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. 2020 May;18(5):1113-1123.
doi: 10.1111/jth.14755. Epub 2020 Mar 5.

The development and characterization of a long acting anti-thrombotic von Willebrand factor (VWF) aptamer

Shuhao Zhu  1 , James C Gilbert  1 , Paul Hatala  2 , Warren Harvey  3 , Zicai Liang  4 , Shan Gao  4 , Daiwu Kang  4 , Bernd Jilma  5
Affiliations

The development and characterization of a long acting anti-thrombotic von Willebrand factor (VWF) aptamer

Shuhao Zhu et al. J Thromb Haemost. 2020 May.

Abstract

Background: Thrombus formation involves coagulation proteins and platelets. The latter, referred to as platelet-mediated thrombogenesis, is predominant in arterial circulation. Platelet thrombogenesis follows vascular injury when extracellular von Willebrand factor (VWF) binds via its A3 domain to exposed collagen, and the free VWF A1 domain binds to platelet glycoprotein Ib (GPIb).

Objectives: To characterize the antiplatelet/antithrombotic activity of the pegylated VWF antagonist aptamer BT200 and identify the aptamer VWF binding site.

Methods: BT100 is an optimized aptamer synthesized by solid-phase chemistry and pegylated (BT200) by standard conjugation chemistry. The affinity of BT200 for purified human VWF was evaluated as was VWF inhibition in monkey and human plasma. Efficacy of BT200 was assessed in the monkey FeCl3 femoral artery thrombosis model.

Results: BT200 bound human VWF at an EC50 of 5.0 nmol/L and inhibited VWF A1 domain activity in monkey and human plasma with mean IC50 values of 183 and 70 nmol/L. BT200 administration to cynomolgus monkeys caused a time-dependent and dose-dependent effect on VWF A1 domain activity and inhibited platelet function as measured by collagen adenosine diphosphate closure time in the platelet function analyzer. BT200 demonstrated a bioavailability of ≥77% and exhibited a half-life of >100 hours after subcutaneous injection. The treatment effectively prevented arterial occlusion in an FeCl3 -induced thrombosis model in monkeys.

Conclusions: BT200 has shown promising inhibition of human VWF in vitro and prevented arterial occlusion in non-human primates. These data including a long half-life after subcutaneous injections provide a strong rationale for ongoing clinical development of BT200.

Keywords: aptamers; arterial thrombosis; platelets; primates; von Willebrand factor.

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Conflict of interest statement

SZ and JG are employees of Guardian Therapeutics, Inc, PH, WH and BJ consultants. Drs SZ, JG, and BJ also have an equity position with Guardian. SG and DW are employees and ZL is the chairman of Suzhou Ribo Life Science Co.

Figures

Figure 1
Figure 1
A, Secondary structure of the anti von Willebrand factor aptamer BT200. The letter “m” stands for methylated and the letters ACGU for the nucleobases adenine, cytosine, guanine, and uracil. B, Cocrystal structure of the unpegylated aptamer BT100 with the von Willebrand factor A1 domain
Figure 2
Figure 2
Binding of BT200 to purified human von Willebrand factor. EC50, Concentration resulting in 50% maximal effect; nmol/L, nanomolar; OD, optical density; data are presented as mean absorbance ± SD for each concentration (N = 6/concentration)
Figure 3
Figure 3
Effect of BT200 on von Willebrand factor (VWF) A1 domain activity in cynomolgus monkey and human plasma as measured using the REAADS® activity assay. VWF A1 domain activity is presented as relative percent concentration, which was determined against a curve made from the reference plasma provided with the kit
Figure 4
Figure 4
Effect of BT200 on VWF activity in monkeys following IV and SC administration as measured using the REAADS® activity assay. VWF A1 domain activity is presented as relative percent concentration which was determined against a curve made from the reference plasma provided with the kit. IV, intravenous infusion; SC, subcutaneous injection; VWF, von Willebrand factor
Figure 5
Figure 5
Effect of BT200 on FeCl3‐induced thrombus formation time in monkeys. Time to reduction of blood flow to <20% of baseline or no blood flow. Time to thrombus formation exceeded the maximum observation period of 2 hours (3600 s) after subcutaneous injection of 1mg/kg BT200. **P = .008, ***P = .005, Compared with PBS control (Kruskal‐Walis analysis of variance followed by U test). SEM, standard error
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