Key Point:

A novel conjugate of urokinase and a monoclonal antibody has enabled researchers to localize a thrombolytic agent to the alveolar capillaries in rats; a similar approach is now being sought in humans.

NANJING, CHINA—Thrombolytic therapy is often used to treat pulmonary embolism (PE), although its inability to distinguish fibrin in lung thrombi from that in protective hemostatic plugs often leads to severe bleeding complications. This problem could be circumvented if thrombolytic agents were delivered only to the pulmonary vasculature, but attempts to provide such targeted treatments have been hindered by the difficulty of finding an appropriate candidate antibody.

However, researchers from Nanjing University in China appear to have answered this challenge.[1] Jian-Ning Liu, PhD, and colleagues recently purified a highly specific endothelial membrane protein from the alveolar capillaries of rats and then used it to create a pulmonary endothelium–specific antibody, which they named RE8F5.

LUNG-SPECIFIC THOMBOLYSIS

A urokinase conjugate of the antibody (UK/RE8F5), when administered to rats with PE, was shown to accumulate overwhelmingly in the lungs, to have robust thrombolytic activity, and to produce limited systemic fibrinogen breakdown. The findings, which represent the first successful attempt at lung-specific thrombolysis, may offer a novel approach for treating PE in humans.

“Since RE8F5 is a monoclonal antibody against rat pulmonary endothelium,” said Dr. Liu, “UK/RE8F5 itself is not suitable for human use.” However, the concept itself can be applied to humans; in fact, his group has already begun the work of identifying a monoclonal antibody with specificity for human pulmonary endothelium.

RE8F5

The researchers’ first objective was to characterize the various monoclonal antibodies they had generated from the rat pulmonary vascular endothelium. Monoclonal antibody RE8F5 quickly stood out because it was the only one that bound exclusively to frozen tissue sections from the rats’ lungs; furthermore, RE8F5 adhered specifically to the alveolar capillaries and not to the large blood vessels or the bronchial epithelium, making it a desirable vehicle for delivering a thrombolytic agent targeted against PE.

In vivo biodistribution assays revealed that both RE8F5 and UK/RE8F5 were taken up mostly by the rats’ lungs; only modest amounts remained in the animals’ livers and circulation. Moreover, the researchers found that lung uptake was rapid. For the conjugate, 23% of the injected dose was detected in the lung tissue after one hour, a finding they described as “expeditious.”

TWO SUPERIOR ATTRIBUTES

To establish the thrombolytic activity of the conjugate, the researchers developed a rat PE model. They injected biotinylated microclots into the rat’s external jugular vein and then assessed the ability of the conjugate, urokinase, and reteplase (a nonglycosylated form of human tissue plasminogen activator) to break the clots down. All three agents exhibited high degradative ability at the highest dose used. Urokinase, at a dose of 100,000 IU, yielded about 92% clot lysis; reteplase, at 150,000 IU, yielded around 97%; and UK/RE8F5, at 8,000 IU, yielded about 90%.

However, UK/RE8F5 had two qualities that made it superior to the other two thrombolytics. First, the residual levels of blood fibrinogen were substantially higher when UK/RE8F5 was used. One hour after injection of the highest doses of each agent, residual levels had dropped to 76% of expected in the rats given urokinase and to 74% of expected in the animals that received reteplase; however, in the rats given the conjugate, residual blood fibrinogen levels did not decline at all. According to Dr. Liu, this finding indicates that UK/RE8F5’s thrombolytic potency was achieved locally, and systemic plasminogen activation was avoided. Bleeding times in the rats’ tails were unchanged before and after thrombolytic treatment only in the UK/RE8F5 model, which also suggests that systemic fibrinolysis was absent.

Second, the conjugate was able to effect PE lysis at a significantly lower dose than was required by the other thrombolytic agents. As a consequence, UK/RE8F5 was found to be 12-fold more effective than urokinase and 16-fold more effective than reteplase in inducing thrombosis.

Dr. Liu and his team are now trying to identify a monoclonal antibody specific for the human pulmonary endothelium. In addition, they are trying to extend their findings by developing a urokinase conjugate with an antibody specific for the middle cerebral artery; their hope is that this conjugate would help lower the rate of intracranial bleeding when thrombolysis is used for the treatment of ischemic stroke.

—Verna L. Schwartz, MS

Reference
1.Ding BS, Zhou YJ, Chen XY, et al. Lung endothelium targeting for pulmonary embolism thrombolysis. Circulation. 2003;108:2892-2898.

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