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Succinobucol-Eluting Stents Increase Neointimal
Thickening and Peri-Strut Inflammation in a Porcine
Coronary Model
Jonathan Watt,
1,2
*MD,MRCP, Simon Kennedy,
3
BSc,PhD,
Christopher McCormick,
1
BEng,EngD, Ejaife O. Agbani,
1
BPharm,MSc,
Allan McPhaden,
4
FRCPath, Alexander Mullen,
1
BSc,PhD,MRPharmS, Peter Czudaj,
5
BSc,
Boris Behnisch,
5
PhD, Roger M. Wadsworth,
1
BPharm,PhD,DSc,
and Keith G. Oldroyd,
2
MD,FSCAI,FRCP
Objective: The aim of this study was to assess the efficacy of stent-based delivery of
succinobucol alone and in combination with rapamycin in a porcine coronary model.
Background: Current drugs and polymers used to coat coronary stents remain subop-
timal in terms of long term efficacy and safety. Succinobucol is a novel derivative of
probucol with improved antioxidant and anti-inflammatory properties. Methods: Poly-
mer-free Yukon stents were coated with 1% succinobucol (SucES), 2% rapamycin
(RES), or 1% succinobucol plus 2% rapamycin solutions (SucRES) and compared with
a bare metal stent (BMS). Results: The in vivo release profile of SucES indicated drug
release up to 28 days (60% drug released at 7 days); 41 stents (BMS, n511; SucES, n
510; RES, n510; SucRES, n510) were implanted in the coronary arteries of 17 pigs.
After 28 days, mean neointimal thickness was 0.31 60.14 mm for BMS, 0.51 60.14
mm for SucES, 0.19 60.11 mm for RES, and 0.36 60.17 mm for SucRES (P<0.05 for
SucES vs. BMS). SucES increased inflammation and fibrin deposition compared with
BMS (P<0.05), whereas RES reduced inflammation compared with BMS (P<0.05).
Conclusion: In this model, stent-based delivery of 1% succinobucol using a polymer-
free stent platform increased neointimal formation and inflammation following
coronary stenting. V
C2012 Wiley Periodicals, Inc.
Key words: angioplasty; stents; antioxidants; restenosis; inflammation
INTRODUCTION
Drug-eluting stents (DES) have decreased the inci-
dence of in-stent restenosis (ISR) compared with bare-
metal stents (BMS). However, polymers and drugs
coated on stents can delay arterial healing and cause
inflammation, which may increase the risk of stent
thrombosis and late ‘‘catch-up’’ restenosis [1]. Thus,
much effort is now being directed towards the develop-
ment of novel DES which inhibit ISR and promote
healing by reducing inflammation.
Preclinical studies have shown that oral probucol
inhibits neointimal hyperplasia and improves re-endo-
thelialization after stent injury via its antioxidant prop-
erties and up-regulation of heme oxygenase-1, which
induces vascular smooth muscle cell (SMC) apoptosis
and promotes endothelial cell (EC) function [2–5].
However, the effects of oral probucol have been
1
Strathclyde Institute of Pharmacy and Biomedical Sciences,
University of Strathclyde, Glasgow, United Kingdom
2
West of Scotland Regional Heart & Lung Centre, Golden
Jubilee National Hospital, Glasgow, United Kingdom
3
University of Glasgow, Institute of Cardiovascular & Medical
Sciences, Glasgow, United Kingdom
4
Department of Pathology, Glasgow Royal Infirmary, United
Kingdom
5
Translumina GmbH, Hechingen, Germany
Conflict of interest: PC and BB are employees of Translumina
GmbH who manufactured the stents. No other authors have a con-
flict of interest.
Grant sponsor: British Heart Foundation Junior Research Fellow-
ship; grant number: FS/05/096/19933; Grant sponsors: Engineering
and Physical Sciences Research Council and Translumina GmbH
*Correspondence to: Jonathan Watt, West of Scotland Regional
Heart & Lung Centre, Golden Jubilee National Hospital, Glasgow,
United Kingdom. E-mail: jonnywatt@hotmail.com
Received 20 January 2012; Revision accepted 5 May 2012
DOI 10.1002/ccd.24473
Published online 14 May 2012 in Wiley Online Library
(wileyonlinelibrary.com)
V
C2012 Wiley Periodicals, Inc.
Catheterization and Cardiovascular Interventions 81:698–708 (2013)
disappointing in clinical trials [6–9]. The use of probu-
col as a stent coating has proven more valuable in a
porcine coronary model [10] and subsequently in the
large ISAR-TEST-2 (Intracoronary Stenting and Angio-
graphic Results: Test Efficacy of three Limus-Eluting
Stents) clinical trial, which showed that a polymer-free
probucol/rapamycin-eluting stent provided improved
efficacy compared with durable polymer-based rapamy-
cin or zotarolimus-eluting stents, with lower rates of
‘‘catch-up’’ restenosis between one and two years
[11,12].
Succinobucol is a novel derivative of probucol,
which has consistently demonstrated improved phar-
macokinetics and superior antioxidant, antiprolifera-
tive, and anti-inflammatory effects compared with pro-
bucol [13–16]. Succinobucol inhibits proinflammatory
cytokine release by monocytes, expression of proin-
flammatory cell adhesion molecules by ECs [13–15]
and platelet aggregation [17], all of which may con-
tribute to restenosis. In the ARISE (Aggressive
Reduction of Inflammation Stops Events) clinical trial,
oral succinobucol reduced the incidence of myocardial
infarction, stroke, and diabetes mellitus; however it
brought about deleterious changes in lipid profiles and
increased the incidence of atrial fibrillation [18]. In
CART-1 (Canadian Antioxidant Restenosis Trial), oral
succinobucol following BMS implantation reduced
ISR, but only when drug compliant patients were ana-
lyzed separately [9]. It is possible that locally targeted
succinobucol therapy achievable by stent-based
delivery might improve efficacy and reduce systemic
adverse effects. However, if negative cellular actions
outweigh its beneficial effects, delivery of succinobu-
col into the artery wall from a stent may lead to
localized toxicity. Bearing in mind the recognized
negative effects of permanent polymers, this preclini-
cal study was designed to test whether local delivery
of succinobucol alone or in combination with rapamy-
cin in the absence of a polymer would have a
favorable effect on vascular healing after stent
implantation.
METHODS
Drugs
Succinobucol (previously AGI-1067), the mono-
succinic acid ester of probucol, was synthesized by
esterification of probucol (Sigma-Aldrich, Poole, Dor-
set). The identity of the product was confirmed by
NMR spectroscopy and purity was in excess of 99%.
Succinobucol is metabolically stable and no significant
active metabolites are formed in vivo [19]. Rapamycin
(sirolimus) is a macrocyclic triene antibiotic with
potent antiproliferative, anti-inflammatory, and immu-
nosuppressive effects. It forms a complex with
FKBP12, which subsequently binds to and inhibits the
molecular target of rapamycin (mTOR), causing arrest
of cell proliferation. Rapamycin (purity 95%) was
purchased from Cfm Oskar Tropitzsch (Marktredwitz,
Germany).
DES Platform
The Yukon DES (Translumina, Hechingen, Ger-
many) used in this study consisted of a pre-mounted,
sandblasted 316L stainless steel microporous stent,
which is designed for on-site stent coating without the
obligate use of a polymer. The detailed process of stent
coating and mechanical stent surface modification for
increased drug storage capacity has been described in
detail previously [20]. All stents used were 3.5 mm in
diameter and 16 mm in length. BMS were uncoated
versions of the Yukon stent. All coating solutions con-
sisted of drug(s) dissolved in 99.5% ethanol. During
bench testing, 0.5% (5 mg/ml), 1% (10 mg/ml), and
2% (20 mg/ml) succinobucol solutions were sprayed
onto a Yukon
V
R
stent and closely examined using scan-
ning electron microscopy (Hitachi S-4800). 1% succi-
nobucol coating solution produced a superior, smooth,
and uniform complete drug layer, optimal for the
Yukon
V
R
DES delivery system and therefore was con-
sidered most appropriate for initial preclinical assess-
ment. Three DES were investigated: a succinobucol-
eluting stent (SucES) which utilized a 1% succinobucol
coating solution; a rapamycin-eluting stent (RES)
which utilized a 2% rapamycin coating solution; and a
dual succinobucol/rapamycin-eluting stent (SucRES)
which utilized a 1% succinobucol/2% rapamycin coat-
ing solution. All stents were coated within 24 hr of
use. The coating concentration of rapamycin was
derived from published data [20,21].
Porcine Coronary Stent Model
Male large, white Landrace pigs (16–22 kg) were
premedicated with aspirin (300 mg oral) and clopidog-
rel (300 mg oral), before sedation by an injection of
tiletamine/zolazepam (Zoletil
V
R
100 mg i.m.) and propo-
fol (Rapinovet
V
R
30 mg i.v.). All animals were intuba-
ted and anesthesia maintained throughout the procedure
using a mixture of isoflurane (1–2%) in oxygen/nitrous
oxide. Unfractionated heparin (70 units/kg i.v.) was
given at the start of the procedure. Access to the coro-
nary arteries was achieved via the left femoral artery,
using standard six French sheaths and coronary guiding
catheters. A total of 2–3 stents were placed under fluo-
roscopic guidance in different coronary arteries (refer-
ence diameter 3–3.5 mm, avoiding excessive tortuosity
and major bifurcations) in either the left anterior
Succinobucol-Eluting Stents in Porcine Model 699
Catheterization and Cardiovascular Interventions DOI 10.1002/ccd.
Published on behalf of The Society for Cardiovascular Angiography and Interventions (SCAI).
descending (LAD), left circumflex (LCx), or right cor-
onary arteries (RCA). Stents were deployed at inflation
pressures necessary to produce a stent to artery ratio of
1.2:1 (10–12 atmospheres). After sheath removal, the
femoral artery was ligated and the leg wound closed
and sutured. All animals were given buprenorphine
(Vetergesic
V
R
0.15 mg i.m.) to provide analgesia and
ampicillin (Amfipen
V
R
350 mg i.m.) for antibiotic cover
immediately after the procedure. Animals were allowed
to recover and received a normal diet, with supplemen-
tation of oral aspirin 75 mg daily and oral clopidogrel
75 mg daily for the duration of the study. All prema-
ture and unexpected deaths were examined by post-
mortem, gross evaluation, and stent examination. Ap-
proval was granted by Strathclyde University Ethics
Review Committee, and the investigation conformed to
the Guiding Principles in the Care and Use of
Animals.
Pharmacokinetic Studies
Drug loading of succinobucol coated stents was
quantified by in vitro elution of SucES in pure ethanol
(n¼4), followed by HPLC analysis. To determine the
in vivo release characteristics of succinobucol, SucES
were deployed in six pigs, using the same techniques
as previously described. Pigs were euthanized by a le-
thal dose of pentobarbital at 1 hr, 1, 3, 7, 14, and 28
days after stent implantation. Two stents were
implanted in each animal into different coronary
arteries, with the exception of the 1 hr time point
where three stents were used. Stents were removed
carefully from freshly isolated arterial segments and
succinobucol in the surrounding artery wall and
remaining on the stent was extracted into acetonitrile.
Samples were chromatographed on a Sphereclone ODS
(2) column (5-lm particle size, 150 4.6 mm
[Phenomenex, UK]). Samples were injected using an
autosampler and pump system (Gynotek 480) in 20-ll
aliquots, at a mobile phase flow rate of 1 ml/min aceto-
nitrile–water (92.5:7.5). The detector (Detector 432,
Kontron Instruments, UK) output was measured at a
wavelength of 242 nm.
Efficacy Study
Nineteen pigs underwent stenting performed by a
single cardiologist blinded to the treatment group using
2–3 stents selected randomly on the morning of the
procedure. Two unexpected premature deaths occurred
within 24 hr of the procedure, and these pigs were not
included in the 28-day efficacy analysis. In both cases,
post-mortem revealed occlusive stent thrombosis with
no evidence of myocardial infarction. It was not possi-
ble to determine whether a particular stent was respon-
sible due to the small number of events. No other clini-
cal events occurred during the study. In total, 17 pigs
completed the 28-day efficacy study and 41 stents were
available for histological evaluation (BMS, n¼11
[five LAD; three LCx; three RCA]; SucES, n¼10
[four LAD; four LCx; two RCA]; RES, n¼10 [three
LAD; three LCx; four RCA]; and SucRES, n¼10
[four LAD; four LCx; two RCA]). Four out of 17 pigs,
each receiving three stents, received two stents from
the same group; all other pigs received different stents
from either two or three groups. The stented coronary
artery segments were dissected from the heart and
flushed with normal saline to remove non-adherent
thrombus. The specimens were fixed in formal saline
and dehydrated in pure acetone before resin embedding
in glycol methacrylate (Technovit 8100, Kulzer). Six
sections were obtained from the proximal to distal por-
tion of the stent using a Buehler Isomet 1000 rotary
saw and mounted on a glass slide. Sections were then
ground and polished using a Buehler Metaserv grinder
to reduce the thickness to 10 lm and provide a uni-
form surface for staining and microscopic evaluation.
Sections were stained using hematoxylin–eosin and
modified Carstairs’ stain. Images were acquired using a
Leica DM LB2 microscope and Leica DFC320 digital
camera. Blinded histological analysis was performed
using computerized morphometry software (Image-Pro
Plus, Cybernetics) according to published methods [22]
and detailed examination by a consultant pathologist.
The injury score for each strut was determined [22]
and a mean score for each artery was calculated. Neo-
intimal thickness was calculated as the mean distance
from each stent strut to lumen; neointimal area was
calculated as stent area minus lumen area; diameter
stenosis was calculated as 100 (1 lumen area/IEL
area). Binary ISR was defined as 50% diameter ste-
nosis. Stent endothelialization score was defined as the
extent of the circumference of the arterial lumen cov-
ered by ECs and graded from 1 to 3 (1 ¼25%; 2 ¼
25–75%; 3 ¼75%). Inflammation was graded as 0,
none; 1, scattered inflammatory cells; 2, inflammatory
cells encompassing 50% of a strut in at least 25–50%
of the circumference of the artery; 3, inflammatory
cells surrounding a strut in at least 25–50% of the cir-
cumference of the artery. The intimal fibrin content
was graded as 0, no residual fibrin; 1, focal regions of
residual fibrin involving any portion of the artery or
moderate fibrin deposition adjacent to the strut involv-
ing <25% of the circumference of the artery; 2, mod-
erate fibrin involving >25% of the circumference of
the artery or heavy deposition involving <25% of the
circumference of the artery; 3, heavy fibrin deposition
involving >25% of the circumference of the artery. All
sections were examined for evidence of uncovered
700 Watt et al.
Catheterization and Cardiovascular Interventions DOI 10.1002/ccd.
Published on behalf of The Society for Cardiovascular Angiography and Interventions (SCAI).
stent struts, and the presence of in-stent luminal throm-
bus was determined throughout the entire stent length.
Cell Culture
Bovine pulmonary artery SMCs, obtained from a
local abattoir, were seeded on to six well plates (up to
passage 4) and allowed to grow to 90% confluence.
Succinobucol or probucol was added to each well at
concentrations (1–20 lmol/L) that have previously been
shown to inhibit SMC proliferation [23]. An additional
well received the maximum amount of vehicle (0.3% di-
methyl sulfoxide). After 24 hr, the well plates were
inspected and photographed, taking account of any evi-
dence of cytotoxicity. The medium was removed, and
adherent cells were dislodged using trypLE Express
(Invitrogen, UK). Trypan blue (0.07%) was added to the
cell suspension and cells counted using a hemocytome-
ter. The percentage of the counted cells not stained by
trypan blue is reported as the percent viable cells. Iden-
tical experiments were performed using abattoir-derived
bovine pulmonary artery ECs, grown to 70% conflu-
ence, and tested with succinobucol. The effect of probu-
col on ECs was not investigated as endothelialization
was complete after 28 days in the pig model, and our
assumption was that the adverse effect of succinobucol
was more likely to be on SMCs, and this was compared
directly to the parent drug (probucol), which acted as a
control. In additional experiments, SIN-1 0.2 lmol/L (3-
morpholinosydnonimine, a peroxynitrite donor) was
used to generate oxidative stress and administered to
bovine aortic SMCs and ECs alone and in combination
with succinobucol or probucol.
Statistical Analysis
Histomorphometric data for each stent were the
mean of six sections from the proximal to distal end.
The endothelial score, inflammatory score, and fibrin
score were the mean of two sections per stent. Data
were assessed for normality using the Shapiro-Wilk
test. Normally distributed data are expressed as mean
SD and groups compared using one-way analysis of
variance with post hoc Dunnett’s test. Non-parametric
data are expressed as median (interquartile range) and
groups compared using the Kruskal–Wallis test. Rates
of binary restenosis were compared using the Fisher’s
exact test. Significance was established by a value of P
<0.05. For the efficacy study, a sample size of nine
per group was calculated to provide 80% power to
detect a treatment difference of 30% between groups at
a two-sided 0.05 significance level, based on the
assumption that the SD of the response variable (neoin-
timal growth) was 20% as shown by similar studies in
the porcine model [20]. Statistical analysis was per-
formed using the SPSS statistical software package
14.0 for Windows (SPSS Inc., Chicago, IL, USA).
RESULTS
Pharmacokinetic studies
The amount of drug loaded on the SucES using a
1% succinobucol coating solution was 465 61 lg
succinobucol per stent. The composition of succinobu-
col coating was assessed using scanning electron mi-
croscopy, which confirmed a smooth uniform drug
layer, optimal for the Yukon DES delivery system
(Fig. 1a and b). The drug loading of the 2% RES using
prior data was 842.7 46 lg per stent [21]. The
SucES provided sustained in vivo drug release for 28
days (Fig. 1c); 59.4% of the total succinobucol loaded
on the SucES was eluted during the first week and
81.0% was eluted after 28 days. Succinobucol concen-
tration in coronary artery tissue immediately surround-
ing the stent quantified at 1 hr, 1, 3, 7, 14, and 28 days
Fig. 1. Characterization of succinobucol coated stents. (a)
Expanded uncoated BMS showing surface modification allow-
ing for drug deposition and release without the use of a poly-
mer (inset, top left shows open cell stent design). (b) Expanded
SucES showing smooth uniform 1% succinobucol coating,
available for controlled release. (c) In vivo release profile of
SucES. Succinobucol elution was controlled over 4 weeks,
with the majority of drug released in the first week. Data points
represent the mean of two measurements of mass of succino-
bucol released as a percentage of total drug loaded on the
SucES (three stents were used for the 1 h time point). (d) Local
succinobucol tissue concentration after SucES implantation.
Succinobucol concentration peaked 1 day after implantation
and was maintained for the duration of the study period. Data
are mean 6SD using two identical stents implanted in the
same pig (three stents were used for the 1 h time point).
Succinobucol-Eluting Stents in Porcine Model 701
Catheterization and Cardiovascular Interventions DOI 10.1002/ccd.
Published on behalf of The Society for Cardiovascular Angiography and Interventions (SCAI).
after stent implantation is displayed in Fig. 1d. Succi-
nobucol tissue concentration peaked at 1 day post im-
plantation (825.9 631.3 ng/mg) and remained over
200 ng/mg during the remainder of the study. At 28
days, succinobucol tissue concentration was 242.2
143.3 ng/mg. No succinobucol was detected in blood
samples obtained from the pulmonary artery at 1 h or
1 day after stent deployment.
In Vivo Efficacy
The injury scores were well matched between groups
(Table I and Fig. 2). Excessive injury (scoring 2 or
Fig. 2. Histomorphometric data. (a) Box and whiskers plot
showing injury scores, which were well matched between
groups (P50.52). (b–d) Dot plots showing individual his-
tomorphometric data. Lines represent group means. SucES
caused a significant increase in neointimal thickening (b),
neointimal area (c) and diameter stenosis (d) (n510–11,
*P<0.01 compared with BMS). The trends for RES to
reduce neointimal thickening (P50.16) and diameter
stenosis (P50.17) versus BMS were not statistically
significant.
TABLE I. Comparison of Histomorphometric Data at 28 Days
Group Injury score Neointimal thickness (mm) Neointimal area (mm
2
) Diameter stenosis (%)
BMS (n¼11) 1.61 (1.36, 1.82) 0.31 0.14 1.93 0.80 35.0 13.6
SucES (n¼10) 1.67 (1.58, 1.82) 0.51 0.14* 2.89 0.61* 51.7 13.1
†
RES (n¼10) 1.49 (1.41, 1.78) 0.19 0.11 1.52 0.60 24.8 11.2
SucRES (n¼10) 1.58 (1.43, 1.66) 0.36 0.17 2.21 0.64 38.4 12.0
Values for injury score are median (IQR), other values are mean SD.
*P<0.01 vs. BMS.
†
P<0.05 vs. BMS.
702 Watt et al.
Catheterization and Cardiovascular Interventions DOI 10.1002/ccd.
Published on behalf of The Society for Cardiovascular Angiography and Interventions (SCAI).
more) was present in less than 20% of cases. Com-
pared with BMS, SucES were associated with a signifi-
cant increase in neointimal thickness, neointimal area,
and diameter stenosis (Table I, Figs. 2 and 3). In all
groups, the neointima was composed almost entirely of
cells with little intercellular matrix. The rate of binary
ISR measured by histological analysis was 9.1%, 60%,
0%, and 20% for BMS, SucES, RES, and SucRES
groups, respectively (P<0.01 between all groups).
Histological findings related to healing and inflamma-
tion are shown in Table II. BMS and SucES were com-
pletely re-endothelialized at 28 days, whereas a non-
significant reduction in endothelial regeneration was
observed in both rapamycin groups (P¼0.21). The
number of stents with any uncovered stent struts was
0/11 in BMS; 1/10 in SucES (one stent with 8.3%
uncovered to total stent struts per section); 3/10 in
RES (three stents with 25.0%, 16.7%, 10.0% uncov-
ered to total stent struts per section), and 0/10 in
SucRES groups (P¼0.08 between all groups). There
were no cases of stent strut malapposition. The number
of stents with in-stent thrombus was 27.3%, 10%,
20%, and 20% for the BMS, SucES, RES, and SucRES
groups, respectively (P¼0.81). Fibrin scores were sig-
nificantly higher in both succinobucol groups. Inflam-
mation was increased in the SucES group and was
characterized by predominantly lymphocytic infiltrates,
with macrophage granuloma formation and foreign
body giant cell reactions near to the stent struts in 6/10
stents (Fig. 4). In the two most severe cases of inflam-
mation, minimal scattered eosinophils were present.
The presence of granuloma formation and giant cells
was identified to a much lesser extent in the BMS
group (1/11 stents), whereas these findings were absent
in the RES and SucRES groups, which were associated
with milder lymphocytic infiltrates only. There was no
significant difference in neovascularization, which was
minimal in all groups.
In Vitro Effects of Succinobucol on Cultured
Endothelial and Smooth Muscle Cells
Succinobucol caused toxicity to cultured bovine pul-
monary artery ECs and SMCs; 24 h after addition of
succinobucol 1 lmol/L, a few cells detached from the
well plate in both EC and SMC cultures; whereas more
than 10 lmol/L of succinobucol caused almost all cells
to lift off the plate (Fig. 5a and b). Trypan blue stain-
ing showed that adherent ECs remained viable up to
succinobucol 5 lmol/L; however, with higher concen-
trations, there was a decline in EC viability (Fig. 5c).
The adherent SMCs had reduced viability at succinobu-
col 1, 5, and 20 lmol/L (Fig. 5d). Probucol was con-
siderably less toxic to SMCs than succinobucol, with
minor detachment of SMCs occurring only at 20 lmol/L
(Fig. 6a). Probucol had no deleterious effect on SMC
viability (Fig. 6b). SIN-1 0.2 lmol/L reduced the
Fig. 3. Representative photomicrographs of BMS (a, e), SucES (b, f), RES (c, g), and SucRES
(d, h) groups. Neointimal thickening was significantly greater in the SucES group. The RES
group displayed less neointimal thickening; however this was not statistically significant.
[Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
TABLE II. Healing and Inflammation
Group Endothelial score Fibrin score Inflammatory score
BMS (n¼11) 3.0 (3.0, 3.0) 0.0 (0.0, 0.0) 2.0 (1.5, 2.0)
SucES (n¼10) 3.0 (3.0, 3.0) 1.0 (0.5, 1.1)* 2.3 (2.0, 2.6)
†
RES (n¼10) 3.0 (2.9, 3.0) 0.0 (0.0, 0.5) 1.5 (1.5, 1.6)
†
SucRES (n¼10) 3.0 (2.9, 3.0) 1.5 (0.4, 1.6)* 2.0 (2.0, 2.0)
All values are median (IQR).
*P<0.005 vs. BMS.
†
P<0.05 vs. BMS.
Succinobucol-Eluting Stents in Porcine Model 703
Catheterization and Cardiovascular Interventions DOI 10.1002/ccd.
Published on behalf of The Society for Cardiovascular Angiography and Interventions (SCAI).
viability of ECs and SMCs. Succinobucol (1 lmol/L)
did not protect against SIN-1-induced toxicity, whereas
probucol provided some protection (Fig. 7).
DISCUSSION
As both reactive oxygen species [24,25] and inflam-
mation [26] are implicated in neointima formation, we
chose to investigate the novel antioxidant and anti-
inflammatory compound, succinobucol, without a poly-
mer to reduce the potential for adverse effects. In our
study, physical stent coating characteristics were opti-
mal, and local delivery of succinobucol was well con-
trolled, with around two-thirds of the drug released
during the first week, matching closely the period after
stent deployment when reactive oxygen species are
released in large quantities [27]. This was very similar
to the release profile of the RES, which retards drug
release for greater than 3 weeks, with around two-
thirds released during the first week [20,21]. However,
stent-based delivery of succinobucol at this dose was
found to increase neointimal growth and inflammation.
Subsequently, we showed that succinobucol destabil-
izes cells in culture leading to cell detachment and loss
of viability. However, cellular damage was much less
marked with probucol. Thus, we postulate that succino-
bucol released from the stent induced direct cellular
deterioration leading to the recruitment of inflamma-
tory cells, as was observed in vivo. The presence of a
widespread granulomatous reaction rich in lympho-
cytes, macrophages, and multinucleated giant cells is
consistent with an unfavorable inflammatory response
to the drug, given that these pathological findings were
rare in the BMS group and mechanical injury scores
were well matched. The extent of inflammation is
known to correlate positively with neointimal growth
[28], suggesting a direct link between increased inflam-
mation and excessive neointimal thickening. The ab-
sence of eosinophils in the majority of artery speci-
mens suggests that hypersensitivity did not play a
major role. The persistent fibrin deposition identified
around the stent struts in vivo also implies that succi-
nobucol was responsible for impaired healing.
Although antioxidants can provide protection against
oxidative stress, they may also lead to generation of
secondary radicals, which can modify important intra-
cellular targets with the potential to cause cytotoxic
effects [29,30]. This potential to act as pro-oxidants
under certain conditions might explain some of the
unfavorable cellular responses identified in our study.
When used in combination with rapamycin, succinobu-
col impaired the antirestenotic effect of rapamycin. It
is likely that the undesirable biological effects of succi-
nobucol on the artery wall negated the favorable
actions of rapamycin, although it is possible that succi-
nobucol impeded the delivery of rapamycin.
Comparison with Previous Studies
In CART-1, oral succinobucol improved coronary ar-
tery dimensions six months after stenting; however, it
failed to influence neointimal growth, measured by
intravascular ultrasound [9]. The ability of oral probu-
col to reduce neointimal growth is variable, with
encouraging results in predominantly animal models
[2–4,23,31], but negative reports in mainly human
studies [6–9,32,33]. First-generation polymer-based
DES are now recognized as being responsible for
impaired healing and inflammation after coronary
stenting [1,34]. Polymeric sirolimus-eluting stents are
known to cause extensive granulomatous inflammatory
Fig. 4. Histological findings in the SucES group. (a) Thick neointima (N) and stent strut (S) sur-
rounded by inflammatory cells and macrophage granuloma (G). L, lumen; E, endothelial layer;
M, medial layer. (b) Presence of a foreign body giant cell reaction (GC) adjacent to a stent strut
(S). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
704 Watt et al.
Catheterization and Cardiovascular Interventions DOI 10.1002/ccd.
Published on behalf of The Society for Cardiovascular Angiography and Interventions (SCAI).
Fig. 5. In vitro effects of succinobucol. ECs (a) and SMCs (b) in culture following 24 h incu-
bation with succinobucol. There was evidence of concentration-dependent toxicity with a
progressive reduction in cell adherence leading to total cell detachment at the highest con-
centration of succinobucol. The viability of the remaining adherent ECs (c) and SMCs (d) after
24 h incubation with succinobucol, showing further evidence of cellular toxicity. Values are
mean 6SD (n54–6, *P<0.05).
Succinobucol-Eluting Stents in Porcine Model 705
Catheterization and Cardiovascular Interventions DOI 10.1002/ccd.
Published on behalf of The Society for Cardiovascular Angiography and Interventions (SCAI).
reactions in the pig model, whereas persistent fibrin
deposition is seen more commonly following exposure
to polymeric paclitaxel-eluting stents [35,36]. Although
difficulties remain in translating these and our findings
to humans, these abnormal pathological responses may
provide a substrate for late adverse clinical events. In
Fig. 6. In vitro effects of probucol. After incubating SMCs (a) for 24 h with probucol, there
was maintained cell adherence with increasing concentrations and only minor cell detach-
ment at the highest concentration of probucol. (b) Probucol had no significant effect on the
viability of remaining adherent SMCs. Values are mean 6SD (n54–5, P5NS).
Fig. 7. Succinobucol (a, b) failed to protect ECs and SMCs from SIN-1 induced toxicity, how-
ever probucol (c) provided some protection. Values are mean 6SD (n54–5, *P<0.01 vs.
control, **P<0.05 for SIN-1 vs. SIN-1 11lM probucol, P5NS all other comparisons).
706 Watt et al.
Catheterization and Cardiovascular Interventions DOI 10.1002/ccd.
Published on behalf of The Society for Cardiovascular Angiography and Interventions (SCAI).
support of this, post-mortem human studies have dem-
onstrated impaired healing and increased arterial
inflammation in cases of stent thrombosis following
first-generation DES implantation [1,37,38]. Polymeric
sirolimus-eluting stents also suffer from progressive re-
stenosis between one and two years follow-up [12].
Thus, avoidance of a persistent inflammatory stimulus
due to the stent, drug, or polymer remains a primary
goal of novel DES to maintain late efficacy and safety.
Notably, the effects of the dual SucRES in our study
contrast with that of a polymer-free probucol/rapamy-
cin stent on an identical platform, which has compared
favorably to the sirolimus- and zotarolimus-eluting
stents [12], with no increase in inflammation detected
during preclinical assessment in pigs [10]. Therefore,
stent-based delivery of succinobucol in combination
with rapamycin clearly does not provide similar
effects. Our study suggests that a critical factor deter-
mining the efficacy of antioxidants used in DES is the
balance of their local cellular actions and toxicity,
which may be dependent on a narrow therapeutic
index. The one-third reduction in neointimal formation
by the RES in our study was similar to previous pre-
clinical data using an identical stent [20], which was
later validated in clinical trials [21,39].
Study Limitations
This investigation of succinobucol-coated stents after
stent injury utilized a standard preclinical model. It is
possible that the effect of local delivery of succinobu-
col in human atherosclerotic arteries would differ from
the effect we have demonstrated in normal pig coro-
nary arteries. A group consisting of BMS sprayed with
solvent (99.5% ethanol) was not included; however, all
coated stents were thoroughly dried with pressurized
air by the coating machine prior to use; therefore a
confounding effect of ethanol seems unlikely. To limit
the number of groups, we chose to test a succinobucol
dose similar to probucol in prior studies that also pro-
vided the most uniform stent coating. Lower concentra-
tion succinobucol solutions produced inferior strut cov-
erage. The relationship between coating concentration,
drug loading, and target tissue concentration is depend-
ent on multiple factors including stent surface affinity
for drug, in vivo release kinetics, and subsequent parti-
tioning into tissues; therefore precise estimation of a
coating concentration required to achieve a pre-speci-
fied local tissue concentration was not achievable. The
local tissue concentration of succinobucol measured in
our study exceeded the threshold for toxic effects in
vitro. However, it is worth considering that the tissue
concentration of rapamycin measured after successful
drug elution in a porcine model [20] is around 1000-
fold greater than that required to inhibit SMC prolifera-
tion [40], yet this stent is currently used in clinical
practice. Further study would be required to determine
the in vivo dose-response and long-term effects of suc-
cinobucol loaded on a stent; however, we think our
results remain noteworthy and a beneficial action is
unlikely.
CONCLUSION
Our results suggest that 1% succinobucol is not a
favorable compound for stent coating in clinical stud-
ies. There was increased neointimal formation and
greater inflammation associated with the succinobucol
groups after 28 days. The mechanism of this adverse
effect may relate to local cell toxicity and resulting
inflammation, or even pro-oxidant effects. The succino-
bucol coating also impaired the effect of rapamycin
from a polymer-free DES, thereby reducing its antires-
tenotic properties. Future studies investigating novel
antioxidants alone or in combination with other agents
loaded on stents will require careful evaluation of their
potential efficacy and local toxicity.
ACKNOWLEDGEMENT
The authors wish to thank Ms. M. MacDonald for
providing anesthetic support for porcine studies.
REFERENCES
1. Joner M, Finn AV, Farb A, et al. Pathology of drug-eluting
stents in humans: Delayed healing and late thrombotic risk. J
Am Coll Cardiol 2006;48:193–202.
2. Tanous D, Brasen JH, Choy K, et al. Probucol inhibits in-stent
thrombosis and neointimal hyperplasia by promoting re-endo-
thelialization. Atherosclerosis 2006;189:342–9.
3. Miyauchi K, Aikawa M, Tani T, et al. Effect of probucol on
smooth muscle cell proliferation and dedifferentiation after vas-
cular injury in rabbits: possible role of PDGF. Cardiovasc Drugs
Ther 1998;12:251–260.
4. Tanaka K, Hayashi K, Shingu T, Kuga Y, Nomura K, Kajiyama
G. Probucol inhibits neointimal formation in carotid arteries of
normocholesterolemic rabbits and the proliferation of cultured
rabbit vascular smooth muscle cells. Cardiovasc Drugs Ther
1998;12:19–28.
5. Deng YM, Wu BJ, Witting PK, Stocker R. Probucol protects
against smooth muscle cell proliferation by upregulating heme
oxygenase-1. Circulation 2004;110:1855–1860.
6. Nunes GL, Abizaid AC, Theodoro MP, et al. Role of probucol
in inhibiting intimal hyperplasia after coronary stent implanta-
tion: A randomized study. Am Heart J 2006;152:914–917.
7. Kim MH, Cha KS, Han J-Y, Kim HJ, Kim JS. Effect of antioxi-
dant probucol for preventing stent restenosis. Catheter Cardio-
vasc Interv 2002;57:424–428.
8. Sekiya M, Funada J, Watanabe K, Miyagawa M, Akutsu H.
Effects of probucol and cilostazol alone and in combination on
Succinobucol-Eluting Stents in Porcine Model 707
Catheterization and Cardiovascular Interventions DOI 10.1002/ccd.
Published on behalf of The Society for Cardiovascular Angiography and Interventions (SCAI).
frequency of poststenting restenosis. Am J Cardiol
1998;82:144–147.
9. Tardif JC, Gregoire J, Schwartz L, et al. Effects of AGI-1067
and probucol after percutaneous coronary interventions. Circula-
tion 2003;107:552–558.
10. Steigerwald K, Merl S, Kastrati A, et al. The pre-clinical assess-
ment of rapamycin-eluting, durable polymer-free stent coating
concepts. Biomaterials 2009;30:632–637.
11. Byrne RA, Mehilli J, Iijima R, et al. A polymer-free dual drug-
eluting stent in patients with coronary artery disease: a random-
ized trial vs. polymer-based drug-eluting stents. Eur Heart J
2009;30:923–931.
12. Byrne RA, Kastrati A, Tiroch K, et al. Two-year clinical and
angiographic outcomes from a randomized trial of polymer-free
dual drug-eluting stents versus polymer-based Cypher and
Endeavor [corrected] drug-eluting stents. J Am Coll Cardiol
2010;55:2536–2543.
13. Meng CQ, Somers PK, Rachita CL, et al. Novel phenolic anti-
oxidants as multifunctional inhibitors of inducible VCAM-1
expression for use in atherosclerosis. Bioorg Med Chem Lett
2002;12:2545–2548.
14. Sundell CL, Somers PK, Meng CQ, et al. AGI-1067: A multi-
functional phenolic antioxidant, lipid modulator, anti-inflamma-
tory and antiatherosclerotic agent. J Pharmacol Exp Ther
2003;305:1116–1123.
15. Murata S, Sundell CL, Lijkwan MA, et al. Effects of AGI-
1096, a novel antioxidant compound with anti-inflammatory and
antiproliferative properties, on rodent allograft arteriosclerosis.
Transplantation 2004;77:1494–1500.
16. Kunsch C, Luchoomun J, Grey JY, et al. Selective inhibition of
endothelial and monocyte redox-sensitive genes by AGI-1067:
A novel antioxidant and anti-inflammatory agent. J Pharmacol
Exp Ther 2004;308:820–829.
17. Serebruany V, Malinin A, Scott R. The in vitro effects of a
novel vascular protectant, AGI-1067, on platelet aggregation
and major receptor expression in subjects with multiple risk fac-
tors for vascular disease. J Cardiovasc Pharmacol Ther
2006;11:191–196.
18. Tardif JC, McMurray JJ, Klug E, et al. Effects of succinobucol
(AGI-1067) after an acute coronary syndrome: A randomised,
double-blind, placebo-controlled trial. Lancet 2008;371:1761–
1768.
19. Muldrew KM, Franks AM. Succinobucol: Review of the meta-
bolic, antiplatelet and cardiovascular effects. Expert Opin Inves-
tig Drugs 2009;18:531–539.
20. Wessely R, Hausleiter J, Michaelis C, et al. Inhibition of neoin-
tima formation by a novel drug-eluting stent system that allows
for dose-adjustable, multiple, and on-site stent coating. Arterios-
cler Thromb Vasc Biol 2005;25:748–753.
21. Hausleiter J, Kastrati A, Wessely R, et al. Prevention of restenosis
by a novel drug-eluting stent system with a dose-adjustable, poly-
mer-free, on-site stent coating. Eur Heart J 2005;26:1475–1481.
22. Schwartz RS, Edelman ER, Carter A, et al. Drug-eluting stents
in preclinical studies: Recommended evaluation from a consen-
sus group. Circulation 2002;106:1867–1873.
23. Lau AK, Leichtweis SB, Hume P, et al. Probucol promotes
functional reendothelialization in balloon-injured rabbit aortas.
Circulation 2003;107:2031–2036.
24. Kanellakis P, Pomilio G, Walker C, et al. A novel antioxidant
3,7-dihydroxy-isoflav-3-ene (DHIF) inhibits neointimal hyperpla-
sia after vessel injury attenuating reactive oxygen species and nu-
clear factor-kappa-B signaling. Atherosclerosis 2009;204:66–72.
25. Lee HM, Jeon BH, Won KJ, et al. Gene transfer of redox fac-
tor-1 inhibits neointimal formation: Involvement of platelet-
derived growth factor-{beta} receptor signaling via the inhibi-
tion of the reactive oxygen species-mediated Syk pathway. Circ
Res 2009;104:219–227.
26. Welt FGP, Rogers C. Inflammation and restenosis in the stent
era. Arterioscler Thromb Vasc Biol 2002;22:1769–1776.
27. Shi Y, Niculescu R, Wang D, Patel S, Davenpeck KL, Zalewski
A. Increased NAD(P)H oxidase and reactive oxygen species in
coronary arteries after balloon injury. Arterioscler Thromb Vasc
Biol 2001;21:739–745.
28. Rogers C, Welt FG, Karnovsky MJ, Edelman ER. Monocyte
recruitment and neointimal hyperplasia in rabbits. Coupled in-
hibitory effects of heparin. Arterioscler Thromb Vasc Biol
1996;16:1312–1318.
29. Tyurina YY, Tyurin VA, Yalowich JC, et al. Phenoxyl radicals
of etoposide (VP-16) can directly oxidize intracellular thiols:
Protective versus damaging effects of phenolic antioxidants.
Toxicol Appl Pharmacol 1995;131:277–288.
30. Kagan VE, Tyurina YY. Recycling and redox cycling of pheno-
lic antioxidants. Ann NY Acad Sci 1998;854:425–434.
31. Wakeyama T, Ogawa H, Iida H, et al. Effects of candesartan and
probucol on restenosis after coronary stenting: results of insight
of stent intimal hyperplasia inhibition by new angiotensin II re-
ceptor antagonist (ISHIN) trial. Circ J 2003;67:519–524.
32. Yokoyama T, Miyauchi K, Kurata T, Sato H, Daida H. Effect
of probucol on neointimal thickening in a stent porcine resteno-
sis model. Japan Heart J 2004;45:305–313.
33. Cote G, Tardif JC, Lesperance J, et al. Effects of probucol on
vascular remodeling after coronary angioplasty. Multivitamins
and Protocol Study Group. Circulation 1999;99:30–35.
34. Farb A, Heller PF, Shroff S, et al. Pathological analysis of local
delivery of paclitaxel via a polymer-coated stent. Circulation
2001;104:473–479.
35. Carter AJ, Aggarwal M, Kopia GA, et al. Long-term effects of
polymer-based, slow-release, sirolimus-eluting stents in a por-
cine coronary model. Cardiovasc Res 2004;63:617–624.
36. Wilson GJ, Nakazawa G, Schwartz RS, et al. Comparison of
inflammatory response after implantation of sirolimus- and
paclitaxel-eluting stents in porcine coronary arteries. Circulation
2009;120:141–149.
37. Finn AV, Nakazawa G, Joner M, et al. Vascular responses to
drug eluting stents. Arterioscler Thromb Vasc Biol 2007;27:
1500–1510.
38. Finn AV, Joner M, Nakazawa G, et al. Pathological correlates
of late drug-eluting stent thrombosis. Circulation 2007;115:
2435–2441.
39. Mehilli J, Kastrati A, Wessely R, et al. Randomized trial of a
nonpolymer-based rapamycin-eluting stent versus a polymer-
based paclitaxel-eluting stent for the reduction of late lumen
loss. Circulation 2006;113:273–279.
40. Rosner D, McCarthy N, Bennett M. Rapamycin inhibits human
in stent restenosis vascular smooth muscle cells independently of
pRB phosphorylation and p53. Cardiovasc Res 2005;66:601–610.
708 Watt et al.
Catheterization and Cardiovascular Interventions DOI 10.1002/ccd.
Published on behalf of The Society for Cardiovascular Angiography and Interventions (SCAI).
