Examination of Anti-Intima Hyperplastic Effect on Cilostazol-Eluting Stent in a Porcine Model

Materials and Methods

Cilostazol-eluting stent. Bare-metal 316L stainless-steel Mustang stents with 0.0040 inch strut thickness were coated with a poly-ester-amides copolymers (PEA) layer. PEA polymer is made by copolymerizing monomers consisting of two alpha amino acids, typically L-leucine and L-lysine, with a diol and a diacid. Drugs can be deposited into the polymer matrix or conjugated onto the PEA polymer backbone, creating a slow-release delivery system. PEA polymer has four primary characteristics: it is biodegradable, noninflammatory, biocompatible and highly absorbable. The kinetics of cilostazol elution from PEA polymer-coated stents was identified in an in vitro system. Approximately 30% of the loaded drug is released from the stent within 100 hours. By 12 days, about 60% of the loaded drug is released from the stent. The highly absorbable property of PEA polymer achieved a smooth stent surface despite the content of 400 µg of cilostazol (Figure 1).
Preparation and stent implantation in the animal study. Twelve domestic juvenile swine weighing 25–35 kg were randomly assigned into two groups receiving either CES or BMS. They were premedicated with 200 mg of acetylsalicyclic acid for 1 day prior to the procedure and 200 mg of ticlopidine for 3 days prior to the procedure and until sacrificed. The swine underwent the procedure under general anesthesia. A 7 Fr sheath introducer was inserted into the right carotid artery via the cutdown technique. A 7 Fr guiding catheter was selected to deliver stents in the artery. Heart rate, blood pressure and electrocardiography were observed throughout the procedure. The operator was not informed of the stent type being implanted. Either 12 BMS or 12 stents coated with a thin layer of polyester amide-PEA containing 400 µg of cilostazol were implanted in the left anterior descending (LAD) artery and the right coronary artery (RCA). Immediately after stent implantation, heparin (3 cc) and nitorol (2 cc) were administered through the arterial sheath. Bare-metal Mustang stents (Microport Medical, Shanghai, China) were used as controls. The 18 mm-length stent was deployed at high pressure (12 atm for 60 seconds) using a balloon measuring 3.5 mm in diameter and 20 mm in length. The stent position was documented angiographically.
Pathologic examination. Neointimal formation in stented pig coronary arteries is maximal at 1 month. The response to healing after placement of a bare stainless-steel stent in a human coronary artery is five to six times longer than in pig or rabbit arteries.¹⁴ In this manner, the swine were sacrificed 28 days after the completion of coronary angiography. After euthanasia, the swines’ hearts were harvested, and the coronary arteries were perfusion-fixed with formalin as described previously.¹⁵
Stented coronary artery segments were dissected and embedded in methyl methacrylate. Specimens were sectioned using a rotational microtome to divide tubular stainless-steel stents. Vessel parameters were measured using methods published previously.^15,16 Intimal area and lumen area were measured. Intimal area was calculated as internal elastic lamina area minus lumen area. Lumen area was calculated as vessel area minus plaque area. An independent observer blinded to treatment regimen inspected all histopathologic analyses. The extent and degree of the injury score was followed by the method of Schwartz et al.¹⁵ Inflammation score was graded based upon the amount and extent of inflammation around the stent struts (0 = no inflammation; 1 = >25% of the neointima; 2 = 25–50% of the neointima; 3 = >50% of the neointima).¹⁷

Quantitative coronary angiography. Angiographic images of implanted stents were measured by a quantitative coronary angiographic (QCA) analysis software program (CMS-MEDIS Medical Imaging Systems, Leiden, The Netherlands). The guiding catheter was utilized as a reference for calibration of all measurements including baseline reference vessel diameter, post-stent minimal lumen diameter, follow-up reference vessel diameter, follow-up minimal lumen diameter and follow-up percent diameter stenosis.
Late loss and loss index were determined at 28 days. Late loss was calculated as the difference between postprocedure and follow-up minimal lumen diameter. Loss index was calculated as the ratio of late loss to acute gain.
Statistical analysis. Data (mean ± standard deviation) were analyzed for overall differences between the CES group and the BMS group using the Student’s t-test. Comparison of the mean values with a p-value <0.05 was considered statistically significant.

Results

A total of 24 stents were successfully implanted in the coronary arteries of 12 swine. Sudden death due to subacute thrombosis occurred in 1 swine in the BMS group 20 days after stent implantation. Otherwise, 5 swine in the BMS group and 6 swine in the CES group survived without clinical or angiographic stent thrombosis. Figure 2 shows the histomorphometric images of the BMS and the CES. The vessel histomorphometric and histopathologic results are shown in Table 1. There were no significant differences in injury and inflammation score between the groups. At 28 days, a significant reduction (23%) in intimal area was observed for the CES group versus the BMS group.
The baseline vessel diameter was similar for both the CES and BMS groups. Table 2 shows the QCA results. At 28 days, the CES group had significantly lower in-stent late loss than the BMS group (0.82 ± 0.81 vs. 1.86 ± 1.04; p = 0.0296). The loss index of the CES group also showed a significant difference compared to the BMS group (0.28 ± 0.83 versus 0.82 ± 0.81; p = 0.05).

Discussion

The present study demonstrated the feasibility of CES to inhibit neointimal formation and suppress restenosis. More importantly, complete endothelialization around the stent struts was observed in all groups at 28 days. There was no significant difference in inflammation scores between the groups, suggesting promising biocompatibility of the drug and the PEA polymer. Additionally, there was a 23% reduction in intimal area between the CES group and the BMS group. Animal studies have shown that DES may increase the presence of fibrin, inflammation and incomplete endothelialization compared with BMS.¹⁸ However, if inflammation is exaggerated in DES, stent thrombosis, malapposition, as well as potential dislodgment may be the consequence.¹⁹ In our study, CES and control stents demonstrated no significant differences in inflammatory and injury scores, but complete endothelialization around the stent struts was observed in both groups at 28 days. It is suggested that the drug, cilostazol, unlike other cytotoxic agents, did not inhibit a natural course of endothelialization. Cilostazol is known to have pleiotropic effects.
Several experimental and clinical studies have identified the antirestenotic property of cilostazol.^6–8 Of these, the U.S. randomized, double-blind, placebo-controlled trial known as the Cilostazol for REstenosis Trial (CREST), was the largest study to demonstrate significantly lower binary restenosis rates in cilostazol-treated patients (a 36% relative-risk reduction). The CREST investigators also identified predictors of increased restenosis including diabetes mellitus, small vessel diameter, lesion length and LAD coronary artery site. The cilostazol-treated group also showed favorable outcomes in these subgroups. Importantly, patients with diabetes mellitus are well known to experience poor prognosis. In the CREST study, the use of cilostazol in oral agent-treated diabetics was associated with a highly significant 63% reduction in restenosis.⁸ The antirestenotic property may be caused by the inhibition of smooth-muscle cell (SMC) proliferation. Cilostazol’s direct inhibition of SMC proliferation may contribute to the significant reduction of late loss after revascularization.⁶ As noted previously, DES thrombosis is one of the primary untoward side effects associated with the sirolimus-eluting stent (SES). DES thrombosis resulted from the possibility of delayed endothelialization and enhanced platelet aggregation after SES implantation. Several in vitro studies have suggested that cilostazol may also affect endothelial cell growth. According to these laboratory studies, cilostazol increased the concentration of hepatocyte growth factor (HGF), which is a novel and potent member of the endothelial cell-specific growth factors. As the result, this activity may attenuate endothelial cell death and stimulate its growth.^20,21 Cilostazol may also control neointimal proliferation by accelerating the regrowth of endothelial cells after balloon angioplasty.⁶ Therefore, local deployment of a cilostazol-coated stent could possibly be expected to enhance early endothelialization as well. If the CES demonstrates acceleration of endothelialization, the need for prolonged antithrombotic therapy could be eliminated. Further studies are required to observe the effects of endothelialization on CES. Virmani et al reported the first case of a localized hypersensitivity reaction caused by a Cypher™ coronary SES (Cordis Corp., Miami, Florida), resulting in an acute myocardial infarction secondary to late in-stent thrombosis at 18 months. They concluded that hypersensitivity to the polymer was the most likely mechanism.²² With current DES, only drug is released, but the polymer remains permanently. In the present study, we utilized a biodegradable PEA polymer. Only BMS remained in the artery after the drug and the polymer were released. Therefore, the problem of current DES may possibly be overcome with this second-generation DES.

Conclusion

In this preliminary preclinical study, stents coated with cilostazol on biodegradable poly-ester-amides copolymers (PEA) suppressed intimal hyperplasia compared to BMS in a porcine model. Further studies are necessary to verify the feasibility of this promising DES.

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