The First In Vitro and In Vivo Assessment Of Anfibatide, a Novel Glycoprotein Ib Antagonist, In Mice and In a Phase I Human Clinical Trial

Platelet adhesion and subsequent aggregation at the site of vascular injury are critical for hemostasis and thrombosis. It has been well accepted that interaction between the GPIb complex and von Willebrand factor (VWF) plays a key role in initiation of platelet adhesion, particularly at high shear. Platelet surface integrin αIIbβ3, through interaction with fibrinogen or other ligands, then mediates platelet aggregation to form a stable hemostatic plug or thrombus. Recently, the indispensable role of the GPIb-VWF interaction in platelet aggregation at extremely high shear (e.g. > 10,000s^-1; areas of stenosis following arteriosclerosis and/or thrombus growth) has been highlighted. Therefore, both the GPIb complex and αIIbβ3 are considered major targets for antithrombotic therapies. Interestingly, although several inhibitors of αIIbβ3 have been developed for antithrombotic therapies, no drug has been developed to target the GPIb complex even though there are limitations for anti-αIIbβ3 therapies. The GPIb complex is, therefore, an attractive target for anti-thrombotic therapy. Here, we evaluated the efficacy and safety in vitro and in vivo of Anfibatide, a novel GPIb antagonist, in mice and in a phase I clinical trial.

Anfibatide was purified from venom of the Agkistrodon acutus snake and its purity was analyzed by mass spectrometry. The effect of Anfibatide on murine platelet function was assessed by in vitro platelet aggregometry, ex vivo perfusion chamber, and two complementary in vivo intravital microscopy models. The effects of Anfibatide on human platelet aggregation and thrombus formation were studied in vitro, and thrombealastography (TEG) was also performed. Most importantly, we evaluated the safety and efficacy of Anfibatide on platelet function and coagulation in a total of 94 healthy human volunteers in a phase I clinical trial.

MALDI-TOF mass spectrometry of Anfibatide showed only one peak and the mass to charge ratio is 29799.7. Anfibatide specifically inhibited ristocetin-induced human platelet aggregation. Interestingly, Anfibatide was not able to inhibit botrocetin-induced murine platelet aggregation in plate-rich plasma (PRP), suggesting that its binding site may differ from other snake venom-derived GPIb antagonists. We found Anfibatide did not affect ADP-, TRAP- or collagen-induced aggregation in PRP, suggesting its specificity to GPIb. In ex vivo perfusion, Anfibatide strongly inhibited murine and human platelet adhesion, aggregation, and thrombus formation on a collagen-coated surface at both high and low shear flow conditions although it is far more sensitive at high shear. Importantly, Anfibatide effectively dissolved the preformed thrombi when we continuously perfused Anfibatide-treated whole blood through perfusion chambers, demonstrating its potential as an anti-thrombotic therapy. In the mesenteric arteriole thrombosis model, Anfibatide strongly inhibited platelet adhesion, thrombus formation, and prevented vessel occlusion in response to FeCl₃ injury (P<0.05). At sites of laser-injured cremaster arterioles, Anfibatide also dramatically inhibited platelet accumulation and thrombus growth. Anfibatide did not cause significant murine platelet activation in vitro and had no significant change in coagulation parameters in TEG when we treated human whole blood with Anfibatide, suggesting it had minimal side effects. In the phase I clinical trial, results showed that Anfibatide can occupy approximately 95% of GPIb and inhibit up to 90% of ristocetin specific platelet aggregation. The inhibitory effect was undetectable four hours after Anfibatide was withdrawn. There were no serious adverse events, or deaths that occurred during the study. Anfibatide did not significantly prolong bleeding time, activated partial thromboplastin time (APTT), prothrombin time (PT), or thrombin time (TT). There was also no spontaneous bleeding or bleeding from blood collection sites. Anfibatide did not significantly affect platelet count and no anti-Anfibatide antibodies were detected in the subjects, suggesting that Anfibatide is well-tolerated in healthy individuals.

These comprehensive studies in mice and human subjects and in the first clinical trial clearly demonstrated that Anfibatide is a safe and potent anti-platelet reagent with great potential for future anti-thrombotic therapy.

Hou:Lee’s pharmaceutical holdings limited: Research Funding. Lei:Lee’s pharmaceutical holdings limited: Research Funding. Zhao:Lee’s pharmaceutical holdings limited: Research Funding. Shen:Lee’s pharmaceutical holdings limited: Research Funding. Zhou:Lee’s pharmaceutical holdings limited: Research Funding. Wang:Lee’s pharmaceutical holdings limited: Research Funding. Marshall:Lee’s pharmaceutical holdings limited: Research Funding. Ni:Lee’s pharmaceutical holdings limited: Research Funding.