A major complication of the surgical treatment of arteries blocked by atherosclerotic plaques is restenosis - a reblockage of the artery. In an effort to prevent restenosis a number of drugs have been administered before, during, or after surgical treatment. These include anticoagulants, antithrombotics, antioxidants, antiproliferative agents, anti-inflammatory and immunosuppressive agents, as well as agents that prevent arterial vasospasm. While many of these drugs have been successful in preventing restenosis in animal models, most, if not all, have had only limited success in clinical trials. The reasons for this lack of clinical success can, in many cases, be attributed to the exact dose and administrative regime used in the animal studies not being applied in the clinical study. Complicating the clinical situation is that in order to be active, the anti-restenotic drug must be present at the site of injury both immediately following injury and for sufficient time afterwards the specific window of opportunity. If too little drug is given, or given too late, for too short at time, or in an interrupted fashion, then restenosis may still occur. A way of preventing the complications of many treatments is to deliver the anti-restenotic agents locally rather than systemically. In this manner, the anti-restenotic drug would be directed only to the site of surgery. Thus systemic effects, as well as the cost of treatment, incidence of bleeding complications and incidence of restenosis will all be minimised.
In the studies described here, we tested the hypothesis that antibodies against cross-linked fibrin (XLF) D-dimer can be used to deliver anti-restenotic agents to areas of fibrin deposition following angioplasty injury of animal arteries. We have called this method DDART - directly delivered anti-restenosis therapy (using anti-D-dimer XLF antibodies).
Using animal models it was found that XLF is deposited onto the artery wall within 10 minutes of injury and remains present for at least 24 weeks. The best antibody to XLF for use in the rabbit model was found to be H93.7C.1D2/48 (1D2), given at a dose of 30-40 µg/kg. 1D2 was conjugated to the anticoagulant heparin using N-succinimidyl 3-(2-pyridyldithio)propionate. The purified 1D2-heparin was administered to rabbits immediately after carotid artery injury, and changes in cross-section arterial areas measured two weeks later. The injured carotid artery of these animals had a significantly smaller percent luminal narrowing and medial area than rabbits given control agents. They also had minimal neointimal development and a smaller intima to media ratio than rabbits given saline, but not those given unconjugated 1D2. Rabbits given the conjugate had positive expansive (adaptive) remodeling of the entire artery, so that any increase in neointimal area was accommodated without loss of functional lumen.
1D2 was also conjugated to low molecular weight heparins (LMWHs) using adipic acid dihydrazide. Two weeks after carotid artery injury, rabbits given 1D2- LMWH had a smaller percent medial area of the area inside the external elastic lamina than control rabbits, and a smaller neointima and intima to media ratio than rabbits given saline or LMWH, but not those given unconjugated 1D2. Possibly the presence of the antibody prevented further fibrin binding and thus SMC stimulation. Although a neointimal thickening was present in arteries given 1D2-LMWH conjugate, there was sufficient positive adjustment in the artery for it to be accommodated without losing luminal area. As with rabbits given 1D2-heparin, the arteries of rabbits given 1D2- LMWH after injury were able to positively remodel and accommodate the (reduced) bulk of neointima.
1D2 was then conjugated to the immunosuppressive agent rapamycin using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide stabilised with N-hydroxysuccinimide. Rapamycin has recently been shown to reduce experimental neointimal thickenings and clinical restenosis. 1D2-rapamycin was given to rabbits after carotid artery injury, and the arterial areas measured 4 weeks later. The medial area in these rabbits was smaller than in control rabbits, in both injured and uninjured arteries. This resulted in an increased intima to media ratio and a decreased luminal diameter, despite the neointima developed after injury being smaller that in control arteries.
Thus it has been shown that antibodies against XLF can site-deliver antirestenotic agents to injured areas of the artery wall. The drugs are able to influence smooth muscle cell biology in the injured region and reducing neointimal formation. It is suggested that DDART treatment may be capable of reducing the restenosis that occurs after surgical treatment of atherosclerotic vessels, without excessive systemic complications.