Stents Eluting 6-Mercaptopurine Reduce Neointima Formation and Inflammation while Enhancing Strut Coverage in Rabbits

<div><p>Background</p><p>The introduction of drug-eluting stents (DES) has dramatically reduced restenosis rates compared with bare metal stents, but in-stent thrombosis remains a safety concern, necessitating prolonged dual anti-platelet therapy. The drug 6-Mercaptopurine (6-MP) has been shown to have beneficial effects in a cell-specific fashion on smooth muscle cells (SMC), endothelial cells and macrophages. We generated and analyzed a novel bioresorbable polymer coated DES, releasing 6-MP into the vessel wall, to reduce restenosis by inhibiting SMC proliferation and decreasing inflammation, without negatively affecting endothelialization of the stent surface.</p><p>Methods</p><p>Stents spray-coated with a bioresorbable polymer containing 0, 30 or 300 μg 6-MP were implanted in the iliac arteries of 17 male New Zealand White rabbits. Animals were euthanized for stent harvest 1 week after implantation for evaluation of cellular stent coverage and after 4 weeks for morphometric analyses of the lesions.</p><p>Results</p><p>Four weeks after implantation, the high dose of 6-MP attenuated restenosis with 16% compared to controls. Reduced neointima formation could at least partly be explained by an almost 2-fold induction of the cell cycle inhibiting kinase p27^Kip1. Additionally, inflammation score, the quantification of RAM11-positive cells in the vessel wall, was significantly reduced in the high dose group with 23% compared to the control group. Evaluation with scanning electron microscopy showed 6-MP did not inhibit strut coverage 1 week after implantation.</p><p>Conclusion</p><p>We demonstrate that novel stents coated with a bioresorbable polymer coating eluting 6-MP inhibit restenosis and attenuate inflammation, while stimulating endothelial coverage. The 6-MP-eluting stents demonstrate that inhibition of restenosis without leaving uncovered metal is feasible, bringing stents without risk of late thrombosis one step closer to the patient.</p></div

Characteristics of 6-MP eluting stents: 6-MP release, coating quality, and biological activity of eluted 6-MP.

<p>The cumulative release of 6-MP was assayed in vitro for the 30 μg DES (diamonds, n = 3) and the 300 μg 6-MP eluting stents (squares, n = 3) up to 38 days, and expressed as absolute release (A) as well as percentage release (B). A polymer-only stent (C) and a stent loaded with 300 μg 6-MP before (D,E) and after expansion (F) show smooth coating surfaces by SEM. The stability of 6-MP after storage was measured after elution from the stents and quantified by its ability to activate Nur77 in a luciferase reporter assay (G). 6-MP was retrieved from stents after 4 (4m, n = 6 measurements) and 16 months (16m, n = 6) and the activity was compared with a negative control (white bar, n = 9) and freshly dissolved 6-MP at 50 μM (black bar, n = 9). * depicts value that is statistically different from other groups analyzed by ANOVA. Error bars represent standard error.</p

6-MP inhibits inflammation in the lesion.

<p>Macrophages, visualized in sections by immunohistochemistry with the RAM-11 antibody, were mainly localized around stent struts (A), as indicated by the arrows. The inflammation score was significantly lower in the high dose stents compared with the controls (B). White bars depict polymer-only (n = 10), light bars 30 μg 6-MP eluting stents (n = 10), dark bars 300 μg 6-MP eluting stents (n = 9). Error bars depict standard error, * indicates significant difference compared to control group (Mann-Whitney U test).</p

SMCs, apoptosis and expression of the cell cycle inhibitor p27^Kip1 in arteries stented for 4 weeks.

<p>Masson trichrome staining (A) revealed collagen (blue), SMCs (red) and nuclei (black). The lesion predominantly consists of SMC, as was visualized by immunohistochemistry with the 1A4 antibody directed against SM-α actin (B). To assess the extent of apoptosis in the stented vessel segments, immunohistochemistry for cleaved caspase-3, an apoptosis marker, was performed (C, magnification 200x). Examples of positive and negative cells are presented in the insert. Apoptosis was low and similar between the groups (D). As a marker of cell cycle inhibition, p27^Kip1 positive cells were shown by immunohistochemistry (E, magnification 200x, examples shown in insert). The ratio of positive cells was significantly higher in the high dose group compared to controls (F). PCNA was used as a marker for proliferation (G). No differences were found between the groups (H). N = 10 for 0 μg, N = 9 for 300 μg. Lines and error bars depict mean and standard error, respectively. * indicates significant difference with control group (Mann-Whitney U test).</p

Scanning electron microscopy of stent surface 1 week after implantation.

<p>For qualitative <i>en face</i> assessment of stent coverage, high dose 6-MP eluting stents as well as Cypher rapamycin-eluting stents were implanted (N = 2). Stents excised after one week were cut longitudinally and sputtered with gold particles. 6-MP eluting stents (A,B) show good coverage, as opposed to rapamycin-eluting stents (C, D), which are completely exposed.</p

Coating formulations.

<p>6-MP, 6-mercaptopurine; GLL, multiblock copolymer 50% (w/w) poly(DL-lactide-co-glycolide), 50% (w/w) poly(DL-lactide); GPCGL, multiblock copolymer 15% (w/w) poly(glycolide-PEG600-ε-caprolactone), 85% (w/w) poly(DL-lactide-co-glycolide)</p><p>Coating formulations.</p

Morphology and morphometric analyses of sectioned arteries stented for 4 weeks.

<p>Representative HE-stained sections of arteries with stents eluting 0 (A), 30 (B) or 300 μg 6-MP (C). HE staining at low (D) and high (E) magnification with adventitia (a), media (m) and intima (i); strut holes are indicated by (s). Lawson-Van Gieson staining clearly reveals the lamina and thus the different layers of the vessel wall (F). The outer diameter of the stented arteries (G) was measured and found to be highly similar within and between the groups, indicating high reproducibility of stent implantation and expansion. The injury score was low in all stents and similar between groups (H). Lumen stenosis (I) was significantly different across the groups (Jonkheere’s independent samples trend test), with 16% reduction of stenosis in the 300 μg 6-MP eluting stents compared with the polymer-only group (Mann-Whitney U test). N = 10 for 0 and 30 μg, N = 9 for 300 μg. * depicts value that is statistically different from other groups. Error bars represent standard error.</p

Adventitial capillaries.

<p>Higher 6-MP release resulted in higher numbers of capillaries (Jonkheere’s independent samples trend test, P = 0.02). Stents loaded with 300 μg 6-MP (n = 9) induced 30% more capillaries than polymer-only stents (N = 10, Mann-Whitney U test, P = 0.02). Low 6-MP dose (N = 10) failed to increase the number.</p

Scanning electron microscopy overview.

<p>SEM images of a 28-day BMS (upper half) and anti- ApoA-I coated stent (lower half) implanted in the rabbit common iliac artery. High magnification details of stent strut coverage are shown of three randomly chosen spots, next to the low magnification overview in the centre. Complete endothelial lining of the lumen is shown in all high-magnification details, with similar endothelial cell aspect.</p

Thrombin generation on coated discs.

<p>Thrombin generation for discs coated with either ApoA-I-, ApoB-, Isotype- or no antibody (empty discs), compared to thrombin generation in absence of a disc (negative control). (n = 4).</p