GFP) by tirofiban as measured by monoclonal antibodies mab3-758, mab10-758, and mab upon ligand binding. GFP ( platelets/ ml) were reacted with 100 nmol of antibody in the presence or absence of 1 M tirofiban. After washing and resuspension in flow cytometric buffer containing 1:100 fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulin G, fluorescence was measured in a flow cytometer. The fluorescence ratio represents the mean relative fluorescence of secondary antibody to the control (resting platelets) for six platelet donors (mean ± S.E.M.). Consistent with fibrinogen binding to platelet GPIIb/IIIa constituting a common pivotal event in platelet aggregation, tirofiban has been shown to inhibit aggregation of in vitro human gel-filtered platelets (GFP) induced by ADP, collagen, -thrombin, and the thromboxane analog, U46619, with IC 50 s ranging from 12 to 31 nmol (26). Likewise, tirofiban inhibited human PRP platelet aggregation induced by ADP, collagen, -thrombin, U46619, epinephrine, and arachidonic acid with IC 50 s ranging from 31 to 66 nmol (26,47). The antiaggregatory activity of tirofiban has been compared across several mammalian species by assaying inhibition of ADP-induced platelet aggregation in vitro in whole blood. The IC 50 s of tirofiban for inhibition of platelet aggregation in human, rhesus monkey, and dog whole blood were 81 ± 13 (S.E.M.) nmol, 140 ± 25 nmol, and 353 ± 48 nmol, respectively. Tirofiban was less potent in inhibiting aggregation in guinea pig, minipig, rabbit, and hamster, with IC 50 s 1 mol (14). The potential for tirofiban to elicit nonspecific vasorelaxant effects has been assessed in vitro in precontracted canine coronary rings. Tirofiban at concentrations up to 1 mmol did not alter tension of PGF 2 - precontracted coronary rings, indicating no intrinsic vasorelaxant activity. IN VIVO PRECLINICAL PHARMACOLOGY The preclinical in vivo pharmacology of tirofiban has been characterized most extensively in the dog. Bolus i.v. administration of tirofiban (1 mg/kg) in dogs resulted in

6 204 J. J. COOK ET AL. short-duration, significant inhibition of ex vivo ADP-induced platelet aggregation in PRP and prolongation of template bleeding time (14). Continuous i.v. infusions of 0.1 to 10 g of tirofiban per kg per min dose dependently inhibited ex vivo platelet aggregation in PRP induced by ADP (Fig. 4) and collagen and dose dependently elevated template bleeding times. Platelet function and bleeding times normalized rapidly after termination of tirofiban infusion, indicating a short functional half-life and no protracted effect on platelet sensitivity to agonists (37). The relationship between template bleeding time prolongation and degree of inhibition of platelet aggregation response to ADP was assessed using sequential acute i.v. doses of 10 to 500 g of tirofiban per kg in dogs. These studies demonstrated that the platelet response to ADP could be inhibited completely prior to a significant extension in bleeding time (Fig. 5) (37). The antithrombotic efficacy of tirofiban alone and in combination with heparin has been assessed in several canine experimental thrombosis preparations. In a canine model of platelet-dependent cyclic flow reductions in the injured, stenosed coronary artery, the bolus i.v. administration of 0.3 and 1 mg of tirofiban per kg alone completely inhibited coronary artery cyclic flow reductions (Fig. 6) (37). In a canine model of coronary artery electrolytic intimal injury leading to occlusive thrombus formation, the i.v. infusions of 3 FIG. 4. Inhibition of ex vivo platelet aggregation response to ADP (10 mol plus 1 M epinephrine; percentage of inhibition of baseline response) during and after 360-min continuous i.v. infusions of 0.1 to 10 g of tirofiban per kg per min in anesthetized dogs. Data are the mean ± S.E.M. with n 4to5.

7 TIROFIBAN 205 FIG. 5. Relation between inhibition of ex vivo platelet aggregation response to ADP (10 mol plus 1 M epinephrine; percentage of inhibition of baseline response) and template bleeding time following increasing sequential i.v. bolus doses of 10, 30, 50, 80, 100, 300, and 500 g of tirofiban per kg in anesthetized dogs. Data are the mean ± S.E.M. with n 4to5. to 10 g of tirofiban per kg per min alone reduced the incidence of occlusive thrombosis, significantly delayed thrombus formation and reduced thrombus mass, and preserved coronary blood flow (37,60). Administered as an adjunct to tissue-derived plasminogen activator (tpa) or streptokinase on a background of heparin for lysis of electrolytic injury-induced coronary artery occlusive thrombus in the dog, 3 and 10 g of tirofiban per kg per min increased the incidence of and reduced the time to thrombolysis and reduced the incidence of post-lysis thrombotic reocclusion during continued tirofiban infusion FIG. 6. Inhibition of left circumflex coronary artery cyclic flow reductions induced by coronary artery injury and stenosis following the bolus i.v. administration of 300 g of tirofiban per kg in anesthetized dogs.

8 206 J. J. COOK ET AL. (37). In the latter canine thrombolysis model, the efficacy of tirofiban as an adjunct to t-pa or streptokinase was reduced in the absence versus the presence of background heparin (37). Hence, multiple studies in canine experimental thrombosis models demonstrate antithrombotic efficacy with tirofiban in both the absence and the presence of heparin, with the potential for enhanced efficacy with background heparin dependent on the model and presumably the type of thrombus produced in the model. The in vivo activity of tirofiban also has been assessed in the baboon. In baboons, i.v. infusion doses similar to those used in dogs dose dependently inhibited ex vivo platelet aggregation responses in PRP to ADP and collagen and elevated template bleeding time (28). In a baboon preparation instrumented with an arteriovenous shunt containing a thrombogenic Dacron graft, continuous i.v. infusions of 0.3 to 3 g of tirofiban per kg per min reduced platelet deposition onto and preserved blood flow through the graft, whereas aspirin displayed no significant activity (27). In baboons placed on cardiopulmonary bypass, a procedure characterized by significant platelet activation, altered function, and loss with concomitant elevation in template bleeding time, the continuous i.v. infusion of 0.3 g of tirofiban per kg per min preserved platelet number, improved platelet function during bypass, and significantly accelerated the normalization of bleeding times after bypass (30). Also in the baboon cardiopulmonary bypass preparation, tirofiban (0.1 and 0.3 g/kg/min) i.v. infusion inhibited thrombin generation as measured by assay of plasma prothrombin fragment F1.2 and thrombin-antithrombin (TAT) complexes. The latter observation indicates that tirofiban inhibited not only platelet aggregation but also the platelet role in coagulation mechanisms leading to prothrombin activation during cardiopulmonary bypass (49). Finally, in vivo in guinea pigs, 1 and 3 mg of bolus tirofiban per kg i.v. transiently elevated mesenteric artery bleeding time and significantly inhibited ex vivo ADP-induced platelet aggregation in PRP (14). Tirofiban also has been reported to delay carotid artery thrombotic occlusion induced by photochemical vessel injury and to prolong ear incision bleeding time in guinea pigs at an i.v. dose that completely inhibited ex vivo collageninduced platelet aggregation in PRP (31). The clinical development and use of a novel antithrombotic agent necessarily involve comparison to and coadministration with conventional as well as newly approved antiplatelet and anticoagulant agents. As a consequence, potential interactions among mechanistically diverse antithrombotic interventions are a concern. Accordingly, a series of preclinical studies were conducted to assess the effects of individual versus combined administrations of tirofiban, unfractionated heparin, aspirin, ticlopidine, and low molecular weight heparin (Enoxaparin ). In an initial study of potential pharmacodynamic and pharmacokinetic interactions among tirofiban and standard antithrombotic therapies, anesthetized dogs were randomized to receive intravenous treatments (single, double, or triple treatments) from a combination of tirofiban (1 g/kg/min for 120 min), unfractionated heparin (100 units/kg bolus plus 1 unit/kg/min for 120 min), and aspirin (5 mg/kg bolus). No combination of tirofiban, unfractionated heparin, or aspirin altered the pharmacokinetic profile of tirofiban nor did any combination affect the anticoagulant activity of heparin or the antiplatelet effects of aspirin. Additionally, no combination of these three agents altered platelet count. However, combination therapies did display interactive effects on platelet function and template bleeding time. The addition of aspirin to tirofiban increased the inhibition of ex

9 TIROFIBAN 207 vivo arachidonic acid-induced but not ADP-induced platelet aggregation in PRP elicited by tirofiban alone. Inhibition of ex vivo collagen-induced platelet aggregation in PRP was greater in the combined tirofiban plus aspirin group compared with either agent alone. The greatest effect of combination therapies was on template bleeding time. Since the cessation of bleeding following incision by the type of device used for template bleeding time measurement involves the formation of a platelet-rich hemostatic plug as well as initiation of coagulation, it was not unexpected that tirofiban, aspirin, and unfractionated heparin could all contribute to prolongation of bleeding time (Fig. 7). Template bleeding time prolongation with the triple combination of tirofiban plus aspirin plus heparin was greater than with all other combinations of single and double treatments. Additionally, bleeding time prolongation in the combined tirofiban plus heparin group was greater than those observed in either the tirofiban or heparin alone groups, and bleeding time prolongation with combined tirofiban plus aspirin was greater than that observed with aspirin alone but not tirofiban alone. A second interaction study in dogs has compared individual versus combined treatments with tirofiban (1 g/kg/min i.v. for 120 min) and ticlopidine (20 mg/kg/day p.o. for 4 days). The combined administration of tirofiban and ticlopidine did not alter the pharmacokinetic profile of tirofiban nor did the combined administration of these agents alter FIG. 7. Maximal elevation in template bleeding time (fold increase over baseline value) following intravenous treatments (single, double, or triple treatments) from a combination of tirofiban (TIR; 1 g/kg/min for 120 min), unfractionated heparin (HEP; 100 units/kg bolus plus 1 unit/kg/min for 120 min), and aspirin (ASA; 5 mg/kg bolus) in anesthetized dogs. Data are the mean ± S.E.M. with n 5to6.

10 208 J. J. COOK ET AL. platelet count. Combined administration of tirofiban plus ticlopidine did display an additive effect on inhibition of ex vivo ADP-induced platelet aggregation in PRP, and combined treatment with these two agents resulted in a bleeding time prolongation that was greater than that produced by ticlopidine alone but only slightly and not significantly greater than that produced by tirofiban alone. Low molecular weight heparins recently have been demonstrated effective, have been approved, and are rapidly gaining acceptance for use in venous (44,59) and coronary arterial (13) thrombotic disorders. Accordingly, an interaction study in dogs has compared intravenous treatments (single, double, or triple treatments) from a combination of tirofiban (1 g/kg/min for 120 min), low molecular weight heparin (three dosing levels: 75 g/kg plus 2 g/kg/min; 100 g/kg plus 3 g/kg/min; 700 g/kg plus 7 g/kg/min; all for 120 min), and aspirin (5 mg/kg bolus). No combination of these three agents altered the pharmacokinetic profile of tirofiban nor did any combination alter platelet count. Low molecular weight heparin did not alter inhibition of ex vivo ADP-induced platelet aggregation in PRP by tirofiban nor did tirofiban alter the anticoagulant activity of low molecular weight heparin. Template bleeding time prolongation with the triple combination of tirofiban plus aspirin plus low molecular weight heparin (highest dose level) was not significantly different from that produced by tirofiban plus aspirin, but it was greater than those elicited by both tirofiban plus low molecular weight heparin and aspirin plus low molecular weight heparin. Additionally, bleeding time prolongation with tirofiban plus low molecular weight heparin was not significantly different from that elicited by tirofiban alone. Therefore, the addition of low molecular weight heparin to tirofiban plus aspirin did not further extend template bleeding time, as had been observed with unfractionated heparin in the earlier interaction study. These findings demonstrate the feasibility of combined administration of tirofiban with aspirin, ticlopidine, unfractionated heparin, and low molecular weight heparin, but they also demonstrate the need for careful dose selection and patient monitoring when coadministering mechanistically diverse antithrombotic agents. CLINICAL DEVELOPMENT The acute coronary syndromes of unstable angina/non-q-wave myocardial infarction (UAP/NQWMI) share a common underlying pathophysiology: disruption of atherosclerotic plaque in a coronary artery (3,11,23,24,51). Plaque rupture results in exposure of thrombogenic material to circulating blood, leading to clot formation with partial or complete occlusion of coronary blood flow and myocardial ischemia or infarction. Similarly, balloon inflation during percutaneous transluminal coronary angioplasty (PTCA) results in plaque rupture that can lead to clot formation and acute closure of the coronary artery. Regardless of whether the plaque rupture occurs spontaneously or iatrogenically, the severity of the resultant clinical syndrome is dependent to a large extent on the ensuing nascent coronary thrombus. Platelet activation, adhesion, and aggregation represent the critical initiating steps in the formation of the arterial thrombus overlying the disrupted arterial plaque. Thus, effective antithrombotic therapy and, in particular, pharmacological antagonism of platelet function have been central to the management of patients presenting with acute coronary syndromes and patients undergoing PTCA (3,4,10,16,25,57). Tirofiban was developed as a potent inhibitor of platelet aggregation to prevent the acute cardiac ischemic events associated with coronary thrombus formation in these clinical settings.

11 TIROFIBAN 209 Clinical Pharmacology Any new antithrombotic drug for use in patients presenting with UAP/NQWMI must fit into the clinical paradigm used to manage these patients. In general, patients presenting with UAP/NQWMI are stabilized with medical therapy for a variable period of time (hours to days) followed, in most instances, by angiography and revascularization with percutaneous interventions (PCIs) or coronary artery bypass graft (CABG) surgery. Patients who remain unstable may require urgent intervention (including CABG surgery). Because the revascularization strategy chosen for the management of a particular patient is not defined until angiography, it was important to develop a drug that would provide rapid onset inhibition of platelet aggregation, but that would also have a short half-life, so that the potent antithrombotic effect could be reversed in the event of a requirement for CABG surgery. The pharmacokinetic and pharmacodynamic profiles of tirofiban are highly consistent with this desired profile. Clinical pharmacokinetic and pharmacodynamic (PK/PD) studies (5,42,43,47) included those performed to address distribution, metabolism, and excretion of tirofiban in healthy subjects and in coronary artery disease patients; the effect of age, gender, and renal and hepatic insufficiency on tirofiban pharmacokinetics; and drug-drug interactions. In the Phase I studies, a total of 84 healthy volunteers, 12 hepatic insufficiency patients, and 22 renal insufficiency patients received tirofiban dosage levels ranging from 0.05 to 0.4 g/kg/min. An additional 12 patients with stable coronary artery disease received tirofiban as a 0.3- g/kg/min loading infusion over 30 min followed by a g/kg/min maintenance infusion. Potential drug interactions, pharmacokinetic analyses, and the effect of renal function were also examined in a subset of UAP/NQWMI patients (762 patients total for clearance studies) in the Platelet Receptor Inhibition in Ischemic Syndrome Management (PRISM) trial. In healthy subjects, the mean plasma clearance of tirofiban determined in various states ranges from 213 to 314 ml/min. Half-life ranges from 1.4 to 1.8 h. In coronary artery disease patients, the plasma clearance of tirofiban ranges from 152 to 267 ml/min. Renal clearance accounts for approximately 40% of plasma clearance. Half-life ranges from 1.9 to 2.2 h. Plasma clearance appears to be slightly lower than in healthy subjects, possibly because of age-related reduction in renal function. Plasma clearance of tirofiban in coronary artery disease patients is similar in males and in females. Both urinary and biliary excretion contribute significantly to the elimination of tirofiban. In patients with mild-tomoderate hepatic insufficiency (42), plasma clearance of tirofiban is not significantly different compared with that in healthy subjects. Plasma clearance of tirofiban was lower to a clinically significant extent ( 50%) in patients with creatinine clearance <30 ml/min, including those requiring hemodialysis (43). In patients with severe renal insufficiency (creatinine clearance, <30 ml/min), 50% dose reduction of tirofiban is warranted. Tirofiban is removed by hemodialysis. In the PRISM study (53), there did not appear to be clinically significant pharmacokinetic interactions with many commonly prescribed antianginal and antihypertensive drugs. Phase II Studies Three dose-finding Phase II studies (33,40,41) provided pharmacokinetic and pharmacodynamic information on tirofiban alone or in combination with heparin in 164 patients

12 210 J. J. COOK ET AL. with unstable coronary artery disease. Two of the studies randomized patients with UAP/ NQWMI; the third study was in high risk patients undergoing PTCA or atherectomy either in the setting of acute coronary syndromes or complex coronary artery lesion morphology. In these studies, tirofiban was administered either as a loading infusion given over 30 min followed by a maintenance infusion or as a bolus dose given over 5 min followed by a maintenance infusion. The maintenance doses ranged from 0.05 to 0.15 g/kg/min. Tirofiban was dosed either with or without concomitant heparin. The primary goal of these studies was to identify a dose regimen that results in high level inhibition of ADP-induced platelet aggregation (IPA) in most patients without excessive prolongation in bleeding time. The two studies in UA/NQWMI patients (40,41) (Table 2) demonstrate that a twostaged i.v. infusion regimen of tirofiban (loading infusion of 0.4 g/kg/min for 30 min followed by 0.1 g/kg/min), with aspirin and heparin, produces approximately 90% median inhibition of ex vivo ADP- induced platelet aggregation with bleeding times in the range of 14 to 20 min during the infusion. Near maximal inhibition is achieved rapidly with the 30-min loading infusion and is maintained over the duration of the infusion. Coadministration of tirofiban and heparin (and aspirin) in such patients results in a similar percentage of inhibition of ADP-induced platelet aggregation as seen with tirofiban alone. The Phase II study in patients undergoing PTCA (33) showed that a two-staged infusion regimen of tirofiban (loading bolus of 10 g/kg over 3 min followed by maintenance infusions of 0.15 g/kg/min) produces >90% median inhibition of ex vivo ADP-induced platelet aggregation in nearly all patients. Near maximal inhibition is achieved rapidly with the 3-min bolus and is maintained over the duration of the infusion. Following discontinuation of tirofiban infusion, platelet function returns to baseline within 4 to 8 h in most patients. Phase III Studies in UAP/NQWMI and PTCA Based on the Phase II studies, the estimated highest yet safe dose for use with and without heparin, which resulted in consistent inhibition across the population, was chosen for the respective Phase III studies. These regimens are shown in Table 3. Time point (h) TABLE 2. Median percentage of inhibition of platelet aggregation and median template bleeding times with tirofiban, with or without heparin (unstable angina) a Maintenance infusion rates ( g/kg/min) Inhibition of platelet aggregation (%) Bleeding times (min) No heparin Heparin No heparin Heparin 24 b (19) c 82.1 (16) 9.8 (22) 14.0 (19) 48 b 86.3 (19) 89.1 (14) 13.0 (21) 20.0 (17) 24 d (20) 95.8 (14) 19.8 (20) 25.7 (14) 48 d 92.1 (19) 94.6 (13) 15.4 (17) 30.0 (12) a From refs. 33, 40, and 41. b All patients received concomitant aspirin based on prior aspirin use. c Numbers in parentheses, number of patients. d All patients received aspirin pretreatment, unless contraindicated.

13 TIROFIBAN 211 TABLE 3. Dosing regimens selected for phase III studies Trial name Design Tirofiban regimen a PRISM b,c Tirofiban vs. heparin 0.6 g/kg/min loading (30 min) (UAP/NQWMI) 0.15 g/kg/min maintenance (47.5 h) PRISM-PLUS d Tirofiban or tirofiban + heparin vs. heparin (Same as above, up to 108 h) (UAP/NQWMI) 0.4 g/kg/min loading (30 min) 0.10 g/kg/min maintenance up to 108 h RESTORE e Tirofiban vs. placebo (all patients received heparin) 10 g/kg bolus over 5 min (PTCA) 0.15 g/kg/min maintenance a In addition, all patients received concomitant aspirin (generally 300 to 325 mg q.d. during the study drug infusion period), unless contraindicated. b Abbreviations: PRISM, Platelet Receptor Inhibition in Ischemic Syndrome Management; UAP/NQWMI, unstable angina pectoris/non-q-wave myocardial infarction; PRISM-PLUS, Platelet Receptor Inhibition in Ischemic Syndrome Management in Patients Limited by Unstable Signs and Symptoms; RESTORE, Randomized Efficacy Study of Tirofiban for Outcomes and Restenosis; PTCA, percutaneous transluminal coronary angioplasty. c From ref. 53. d From ref. 54. e From refs. 55 and 56. Clinical Program in Unstable Angina/Non-Q-Wave Myocardial Infarction Two independent, double-blind, active-controlled studies, comprising 5147 randomized patients, tested the fundamental concept that tirofiban, as a potent antiplatelet agent, could prevent acute cardiac ischemic events resulting from thrombus formation and propagation on disrupted atherosclerotic plaque in the coronary circulation. The PRISM trial (53) was designed to demonstrate that tirofiban alone could reduce acute cardiac ischemic events in patients with UAP/NQWMI in a treatment strategy that involved medical stabilization only. The trial was thus designed to demonstrate drug activity unconfounded by concomitant use of heparin or cardiac interventions. A separate trial (PRISM-PLUS) (54) was designed to study the clinical use of tirofiban with heparin and of tirofiban alone compared with heparin alone in a comprehensive treatment strategy, including medical stabilization, angiography, and angioplasty, if indicated. The trial would provide information on the use of a potent antiplatelet agent, not only for the initial medical management, but for the decision tree of how patients with UAP/NQWMI are managed. Thus, this trial provides a comprehensive framework for understanding the benefits of tirofiban in reducing acute and long-term cardiac ischemic events related to plaque disruption and coronary thrombus formation. The demographics of patients enrolled in the PRISM and PRISM-PLUS trials are presented in Table 4. The PRISM-PLUS trial randomized a larger number of patients with NQWMI compared with PRISM, where the patient population primarily had unstable angina. Overall, the baseline demographics of the populations studied were similar. PRISM: Efficacy of Tirofiban Alone versus Heparin in Patients with UAP/NQWMI The PRISM trial was a randomized, parallel, double-blind study in 3232 patients. It was designed to investigate the clinical safety and efficacy of tirofiban alone compared with heparin for the initial medical stabilization of UAP/NQWMI patients. This study was the first large endpoint trial of a GPIIb/IIIa inhibitor not used on a background of heparin. To enter the trial, patients had to present with chest pain and either documented electrocar-

14 212 J. J. COOK ET AL. TABLE 4. Overall demographics for tirofiban UAP/NQWMI efficacy studies a PRISM b (n 3232) PRISM-PLUS (n 1915) Total randomized No. % No. % Gender Female Male Common diagnoses Hypertension Hypercholesterolemia History of myocardial infarction Diabetes Presentation NQWMI Unstable angina Age, years (mean ± S.D.) 62.4 ± ± 11.6 a From refs. 53 and 54. b Abbreviations: PRISM, Platelet Receptor Inhibition in Ischemic Syndrome Management; PRISM-PLUS, Platelet Receptor Inhibition in Ischemic Syndrome Management in Patients Limited by Unstable Signs and Symptoms; NQWMI, non-q-wave myocardial infarction. diographic (ECG) evidence of ischemia, elevated cardiac enzymes, or a documented history of coronary artery disease. Patients randomized to receive tirofiban received a loading dose at a rate of 0.6 g/kg/min over 30 min, followed by 0.15 g/kg/min for the next 47.5 h. Patients randomized to receive heparin received a bolus of 5000 units i.v. followed by a maintenance infusion of 1000 units/h for 48 h. Heparin was adjusted as needed by an unblinded coinvestigator to maintain the increase in activated partial thromboplastin time (aptt) twofold control value (2X-control). For patients receiving a placebo for heparin, dummy dose adjustment instructions were provided to the unblinded coinvestigator. All patients received aspirin unless contraindicated. The primary endpoint of this study was at 48 h (the end of study drug infusion) and was the composite occurrence of refractory ischemia, new myocardial infarction, or death. At the time of the 48-h primary endpoint, 91 patients in the heparin group (5.6%) and 61 patients in the tirofiban group (3.8%) experienced a primary event (Table 5). This risk reduction in events by tirofiban was 33% (P 0.01). The secondary endpoints were the composite occurrence of refractory ischemia, new myocardial infarction, or death at 7 and 30 d. Readmissions for unstable angina were included in the 30-d endpoint. While the TABLE 5. PRISM a Study: clinical event results at 48 h (primary endpoint) b Tirofiban (n 1616) Heparin (n 1616) Risk 95% Confidence bounds Event No. % No. % ratio Lower Upper P value Composite endpoint MI/death a Abbreviations: PRISM, Platelet Receptor Inhibition in Ischemic Syndrome Management; MI, myocardial infarction. b From ref. 53.

15 TIROFIBAN 213 results for the composite endpoint favored tirofiban at both time points, the benefits were small. At 7 d, the risk reduction for the composite endpoint was 10% (P 0.33) and, at 30 d, the odds reduction was 8% (P 0.34). The absolute difference in the composite endpoint event rates at 7 d (1.0%) was maintained at 30 d (1.2%). Of note, the benefit of tirofiban with respect to death at 30 d was greater than with respect to the other components. The risk reduction for death at 30 d was 38% and achieved statistical significance (P 0.02). Overall, the PRISM study demonstrated that potent platelet inhibition with tirofiban can stabilize patients following presentation with UAP/NQWMI. PRISM-PLUS: Efficacy of Tirofiban plus Heparin versus Heparin in UAP/NQWMI Patients The PRISM-PLUS trial was a randomized, double-blind, multicenter study that documented the efficacy of tirofiban with heparin and aspirin for the management of patients with UAP/NQWMI. In this study, 1915 patients with documented UAP/NQWMI (i.e., supported by ECG changes or cardiac enzyme elevations) were randomized to receive tirofiban with heparin, tirofiban alone, or heparin alone; all patients received aspirin unless contraindicated. Early in the trial, the tirofiban-alone arm was dropped because of an excess mortality in this arm at 7 d (4.6% vs. 1.1%, tirofiban alone vs. heparin alone, respectively). The findings from the dropped arm relate only to the use of tirofiban monotherapy and not to tirofiban with heparin, as each arm in PRISM-PLUS was adequately powered to separately compare tirofiban or tirofiban plus heparin with heparin in UAP/NQWMI patients. The primary efficacy endpoint of the trial was the composite occurrence of refractory ischemic conditions, new myocardial infarction, or death at 7 d. The secondary efficacy endpoints were the composites of the same events at 48 h and 30 d (the 30-d secondary composite endpoint also included readmissions for unstable angina). A prospective analysis examined 30-d clinical outcomes of the patients undergoing PTCA. As specified in the protocol, patients were also followed up at 6 mo. Patients randomized to tirofiban and heparin received a loading dose of tirofiban of 0.4 g/kg/min over 30 min followed by a maintenance infusion at 0.1 g/kg/min and a 5000-unit i.v. bolus of heparin followed by an initial maintenance infusion of 1000 units/h. Patients randomized to receive heparin alone received a 5000-unit i.v. bolus of heparin followed by an initial maintenance infusion of 1000 units/h. Heparin was adjusted by an unblinded coinvestigator to maintain the aptt approximately 2 control. All aptt results remained blinded to individuals directly responsible for patient care. The study drug was to start within 12 h of the last episode of chest pain and could be continued for up to 108 h; on average, patients received 71.3 h of study drug. Patients were to receive a minimum of 48 h of therapy; during the initial 48-h infusion period, patients were not to undergo angiography unless they developed refractory ischemia or a new myocardial infarction. After 48 h, patients were allowed to undergo angiography with study drug continuing during the procedure. After angiography, patients undergoing PTCA were to have the study drug continued for 12 to 24 h following the procedure. Patients undergoing CABG surgery or selected for medical management were to have the study drug discontinued after angiography. The decision to proceed to PTCA or CABG was left to the physician s discretion.

16 214 J. J. COOK ET AL. PRISM-PLUS Results Tirofiban Monotherapy Arm The event rates observed at 7dinthetirofiban-alone arm of PRISM-PLUS must be reviewed in connection with the data from the PRISM study. In PRISM, the incidence of death was lower in the tirofiban arm than in the heparin arm at 7 d (1.0% vs. 1.6%, respectively). In addition, there was a statistically significant reduction in mortality with tirofiban at 30 d (P 0.02) in PRISM. When the data from PRISM-PLUS are considered in light of the larger PRISM study, the excess mortality cannot be substantiated. These findings suggest that mortality in the tirofiban-alone arm is likely to be comparable to that with heparin. PRISM-PLUS Results Tirofiban plus Heparin versus Heparin Alone The main results of the trial are presented in Table 6. Tirofiban, administered with heparin, reduces the composite endpoint of refractory ischemic condition, new myocardial infarction, or death by 32%, compared with heparin alone at 7 d after the start of therapy (P 0.004). Addition of tirofiban to heparin also reduces the composite endpoint of myocardial infarction/death by 43% compared with heparin alone at 7 d after the start of therapy (P 0.006). This beneficial effect is seen as early as 48 h after the start of therapy. The early benefit of combination therapy on the composite endpoint is sustained through 6 mo. An overall benefit for the composite endpoint of myocardial infarction or death also persists for 6 mo: the early absolute difference in the incidence of death/ myocardial infarction at 7 d (3.4%) was maintained through 30 d (3.2%) and 6 mo (3.0%). A Kaplan-Meier curve for the composite endpoint at 6 mo is displayed in Fig. 8. The curves for the composite endpoint separate early and remain parallel for the 180-d period. This is consistent with the drug effect on the early pathophysiology with the early benefit TABLE 6. PRISM-PLUS a clinical event summary b Tirofiban + heparin (n 773) Heparin (n 797) Tirofiban + heparin vs. heparin 95% confidence bounds Event No. % No. % Risk ratio Lower Upper P value 48 h Composite endpoint MI/death d (one endpoint) Composite endpoint MI/death d Composite endpoint a MI/death d Composite endpoint c MI/death a Abbreviations: PRISM-PLUS, Platelet Receptor Inhibition in Ischemic Syndrome Management in Patients Limited by Unstable Signs and Symptoms; MI, myocardial infarction. b From ref. 54. c Includes readmission for unstable angina pectoris.

17 TIROFIBAN 215 FIG. 8. PRISM-PLUS composite endpoint with 180-d follow-up: heparin versus combination (tirofiban plus heparin) (54). of tirofiban plus heparin being sustained through 6 mo. The benefit of tirofiban plus heparin was consistent among patients regardless of age and gender. A consistent reduction in clinical events was also seen in diabetic patients. Benefit was also observed regardless of the revascularization strategy (PTCA, CABG, or no revascularization) chosen for the in-hospital management of the patient. Summary Phase III Program in Patients with UA/NQWMI Overall, the Phase III unstable angina clinical trials demonstrate the efficacy of tirofiban at reducing acute and long-term cardiac ischemic complications in patients with UAP/ NQWMI. As demonstrated in PRISM-PLUS, a strategy of adding tirofiban to heparin (and aspirin) through medical stabilization, angiography, and angioplasty reduces cardiac ischemic complications compared with current standard therapy. Tirofiban plus heparin also reduces the risk of the combined endpoint of death and myocardial infarction that represents irreversible cardiac morbidity. The benefit of this combination therapy persists to 6 mo. The findings are consistent with the pathophysiology of the disease process, as it relates to platelet aggregation and arterial thrombus formation and to the demonstrated activity of tirofiban as a potent inhibitor of platelet aggregation. Phase III Program in Patients Undergoing PTCA Two trials, PRISM-PLUS and RESTORE (54 56), evaluated the benefit of tirofiban plus heparin in patients undergoing PTCA. In the PRISM-PLUS trial, approximately 30%

18 216 J. J. COOK ET AL. of the patients underwent interventions during the initial hospitalization. Thus, an examination of this cohort allows for evaluation of the effect of therapy with tirofiban plus heparin before and after PCI on clinical outcomes. The RESTORE trial evaluated the benefit of tirofiban plus heparin when such therapy is initiated at the time of the PTCA. PRISM-PLUS Patients Undergoing PTCA The clinical outcomes of the PRISM-PLUS patients undergoing PTCA were compared as a prospectively planned exploratory analysis (Table 7) of the patients who underwent PTCA at any time during the trial. Note that patients undergoing PTCA were not necessarily comparable between treatment groups, since study therapy outcomes could potentially influence the decision to proceed with PTCA. For this reason, formal statistical tests were not performed. However, the fact that the number of patients undergoing angioplasty was almost equal between treatment groups, despite clear differences in antecedent course, suggests that the decision to proceed with PTCA is likely to be driven more by the patient s angiographic anatomy than by clinical outcome. With these limitations in mind, the outcomes (Day 30) of patients after angioplasty performed during the initial hospitalization were clearly improved in patients receiving combination therapy: 8.8% of the patients in the tirofiban plus heparin group experienced any clinical event, compared with 15.2% of the patients in the heparin group, a risk reduction of 46%. There were similar reductions in the composite of myocardial infarction and death (risk reduction 43%) in patients undergoing PTCA. The absolute reduction in events for the composite endpoint and for the composite of myocardial infarction/death was 6.4% and 4.3%, respectively. Similar results were observed at 180 d (Fig. 9). RESTORE: Efficacy of Tirofiban plus Heparin in Patients Undergoing PTCA or Atherectomy The RESTORE trial was a randomized, placebo-controlled, multicenter study. It was designed to investigate the safety and effects of tirofiban on subsequent cardiac events when used in combination with heparin and aspirin in patients undergoing PTCA or directional atherectomy, within 72 h of presentation with an acute coronary syndrome. TABLE 7. Clinical events following PTCA a performed during the initial hospitalization b Combination Heparin Combination vs. heparin (n 239) (n 236) Odds 95% Confidence bounds Event No. % No. % ratio Lower Upper 30 d Composite endpoint MI/death d Composite endpoint c MI/death c a Abbreviations: PTCA, percutaneous transluminal coronary angioplasty; MI, myocardial infarction. b From ref. 54. c Risk ratio.

19 TIROFIBAN 217 FIG. 9. Outcomes following in-hospital percutaneous transluminal coronary angioplasty (PTCA) in PRISM- PLUS: heparin versus combination (tirofiban plus heparin) (54). Patients were randomly assigned to either tirofiban (as a bolus of 10 g/kg i.v. over 3 min, followed by an infusion of 0.15 g/kg/min) or placebo initiated at the time of the procedure and continued for 36 h. All patients received open-label heparin and aspirin (325 mg) daily. The heparin was administered according to the catheterization laboratory s standard procedure, but the investigator was asked to adhere to specified heparin-dosing guidelines, if possible, to maintain the activated clotting time (ACT) in the range of 300 to 400 s during the procedure. In general, heparin was discontinued at the conclusion of the PTCA/atherectomy procedure, and sheaths were removed when ACT was <180 s. The study drug infusion continued while the sheath was being removed. If necessary, at the treating physician s discretion, heparin could be restarted after either sheath removal or PTCA/atherectomy before sheath removal, for medical reasons. RESTORE Results Overall, the baseline demographics of the population studied in RESTORE were quite similar to those of the unstable angina trials, as one might expect from a population of patients who present to the hospital with acute coronary syndromes. The primary outcome measure was defined as a composite of the following: death from any cause, nonfatal myocardial infarction, CABG, repeat percutaneous intervention (urgent and nonurgent) of the target vessel for recurrent ischemia, or insertion of a coronary endovascular stent because of procedural failure. Outcomes were also to be measured at the prospectively designated times of 2, 7, and 180 d after randomization.