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Contrast media as carriers for local drug delivery

Bruno Scheller , Ulrich Speck , Bernd Romeike , Alexander Schmitt , Milos Sovak , Michael Böhm , Hans-Peter Stoll
DOI: http://dx.doi.org/10.1016/S0195-668X(03)00317-8 1462-1467 First published online: 1 August 2003

Abstract

Background Lipophilic taxanes can be dissolved in contrast media at significantly higher concentration than in saline. As contrast media have occasionally been observed to delineate the contour of coronary arteries for some seconds they may serve as a matrix for an antiproliferative drug aimed at preventing restenosis. The aim of this study was to test a novel taxane-contrast agent formulation for this new approach in the setting of coronary stenting.

Methods and results In cell culture experiments (bovine vascular smooth muscle cells), 60-min incubation with contrast agent-taxane formulations (iopromide–paclitaxel, iopromide–protaxel) induced a significant, concentration-dependent inhibition of vascular smooth muscle cell (VSMC) proliferation over 12 days. Shorter incubation times of 10 and 3min showed the same efficacy. For in vivo investigation, 16 stents were implanted into the coronary arteries of eight pigs using a 1.3 to 1 overstretch ratio. A control group received iopromide 370 alone while the treatment group was injected with a iopromide-protaxel formulation at a dose of 74μmol/l, which is far below protaxel levels inducing systemic toxicity. Quantitative angiography and histomorphometry of the stented arteries asserted statistic equality of the baseline parameters between the control and treatment groups. After 28 days, the treatment group showed a marked reduction of the parameters characterizing in-stent restenosis, especially a 34% reduction of the neointimal area.

Conclusions First evidence is provided that using a contrast agent as solvent for a taxane constitutes a new drug delivery mechanism able to inhibit in-stent restenosis in the porcine restenosis model.

  • Restenosis
  • Stents
  • Angioplasty
  • Contrast media
  • Taxanes
  • Protaxel
  • Iopromide

1 Introduction

Numerous, initially promising, approaches using systemic antiproliferative agents have so far failed to prevent restenosis.1The inhibition of restenosis thus continues to present a challenge to interventional cardiologists. Coronary radiation therapy has been considered a breakthrough in preventing in-stent restenosis2but the method crucially relies on the availability of the radiotherapeutic armamentarium. More recently drug-eluting stents suppressing neointimal proliferation by the sustained release of antiproliferative drugs have shown very promising results in preclinical and clinical trials.3,4However, concerns have been raised that such drug-releasing stents while being effective may, just like irradiation, be associated with thrombotic complications. Prior studies showed that polymeric matrixes on the stent embedding the antiproliferative drug could induce inflammation and thrombosis.5Nevertheless, such stents are currently well accepted.

Contrast agents have occasionally been observed to delineate the contours of coronary arteries for some seconds after their initial washout. Based on this observation, we speculated that such a layer of contrast medium close to the endothelium where flow velocity is very low could possibly act as a matrix for antiproliferative drugs. The key requirement for such a mode of drug delivery would be a rapid uptake by the endothelium to compensate for the short contact time. Antiproliferative taxanes like paclitaxel seem to be suitable due to their high lipophilicity and tight binding to various cell constituents6resulting in effective local retention at the site of delivery.7,8In the light of these considerations we hypothesized that a contrast medium–taxane formulation may serve two purposes: first, to visualize the coronary arteries in conventional fluoroscopy, and, secondly, to repeatedly deliver an antiproliferative agent to the site of injury where it is released from the endothelial contrast film with each contrast medium injection. This approach seems to be attractive because it does not require a particular delivery device or a special stent, which is associated with the above-mentioned disadvantages. On the other hand, the low water solubility of taxanes and the unpredictable acute toxicity of the preparation may result in a low efficacy and adverse reactions.

The aim of this study was to investigate the feasibility, efficacy, and safety of suppressing neointimal proliferation in vitro by taxanes in general and in the porcine coronary overstretch stent model utilizing protaxel and iopromide 370 in particular.

2 Methods

2.1 Study drugs

The contrast agent iopromide 370 (Ultravist®, Schering AG, Berlin, Germany) was used alone or as a solvent for the two taxanes, protaxel and paclitaxel, investigated in this study in cell culture experiments and in the porcine coronary stent model (both substances were provided by Interpharma Praha, a.s., Czech Republic). Paclitaxel is widely used in antineoplastic chemotherapy.6Protaxel, a derivative of paclitaxel with an improved tolerance and efficacy,9,10was chosen for the animal study.

For the in-vitro studies, iopromide 370 was diluted with water for injection to yield a 40.5% v/v solution, which is isoosmolar. The taxanes were dissolved in the contrast agent, which contained 1% v/v ethanol and was further diluted 1:1 with cell culture medium to a final taxane concentration of 1.46μmol/l (low concentration) or 14.6μmol/l (high concentration) in the incubation solution. In the porcine study, protaxel was administered in a final concentration of 74μmol/l of iopromide (370mg iodine/ml) containing 0.5% v/v ethanol. The total dose of protaxel each pig received was about 5.8mg.

2.2 Cell culture experiments

Bovine aortic smooth muscle cells (passage <10) were cultured using Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% foetal bovine serum (FBS). Cells were characterized immunohistochemically by smooth muscle α-actin staining.

For assessment of the response to the two taxanes, cells were seeded at 10 000/cm2and incubated for 60min with 0.9% saline alone (control I), iopromide-ethanol (control II), and iopromide containing high or low concentrations of protaxel or paclitaxel. Subsequently, the supernatant was removed, the cells washed with 0.9% saline, and further cultured with DMEM+10% FBS. At days 0, 3, 6, 9, and 12, cultures were exposed to trypsination, and cell densities were determined by counting under the microscope. Iopromide containing the high concentration of protaxel was used to investigate the effect in relation to the contact length (60, 10, and 3min) and was compared with 0.9% saline as control.

2.3 Animal study

All experiments were conducted in accordance with the guidelines for animal experiments set forth by the animal protection committee of the Sachsen-Anhalt government. Eight domestic pigs (weight: 26.6±1.7kg; 4 male, 4 female) were pre-sedated by intramuscular injection of ketamine, xylazine, and atropine. After establishment of a venous access, anaesthesia was initiated by intravenous injection of propofol followed by orotracheal intubation and maintained with 1.0–2.0 vol% isoflurane, 70 vol% N2O, and 30 vol% oxygen.

All animals received 5000IU of heparin, 250mg aspirin, and 200μg intracoronary nitro-glycerine. The coronary arteries were imaged using a standard angiographic technique via the left carotid artery. Target segments were selected in the left anterior descending (LAD) and circumflex (CX) arteries, and the vessel diameter was estimated by comparison with the diameter of the angiographic catheter. Stents (diameter 3.0 or 3.5mm according to the vessel diameter, length 16mm; Coronary Wave Stent, Jomed, Rangendingen, Germany) were implanted with an oversize ratio of 1.3:1. During the procedure, each pig received a constant volume of 3ml of the iopromide-protaxel composition/kg body weight by multiple intracoronary injection. After stent implantation prophylactic intramuscular streptomycin and penicillin were given; pigs were allowed to recover and survive for 28 days. Oral aspirin at a dose of 100mg and ticlopidine at 250mg per day were administered starting 3 days before the procedure and continued until sacrifice.

Follow-up angiography was performed after 28 days and the animals were subsequently sacrificed using 3M KCl in deep anaesthesia. Hearts were rapidly excised, the coronary systems flushed with 0.9% saline and the arteries fixed by perfusion with 4% buffered formalin under physiological pressure and overnight immersion. The target segments were then dissected and samples for histology obtained.

Coronary imaging was done with a Philips PolyArc fluoroscope connected to a digitizer using an Apple Macintosh Power PC. The CAAS II System (Pie Medical, the Netherlands) was used for quantitative coronary analysis by two experienced observers blinded to the treatment groups. Discrepancies were resolved by mutual consensus. The following parameters were evaluated: reference diameter, stent diameter (baseline angiography), minimal luminal diameter (MLD) (follow-up angiography).

Stented coronary arteries were dissected from the formalin-fixed hearts and immersed in methyl-methacrylate (Merck, Darmstadt, Germany) as previously described.11Four representative cross-sections per stent were separated from the blocks with a coping saw, polished, and glued on acrylic plastic slides. Final specimens, about 5–10μm thick, were stained by Gieson's technique.

Stented arteries were measured histomorphometrically in four cross-sectional planes per stent using an Olympus microscope connected via a video chain to the image digitizer. Measurements were performed using the NIH image program (PC version ‘Scion Image’, Scion Corporation, Maryland, USA). The evaluated parameters were: luminal diameter, external elastic lamina (EEL) diameter, maximal neointimal thickness, EEL area, luminal area, and neointimal area.

2.4 Statistical analysis

Histomorphometric variables of the four cross-sectional planes were averaged to obtain a mean value per stent. Continuous variables of quantitative coronary angiography and histomorphometry were compared by Student’s t-test after normal distribution had been assured by software SPSS 10.0 for Windows (SPSS Inc., Chicago, IL, USA).

3 Results

3.1 Solubility

After adding 0.5% ethanol, protaxel is soluble in iopromide 370 at concentrations up to >200μM, which is far above the solubility of about 20μM in saline under identical conditions.

3.2 Cell culture

Iopromide 370 containing 0.5% v/v ethanol had the same effects on vascular smooth muscle cell (VSMC) proliferation as saline, while the iopromide-paclitaxel and iopromide-protaxel combinations significantly inhibited VSMC proliferation in a concentration-dependent manner at all time points (Table 1). Incubation with iopromide-protaxel for 10 or 3min showed the same efficacy as incubation for 60min (Table 2).

View this table:
Table 1

Effects on cell density of saline (control), iopromide-ethanol, and of low and high concentrations of iopromide–paclitaxel and of iopromide–protaxel. Incubation time was 60min. The final taxane concentrations in the incubation medium after dilution with the culture medium were half those given in the Table

AgentDay 0Day 3Day 6Day 9Day 12
Saline (control)1.03.86.37.38.8
Iopromide-ethanol1.03.56.47.58.3
Iopromide-protaxel 2.84μg/ml1.02.73.55.67.1
Iopromide-protaxel 28.4μg/ml1.01.11.31.62.0a
Iopromide-paclitaxel 2.57μg/ml1.02.13.55.86.4
Iopromide-paclitaxel 25.7μg/ml1.01.51.82.22.9a
  • a P<0.05 compared to control.

View this table:
Table 2

Effects on cell density of different incubation times (60 10, and 3min) with the high concentration of iopromide–protaxel. The final taxane concentrations in the incubation medium after dilution with the culture medium were half those given in the Table

Agent —incubation timeDay 0Day 3Day 6Day 9Day 12
Saline (control)—60min1.03.26.47.27.9
Iopromide-protaxel 28.4μg/ml—60min1.00.81.01.81.8a
Iopromide-protaxel 28.4μg/ml—10min1.01.11.41.71.4a
Iopromide-protaxel 28.4μg/ml—3min1.01.51.61.72.1a
  • a P<0.05 compared to control.

3.3 Animal study

Sixteen stents were implanted into the LAD and CX coronary arteries of eight pigs. Quantitative coronary angiography documented no differences in the baseline parameters. At day 28, narrowing of the lumen was found within the stented areas of all animals. There was, however, a marked reduction of in-stent restenosis in the group treated with iopromide-protaxel as reflected by the angiographic parameters. In particular, late lumen loss was reduced by 53% (0.9±0.6mm vs 1.9±0.8mm, P=0.01) and the minimal luminal diameter was larger (2.6±0.6mm vs 1.5±0.8mm, P=0.01) compared to the group which received iopromide alone (Table 3). Edge effects were not observed.

View this table:
Table 3

Quantitative coronary angiography: stent diameter, vessel reference diameter, and overstretch ratio at baseline; minimal luminal diameter and late lumen loss at follow-up. Mean±standard deviation

Group I (iopromide)Group II (iopromide-protaxel)P
No. of pigs44
No. of stented vessels88
Baseline
Stent diameter3.4±0.3mm3.5±0.2mm0.66
Vessel reference diameter2.7±0.3mm2.8±0.3mm0.79
Overstretch ratio1.26±0.091.26±0.130.97
Follow-up (28 days)
Minimal luminal diameter1.5±0.8mm2.6±0.6mm0.006
Late lumen loss1.9±0.8mm0.9±0.6mm0.01

Histologic evaluation showed that all stents were sufficiently expanded, and a mural thrombus, which was thin, was seen in only one out of 16 stents. Both groups had a similar coronary artery size by EEL diameter and area. There was a significant reduction of restenosis, which confirmed the angiographic measurements using the parameters listed in Table 4. The neointimal area was reduced by 34%, the diameter of stenosis by 34%, maximal neointimal thickness by 38%, and the luminal area was 42% larger. A typical histology of a stented vessel after 28 days is shown in Fig. 1. Histologically, there was no evidence of an inflammatory response in the neighbourhood of the stent struts. In particular, there was no evidence of fibrin deposits and/or mononuclear cells. Despite the marked reduction of neointimal proliferation, all stent struts were covered with neointima. Endothelialization of the complete luminal surface including the areas of stent struts was present in all samples (Fig. 1, panel B).

View this table:
Table 4

Histomorphometry of the stented porcine coronary arteries after 28 days. Injury score, luminal diameter, external elastic lamina (EEL) diameter, maximal neointimal thickness, EEL area, luminal area, and neointimal area. Mean±standard deviation.

Group I (iopromide)Group II (iopromide–protaxel)PΔ
No. of stented vessels88
Injury score1.03±0.191.08±0.180.594
EEL diameter3.20±0.10mm3.25±0.17mm0.464+2%
Minimal luminal diameter2.01±0.24mm2.46±0.44mm0.025+22%
EEL area8.06±0.61mm27.89±1.00mm20.690−2%
Luminal area3.41±0.85mm24.84±1.43mm20.029+42%
Diameter stenosis37.0±7.8%24.3±12.5%0.029−34%
Area stenosis57.8±9.4%38.7±16.7%0.014−30%
Neointimal area4.65±0.86mm23.05±1.29mm20.011−34%
Maximal neointimal thickness0.90±0.16mm0.55±0.26mm0.006−38%
Fig. 1

Examples of histology of stented porcine coronary arteries after 28 day: control (left) versus iopromide-protaxel (right).

4 Discussion

Coronary stents coated with antiproliferative agents like sirolimus or taxane compounds such as paclitaxel have shown promising anti-restenotic effects in preclinical and clinical trials,3,4,12and such results fuel an enormous expectation towards this technology. This study for the first time investigates an alternative concept of local taxane administration using a novel combination of a taxane compound with a non-ionic contrast agent (iopromide). The concept is based on the assumption that a thin film of contrast agent outlining the inner luminal surface of a coronary artery may act as a reservoir for local taxane delivery. Moreover, the high lipophilicity oftaxane formulations6should ensure rapid and sufficient local delivery. Using contrast agents injected duringangiographic procedures as drug carriers ensures a more selective administration than intravenous or oral administration but a less selective one compared to drugs coated on stents since a large proportion of the drug will inevitably stay in the vessel lumen and be washed away with the blood stream.

We found a substantial anti-restenotic effect in the porcine coronary stent model with a statistically significant reduction of neointimal proliferation. This result supports the underlying hypothesis that the antiproliferative effect occurs in spite of the very short timespan of agent contact with the endothelium.

The cell culture experiments were performed using paclitaxel concentrations in the range of human plasma concentrations measured during and shortly after intravenous infusion in cancer therapy (∼5–10μm). Exposure times ranged from 3 to 60min: the effect of the taxane on the proliferation of vascular smooth muscle cells was found to be concentration-dependent but of equal magnitude for all exposure times tested. These results suggest that a contact time of three minutes, and probably less, will be sufficient for the pharmacologic effect to occur. To compensate for the short contact time and to ensure rapid uptake into the tissue due to a high concentration gradient a significantly higher paclitaxel concentration was chosen for the in vivo study. Local delivery of 10μM paclitaxel was not efficacious in reducing restenosis in a similar study in porcine coronary arteries.13Because of the low water solubility of the taxanes a control experiment applying the same taxane concentration in a different solvent such as saline was not feasible. Thus, the original hypothesis assuming a contribution of the contrast agent to the efficacy of the preparation beyond improving solubility e.g. by coating the vessel could not be tested.

Paclitaxel has already been investigated in previous studies employing a variety of catheter-based local drug delivery approaches. The ‘double-balloon’ catheter,13the ‘porous balloon’,14and even intrapericardial administration15were utilized. Although all of these approaches showed efficacy in preclinical trials, they require special and sometimes cumbersome devices, involve blockage of coronary blood flow, or induce additional vascular injury.

Stents coated with polymers releasing paclitaxel effectively suppress in-stent restenosis in humans.12However, concerns have been raised that such matrix polymers may be thrombogenic,5and more importantly, that incomplete healing of the stent struts with intimal fibrin deposits, intra-intimal haemorrhage, and increased intimal and adventitial inflammation may jeopardize outcome16and may even induce sudden thrombotic occlusion. Our technique takes advantage of standard coronary angiography. No special catheters, radiation, special polymers or other sustained-release techniques are involved. All stent struts were covered with a thin layer of neointima in all treated animals and no adverse histologic phenomena were seen. We therefore expect that acute, subacute, or late stent thrombosis should not be a limitation in a clinical setting.

The decrease of neointimal growth observed in our study was comparable to that achieved earlier with paclitaxel-coated stents.4However, a major limitation of our experiments is the lack of direct pharmacokinetic evidence for uptake of the active compound into the vessel wall. Nevertheless, we assume that taxanes are effectively accumulated in the coronary wall and that this uptake is the reason for the antiproliferative effect documented in our study. This hypothesis is supported by two recently published papers demonstrating effective convection and diffusion mechanisms of paclitaxel from the lumen into the arterial wall.7,17In addition, competitive binding, e.g. by albumen and other plasma proteins, was identified as the main reason for diminished paclitaxel accumulation.17We speculate that arterial uptake in our study was not hindered by protein binding since there was direct exposure of the drug to the luminal surface. We also assume that preceding diagnostic contrast medium injections do not interfere with taxane uptake into the vessel wall due to the rapid contrast washout. Conversely, the rapid washout reduces the potential exposure times for the drug to only a few seconds. However, this is most likely compensated for by the repeated intracoronary bolus injections usually required in interventional coronary procedures.

In contrast to previously established techniques for preventing restenosis, our approach is characterized by an organ-directed and minimal systemic exposure to an antiproliferative agent. The risk of systemic toxicity depends on the taxane concentration in the contrast agent and the total volume administered during the procedure. In the clinical setting, varying amounts of contrast medium are required for interventions of different complexity. Volumes ranging from 60 to 200ml would translate into a dose range per patient of 4.4mg to 14.5mg. These values compare favourably with the paclitaxel dose of 150mg/m2typically administered in tumour therapy. Thus, only 2–6% of a single dose administered in tumour therapy would be required for preventing restenosis. While the initial intracoronary taxane concentration during passage of the bolus is substantially higher than plasma levels in cancer therapy (5μmol/l),6,18the taxane plasma concentration would not exceed 0.3μg/ml if 200ml of the taxane-containing contrast medium were injected. It remains to be seen whether this is of clinical relevance, but we have not observed any adverse effects on myocardial contractility. Paclitaxel-coated stents usually contain less than 100μg of the drug which is less than 1% of the dose administered with the contrast agent.

From a clinician's perspective systemic toxicity remains the key concern of this new approach to the prevention of restenosis. To date there are no data available characterizing the risk of the low systemic taxane doses used in our study that are ineffective against malignant diseases. No cardiotoxicity attributable to paclitaxel was noted in cancer patients with pre-existing cardiomyopathy undergoing chemotherapy.19

Haematologic, neurologic, and gastrointestinal side effects are known to occur in patients undergoing paclitaxel chemotherapy, but such effects are seen only at 40 times higher plasma levels than those we conceivably could produce and after several infusion cycles.6Furthermore, protaxel was reported to have at least a two to three times higher tolerance than paclitaxel.10We would therefore expect that the use of taxanes, and particularly of protaxel, administered as described here, is safe.

5 Conclusion

This study provides first evidence that a combination of protaxel with a contrast agent inhibits in-stent restenosis. Since only a small dose of the taxane is delivered selectively, no discernable systemic or local toxicity is expected, making this approach potentially suitable for the prevention and treatment of restenosis in interventional cardiology.

Acknowledgments

The authors thank Dirk Mahnkopf, Dirk Amelang, and Antje Mittag from the Institute of Medical Technology, Magdeburg, Germany, for their excellent support in conducting the animal experiments and Nicole Karthein for excellent assistance in cell culture experiments. We also thank Schering AG, Berlin, Germany, Interpharma, Prague, Czech Republic, and JOMED GmbH, Rangendingen, Germany, for their support.

References

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