The Toxicologist - Society of Toxicology

215 HUMAN INDUCED PLURIPOTENT STEM CELL
DERIVED CARDIOMYOCYTES FOR ASSESSING DRUG
INDUCED CARDIAC ARRHYTHMIAS.
B. Anson 1 , J. Ma 1 , L. Guo 2 , S. Fiene 1 , R. Adams 2 , T. Weiser 3 , T. Singer 3 and
K. Kolaja 2 . 1 Cellular Dynamics International, Madison, WI, 2 Non-Clinical Safety, F.
Hoffman-La Roche, Nutley, NJ and 3 Pharmacology Research and Early Development,
F. Hoffman-La Roche AG, Basel, Switzerland.
The potential for drug-induced cardiac arrhythmias is a vital component of the toxicological
and safety pharmacological profile of new chemical entities (NCEs).
Cardiomyocytes contain multiple ion channels and ion transporters and cardiac
electrical activity arises from the temporal and spatial activity of these proteins.
Commonly employed methods fail to recapitulate the human condition as the predominant
systems typically express a single channel without ancillary proteins and
may therefore miss exacerbating or compensatory protein interactions, while animal
models may ultimately miss species-specific effects. Human induced pluripotent
stem cell (hIPSC) -derived cardiomyocytes provide a relevant human system in
which to assess the arrhythmogenic nature of an NCE. We present data characterizing
the electrophysiological profile of hIPSC-derived cardiomyocytes and demonstrating
their suitability for assessing NCE-induced arrhythmogenicity. Single cell
voltage and current clamp experiments demonstrated the presence of multiple endogenous
ionic currents present in human cardiomyocytes including I Na , I Ca , I to ,
I Kr , I f , and I K1 while drug application produced the expected changes in action potential
phenotypes including an approximate 70% increase in duration in response
to hERG channel block (100nM of E-4031), 80% decrease in duration in response
to Ca 2+ channel block (100nM nifedipine), and a 60% slowing of the action potential
upstroke in response to Na + channel block (10μM TTX). These results were
verified through organotypic extracellular recordings made with microelectrode
array (MEA) techniques that also demonstrated a 35% increase in field potential
duration in response to block of IKs by 10μM chromanol 293B. This dataset
demonstrates that hIPSC-derived cardiomyocytes are a suitable and novel humanbased
model for assessing NCE arrhythmogenic potential.
216 ISIS 416858, AN ANTISENSE INHIBITOR OF FACTOR
XI, IS WELL TOLERATED AND PRODUCES NO RISK
OF BLEEDING IN THE CYNOMOLGUS MONKEY FOR
UP TO 13 WEEKS OF TREATMENT.
H. S. Younis 1 , J. Crosby 2 and S. P. Henry 1 . 1 Preclinical Development, ISIS
Pharmaceuticals, Carlsbad, CA and 2 Drug Discovery, ISIS Pharmaceuticals,
Carlsbad, CA.
Complications associated with increased bleeding risk are the main limitations with
current anticoagulant therapy. A strategy to produce sufficient anticoagulant properties
with reduced bleeding risk may be the inhibition of Factor XI in the intrinsic
coagulation cascade. The objective of this work was to determine the safety profile
of ISIS 416858, a 2’-methoxyethoxy antisense oligonucleotide inhibitor of Factor
XI, with special focus on assessing bleeding risk. Cynomolgus monkeys were administered
ISIS 416858 (4, 8, 12 and 40 mg/kg/wk, SC) for up to 13 weeks. ISIS
416858 produced a dose-dependent reduction in systemic Factor XI concentrations
and a concomitant increase in aPTT. Plasma Factor XI was reduced by 80% at 4
weeks of treatment (40mg/kg/wk) that resulted in a 33% increase in aPTT by 13
weeks. No effects on PT or platelets were observed during this time period confirming
that the effects of ISIS 416858 on Factor XI inhibition were limited to the
intrinsic coagulation cascade. Skin laceration bleeding time, a measure of bleeding
risk, was unchanged following 13 weeks of treatment. ISIS 416858 produced typical
changes anticipated for an antisense oligonucleotide in the monkey including
acute and transient complement activation, basophilic granules and mononuclear
cell infiltrates in multiple tissues, which increased in incidence and severity in a
dose-dependent manner. Collectively, these results suggest that ISIS 416858 produces
antisense inhibition of Factor XI, is well tolerated and produces no risk of
bleeding at clinically relevant doses.
217 BENZO[A]PYRENE AUGMENTS INFLAMMATORY AND
PROTEOLYTIC CHANGES IN MOUSE MODEL OF
ABDOMINAL AORTIC ANEURYSM.
P. A. Prins 1 , P. R. Perati 1 , A. Ramesh 2 , Z. Guo 2 and U. K. Sampson 1 .
1 Cardiovascular Medicine, Vanderbilt University, Nashville, TN and 2 Meharry
Medical College, Nashville, TN.
Background: Benzo[a]pyrene [B(a)P] is an abundant environmental polycyclic aromatic
hydrocarbon (PAH) known to promote atherosclerosis—a predisposing factor
for the development of abdominal aortic aneurysms (AAA). Since experimental
46 SOT 2011 ANNUAL MEETING
evidence suggests that inflammation and enzymatic degradation of the vascular wall
are central to the pathobiology of AAA, we sought to determine the effects of BaP
exposure on these processes. Methods: A total of 112 male, 5 weeks of age, Apo E-
/- mice were evaluated in 4 experimental groups: B(a)P (n=39), AngII (n= 30),
B(a)P+AngII (n=29) and control (n=24). Exposure to B(a)P (0.71mg/kg/day x
60days) was via oral gavage and AngII exposure (1000ng/kg/min x 2 wks) occurred
via subcutaneous osmotic mini pumps. In the B(a)P+AngII group, AngII exposure
overlapped with the last two weeks of B(a)P treatment. Results: There were no incidence
of AAA in either control or B(a)P groups. There was no difference in the
number of intact (Ni) and ruptured (Nr) AAA between the AngII (Ni = 10, Nr = 7)
and B(a)P + AngII (Ni = 8, Nr = 8) groups. Intact AAA from mice in the
B(a)P+AngII group were significantly larger in cross-sectional diameter (2.30 ±
0.09, N=8) compared to those of the AngII group (1.9 ± 0.09, N=10) (p < 0.05).
Immunohistochemical and molecular evaluation of aortic tissue from B(a)P+AngII,
and AngII treatment groups demonstrated higher levels of tumor necrosis factor
alpha (TNF-α) and cyclophilin A (Cyp A) proteins, and of matrix metalloproteinase
(MMP9) in the B(a)P+AngII group. Accordingly, serum inflammatory cytokine
levels (TNF-α) and interleukin (IL-6) were higher in the B(a)P+AngII
group. Conclusions: These findings suggest that B(a)P potentiates the inflammatory
and proteolytic processes that attend AAA formation. Given the abundance of
B(a)P (e.g. in charcoaled foods and smoke), the possible role of B(a)P in AAA evolution
merits further evaluation for mechanistic insights.
218 ARYL HYDROCARBON RECEPTOR AGONIST - PCB 77
- INCREASES THE INCIDENCE AND SEVERITY OF
ABDOMINAL AORTIC ANEURYSM.
V. Arsenescu, R. Arsenescu and L. Cassis. GCNS, University of Kentucky,
Lexington, KY. Sponsor: H. Swanson.
Rationale: Male sex and smoking are the main risk factors for abdominal aortic
aneurysm (AAA). PCB 77, a dioxin-like component of cigarette smoke, triggers
AhR dependent inflammatory response in the cardiovascular system. We hypothesized
that PCB77 will induce inflammation in the perianeurysmal adipose tissue
and promote the development of Angiotensin II (AngII) - induced AAA. Methods:
We injected male, apoE-/- mice with vehicle or PCB 77 (150mmol/Kg, i.p) at the
beginning and at the end of 1 month AngII infusion. We monitored body weight,
blood pressure and the aneurysm formation and progression using ultrasonography.
The incidence of abdominal aortic aneurysm was analyzed in a cohort of patients
with/without exposure to cigarette smoke. CYP1A1 induction was determined in
aortic wall specimens from patients that underwent AAA surgical repair. Results:
PCB 77 increased body weight by expanding visceral adipose tissue and liver mass
in AngII + PCB 77 - infused mice. In addition, total cholesterol and free fatty acids
(NEFA) were increased in mice receiving PCB 77. AAA incidence and severity were
increased in mice exposed to PCB 77 (9 out of 10) compared to AngII group (5 out
of 11). PCB77 was preferentially accumulated in the mesenteric fat. Furthermore,
PCB 77 treatment induced proinflammatory adipokines, Angiotensin, AT1 and
macrophage accumulation in the perianeurysmal adipose tissue while decreasing
the antiinflammatory adiponectin. Patients exposed to cigarette smoke had twice
the incidence of AAA and it was correlated with high CYP1A1 expression in the
aneurysmal aortic wall. Conclusions: Persistent exposure to a dioxin like toxicant
was associated with increase AAA severity and mortality. Metabolic syndrome and
inflammatory response in the perivascular fat could explain this association. The
strong correlation between smoking and AAA in human subjects further supports
our observations. Smoking cessation and avoidance of fatty foods containing
dioxin-like toxicants represent viable approaches for prevention and early, non-surgical
management of patients with AAA.
219 NNK, A TOBACCO-SPECIFIC CARCINOGEN, INHIBITS
THE EXPRESSION OF LYSYL OXIDASE: A TUMOR
SUPPRESSOR GENE.
Y. Zhao, S. Gao, J. Zhou, P. Toselli and W. Li. Biochemistry, Boston University
School of Medicine, Boston, MA.
4-(methyl nitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a tobacco-specific carcinogen,
is believed to contribute to the cancer burden in cigarette smokers. To
evaluate NNK effects on the expression of lysyl oxidase (LO), a tumor suppressor
gene, we examined this enzyme at promoter, mRNA, protein and catalytic levels in
NNK-treated rat fetal lung fibroblasts (RFL6). Exposure of cells to NNK at 10, 30,
100, and 300 μM for 48 h reduced LO transcript levels to 79.3, 56.4, 8.6 and
1.8% of the control, respectively. NNK decreased levels of all LO protein species
including the 46 kDa preproenzyme, the 50 kDa proenzyme and the 32 kDa mature
enzyme, in a dose-dependent manner. Notably, the 50 kDa and the 32 kDa
species of LO were more sensitive to NNK. Although 300 μM NNK, markedly de-