Favorable therapeutic index of the direct factor Xa inhibitors, apixaban and rivaroxaban, compared with the thrombin inhibitor dabigatran in rabbits.

BACKGROUND: Apixaban is an oral, direct factor Xa (FXa) inhibitor in late-stage clinical development. This study assessed effects of the direct FXa inhibitors, apixaban and rivaroxaban, vs. the direct thrombin inhibitor, dabigatran, on venous thrombosis (VT), bleeding time (BT) and clotting times in rabbits. METHODS: We induced the formation of non-occlusive thrombus in VT models by placing threads in the vena cava, and induced bleeding by the incision of cuticles in anesthetized rabbits. Apixaban, rivaroxaban and dabigatran were infused IV to achieve a stable plasma level. Clotting times, including the activated partial thromboplastin time (aPTT), prothrombin time (PT), modified PT (mPT) and thrombin time (TT), were measured. RESULTS: Apixaban, rivaroxaban and dabigatran exhibited dose-related efficacy in preventing VT with EC(50) of 65, 33 and 194 nm, respectively. At doses for 80% reduction of control thrombus, apixaban, rivaroxaban and dabigatran prolonged BT by 1.13 +/- 0.02-, 1.9 +/- 0.1-* and 4.4 +/- 0.4-fold*, respectively (*P < 0.05, vs. apixaban). In the treatment model, these inhibitors equally prevented growth of a preformed thrombus. Antithrombotic doses of apixaban and rivaroxaban prolonged aPTT and PT by <3-fold with no effect on TT. Dabigatran was > or = 50-fold more potent in prolonging TT than aPTT and PT. Of the clotting assays studied, apixaban, rivaroxaban and dabigatran responded the best to mPT. CONCLUSION: Comparable antithrombotic efficacy was observed between apixaban, rivaroxaban and dabigatran in the prevention and treatment of VT in rabbits. Apixaban and rivaroxaban exhibited lower BT compared with dabigatran at equivalent antithrombotic doses. The clinical significance of these findings remains to be determined.

Arterial antithrombotic and bleeding time effects of apixaban, a direct factor Xa inhibitor, in combination with antiplatelet therapy in rabbits.

BACKGROUND: Optimal treatment of arterial thrombosis may include a combination of antiplatelet and anticoagulant drugs. We evaluated apixaban, a direct and highly selective factor Xa inhibitor, in combination with clinically relevant doses of aspirin and/or clopidogrel for prevention of arterial thrombosis in rabbits. METHODS: Studies were conducted in rabbit models of electrically induced carotid artery thrombosis and cuticle bleeding time (BT). Apixaban 0.04 and 0.3 mg kg(-1) h(-1) or aspirin 1 mg kg(-1) h(-1) was infused intravenous (i.v.) continuously from 1 h before artery injury or cuticle bleed until the end of the experiment. Clopidogrel at 3 mg kg(-1) was dosed orally once daily for three days, with the last dose given 2 h before injury. RESULTS: Control thrombus weight and BT averaged 8.6 +/- 0.9 mg and 181 +/- 12 s, respectively (n = 6 per group). Effective doses of apixaban that reduced thrombus weight by 20 and 50% (ED(20) and ED(50)) were 0.04 and 0.3 mg kg(-1) h(-1) i.v., respectively. Addition of aspirin to apixaban ED(20) and ED(50) significantly reduced the thrombus weight from 7.4 +/- 0.5 to 5.3 +/- 0.3 and 3.6 +/- 0.3 mg, respectively, with no significant increases in BT (190 +/- 7 s vs.181 +/- 9 and 225 +/- 11 s, respectively). Addition of aspirin and apixaban (ED(20) dose) to clopidogrel produced a further significant reduction in thrombus weight from 5.3 +/- 0.3 to 0.7 +/- 0.1 mg. This combination of clopidogrel and aspirin with apixaban (ED(20) dose) produced a significant but moderate BT increase of 2.1 times control. CONCLUSIONS: The combination of apixaban and aspirin or apixaban, aspirin and clopidogrel can reduce formation of occlusive arterial thrombosis without excessive increases in BT in rabbits.

Apixaban, an oral, direct and highly selective factor Xa inhibitor: in vitro, antithrombotic and antihemostatic studies.

BACKGROUND: Apixaban is an oral, direct and highly selective factor Xa (FXa) inhibitor in late-stage clinical development for the prevention and treatment of thromboembolic diseases. OBJECTIVE: We evaluated the in vitro properties of apixaban and its in vivo activities in rabbit models of thrombosis and hemostasis. METHODS: Studies were conducted in arteriovenous-shunt thrombosis (AVST), venous thrombosis (VT), electrically mediated carotid arterial thrombosis (ECAT) and cuticle bleeding time (BT) models. RESULTS: In vitro, apixaban is potent and selective, with a K(i) of 0.08 nm for human FXa. It exhibited species difference in FXa inhibition [FXa K(i) (nm): 0.16, rabbit; 1.3, rat; 1.7, dog] and anticoagulation [EC(2x) (microm, concentration required to double the prothrombin time): 3.6, human; 2.3, rabbit; 7.9, rat; 6.7, dog]. Apixaban at 10 microm did not alter human and rabbit platelet aggregation to ADP, gamma-thrombin, and collagen. In vivo, the values for antithrombotic ED(50) (dose that reduced thrombus weight or increased blood flow by 50% of the control) in AVST, VT and ECAT and the values for BT ED(3x) (dose that increased BT by 3-fold) were 0.27 +/- 0.03, 0.11 +/- 0.03, 0.07 +/- 0.02 and > 3 mg kg(-1) h(-1) i.v. for apixaban, 0.05 +/- 0.01, 0.05 +/- 0.01, 0.27 +/- 0.08 and > 3 mg kg(-1) h(-1) i.v. for the indirect FXa inhibitor fondaparinux, and 0.53 +/- 0.04, 0.27 +/- 0.01, 0.08 +/- 0.01 and 0.70 +/- 0.07 mg kg(-1) day(-1) p.o. for the oral anticoagulant warfarin, respectively. CONCLUSIONS: In summary, apixaban was effective in the prevention of experimental thrombosis at doses that preserve hemostasis in rabbits.

Nonpeptide factor Xa inhibitors II. Antithrombotic evaluation in a rabbit model of electrically induced carotid artery thrombosis.

SK549 (mol. wt. 546 Da) is a synthetic, selective inhibitor of human coagulation factor Xa (fXa) (K(i) = 0.52 nM). This study compared the antithrombotic effects of SK549 and a series of benzamidine isoxazoline fXa inhibitors with aspirin, DuP 714 (a direct thrombin inhibitor), recombinant tick anticoagulant peptide, or heparin in a rabbit model of electrically induced carotid arterial thrombosis. Compounds were infused i.v. continuously from 60 min before electrical stimulation to the end of the experiment. Values of ED(50) (dose that increases the carotid blood flow to 50% of the control) were 0.12 micromol/kg/h for SK549, 0.56 micromol/kg/h for aspirin, 0.14 micromol/kg/h for DuP 714, 0.06 micromol/kg/h for recombinant tick anticoagulant peptide, and >100 U/kg/h for heparin. The EC(50) (plasma concentration that increased blood flow to 50% of the control) for SK549 was 97 nM. Unlike aspirin and heparin, SK549 was efficacious and, at 1.5 micromol/kg/h i.v. (n = 9), maintained carotid blood flow at 87 +/- 6% of control level for greater than 90 min. Unlike heparin, SK549 inhibited ex vivo fXa activity but not ex vivo thrombin activity. There was a highly significant correlation between K(i) (fXa) and ED(50) of a series of fXa inhibitors (r = 0. 85, P <.001). Therefore, these results suggest that SK549 is a novel, potent, and effective antithrombotic agent in a rabbit model of arterial thrombosis. It is likely that SK549 exerts its antithrombotic effect through selective inhibition of fXa. Furthermore, SK549 may be clinically useful for the prevention of arterial thrombosis.

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Nonpeptide factor Xa inhibitors: I. Studies with SF303 and SK549, a new class of potent antithrombotics.

A series of benzamidine isoxazoline derivatives was evaluated for their inhibitory potency against purified human factor Xa (fXa) and in a rabbit model of arteriovenous shunt thrombosis for their antithrombotic activities, expressed as K(I) and IC(50), respectively. A highly significant correlation was found between K(I) and IC(50) (r = 0.93, P <.0001). The antithrombotic effects of SF303 [mol. wt. 536; K(I): fXa, 6.3 nM; thrombin, 3,100 nM; trypsin, 110 nM; tissue plasminogen activator >20,000 nM; plasmin, 2,500 nM] and SK549 [mol. wt. 546; K(I): fXa, 0.52 nM; thrombin, 400 nM; trypsin, 45 nM; tissue plasminogen activator >33,000 nM; plasmin, 890 nM] were compared with recombinant tick anticoagulant peptide [K(I)(fXa) = 0.5 nM], DX-9065a [K(I)(fXa) = 30 nM], and heparin or low molecular weight heparin (dalteparin) in a rabbit model of arteriovenous shunt thrombosis. ID(50) values for preventing arteriovenous shunt-induced thrombosis were 0.6 micromol/kg/h for SF303, 0.035 micromol/kg/h for SK549, 0.01 micromol/kg/h for recombinant tick anticoagulant peptide, 0.4 micromol/kg/h for DX-9065a, 21 U/kg/h for heparin, and 23 U/kg/h for low molecular weight heparin. SK549 produced a concentration-dependent antithrombotic effect with an IC(50) of 0.062 microM. To evaluate its potential oral efficacy, SK549 was given intraduodenally at a dose of 5 mg/kg; it produced a peak antithrombotic effect of 59 +/- 4% with a duration of action greater than 6.7 h. Therefore, our study suggests that SF303, SK549, and their analogs represent a new class of synthetic fXa inhibitors that may be clinically useful as antithrombotic agents.

Antithrombotic actions of selective inhibitors of blood coagulation factor Xa in rat models of thrombosis.

The antithrombotic actions of selective factor Xa (FXa) inhibitors, recombinant tick anticoagulant peptide (rTAP) and DX-9065a, were evaluated in experimental thrombosis models in anesthetized rats. In the first model, thrombosis was induced by exposing flowing blood to a silk thread anchored in an arteriovenous (AV) shunt. rTAP, DX-9065a and heparin, given as an iv infusion 1 hr before blood was circulated in the AV shunt, had ID50s of 0.007, 0.6 mumol/kg/hr and 16 U/kg/hr, respectively. In the model of venous thrombosis which was induced by hypotonic saline (0.225%) followed by 15-min stasis of abdominal vena cava, rTAP and heparin had ID50s of 0.007 mumol/kg/hr and 3.5 U/kg/hr, respectively. In both models, full inhibition of thrombus formation was achieved with FXa inhibition at doses which only modestly increased ex vivo plasma clotting time APTT (1.26 to 1.82 over the baseline). By contrast, the maximum antithrombotic effect of heparin was associated with high and significant APTT prolongation (> 5 fold over the baseline). Therefore, our study suggests that FXa inhibitors are effective agents in preventing thrombosis in both rat thrombosis models and may have therapeutic antithrombotic potential.

Pharmacology of XR510, a potent orally active nonpeptide angiotensin II AT1 receptor antagonist with high affinity for the AT2 receptor subtype.

The angiotensin II (Ang II) type 1 receptor (AT1) mediates all known physiological effects of ANG II, whereas functions of the type 2 (AT2) receptor are not clear. Should undesirable AT2 effects be identified, it may be advantageous to combine antagonism of AT1 and AT2 receptors. XR510 was shown to inhibit the specific binding of [125I]Sar1,Ile8-Ang II for AT1 and AT2 subtype binding sites in rat adrenal membranes with IC50 of 0.26 and 0.28 nM, respectively, and in human tissues with subnanomolar binding affinity. In isolated rabbit aorta, XR510 exerted insurmountable Ang II antagonism with a Kb value of 4 nM. In conscious renal hypertensive rats, XR510 decreased blood pressure (BP) with intravenous (i.v.) and oral (p.o.) ED30 of 0.08 and 0.27 mg/kg, respectively. In spontaneously hypertensive rats (SHR), repeated daily oral dosing of XR510, losartan, and enalapril at 30 mg/kg/day decreased BP similarly. In conscious furosemide-treated dogs, XR510, given either intravenously or orally, decreased BP. These results suggest that XR510 is an orally active and selective Ang II receptor antagonist with equal binding affinities for AT1 and AT2 receptor binding sites.