The humanized anti-glycoprotein Ib monoclonal antibody h6B4-Fab is a potent and safe antithrombotic in a high shear arterial thrombosis model in baboons

2008 ◽  
Vol 100 (10) ◽  
pp. 670-677 ◽  
Author(s):  
Alexandre Fontayne ◽  
Muriel Meiring ◽  
Seb Lamprecht ◽  
Jan Roodt ◽  
Eddy Demarsin ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Side Effects ◽  
Blood Loss ◽  
Bleeding Time ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Plasma Concentrations ◽  
Platelet Glycoprotein ◽  
Fab Fragment ◽  
Platelet Receptor

SummaryThe Fab-fragment of 6B4, a murine monoclonal antibody targeting the human platelet glycoprotein (GP) Ibα and blocking the binding of von Willebrand factor (VWF), is a powerful antithrombotic. In baboons, this was without side effects such as bleeding or thrombocytopenia. Recently, we developed a fully recombinant and humanized version of 6B4-Fab-fragment, h6B4-Fab, which maintains its inhibitory capacities in vitro and ex vivo after injection in baboons. We here investigated the antithrombotic properties, the effect on bleeding time and blood loss and initial pharmacokinetics of h6B4-Fab in baboons. The antithrombotic effect of h6B4-Fab on acute platelet-mediated thrombosis was studied in baboons where thrombus formation is induced at an injured and stenosed site of the femoral artery, allowing for cyclic flow reductions (CFRs) which are measured on an extracorporeal femoral arteriovenous shunt. Injection of 0.5 mg/kg h6B4-Fab significantly reduced the CFRs by 80%, whereas two extra injections, resulting in cumulative doses of 1.5 and 2.5 mg/kg, completely inhibited the CFRs. Platelet receptor occupancy, plasma concentrations and effects ex vivo were consistent with what was previously observed. Finally, minimal effects on bleeding time and blood loss, no spontaneous bleeding and no thrombocytopenia were observed. We therefore conclude that h6B4-Fab maintains the antithrombotic capacities of the murine 6B4-Fab, without causing side effects and therefore can be used for further development.

Antiplatelet and Antithrombotic Effects of YM337, the Fab Fragment of a Humanized Anti-GPIIb/IIIa Monoclonal Antibody in Monkeys

1996 ◽  
Vol 75 (04) ◽  
pp. 679-684 ◽  
Author(s):  
Seiji Kaku ◽  
Tomihisa Kawasaki ◽  
Nami Hisamichi ◽  
Yumiko Sakai ◽  
Yuta Taniuchi ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Arachidonic Acid ◽  
Platelet Aggregation ◽  
Bleeding Time ◽  
Bolus Injection ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Fab Fragment ◽  
Inhibition Of Platelet Aggregation ◽  
Antithrombotic Effects

SummaryThe antiplatelet and antithrombotic effects of the Fab fragment of the humanized antiplatelet glycoprotein (GP) IIb/IIIa monoclonal antibody C4G1 (YM337) were investigated in monkeys. First, the relationship between the inhibition of platelet aggregation and the prolongation of bleeding time was studied in rhesus monkeys. YM337 dose-dependently inhibited ex vivo platelet aggregation, with complete inhibition at doses higher than 0.25 mg/kg intravenous injection or 1.5 μg/kg/min infusion. At 0.25 mg/kg bolus injection followed by 1.5 μg/kg/min infusion, YM337 immediately and continuously inhibited platelet aggregation during the 6-h infusion period with platelet aggregation rapidly returning to over 50% of baseline within 1 h after the cessation of infusion. Template-bleeding time was significantly prolonged during the period of complete inhibition of platelet aggregation.Second, the antithrombotic effects of YM337 were investigated in a photochemically-induced thrombosis model in squirrel monkeys. YM337 at a dose of 1 mg/kg intravenous injection followed by 6 μg/kg/min infusion for 60 min prevented occlusive thrombus formation in all 4 monkeys. In contrast, time to occlusive thrombus formation did not change on intravenous bolus injection of aspirin 17 mg/kg (11.3 ± 5.2 min) or sodium ozagrel (9.4 ± 3.0 min) compared with saline (13.3 ± 4.0 min). YM337 but not aspirin or sodium ozagrel significantly inhibited ex vivo ADP-induced platelet aggregation, while all drugs completely inhibited arachidonic acid-induced platelet aggregation. However, while aspirin and sodium ozagrel inhibited the thromboxane B2 generation accompanying arachidonic acid-induced platelet aggregation, YM337 had no effect on this variable. Platelet counts and bleeding time showed no significant change in any group in this squirrel monkey model.These results indicate that YM337, with a short half-life, may be a useful therapeutic agent in patients with thrombotic disorders.

scholarly journals Antithrombotic effect of a monoclonal antibody to the platelet glycoprotein IIb/IIIa receptor in an experimental animal model

Blood ◽  
1986 ◽  
Vol 68 (3) ◽  
pp. 783-786 ◽  
Author(s):  
BS Coller ◽  
JD Folts ◽  
LE Scudder ◽  
SR Smith
Keyword(s):  
Monoclonal Antibody ◽  
Animal Model ◽  
Platelet Aggregation ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Platelet Glycoprotein ◽  
Antithrombotic Effect ◽  
Antithrombotic Agent ◽  
Antithrombotic Effects ◽  
Platelet Thrombus

A murine monoclonal antibody directed at the platelet glycoprotein IIb/IIIa complex, which blocks platelet aggregation ex vivo, was tested for its antithrombotic effects in an established animal model of acute platelet thrombus formation in partially stenosed arteries. Infusion of 0.7 to 0.8 mg/kg of the F(ab')2 fragment of the antibody completely blocked new thrombus formation despite multiple provocations, making it the most potent antithrombotic agent tested in this model.

Ex vivo evaluation of anti-GPVI antibody in Cynomolgus monkeys: Dissociation between anti-platelet aggregatory effect and bleeding time

2006 ◽  
Vol 96 (08) ◽  
pp. 167-175 ◽  
Author(s):  
Yutaka Matsumoto ◽  
Hisao Takizawa ◽  
Kazuhiro Nakama ◽  
Xiaoqi Gong ◽  
Yoshihisa Yamada ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Bleeding Time ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Inhibitory Effect ◽  
Bleeding Tendency ◽  
Fab Fragment ◽  
Dose Escalation Study ◽  
Cynomolgus Monkeys ◽  
Induced Aggregation

SummaryRecent progress in the understanding of thrombus formation has suggested an important role of glycoprotein (GP)VI. In contrast to its pivotal role in collagen-induced platelet activation, it has been suggested that its blockade does not induce massive bleeding tendency. To demonstrate the dissociation between inhibitory effect on platelet aggregation and bleeding by GPVI blockade, we examined the effects of Fab fragment of OM2, an anti-human GPVI monoclonal antibody on ex vivo collagen-induced platelet aggregation and skin bleeding time after intravenous injection in cynomolgus monkeys. In a dose-escalation study, OM2 potently (>80%) inhibited collagen-induced platelet aggregation at the cumulative dose of 0. 2 mg/kg with a slight prolongation of bleeding time (1. 3 times baseline value). Furthermore, at 18. 8 mg/kg, the highest dose tested, prolongation of bleeding time was still mild (1. 9 times). In contrast, abciximab, Fab fragment of anti-GPIIb/IIIa antibody prolonged bleeding time by 5. 0 times at 0. 35 mg/kg, the lowest effective dose on platelet aggregation. Ina pharmacodynamic study,a bolus injection of OM2 at 0. 4 mg/kg produced potent inhibition of collagen-induced aggregation up to six hours after injection, showing longer half-life than that of abciximab. The injection of OM2 Fab did not induce thrombocytopenia and GPVI depletion in monkeys. These results suggest that blockade of GPVI by antibody can exerta potent inhibitory effect on collagen-induced platelet aggregation with a milder prolongation of bleeding time than blockade of GPIIb/IIIa. This study indicates that OM2 has the potential to be developed as a new class of therapeutic tool.

Inhibition of Platelet Glycoprotein β3 by Chimeric Monoclonal Antibody Prevents Arterial Thrombosis in Beagle Dogs,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3357-3357
Author(s):  
Shundong Ji ◽  
Miao Jiang ◽  
Ningzheng Dong ◽  
Xia Bai ◽  
Changgeng Ruan
Keyword(s):  
Bleeding Time ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Dose Range ◽  
Negative Control Group ◽  
Negative Control ◽  
Platelet Glycoprotein ◽  
Control Group ◽  
Beagle Dogs

Abstract Abstract 3357 In this present study in beagle dogs, we evaluated the antithrombotic efficacy of Pulaimab, the chimeric monoclonal antibodies SZ21-F(ab)2 fragments against platelet glycoprotein (GP) β3, in a modified Folts model. The Folts model is widely accepted to be effective and clinically relevant for testing potential anti-thrombotic agents, in this model the cyclic flow reductions (CFRs) are caused by platelet dependent thrombi that form under high-shear conditions at injured stenosed sites of an artery. Thirty beagle dogs of either sex, weighing 7.5 to 14 kg, were randomly divided into five groups of six (three females and three males), the details were following: negative control group (injected normal saline), positive control group (injected 0.2mg/Kg of Reopro), and three experimental groups [injected 0.2, 0.4, 0.8 mg/Kg of SZ21-F(ab)2, respectively]. A dose range from 0.2 to 0.8 mg/kg of Pulaimab significantly reduced the CFRs by 21–73%, without reduction of platelet numbers and prolongation of the bleeding time. Ex vivo ADP-induced platelet aggregation was equally reduced. The present study demonstrates that the inhibition of platelet GP αIIbβ3function by SZ21-F(ab)2 is a powerful intervention to prevent platelet thrombus formation in injured arteries without thrombocytopenia and prolongation of the bleeding time. We therefore conclude that F(ab)2 fragments of inhibitory anti-GPβ3 antibodies may be useful compounds to prevent thrombosis. Disclosures: No relevant conflicts of interest to declare.

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 ◽  
2002 ◽  
Vol 99 (10) ◽  
pp. 3623-3628 ◽  
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.

Evaluation of AR-H067637, the active metabolite of the new direct thrombin inhibitor AZD0837, in models of venous and arterial thrombosis and bleeding in anaesthetised rats

2010 ◽  
Vol 104 (12) ◽  
pp. 1242-1249 ◽  
Author(s):  
Susanne Pehrsson ◽  
Karin Johansson ◽  
Magnus Kjaer ◽  
Margareta Elg
Keyword(s):  
Blood Loss ◽  
Arterial Thrombosis ◽  
Bleeding Time ◽  
Thrombus Formation ◽  
Plasma Concentrations ◽  
Direct Thrombin Inhibitor ◽  
Thrombin Inhibitor ◽  
Coagulation Time ◽  
Dose Dependent ◽  
Anaesthetised Rats

SummaryAZD0837, currently in clinical development, is a once-daily oral anticoagulant that is bioconverted to AR-H067637, a selective, reversible direct thrombin inhibitor (DTI). When developing a new DTI, the anti-thrombotic effects are commonly investigated in in vivo animal models; this report shows the effect of AR-H067637 in venous and arterial thrombosis and bleeding models in anaesthetised rats. Thrombus formation was induced by topical application of ferric chloride to the carotid artery or to the caval vein with partial stasis. Cutaneous incision bleeding time and muscle transection blood loss were assessed, with or without acetylsalicylic acid (ASA). Activated partial thromboplastin time (APTT), ecarin coagulation time (ECT) and thrombin coagulation time (TCT) were used as plasma biomarkers of anticoagulant effect. Dalteparin was used as a reference compound. AR-H067637, given by continuous infusion, displayed a dose-dependent antithrombotic effect, with 50% inhibition (IC50) of thrombus size in venous and arterial thrombosis models obtained at plasma concentrations of 0.13 μM and 0.55 μM, respectively, without increased bleeding. Dose-dependent increased bleeding and blood loss were seen at plasma concentrations ≥1 μM AR-H067637. At the highest AR-H067637 plasma concentration tested, bleeding time and blood loss increased two and four times the vehicle group. Addition of ASA moderately potentiated bleeding time and blood loss. APTT, ECT and TCT were dose-dependently prolonged. These studies demonstrate that the DTI AR-H067637 inhibits thrombus formation in rat venous and arterial thrombosis models with no or minor increases in bleeding.

The von Willebrand factor antagonist (GPG-290) prevents coronary thrombosis without prolongation of bleeding time

2007 ◽  
Vol 98 (08) ◽  
pp. 397-405 ◽  
Author(s):  
Michael Wadanoli ◽  
Dianne Sako ◽  
Gray Shaw ◽  
Robert Schaub ◽  
Qin Wang ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Platelet Adhesion ◽  
Bleeding Time ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Coronary Thrombosis ◽  
Platelet Glycoprotein ◽  
Closure Time ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryThe interaction between von Willebrand factor (VWF) and platelet glycoprotein Ibα (GPIbα) is a critical step that allows platelet adhesion, activation and subsequent thrombus formation to the injured vessel wall under high-shear conditions. In this study, we sought to investigate 1) whether GPG-290, a recombinant human GPIbα chimeric protein, would prevent thrombosis in a canine model of coronary thrombosis by blocking VWFGPIbα interaction; and 2) whether desmopressin (DDAVP), a VWF release stimulant, could reduce the prolonged bleeding time caused by a 10x efficacious dose of GPG-290. The antithrombotic efficacy of GPG-290 was evaluated by the in-vivo ability to prevent cyclic flow reductions (CFRs) and ex-vivo inhibition of platelet adhesion/aggregation reflected by prolongation of Platelet Function Analyzer (PFA-100®) collagen /ADP closure time. The anti-hemostatic effect was assessed by template bleeding time. GPG-290 at doses of 25, 50 and 100 μg/kg abolished CFRs in 67%,100% and 100% of the treated dogs without bleeding time prolongation, respectively; GPG-290 dose-dependently prolonged the ex-vivo collagen/ADP-closure time, while it had no effects on plasma VWF antigen level (VWF:Ag) and VWF-collagen binding activity (VWF:CB); the prolonged template bleeding time caused by 500 μg/kg of GPG-290 was prevented by intravenous infusion of DDAVP (0.3 μg/kg). In conclusion, GPG-290 appears to be an effective agent for treating arterial thrombosis without bleeding time prolongation.

scholarly journals Antithrombotic effect of a monoclonal antibody to the platelet glycoprotein IIb/IIIa receptor in an experimental animal model

Blood ◽  
1986 ◽  
Vol 68 (3) ◽  
pp. 783-786 ◽  
Author(s):  
BS Coller ◽  
JD Folts ◽  
LE Scudder ◽  
SR Smith
Keyword(s):  
Monoclonal Antibody ◽  
Animal Model ◽  
Platelet Aggregation ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Platelet Glycoprotein ◽  
Antithrombotic Effect ◽  
Antithrombotic Agent ◽  
Antithrombotic Effects ◽  
Platelet Thrombus

Abstract A murine monoclonal antibody directed at the platelet glycoprotein IIb/IIIa complex, which blocks platelet aggregation ex vivo, was tested for its antithrombotic effects in an established animal model of acute platelet thrombus formation in partially stenosed arteries. Infusion of 0.7 to 0.8 mg/kg of the F(ab')2 fragment of the antibody completely blocked new thrombus formation despite multiple provocations, making it the most potent antithrombotic agent tested in this model.

The Direct Factor Xa Inhibitor Apixaban Produces Broad Antithrombotic Activity with Limited Effects on Bleeding Time in Rats.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4005-4005
Author(s):  
William A. Schumacher ◽  
Jeffrey S. Bostwick ◽  
Anne B. Stewart ◽  
Thomas E. Steinbacher ◽  
Donald J. Pinto ◽  
...  
Keyword(s):  
Bleeding Time ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Plasma Concentrations ◽  
Vena Cava ◽  
Factor Xa ◽  
Experimental Models ◽  
Factor Xa Inhibitor ◽  
Dependent Manner ◽  
Venous Shunt

Abstract Apixaban is an oral, direct and highly selective factor Xa inhibitor which is in late stage clinical development for the prevention and treatment of thromboembolic diseases. Apixaban is potent against human and rat factor Xa (Ki = 0.08 and 1.3 nM, respectively). The efficacy/safety profile of apixaban was determined in experimental models of thrombosis and hemostasis performed in anesthetized rats. Thrombosis was induced either by topical application of FeCl2 to the vena cava (VT) or carotid artery (AT), tissue factor infusion into the vena cava (TF-VT), or within an extracorporeal arterial-venous shunt (ST). Bleeding time was measured in response to template incision of the renal cortex. Apixaban was administered as a continuous i.v. infusion of 0.1, 0.3, 1 or 3 mg/kg/h starting 60 min before each experimental procedure (n=5 or 6 per dose). These respective doses increased the ex vivo prothrombin time by 1.3, 1.9, 3.0 and 3.9 times control baseline, and achieved plasma concentrations of 0.3, 1.4, 5.0 and 12.2 μM. In comparison to vehicle treatment, the 3 mg/kg/h dose of apixaban decreased thrombus weight by 90 ± 2% in VT, 62 ± 7% in TF-VT, 62 ± 4% in AT, and 79 ± 3% in ST (all p<0.05). A 50% thrombus weight reduction sufficient to prevent vascular occlusion in each model would require projected doses (mg/kg/h) of 0.4 (VT), 1.9 (TF-VT), 0.8 (AT) and 1.1 (ST). The lowest dose level which preserved carotid blood flow at the pre-injury level during thrombus formation was 0.3 mg/kg/h. Apixaban prolonged bleeding time in a dose-dependent manner with no effect detected at 0.1 and 0.3 mg/kg/h, while significant (p<0.05) increases of 1.34 ± 0.12 and 2.13 ± 0.17 times control were observed at 1 and 3 mg/kg/h, respectively. In comparison, aspirin increased bleeding time by 1.42 ± 0.1 times control (n=7, p<0.05) when tested at a 10 mg/kg dose, a dose which produced maximum cyclooxygenase inhibition. These studies predict a wide therapeutic index for apixaban based on the prevention of occlusive thrombosis in a variety of experimental models without excessive bleeding time prolongation. These results are consistent with the safety and efficacy observed with apixaban in phase II clinical trials.

Anti-Human VWF Monoclonal Antibody SZ-123 Prevents Arterial Thrombus Formation by Inhibiting VWF–collagen and VWF-Platelet Interactions in Rhesus Monkeys

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 5194-5194
Author(s):  
Yiming Zhao ◽  
Changgeng Ruan
Keyword(s):  
Monoclonal Antibody ◽  
Platelet Aggregation ◽  
Bleeding Time ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Significant Prolongation ◽  
Arterial Thrombus

Abstract Abstract 5194 Objective: To investigate the in vivo antithrombotic efficacy of an anti-VWF monoclonal antibody SZ-123, and its potential underlying mechanism. Methods and Results: Cyclic flow reductions (CFRs) were measured in the femoral artery of monkeys before and after intravenous administration of SZ-123. Ex vivo VWF binding to collagen, platelet aggregation, platelet count and template bleeding time were performed as measurements of antithrombotic activity. In addition, plasma VWF, SZ-123 levels, and VWF occupancy were measured by ELISA. Administration of 0. 1, 0. 3, and 0. 6 mg/kg SZ-123 resulted in 45. 3%, 78. 2%, and 100% reduction in CFRs, respectively. When 0. 3 and 0. 6 mg/kg SZ-123 were administrated, 100% of VWF was occupied by the antibody. Moreover, 100% ex vivo inhibition of VWF-collagen binding and 60–95% inhibition of platelet aggregation were observed from 15 min to 1h. None of the doses resulted in significant prolongation of bleeding time. In vitro experiment also revealed that SZ-123 not only blocks collagen-VWF A3 interaction but also inhibits indirectly VWF A1 binding to GPIba induced by ristocetin. Conclusions: SZ-123 prevents in vivo arterial thrombus formation under high shear conditions by inhibiting VWF A3–collagen and VWF A1-platelet interactions and does not prolong bleeding time. Disclosures: No relevant conflicts of interest to declare.

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