Rat Tail Transection Model Research Articles

Synthetic, organic compound vepoloxamer (P-188) potentiates tissue plasminogen activator

ObjectivePoloxamer-188 is a synthetic, organic compound that acts by binding hydrophobic pockets on damaged lipid bilayers in the circulation. P-188 reduces blood viscosity and confers anti-inflammatory and cytoprotective effects. Vepoloxamer (Mast Therapeutics, San Diego, Calif) is a purified version of this compound that has limited side effects. The aim of this study was to investigate drug interactions between vepoloxamer and heparin and tissue plasminogen activator (tPA). MethodsAn experimental rat tail transection model was used to study vepoloxamer's interaction with heparin. Sprague-Dawley rats were divided into saline (1 mL/kg; group 1) or vepoloxamer (25 mg/kg; group 2) treatment groups. The rats were then subjected to saline (n = 6), low-dose heparin (125 μg/kg; n = 6), or high-dose heparin (250 μg/kg; n = 6). After 5 minutes, the distal 2 mm of the tail was transected, and time to clot formation was measured as bleeding time. A rat internal jugular vein thrombosis model was used to assess vepoloxamer's interaction with tPA. Sprague-Dawley rats were divided into saline (1 mL/kg; group 1) or vepoloxamer (25 mg/kg; group 2) treatment groups. After internal jugular vein thrombosis, rats were treated with saline (n = 6), systemic low-dose tPA (0.5 mg/kg; n = 6), or systemic high-dose tPA (1.0 mg/kg; n = 6). Clot lysis was assessed using an ultrasound Doppler probe to detect blood flow. No flow up to 15 minutes was recorded as no lysis. ResultsInteraction with heparin: Vepoloxamer by itself, without any heparin, increased tail bleeding time (10.3 vs 7.1 minutes; P = .001). Effects of heparin on tail bleeding time were enhanced by vepoloxamer at low dose (14.2 vs 6.2 minutes; P < .001). At high-dose heparin, vepoloxamer did not prolong bleeding time (17.8 vs 17.0 minutes). Interaction with tPA: No rat exhibited spontaneous clot lysis with either saline or vepoloxamer. The effect of tPA was facilitated by vepoloxamer at low dose, as more rats showed clot lysis (4/6 [66%]) compared with tPA alone, which showed no clot lysis (0/6), although statistical significance was not reached (P = .06). At high-dose tPA, vepoloxamer had no additional effects on clot lysis (5/6 [83% ] vs 4/6 [66%]). ConclusionsVepoloxamer alone modestly increased bleeding time. Vepoloxamer also increased bleeding time in rats treated with low-dose heparin but not with high-dose heparin. Vepoloxamer potentiated clot lysis in the setting of low-dose tPA.

Abstract 19361: Reversal of Anticoagulant-induced Bleeding in External and Internal Bleeding Models by PER977, a Small Molecule Anticoagulant Antidote

The new oral anticoagulants (NOACs) offer significant advantages over heparins and warfarin therapies. However, concern over serious bleeding and potential over-dosage is heightened with the lack of a specific reversal agent. PER977 is a synthetic small molecule in clinical development as an anticoagulant reversal agent. In the current studies, PER977 reversal was evaluated in two preclinical bleeding models, recapitulating the clinical scenarios of reversal in actively bleeding cutaneous wounds and internal large organ lacerations. Reversal of anticoagulation-induced, active bleeding was demonstrated in a rat tail transection model. Rats were dosed with the direct factor Xa inhibitor, edoxaban, resulting in large increases in blood loss mass. Briefly, the rat was orally dosed with edoxaban. After 75 min, rat tails were fully transected 2 mm from the tip. Immediately after tail transection, PER977 solution or saline was administered through the jugular vein. Blood from the tail was collected for 7.5 minutes and then transferred into a second collection tube for an additional 7.5 minutes. PER977 significantly decreased bleeding in vivo in rats treated with edoxaban (Figure 1a) within the first 7.5 minutes after PER977 administration. Additionally, bleeding was evaluated in a rat liver laceration model of internal bleeding. Following calibrated cuts to the medial lobe of the exposed liver, bleeding time was determined to the nearest 15 seconds. This rat liver laceration internal bleeding model demonstrated that edoxaban can significantly increase the bleeding time after the rat liver is lacerated. Moreover, the i.v. administration of PER977 can fully reverse the anticoagulant effect of edoxaban 10 minutes after PER977 administration (Figure 1b). In conclusion, PER977 is a synthetic small molecule that reverses NOAC anticoagulation in animal models of external and internal bleeding, and is currently in phase 2 clinical studies with edoxaban.

Abstract 18809: PER977: A Synthetic Small Molecule Which Reverses Over-Dosage and Bleeding by the New Oral Anticoagulants

With regards to route of administration, drug interactions and predictability of bioactivity, the new oral anticoagulants (NOACs; direct factor Xa and IIa inhibitors) offer significant advantages over heparins and warfarin therapies. However, concern over serious bleeding, emergency procedures and potential over-dosage is heightened with the current lack of a specific reversal agent. PER977 is a synthetic small molecule rationally designed anticoagulant antidote. Reversal of anticoagulation induced bleeding was demonstrated in a rat tail transection model: weight-matched rats were overdosed with rivaroxaban, edoxaban, dabigatran, or apixaban, resulting in large increases in blood loss volume. PER977 was administered IV and after thirty minutes, blood loss volume was quantified. PER977 significantly decreased bleeding in vivo in rats treated with NOAC (Figure 1a). PT (for edoxaban, rivaroxaban, and apixaban) or aPTT (for dabigatran) and thromboelastography (TEG) (TEG- for edoxaban, Figure 1b) were also used to quantify NOAC anticoagulation reversal in vivo in rats; PER977 restored these laboratory values to baseline levels within 20 minutes of administration. Importantly, PER977 has also shown no pro-coagulant properties as measured by TEG, PT and aPTT in rat and human whole blood, nor any interaction with circulating coagulation factor proteins. In vivo and in vitro toxicology and safety studies have been completed and a first-in-human clinical trial to demonstrate safety and efficacy in healthy human volunteers with PER977 and edoxaban is underway. In conclusion, PER977 is a synthetic small molecule new chemical entity under development that reverses NOAC anti-coagulation in vivo as measured by TEG, PT, and aPTT, and also by bleeding volume in a rat tail transection model.

Comparative Antithrombotic and Bleeding Effects of Two U.S. Generic Enoxaparins

Abstract 1172 Background:Low molecular weight heparins (LMWHs) are complex biologic drugs that exhibit heterogeneity in terms of saccharide chain length and in the composition (sulfate, acetyl), content, and location of functional groups. Such heterogeneity impacts the biologic activity of LMWHs as there is a certain threshold chain length required for thrombin inhibitory activity and a particular sequence is required for interaction with antithrombin. In July 2010 the US Food and Drug Administration published requirements necessary to demonstrate the ‘sameness' of generic LMWHs with the originator LMWH. We undertook this study to compare the primary anticoagulant effect of thrombosis prevention and the primary adverse effect of bleeding of two FDA approved generic enoxaparins. Methods:Four batches of commercially available Sandoz US generic enoxaparin (Princeton, NJ) and two batches of Watson US generic enoxaparin (Parsippany, NJ) were compared. All products were obtained from hospital pharmacies as pre-filled syringes containing 40 mg of the drugs. The molecular weight profile of each batch was determined by HPLC in relation to well-defined heparin fractions and by utilizing the US Pharmacopeia method. In vitro activity was determined by supplementing each LMWH batch to normal human plasma over a range of concentrations (0–10 mg/ml) and analyzing these samples using aPTT, anti-FXa and anti-FIIa assays. Hemorrhagic activity was measured using a rat tail transection model five minutes after administration of a 2 mg/kg intravenous dose (n=8 rats/batch). Upon completion of the bleeding model, antithrombotic activity was assessed using a jugular vein clamping model (∼90 minutes post-dosing). Blood samples collected from treated rats were used to estimate circulating blood levels of LMWH using anti-FXa and anti-FIIa assays. Results:The two groups of generic enoxaparins exhibited a similar molecular weight profile with mean molecular weights of 4,270 ± 20 Da for the Sandoz products and 4,420 ± 80 Da for the Watson products. In vitro activities were similar between batches of the same product, but the individual products differed considerably (p=0.01). In the aPTT and anti-FIIa assays, the Watson LMWH produced significantly more activity than the Sandoz LMWH at concentrations ≥5 μg/ml. At 5 μg/ml, a clotting time of 74.0 ± 16.6 sec was observed with Watson batches compared to clotting times ranging from 52.9 to 54.9 sec for Sandoz LMWHs. While a similar pattern was observed with the anti-FXa assay, the differences between products were not statistically significant. In the bleeding model, all LMWHs prolonged the bleeding time compared to vehicle control. One batch of the Watson LMWH, however, produced a significantly longer bleeding time compared to all other samples tested (33.8 ± 5.1 min vs. a range of 11.9 ± 2.4 to 18.3 ± 4.3 minutes for the other samples; p=0.003). The same batch produced significantly more antithrombotic activity (6.3 ± 0.7 clampings) compared to the other samples (range of 4.0 ± 0.6 to 4.6 ± 0.5 clampings; p<0.001). One batch of Sandoz LMWH produced a significantly smaller prolongation of bleeding time compared to other samples. Circulating drug levels determined by anti-FXa and anti-FIIa activities were comparable in all treatment groups but did not appear to correlate with hemorrhagic or antithrombotic activities. Conclusion:The expectation was that all batches of all generic enoxaparins would produce the same in vitro, in vivo, and ex vivo outcomes. The findings of this study suggest that incorporation of traditional animal models in the development of generic enoxaparins may be of value as multiple biological effects of LMWHs, not fully addressed with the anti-FXa and anti-FIIa activities, contribute to the overall antithrombotic activity of these LMWHs. This study demonstrates that all generic enoxaparins available today may not necessarily be the same. These findings underscore the importance of in vivo studies in animal models to demonstrate the bioequivalence of the generic products. Disclosures:Jeske:Sanofi-Aventis, Paris, France: Research Funding. Walenga:Sanofi-Aventis, Paris, France: Research Funding. Escalante:Sanofi-Aventis, Paris, France: Research Funding. Hoppensteadt:Sanofi-Aventis, Paris, France: Research Funding. Cunanan:Sanofi-Aventis, Paris, France: Research Funding. Kahn:Sanofi-Aventis, Paris, France: Research Funding. Paulus:Sanofi-Aventis, Paris, France: Research Funding. Fareed:Sanofi-Aventis, Paris, France: Research Funding. Bakhos:Sanofi-Aventis, Paris, France: Research Funding.

PRT064445 but Not Recombinant Fviia Reverses Rivaroxaban Induced Anticoagulation As Measured by Reduction in Blood Loss in a Rabbit Liver Laceration Model

AbstractAbstract 3414We used a modified rabbit liver laceration model to demonstrate the effects of PRT064445, a recombinant fXa derivative, to reverse rivaroxaban induced anticoagulation as measured by reduction of blood loss, decrease of unbound fraction of rivaroxaban in plasma and relevant pharmacodynamic markers: anti-fXa activity, prothrombin (PT) and activated thromboplastin (aPTT) times. Recombinant fVIIa (rfVIIa) was tested in the same model for comparison.Anesthetized rabbits were administered vehicle or 1 mg/kg rivaroxaban via IV bolus injection. After 30 minutes to allow the rivaroxaban to biodistribute, vehicle, PRT064445 or rfVIIa was administered as a bolus injection followed by laceration of two liver lobes (5× each lobe: 1-cm long and 3-mm deep incisions with scalpel blade) and blood loss collected on pre-weighed gauze over 15 minutes. Blood loss due to rivaroxaban anticoagulation represented approximately 10% of the animal’s total blood volume. Total rivaroxaban concentration in plasma was measured by LC-MS/MS. Free fraction of rivaroxaban (non-protein, non-PRT064445 bound) was assessed using equilibrium dialysis method followed by LC-MS/MS quantitation. Anti-fXa activity was measured using a modified chromogenic LMW heparin kit. PT and aPTT was measured using commercial reagents (HemosIL). Anticoagulation by rivaroxaban (1.65 μM average plasma concentration at 30 min time point) resulted in 2.3-fold and 1.9-fold prolongation of PT and aPTT, respectively, and increased blood loss by 3.2-fold over vehicle. PRT064445 (75 mg/rabbit) administration reduced blood loss due to rivaroxaban anticoagulation by >85% and decreased peak anti-fXa activity by 98%, PT by 74%, aPTT by 66% and the free fraction of rivaroxaban in plasma from 26% to <0.5%. In contrast, rfVIIa (150 μg/kg) had no effect on blood loss but reversed PT by 85% and aPTT by 54%. PRT064445 and rfVIIa alone had no effect on blood loss, but rfVIIa decreased PT by 35%.These data demonstrate that PRT064445 can reduce blood loss due to rivaroxaban induced anticoagulation using a single bolus administration. Reduction of blood loss with PRT06445 correlated to the decrease in the free fraction of rivaroxaban in plasma as well as PD markers anti-fXa activity and PT, while rfVIIa decreased PT but had no effect on blood loss. These and our previous data with reversal of enoxaparin-induced anticoagulation in a rat tail transection model indicate that PRT064445 can be used as a universal antidote for both direct and indirect fXa inhibitors. Disclosures:Hollenbach:Portola Pharmaceuticals: Employment. Lu:Portola Pharmaceuticals: Employment. Tan:Portola Pharmaceuticals: Employment. Lee:Portola Pharmaceuticals: Employment. Athiwat:Portola Pharmaceuticals: Employment. Inagaki:Portola Pharmaceuticals: Employment. Sinha:Portola Pharmaceuticals Inc.: Employment.

Novel Antiplatelet Activity of Protocatechuic Acid through the Inhibition of High Shear Stress-Induced Platelet Aggregation

Bleeding is the most common and serious adverse effect of currently available antiplatelet drugs. Many efforts are being made to develop novel antithrombotic agents without bleeding risks. Shear stress-induced platelet aggregation (SIPA), which occurs under abnormally high shear stress, plays a crucial role in the development of arterial thrombotic diseases. Here, we demonstrate that protocatechuic acid (PCA), a bioactive phytochemical from Lonicera (honeysuckle) flowers, selectively and potently inhibits high shear (>10,000 s(-1))-induced platelet aggregation. In isolated human platelets, PCA decreased SIPA and attenuated accompanying platelet activation, including intracellular calcium mobilization, granule secretion, and adhesion receptor expression. The anti-SIPA effect of PCA was mediated through blockade of von Willebrand factor binding to activated glycoprotein Ib, a primary and initial event for the accomplishment of SIPA. Conspicuously, PCA did not inhibit platelet aggregation induced by other endogenous agonists like collagen, thrombin, or ADP that are important in both pathological thrombosis and normal hemostasis. Antithrombotic effects of PCA were confirmed in vivo in a rat arterial thrombosis model, where PCA significantly delayed the arterial occlusion induced by FeCl(3). Of particular note, PCA did not increase bleeding times in a rat tail transection model, whereas conventional antiplatelet drugs, aspirin, and clopidogrel substantially prolonged it. Collectively, these results suggest that PCA may be a novel antiplatelet agent that can prevent thrombosis without increasing bleeding risks.

Abstract 414: Combined Tissue Factor/Factor VIIa Inhibitor and Acetylsalicylic Acid Show Increased Antithrombotic Potency Without Affecting Bleeding Time

Since increased tissue factor (TF) in human atherosclerotic plaques is believed to trigger thrombotic events, TF/factor VIIa (FVIIa) inhibition might have prophylactic potential for atherothrombosis such as acute coronary syndrome (ACS). Although recent clinical observation by rivaroxaban provides a scientific rationale for the addition of anticoagulant to current antiplatelet treatment, these might have potential to increase bleeding. It is therefore important that emerging anticoagulants demonstrate additive antithrombotic effects with no concomitant increase in bleeding for ACS indication. We recently discovered ER-410660, a potent TF/FVIIa inhibitor. It suppressed venous thrombosis without affecting bleeding time and suggested that ER-410660 could act as a novel anticoagulant with a low bleeding risk. In the photochemically-induced thrombosis (PIT) model, the thrombus after vessel-wall injury has been developed by the deposition of TF, platelets, and fibrin. Therefore, we hypothesized that ER-410660 would be effective in the PIT model and could show enough margin against bleeding. The antithrombotic effects of ER-410660 and acetylsalicylic acid (ASA), both alone and in combination, was assessed using a rat PIT model. We also measured the bleeding time of ER-410660 in combination with ASA using a rat tail-transection model. Intravenous (iv) pretreatment with ER-410660 prolonged the time to occlusion (TTO) of the artery. The TTO was doubled at 0.99 mg/kg (95% CI, 0.51-1.9). Orally administered ASA also showed antithrombotic effect, and the TTO was doubled at approximately 12 mg/kg. Combined administration of 1 mg/kg ER-410660 (iv) and 10 mg/kg ASA (orally) showed an additive antithrombotic effect, with no cessation of blood flow during the 30-min observation period. Intravenous administration of ER-410660 (0.6, 2, and 6 mg/kg) to rats treated with ASA (10 mg/kg) did not significantly increased when compared with ASA alone, even at the highest dose. In conclusion, a TF/FVIIa inhibitor suppressed arterial thrombosis without affecting the bleeding time associated with ASA. Owing to these favorable characteristics, anticoagulation via TF/FVIIa inhibition may be a superior approach to the prophylaxis of atherothrombotic diseases.

Synergistic effect of a factor Xa inhibitor, TAK-442, and antiplatelet agents on whole blood coagulation and arterial thrombosis in rats

IntroductionActivated platelets facilitate blood coagulation by providing factor V and a procoagulant surface for prothrombinase. Here, we investigated the potential synergy of a potent factor Xa/prothrombinase inhibitor, TAK-442, plus aspirin or clopidogrel in preventing arterial thrombosis and whole blood coagulation. MethodsThrombus formation was initiated by FeCl3-induced rat carotid injury. Bleeding time was evaluated with the rat tail transection model. Whole blood coagulation was assessed by thromboelastographic examination (TEG) for which blood obtained from control, aspirin-, or clopidogrel-treated rats was transferred to a TEG analyzer containing, collagen or adenosine diphosphate (ADP), and TAK-442 or vehicle. ResultsTAK-442 (3mg/kg, po), aspirin (100mg/kg, po) or clopidogrel (3mg/kg, po) alone had no significant effect on thrombus formation, whereas the combination of TAK-442 with aspirin and clopidogrel remarkably prolonged the time to thrombus formation without additional significant prolongation of bleeding time. TEG demonstrated that the onset of collagen-induced blood coagulation were slightly longer in aspirin-treated rats than control; however, when the blood from aspirin-treated rats was subsequently treated in vitro with 100nM TAK-442, the onset of clotting was significantly prolonged. In contrast, only marginal prolongation was observed with TAK-442 treatment of blood from control animals. The onset time of ADP-induced blood coagulation was slightly longer in clopidogrel-treated rats compared with control, and it was further extended by TAK-442 treatment. ConclusionThese results demonstrate that blood coagulation can be markedly delayed by the addition of TAK-442 to antiplatelets treatment which could contribute to synergistic antithrombotic efficacy in these settings.

Antithrombotic Potential of GW813893: A Novel, Orally Active, Active-site Directed Factor Xa Inhibitor

Factor Xa (FXa) has been a target of considerable interest for drug development efforts aimed at suppressing thrombosis. In this report, a new orally active, small molecule, active-site directed FXa inhibitor, GW813893, has been profiled in a succession of in vitro and in vivo assays involved in its preclinical characterization as a potential antithrombotic therapeutic. In vitro profiling of GW813893 consisted of assessing its inhibitory potential against FXa and a broad panel of related and unrelated enzymes and receptors. Additionally, the FXa inhibition potential of GW813893 was assessed in prothrombinase and plasma-based clotting assays. In vivo characterization of GW813893 consisted of thrombosis studies in a rat inferior vena cava model, a rat carotid artery thrombosis model, and a rabbit jugular thrombosis model. Bleeding studies were conducted in a rat tail transection model. Ex vivo determinations of compound effects on FX and clotting activity were also undertaken. GW813893 was more than 90-fold selective over all enzymes tested, and it inhibited FXa and prothrombinase activity with a Ki of 4.0 nM and 9.7 nM, respectively. In vivo, GW813893 concentration-dependently suppressed thrombotic activity in all models tested. The antithrombotic activity correlated with the suppression of plasma-based clotting activity and the inhibition of plasma FX activity (P < 0.02). Over the antithrombotic dose-range, an increased bleeding diathesis was not observed. These experiments demonstrate that GW813893 is a potent, selective, orally active inhibitor of FXa. The data suggest that GW813893 has robust antithrombotic potential at doses that have no detectable hemostasis liability. Collectively, the profile suggests that GW813893 has the preclinical pharmacology underpinnings of an oral antithrombotic therapeutic.

Comparison of YM150, an Oral, Direct Factor Xa Inhibitor, with Other Antithrombotic Agents in Rodent Venous and Arterial Thrombosis Models.

YM150, an oral, direct factor Xa inhibitor, is currently being evaluated in Phase II studies as prophylaxis for venous thromboembolism in patients undergoing orthopedic surgery. In the present study, we compared the antithrombotic effect of YM150 with the effects of antithrombin-dependent indirect factor Xa inhibitors, enoxaparin and fondaparinux, and a direct thrombin inhibitor, ximelagatran, in ferric chloride (FeCl3)-induced venous and arterial thrombosis models in rats. We also evaluated the bleeding time in a rat tail transection model. Prior to any experimentation, male Sprague-Dawley rats, which had been fasting for at least 12 h, were anesthetized with urethane (1 g/kg, i.p.) or sodium pentobarbital (50 mg/kg, i.p.). YM150 and ximelagatran were administered intra-duodenally, and both enoxaparin and fondaparinux were given subcutaneously, 30 min prior to induction of thrombus or tail transection. All animals were kept warm with a heating pad during the experiments. Venous and arterial thromboses were produced, respectively, by the 5 min application of 8% FeCl3 soaked filter paper to the external surface of the inferior vena cava and 35% FeCl3 soaked filter paper to the abdominal aorta. The venous thrombosis model was supplemented by using a silk thread venous stenosis. To measure bleeding time, the tail was transected 5 mm from its tip. Blood was carefully blotted each 30 sec with a filter paper. Once a blood stain was observed, we defined bleeding as blood flow sustained over 30 sec. Bleeding time was defined as the sum of the bleeding periods during the 60 min observation in each animal. Administration of intra-duodenal YM150 significantly inhibited both venous and arterial thrombus formation at doses of 10 mg/kg or greater, and 3 mg/kg or greater, respectively. This indicated that YM150 promoted an antithrombotic effect at similar dose ranges for venous and arterial thromboses. In contrast, YM150 did not prolong the bleeding time at doses up to 30 mg/kg. Venous thrombus formation was inhibited by subcutaneous enoxaparin at doses of 100 IU/kg or greater and fondaparinux at doses of 0.03 mg/kg or greater. Arterial thrombus formation was inhibited by subcutaneous administration of 1000 IU/kg enoxaparin and 3 mg/kg fondaparinux. The results indicated that 10–100 times higher doses of these antithrombotics were needed to inhibit arterial thrombosis. Furthermore, enoxaparin at doses of 300 IU/kg or greater and fondaparinux at doses of 1 mg/kg or greater, significantly prolonged the bleeding time, suggesting that these two medications may be associated with increased risk of hemorrhage at concentrations used to prevent arterial thrombosis. At doses of 1 mg/kg or greater, intra-duodenal ximelagatran inhibited both venous and arterial thrombus formation. The dose-response curve for ximelagatran tended to be steeper than that for other anticoagulants tested. Antithrombotic doses of ximelagatran (1 mg/kg or greater), produced similar prolongations of bleeding time as those seen with administration of enoxaparin and fondaparinux. In conclusion, YM150, an oral direct factor Xa inhibitor, shows promise as an antithrombotic drug with potentially wider safety margins than current antithrombin-dependent factor Xa inhibitors and a thrombin inhibitor.

The Antithrombotic Efficacy of AT-1459, a Novel, Direct Thrombin Inhibitor, in Rat Models of Venous and Arterial Thrombosis

The antithrombotic efficacy of AT-1459, a novel, direct thrombin inhibitor (Ki = 4.9 nM) was evaluated in rat models of venous thrombosis combined with a bleeding time test and arterial thrombosis. After drugs were given by i. v. bolus injection plus a continuous infusion, the ID50, (a dose that exhibits 50% inhibition of thrombus formation over each vehicle group) values of AT-1459, argatroban, and dalteparin were 0.04 mg/kg plus 0.04 mg/kg/h, 0.1 mg/kg plus 0.4 mg/ kg/h, and 13.0 IU/kg plus 26.0 IU/kg/h, respectively, in the venous thrombosis study. The BT2 (a dose that causes 2-fold prolongation of bleeding time over each vehicle group) values of AT-1459, argatroban, and dalteparin were 0.9 mg/kg plus 0.9 mg/kg/h, 1.0 mg/kg plus 0.6 mg/kg/h, and 345.5 IU/kg plus 691.0 IU/kg/h in the rat tail transection model. The ratios of BT2/ID50 of AT-1459, argatroban, and dalteparin were 22.5, 10.0, and 26.6, respectively. In a rat model of arterial thrombosis induced by topical FeCl2 application, intravenous administration of AT-1459, argatroban, and dalteparin improved the vessel patency significantly (P < 0.01) at 0.6 mg/kg plus 0.6 mg/kg/h, 0.6 mg/kg plus 2.4 mg/kg/h, and 300 IU/kg plus 600 IU/kg/h, respectively. The oral antithrombotic effect of AT-1459 lasted for 6 after administering 30 mg/kg and improved the vessel patency significantly 1 h after administering the same dose in venous and arterial thrombosis models, respectively, with a rapid onset of action. Warfarin also inhibited thrombus weight and improved the vessel patency significantly after oral administration of 0.3 mg/kg for three consecutive days in the same study. The antithrombotic and hemorrhagic effects of all drugs studied were correlated with plasma concentration or clotting times. These results suggest that AT-1459 may be clinically useful as an orally available antithrombotic agent for the prevention of venous and arterial thrombosis.

In Vitro and In Vivo Studies of AT-1362, a Newly Synthesized and Orally Active Inhibitor of Thrombin

AT-1362 was found to be a potent, selective, and competitive inhibitor of thrombin, with a Ki value of 6.7 nM. In a rat model of venous thrombosis induced by partial stasis and endothelial disruption, the ID 50 values (a dose required to obtain 50% inhibition of thrombus formation over each vehicle group) of AT-1362 and argatroban were 0.03 mg/kg i.v. plus 0.5 μg/kg/minute and 0.13 mg/kg i.v. plus 8.7 μg/kg/minute, respectively, and the antithrombotic effect of AT-1362 without prolongation of bleeding time lasted for 2 hours and disappeared 4 hours after oral administration of 30 mg/kg. In the rat tail transection model, the BT 2 values (a dose causing two-fold prolongation of the bleeding time over each vehicle group) of AT-1362 and argatroban were 0.56 mg/kg i.v. plus 9.3 μg/kg/minute and 1.1 mg/kg i.v. plus 73.3 μg/kg/minute, respectively. The reduction of thrombus formation and the prolongation of bleeding time were correlated with an ex vivo activated partial thromboplastin time (APTT) for both drugs. AT-1362 at 0.3 mg/kg i.v. plus 5 μg/kg/minute and argatroban at 0.6 mg/kg i.v. plus 40 μg/kg/minute significantly (p<0.05 and p<0.01, respectively) improved the vessel patency in a FeCl 2-induced carotid artery thrombosis model in rats. These results suggest that AT-1362 may be a potent antithrombotic agent for the treatment of thrombotic diseases.

DX-9065a, an Orally Active Factor Xa Inhibitor, Does not Facilitate Haemorrhage Induced by Tail Transection or Gastric Ulcer at the Effective Doses in Rat Thrombosis Model

DX-9065a is an antithrombin III (AT III)-independent and selective inhibitor of activated blood coagulation factor X (FXa). We evaluated the effects of DX-9065a and warfarin on bleeding time and blood loss in rat tail transection model and on blood loss in hydrochloride (HCl)-induced rat gastrointestinal haemorrhage model. The blood loss was determined by measuring the haemoglobin content in saline immersed with transected tail or hematin chloride content in the gaster after HCl administration. DX-9065a or warfarin was administered orally at 1 h or 15-21 h before the haemorrhagic stimuli, respectively. The dose required for 50% inhibition of thrombus formation (ID50) was 21 mg/kg for DX-9065a and 0.75 mg/kg for warfarin in a copper wire-inserted arteriovenous (AV) shunt model. In contrast to DX-9065a (10 or 30 mg/kg), warfarin (0.75 mg/kg) significantly prolonged the bleeding time. In rat tail transection model, the blood loss for the control group was 102+/-41 microl at 20 min after the transection. While warfarin (0.75 mg/kg) facilitated the blood loss about 5 times as much as the control, DX-9065a (10 or 30 mg/kg) did not. In rat gastrointestinal model, the blood loss for the control group was 15.9+/-5.6 microl at 15 min after HCl administration. In contrast to DX-9065a (10 or 30 mg/kg), warfarin (0.75 mg/kg) increased the blood loss about twice as much as the control. Thus, compared with warfarin, DX-9065a only increased bleeding time or blood loss to a minor extent in the doses tested. These observations suggest that direct inhibition of FXa could be preferable to warfarin in the suppression of thrombosis without haemorrhagic complications.

Formula omitted] activity of heparin is not predictive of blood loss after neutralization by protamine

To study whether the ex vivo activity of heparin and Fraxiparin R correlates to and predicts the extent of blood loss induced by the heparins (pre-and post neutralization by protamine), a rat tail transection model and a rabbit ear bleeding model were used. In the rat model heparin (2 mg/kg i.v.) significantly prolonged the bleeding time, while this dose of Fraxiparin R had no effect. In the rabbit ear blood loss model, heparin (2 mg/kg i.v.) produced significant increases in blood loss while Fraxiparin R (2 mg/kg i.v.) produced approximately 30 % of the blood loss induced by heparin. Equigravimetric protamine reduced the heparin-induced blood loss by approximately 50%, however, significant blood loss, thrombin time and Heptest R activity remained. Heparin and Fraxiparin R (3 mg/kg s.c.) did not cause any increased bleeding. While, all activities of heparin were completely neutralized by protamine, the Heptest R activity of Fraxiparin R was resistant to neutralization. The ex vivo activity of heparins after neutralization by protamine does not correlate to the extent of blood loss which suggests it may not be necessary to neutralize all ex vivo activities of the heparins to baseline values to be assured that blood loss is reversed.

Pharmacological Properties of a Low Molecular Weight Butyryl Heparin Derivative (C4-CY 216) with Long Lasting Effects

We have investigated the pharmacological properties of an O-acetylated butyryl derivative of the low molecular weight heparin CY 216 (C4-CY 216). In a purified system the ability of C4-CY 216 to catalyze thrombin and factor Xa inhibition was comparable to that of CY 216. The antithrombin and antifactor Xa catalytic efficiencies of C4-CY 216 were reduced 217 and 12 times respectively when albumin (10 mg ml-1) was added to the reagents, while those of CY 216 were essentially unchanged. In plasma, the antifactor Xa specific activity of C4-CY 216 was close to that of CY 216 but the antithrombin specific activity was 2 times lower. After bolus and continuous intravenous injection to rabbits, the clearances of the two activities of C4-CY 216 were on average half the corresponding values of CY 216. After subcutaneous injection, the bioavailability of C4-CY 216 was comparable to that of CY 216. C4-CY 216 was as potent as CY 216 in preventing venous thrombosis in the thromboplastin-Wessler model and the duration of the antithrombotic effect was longer than that of the parent compound. The chemical alteration of CY 216 did not enhance the prohaemorrhagic effect in the rat tail transection model. Therefore, the new concept of heparin derivative having a low clearance and long lasting effects that we have recently reported for unfractionated heparin may also be applied to a low molecular weight heparin.

Neutralization of dermatan sulfate [formula omitted] and [formula omitted] by protamine sulfate and polybrene

The neutralization of the anticoagulant, anti-thrombin, and bleeding effects of dermatan sulfate (DS), a potential antithrombotic agent, was investigated. Protamine sulfate (PS) and hexadimethrine bromide (Polybrene), which reverse the anticoagulant effect of heparin, also neutralized DS in vitro . In human plasma, polybrene was approximately 3 times more active on a weight basis than PS for neutralizing DS (1.5 ug polybrene inhibits 1 ug DS). Intravenous administration of polybrene to rabbits pretreated with DS in a 1:1 weight ratio immediately neutralized 90% of DS and this effect was stable with time. In contrast, PS in a weight ratio of 6:1 (PS to DS) only neutralized 50% of DS injected. When plasma DS concentrations were maintained by continuous infusion between 3 and 15 ug/ml, a bolus of polybrene 0.25 mg/kg induced an immediate drop of about 4 ug/ml but initial values of DS were recovered within 20 min. PS was again much less effective than polybrene for neutralizing DS. The bleeding effect of DS and its correction by polybrene was studied by using the rat tail transection model. Very large doses of DS (> 10 mg/kg) were required to get a modest prolongation of bleeding time. The injection of equivalent doses of polybrene in animals pretreated by DS induced a strong bleeding effect associated with a drop in platelet and leukocyte counts. Animal models are thus inappropriate for investigating the correction of DS-induced bleeding, because high doses of both DS and neutralizing agents are required in these models. Our results indicate that, provided the doses of neutralizing agents remain below their established levels of toxicity in man, DS could if necessary be neutralized completely by polybrene and partially by PS.