Safety of Zilver PTX Drug-Eluting Stent Implantation Following Drug-Coated Balloon Dilation in a Healthy Swine Model

Abstract

Purpose: To compare the safety of Zilver PTX drug-eluting stents (DES) following drug-coated balloon (DCB) angioplasty or conventional balloon angioplasty (BA) in a healthy porcine iliofemoral artery model. Methods: DES implantation following DCB (DCB+DES) or BA (BA+DES) was assessed by angiography and histology in the nondiseased iliofemoral arteries of 20 animals, with sacrifice at 1, 3, and 6 months. Safety assessment compared quantitative measures of vessel integrity (eg, preservation of artery geometry, structure, and lumen dimensions; absence of aneurysm; malapposition) and histological parameters (eg, excessive inflammation). The percentage of uncovered struts could not be >30% per section and the endothelial cell loss had to be <50%. The vascular and skeletal muscle changes in the downstream regions were also assessed histologically for evidence of emboli. Results: No significant differences in safety parameters, including inflammation and endothelial cell loss, were observed between the 2 groups at all time points. Percentage of fibrin was significantly higher in DCB+DES at 3 months [20.0% (IQR 11.6, 28.4) vs BA+DES 4.2% (IQR 1.4, 9.6), respectively; p=0.04], with consistent trends between groups at all time points. Medial smooth muscle cell loss peaked at 1 month and was not statistically different between groups at any time point, although the loss was greater in the DCB+DES group. Sections with arterioles exhibiting paclitaxel-associated fibrinoid necrosis in downstream tissues were observed exclusively in the DCB group at 1 month (14.3% of sections) and 3 months (11.5%). Conclusion: This preclinical study suggests that Zilver PTX stent implantation is a safe strategy after DCB angioplasty and might be considered for patients who require stenting after DCB treatment.

Keywords

animal model balloon angioplasty drug-coated balloon drug-eluting stent iliofemoral artery percutaneous transluminal angioplasty peripheral artery disease porcine model safety evaluation

Introduction

Paclitaxel-coated drug-eluting stents (DES) have shown promising results vs bare metal stents (BMS) for the treatment of obstructive femoropopliteal peripheral artery disease (PAD).^1–3 In the Zilver PTX randomized clinical trial (RCT),¹ the DES demonstrated superior clinical outcomes including primary patency and freedom from target lesion revascularization compared with standard of care [balloon angioplasty (BA) and provisional BMS)] through 5 years. However, with the advent of drug-coated balloons (DCBs), another alternative to BA has become available for the treatment of superficial femoral artery (SFA) disease,⁴ especially short, noncalcified lesions. The use of DCBs, which avoids implantation of a permanent metallic device, has also produced better clinical outcomes against conventional BA in RCTs.^5,6

After DCB treatment in the femoropopliteal arteries, provisional stenting is still necessary in ~10% to 40% of patients, mainly due to flow-limiting dissection or refractory residual stenosis.^5–10 In current clinical practice, physicians often choose BMS as the provisional stent after DCB treatment because its safety has been established in many trials.^5–12 Although DCBs have been shown to reduce restenosis in the femoropopliteal segment by more than half vs uncoated balloons regardless of stent placement,¹³ the drug effect may not be sufficient to prevent restenosis in lesions with severe calcification,¹⁴ which is a known risk factor for vessel dissection and higher rates of restenosis after angioplasty.^15,16 DES might be the device of choice for such lesions, since calcification in the SFA is not itself an independent predictor of restenosis after treatment with DES.³ Therefore, provisional DES implantation rather than BMS after DCB treatment might be the strategy of choice, especially in calcified lesions. However, this strategy is not yet standard of care because of potential safety concerns regarding the effects of increased drug levels at the treated lesion, which if severe could theoretically lead to aneurysm formation or thrombosis. The aim of the current study was to evaluate the safety of Zilver PTX DES (Cook Medical, Bloomington, IN, USA) following DCB treatment in a healthy swine iliofemoral artery model compared with Zilver PTX DES implantation following BA treatment.

Methods

Experimental Model

The study protocol to compare the safety of DES implantation following DCB or uncoated balloon angioplasty was reviewed and approved by the Institutional Animal Care and Use Committee at Synchrony Labs (Durham, NC, USA). A sample size calculation based on 80% power, a 2-sided alpha of 0.05, and an expected difference in the endpoint of ~30%±15% determined group sizes of 6 to 8 animals. Thus, 20 nonatherosclerotic, healthy Yucatan Miniature Swine were randomized to the 1-month (n=6), 3-month (n=6), or 6-month (n=8) follow-up groups. All animals received dual antiplatelet therapy consisting of aspirin (325 mg/d) and clopidogrel (75 mg/d) starting at least 2 days prior to the procedures and continuing throughout the experiment. Animals were feed a normal diet and received humane care in accordance with the guidelines from Principles of Laboratory Animal Care formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health.

Angioplasty

For the angioplasty procedure, the animals were fasted overnight, given antibiotics (ceftiofur 5 mg/kg), anesthetized with intramuscular tiletamine (3−6 mg/kg), intubated, and maintained under general anesthesia with isoflurane. Open carotid artery access was secured, and heparin (100−300 U/kg) was administered intravenously. Quantitative vessel angiography (QAngio XA software, version 7.3; Medis Inc, Raleigh, NC, USA) was performed prior to device introduction and immediately following stent implantation. Percent acute lumen gain and percent late lumen loss were calculated using the following formulas: [(poststent diameter/prestent diameter)–1]×100 and [(follow-up diameter/poststent diameter)–1]×100, respectively.

Each animal served as its own control and underwent balloon inflation in both iliofemoral arteries using the 5-×40-mm IN.PACT Admiral balloon coated with 3.5 µg/mm² paclitaxel (Medtronic, Minneapolis, MN, USA) on one side and the uncoated 5-×40-mm Cook Advance 35LP balloon (Cook Medical) on the other. Vessel segments and balloon inflation pressures was selected to target a balloon to artery ratio of ~1.1. Balloons remained inflated for ~1 minute. Following balloon inflation, a 6-×40-mm Zilver PTX DES (eluting 3.0 µg/mm² paclitaxel) was deployed across each previously ballooned region (1 stent per vessel, 2 stents per animal). Stents were sized ~1 to 2 mm larger than the pretreated vessel diameter. After the procedure, the carotid artery access was ligated, and the animals were recovered from anesthesia and returned to their housing.

Histological Assessment

At the scheduled time of termination, each animal was anesthetized as described above to perform peripheral angiography. After the animal was euthanized, the stent and the adjoining 5 to 10 mm of non-stented proximal and distal artery were removed following perfusion fixation. The stented vessel segments were shipped to CVPath Institue (Gaithersburg, MD, USA), where they were radiographed, stripped of adventitial fat, and processed for plastic embedding. The proximal and distal nonstented segments were embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) and Movat pentachrome. The stented segments were dehydrated in a graded ethanol series and embedded in Spurr resin. After the resin polymerized, the stented segments were cut perpendicular to the stent at its midpoint and again close to the proximal and distal ends of the stent, creating 4 specimens per stent. The plasticized specimens were cut with a microtome into 4- to 6-µm thick sections from the 4 regions of each stent and stained with H&E and Movat pentachrome for light microscopy and morphometric analysis.

Histomorphometry of the stented sections was performed using computer-assisted software (IPLab, Scanalytics, Rockville, MD, USA) as previously described.¹⁷ Measurements included the cross-sectional areas for the external elastic lamina (EEL), internal elastic lamina (IEL), and lumen. The percent area stenosis was calculated as: [1−(lumen area/IEL area)]×100. Neointimal thickness was measured from the inner surface of each strut site to the lumen border. The histological sections were examined for inflammation, fibrin, giant cell reaction, and vessel injury as listed in Supplemental Table 1.

In addition to the vessels, the skeletal muscle and animal’s coronary band downstream from the femoral arteries were examined; in total, proximal and distal regions of 6 skeletal muscle territories (gluteus maximus, gastrocnemius, cranial tibialis, rectus femoris, semimembranosus, and semitendinosus) and a single coronary band section were collected from each leg and immersion-fixed in 10% neutral buffered formalin, dehydrated, embedded in paraffin, cut into 4- to 6-µm thick sections, and stained with H&E for histological evaluation to assess arterial changes as well as the presence of emboli.

Safety Endpoints

Safety assessment in this preclinical model involved comparing quantitative measures of vessel integrity (eg, preservation of artery geometry, structure, and lumen dimensions; absence of aneurysm; malapposition) and histological parameters (eg, excessive inflammation). The percentage of uncovered struts could not be >30% per section and the endothelial cell loss had to be <50% (endothelial cell loss score <3).

Statistical Analysis

Results for continuous variables with normal distribution were expressed as mean ± standard deviation. Normality of the distribution was determined using the Shapiro-Wilk test. Non-normally distributed variables were expressed as median and 25th to 75th percentiles (interquartile range, IQR). Categorical data were analyzed using the chi-square or Fisher exact test. The Student t test was used to analyze continuous parametric variables, whereas the Kruskal-Wallis test was used for nonparametric data. P<0.05 was considered statistically significant. JMP software (version 13.0; SAS Institute, Inc, Cary, NC, USA) was used for statistical analysis.

Results

All animals survived the procedure and scheduled observation period with equivalent weight gain and no significant adverse events. There were no vessel dissections, ectasia/aneurysms, early or late thromboses, or hemorrhagic foci noted on angiography or gross examination. All radiographs of the stented arteries documented full expansion and absence of stent fracture.

Quantitative vessel angiography measurements showed that percent acute lumen gain and percent late lumen loss were similar between both groups at all time points (Supplemental Table 2). Light microscopy revealed that the vascular responses to DCB+DES were largely comparable to BA+DES at all time points, with patent stents in both groups (Figure 1). Vessel dimensions relative to the EEL, stent area, lumen area, and percent area stenosis were similar in both groups, with no stent strut malapposition at any time point (Figure 2 and Supplemental Table 3). No animal in either the DCB+DES or BA+DES group showed evidence of lumen thrombosis.

While the percentage of uncovered struts was nonsignificantly higher at 1 month in the DCB+DES vs BA+DES group [15.3% (IQR 8.8, 19.2) vs 8.5% (IQR 4.3, 13.2), respectively; p=0.6], strut coverage of both groups was almost complete by 3 months. All sections in both groups met the safety endpoint (<30% uncovered struts per section) at all time points (Figure 3 and Supplemental Table 3). Endothelial cell loss (score <3), the other safety endpoint, was similar in both groups at all time points, peaking at 1 month [1.3 (IQR 1.1, 1.6) vs 1.3 (IQR 1.1, 1.6), respectively; p>0.99] and decreasing thereafter.

Inflammation peaked at 1 month and was similar in both groups at all time points. A rare finding was seen in a single DCB+DES section that showed the presence of granulomatous reaction involving 2 struts at 6 months (Supplemental Table 4). The medial injury score was also similar in both groups at all time points.

Signs of drug effect, such as medial smooth muscle cell (SMC) loss, peaked at 1 month and did not differ significantly at any time point, although there was a trend toward greater loss in the DCB+DES animals compared with BA+DES. At 3 months, the transmural SMC loss score was 3.3 (IQR 2.8, 3.9) for the DCB+DES group vs 2.5 (IQR 2.3, 2.9) for the BA+DES groups (p=0.09; Figure 4 and Supplemental Table 5). Fibrin deposition was significantly higher in the DCB+DES group at 3 months [20.0% (IQR 11.6, 28.4) struts with moderate to severe fibrin deposition vs 4.2% (IQR 1.4, 9.6) in the vs BA+DES group; p=0.04], with similar nonsignificant trends at 1 and 6 months. Medial proteoglycan and collagen deposition peaked at 3 months and was similar in both groups at all time points.

Histological analysis of vascular changes in non-target skeletal muscle and coronary band tissues revealed evidence of arterial changes (Figure 5), mainly characterized by fibrinoid necrosis and/or inflammation in small arteries and arterioles only in the DCB+DES group at 1 month [median 14.3% (IQR 0.0, 28.6)] and 3 months [median 11.5% (IQR 1.9, 15.4)]. Thrombotic emboli were observed in 1 section of the DCB+DES group at 1 month and 1 section at 3 months but none in the BA+DES at any time point.

Discussion

Provisional stenting after DCB angioplasty for femoropopliteal occlusive disease is frequently required, though it has not been previously demonstrated that using DES for this purpose is safe. The drug dose is one important factor for the safety of DES because higher drug doses may cause medial wall necrosis (which could theoretically lead to aneurysm formation), hemorrhage, calcium deposits, excessive inflammation, and slower strut coverage.^18,19 Thus, it is important to assess the safety of using a combination treatment of DCB and DES, both of which transfer paclitaxel to the arterial wall. The safety of a 4 times dose of paclitaxel in the Lutonix DCB (100% overlap of 2 double dose DCBs, each coated with 4 µg/mm²; Lutonix Inc, New Hope, MN, USA) was previously reported in the same swine preclinical model.²⁰ Although previous preclinical studies demonstrated delayed healing with overlapping DES in the rabbit iliac artery model,¹⁹ no safety concerns were found in DCB+DES compared with BA+DES in the current study. Measures of biological effect tended to be slightly greater in the DCB+DES group, though markers of healing, such as uncovered struts and endothelial cell loss, were not different between groups at any time point. In the current study, DCB+DES showed significantly higher fibrin deposition at 3 months, which peaked at 1 month and decreased at 3 months followed by absent or minimal deposition at 6 months in both groups. There was also a trend toward greater medial SMC loss in the DCB+DES group, especially in terms of transmural SMC loss at 3 months.

In preclinical models, DCB drug levels in the arterial wall throughout the first 30 days were ~5 to 25 times less than what was seen in the femoral arteries of pigs treated with Zilver PTX,^20,21 but the specific levels after DCB+DES vs BA+DES have not been studied. The safety results in the current study appear promising regarding the use of DCB+DES. Nonetheless, it is our belief that a cautious strategy should be taken; specifically, longer dual antiplatelet therapy should be considered for patients treated with DCB+DES to allow adequate time for vascular healing.

Although the percentage of provisional stenting after BA or DCB treatment varies widely in clinical studies (40% to 50% in RCTs of stents,^22,23 10% to 20% in DCB RCTs,^5,6 and up to 25% in real-world registries^7,12,24,25), provisional stenting is thought to be mandatory in lesions with flow-limiting dissection and refractory stenosis after balloon dilation. The protocols of DCB RCTs suggest BMS implantation as the provisional stenting approach of choice if angiographic results are unsatisfactory. However, there are little data to support this or any other approach.

Severe arterial calcification is one of the independent predictors of arterial dissection and restenosis after balloon angiography,^15,16 and DCB alone are not as efficacious in heavily calcified lesions.¹⁴ Yokoi et al² showed favorable clinical outcomes of Zilver PTX in such high-risk lesions in a real-world, all-comers trial. Although the patients in the trial were older and the lesion length was longer compared with the Zilver RCT trial,²² 12-month freedom from target lesion revascularization was comparable (91.4% vs 91.6%, respectively). Therefore, if shown to be safe, provisional DES implantation after DCB treatment, rather than provisional BMS implantation, should be considered the gold standard, especially in calcified lesions.

One potential caveat to the approach of using the DCB+DES approach vs BA+DES concerns the potential embolic effects caused by downstream release of paclitaxel into nontarget tissues. Indeed, such effects were exclusively seen in the DCB group. The percentages of sections and arteries exhibiting fibrinoid necrosis in downstream muscles were significantly greater in the DCB arm at 1 and 3 months, and results are similar to a previous preclinical study of IN.PACT DCB without provisional Zilver PTX stenting.²⁶ The excipient coating and crystallinity of the drug/excipient combination are the main proprietary features responsible for the downstream emboli.^20,26 Although no complications due to distal emboli have been reported in large RCTs of DCB, there are 2 case reports of painful rash due to distal embolization at 1 to 2 weeks after the procedure.^27,28 Conversely, there were no sections with fibrinoid necrosis in the BA+DES group in the current study. Zilver PTX uses a unique coating of paclitaxel alone, without polymers, binders, or carriers,²¹ without any reports of distal emboli from the Zilver PTX. In this study, signs of biological drug effect in the treated vessels were largely equivalent between the DCB+DES and BA+DES groups. Thus, in lesions at high risk for vessel dissection or recoil, an approach that uses BA followed by DES rather than DCB+DES may avoid potential complications associated with emboli from DCB while still minimizing restenosis risk.

Limitations

In the study, the pathological findings are limited to healthy swine and do not take into consideration all the complexities of human PAD that may be observed in patients with diabetes, obesity, and other associated factors. Moreover, transferring preclinical findings observed in healthy swine arteries to PAD in humans is clearly more complex, as the culprit lesions in humans are often complicated by atherosclerosis, fibrosis, and intimal calcification together with extensive medial calcification. DCB+BMS, the standard provisional stenting strategy, was not used as a control. Instead BA+DES, a standard DES stenting strategy, was evaluated in the current study. This may also represent a limitation to the interpretation of the present study.

Conclusion

Provisional Zilver PTX stent implantation following DCB use is a safe strategy and might be considered in place of BMS as a provisional stenting approach if angiographic results are unsatisfactory following balloon angioplasty of femoropopliteal occlusive lesions.

Footnotes

Declaration of Conflicting Interests

The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: Sho Torii receives honoraria from Abbott Vascular Japan and Terumo Corporation and research grants from SUNRISE Laboratory. Renu Virmani and Aloke V. Finn have received institutional research support from Abbott Vascular, BioSensors International, Biotronik, Boston Scientific, Cook Medical, Medtronic, MicroPort Medical, OrbusNeich Medical, SINO Medical Technology, and Terumo Corporation. Renu Virmani has speaking engagements with Merck; receives honoraria from Abbott Vascular, Boston Scientific, Cook Medical, Lutonix, Medtronic, and Terumo Corporation; and is a consultant for 480 Biomedical, Abbott Vascular, Medtronic, and W.L. Gore. Brandt Young and Anthony Ragheb are employees of Cook Research Incorporated.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

Supplemental Material

The online material is available at

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