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Cardiovascular Research 2000 46(3):585-594; doi:10.1016/S0008-6363(00)00042-0
© 2000 by European Society of Cardiology
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Copyright © 2000, European Society of Cardiology

In vitro evaluation of c7E3-Fab (ReoProTM) eluting polymer-coated coronary stents

Julia H. Barona, Anthony H. Gershlicka,*, Kai Hogrefea, Johanna Armstrongb, Cathy M. Holtb, Rajesh K. Aggarwala, Michael Azrinc, Michael Ezekowitzd and David P. de Bonoa

aDivision of Cardiology, Department of Medicine and Therapeutics, University of Leicester, Glenfield Hospital, Leicester, UK
bCardiovascular Medicine, Division of Clinical Sciences, University of Sheffield, Sheffield, UK
cDivision of Cardiology, University of Connecticut, Farmington, CT, USA
dYale University School of Medicine, New Haven, CT, USA

* Corresponding author. Tel.: +44-116-2875792; fax: +44-116-2563038

Received 2 December 1999; accepted 26 January 2000


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: Stent thrombosis and in-stent restenosis remain problematic in certain patient sub-groups. c7E3-Fab (ReoProTM, abciximab) inhibits the platelet glycoprotein IIb/IIIa receptor as well as the smooth muscle cell {alpha}vβ3 receptor, and thus may influence both processes, especially if high local concentrations could be achieved. We have studied the adsorption and elution characteristics of c7E3-Fab on commercially available polymer-coated stents. We have also investigated the effect of such antibody binding on platelet deposition in vitro, and on antibody deposition into ex vivo human saphenous vein wall to assess whether such stents may influence stent thrombosis and restenosis. Methods and results: Adsorption was measured using a radioisotope technique after immersing segments of polymer-coated stents in c7E3-Fab solutions. Uptake was dependent on antibody concentration and duration of immersion of wire in the solution. After 22 h (at 5 mgml–1), 1146±101 ngcm–1 wire was adsorbed. In an in vitro perfusion circuit, the antibody eluted slowly, with 53% remaining after 12 days washing. To determine the value that such stents might have in clinical practise, adsorption to balloon-mounted stents was assessed at room temperature, using commercially available c7E3-Fab (2 mgml–1). Efficacy of eluting c7E3-Fab was determined by measuring deposition of 111-Indium platelets. Immersing stents in c7E3-Fab for 20 min inhibited platelet deposition by 82.3% compared to controls (P=0.018). Deployment of treated stents in ex vivo saphenous vein resulted in the deposition of c7E3-Fab in the intima and media. Conclusions: c7E3-Fab can be passively adsorbed onto polymer-coated stents. It elutes slowly and in a predictable manner, significantly inhibiting platelet deposition in vitro. These studies pave the way to developing stent-based delivery of a potent anti-platelet agent that may additionally affect smooth muscle cell activity.

KEYWORDS Stents; Platelets; Monoclonal antibodies; Restenosis; Thrombosis/embolism


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
The use of intracoronary stenting has increased significantly in recent years [1]. Thrombotic stent occlusion, although much less common with current anti-platelet regimens, may still occur in certain high-risk patients with significant adverse consequences such as myocardial infarction [2]. This complication, together with in-stent restenosis, increases morbidity, mortality and financial expense and may limit the use of stents in an unselected patient population. c7E3-Fab (ReoProTM, abciximab) is a potent anti-platelet agent. Its systemic use has significantly reduced thrombotic complications following complex angioplasty and stenting [3–6]. In the EPIC trial, the incidence of further coronary events and the need for revascularisation was reduced at 6 months and beyond [6]. Recently presented data from the EPISTENT trial also suggested a beneficial effect on restenosis, although this was limited to patients with diabetes [7].

Delivering agents loaded onto the stent may allow site-specific delivery of an appropriate dose over a prolonged period, reducing potential unwanted systemic effects [8,9]. Drugs can be covalently bound to the stent surface, incorporated into a biodegradable coating or stent, or a biostable coating can be used as a depot for the agent, which elutes down a concentration gradient. In the Benestent-II trial [10], covalently bonded heparin-coated stents were deployed in 600 patients (200 in the pilot study). Stent thrombosis occurred in just one patient, giving a thrombosis rate of 0.2%. Unfortunately, the design was such that this could not be attributed to the heparin coating as there was no randomisation to receive uncoated stents. The low thrombosis rate could also be due to the large vessel size, increasing operator experience and high-pressure deployment. Other heparin-containing coatings have been studied [11,12]. Although there are four heparin-coated stents commercially available, their high initial costs and doubts over whether they are truly non-thrombogenic have limited their utilisation. In all these, the heparin is irreversibly attached to the stent. For this reason, the heparin is not available to elute to influence the surrounding milieu, but instead can be regarded as a coating. Heparin is also unable to inactivate clot-bound thrombin [13], and despite promising animal studies [14,15], no inhibition of restenosis has been demonstrated with systemic administration [16].

To overcome some of these disadvantages, more potent anti-thrombins and anti-platelet agents have been applied to drug-releasing stent coatings. Hirudin and iloprost (a prostacyclin analogue) have been applied to a biodegradable polyactic acid-polyethylene glycol coating, and retain anti-thrombotic effects in flowing human plasma for 30 days [17].

We have previously published studies demonstrating the anti-thrombotic efficacy of AZ1, loaded onto stents coated with a cellulose polymer, implanted in balloon-injured rabbit internal iliac vessels [8]. AZ1 is an antibody active in rabbits, which blocks the glycoprotein IIb/IIIa receptor, preventing platelet aggregation [18]. These studies demonstrated a 50% reduction in stent thrombosis with no evidence of systemic effects from release of the drug (bleeding times and systemic platelet aggregation studies). c7E3-Fab, active against this receptor in humans, could not be used in these early experiments as it does not cross-react with integrins in small animal models. The current studies extend the concept to c7E3-Fab (abciximab), a drug with established clinical use, in order to assess the potential in man.

The aims of the current work were therefore as follows:

(A) To establish the adsorption and elution kinetics of c7E3-Fab adsorbed onto polymer-coated stent wires.
(B) To assess the effect of adsorbed c7E3-Fab on platelet deposition to stent wires.
(C) To determine whether c7E3-Fab-treated wires retained anti-platelet properties over time.
(D) To examine whether balloon-mounted stents immersed in c7E3-Fab adsorbed sufficient antibody to inhibit platelet deposition when deployed in an in vitro circuit, and thus assess their potential clinical application.
(E) To examine ex vivo vessel wall deposition of c7E3-Fab eluting from the stent in a saphenous vein graft model to assess potential delivery into the vessel wall.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
The polymer-coated GRII stents (3 mmx20 mm) were provided by Cook Inc, USA. The polymer is a biocompatible, biostable proprietary chlorohydrocarbon of approximate volume 2 mm3 per stent

The c7E3-Fab was supplied at a concentration of 2 mgml–1 in a transport buffer (0.01 M sodium phosphate, 0.15 M NaCl and 0.001% Tween-80, pH 7.2) by Centocor, USA. Concentration was achieved by centrifugation in microsep filtrons (Filtron Technology). c7E3-Fab radiolabelled with 125iodine (125I) was prepared using the iodogen method [19].

The control irrelevant antibody (CBL 600, IgG) was purchased from Cymbus Biotechnology, UK.

All investigations conformed with the principles outlined in the Declaration of Helsinki [20].

2.1 Loading of c7E3-Fab onto polymer-coated stent wires
2.1.1 c7E3-Fab adsorption
Ten-millimetre lengths of stent wires were cut from polymer-coated GRII stents. Adsorption at concentrations of 2 mgml–1 and 5 mgml–1 were compared. To allow quantification, 5 µl of radiolabelled c7E3-Fab (specific activity 30 µCimg–1) was added to the antibody solution. The effects of antibody concentration, temperature and duration of immersion were studied.

Antibody solutions were placed in polypropylene tubes. The tubes were pre-treated with 1% bovine serum albumin (BSA) in PBS for 48 h, and then rinsed. This prevented the radiolabelled antibody from adhering to the tube, but did not reduce antibody adsorption on the stent wires (data not shown). The wires were immersed at 25°C, 37°C or 45°C for periods of between 20 min and 120 h, after which time they were rinsed three times with 5 ml of PBS. Six wires were tested under each set of conditions. The radioactivity associated with each wire was determined in a gamma counter (Packard Cobra series Auto-gamma counting system, 15–75 keV window). Five microlitres of the corresponding antibody solution (containing a known amount of c7E3-Fab) was also counted to act as a standard for antibody quantification and to correct for radioactive decay. The same standard was kept for the elution studies.

2.1.2 c7E3-Fab elution in vitro
Wires immersed and quantified as described above were perfused continuously (as previously described in detail [8]) at either 20 or 40 mlmin–1 in a closed-loop circuit with PBS containing 1% bovine serum albumin (BSA) (Fig. 1). The wires were removed from the manifold at set intervals, and the c7E3-Fab remaining was quantified by gamma counting. Six wire lengths were assessed for each set of conditions. The perfusate was maintained at 37°C, and was changed every 48 h. By calculation, the highest concentration of c7E3-Fab in the perfusate at any time was less than 15 ngml–1.


Figure 1
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  		Fig. 1 In vitro perfusion circuit for elution of c7E3-Fab from polymer-coated stent wires. The standard flow-rates used were 20 and 40 mlmin–1. In the platelet experiments, the PBS was replaced by blood containing 111-In labelled platelets.

 
2.2 The effect of antibody eluting wires on platelet deposition
Ten-millimetre lengths of polymer-coated stent wire were immersed in a solution of c7E3-Fab (25°C, 2 mgml–1, containing a known quantity of radioiodinated c7E3-Fab) for between 20 min and 18 h. The wires were rinsed, and the c7E3-Fab adsorption was quantified as above. Control wires were immersed in an irrelevant antibody (CBL600) under similar conditions.

Seventeen millilitres of blood was drawn without stasis from a volunteer who had taken aspirin 300 mg daily for 3 days. The platelets were labelled with 111indium (111In) using a standard technique [21]. Labelling efficiency was 82±6%. The radiolabelled platelets were added to a further 100 ml of blood containing heparin (10 uml–1).

The stent wire segments, pre-treated with c7E3-Fab, an irrelevant antibody or PBS alone, were placed in the perfusion circuit (Fig. 1), with the blood containing the 111In-labelled platelets as perfusate. The wires were washed continuously for 3 h at 40 mlmin–1, the calculated sheer rate at the surface of the stent being approximately 850 s–1. Wires were rinsed and the radioactivity associated with each wire was counted and quantified as above. The 111In and 125I counts could be distinguished by their different spectra (171–295 keV for 111In, 15–75 keV window for 125I).

At 30-min intervals, samples of the blood perfusate were taken, and the ability of the platelets to aggregate to ADP (3 µmol) was assessed using a whole blood impedance aggregometry technique [22].

2.3 Persistence of the anti-platelet properties of the drug-eluting wires
A further experiment examined the anti-platelet properties of 10-mm wires that had been loaded with c7E3-Fab and washed continuously with PBS (according to the elution experiments described above) for 7 days prior to the platelet deposition experiment. The intention was to assess the persistence of the biological efficacy of the c7E3-Fab after the stent has been in the ‘circulation’ for some time. Six wires adsorbed in c7E3-Fab (2 mgml–1, room temperature) for 20 min, and six control wires treated with irrelevant mouse IgG were assessed under these conditions.

2.4 Balloon-mounted whole stent experiments
Stents are normally pre-mounted on delivery balloons. To evaluate the amount of antibody that could be adsorbed onto balloon-mounted stents, whole 3 mmx20 mm balloon mounted stents were compared with unmounted stents. They were immersed in c7E3-Fab (2 mgml–1, room temperature) for 20 min or 24 h, and the adsorption was quantified as above.

In a further experiment, platelet deposition on such stents (immersed in c7E3-Fab while balloon-mounted) was compared with control stents immersed in irrelevant IgG. These were deployed in a 15-cm length of PVC tubing (I.D., 3 mm) at 10 atm for 1 min. This tubing was connected to the perfusion circuit as above. A flow-rate of 120 mlmin–1 was used, giving an estimated shear rate of 850 s–1. Platelet aggregometry throughout the experiments was monitored as previously described. After 3 h, the stents were carefully removed from the tubing, rinsed and gamma counted. The experiment was repeated seven times for each period of immersion.

2.5 Local deposition in a vein graft model
To assess qualitatively whether c7E3-Fab eluting from the stent was retained in tissue, balloon-mounted stents immersed in c7E3-Fab (2 mgml–1, room temperature) were deployed in fresh balloon-injured saphenous vein mounted in an ex vivo circuit [23]. The vein was bathed in culture medium at 37°C, and perfused with similar medium at 70 mlmin–1 for 24 h. The vein was cut longitudinally, and the stent removed. Sections were stained using a previously described technique, with minor modifications [24]. They were incubated with FITC-rabbit polyclonal anti-c7E3-Fab (Centocor, USA, 1:50). To overcome autofluorescence, this was followed by biotinylated goat anti-rabbit secondary antibody (Vector, 1:200) and horseradish peroxidase-conjugated ABC (Vector). Diaminobenzidine (Sigma) was used to detect the c7E3-Fab, and the tissue was counterstained with Carazzi's haematoxylin.

2.6 Statistical evaluation
An unpaired t-test or ANOVA was used to analyse the adsorption and elution data.

The Mann–Whitney non-parametric test was used to analyse the platelet deposition data for the segments of stent wire. Because of the variability in absolute platelet deposition, the results are presented as a percent reduction in 111In counts on treated wires compared to control wires exposed to labelled platelets during the same experiment.

The Wilkinson matched-pairs signed ranks test was used to analyse the data for the whole stent platelet deposition experiments.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Loading of c7E3-Fab onto polymer-coated stent wires
3.1.1 Antibody adsorption characteristics
The quantity of c7E3-Fab adsorbed onto the polymer-coated wire lengths was dependent on the duration of immersion in the antibody solution. The adsorption curve is initially steep, but begins to level off after 6 h.

The adsorption of c7E3-Fab from solution at concentrations of 2 mgml–1 (commercial concentration) and 5 mgml–1 of c7E3-Fab was compared. At 2 h, there was already a significant difference in the adsorption [543 vs. 754 ngcm–1 (P=0.002)]. After 120 h, those immersed in 5 mgml–1 had adsorbed 1493 ngcm–1, compared to 1257 ngcm–1 with 2 mgml–1 (P=0.018). These results are shown in Fig. 2.


Figure 2
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  		Fig. 2 The effect of altering the conditions under which the adsorption experiments were carried out: comparison of adsorption using c7E3-Fab concentrated to 5 mgml–1, vs. adsorption with no previous preparation of the antibody (2 mgml–1).

 
Adsorption of c7E3-Fab (2 mgml–1) at 25°C, 37°C and 45°C was compared. There was no significant difference in the quantity of antibody adsorbed at different temperatures at 20 min (P=0.41), 2 h (P=0.74) or 24 h (P=0.48) (ANOVA) (Table 1).


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  		Table 1 Effect of temperature on c7E3-Fab (2 mgml–1) uptake following immersion for different time periods (ngcm–1 wire±1 S.D.)

 
3.1.2 Elution
Elution of c7E3-Fab from polymer-coated stent wires is shown in Fig. 3. The wires were adsorbed for 48 h in c7E3-Fab (2 mgml–1), and perfused at 20 mlmin–1. The curve is biphasic with initial rapid c7E3-Fab elution for the first 2 h followed by a period of gradual slow elution. At 12 days, 53% of the c7E3-Fab was still present on the polymer-coated stent wire. Antibody elution from wires adsorbed for 2 h (2 mgml–1 room temperature) was compared using two different flow-rates. Increasing the flow-rate to 40 mlmin–1 had no significant effect on the amount of c7E3-Fab remaining on the stent (Table 2). Elution from wires immersed in a higher concentration of antibody (5 mgml–1) showed a similar curve. More rapid initial elution was noted, but a significantly greater quantity still remained on the stents at 7 days (Fig. 4).


Figure 3
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  		Fig. 3 The elution of c7E3-Fab from polymer-coated stent wires in an in vitro circuit. The wires were continuously washed with PBS+1% BSA at 20 mlmin–1, 37°C.

 

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  		Table 2 Comparison of antibody elution at 20 and 40 mlmin–1 flow-ratesa

 

Figure 4
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  		Fig. 4 Comparison of elution rates of c7E3-Fab from stents immersed in different concentrations (2 mgml–1 or 5 mgml–1, 2 h, room temperature). Although the differences at 180 h are small, they remain significant.

 
3.2 Platelet deposition on polymer-coated c7E3-Fab eluting stent wires
There was a significant reduction in platelet deposition onto polymer-coated stent wires treated with c7E3-Fab as compared with controls (Table 3). In each experiment undertaken the average platelet deposition was inhibited by greater than 90% on c7E3-Fab treated wires compared to controls. Unlike the control wires, none of the treated wires had visible thrombus attached to them. The reduction in platelet deposition remained significant even when the immersion in the antibody solution was reduced to 20 min, a time period potentially acceptable for use in the catheter suite.


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  		Table 3 Results of four platelet deposition studiesa

 
Aggregometry undertaken during the platelet deposition was carried out to confirm both the platelet viability, and that the c7E3-Fab concentration in the perfusate had not reached such a level as to influence aggregation in the general blood pool. The platelets in the perfusate retained their ability to aggregate to ADP (to within ±6% of the control value) throughout the experiments, confirming low concentrations of c7E3-Fab in the perfusate. The maximum calculated concentration of c7E3-Fab in the perfusate at any time was 11.9 ngml–1. This is well below the concentration required to influence platelet aggregation [25].

3.3 Persistence of platelet inhibitory activity
After 20 min exposure to c7E3-Fab (2 mgml–1), 282±23 ngcm–1 of c7E3-Fab were adsorbed. Forty-eight percent (135±14 ngcm–1) of this remained after 7 days continuous elution with PBS in the perfusion circuit. Even following such a short immersion and prolonged perfusion, there was a 98% reduction in 111In-labelled platelet deposition compared with controls when the wires were subsequently placed in the blood perfusion circuit for 3 h (P=0.004).

3.4 Adsorption, elution and platelet deposition on whole balloon mounted stents
The adsorption of c7E3-Fab (2 mgml–1) onto balloon-mounted stents was reduced compared with that on unmounted stents. After a 20-min immersion, 3.04±0.10 µg were adsorbed onto mounted stents, compared with 5.16±0.27 µg for unmounted stents (P<0.001). With a 24-h immersion under the same conditions, adsorption onto mounted stents was reduced by 38% (8.73±0.41 vs. 14.0±0.77 µg/stent, P<0.001).

Despite reductions in the quantity of c7E3-Fab adsorbed on balloon-mounted stents, platelet deposition was still significantly reduced compared to controls. 111In-counts were reduced by 87.2% for stents immersed for 20 min (P=0.018), and by 87.9% for those immersed for 24 h (P=0.018).

At the end of the 3-h platelet circuit experiments, 2.32±0.23 µg (76.3%) and 5.37±0.65 µg (61.5%) c7E3-Fab per stent remained on 20-min stents and 24-h stents, respectively.

3.5 Tissue deposition
Fig. 5(a) and (b) shows the immunohistochemical localisation of c7E3-Fab eluting from stents into saphenous vein samples ex vivo. Compared to photomicrograph Fig. 5(a) (control stent), the histological section shown in Fig. 5(b) (c7E3-Fab eluting stent) demonstrates brown staining of the media, indicating that the drug has eluted from the stent into the vessel wall. Such penetration would be required if c7E3-Fab delivered locally were to influence smooth muscle cell activity.


Figure 5
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  		Fig. 5 Immunohistochemical localisation of c7E3-Fab in saphenous vein graft model. Photograph (a) shows a section taken through the vessel wall that had contained a stent immersed in control antibody. In comparison, section (b), taken from a vessel that had a c7E3-Fab treated stent deployed in it, demonstrates brown staining (indicating the presence of the antibody) into the media layer.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
Current antiplatelet regimens, combined with improvements in stent design and deployment, have reduced the incidence of sub-acute thrombosis [26,27]. Complications may still occur when stents are deployed in small arteries, or in the setting of acute coronary syndromes or ‘bailout’ [28]. In addition 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 sub-groups [29–31]. Treatment of ISR is difficult, although Rotablator and brachytherapy may have a role. Recently, concerns have been raised that thrombosis may occur as a late complication of brachytherapy [32].

Our aim is to develop a stent that is truly non-thrombogenic and may also influence restenosis, while remaining as user friendly as current stents with little increase in cost. Local delivery of drugs reduces the systemic concentrations seen with intravenous therapy, reducing potential unwanted side effects, but balloon-based delivery systems have been shown to be inefficient at delivering agents to the vessel wall, with poor local retention [33]. The advantage of stent-based delivery is that higher local concentrations can be maintained for longer, allowing the agent to influence the milieu.

Polymer coatings can themselves be responsible for local inflammation and neointimal proliferation [34,35]. The choice of the coating is therefore important. The chlorohydrocarbon polymer currently used to coat the GR II stent has been shown to have no adverse effects on the vessel wall in animal studies [36]. Although higher restenosis rates have been reported with the Cook GR II, these are associated with stent underdeployment and recoil [37]. The principle investigated was the application of polymer coatings as a vehicle for drug delivery, rather than to study a specific stent. More recently this polymer has been applied to a new slotted tube stent. c7E3-Fab is a chimeric human/mouse Fab monoclonal antibody fragment. It was developed primarily to inhibit platelet aggregation while minimising its antigenic properties [38]. It blocks the glycoprotein IIb/IIIa receptors on platelets, preventing the formation of fibrinogen bridges, the final common pathway in platelet thrombus formation. It also influences SMC function by cross-reacting with the {alpha}vβ3 integrin [39,40]. This is one of the main integrins on SMCs responsible for mediating their migration [41,42], a key step necessary for neointimal formation [43]. Inhibition of the {alpha}vβ3 integrin using peptides significantly reduces neointimal formation following arterial injury in animal models [44]. Additional effects may be mediated through the prevention of non-occlusive 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 [45].

We have demonstrated that the adsorption of c7E3-Fab to polymer-coated stents is influenced by the concentration of the antibody in solution, and by the duration of the immersion. Two concentrations of c7E3-Fab were tested to determine if a difference in concentration of drug influenced in-vitro platelet deposition. It became clear that even short immersion times using commercially available concentrations of c7E3-Fab (2 mgml–1) significantly reduced deposition of platelets on the wires and whole stents in vitro. Reducing the platelet deposition by over 85% indicates that c7E3-Fab loaded stents are likely to have at least the same degree of biological efficacy in vivo as with our previously loaded AZ1 stents. The biological effect appears long lasting, even after short exposure times. Passive adsorption (as opposed to covalent bonding, as is used for heparin-loaded stents) allows the antibody to elute from the stent, which may penetrate and influence the surrounding milieu, as shown in the vein graft model experiment. The simplicity of the technique is also important, allowing the loading to be undertaken in the catheter laboratory or even at the company source prior to distribution.

Data from the EPIC trial would suggest that 80% of all platelet receptors should be blocked for the beneficial effect of c7E3-Fab to be seen [46]. It is therefore perhaps surprising that we have seen such a profound benefit with local delivery and elution of this drug. A likely explanation is that a layer of platelets still becomes adherent to the stent wire via other receptors, as would initially happen in a damaged vessel. The recruitment of subsequent platelet layers (mediated via the Gp IIb/IIIa receptor) appears to be prevented by the high local concentration of c7E3-Fab. Local platelet degranulation will also be reduced, suppressing release of smooth muscle cell chemotactic factors such as platelet-derived growth factor.

With approximately 50% of the antibody still available on the stent at 12 days, antibody should be available locally throughout the period of time that SMCs are more active. Upregulation of the {alpha}vβ3 integrin following arterial injury has been shown to be greatest within the first 2 weeks [44], and it may be advantageous to inhibit its action throughout this period to achieve a beneficial effect on restenosis. The ERASER trial [47] failed to show any reduction in late loss with c7E3-Fab administration after angioplasty, but the concentration in the vessel wall was likely to be low as the agent was administered systemically for only a 24-h maximum. Our data has shown that the drug will still be available for up to 2 weeks at least. Recently published animal experiments [48] suggest a very high systemic dose, which would not be tolerated by man, can influence intimal hyperplasia. Such a local dose may be achievable with local stent-based delivery without risk of systemic complications.

In summary, we have shown that immersion of polymer-coated stents in c7E3-Fab for a short duration loads sufficient agent to significantly reduce platelet deposition. Benefit in conditions such as stenting of diffuse disease in small vessels may be realised. We believe we are nearing the development of a drug eluting stent that will control thrombosis in any situation. Through separate effects (inhibiting platelet-rich thrombus stimulation of smooth muscle cells and direct effects on the {alpha}vβ3 integrin itself), they may well reduce restenosis, reducing the need for reintervention.

Local drug delivery using the stent as the delivery device may be an important next step in stent development. The future impact of drug-eluting stents on thrombosis and in-stent restenosis in high-risk patients, as well as an assessment of their potential to reduce or remove the need for adjunctive systemic heparin and oral antiplatelet therapy, needs to be investigated. The authors are currently planning such clinical trials.

Time for primary review 35 days.


   Acknowledgements
 
We are grateful for the support provided by Dr. Bob Jordan at Centocor. The work was supported by the British Heart Foundation and Cook Corporation, USA.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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