Reversal Activity and Toxicity of Heparin-Binding Copolymer after Subcutaneous Administration of Enoxaparin in Mice

Abstract

Uncontrolled bleeding after enoxaparin (ENX) is rare but may be life-threatening. The only registered antidote for ENX, protamine sulfate (PS), has 60% efficacy and can cause severe adverse side effects. We developed a diblock copolymer, heparin-binding copolymer (HBC), that reverses intravenously administered heparins. Here, we focused on the HBC inhibitory activity against subcutaneously administered ENX in healthy mice. BALB/c mice were subcutaneously injected with ENX at the dose of 5 mg/kg. After 110 min, vehicle, HBC (6.25 and 12.5 mg/kg), or PS (5 and 10 mg/kg) were administered into the tail vein. The blood was collected after 3, 10, 60, 120, 360, and 600 min after vehicle, HBC, or PS administration. The activities of antifactors Xa and IIa and biochemical parameters were measured. The main organs were collected for histological analysis. HBC at the lower dose reversed the effect of ENX on antifactor Xa activity for 10 min after antidote administration, whereas at the higher dose, HBC reversed the effect on antifactor Xa activity throughout the course of the experiment. Both doses of HBC completely reversed the effect of ENX on antifactor IIa activity. PS did not reverse antifactor Xa activity and partially reversed antifactor IIa activity. HBC modulated biochemical parameters. Histopathological analysis showed changes in the liver, lungs, and spleen of mice treated with HBC and in the lungs and heart of mice treated with PS. HBC administered in an appropriate dose might be an efficient substitute for PS to reverse significantly increased anticoagulant activity that may be connected with major bleeding in patients receiving ENX subcutaneously.

Keywords: anticoagulation; antidote; block copolymer; enoxaparin; heparins; macromolecules; mice; protamine; rodents; toxicity.

Figure 1

The time-course of the neutralization of enoxaparin (ENX) by HBC (6.25 and 12.5 mg/kg) or protamine sulfate (PS) (5 and 10 mg/kg) in mice, measured by antifactor Xa activity 3, 10, 60, 120, 360, and 600 min after antidote administration. * p < 0.05, ** p < 0.01 vs. ENX + PBS (phosphate buffered saline), # p < 0.05, ## p < 0.01 vs. PS 5 mg/kg, ^ p < 0.05, ^^ p < 0.01 vs. PS 10 mg/kg, Mann–Whitney test. Results are shown as median with lower and upper limits, n = 6.

Figure 2

The time-course of neutralization of enoxaparin (ENX) by HBC (6.25 and 12.5 mg/kg) or protamine sulfate (PS) (5 and 10 mg/kg) in mice measured by antifactor IIa activity 3, 10, 60, 120, 360, and 600 min after antidote administration. * p < 0.05, ** p < 0.01 vs. ENX + PBS (phosphate buffered saline), # p < 0.05, ## p < 0.01, vs. PS 5 mg/kg, ^ p < 0.05, ^^ p < 0.01 vs. PS 10 mg/kg, Mann–Whitney test. Results are shown as median with lower and upper limits, n = 6.

Figure 3

Representative micrographs of tissue sections of kidney, lung, heart, liver, spleen, and brain sections of mice treated subcutaneously with enoxaparin (ENX) (5 mg/kg) and an intravenous bolus of vehicle, HBC (6.25 and 12.5 mg/kg) or protamine sulfate (PS) (5 and 10 mg/kg). Organs were harvested 10 h after antidote administration. Original magnification, 100×; hematoxylin and eosin staining. PBS, phosphate buffered saline.

Figure 4

Representative micrographs of tissue sections of kidney, lung, heart, liver, spleen, and brain sections of mice treated subcutaneously with enoxaparin (ENX) (5 mg/kg) and an intravenous bolus of vehicle, HBC (6.25 and 12.5 mg/kg) or protamine sulfate (PS) (5 and 10 mg/kg). Organs were harvested 10 h after antidote administration. Original magnification, 400×; hematoxylin and eosin staining. PBS, phosphate buffered saline.

Figure 5

Schematic representation of the study protocol. The diagram shows the time-course of events from enoxaparin (ENX) subcutaneous administration and injection with vehicle (PBS) or antidotes: HBC (6.25 and 12.5 mg/kg) or protamine sulfate (5 and 10 mg/kg) into the mice tail vein.