News Tips for Wednesday, Feb. 9, 2011
Embargoed for 7:30 a.m. PT/10:30 a.m. ET, Wednesday, Feb. 9, 2011 – Abstract 215
Control #: 11-ISC-A-2474-AHA
201 - Plenary Session II (Presentation #: 215 ; Speaking Time: 2/10/2011 11:02:00 AM 2/10/2011 11:14:00 AM)
Title
Familial Intracranial Aneurysms: Is Anatomic Vulnerability Heritable?
Author Block
Jason Mackey, Univ of Cincinnati, Cincinnati, OH; Robert D. Brown Jr., Mayo Clinic, Rochester, MN;
Charles J. Moomaw, Univ of Cincinnati, Cincinnati, OH; Dheeraj Gandhi, Johns Hopkins Univ,
Baltimore, MD; Laura Sauerbeck, Univ of Cincinnati, Cincinnati, OH; Richard Hornung, Cincinnati
Children's Hosp, Cincinnati, OH; Daniel Woo, Dawn Kleindorfer, Matthew L. Flaherty, Univ of
Cincinnati, Cincinnati, OH; Irene Meissner, Mayo Clinic, Rochester, MN; Tatiana Foroud, Indiana Univ,
Indianapolis, IN; Craig Anderson, Univ of Sydney, Sydney, Australia; Guy Rouleau, Notre Dame Hosp,
Montreal, QC, Canada; E. Sander Connolly, Columbia Univ, New York, NY; Ranjan Deka, Univ of
Cincinnati, Cincinnati, OH; Daniel L. Koller, Indiana Univ, Indianapolis, IN; Todd Abruzzo, Univ of
Cincinnati, Cincinnati, OH; John Huston III, Mayo Clinic, Rochester, MN; Joseph P. Broderick, Univ of
Cincinnati, Cincinnati, OH; for the FIA Investigators
Disclosure Block:
J. Mackey: None. R.D. Brown: None. C.J. Moomaw: None. D. Gandhi: None. L. Sauerbeck:
None. R. Hornung: None. D. Woo: None. D. Kleindorfer: None. M.L. Flaherty: None. I. Meissner:
None. T. Foroud: None. C. Anderson: Employment; Significant; The George Institute for Global
Health; The National Health and Medical Research Council of Australia. G. Rouleau: None. E.S.
Connolly: None. R. Deka: None. D.L. Koller: None. T. Abruzzo: None. J. Huston: None. J.P.
Broderick: Research Grant; Significant; NIH R01 NS39512.
Abstract Content
Introduction: Previous studies have suggested that family members with intracranial
aneurysms (IAs) often harbor aneurysms in similar anatomic locations, though data are limited.
To test the hypothesis that anatomic vulnerability in families might be heritable, we investigated
whether IA arterial territory concordance within families of the Familial Intracranial Aneurysm
(FIA) Study was greater than between-family concordance.
Methods: The FIA Study is a multicenter study whose goal is to identify genetic and other risk
factors for formation and rupture of IAs. The study required at least three affected family
members or an affected sibling pair for inclusion. After identifying all affected subjects with a
“definite” or “probable” phenotype, we identified the affected probands and first degree relatives
(FDRs) in each family. We stratified each IA of the probands by major arterial territory (ICA,
MCA, ACA, and VB) and calculated each family’s proband-FDR territory concordance and
overall contribution to the concordance analysis. IAs without a documented location were
excluded from analysis. The analysis weighted each family’s contribution by the number of
affected FDRs since the probability of concordance increases with the number of family
members. For the between-family analysis we compared each within-family proband with a
randomly selected family from the cohort using sampling with replacement. Concordance was
calculated for the four arterial distributions. We then compared within-family to between-family
concordance using logistic regression.
Results: There were 535 families in the study, of which 134 were excluded due to lack of an
affected FDR. Also excluded were families with lack of proband/FDR pairs due to uncertain
phenotype (30) or unknown IA location (53). Of the 318 remaining probands, 230 had one
affected FDR, 66 had two FDRs, 19 had three FDRs, 2 had four FDRs, and 1 had five FDRs. Of
the 318 probands, some of whom had more than one IA, 157 had an ICA IA, 118 had a MCA IA,
80 had an ACA IA, and 48 had a VB IA. Concordance was greater within families than between
families in the ICA and MCA distributions and in the overall analysis (Table).
Conclusions: In a highly enriched sample with familial predisposition to IA development, we
found that IA territorial concordance was more likely within families than between families. The
study supports the hypothesis that genetic predisposition for IAs in the same arterial territory
within families exists. Future studies of IA genetics should consider stratifying cases by IA
location.
Embargoed for 7:30 a.m. PT/10:30 a.m. ET, Wednesday, Feb. 9, 2011 – Abstract 1
Control #: 11-ISC-A-2486-AHA
Session Assignment
A1 - Aneurysm Oral Abstracts (Presentation #: 1 ; Speaking Time: 2/9/2011 7:30:00 AM 2/9/2011 7:42:00 AM)
Slotter Assignment(s)
Title
Unruptured Intracranial Aneurysms in the FIA and ISUIA Cohorts: Differences in Multiplicity and
Location
Author Block
Jason Mackey, Univ of Cincinnati, Cincinnati, OH; Robert D. Brown Jr., Mayo Clinic, Rochester, MN;
Charles J. Moomaw, Univ of Cincinnati, Cincinnati, OH; Dheeraj Gandhi, Johns Hopkins Univ,
Baltimore, MD; Laura Sauerbeck, Univ of Cincinnati, Cincinnati, OH; Richard Hornung, Cincinnati
Children's Hosp, Cincinnati, OH; Daniel Woo, Dawn Kleindorfer, Matthew L. Flaherty, Univ of
Cincinnati, Cincinnati, OH; Irene Meissner, Mayo Clinic, Rochester, MN; Craig Anderson, Univ of
Sydney, Sydney, Australia; E. Sander Connolly, Columbia Univ, New York, NY; Guy Rouleau, Notre
Dame Hosp, Montreal, QC, Canada; David F. Kallmes, Mayo Clinic, Rochester, MN; James C. Torner,
Univ of Iowa, Iowa City, IA; John Huston III, Mayo Clinic, Rochester, MN; Joseph P. Broderick, Univ of
Cincinnati, Cincinnati, OH; for the FIA Investigators
Disclosure Block:
J. Mackey: None. R.D. Brown: Research Grant; Significant; NIH R01 NS028492. C.J. Moomaw:
None. D. Gandhi: None. L. Sauerbeck: None. R. Hornung: None. D. Woo: None. D. Kleindorfer:
None. M.L. Flaherty: None. I. Meissner: None. C. Anderson: Employment; Significant; The George
Institute for Global Health; The National Health and Medical Research Council of Australia. E.S.
Connolly: None. G. Rouleau: None. D.F. Kallmes: None. J.C. Torner: Research Grant; Significant;
NIH R01 ISUIA Statistical PI. J. Huston: None. J.P. Broderick: Research Grant; Significant; NIH R01
NS39512.
Abstract Content
Introduction: Familial predisposition is an important non-modifiable risk factor for the formation
and rupture of intracranial aneurysms (IAs), though the data regarding the characteristics of
familial IAs are currently limited. We sought to characterize aneurysm location and IA multiplicity
in the Familial Intracranial Aneurysm (FIA) study and to compare this cohort with the cohort of
non-familial IA subjects in the International Study of Unruptured Intracranial Aneurysms (ISUIA)
study.
Methods: The FIA Study is a multicenter international study whose goal is to identify genetic
and other risk factors for formation and rupture of IAs. The study required at least three affected
family members or an affected sibling pair for inclusion. We analyzed subject demographics,
aneurysm location, and multiplicity in the unruptured cohort of the FIA Study and then compared
the FIA cohort to the ISUIA cohort. To improve comparability of the datasets, we excluded all
ISUIA subjects with a family history of IAs or subarachnoid hemorrhage and all subjects with a
ruptured aneurysm in either cohort.
Results: Among subjects enrolled in the FIA study, 983 had “definite” or “probable” phenotypes
for IA. Of these 983 subjects, 476 subjects were excluded: 391 had a ruptured aneurysm, 79
subjects had unknown rupture status, 4 subjects had aneurysms <2mm (an exclusion criterion
for ISUIA), 1 subject’s aneurysm was on an AVM feeding artery, and 1 subject had an unknown
aneurysm location. The remaining 507 subjects with unruptured aneurysms were included in the
analysis. Of the 4059 subjects in the ISUIA study, 983 had a previous aneurysm rupture and
657 of the remainder had a positive family history, leaving 2419 subjects in the analysis.
Multiplicity was more common in the FIA cohort compared with the ISUIA cohort (Table), with
156 FIA subjects harboring 2-3 aneurysms and 24 subjects with ≥4 aneurysms. With regard to
aneurysm location, FIA subjects had a slightly higher proportion of MCA aneurysms, while
ISUIA subjects had a higher proportion of PComm aneurysms (Table).
Conclusions: Subjects with a strong familial predisposition are more likely to have multiple
aneurysms and an aneurysm in the MCA territory than subjects without a family history of IAs.
Heritable structural vulnerability is a possible explanation for these findings and emphasizes the
importance of ongoing investigations into the underlying genetic mechanisms of IA formation.
Embargoed for 9:39 a.m. PT/12:39 p.m. ET, Wednesday, Feb. 9, 2011 – Abstract 24
Control #: 11-ISC-A-1846-AHA
Session Assignment
A4 - Community/Risk Factors Oral Abstracts I (Presentation #: 24 ; Speaking Time: 2/9/2011
9:39:00 AM - 2/9/2011 9:51:00 AM)
Title
Baseline Pre-Hypertension and Risk of Incident Stroke: A Meta-analysis
Author Block
Meng Lee, Jeffrey L Saver, UCLA Stroke Ctr, Los Angeles, CA; Brian Chang, UCSD Medical Sch, Los
Angeles, CA; Kuo-Hsuan Chang, Chang Gung Memorial Hosp, Taipei, Taiwan; Katrina Lin, UCLA
Medical Sch, Los Angeles, CA; Bruce Ovbiagele, UCLA Stroke Ctr, Los Angeles, CA
Disclosure Block:
M. Lee: None. J.L. Saver: None. B. Chang: None. K. Chang: None. K. Lin: None. B. Ovbiagele:
None.
Abstract Content
Background: Prevalence of pre-hypertension (systolic blood pressure [SBP] of 120-139 mmHg
or diastolic blood pressure [DBP] of 80-89 mmHg) among adults in the United States is roughly
31%. However, the role of pre-hypertension in conferring greater stroke risk is unsettled: while
some studies linked pre-hypertension to higher stroke risk, others did not. We conducted a
systematic review and meta-analysis to properly investigate the strength and direction of the
relation of pre-hypertension with stroke risk.
Methods: A systematic literature search was performed. We included studies that prospectively
collected data within cohort studies or clinical trials, assessed incident stroke, had follow-up of at
least one year, and reported quantitative estimates of the multivariate adjusted relative risk (RR)
and 95% confidence interval (CI) for stroke associated with pre-hypertension. We combined
data using the inverse variance approach.
Results: The final primary analysis included 12 prospective cohort studies comprising 525,246
participants with 5,467 incident stroke events. Pre-hypertension was associated with higher risk
of subsequent stroke (RR 1.58, 95% CI 1.40 to 1.79, P<0.001) (Fig 1). Heterogeneity existed
among these studies (P<0.001, I2=64%). Seven studies further distinguished low prehypertensive patients (SBP 120-129 mmHg or DBP 80-84 mmHg) and high pre-hypertensive
patients (SBP 130-139 mmHg or DBP 85-89 mmHg). Among low range pre-hypertensive
patients, incident stroke risk was not increased (RR 1.22, 95% CI 0.95 to 1.57, P=0.11).
However, for higher values within the pre-hypertensive range, stroke risk was substantially
boosted (RR 1.79, 95% CI 1.49 to 2.16, P<0.001). In subgroup analyses, pre-hypertension
significantly predicted higher stroke risk across race-ethnicity, stroke severity (fatal vs. all
stroke), and stroke subtypes (ischemic vs. non-specified stroke).
Conclusion: Baseline pre-hypertension substantially raises future stroke risk. This risk appears
largely driven by higher SBP or DBP values within the pre-hypertensive range. Given the high
prevalence of pre-hypertension, randomized trials to evaluate the efficacy of blood pressure
lowering therapies in persons with this condition are warranted.
Abstract 214 - Embargoed for 11:51 a.m. PT/2:51 p.m. ET, Wednesday, Feb. 9, 2011
Control #: 11-ISC-A-2513-AHA
Session Assignment
200 - Plenary Session I (Presentation #: 214 ; Speaking Time: 2/9/2011 11:51:00 AM 2/9/2011 12:03:00 PM)
Title
How the Heart Whispers to the Brain: Serotonin as Neurovascular Mediator in Patent Foramen Ovale
Related Stroke
Author Block
MingMing Ning, CardioNeurology Clinic, Clinical Proteomics Res Ctr, Dept of Neurology,
Massachusetts General Hosp / Harvard Medical Sch, Boston, MA; Deepti Navaratna, Neuroprotection
Res Lab, Dept of Radiology, Massachusetts General Hosp / Harvard Medical Sch, Boston, MA; Zareh
Demirjian, Dept of Hematology, Massachusetts General Hosp / Harvard Medical Sch, Boston, MA;
Ignacio Inglessis-Azuaje, Dept of Cardiology, Massachusetts General Hosp / Harvard Medical Sch,
Boston, MA; David McMullin, New York Univ, New York, NY; G William Dec, Igor Palacios, Dept of
Cardiology, Massachusetts General Hosp / Harvard Medical Sch, Boston, MA; Ferdinando Buonanno,
CardioNeurology Clinic, Clinical Proteomics Res Ctr, Dept of Neurology, Massachusetts General Hosp /
Harvard Medical Sch, Boston, MA; Eng H Lo, Neuroprotection Res Lab, Dept of Radiology, and Clinical
Proteomics Res Ctr, Dept of Neurology, Mass General Hosp / Harvard Medical Sch, Boston, MA
Disclosure Block:
M. Ning: None. D. Navaratna: None. Z. Demirjian: None. I. Inglessis-Azuaje: None. D. McMullin:
None. G. Dec: None. I. Palacios: None. F. Buonanno: None. E.H. Lo: None.
Abstract Content
With strong epidemiologic data establishing PFO as an independent risk factor for stroke, there
is compelling reason to investigate the molecular mechanisms underlying PFO-related
neurovascular injury. PFO enables direct mixing of venous and arterial circulation, thereby not
only serving as a conduit for venous clots, but also enabling harmful factors such as serotonin
(5HT) to avoid pulmonary filtration and to remain in circulation at elevated levels, potentially
creating a prothrombotic state that increases risk of stroke. We hypothesize that PFO-related
neurovascular injury may involve cerebral endothelial response to active 5HT
inappropriately kept in circulation by PFO. 5HT is vasoactive, prothrombotic, and can cause
oxidative stress in a dose-dependent manner in the heart, but its role in stroke is unclear. MMP9
is a well-documented marker of neurovascular injury. Endovascular PFO closure provides a rare
opportunity to explore a bedside model to dissect PFO circulatory signaling. We use a combined
cell biology and translational clinical approach to test our heart-brain signaling hypothesis.
Methods: Non-migraine stroke patients were recruited in accordance with IRB approval, plasma
was sampled from venous blood before PFO closure, and from left and right atria pre and post
closure. Active 5HT was measured by ELISA and MMP9 by zymography. Human brain
endothelial cell (HBEC) cultures were exposed for 24 hours to various levels of 5HT (0.1-10
µM), ketanserin (a 5HT inhibitor), and U83836 (a free-radical scavenger). Results: In human
plasma, peripheral 5HT is higher in PFO stroke than in stroke without PFO (14 vs 10 ng/ml,
p<0.05, n=55). 5HT and MMP9 levels decrease in the left atrium immediately post successful
PFO closure (A, B), but venous 5HT remains elevated if there is persistent residual venousarterial mixing (C). In cell cultures, 5HT-treated HBEC media show an increase of MMP9 (D)
that is blocked by ketanserin and by U83836E (E). Conclusion: Clinically, the correlation of
venous and arterial 5HT levels to the presence or absence of PFO-related shunting strongly
supports our hypothesis that venous-arterial mixing via PFO contributes to a persistent
imbalance of circulatory signaling factors. The upregulation of MMP9 by 5HT in HBEC - a
response that is dose-dependent, stimulus-specific (blocked by 5HT inhibitor) and may be
mediated by oxidative stress (blocked by free radical scavenger) - supports our hypothesis that
5HT may mediate injury to the brain vasculature. Together these data suggest circulatory 5HT
as a novel potential stroke risk mediator responsive to mechanical PFO closure.
g
Abstract MP14 - Embargoed for 3 p.m. PT/6 p.m. ET, Wednesday, Feb. 9, 2011
Control #: 11-ISC-A-3386-AHA
Session Assignment
MP3 - Community/Risk Factors Moderated Poster Tour I (Presentation #: W MP14 ; Speaking
Time: 2/9/2011 5:50:00 PM - 2/9/2011 5:55:00 PM)
Title
Poorer Patients Have More Severe Ischemic Strokes
Author Block
Dawn Kleindorfer, Christopher Lindsell, Kathleen A Alwell, Charles J Moomaw, Daniel Woo, Matthew
L Flaherty, Pooja Khatri, Opeolu Adeoye, Simona Ferioli, Brett M Kissela, UNIV OF CINCINNATI
HOSP, Cincinnati, OH
Disclosure Block:
D. Kleindorfer: Research Grant; Significant; NIH R-01 grant support. C. Lindsell: None. K.A. Alwell:
None. C.J. Moomaw: None. D. Woo: None. M.L. Flaherty: None. P. Khatri: None. O. Adeoye:
None. S. Ferioli: None. B.M. Kissela: None.
Abstract Content
Introduction: Initial stroke severity is one of the strongest predictors of eventual stroke
outcome. However, predictors of initial stroke severity have not been well-described within a
population. We hypothesized that poorer patients would have a higher initial stroke severity
upon presentation, even after controlling for other known predictors of stroke outcome.
Methods: Using previously published surveillance methodology, we identified all cases of
hospital-ascertained ischemic stroke (IS) occurring in 2005 within a biracial population of 1.3
million. “Community” socioecomic status (SES), a well-validated aggregate variable for
estimating individual SES, was determined for each patient based on the % below poverty in the
census tract in which the patient resided (determined through geocoding) Community SES for
the 346 census tracts in the region was collapsed into 4 categories: % below poverty of 0%-5%
(richest), 6%-10%, 11%-25%, and >25% (poorest). Patients who lived in institutions were
excluded. Severity of stroke was estimated with a retrospective NIH Stroke Scale Score. Linear
regression was used to model the effect of SES on stroke severity. Models were adjusted for
race, gender, age, pre-stroke disability, and history of medical comorbidities (see table).
Results: There were 2264 ischemic stroke events detected in 2005; 25 were excluded due to
missing data or age<18, and 306 were excluded due to residence within institutions. Among
1933 remaining cases, 21.9% were black, 52.3% were female, and the mean age was 71 years
(range 19-104). The median NIHSSS was 3 (range 0-40). The poorest community SES was
associated with a significantly increased initial NIHSSS by 1.6 points (95% CI 0.5-2.6 p<0.001)
compared with the richest category in the univariate analysis, which remained significant even
after adjustment for demographics and comorbidities (Table).
Conclusion: We found that increasing community poverty was associated with worse stroke
severity at presentation, independent of other known factors associated with stroke outcomes.
In fact, the magnitude of change in the NIHSSS severity for the poorest patients was similar to
the effect seen for a history of coronary artery disease or hypertension. SES may impact stroke
severity via medication compliance, access to care, cultural factors, or may be a proxy measure
for undiagnosed disease states. Understanding how socioeconomic factors contribute to initial
stroke severity is critical to improving outcomes among stroke patients in the U.S.
Abstract MP63 - Embargoed for 3 p.m. PT/6 p.m. ET, Wednesday, Feb. 9, 2011
Control #: 11-ISCN-A-2300-AHA
Session Assignment
MP11 - Nursing Symposium Moderated Poster Tour I (Presentation #: W MP63 ; Speaking
Time: 2/9/2011 5:55:00 PM - 2/9/2011 6:00:00 PM)
Title
Stroke Laterality and Short Term Behavioral Outcomes in Pediatric Stroke
Author Block
Patricia A Plumb, Peter L Stavinoha, Rong Huang, Michael M Dowling, Children's Medical Ctr Dallas,
Dallas, TX
Disclosure Block:
P.A. Plumb: None. P.L. Stavinoha: None. R. Huang: None. M.M. Dowling: None.
Abstract Content
Background and Purpose: Stroke affects approximately 2-13 per 100,000 children under age
18 every year and ranks among the top ten causes of death in children. Long-term motor and
cognitive deficits that interfere with daily life and academic attainment affect 40-60% of
survivors. Parents and caregivers ask what behavior changes to expect and whether or not their
child with be “normal” after stroke. We noted that parents report behavioral changes in their
children after stroke, such as short temper, irritability, easy frustration, or symptoms of
depression. As a first step in assessing the etiology of these changes, we examined the
relationship between hemispheric lateralization and the emotional/behavioral changes reported
by parents.
Methods: Over a two year period, we interviewed parents of 25 children, ages 2-18 who had
suffered an acute arterial ischemic stroke. Utilizing the Pediatric Stroke Outcome Measure
(PSOM), we asked parents about their child’s behavior during a 3 month post stroke interview.
This questionnaire asks direct questions regarding the presence or absence of emotional
changes or depression. Strokes were categorized as involving the left, right, or bilateral
hemispheres by MRI review.
Results: Strokes were lateralized to the left hemisphere in 13, the right hemisphere in 8, with 4
bilateral. Parents noted emotional changes following stroke in 10 (40%), with parents reporting
depression in 8 (32%). There was no significant difference in the hemispheric lateralization for
children with emotional changes (p=0.18, Fischer’s Exact Test). However, left hemisphere
strokes were associated with depression (p=0.046) when excluding those with bilateral stroke.
Interestingly, none of the children with right hemisphere stroke were noted to have symptoms of
depression. This lateralization was more pronounced when the children with any left
hemispheric stroke (including bilateral stroke) were included in the analysis (p=0.026).
Conclusions: Emotional changes and symptoms of depression affect a large proportion of
children after stroke. We found an association between parent reported depression and left
hemispheric stroke. The etiology of this association is unclear but could be due to impaired
language function. This study is limited by its small sample size and limited behavioral
evaluation, but demonstrates the need for assessment of emotional changes and depression in
pediatric stroke survivors and the need to educate parents about the potential emotional
struggles as their children recover. These emotional changes could impede cognitive, language,
and motor therapies and inhibit maximal recovery.
Abstract P42 - Embargoed for 3 p.m. PT/6 p.m. ET, Wednesday, Feb. 9, 2011
Control #: 11-ISC-A-1888-AHA
Session Assignment
P3 - Basic and Translational Neuroscience of Stroke Recovery Posters I (Presentation #: W
P42 ; Speaking Time: 2/9/2011 6:15:00 PM - 2/9/2011 6:45:00 PM)
Title
Translational Stroke Research III: In Vivo Activity and Mechanism of Action of a New Curcumin Hybrid
Neurotrophic-Neuroprotective Compound
Author Block
Paul A. Lapchak, Cedars-Sinai Medical Ctr Dept. Neurology, Los Angeles, CA; Dave Schubert, Pamela
Maher, SALK Inst Neurobiology, La Jolla, CA
Disclosure Block:
P.A. Lapchak: None. D. Schubert: None. P. Maher: None.
Abstract Content
We have shown that the curcumin analog CNB-001 has neuroprotective and neurotrophic properties in vitro and
has a large safety margin using comparison studies done using HT22 and H4IIE cells. For in vivo characterization
of CNB-001, we used the rabbit small clot embolic stroke model (RSCEM), which is a useful translational stroke
model and predictor of clinical efficacy (Lapchak, TSR 1(2), 96-107, 2010). We tested the hypothesis that CNB001 may be neuroprotective following cerebral ischemia using clinical rating scores as the endpoint. CNB-001 or
DMSO were administered SC following the injection of small blood clots into the brain vasculature. Behavior was
measured 24 hours following embolization in order to calculate the effective stroke dose (P50) that produces
neurological deficits in 50% of the rabbits. A treatment is considered beneficial if it significantly increases the P50
compared to control. The initial study used a 5 min post-embolization treatment time, to ensure that the dose and
route of administration of the drug can produce a neuroprotective or positive signal. There was a significant
improvement in behavior and the P50 value for the CNB-001-treated group was increased by 157% compared with
control. At this dose there was no overt behavioral response or toxicity within the time frame of the study. In
addition, when tested 1 hour post-embolization, CNB-001 significantly (P<0.05) reduced stroke-induced behavioral
deficits and increased the P50 value by 233% compared with the control group.
To obtain insight into how CNB-001 functions in vivo, we examined a variety of signaling pathways previously
implicated in stroke. Brain extracts were taken at 6 h post-embolism from control, stroked and CNB-001 embolized
rabbits. Since the Ras-ERK cascade has been implicated in nerve cell survival in ischemia, we initially examined
this pathway. A significant decrease in ERK phosphorylation in the stroked animals relative to control animals was
observed, and this decrease was largely prevented in the presence of CNB-001 treatment. PI3K-Akt signaling,
which is also implicated in neuronal cell survival following stroke was decreased in the stroked animals but was
maintained in the presence of CNB-001. CNB-001 also has BDNF-like activity, and BDNF causes the activation of
the ERK and Akt pathways. Since the expression of CaMKII is also increased by BDNF, we assayed CaMKII
expression in CNB-001-treated animals. In this study, we found that CNB-001 increased CaMKII expression.
Finally, the ER-associated chaperone ORP150 has been linked to the PI3K/Akt and BDNF signaling pathways.
ORP150 expression was decreased in the stroked rabbit brains and this decrease was again prevented by CNB-001.
In conclusion, CNB-001 promotes behavioral improvement following embolic strokes in rabbits, an effect that may
be associated with the normalization of Ras-ERK and PI3K-Akt signaling pathways.
Abstract P40 - Embargoed for 3 p.m. PT/6 p.m. ET, Wednesday, Feb. 9, 2011
Control #: 11-ISC-A-1864-AHA
Session Assignment
P3 - Basic and Translational Neuroscience of Stroke Recovery Posters I (Presentation #: W
P40 ; Speaking Time: 2/9/2011 6:15:00 PM - 2/9/2011 6:45:00 PM)
Title
Translational Stroke Research I: Synthesis and In Vitro Optimization of a New Curcumin Hybrid
Neurotrophic-Neuroprotective Compound
Author Block
Paul A. Lapchak, Cedars-Sinai Medical Ctr Dept. Neurology, Los Angeles, CA; Pamela Maher, Dave
Schubert, SALK Inst Neurobiology, La Jolla, CA
Disclosure Block:
P.A. Lapchak: None. P. Maher: None. D. Schubert: None.
Abstract Content
The plant polyphenolic curcumin is a multifunctional molecule with upwards of 10 separate activities. However,
there are some properties of curcumin that could be improved, including bioavailability and its lack of neurotrophic
activity and the ability to inhibit excitotoxicity. To circumvent some of these problems, we synthesized a library of
hybrid molecules between cyclohexyl bisphenol A (CBA), a molecule with neurotrophic activity and curcumin. A
simple pyrazole derivative, called CNB-001, that eliminates the labile dicarbonyl group of curcumin and stabilizes
the molecule, was identified and further characterized as follows.
For the in vitro studies, we used (1) an in vitro ischemia assay with HT22 cells treated with 20 µM IAA iodoacetic
acid (IAA), an irreversible inhibitor of glyceraldehyde 3-phosphate dehydrogenase (G3PDH), (2) an oxidative
stress (Oxytosis) assay with HT22 cells treated with the excitotoxic amino acid glutamate (5 mM), which induces
cell death mediated by the depletion of intracellular glutathione and (3) a trophic factor withdrawal (TFW) assay
with primary cortical neurons from 18-day-old rat embryos cultured at a low cell density in DMEM/F12 with N2
supplement. For the IAA and oxytosis assays, cell survival was measured using an MTT colorimetric assay. For the
TFW assay, viability was determined after 2 days using a fluorescent live-dead assay (and the data presented as the
percentage of input cells surviving.
First, there was a dose-dependent effect of CNB-001 on the survival of HT22 cells using both the in vitro ischemia
(EC50 0.3 µM) and oxidative stress (EC50 0.7 µM) assays. CNB-001 promoted the survival of freshly plated, lowdensity cultured rat cortical neurons in a dose-dependent manner with an EC50 value of 0.7 µM. Thus, CNB-001 has
neurotrophic activity in addition to neuroprotective activity. Second, we used the IAA assay to elucidate
mechanisms underlying the neuroprotective effects of CNB-001. After IAA treatment, there were decreases in both
ERK and Akt phosphorylation that were prevented by treatment with CNB-001. We also found that KN62, a
CaMKII inhibitor, reduce CNB-001 neuroprotection and blocked the effects of CNB-001 on both ERK and Akt
phosphorylation suggesting that CaMKII regulates the ERK and Akt pathways and is a more proximal target of
CNB-001. Since CNB-001 rescues HT-22 cells from ischemia, and both ERK and Akt activation and CaMKII
expression are downstream pathways activated by neurotrophic molecules such as BDNF, we determined if BDNF
could rescue HT-22 cells expressing the BDNF receptor, TrkA using the in vitro ischemia model. We found that
BDNF rescues cells transfected with wild type TrkA (TK4), but not cells containing a mutated nonfunctional
receptor (TK1) and this rescue is blocked by KN62. The rescue is not, however, as complete as with CNB-001,
suggesting that CNB-001 also functions by other mechanisms that remain to be defined.
Abstract P41 - Embargoed for 3 p.m. PT/6 p.m. ET, Wednesday, Feb. 9, 2011
Control #: 11-ISC-A-1887-AHA
Session Assignment
P3 - Basic and Translational Neuroscience of Stroke Recovery Posters I (Presentation #: W
P41 ; Speaking Time: 2/9/2011 6:15:00 PM - 2/9/2011 6:45:00 PM)
Title
Translational Stroke Research II: In Vitro Cellular Toxicity Screening of an Optimized Curcumin Hybrid
Neurotrophic-Neuroprotective Compound
Author Block
Paul A. Lapchak, Cedars-Sinai Medical Ctr Dept. Neurology, Los Angeles, CA
Disclosure Block:
P.A. Lapchak: None.
Abstract Content
In the present study, we used a comprehensive cellular toxicity (CeeTox) analysis panel to
determine the toxicity profile for CNB-001 [4-((1E)-2-(5-(4-hydroxy-3-methoxystyryl-)-1-phenyl1H-pyrazoyl-3-yl)vinyl)-2-methoxy-phenol)], which is a hybrid molecule created by combining
cyclohexyl bisphenol A, a molecule with neurotrophic activity and curcumin, a spice with
neuroprotective activity. CNB-001 is a lead development compound, since we have recently
shown that CNB-001 has significant preclinical efficacy in vitro in 3 separate assays. The
industry standard CeeTox analysis assay allows investigators to determine the predicted Ctox
value (an estimated concentration where toxicity would be expected to occur in a rat 14-day invivo repeat dose study) for a drug. For this, rat hepatoma derived H4IIE cells were used as the
test system.
The CeeTox assay measures of cellular toxicity including mitochondrial function, apoptosis,
oxidative stress, protein binding, solubility and microsomal metabolic stability. CNB-001 was
soluble up to 100 µM using the CeeTox assay system. The drug was not acutely toxic and no
effect on membrane integrity or subcellular markers of acute toxicity, with the exception of a
decrease in cell proliferation observed with a concentration that produced a half maximal
response (TC50) value of 88 µM, suggesting a cytostatic effect. CNB-001 did not affect
measures of oxidative stress or apoptosis (i.e. Caspase 3 activity).
We found that CNB-001 resulted in an inhibition of Ethoxyresorufin-0-deethylase activity,
indicating that the drug may affect cytochrome P4501A activity and that CNB-001 was
metabolically unstable using a rat microsome preparation assay system. CNB-001 was
metabolized via phase 1 metabolism, since only 42% of parent remained after a 30 minute
incubation with rat microsomes. The only "toxicity" of CNB-001 was a cytostatic effect effect was
only observed at 183-643 fold the EC50 for neuroprotective/neurotrophic activity using HT22 cell
and primary cortical neuron in vitro assays. Based upon a proprietary CeeTox algorithm, the
Ctox Ranking (µM) for CNB-001 was estimated to be 42 µM which places it in the CeeTox Inc.
category of moderate to low probability of in vivo effects. However, it is important to realize that
the Ctox ranking, a treatment regimen that may not be necessary for stroke patients. Taken
together, the results indicate that there is a significant therapeutic safety window for CNB-001
and that it should be further developed as a novel neuroprotective agent to treat stroke.