Comparison of PD0348292, a selective factor Xa inhibitor, to antiplatelet agents for the inhibition of arterial thrombosis

SummaryThe objective of this study was to determine if orally-administered PD0348292, a direct specific factor Xa inhibitor, inhibits thrombosis following porcine carotid arterial injury comparably to aspirin or clopidogrel alone or in combination. We further sought to determine whether the antithrombotic efficacy in vivo could be predicted using an ex-vivo perfusion chamber. Oral treatments included: PD0348292 (0.4, 0.9, or 4.3 mg/kg); PD0348292 (0.4 mg/kg) plus aspirin (325 mg); aspirin; clopidogrel (75 mg); aspirin plus clopidogrel; or vehicle (n=6–10/group). Aspirin and clopidogrel were administered 27 and four hours pre-injury and PD0348292 or vehicle was administered four hours pre-injury. Both carotid arteries were crush-injured, and thrombus was measured by detection of 111In-platelets over 30 minutes. Prior to injury, the antithrombotic efficacy was assessed by ex-vivo perfusion chamber platelet deposition. PD0348292 produced dose-dependent prothrombin time (0.9- to 2.9-fold) and aPTT (1.4- to 2.5-fold) prolongations. Bleeding times were significantly prolonged in each active drug group compared to vehicle, but were not significantly different between drug groups. PD0348292 significantly inhibited arterial platelet deposition (x106/cm2) at 4.3(549 ± 1,066), 0.9 (399 ± 162) and 0.4 mg/kg (531 ± 470) compared to vehicle (2,242 ± 1,443). Aspirin (992 ± 973), clopidogrel (537 ± 483), clopidogrel plus aspirin (228 ± 66) or PD0348292 plus aspirin (558 ± 317) also significantly inhibited platelet deposition, although these values were not significantly different than with any dose of PD348292. Perfusion chamber platelet deposition correlated significantly with in-vivo anti-thrombotic response. In conclusion, PD0348292 inhibited arterial thrombosis comparable to aspirin plus clopidogrel. Perfusion chamber methodology may be useful in predicting in-vivo antithrombotic efficacy.

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Comparison of antithrombotic efficacy between edoxaban, a direct factor Xa inhibitor, and fondaparinux, an indirect factor Xa inhibitor under low and high shear rates

Thrombosis and Haemostasis ◽

10.1160/th11-07-0451 ◽

2011 ◽

Vol 106 (12) ◽

pp. 1062-1068 ◽

Cited By ~ 10

Author(s):

Naoki Tsuji ◽

Yuko Honda ◽

Chikako Kamisato ◽

Yoshiyuki Morishima ◽

Toshiro Shibano ◽

...

Keyword(s):

Shear Rate ◽

Arterial Thrombosis ◽

Factor Xa ◽

High Shear ◽

Shear Rates ◽

Thrombosis Model ◽

Perfusion Chamber ◽

High Shear Rates ◽

Antithrombotic Effects ◽

Antithrombotic Efficacy

SummaryEdoxaban is an oral, direct factor Xa (FXa) inhibitor under late-phase clinical development. This study compared the antithrombotic efficacy of edoxaban with that of an indirect FXa inhibitor, fondaparinux, in in vivo venous and arterial thrombosis models and in ex vivo perfusion chamber thrombosis model under low and high shear rates in rats. Venous and arterial thrombi were induced by platinum wire insertion into the inferior vena cava and by application of FeCl3 to the carotid artery, respectively. The perfusion chamber thrombus was formed by blood perfusion into a collagen-coated capillary at 150 s-1 (low shear rate) and 1,600 s-1 (high shear rate). Effective doses of edoxaban that reduced thrombus formation by 50% (ED50) in venous and arterial thrombosis models were 0.076 and 0.093 mg/kg/h, respectively. In contrast, ED50 of fondaparinux in the arterial thrombosis model (>10 mg/kg/h) was markedly higher compared to ED50 in the venous thrombosis model (0.021 mg/kg/h). In the perfusion chamber thrombosis model, the ratio of ED50 under high shear rate (1.13 mg/kg/h) to that under low shear rate (0.63 mg/kg/h) for edoxaban was 1.9, whereas that for fondaparinux was more than 66. While the efficacy of fondaparinux markedly decreased in arterial thrombosis and in a high-shear state, edoxaban exerted consistent antithrombotic effects regardless of flow conditions. These results suggest that shear rate is a key factor in different antithrombotic effects between edoxaban and fondaparinux.

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Enhanced endogenous thrombolysis induced by a specific factor Xa inhibitor, DX-9065a, evaluated in a rat arterial thrombolysis model in vivo

Thrombosis Research ◽

10.1016/s0049-3848(02)00088-9 ◽

2002 ◽

Vol 106 (2) ◽

pp. 165-168 ◽

Cited By ~ 6

Author(s):

Masaru Hashimoto ◽

Yuko Onobayashi ◽

Kazuhiro Oiwa ◽

John C Giddings ◽

Junichiro Yamamoto

Keyword(s):

Factor Xa ◽

Specific Factor ◽

Factor Xa Inhibitor ◽

Arterial Thrombolysis

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The effect of a dual or a triple antithrombotic therapy with apixaban on thrombus formation in vivo and in an ex vivo perfusion chamber model

Medicine ◽

10.1097/md.0000000000004145 ◽

2016 ◽

Vol 95 (27) ◽

pp. e4145 ◽

Cited By ~ 1

Author(s):

Stefan Weisshaar ◽

Brigitte Litschauer ◽

Sebastian Bucher ◽

Martin Riesenhuber ◽

Stylianos Kapiotis ◽

...

Keyword(s):

Antithrombotic Therapy ◽

Ex Vivo ◽

Thrombus Formation ◽

Ex Vivo Perfusion ◽

Perfusion Chamber ◽

Chamber Model ◽

Triple Antithrombotic Therapy

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A specific factor Xa inhibitor, DX-9065a, prevents experimental DIC in primates by the inhibition of factor Xa in vivo

The Japanese Journal of Pharmacology ◽

10.1016/s0021-5198(19)40826-3 ◽

1998 ◽

Vol 76 ◽

pp. 179

Author(s):

Toshio Fukuda ◽

Yoshiyuki Morishima ◽

Tsuyoshi Hara ◽

Satoshi Kunitada

Keyword(s):

Factor Xa ◽

Specific Factor ◽

Factor Xa Inhibitor

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In Vitro Characteristics, Anticoagulant Effects and In Vivo Antithrombotic Efficacy of a Novel, Potent and Orally Active Direct Factor Xa Inhibitor, DU-176b.

Blood ◽

10.1182/blood.v104.11.1862.1862 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1862-1862 ◽

Cited By ~ 3

Author(s):

Yoshiyuki Morishima ◽

Taketoshi Furugohri ◽

Koji Isobe ◽

Yuko Honda ◽

Chikako Matsumoto ◽

...

Keyword(s):

Oral Administration ◽

Factor Xa ◽

Coagulation Cascade ◽

Venous Stasis ◽

Factor Xa Inhibitor ◽

Fxa Inhibitor ◽

Antithrombotic Efficacy ◽

Orally Active

Abstract Factor Xa (FXa) is a key serine protease in the coagulation cascade and is a promising target enzyme for developing a new antithrombotic agent. Our first clinical candidate for a small molecular direct FXa inhibitor DX-9065a potently inhibits FXa (Ki = 41 nM) and exerts antithrombotic effects in animal models. However, due to its poor bioavailability (10% in monkeys) the compound is used only as an injectable formulation in clinical studies. Here we report in vitro characteristics of serine proteases inhibition, anticoagulant effects and in vivo antithrombotic efficacy of DU-176b, a novel, potent and orally active direct FXa inhibitor. DU-176b competitively inhibited human FXa with a Ki value of 0.561 nM, indicating 70-fold increase in FXa inhibitory activity compared with DX-9065a. DU-176b demonstrated 10,000-fold selectivity relative to inhibition of thrombin (Ki = 6.00 μM), and had no effects on the enzymatic activities of factor VIIa, t-PA, plasmin, trypsin and chymotrypsin. In human plasma, DU-176b prolonged prothrombin time (PT) and activated partial thromboplastin time (APTT) in a concentration-dependent manner. Its concentrations for doubling these clotting times were 0.256 and 0.508 μM, respectively. After oral administration of DU-176b to rats, significant anti-Xa activity was observed in plasma over 4 h. The oral bioavailability of DU-176b (approximately 50%) was significantly higher than that of DX-9065a (10%) in monkeys. The antithrombotic efficacy of DU-176b was examined by oral administration to rats 30 minutes prior to thrombogenic stimuli. In a venous stasis thrombosis model, DU-176b (0.5 – 12.5 mg/kg, p.o.) dose-dependently inhibited thrombus formation, prolonged PT, and revealed plasma anti-Xa activity. DU-176b also exerted significant anticoagulant effect in a rat model of tissue factor-induced disseminated intravascular coagulation at doses of 0.1 – 2.5 mg/kg, p.o. These results demonstrate that DU-176b is a potent and selective factor Xa inhibitor that possesses antithrombotic effect after oral administration. DU-176b has the potential to be clinically useful for prophylaxis and treatment of several thrombotic diseases.

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Specific Pharmacological Targeting of the Syk Kinase Activity in Platelets: A Novel, Safe Anti-Thrombotic Strategy

Blood ◽

10.1182/blood.v112.11.409.409 ◽

2008 ◽

Vol 112 (11) ◽

pp. 409-409 ◽

Cited By ~ 2

Author(s):

Suzanne Delaney ◽

Uma Sinha ◽

Nisha Nanda ◽

Yibing Yan ◽

Anjali Pandey ◽

...

Keyword(s):

Intracellular Calcium ◽

Arterial Thrombosis ◽

Kinase Activity ◽

Ex Vivo ◽

Thrombus Formation ◽

Wild Type ◽

Primary Hemostasis ◽

Syk Kinase ◽

Perfusion Chamber

Abstract Studies of the Syk −/− mouse have implicated spleen tyrosine kinase (Syk), a signaling protein with both kinase and scaffolding activities, in platelet signaling following engagement of GPVI and αIIbβ3 by collagen and fibrinogen, respectively. The present study was designed to determine whether specific inhibition of the kinase activity of Syk, without targeting the Syk scaffolding function, affected in vivo arterial thrombosis. In preliminary experiments, blood from wild-type and Syk−/− mice was perfused through collagen-coated capillaries under arterial shear rates to study ex vivo thrombosis. While blood from wild-type mice formed robust thrombi (37±4.7 μm3/μm2), none was observed in Syk−/− mice. Thrombi intermediate in size (16±3.9 μm3/μm2) developed in Syk+/− mice. To achieve specific pharmacological targeting of the kinase activity of Syk, P142-76, a potent (IC50 = 4 nM) and selective Syk kinase inhibitor was utilized. P142-76 was screened against a broad panel of 139 purified kinases at 50 nM. While Syk kinase was inhibited by 92%, all other kinases retained more than 70% of their activity. In washed human platelets, P142-76 inhibited convulxin (CVX)-induced phosphorylation of LAT (linker for activation of T-cells; IC50 = 111 nM) and intracellular calcium increases (IC50 = 31 nM). The GPVI/Syk-specificity of P142-76 activity was confirmed by its inability to inhibit intracellular calcium increases induced by the PAR1 thrombin receptor agonist TRAP. P142-76 also inhibited CVX-induced aggregation of both human washed platelets (IC50 = 87 nM) and platelet-rich plasma (IC50 = 2.5 μM). Considering the controversial data in respect to the participation of GPVI in arterial thrombosis in murine models, the dependence of arterial thrombosis on Syk function was studied in vivo in pigs. Cross-species activity of P142-76 was confirmed in vitro (CVX-induced PRP aggregation IC50= 350 nM; 5 μM P142-76 completely inhibited thrombosis triggered by collagen in the perfusion chamber assay). At a plasma concentration which abolished ex vivo CVX-induced but not ADP-induced pig platelet aggregation, P142-76 significantly inhibited the deposition of [111In]-labeled platelets in a carotid artery crush swine thrombosis model, without compromising primary hemostasis. % aggregation Swine (n=3) Platelet Deposition % inhibition Plasma Conc (ng/ml) Bleed Time (min) Activated Clotting Time (sec) ADP (20 μM) CVX (250 ng/ml) Control Artery 0 0 3±0.9 133±22 100 100 Treated Artery 76±6.5 1343±304 3.5±0.3 130±13 100 0 To clarify further the contribution of the kinase activity of Syk to arterial thrombosis, effects of P142-76 on human blood were evaluated in real time in the collagen-coated perfusion chamber. Low concentrations of P142-76 (0.3 μM) affected thrombus stability, while increasing concentrations (1–5 μM) delayed and then completely inhibited thrombus formation. Furthermore, P142-76 destabilized pre-formed thrombi, indicating a critical role for Syk in conferring strength to platelet-platelet interactions, i.e. αIIbβ3-mediated cohesion. Our data indicate that the kinase activity of Syk acts in arterial thrombosis through at least two distinct mechanisms. First, Syk kinase confers stability to platelet-platelet interactions downstream of αIIbβ3. Second, it initiates thrombus formation on collagen surfaces. This dual activity of the kinase activity of Syk makes it a preferred target for inhibition of arterial thrombosis, as it does not compromise primary hemostasis.

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Inhibition of the von Willebrand (VWF)–collagen interaction by an antihuman VWF monoclonal antibody results in abolition of in vivo arterial platelet thrombus formation in baboons

Blood ◽

10.1182/blood.v99.10.3623 ◽

2002 ◽

Vol 99 (10) ◽

pp. 3623-3628 ◽

Cited By ~ 79

Author(s):

Dongmei Wu ◽

Karen Vanhoorelbeke ◽

Nancy Cauwenberghs ◽

Muriel Meiring ◽

Hilde Depraetere ◽

...

Keyword(s):

Monoclonal Antibody ◽

Arterial Thrombosis ◽

Bleeding Time ◽

Ex Vivo ◽

Thrombus Formation ◽

High Shear ◽

Pharmacologic Effect ◽

Antithrombotic Efficacy ◽

Von Willebrand

The interaction between collagen, von Willebrand factor (VWF), and glycoprotein Ib is the first step in hemostasis and thrombosis especially under high shear conditions. We studied the inhibition of the VWF-collagen interaction by using an antihuman VWF monoclonal antibody 82D6A3 to prevent arterial thrombosis in baboons to develop a new kind of antithrombotic strategy and determine for the first time experimental in vivo data concerning the importance of the collagen-VWF interaction. We used a modified Folts model to study the antithrombotic efficacy of 82D6A3, where cyclic flow reductions (CFRs) were measured in the femoral artery. Administering a dose of 100, 300, and 600 μg/kg resulted in a 58.3%, 100%, and 100% reduction in the CFRs, respectively. When 100 μg/kg 82D6A3 was infused into the baboons, 80% of VWF-A3 domain was occupied, corresponding to 30% to 36% ex vivo inhibition of VWF binding to collagen, with no prolongation of the bleeding time. The bleeding time was also not significantly prolonged when the CFRs were abolished at doses of 300 μg/kg and 600 μg/kg. At these doses 100% of VWF was occupied by the antibody and 100% ex vivo inhibition of the VWF-collagen binding was observed. 82D6A3 has a high affinity for VWF; after 48 hours still 68% VWF (300μg/kg) was occupied with a pharmacologic effect up to 5 hours after administration (80%-100% occupancy). In conclusion, these results clearly indicate that the VWF-collagen interaction is important in vivo in thrombosis under high shear conditions and thus might be a new target for preventing arterial thrombosis.

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Anticoagulant Efficacy and Immunogenicity of the Selective Factor Xa Inhibitor Antistasin Following Subcutaneous Administration in the Rhesus Monkey

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648449 ◽

1992 ◽

Vol 67 (03) ◽

pp. 371-376 ◽

Cited By ~ 16

Author(s):

Christopher T Dunwiddie ◽

Elka M Nutt ◽

George P Vlasuk ◽

Peter K S Siegl ◽

Linda W Schaffer

Keyword(s):

Rhesus Monkey ◽

Neutralizing Antibodies ◽

Ex Vivo ◽

Anticoagulant Activity ◽

Subcutaneous Administration ◽

Factor Xa ◽

Specific Factor ◽

Factor Xa Inhibitor ◽

Duration Of Action ◽

Monkey Model

SummaryThe antithrombotic efficacy and duration of action of a single subcutaneous administration of the selective factor Xa inhibitor recombinant antistasin (rATS) was evaluated in a rhesus monkey model of mild disseminated intravascular coagulation. rATS (1 mg/kg) was shown to be fully effective and comparable to standard heparin (1,000 U/kg) in the suppression of thromboplastin-induced fibrinopeptide A generation for at least 5 h following a single subcutaneous administration. The absorption rate of rATS, as measured by ex vivo activated partial thromboplastin times (aPTT), mirrored that of standard heparin exhibiting peak anticoagulant activity between 1 and 2 h post administration. The anticoagulant effects of a single rATS dose lasted for longer than 30 h maintaining an aPTT value at least 2-fold higher than baseline. Repeated subcutaneous administrations of rATS resulted in the generation of fully neutralizing antibodies. These results suggest that specific factor Xa inhibition may be as effective as standard heparin in the treatment of venous thrombosis. Due to its antigenicity however, rATS is probably not suitable for chronic subcutaneous anticoagulant therapy.

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Studies on the Haemostatic Defect in a Complicated Syndrome

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649920 ◽

1995 ◽

Vol 74 (05) ◽

pp. 1244-1251 ◽

Cited By ~ 37

Author(s):

H Stormorken ◽

H Holmsen ◽

R Sund ◽

K S Sakariassen ◽

T Hovig ◽

...

Keyword(s):

Ex Vivo ◽

Bleeding Tendency ◽

Clot Retraction ◽

Abnormal Finding ◽

Shear Rates ◽

Perfusion Chamber ◽

Coagulant Activity ◽

5 Ht Uptake ◽

And Storage

SummaryThe Stormorken syndrome is a multifacetted syndrome including a bleeding tendency. No deviations were found in the coagulation- or fibrinolytic systems. Platelet number was low normal, and size abnormal, whereas EM findings were unremarkable. Survival time was half normal. Clot retraction was initially rapid, but clearly decreased, whereas prothrombin consumption was also initially rapid, but complete. Membrane GP’s were normal, so was AA metabolism, PI-cycle, granule storage and secretion, and c-AMP function, whereas 5-HT uptake and storage was decreased. Optical platelet aggregation was low normal with all physiological agonists. The only clearly abnormal finding was that coagulant activity was present on non stimulated platelets at the same level as kaolin-stimulated normal platelets. This indicated a platelet abnormality which should lead to a thrombogenic, not to a haemorrhagic trait. This paradox may have its origin in rheology, because when challenged with in vivo shear rates in an ex vivo perfusion chamber, platelet cohesion was abnormally low. Further studies to better delineate the membrane abnormality are underway.

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