Open Access Research

Safety evaluation of a recombinant plasmin derivative lacking kringles 2-5 and rt-PA in a rat model of transient ischemic stroke

R Christian Crumrine1*, Victor J Marder2, G McLeod Taylor1, Joseph C LaManna3, Constantinos P Tsipis3, Valery Novokhatny1, Philip Scuderi1, Stephen R Petteway1 and Vikram Arora1

Author Affiliations

1 Research and Pre-clinical Development, Grifols Therapeutics, Inc., Research Triangle Park, North Carolina, USA

2 Division of Hematology/Medical Oncology, Department of Medicine, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA

3 Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, OH, USA

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Experimental & Translational Stroke Medicine 2012, 4:10  doi:10.1186/2040-7378-4-10

Published: 16 May 2012

Abstract

Background

Tissue type plasminogen activator is the only approved thrombolytic agent for the treatment of ischemic stroke. However, it carries the disadvantage of a 10-fold increase in symptomatic and asymptomatic intracranial hemorrhage. A safer thrombolytic agent may improve patient prognosis and increase patient participation in thrombolytic treatment. A novel direct-acting thrombolytic agent, Δ(K2-K5) plasmin, promising an improved safety profile was examined for safety in the snare ligature model of stroke in the rat.

Methods

Male spontaneously hypertensive rats were subjected to 6 hours middle cerebral artery occlusion followed by 18 hours reflow. Beginning 1 minute before reflow, they were dosed with saline, vehicle, Δ(K2-K5) plasmin (0.15, 0.5, 1.5, and 5 mg/kg) or recombinant tissue-type plasminogen activator (10 and 30 mg/kg) by local intra-arterial infusion lasting 10 to 60 minutes. The rats were assessed for bleeding score, infarct volume, modified Bederson score and general behavioral score. In a parallel study, temporal progression of infarct volume was determined. In an in vitro study, whole blood clots from humans, canines and rats were exposed to Δ(K2-K5). Clot lysis was monitored by absorbance at 280 nm.

Results

The main focus of this study was intracranial hemorrhage safety. Δ(K2-K5) plasmin treatment at the highest dose caused no more intracranial hemorrhage than the lowest dose of recombinant tissue type plasminogen activator, but showed at least a 5-fold superior safety margin. Secondary results include: temporal infarct volume progression shows that the greatest expansion of infarct volume occurs within 2–3 hours of middle cerebral artery occlusion in the spontaneously hypertensive rat. A spike in infarct volume was observed at 6 hours ischemia with reflow. Δ(K2-K5) plasmin tended to reduce infarct volume and improve behavior compared to controls. In vitro data suggests that Δ(K2-K5) plasmin is equally effective at lysing clots from humans, canines and rats.

Conclusions

The superior intracranial hemorrhage safety profile of the direct-acting thrombolytic Δ(K2-K5) plasmin compared with recombinant tissue type plasminogen activator makes this agent a good candidate for clinical evaluation in the treatment of acute ischemic stroke.

Keywords:
Ischemic stroke; Δ(K2-K5) plasmin; Intracranial hemorrhage; Spontaneously hypertensive rat model; Recombinant tissue-type plasminogen activator (rt-PA); Middle cerebral artery occlusion (MCAo)