Targeted delivery of thrombolytics to newly formed thrombi

Cardiovascular diseases have been the most common cause of death in the US each year with the exception of 1918. In 1998, this group of diseases claimed the lives of nearly one million Americans, which translates to 40% of all deaths and 1 death every 30 seconds. Strikingly, more people die from cardiovascular disease than the next six leading causes of death combined, and if cardiovascular disease were to be eliminated, the average life expectancy would rise by 7 years. Atherosclerotic diseases including stroke, myocardial infarction and limb-threatening ischemia associated with peripheral vascular disease (PAOD), result from the formation of plaques which rupture, resulting in the formation of blood clots. These can then either occlude vessels at the site of plaque rupture or they can become detached occluding vessels at a distant site. This clot formation contributes heavily to the mortality and economic cost of cardiovascular disease.

Deep venous thrombosis affects approximately 0.2% of the population, most commonly adults over age 60 but also anyone who is immobilized for long periods; recovering from recent surgery, trauma, or childbirth; obesity; use of medications such as estrogen and birth control pills. Deep venous thrombosis causes leg pain and swelling. Although self-resolution is the norm, thrombi may detach and embolize to other major organs such as the lung - known clinically as pulmonary embolism. Pulmonary embolism is the third most common cause of death in the US and has been termed the "silent killer". Autopsy results show that as many as 60% of patients dying in the hospital have had a pulmonary embolism, but the diagnosis is missed in about 70% of the cases. As a result, of the 650,000 cases occurring annually, over 200,000 victims die. Acute pulmonary embolism is a dynamic process. Thrombi begin to lyse immediately after reaching the lung. Usually, lysis is complete within several weeks in the absence of preexisting cardiopulmonary disease; in some instances, even large thrombi may lyse in a few days. The physiologic alterations lessen over hours or days as pulmonary circulation improves. However, massive emboli may cause death within minutes or hours, before infarction has time to develop.

Preventative options for cardiovascular diseases normally include anti-thrombotic and/or anti-coagulant therapies, often in conjunction with percutaneous interventions (for example, coronary angioplasty followed by stent placement). Thrombolytic treatments generally become necessary in acute disease (ie stroke with presentation within 3 hours of onset, myocardial infarction within 12 hours of onset, and acute limb-threatening ischemia).

Streptokinase remains the most widely prescribed thrombolytic agent for acute myocardial infarction (AMI) in many parts of the world due to it's low cost. This is despite the lower efficacy compared to more expensive agents such as t-PA and third generation thrombolytics related to t-PA. At present, rt-PA is the only agent approved for the treatment of stroke, and for only a brief window of time (onset of symptoms less than three hours). Only 3% of American stroke victims receive t-PA since they present outside of this window or because of fears of hemorrhage.

The use of thrombolytics as a preventative approach is precluded by a number of factors including the risk of hemorrhage and tissue damage. Hemorrhage also presents a significant risk to patients being treated by standard antithrombotic such as aspirin and new generation GPIIb/IIIa blockers (e.g. Reopro). The development of thrombolytics which do not produce hemorrhage would therefore revolutionize not only acute treatment of cardiovascular disorders but also preventative measures.

The formation of blood clots is a key defense mechanism to prevent blood loss following trauma, surgery or various invasive interventions. Currently used thrombolytics and antithrombotic agents do not dissociate between these hemostatic clots and those that form following plaque rupture and thus they are associated with hemorrhage.

In their recent paper Murciano et al have developed a strategy for targeting t-PA to nascent thrombi. This involved linking a large number of biotinylated t-PA molecules to the surface of erythrocytes. In an in vitro system, erythrocytes engineered in this fashion were able to dissolve approximately 90% of nascent clots but less than 5% of less permeable, preexisting clots indicating throbus selectivity. Two hours after intravenous administration to rats at least 10-fold more t-PA remained in the circulation when administered linked to erythrocytes compared to when given alone. This was mirrored by an increase in the fibrinolytic activity of blood sampled from animals treated with engineered erythrocytes. This increase in t-PA half-life as well as the increase in selectivity addresses two of the most serious hurdles precluding the prophylactic use of t-PA.

A model of pulmonary microembolism was then employed to evaluate the therapeutic potential of the engineered erythrocytes. While thrombolysis was very short lived when t-PA alone was employed as a pre-treatment, the administration of t-PA linked erythrocytes caused a dramatic fibrinolysis of emboli even when administered prior to the formation of emboli. Post-treatment with engineered cells however had little effect. This confirms in vitro findings and demonstrates once again that erythrocyte linked t-PA has a half-life and thrombus selectivity consistent with prophylactic use. Similar findings were reported in a second model of thrombosis characterized by larger clots, characteristic of the life-threatening occlusive clots that form under clinical conditions.

This study therefore demonstrates the feasibility of developing standard thrombolytics into thromboprophylactics and if such a strategy can be used to limit the formation of new clots in patients the clinical potential could be immense. Future steps in the development of this technology will include the optimization of the linkage between t-PA (or indeed other thrombolytics) and the carrier - this will not only improve efficacy but it will also improve the proprietary position paving the way towards commercial development of this technology.

Entry date Friday, September 19, 2003

Adapted from Murciano et al, Nat Biotechnol. 2003 Aug;21(8):891-6. Epub 2003 Jul 06.