Inhibitors of blood coagulation factors Xa and IIa synergize to reduce thrombus weight and thrombin generation in vivo and in vitro.

BACKGROUND: Many compounds currently in development for treatment of thrombotic disorders demonstrate high specificity for single targets of blood coagulation such as factor Xa (FXa) or thrombin. AIM: The aim of this study is to determine if inhibition of both FXa and thrombin by simultaneous administration of PD0313052 and argatroban, respectively, synergistically increases the effect of either drug alone in vivo and in vitro. METHODS AND RESULTS: Analyses of thrombin generation from combined inhibition in human plasma using statistical methods of Bliss independence identified a synergistic reduction in thrombin production 30% lower than predicted by simple additivity. The greatest synergy occurred at concentrations of each compound below their individual IC50 values. In a rabbit arterio-venous shunt model (RAV) of thrombosis, co-administration of PD0313052 and argatroban reduced thrombus weight (TW) to a much greater degree than expected by additivity alone producing a synergistic decrease of 45% over the level predicted by additivity. Analyses of thrombin generation in plasma samples from the RAV also demonstrated 38% synergy ex vivo. Furthermore, at plasma concentrations with the greatest synergistic effect, no increase in bleeding or appreciable change in prothrombin time, activated partial thromboplastin time, or activated clotting time was observed, but thrombus weight reduction was greater than twofold higher than that expected from simple additivity. CONCLUSIONS: These results demonstrate a significant synergistic antithrombotic effect of combining low doses of PD0313052 and argatroban and support the hypothesis that simultaneous targeting of multiple coagulation enzymes may offer an improved therapeutic index in the prevention and treatment of thrombosis.

Gas6 receptors Axl, Sky and Mer enhance platelet activation and regulate thrombotic responses.

Gas6 (encoded by growth arrest-specific gene 6) is a vitamin-K dependent protein highly homologous to coagulation protein S that is secreted from platelet alpha-granules and has recently been demonstrated to participate in platelet thrombus formation. The current study evaluated the contribution of each of the three known Gas6 receptors (Axl, Sky and Mer) in human and mouse platelet function. Flow cytometry analyses confirmed that all three receptors are present on both human and mouse platelets. Pre-incubation of human platelets with either an anti-Gas6 antibody or blocking antibodies to Sky or Mer inhibited platelet aggregation and degranulation responses to both ADP and the PAR-1 activating peptide, SFLLRN, by more than 80%. In contrast, a stimulatory anti-Axl antibody increased activation responses to these agonists, suggesting a potentiating role for Gas6 in platelet activation. Moreover, in a mouse model of thrombosis, administration of Gas6 or Sky blocking antibodies resulted in a decrease in thrombus weight similar to clopidogrel but, unlike clopidogrel, produced no increase in template bleeding. Thus, Gas6 enhances platelet degranulation and aggregation responses through its known receptors, promoting platelet activation and mediating thrombus formation such that its inhibition prevents thrombosis without increasing bleeding.

Inhibition and reversal of platelet-rich arterial thrombus in vivo: direct vs. indirect factor Xa inhibition.

BACKGROUND/OBJECTIVE: The efficacy of a direct factor (F)Xa inhibitor, ZK-807834, was compared with indirect inhibition by enoxaparin for inhibition and deaggregation of acute platelet-rich thrombi in a well-characterized porcine carotid injury model. METHODS: A crush injury was performed on a randomly chosen carotid artery and the thrombus allowed to propagate for 30 min. Pigs then received intravenous drug for 35 min: ZK-807834-Dose 1 (40 microg kg(-1) bolus + 1.5 microg kg(-1) min(-1) infusion, n=6); ZK-807834-Dose 2 (20 microg kg(-1) bolus + 0.75 microg kg(-1) min(-1) infusion; n=6); enoxaparin (1 mg kg(-1) bolus; n=6); or saline (n=6). Five minutes after drug initiation, the contralateral artery was injured. Thrombus size was monitored by scintillation detection of autologous 111In-platelets. RESULTS: The prothrombin time ratio was 2.2 +/- 0.1; 1.4 +/- 0.3; 1.2 +/- 0.9 and 1.1 +/- 0.2, respectively. ZK-807834-Dose 1 significantly inhibited carotid platelet deposition (525 +/- 226 x 10(6) cm(-2); P = 0.008), whereas ZK-807834-Dose 2 (2325 +/- 768) and enoxaparin (1236 +/- 383) were not different from saline (2776 +/- 642). Thrombus deaggregation was greatest for animals receiving ZK-807834-Dose 1 (473 +/- 185). Neither ZK-807834-Dose 2 (1588 +/- 480) nor enoxaparin (1618 +/- 686) was different from saline control (2222 +/- 598). CONCLUSIONS: Direct FXa inhibition with ZK-807834, at a prothrombin time ratio of 2.2, effectively inhibits thrombosis and promptly deaggregates thrombi induced by arterial injury. In contrast, indirect FXa inhibition with enoxaparin was ineffective.

Recent advances in the discovery and development of direct coagulation factor Xa inhibitors.

Coronary heart disease (CHD) is the leading cause of mortality and morbidity in the United States. Currently, there are approximately 12 million Americans with CHD, which is most frequently caused by atherosclerosis. The thrombotic complications of atherosclerosis, such as acute coronary syndrome and ischemic stroke, can be fatal and those who survive such events have a far greater risk of future cardiovascular events. This huge medical need cries out for improved novel anticoagulants, antiplatelet agents, and profibrinolytic agents. These agents will successfully respond to the medical need by providing safe, effective, and easily administered treatments that have little, if any, drug and food interactions and that require minimal monitoring. The currently approved antiplatelet agent, clopidogrel, has satisfied some of these requirements and has played a large role in expanding the antithrombotic market over the past few years. New antithrombotics approaching the marketplace, such as the prodrug thrombin inhibitor ximelagatran, have promise in expanding the antithrombotic market further. Over the past two decades, the pharmaceutical industry has mounted a huge effort to develop antithrombotics that function by inhibiting key enzymes positioned at "higher" levels of the coagulation system. Direct inhibitors of factor Xa, which may provide a better safety and efficacy profile than currently available agents, appear to be the next major class of antithrombotic agents poised to take the pharmaceutical industry one step closer to delivering the ideal antithrombotic agent. This review focuses on recent innovations in the discovery and development of potent parenteral and oral direct factor Xa inhibitors.

Non-hemostatic activity of coagulation factor Xa: potential implications for various diseases.

Because of its unique position at the convergence point of the intrinsic (contact) and extrinsic (tissue factor/factor VIIa) pathways in the coagulation system, coagulation factor Xa (FXa) has been a theoretically interesting therapeutic target for antithrombotic drugs for many years. More recently, the discovery of naturally occurring FXa inhibitors, such as tick anticoagulant peptide and antistasin, has helped substantiate FXa as a desirable target by demonstrating the efficacy and potential safety advantages of FXa inhibition over conventional antithrombotic therapy. These discoveries led to the design and development of many small-molecule inhibitors of FXa, which have provided potent and selective tools for evaluating the potential role of FXa in various diseases. In addition, these advances have been instrumental in defining the biology of FXa and have aided in the discovery of specific receptors and intracellular signaling pathways for FXa that may be important in the progression of, or the response to, various diseases.

Pharmacological characterization of a novel factor Xa inhibitor, FXV673.

FXV673 is a novel, potent, and selective factor Xa (FXa) inhibitor. FXV673 inhibited human, dog, and rabbit FXa with a K(i) of 0.52, 1.41, and 0.27 nM, respectively. FXV673 also displayed excellent specificity toward FXa relative to other serine proteases. It showed selectivity of more than 1000-fold over thrombin, activated protein C (aPC), plasmin, and tissue-plasminogen activator (t-PA). FXV673 prolonged plasma activated partial thromboplastin time (APTT) and prothrombin time (PT) in a dose-dependent fashion. In the APTT assays, the concentrations (microM) required for doubling coagulation time were 0.41 (human), 0.65 (monkey), 1.12 (dog), 0.25 (rabbit), and 0.80 (rat). The concentrations (microM) required in the PT assays were 1.1 (human), 1.32 (monkey), 2.31 (dog), 0.92 (rabbit), and 1.69 (rat). A coupled-enzyme assay was performed to measure thrombin activity following prothrombinase conversion of prothrombin to thrombin. FXV673 showed IC(50)s of 1.38 and 2.55 nM, respectively, when artificial phosphatidylserine/phosphatidylcholine (PS/PC) liposomes or fresh platelets were used as the phospholipid source for prothrombinase complex formation. It was demonstrated that FXV673 could inhibit further thrombin generation in the prothrombinase complex using PS/PC liposomes. FXV673 dose-dependently prolonged the time to vessel occlusion and inhibited thrombus formation in well-characterized canine models of thrombosis. Interspecies extrapolation (approximately 2.5-fold higher sensitivity for FXa inhibition in human than in dog) suggested that 100 ng/ml of FXV673 would be an effective plasma concentration for clinical studies. Currently FXV673 is undergoing clinical studies to be developed as an antithrombotic agent.

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.

Coagulation factor Xa inhibition: biological background and rationale.

Ischemic heart disease and cerebrovascular disease are the leading causes of death in the world. Surprisingly, these diseases are treated by relatively antiquated drugs. However, due to our improved understanding of the underlying pathology of these diseases, and a number of technological advances in tools for drug discovery and chemical optimization, an exciting new wave of antithrombotic compounds is beginning to emerge in clinical trials. These agents, referred to as direct coagulation factor Xa inhibitors, appear to provide an enhanced risk-benefit margin compared to conventional therapy. Preclinical and early clinical data gathered over the past few years suggests that direct fXa inhibitors will provide the necessary advancements in efficacy, safety, and ease of use required to displace conventional therapy. Whether or not these agents will succeed will be determined as this class of agents advances through clinical trials in the near future. This review describes some of the key studies that sparked an interest in fXa as a therapeutic target, highlighting the findings that provided important rationale for continuing the development of potent and selective direct fXa inhibitors.

In vitro characterization of a novel factor Xa inhibitor, RPR 130737.

RPR 130737 inhibited factor Xa (FXa) with a Ki of 2.4 nM and also displayed excellent specificity toward FXa relative to other serine proteases. It showed selectivity of more than 1000-fold over thrombin, activated protein C, plasmin, tissue-plasminogen activator and trypsin. RPR 130737 prolonged plasma activated partial thromboplastin time and prothrombin time in a dose-dependent fashion. In the activated partial thromboplastin time assay, the concentrations required for doubling coagulation time were 0.32 microM (human), 0.61 microM (monkey), 0.44 microM (dog), 0.15 microM (rabbit), and 0.82 microM (rat). The concentrations required to double prothrombin time were 0.86 microM (human), and 1.26 microM (monkey), 1.15 microM (dog), 0.39 microM (rabbit) and 7.31 microM (rat). Kinetic studies revealed that RPR 130737 was a fast binding, reversible and competitive inhibitor for FXa when Spectrozyme FXa, a chromogenic substrate, was used. A coupled-enzyme assay measuring thrombin activity following prothrombinase conversion of prothrombin to thrombin indicated that RPR 130737 was a potent inhibitor for prothrombinase-bound FXa. In this assay, RPR 130737 showed IC50s of 17 nM and 35.9 nM, respectively when artificial phosphatidylserine/phosphatidylcholine (PS/PC) liposomes or gel-filtered platelets were used as the phospholipid source. An FX-deficient plasma clotting-time correction assay further demonstrated that RPR 130737 was a specific inhibitor of FXa. RPR 130737 showed no effect on platelet aggregation in vitro. These results indicate that RPR 130737 has the potential to be developed as an antithrombotic agent based on its potent and selective inhibitory effect against FXa.

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.

Pharmacodynamic activity and antithrombotic efficacy of RPR120844, a novel inhibitor of coagulation factor Xa.

These studies were designed to examine the pharmacodynamic profile and antithrombotic efficacy of RPR120844, a competitive inhibitor of coagulation factor Xa, with a K(i) of 7 nM against human factor Xa. In vitro, RPR120844 doubled activated partial thromboplastin time (APTT) at concentrations of 1.54, 1.48, and 0.74 microM in plasma obtained from humans, dogs, and rats, respectively. Intravenous bolus administration of RPR 120844 at 0.3, 1, and 3 mg/kg to rats resulted in maximal increases in APTT of 1.8-, 2.6-, and 8.4-fold over baseline, respectively. The effect on prothrombin time (PT) was less pronounced, resulting in a 4.4-fold increase at 3 mg/kg. These effects were rapidly reversible; APTT and PT returned to control values by 30 min after dosing. Intragastric administration to rats at 50, 100, and 200 mg/kg resulted in modest increases in APTT and PT of 1.5- and 1.3-fold over baseline at the highest dose. Plasma levels were estimated by anti-Xa activity by using an amidolytic, chromogenic assay. Plasma levels were 0.65, 1.29, and 2.45 microM at 30 min after dosing at 50, 100, and 200 mg/kg, respectively. Intravenous administration to dogs at 0.1 and 0.3 mg/kg produced maximal increases in APTT of 1.7- and 2.4-fold over baseline, respectively. Intragastric administration to dogs at 50 mg/kg resulted in maximal increases in APTT and PT of 1.7- and 1.1-fold over baseline, with peak plasma levels of 3.9 microM observed at 15 min after dosing. In a rat model of FeCl2-induced carotid artery thrombosis, RPR120844 (3 mg/kg, i.v. bolus + 300 microg/kg/min constant infusion; n = 4) significantly increased time-to-occlusion from 18+/-1 min (vehicle, n = 4) to 60 min (maximal observation time) and reduced thrombus mass from 5.5 +/- 0.2 mg (vehicle) to 1.4 +/- 0.2 mg. These results indicate that RPR120844 is a potent, selective inhibitor of Xa that exhibits oral activity and is efficacious in a standard model of arterial thrombosis.

Synthesis, SAR and in vivo activity of novel thienopyridine sulfonamide pyrrolidinones as factor Xa inhibitors.

Thienopyridine sulfonamide pyrrolidinones were found to be potent and selective inhibitors of the coagulation cascade enzyme factor Xa. SAR studies led to several compounds that were selected for further in vivo investigation. These novel aryl binding pocket moieties represent a structural modification to a series of fXa inhibitors. Several compounds proved to be efficacious i.v. antithrombotic agents.

Design and structure-activity relationships of potent and selective inhibitors of blood coagulation factor Xa.

The discovery of a series of non-peptide factor Xa (FXa) inhibitors incorporating 3-(S)-amino-2-pyrrolidinone as a central template is described. After identifying compound 4, improvements in in vitro potency involved modifications of the liphophilic group and optimizing the angle of presentation of the amidine group to the S1 pocket of FXa. These studies ultimately led to compound RPR120844, a potent inhibitor of FXa (K(i) = 7 nM) which shows selectivity for FXa over trypsin, thrombin, and several fibrinolytic serine proteinases. RPR120844 is an effective anticoagulant in both the rat model of FeCl(2)-induced carotid artery thrombosis and the rabbit model of jugular vein thrombus formation.

Sulfonamidopyrrolidinone factor Xa inhibitors: potency and selectivity enhancements via P-1 and P-4 optimization.

Sulfonamidopyrrolidinones were previously disclosed as a selective class of factor Xa (fXa) inhibitors, culminating in the identification of RPR120844 as a potent member with efficacy in vivo. Recognizing the usefulness of the central pyrrolidinone template for the presentation of ligands to the S-1 and S-4 subsites of fXa, studies to optimize the P-1 and P-4 groups were initiated. Sulfonamidopyrrolidinones containing 4-hydroxy- and 4-aminobenzamidines were discovered to be effective inhibitors of fXa. X-ray crystallographic experiments in trypsin and molecular modeling studies suggest that our inhibitors bind by insertion of the 4-hydroxybenzamidine moiety into the S-1 subsite of the fXa active site. Of the P-4 groups examined, the pyridylthienyl sulfonamides were found to confer excellent potency and selectivity especially in combination with 4-hydroxybenzamidine. Compound 20b (RPR130737) was shown to be a potent fXa inhibitor (K(i) = 2 nM) with selectivity against structurally related serine proteinases (>1000 times). Preliminary biological evaluation demonstrates the effectiveness of this inhibitor in common assays of thrombosis in vitro (e.g. activated partial thromboplastin time) and in vivo (e.g. rat FeCl(2)-induced carotid artery thrombosis model).

RPR120844, a novel, specific inhibitor of coagulation factor Xa inhibits venous thrombosis in the rabbit.

The in vivo antithrombotic activity of RPR 20844, a novel synthetic coagulation factor Xa (fXa) inhibitor (Ki = 7 nM), was assessed by its ability to inhibit thrombus formation in a damaged segment of the rabbit jugular vein. Intravenous dose-response studies were performed and thrombus mass (TM), activated partial thromboplastin time (APTT), prothrombin time (PT), inhibition of ex vivo fXa activity and plasma drug levels (PDL) were determined. TM, measured at the end of a 50 min infusion, was significantly reduced (p<0.05 vs. saline-treated animals) by RPR120844 at 30 and 100 microg/kg/min. At doses of 10, 30 and 100 microg/kg/min, APTT was prolonged by 2.1, 4.2 and 6.1-fold, and PT was prolonged by 1.4, 2.2 and 3.5-fold, respectively. PDL were determined by measuring anti-fXa activity using an amidolytic assay. Peak PDL were 0.8+/-0.3, 1.5+/-0.9 and 2.4+/-0.6 microM, respectively. The drug effect was reversible with APTT, PT and PDL returning toward pretreatment values 30 min after termination of treatment. The results suggest that RPR 120844, or similar compounds, may provide an efficacious, yet easily reversible, means of inhibiting thrombus formation.

Design of a new class of orally active fibrinogen receptor antagonists.

The integrin receptor recognition sequence Arg-Gly-Asp was successfully used as a template from which to develop a series of potent, selective, orally active, peptide-based fibrinogen receptor antagonists with a long duration of action. Simple modifications centered on the Arg and Gly residues quickly led to a modified peptide (1) with significantly enhanced ability to inhibit in vitro platelet aggregation. Substitution of the guanidino group in 1 by piperidine provided 3, which showed not only a further increase in potency but also a modest degree of oral efficacy. Finally, exploration of the nature of the C-terminal amino acid, with respect to its side-chain functionality and the carboxy terminus, yielded a group of molecules that showed excellent in vitro potency for inhibiting platelet aggregation, excellent integrin selectivity, a high level of oral efficacy, and an extended duration of action.

Inhibition of repetitive thrombus formation in the stenosed canine coronary artery by enoxaparin, but not by unfractionated heparin.

Experiments were designed to compare the antithrombotic efficacy of enoxaparin and unfractionated heparin (UH) in a model of platelet-dependent cyclic flow reductions (CFRs) in the stenosed canine circumflex coronary artery. Low-molecular-weight heparins (LMWHs) are safe and effective in the prevention and treatment of venous thromboembolism. The present experiments were designed to evaluate the potential use of LMWHs in arterial thrombotic indications by comparing the antithrombotic effect of an LMWH with that of UH in an animal model of unstable angina. After establishment of consistent CFRs by experimentally induced vascular stenosis and damage, vehicle (saline), enoxaparin, or UH was administered intravenously as a loading dose plus a continuous infusion for 1 hour. The inhibition of CFRs was taken as an indicator of antithrombotic efficacy. Enoxaparin inhibited repetitive platelet thrombus formation in a dose-dependent manner, with significant inhibition of CFRs achieved at 0.5 mg/kg + 5 microg/kg per minute. This dose of enoxaparin resulted in anti-Xa levels of 0.9 to 1.0 IU/mL, anti-IIa levels of 0.2 to 0.3 IU/mL, activated partial thromboplastin time (APTT) of 1.3-fold over baseline, and a 1.4-fold increase (NS) in template bleeding time. Near-complete abolishment of CFRs was achieved with enoxaparin at 1.0 mg/kg + 10 microg/kg per minute. This dose of enoxaparin produced anti-Xa levels of 2 to 2.2 IU/mL, anti-IIa levels of 0.5 to 0.6 IU/mL, an increase in APTT of 1.4- to 1.5-fold over baseline, and a 1.9-fold increase (P<0.05) in template bleeding time. In contrast, UH had no significant effect on CFRs at a dose (100 U/kg + 10 U/kg per minute) that resulted in anti-Xa levels of 1.2 to 1.6 IU/mL, anti-IIa levels of 1.8 to 2.4 IU/mL, an increase in APTT greater than 10-fold over baseline, and a 2.5-fold increase (P<0.05) in template bleeding time. Compared with the vehicle group, circulating platelet count and hematocrit were not changed significantly by any dose of enoxaparin or UH tested. Enoxaparin, unlike UH, prevented repetitive platelet-dependent thrombus formation in the dog, thereby supporting the potential use of enoxaparin as a replacement for heparin in the treatment of arterial thrombotic disorders such as unstable angina.

Identification and initial structure-activity relationships of a novel class of nonpeptide inhibitors of blood coagulation factor Xa.

The discovery and some of the basic structure-activity relationships of a series of novel nonpeptide inhibitors of blood coagulation Factor Xa is described. These inhibitors are functionalized beta-alanines, exemplified by 2a. Docking experiments placing 2a in the active site of Factor Xa implied that the most expeditious route to enhancing in vitro potency was to modify the group occupying the S3 site of the enzyme. Increasing the hydrophobic contacts between the inhibitor and the enzyme in this region led to 8, which has served as the prototype for this series. In addition, an enantioselective synthesis of these substituted beta-alanines was also developed.