Page 130 - Haematologica Vol. 107 - September 2022

ARTICLE - Effects of BT200 on VWF/FVIII in humans
K.D. Kovacevic et al.
site of the venous catheter (median cubital vein). This was diagnosed with color-coded duplex sonography and both events were treated with enoxaparin. One subject received 8000 IE SC once daily, and the other 4000 IE SC once daily. Additionally, one volunteer developed venous throm- bosis of the limb (lower left leg) and was treated with enoxaparin 6000 IE SC for 4 days, and then with edoxaban 60 mg once a day for 11 weeks. Furthermore he was pre- scribed compression stockings. Lastly, one subject devel- oped thrombophlebitis of the cubital vein and a superficial thrombophlebitis which extended into the great saphen- ous vein. In this subject factor V Leiden, which would pre- dispose him to thrombotic events, was diagnosed. He was treated with enoxaparin 8000 IE SC 2x1 for 5 days and then with edoxaban 60 mg orally 1x1. Subjects who experi- enced these events received a single dose of BT200 ≥36 mg (3 of 4 subjects and 1 of them also received desmo- pressin), or repeated weekly doses of 12 mg (in the re- maining 1 subject).
D-dimer and prothrombin fragment levels were measured in 28 subjects. Although no significant difference between levels in the placebo and BT200 groups was seen, two out of seven subjects in the placebo arm had elevated D- dimer levels at baseline (0.95 and 1.61 mg/mL), and three more placebo-treated subjects showed elevated D-dimer levels in at least one blood sample (0.75, 1.07, 1.43 0mg/mL). Therefore, due to its low specificity, we decided not to continue measuring D-dimer levels. However, D- dimer levels do demonstrate a good negative predictive value for ruling out pulmonary embolism when in the nor- mal range.26
Three serious adverse events occurred, all of which were considered to be unrelated to the study drug. One subject on placebo had severe back pain which was treated in hospital, one subject receiving 24 mg suffered a bone frac- ture 24 days after dosing and underwent surgery, and one subject had an intervertebral disc protrusion 17 days after treatment with 48 mg BT200. She was managed with re- peated desmopressin infusions to restore VWF-dependent platelet function before undergoing surgery.
Discussion
This first-in-human trial examined the tolerability, safety, pharmacokinetics and pharmacodynamic effects of the VWF A1-binding aptamer, BT200. In previous non-human primate studies the median half-life was found to be 3-4 days for BT200 doses ranging from 0.5-10 mg/kg.21 In our healthy volunteers, the estimated half-life ranged from 7- 12 days for BT200 doses approximately ≥0.02 mg/kg, which is 2- to 3-fold longer than the dose in monkeys. Single SC doses of ≥12 mg BT200 yielded plasma concentrations of ~1 mg/mL in humans which dose-dependently decreased
VWF activity and shear-dependent platelet function by >50%. This indicates that the concentration-effect relation- ships seen in recent in vitro studies using human blood7,27,28 are directly applicable to the in vivo clinical pharmacology. Treatment with ≥1 mg/kg BT200 inhibited VWF-dependent platelet function and thrombus formation in non-human primates,21 suggesting a possible therapeutic role for BT200 in the secondary prevention of cardio- and cer- ebrovascular diseases.27 Interestingly, as this was not seen in the preliminary monkey studies, BT200 increased VWF antigen levels and FVIII activity up to 4-fold above base- line. As BT200 did not increase VWF propeptide levels, this increase in VWF/FVIII can be explained by decreased clearance of VWF. The mechanism of VWF clearance is complex and involves different receptors,29 and VWF clear- ance dominates FVIII clearance.30 While this trial was still ongoing, it was published that pegylation of VWF A1-do- mains with 40 kDa polyethylene glycol decreases their clearance by the macrophage LRP1.23 BT200 binding to the A1-domain of VWF indirectly “pegylates” the A1-domain, and decreased clearance is the likely underlying mechan- ism of action for the observed increase in VWF/FVIII levels. X-ray crystallography demonstrates that BT200 binds to the VWF A1-domain with multiple contacts in the region from R1395 to Q1402.21 This region includes a critical bind- ing site on VWF-A1 for LRP1, the primary clearance recep- tor for the VWF/FVIII complex, i.e., K1408 on VWF A1.31 This same region from R1395 to Q1402 on VWF-A1 partially overlaps with that of the corresponding binding site for glycoprotein Ib, providing a biophysical basis for the ob- served pharmacological competition between BT200 and platelet glycoprotein Ib.21
The FVIII was hemostatically active, which was not only reflected by a shortening of the activated partial throm- boplastin time, but also by an increase in the endogenous thrombin potential. While the overall safety and tolerabil- ity profiles of BT200 were good and similar to nonclinical safety experience in non-human primates, this heightened hemostatic activity resulted in the occurrence of venous thrombosis and thrombophlebitis at the inser- tion site of venous catheters in two subjects. Additionally, distal venous thrombosis/thrombophlebitis occurred in two further subjects, one of whom had a medical history of previous thrombosis associated with the factor V Leiden mutation. One may potentially be able to explain the otherwise rare events of thrombosis of the upper limbs as follows. After venous puncture, normal hemo- stasis induces a localized platelet plug to stop bleeding. Normally, thrombus growth is stopped by endogenous fi- brinolysis. However, after the administration of BT200 there is an increase in endogenous thrombin potential due to supra-normal FVIII levels. This leads to the local- ized plug growing, and it then becomes clinically visible thrombosis.
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