Eptifibatide-Eluting Stent as an Antiproliferative and Antithrombotic Agent: In Vitro Evaluation

Coronary stent placement is the most commonly employed technique for percutaneous treatment of atherosclerotic heart disease. It accounts for about 75% of the procedures performed worldwide.¹ In comparison to conventional balloon angioplasty, stents have improved the efficacy of percutaneous coronary interventions (PCI) by reducing abrupt or threatened vessel closure^2,3 and by reducing restenosis.^4,5 Notwithstanding, coronary stent thrombosis has remained an uncommon, but serious complication of PCI.⁶ The newer drug-eluting stent (DES) inhibits restenosis by inhibiting SMC proliferation, but there are concerns that the drug eluted from the stents may delay the endothelialization process⁷ around the stent struts and thus increase the risk of prolonged thrombogenicity of the stent leading to late stent thrombosis.⁸ Therefore, stent thrombosis may be more common after DES use, with consequent danger to the patients. For this reason, patients who have received DES are often prescribed aspirin and clopidogrel therapy for at least 6 months. Despite dual antiplatelet therapy, stent thrombosis persists at a rate of 0.5–2% in elective cases,^9–12 and up to 6% in patients with acute coronary syndromes.^10,13 Eptifibatide¹⁴ is a synthetic cyclic heptapeptide inhibitor of the platelet glycoprotein (GP) IIb/IIIa receptor. It is derived from the structure of barbourin, which has been isolated from the venom of the rattlesnake. The GP IIb/IIIa receptor is a member of the integrin super-family of cell surface adhesive protein receptors.¹⁵ The GP IIb/IIIa receptor binds specifically to one anchorage region on each end of the fibrinogen protein, facilitating cross-links and platelet aggregation. Preclinical pharmacological studies have established that eptifibatide can inhibit thrombosis effectively when given intravenously. Pharmacokinetics and pharmacodynamic studies¹⁶ in both animal models and humans have shown that the antiplatelet effect of eptifibatide has a rapid onset of action, > 80% inhibition of platelet aggregation within 15 minutes and a short plasma life (2.5 hours), as well as being rapidly reversible. It is licensed for use in patients with acute coronary syndromes and for use in PCI.^17–19 The GP IIb/IIIa receptor is closely related to the avb3 receptor^20–23 which binds matrix proteins deposited on injured tissues, including vitronectin, von Willebrand Factor, fibrinogen, fibronectin, collagen and thrombospondin. This receptor mediates a number of processes, which include smooth muscle cell adhesion, migration and proliferation. Previous studies²⁴ have shown that eptifibatide alone can inhibit avb3-mediated attachments of human arterial smooth muscle cells (HASMCs) to thrombospondin (TSP) and prothrombin. In cell proliferation assays, eptifibatide compound inhibited avb3-mediated responses to soluble TSP by HASMCs and b3 integrin-expressing human embryonic kidney cells. With these findings on the effectiveness of eptifibatide on the avb3 receptor, it was considered that it might be possible to inhibit neointimal hyperplasia with such an antiplatelet drug if the local dose were sufficient (which might not be safely achievable using systemic administration) and reduces the risk of both restenosis and thrombosis. We hypothesized that a polymer-coated stent could deliver eptifibatide locally and for a sustained period. To test this, the absorption and elution characteristics of eptifibatide on this stent were studied, as well as its effects on platelet aggregation and SMC proliferation. The current studies extend the concept of DES to eptifibatide, a drug with established clinical use, in order to assess its potential in man as a DES agent with dual properties, both antithrombotic and possibly antirestenotic as well. Materials and Methods The polymer polyvinyl butyrate (PVB) is a derivative of polyvinyl alcohol, used as a delivery vehicle for eptifibatide on the bare metal stainless steel stents. PVB is a biocompatible resin and contains only carbon, hydrogen and oxygen. PVB is nonthrombogenic and has neutral effect on the platelets.^25–29 All bare metal stainless stents were expanded at low atmospheric pressure (6 atm). Eptifibatide was supplied at a concentration of 2 mg ml by Schering-Plough and was freeze-dried and later radiolabeled with tritium (Pepceuticals Ltd.). Human vascular SMCs (HVSMCs) originated from primary explants culture of vein fragments. Fragments of veins with normal morphology were obtained from healthy margins of veins removed from patients undergoing varicose vein surgery. Leicester Research Ethics Committee approved these experiments. All investigations conformed to the principles outlined in the Declaration of Helsinki.³⁰Absorption and elution studies with eptifibatide — Radiolabeling experiment. Eptifibatide absorption. All bare metal stents (3 x 18 mm) were weighed prior to stent coating. The bare metal stents were sprayed with hydrophobic PVB. Polymer and unlabeled drug (15%) were dissolved in methanol/chloroform (50% w/w), and 80 µl of 3H-radiolabeled eptifibatide (specific activity 20 ci/mmol)) was added to the solution and used as a “spike” to allow detection of the drug. The stents were sprayed using ultrasonic micro spray to allow uniform distribution of the drug on the stent. Absorption was by diffusion, in a hydrophilic solvent such as ethanol, into the substance of the polymer, where the drug is held by hydrophilic-hydrophobic interaction between the drug and the polymer. Each stent had 6 drug/polymer coating passes, dried in a vacuum oven for 1 hour (to remove any remaining solvent), and was then weighed to obtain drug loadings. Each stent then had a top-coating with pure polymer (2 passes) to retard drug elution. The stents were stored under vacuum desiccant over 2 days. Eptifibatide elution in vitro. Radiolabeled eptifibatide-coated stents were perfused continuously (as previously described in detail)³¹ at 25 ml min-1 in a closed-loop circuit with PBS solution (Figure 2). The reservoirs were pretreated with 1% bovine serum albumin (BSA) in PBS for 48 hours and then rinsed. This reduced nonspecific binding of radiolabeled eptifibatide from adhering to the surface of the reservoir. The perfusate was maintained at 37°C and was changed every 24 hours. The radioactive drug eluted from the stent in each PBS solution was quantified by beta counting in a beta-counter (Tricarb® Packard Liquid Scintillation Analyzer). Three stents were tested under each set of conditions. Platelet aggregometry. Samples from the perfusate were taken after elution of the eptifibatide-coated stents, and the ability of the platelets to aggregate to ADP (4 µM) was assessed using a whole blood impedance aggregometry technique.³²The effect of eptifibatide-eluting stents on platelet deposition. Human platelet-rich plasma (PRP) was obtained from the blood bank and the standard method of Hawker³³ was used to label human platelets with indium oxine (111In). The bare metal stents and stents coated with eptifibatide were placed in the perfusion circuit, with the blood containing the 111In-labeled platelets as perfusate. The stents were perfused continuously for 1 hour at 40 ml min-1, the calculated shear rate at the surface of the stent being approximately 850 s-1. Stents were then rinsed and the radioactivity associated with each stent was counted in a gamma counter (Packard Cobra series Auto-gamma counting system, 15–75keV window). The effect of eptifibatide-eluting stents in cell culture proliferation. Human smooth muscle cells were cultured in medium 199 with Earl’s salt and 10% fetal bovine serum (FBS). Cells were passaged 4 to 6 times. SMC phenotype was verified by immuno-precipitation with anti-SM alpha-actin antibody (1A4, Sigma) and by a characteristic hill-and-valley growth pattern, and were 80% confluent prior to assay. Cells were starved for 18 hours in Earl’s medium 199 prior to assay in order to “equilibrate” them to stop the proliferation of cells and then reach the same cell cycle point. SMCs were detached with trypsin, washed in the presence of trypsin inhibitor (Sigma-Aldrich), resuspended in Earl’s medium 199 containing 5% BSA and centrifuged for 5 minutes at 1,500 rpm. Cells pellet were resuspended in Earl’s medium 199 in 5% BSA and then cells were plated at a density of 3 x 105 cells per 60 mm dish. Each of the bare metal and eptifibatide-loaded stents were fixed in the center of the dishes with sterile surgical bone cement in separate experiments. All culture dishes were incubated at 37°C in a 5% CO2 incubator. Culture medium was replaced every 48 hours. The zone of cell growth inhibition was measured at 7 days after initial plating with a micrometer inserted into the eyepiece of a standard inverted microscope (Olympus). In a subset of the cultures (at 7 days), the cells were stained with toluidine blue to allow clear-cut demarcation of the zone of cell growth inhibition at low levels of magnification.³⁴Statistical evaluation. Results are presented as mean ± standard deviation (SD). Analysis of statistical significance was performed using the Student’s t-test for parametric and the Mann-Whitney U-tests for nonparametric results. A p-value of Results Loading of eptifibatide onto polymer-coated stents. Eptifibatide was successfully loaded onto bare metal stents using polyvinyl butyrate polymer. A maximum of 111 µg of eptifibatide was loaded onto 3.0 x 18 mm bare metal stents, with less total amount of eptifibatide loaded on shorter stents (Figure 1). Elution in vitro. Elution of eptifibatide from polymer-coated stents in the perfusion circuit (Figure 2) is shown in Figure 3. These stents were perfused at 25 ml min-1 at 37°C. Drug elution characteristics were tested in a phosphate buffer saline perfusion circuit for up to 4 weeks. The curve is biphasic and consisted of an early rapid elution phase (24% ± 0.03 loss over 1 hour), followed by a sustained release with 44% ± 2.30 still present on the stent after 30 days. Platelet aggregometry. Aggregometry undertaken during the platelet deposition experiment was carried out to confirm both the platelet viability and that the eptifibatide eluted from stent had retained antiplatelet properties in the perfusate. Eluted eptifibatide significantly inhibited platelet aggregation by 95% ± 0.70 in response to ADP (4 µM) at 10 minutes (p Effect of eptifibatide-coated stents on platelet deposition. There was a significant reduction in platelet deposition onto polymer-coated stent treated with eptifibatide as compared with controls (Figure 5). Platelet deposition on stents eluting eptifibatide was significantly reduced by 48% ± 6 compared with controls (p = 0.0065), when stents were perfused continuously in blood containing the 111In-labeled platelets for 1 hour at 40 ml min-1. Unlike the control stents, none of the treated stents had visible thrombus attached to them. Effect of eptifibatide-eluting stents on SMC proliferation. In the cell culture experiment, SMC proliferation was measured around the stents and the cells were stained with toluidine blue to allow clear-cut demarcation at 7 days. Eptifibatide-loaded stents (50 µg/stent) placed in SMC culture showed a distinct zone of cell growth inhibition within 1 mm2 of stent (88 ± 22 vs. 208 ± 23 SMCs in control), and within 2 mm2 of stent (131 ± 32 vs. 191 ± 23 SMCs in control) (both p Discussion Stent implantation has become the standard treatment during PCI.35–37 This is because it has overcome most of the major complications of a standard balloon angioplasty procedure which include acute coronary occlusion secondary to intimal dissection following balloon angioplasty and a superior long-term outcome in comparison to balloon angioplasty.^38–41 In-stent restenosis (ISR) is not uncommon, affecting approximately 20% of patients at 6 months with BENESTENT-type lesions, and rising to almost 40% in high-risk subgroups.^42–44 Current antiplatelet regimens, combined with improvements in stent design and deployment, have reduced the incidence of subacute thrombosis.⁴⁵ As restenosis becomes less problematic with new DES (rapamycin, paclitaxel),^46–49 the acute and long-term outcomes have become the focus of current attention. Clinical experience of the long-term effects of DES in coronary arteries suggest that they maintain their clinical benefits,^50–57 but doubts remain about the long-term risk of stent thrombosis. A recent case series has highlighted the complications even over a year later of interrupting antiplatelet therapy in DES patients. Four cases of angiographically confirmed late thrombosis were reported between 335 and 442 days after DES implantation, resulting in myocardial infarction.⁵⁸ Further potential side effects could be late positive remodeling and aneurysmal formation. The search for an ideal stent has resulted in a huge variety of stents, differing in design, material, surface and coatings.⁵⁹ Polymer coatings have shown conflicting results and can themselves be responsible for local inflammation and excessive neointimal proliferation.^60,61 A number of other coatings like phosphorylcholine,^62,63 inert polymer64 or heparin,^65,66 demonstrated a reduction in subacute stent thrombosis rate and neointimal hyperplasia in a pig model, but have shown no benefit in human studies.⁶⁶ Therefore, the choice of coating is important. Polyvinyl butyrate is a biocompatible resin and has shown to have no adverse effects on the vessel wall in animal studies.⁶⁷ We have demonstrated polyvinyl butyrate as a delivery vehicle in vitro for eptifibatide elution from the stent. A biphasic elution curve is characteristic of the release of drugs from many polymer-coated stents, including eptifibatide-eluting stent. This is thought to be due to a very rapid wash-off of very lightly adherent drug molecules on or near the surface of the polymer, followed by a slower release of drug from within the substance of the polymer. Elution seen with other drug/stent combinations, including the antithrombotic medications activated protein C and abciximab,^68,69 also followed a similar rapid initial release and then a slower, more sustained release over several days. The elution characteristics seen are an approximation of those that occur in vivo, since the model used is not identical to the conditions found in an atherosclerotic human coronary artery. In particular, the stent is not perfused with blood and has not been deployed into an artery segment to examine stent-vessel wall interaction. The model used has been used previously to predict in vivo effectiveness. It was found that eptifibatide elutes rapidly in the first hour from the stent in a perfusion circuit followed by sustained release over days. Up-regulation of the avb3 integrin following arterial injury has been shown to be greatest within the first 2 weeks, and inhibition of the avb3 integrin-using peptides significantly reduces neointimal formation following arterial injury in animal models.⁷⁰ With approximately 55% of eptifibatide still available on the stent at 15 days, it should be present locally throughout the period of time that SMCs are more active. Our data on SMC proliferation assay have shown that eptifibatide-eluting stents inhibit SMC proliferation within 2 mm² of stent, which may have effects on neointimal hyperplasia. Additional effects may be mediated through the prevention of nonocclusive platelet thrombus deposition locally. Animals rendered thrombocytopenic have a reduced neointimal response to vessel injury, presumably through reduction in the release of growth factors and a reduction in the amount of thrombin present.⁷¹ Eptifibatide is a selective high-affinity inhibitor of the platelet GP IIb/IIIa receptor. It produces dose-dependent ex vivo inhibition platelet aggregation induced by adenosine diphosphate (ADP) by preventing the binding of fibrinogen, von Willebrand factor and other adhesive ligands to GP IIb/IIIa.^72,73 We have demonstrated that eptifibatide-eluting stents significantly inhibit platelet adhesion onto stents and eluted drug from the stent effectively inhibits platelet aggregation in response to ADP. It also needs to be noted that early inhibition of platelet adhesion does not necessarily mean there will be a benefit of stent thrombosis prevention, which often does not occur several weeks or months later. Overall, the inhibitory results of eptifibatide on platelet deposition, the possibility of an inhibitory effect on SMC proliferation and involvement of avß3 in the restenosis process show that stent-based local drug delivery of eptifibatide may be the way forward to ensure higher local concentration, local inhibition of platelet deposition and SMC activity, reducing the need for antiplatelet agents post-procedure. The possibility of an inhibitory effect of eptifibatide on SMC proliferation needs further examination in SMC studies. In order to establish whether eptifibatide can be an effective antirestenotic agent, further studies to demonstrate the effect of eptifibatide-eluting stents in an in vivo model have now been commenced. Acknowledgement. We are grateful for the support provided by PolyBiomed Ltd., United Kingdom. This work was supported by British Heart Foundation Fellowship No. FS/02/022.

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