It's old news that the biggest perpetrator of severe disease and premature death in Western societies is the blockage of blood vessels by abnormal clots.

"Basically," observed molecular biologist/biochemist Christopher Rusconi, "it's the kind of cardiovascular disease that results in formation of a thrombus. I think it kills more people than the next three to five possible causes of death in the industrial world. So when you think of heart attack, stroke, pulmonary embolism - anything forming a clot that stops the blood flow - [it] far outstrips any other cause of death.

"That's obviously a big deal," Rusconi continued. "There's also a huge number of interventions, both medical and surgical, each year to help treat the diseases associated with clotting. In cardiovascular treatments you have more than 600,000 angioplasties a year. Coronary artery bypass grafts, where you sew in a new vessel to patch around, also exceed 600,000 a year. And then there's unstable angina, the chest-pain people, who have partial occlusion of a vessel that can readily lead to forming a blood clot and a heart attack - 1.4 million new cases a year. Those annual numbers are on a huge upswing. They're not just a huge cause of death, but a huge burden medically to society and to people."

Rusconi is director of Duke University's Research Program in Combinatorial Therapeutics in Durham, N.C. He is lead author of an article in the current issue of Nature, dated Sept. 5, 2002, titled: "RNA aptamers as reversible antagonists of coagulation factor IXa." Its senior author is Bruce Sullenger, vice chair of surgery at Duke.

"I think the biggest-picture principal finding of this paper," Rusconi told BioWorld Today, "is that we've created a blood-thinning drug with the intent of making a matching antidote that could counteract the effects of the initial drug when needed. That's the fundamentals of our technology. And then we applied it to an area of obvious need."

Just What The Doctors Ordered

"We asked Duke cardiovascular clinicians what they needed in terms of therapeutics, and they described a need to have a blood thinner with activities that could be controlled by an antidote," he said. "Now we believe that we have a workable solution to designing such antidotes. I think one novel outcome is the first anticoagulant/antidote pair since heparin became the clot-controlling drug of choice in the 1920s - and later its antidote, protamine, came into play, and joined it.

"When patients undergo cardiac surgical procedures," Rusconi explained, "such as angioplasty or coronary artery bypass, heparin is commonly administered prior to the procedure to prevent blood clots from closing off the arteries. Blood tends to clot when subjected to foreign instruments," he went on, "such as a bypass machine or balloons used in angioplasty. Heparin is sometimes administered after a sudden cardiac arrest to ensure blood flow through the heart," Rusconi added. "It's been proven to be a lifesaver, but during and after surgery, blood thinners can put a patient at risk from hemorrhaging, other complications and death."

The co-authors focused their efforts on a class of drugs called aptamers, which are compounds made of RNA nucleic acid that bind directly to a target protein, Factor IXa, and inhibit that protein's activity.

"There's a massive industry around making new anticoagulants," Rusconi pointed out. "One of those targets is Factor IXa, a member of the coagulation cascade, which is given to hemophiliacs who lack IXa..

"We chose Factor IXa as the target to inhibit by our aptamer drug," Rusconi recalled. "In the aptamer generation process we made a very large combinatorial library, like 10¹⁴ - 100 trillion - different compounds from which to find our aptamers. Ours were nucleic acid libraries, actually bigger than a phage-display library. They formed a sheet based on the aptamer's sequence. By an iterative fashion, we contacted a protein with that library, a very small portion of which bound to our protein. By a simple screening we constructed the antidote."

Patients' Plasma Provided Tissue Samples

To try out their matching compounds, Rusconi and his colleagues conducted in vitro experiments, as they reported in Nature. "We first made this inhibitor to Factor IXa, and figured out how to prevent it from doing its coagulant job. We then designed and tested a panel of potential antidotes that could bind to and neutralize that activity. In biochemical experiments, we found one that did that the best. We then looked in the normal, healthy human plasma system, which is a tissue system - really an ex vivo system - and isolated an antidote that could effectively neutralize our factor IXa inhibitor, within 10 minutes, and at doses that made sense in eventually moving forward to animal models.

"Then to make it a little more relevant," Rusconi continued, "we did the same experiment in blood samples isolated from patients who could not tolerate heparin. We first thinned their plasma with the IX aptamer inhibitor. Then we added the antidote and measured various data points in terms of how it neutralizes. How fast? How much to neutralize? How stable? Data showed that within 10 minutes we could completely neutralize the drug's activity. And that set the stage for our rationally moving into the animals.

"We're working right now with pigs, which are a standard cardiovascular anticoagulant model. They have big blood vessels; they eat like humans. If we inject the aptamer into a pig, does the animal respond to it? We can measure changes in clotting parameters. And we're doing exactly the same experiments we reported in vitro. To date, the initial in vivo data in the pigs are encouraging.

"Right now our goal is to go to Phase I clinical trials within two years - 20 to 24 months. We still have a little optimization of the aptamer to do," Rusconi said. "What we would like is an injectable drug that has a longer circulating half-life, probably in the nine- to 12-hour range. Then we will move into preclinical toxicity studies.

"Duke has filed patent applications covering the aptamer compositions and the concept of the antidotes, for which Sullenger and I are the sole inventors," Rusconi noted. "The sales of injectable antithrombotics - anticoagulants, platelet inhibitors and clot-busting drugs - were projected to be a $3 billion market in 2001. And our technology," he concluded, "fits in nicely with markets that are projected to grow to $21 billion or $22 billion by 2007."