Antithrombotic efficacy of a novel factor Xa inhibitor, FXV673, in a canine model of coronary artery thrombolysis.

We compared the antithrombotic efficacy of a potent factor Xa inhibitor, FXV673, to heparin and RPR109891, a GPIIb/IIIa antagonist, when used as adjunctive therapy in a canine model of rt-PA-induced coronary thrombolysis. Thrombus formation was induced by electrolytic injury to stenosed coronary artery. After thrombotic occlusion, a 135 min infusion of saline (n=8), FXV673 (10, 30 or 100 microg kg(-1)+1, 3, or 10 microg kg(-1) min(-1), respectively; n=8 per dose), heparin (60 u kg(-1)+0.7 u kg(-1) min(-1), n=8), or RPR109891 (30 microg kg(-1)+0.45 microg kg(-1) min(-1), n=8), was initiated. Aspirin (5 mg kg(-1), i.v.) was administered to all animals. Fifteen minutes after the start of drug infusion, rt-PA was administered (100 microg kg(-1)+20 microg kg(-1) min(-1) for 60 min). The incidence of reperfusion in the high dose FXV673 (8/8, 100%) was significantly greater than that in the heparin group (4/8, 50%), with a trend to faster reperfusion (23+/-5 min for FXV673 versus 41+/-11 min for heparin). Only 2/8 (25%) of the vessels reoccluded in the high dose FXV673 group, compared to 4/4 (100%) and 5/5 (100%) vessels in the heparin and RPR109891 groups, respectively (P<0.05). Throughout the protocol, blood flow was higher in the FXV673 treated group compared to other groups. FXV673 enhanced vessel patency in a dose-dependent manner. Compared to vehicle and heparin groups, the thrombus mass was decreased by 60% in the high dose FXV673. FXV673, heparin and RPR109891 increased the bleeding time by 2.7, 1.7 and 4 fold, and APTT by 2.8, 2.7 and 1.2 fold, respectively. In conclusion, FXV673 is more effective than heparin and at least as effective as RPR109891 when used as an adjunct during rt-PA-induced coronary thrombolysis.

Role of short-term inhibition of factor Xa by FXV673 in arterial passivation: a study in a chronic model of thrombosis in conscious dogs.

Factor Xa (fXa) plays a pivotal role in the activation of the coagulation system during thrombosis, but, unlike GPIIb/IIIa receptor antagonists, the role of fXa inhibition in arterial passivation is not well defined. We compared the long-term antithrombotic efficacy of a direct fXa inhibitor, FXV673, and heparin after short-term infusion in conscious dogs. Dogs were instrumented surgically to induce carotid artery thrombosis by electrolytic injury. On day 1, dogs received a 3-h infusion of placebo (n = 10), FXV673 (100 microg/kg + 10 microg/kg/min, n = 7), or heparin (60 U/kg + 0.7 U/kg/min, n 7). Injury (100 microA) was initiated concomitantly for 1 h. The procedure was repeated on day 2 with injury of 200 microA for 3 h. Carotid artery blood flow (CBF) and coagulation parameters were monitored continuously for 3 h on days 1 and 2 and for 30 min on days 3, 4, and 5. On day 1 at 3 h, CBF in the placebo-treated group was 26% of baseline with 70% incidence of occlusion. None of the vessels occluded in the heparin and FXV673 groups; however, the CBF was significantly higher in the FXV673 group (92+/-8 ml/min versus 39+/-12 ml/min). Before injury on day 2, CBF recovered in all groups to 71-89% of baseline. After the second injury, all vessels in the placebo-treated group progressed to complete occlusion by 3 h. CBF was significantly higher in FXV673 group compared with heparin throughout the 3-h period. On days 3, 4, and 5 the placebo-treated vessels remained occluded, but the CBF in the heparin group was 33+/-20 ml/min, 55+/-11 ml/min and 68+/-12 ml/min, respectively, compared with 84+/-10 ml/min, 98+/-7 ml/min, and 99+/-10 ml/min in the FXV673 group. The arterial thrombus mass was significantly lower in FXV673 group (13+/-4 mg) compared with placebo (103+/-10 mg) and heparin (44+/-11 mg). In summary, these data demonstrate that short-term infusion of FXV673 was associated with long-term efficacy that was superior to standard heparin and underscore the role of direct fXa inhibition in arterial passivation.

Superiority of enoxaparin over heparin in combination with a GPIIb/IIIa receptor antagonist during coronary thrombolysis in dogs.

It is known that a low-molecular-weight heparin (LMWH) is more effective than unfractionated heparin in unstable angina/non-Q-wave myocardial infarction (UA/NQMI) and the platelet GPIIb/IIIa receptors play an important role in acute myocardial infarction (AMI). Therefore, enoxaparin might have a similar advantage over heparin when used with a GPIIb/IIIa antagonist (RPR109891) in coronary thrombolysis. After induction of coronary thrombosis in anesthetized dogs, infusion of saline, enoxaparin, heparin, RPR109891, enoxaparin+RPR109891, or heparin+RPR109891 was initiated followed 15 min later by recombinant tissue plasminogen activator (rt-PA). The incidence of reperfusion in the enoxaparin+RPR109891- and the heparin+RPR109891-treated groups was similar, but time to reperfusion tended to be shorter for enoxaparin versus heparin. Only 43% of the vessels reoccluded in the enoxaparin+RPR109891 group, compared to 100% vessels in the heparin+RPR109891 group. Enoxaparin+RPR109891 maintained flow for a significantly longer time compared to saline, enoxaparin, heparin, and heparin+RPR109891. Enoxaparin+RPR109891 and heparin+RPR109891 increased the template bleeding time by 2- and 3-fold and activated partial thromboplastin time (APTT) by 1.3- and 3-fold, respectively. These data suggest that enoxaparin is more effective and potentially safer than heparin when combined with a GPIIb/IIIa receptor antagonist during rt-PA-induced coronary thrombolysis.

Antithrombotic efficacy of single subcutaneous administration of a recombinant nematode anticoagulant peptide (rNAP5) in a canine model of coronary artery thrombolysis.

We examined the adjunctive benefit of recombinant nematode anticoagulant peptide (rNAP5), a factor Xa inhibitor, in a canine model of recombinant (rt)-PA-induced thrombolysis. In anesthetized dogs, a stable occlusive thrombus was formed by electrolytic injury of the vessel wall, after which the animals were administered rt-PA (1.44 mg/kg, i.v.) and rNAP5 (0.1 mg/kg, s.c.: n=13), or rt-PA plus vehicle (1-2 ml, s.c.; n=13). Hemodynamic and coagulation parameters were monitored for 360 minutes. Single subcutaneous administration of rNAP5 resulted in a prolonged and sustained increase in the activated partial thromboplastin time (>100-fold), whereas prothrombin time was unchanged. The template bleeding time was not altered significantly throughout the protocol (maximum 1.4-fold). The incidence of reperfusion was similar in the two groups with a trend toward faster reperfusion in the rNAP5 group (34+/-4 minutes) compared to the vehicle group (63+/-15 minutes; p=0.07). After reperfusion, 80% of the vessels in the vehicle group reoccluded, whereas only 14% of vessels reoccluded in the rNAP5-treated group. Times to reocclusion were 65+/-21 minutes and 221+/-28 minutes, respectively (p<0.05). Single subcutaneous administration of rNAP5 sustained the coronary artery blood flow after reperfusion, such that at the end of protocol the flow was 47% of the preocclusion value as compared to the vehicle group in which the flow was 11% (p<0.05). Cyclic flow reductions were most prominent during rt-PA-induced reperfusion and were similar in both groups. The results indicate that a single subcutaneous administration of rNAP5 provides a sustained antithrombotic effect in maintaining the coronary artery patency during rt-PA-induced thrombolysis.

Contribution of in vivo models of thrombosis to the discovery and development of novel antithrombotic agents.

Cardiovascular and cerebrovascular diseases continue to be the leading cause of death throughout the world. Over the past two decades, great advances have been made in the pharmacological treatment and prevention of thrombotic disorders (e.g., tissue plasminogen activators, platelet GPIIb/IIIa antagonists, ADP receptor antagonists such as clopidogrel, low-molecular weight heparins, and direct thrombin inhibitors). New research is leading to the next generation of antithrombotic compounds such as direct coagulation FVIIa inhibitors, tissue factor pathway inhibitors, gene therapy, and orally active direct thrombin inhibitors and coagulation Factor Xa (FXa) inhibitors. Animal models of thrombosis have played a crucial role in discovering and validiting novel drug targets, selecting new agents for clinical evaluation, and providing dosing and safety information for clinical trials. In addition, these models have provided valuable information regarding the mechanisms of these new agents and the interactions between antithrombotic agents that work by different mechanisms. This review briefly presents the pivitol preclinical studies that led to the development of drugs that have proven to be effective clinicallly. The role that animal models of thrombosis are playing in the discovery and development of novel antithrombotic agents is also described, with specific emphasis on FXa inhibitors. The major issues regarding the use of animal models of thrombosis, such as the use of positive controls, appropriate pharmacodynamic markers of activity, safety evaluation, species-specificity, and pharmacokinetics, are highlighted. Finally, the use of genetic models in thrombosis/hemostasis research and pharmacology is presented using gene-therapy for hemophilia as an example of how animal models have aided in the development of these therapies that are now being evaluated clinically. In summary, animal models have contributed greatly to the discovery of currently available antithrombotic agents and will play a primary role in the discovery and characterization of the novel antithrombotic agents that will provide safe and effective pharmacological treatment for life-threatening thrombotic diseases.

Role of extracellular ionized calcium in the in vitro assessment of GPIIb/IIIa receptor antagonists.

Several preclinical studies have found a poor correlation between the ex vivo platelet inhibitory potency and the in vivo antithrombotic efficacy of GPIIb/IIIa receptor antagonists. The present study was designed to examine the differential in vitro potencies of c7E3, MK-383, DMP-728, and SM-20302 in inhibiting ex vivo platelet aggregation under normocalcemic and hypocalcemic conditions. Human blood was collected in either trisodium citrate (0. 37%) or PPACK (20 microg/mL). Platelet aggregation assays were performed in platelet-rich plasma from citrate-anticoagulated blood (cPRP) and PPACK-anticoagulated blood (pPRP) using ADP (20 microM) and TRAP (10 microM) as agonists in the presence of c7E3, MK-383, DMP-728, or SM-20302. The concentration of ionized calcium in cPRP was 16-19 times lower than that in pPRP. The IC(50) of c7E3 for inhibiting ADP-induced platelet aggregation in cPRP (2.76 +/- 0.11 microg/mL) was 1.6 times lower than that in pPRP (4.46 +/- 0.48 microg/mL; P < 0.05). Similarly, the IC(50) for c7E3 for inhibiting TRAP-induced platelet aggregation in cPRP (4.52 +/- 0.34 microg/mL) was 1.7 times lower than that in pPRP (7.69 +/- 0.43 microg/mL; P < 0.05). MK-383, DMP-728, and SM-20302 also demonstrated 1.96-, 1.15-, and 1.43-fold lower IC(50) values, respectively, in cPRP as compared with pPRP. Chelation of ionized calcium in pPRP led to a progressive increase in platelet inhibition by all the antagonists. These results suggest that the observed in vitro inhibitory potency of a GPIIb/IIIa receptor antagonist is markedly enhanced when trisodium citrate is used as an anticoagulant to collect blood for ex vivo assay. These findings indicate that dosing regimens for GPIIb/IIIa receptor antagonists based on the platelet inhibition profile in citrate may provide misleading information with respect to their true in vivo antithrombotic efficacy.

Thrombostatin inhibits induced canine coronary thrombosis.

Thrombostatin (RPPGF), an angiotensin converting enzyme metabolite of bradykinin, is an inhibitor of alpha-thrombin's ability to activate platelets. We examined the in vivo pharmacokinetics and pharmacodynamics of thrombostatin in rabbits and its ability to inhibit coronary thrombosis induced by electrolytic injury in dogs. Plasma half-life of thrombostatin had a t1/2alpha of 2.6 min and a t1/2beta of 24 min in rabbits. Ligating the renal arteries did not prolong clearance (t1/2alpha = 2.4 min; t1/2beta = 12 min). Thrombostatin produced a prolonged in vivo antiplatelet effect. At 30 min after a single intravenous administration in rabbits, thrombostatin's plasma concentration was <8.7 microM (5 microg/ml). However, ex vivo 20 and 40 nM gamma-thrombin-induced platelet aggregation of these rabbits' platelets was inhibited 40% for 2.75 and 1 h, respectively. In vitro, flow cytometry studies revealed that thrombostatin specifically bound to human platelets and washed human platelets treated with thrombostatin were less responsive to gamma-thrombin than control platelets. Using electrolytic injury to induce coronary artery thrombosis, dogs treated with thrombostatin, aspirin, or combined thrombostatin and aspirin occluded in 62+/-25 (mean +/- SD), 62+/-36, or 89+/-32 min versus untreated animals which occluded at 39+/-27 min, (p<0.01, p<0.01 and p<0.001, respectively). These studies show that thrombostatin binds to platelets and can delay coronary occlusion in vivo.

Effect of time of 7E3 administration on rt-PA-induced reperfusion: study in a canine model of thrombus-based occlusion-reperfusion.

Chimeric version of the murine monoclonal antibody, 7E3 has been proposed for the early restoration of coronary artery patency during thrombolytic therapy. We determined the optimal time for administration of 7E3 during recombinant tissue plasminogen activator (rt-PA)-induced thrombolysis using a canine model of coronary artery thrombosis. After 30 min of thrombotic occlusion, microspheres were injected to assess regional myocardial blood flow, followed by a 90-min rt-PA infusion. Dogs were randomized to three groups wherein 7E3 (0.8 mg kg(-1), i.v.) was administered either 5 min before rt-PA (Group I), at the first evidence of thrombolysis (Group II), or after the completion of rt-PA infusion (Group III). Hemodynamic parameters were monitored for 6 h after which infarct size was estimated. Time to occlusion/reperfusion was similar in all groups. In the rt-PA alone group, 78% arteries reoccluded after 60 min of reperfusion. The incidence of reocclusion was lower in Groups II (25%, P = 0.04) and III (0%. P < 0.01). All arteries (100%) were patent at the end of the protocol in Group III vs 50% remaining patent in Group I (P = 0.01). Arterial patency was maintained longer in Group III (301 min, n = 10), compared with Groups I (124 min, n = 5) and II (124 min, n = 6). Arterial flow was greater in Group III (82%) compared with Groups I (27%) and II (35%) (P < 0.01). Regional myocardial blood flow and infarct size were similar in all groups. The data indicate that the time of administration of 7E3 in conjunction with rt-PA-induced thrombolysis influences patency status. The experimental results suggest that in the absence of aspirin and heparin, optimal thrombolysis is obtained when 7E3 is administered after the completion of rt-PA infusion regimen.

Correlation between the in vivo efficacy of GPIIb/IIIa receptor antagonists (m7E3, MK-383 and DMP-728) and ex vivo platelet inhibition.

In vivo antithrombotic efficacy of GPIIb/IIIa receptor antagonists (m7E3, MK-383 and DMP-728) was studied with respect to their ex vivo platelet inhibition in heparinized platelet-rich plasma (hPRP) and citrated platelet-rich plasma (cPRP) using a canine model of carotid artery thrombosis. For each drug group (n = 6), the right carotid artery was used as control vessel and resulting occlusive thrombus was kept in situ to examine the direct thrombolytic efficacy of the antagonists. Thirty minutes after occlusion of control vessel, a low or high dose of each antagonist was administered and the left carotid artery was used as test vessel. All control vessels occluded within 86-96 min in response to electrolytic injury. The incidence of occlusion with lower doses of m7E3, DMP-728, and MK-383 was 100, 33 and 100%, respectively; corresponding times to occlusion were 174, 220 and 118 min. Lower doses inhibited ADP- or AA-induced platelet aggregation in cPRP (>80%). Incidence of occlusion with high doses of m7E3, DMP-728 and MK-383 was 33, 0 and 0%, respectively; corresponding times to occlusion were 209, >240 and >240 min. Higher doses inhibited aggregation in cPRP (>80%), but only partially in hPRP (45-66%). Dose-dependent prolongation of bleeding time occurred with all antagonists. None of the antagonists lyzed preformed thrombi in control vessels. The results indicate that ex vivo platelet aggregation conducted in hPRP, as opposed to conventional cPRP, provides a better assessment of the in vivo efficacy of GPIIb/IIIa receptor antagonists.

Temporary and partial inhibition of platelets by SM-20302 prevents coronary artery thrombosis in a chronic canine model.

We proposed that temporary and partial platelet inhibition by a GPIIb/IIIa receptor antagonist, SM-20302, would provide sustained antithrombotic efficacy in a chronic model of coronary artery thrombosis. Instrumented, conscious dogs received vehicle (Group I, n = 7), low dose SM-20302 (30 microg/kg bolus + 1 microg/kg/min infusion for 6 h) (Group II, n = 7), or high dose SM-20302 (100 microg/kg bolus + 1 microg/kg/min infusion for 6 h) (Group III, n = 7). Thrombosis was initiated by electrolytic injury to the circumflex coronary artery. Coronary blood flow was monitored for 6 h on day 1 and days 2-6. Platelet aggregation was performed in platelet-rich plasma prepared from citrated or heparinized blood. At 6 h, both doses of SM-20302 inhibited adenosine diphosphate-induced platelet aggregation completely (> 90%) in citrated platelet-rich plasma, but incompletely (57-59%) in heparinized platelet-rich plasma. Platelet reactivity returned to baseline values at 24 h. Control animals developed thrombotic occlusion on Day 1. Both doses of SM-20302 maintained vessel patency during the infusion period (Day 1) and the subsequent 5 days. Myocardial infarct size and mortality in the drug treated groups were reduced compared to the vehicle group. Thus, temporary inhibition of platelet reactivity by SM-20302 is associated with sustained prevention of primary thrombus formation, and reduction in infarct size and mortality.

Pharmacokinetics and pharmacodynamics of SM-20302, a GPIIb/IIIa receptor antagonist, in anesthetized dogs.

We examined the pharmacokinetic and pharmacodynamic properties of SM-20302, a GPIIb/IIIa receptor antagonist, in anesthetized dogs. SM-20302 was administered intravenously in doses of 30 (n=2), 100 (n=4), 300 (n=4), and 1,000 microg/kg (n=4). The half-life of the initial phase was 4 min, and that of the terminal phase was 162-209 min. SM-20302 produced a dose-dependent increase in the initial plasma concentration and the area under concentration-time curve but did not alter the volume of distribution, mean residence time, or plasma clearance. Plasma clearance for SM-20302 ranged from 6.58 to 9.73 ml/min/kg. All doses of SM-20302 inhibited (> or =90%) the ex vivo platelet aggregation induced by adenosine diphosphate (ADP) or arachidonic acid (AA) in citrated platelet-rich plasma (cPRP). In heparinized PRP (hPRP), a dose-dependent (44-89%) inhibition was observed. By using a sigmoid Emax model, the in vivo median inhibitory concentration (IC50) for SM-20302 was estimated to be 14-19 ng/ml in cPRP and 79-89 ng/ml in hPRP. To validate the calculated parameters, an infusion regimen was designed for the prevention of coronary artery thrombosis. Infusion of SM-20302 produced 64-67% inhibition of platelets in hPRP and maintained vessel patency despite vessel wall injury. The results suggest that SM-20302 exhibits linear pharmacokinetics and that its ability to inhibit platelet aggregation in hPRP may correlate more accurately with its in vivo antithrombotic efficacy.

In vivo efficacy of SM-20302, a GP IIb/IIIa receptor antagonist, correlates with ex vivo platelet inhibition in heparinized blood but not in citrated blood.

We tested the hypothesis that the in vivo antithrombotic efficacy of SM-20302, a GP IIb/IIIa receptor antagonist, correlates with the ex vivo platelet inhibition in heparinized platelet rich plasma (hPRP) but not in citrated PRP (cPRP). The studies were performed in a canine model of carotid artery thrombosis in which thrombus formation was induced by electrolytic injury. Thrombosis of the right carotid artery was induced immediately after the administration of saline (n=12). Thirty minutes after persistent occlusive thrombosis was obtained, the vessel segment was ligated, and the time to occlusion and thrombus weight were noted. Subsequently, thrombosis of the left carotid artery was initiated in the presence of SM-20302 (100, 300, 600, or 1000 microg/kg i.v.; n=4 to 6). All the doses of SM-20302 inhibited (by > or = 90%) the ex vivo platelet aggregation induced by ADP and arachidonic acid (AA) in cPRP. In hPRP, a dose-dependent inhibition of ex vivo platelet aggregation was observed. The maximal inhibition produced by 100 to 1000 microg/kg SM-20302 ranged from 18% to 80% for ADP and 44% to 88% for AA. Maximal prolongation of the template bleeding time induced by the 100-, 300-, 600-, and 1000-microg/kg doses were 2.5-, 9.5-, 10-, and > 10-fold, respectively. All the injured carotid arteries (n=12) in the saline-treated group occluded. SM-20302 pretreatment produced a dose-dependent maintenance of the carotid artery patency, and the incidence of occlusion at 4 hours was 5/6, 3/6, 0/6, and 0/6 for the 100-, 300-, 600-, and 1000-microg/kg doses, respectively. The results indicate that SM-20302 prevents carotid artery thrombosis in response to electrolytic arterial wall injury and that its in vivo antithrombotic efficacy can be correlated accurately with the ex vivo platelet inhibition in PRP prepared from heparinized blood but not from citrated blood.

Antithrombotic efficacy of a recombinant nematode anticoagulant peptide (rNAP5) in canine models of thrombosis after single subcutaneous administration.

We describe the antithrombotic effects of recombinant nematode anticoagulant peptide (rNAP5), a selective and direct factor Xa inhibitor, after a single s.c. administration in canine models of arterial and venous thrombosis. The systemic anticoagulant effects of rNAP5 were evaluated initially in conscious dogs after s.c. dosing (0.03, 0.1 and 0.3 mg/kg) that resulted in a dose-dependent increase in the activated clotting time and the activated partial thromboplastin time. The antithrombotic effects of rNAP5 were evaluated in anesthetized dogs where saline or rNAP5 (0.03, 0.1 and 0.3 mg/kg s.c.) was administered 1 hr before the left circumflex coronary artery was subjected to electrolytic injury. In the saline group (n = 10), the left circumflex artery occluded in 79 +/- 9 min, and 5 of 10 animals progressed to sudden death due to ventricular fibrillation. rNAP5 significantly prolonged the time to occlusion in the 0.03 mg/kg (163 +/- 62 min) and 0.1 mg/kg (327 +/- 62) treatment groups (n = 6). In the 0.3 mg/kg group (n = 5), all of the injured vessels remained patent for 8 hr. There was a dose-dependent reduction in the thrombus mass in the rNAP5-treated animals as compared with controls, as well as a lower mortality rate. rNAP5, in the doses of 0.03 and 0.1 mg/kg, did not alter the bleeding time, whereas 0.3 mg/kg produced a 5-fold increase. In a separate study, we evaluated the efficacy of rNAP5 (0.1 mg/kg) in the prevention of carotid artery and jugular vein thrombosis. In response to endothelial injury, the carotid artery and jugular vein in the saline group (n = 6) occluded in 142 +/- 16 and 100 +/- 11 min, respectively, compared with rNAP5, which maintained vessel patency in the carotid artery (6/6) and jugular vein (5/6) and significantly decreased the thrombus weights. The results demonstrate that rNAP5 has antithrombotic efficacy in canine models of arterial and venous thrombosis after a single s.c. administration.

TP-9201, a glycoprotein IIb/IIIa platelet receptor antagonist, prevents rethrombosis after successful arterial thrombolysis in the dog.

BACKGROUND AND PURPOSE: We examined the ability of TP-9201, a platelet glycoprotein IIb/IIIa receptor antagonist, to prevent carotid artery rethrombosis in the anesthetized dog. METHODS: Occlusive thrombosis was induced by electrolytic injury of the left carotid artery. Thirty minutes later, 0.05 U/kg of anisoylated plasminogen streptokinase activator complex (APSAC) was infused locally to achieve clot lysis. Carotid artery recanalization was followed immediately by the infusion of either saline (10 mL/h, 240 minutes; n = 9), low-dose TP-9201 (120 micrograms/kg plus 3 micrograms.kg-1.min-1, 240 minutes; n = 7), or high-dose TP-9201 (185 micrograms/kg plus 5 micrograms.kg-1.min-1, 240 minutes; n = 7). Ex vivo platelet aggregation responses to ADP or arachidonic acid were determined. RESULTS: TP-9201 produced complete inhibition of platelet aggregation in citrated platelet-rich plasma but a partial and dose-dependent inhibition in heparinized platelet-rich plasma. A twofold and eightfold increase in the template bleeding time was associated with the infusion of low-dose and high-dose TP-9201, respectively. There were frequent cyclic flow reductions in both the saline and low-dose TP-9201-treated groups after thrombolysis. However, the high-dose TP-9201-treated group exhibited a sustained flow with minimal evidence of cyclic flow reductions. At the conclusion of the protocol, patent vessels were found more frequently in the high-dose TP-9201 (5/7; P = .0048) than in the low-dose TP-9201 treatment group (2/7; P = .17) when compared with the saline group (0/9). Infusion of high-dose TP-9201 was associated with a significant reduction in the thrombus mass as compared with the control vessels. CONCLUSIONS: Administration of TP-9201 in conjunction with successful thrombolysis inhibited ex vivo platelet aggregation and prevented rethrombosis of the canine carotid artery. This study demonstrates that TP-9201, an inhibitor of the platelet GPIIb/IIIa receptor, can inhibit platelet-vessel wall interaction and thus prevent rethrombosis.

CVS-1123, a direct thrombin inhibitor, prevents occlusive arterial and venous thrombosis in a canine model of vascular injury.

CVS-1123, low-molecular-weight, direct thrombin inhibitor was studied in an anesthetized canine model of arterial and venous thrombosis to determine whether thrombin inhibition could reduce the incidence of occlusive thrombosis in response to vessel-wall injury. The left carotid artery (LCA) and right jugular vein (RJV) were instrumented with a flow probe, intraluminal electrode, and critical stenosis. Either saline (n = 9), or CVS-1123 (n = 12) was administered in a loading dose of 2 mg/kg i.v., followed by an infusion (2.46 mg/kg/h for 180 min). Vessel-wall injury was initiated by applying a 300-microA anodal current to the intimal surface of the LCA and RJV. Platelet aggregation in response to gamma-thrombin remained inhibited by CVS-1123 for 8 h. The activated partial thromboplastin time (aPTT) was increased and remained elevated for the duration of the protocol. The prothrombin time (PT) showed an initial increase and then a rapid decrease after the infusion was discontinued. There was a twofold increase in the bleeding time (BT) at 2 h. The time to occlusion of the LCA was prolonged (380 +/- 22 min in the CVS-1123 group vs. 152 +/- 18 min in the saline group) with seven of 12 patent arteries at 8 h. Similarly, the time to occlusion for RJV was prolonged (415 +/- 16 min in the CVS-1123 group vs. 99 +/- 8 min in the saline group) with eight of 12 veins remaining patent at 8 h. CVS-1123 administration was associated with a decrease in the thrombus weights in both the LCA and RJV as compared with the saline-treated animals. In summary, CVS-1123 modifies the thrombogenic response to deep vessel-wall injury in both the arterial and venous circulations. The results suggest that CVS-1123 is an effective antithrombin and may offer a therapeutic alternative to current antithrombins in the management of arterial and venous thrombosis.

Orally effective CVS-1123 prevents coronary artery thrombosis in the conscious dog.

BACKGROUND: We examined the oral efficacy of a direct thrombin inhibitor, CVS-1123 [(CH3CH2CH2)(2)-CH-CO-Asp (OCH3)-Pro-Arg-CHO; MW, 575]. The object was to determine whether thrombin inhibition could reduce the incidence of occlusive coronary artery thrombosis in response to arterial wall injury. METHODS AND RESULTS: Arterial wall injury was induced in conscious dogs by a 150-muA anodal current applied to the intimal surface of the circumflex coronary artery 30 minutes after oral CVS-1123 (20 mg/kg every 8 hours for three doses; n = 11) or placebo containing diluent (n = 10). Dogs were monitored for 8 hours and at 24 hours. The coronary artery remained patent for 24 hours in 8 of 11 CVS-1123-treated dogs. All dogs (n = 10) in the placebo group developed a sustained, occlusive arterial thrombus. Two hours after the initial oral dose, the plasma CVS-1123 concentration was 13 +/- 1 microgram/mL, reaching a maximum of 15 +/- 1 micrograms/mL after the second dose and 4.4 +/- 0.5 micrograms/mL at 24 hours. Ex vivo platelet aggregation to gamma-thrombin was inhibited and activated partial thromboplastin time was increased after treatment with CVS-1123 (P < .05). CONCLUSIONS: The direct thrombin inhibitor CVS-1123 is effective after oral administration in reducing the incidence of primary thrombus formation in an experimental model of arterial wall injury. Thrombin-specific inhibitors, such as CVS-1123, may be alternative antithrombotic agents in clinical settings in which heparin-associated thrombosis is a complicating factor or when long-term anticoagulation is required.