Poster Presentations P3 S545

(v/v) fetal bovine serum. Experiments were performed in 6-day-old cul-

tures. Results: The 24-hour exposure of cultured hippocampal cells to

Aß1-42 (15 mM) alone or in combination with either resveratrol (20 mM)

or EGCG (10 mM) reduced Aß1-42-mediated increased expression of the

57 kDa death-inducing form of AIF. Moreover, EGCG completely in-

hibited the activation of the key apoptotic executioner, caspase-3, and re-

duce the number of apoptotic cells, whereas resveratrol was less

effective. Conclusions: Our findings show that these polyphenols do not

share the same mechanism of action, suggesting that a combination of

EGCG and resveratrol might provide additional neuroprotection against

Aß-associated cell death.

P3-317 TAUTOMERIC STRUCTURES OF CURCUMIN

DERIVATIVES IS INVOLVED IN THEIR AMYLOID-

BINDING ACTIVITY IN VITRO AND IN VIVO

Daijiro Yanagisawa1, Nobuaki Shirai2, Tomone Amatsubo1,

Hiroyasu Taguchi1, Koichi Hirao2, Makoto Urushitani1,

Shigehiro Morikawa1, Toshiro Inubushi1, Yoshiko Wada3, Ken-

nosuke Wada3, Akitsugu Yamamoto3, Ikuo Tooyama1, 1Shiga University of

Medical Science, Otsu, Japan; 2Industrial Research Center of Shiga Pre-

fecture, Ritto, Japan; 3Nagahama Institute of Bioscience and Technology,

Nagahama, Japan. Contact e-mail: daijiroy@belle.shiga-med.ac.jp

Background: Curcumin, which can exist in an equilibrium between keto

and enol tautomers, binds to b-amyloid (Ab) fibrils/aggregates. The aim

of this study was to assess the relationship between the tautomeric struc-

tures of curcumin derivatives and their amyloid-binding activities. We fur-

ther investigated the brain-blood barrier permeability and amyloid-binding

activity of curcumin derivatives in vivo. Methods: We synthesized curcu-

min derivatives, F-H-Cur, and F-Mecar-Cur that were substituted at the C-4

position. We further synthesized keto form analogue, F-diMe-Cur. The

binding activities to Ab aggregates of the curcumin derivatives measured

in ThT competition study. In order to determine the (keto or enol) forms

of the curcumin derivatives upon binding to the Ab aggregates, NMR spec-

tra and UV-visible absorption spectra were measured. To test whether cur-

cumin derivatives cross the blood-brain barrier and bind to senile plaques

in vivo, APP transgenic mice were intravenously injected with F-Mecar-

Cur. Results: Curcumin derivatives with keto-enol tautomerism showed

high levels of binding to Ab aggregates but not to Ab monomers. The

binding activity of the keto form analogue of curcumin to Ab aggregates

was found to be much weaker than that of curcumin derivatives with

keto-enol tautomerism. The color of a curcumin derivative with keto-

enol tautomerism, which was substituted at the C-4 position, changed

from yellow to orange within 30 minutes of being combined with Ab ag-

gregates in physiological buffer. This resulted from a remarkable increase

in the enol form with extended conjugation of double bonds upon binding.

When APP transgenic mice were intravenously injected with F-Mecar-Cur,

fluorescence that derived from the enol form of F-Mecar-Cur was detected

in senile plaques in the brain. Conclusions: Curcumin derivatives exist

predominantly in the enol form during binding to Ab aggregates, and

that the enolization of curcumin derivatives is crucial for binding to Ab ag-

gregates. Furthermore, F-Mecar-Cur can cross the blood-brain barrier, and

binds to senile plaques in vivo. These findings suggest that the keto-enol

tautomerism of curcumin derivatives may be a novel target for the design

of amyloid-binding agents that can be used both for therapy and for amy-

loid detection in Alzheimer’s disease.

P3-318 THE SAFETY, TOLERABILITY,

PHARMACOKINETICS AND

PHARMACODYNAMICS OF SINGLE AND

MULTIPLE DOSES OF BMS-708163 IN YOUNG AND

ELDERLY JAPANESE SUBJECTS

George Ono1, Kaoru Kondo1, Naomi Yamahira1, Hiroyuki Yoshitsugu1,

Yasuhiko Imai1, Masaki Hiraoka1, Randy Dockens2, Gary Tong2,

Jun-Sheng Wang2, Noboru Nakamichi3, 1Bristol-Myers Squibb, Tokyo,

Japan; 2Bristol-Myers Squibb, Princeton, NJ, USA; 3Yokohama Minoru

Clinic, Tokyo, Japan. Contact e-mail: george.ono@bms.com

Background: BMS-708163, in Phase II development for Alzheimer’s

Disease, selectively inhibits Ab synthesis relative to Notch, and is a potent

and orally active g-secretase inhibitor. We evaluated the safety, tolerabil-

ity, pharmacokinetics, and pharmacodynamics of single ascending doses

(SAD) and multiple ascending doses (MAD) of BMS-708163 in healthy

young and elderly Japanese subjects in 2 randomized, double-blind,

placebo-controlled studies. Methods: SAD: Healthy young Japanese

men (n ¼ 24) were randomized to single doses of BMS-708163 50,

100, or 200 mg, or placebo (sequential panels); MAD: Young Japanese

men (n ¼ 16) and elderly Japanese healthy volunteers (n ¼ 16) were ran-

domized to BMS-708163 50 or 100 mg or placebo for 14 days. Results:

SAD: There were no gastrointestinal tract-related adverse events (AEs) or

clinically significant abnormalities in vital signs, electrocardiograms, clin-

ical laboratory tests, fecal occult blood tests, stool frequency and consis-

tency evaluations, urinary and salivary cortisol, thyroid function tests, or

lymphocyte immunophenotyping. BMS-708163 was quickly absorbed

(mean Tmax 1.0-1.3h); plasma profiles were biphasic; mean terminal T1/

2 was ?30h; plasma AUC was dose-proportional in the range 50-200

mg. BMS-708163 decreased plasma Ab1-40 dose-dependently after dosing.

MAD: The most common AE was positive fecal occult blood test (n ¼ 7,

all mild). BMS-708163 was quickly absorbed (median Tmax 1.0-1.5h);

the exposure at 100 mg was 2- to 3-fold higher than that at 50 mg;

mean terminal T1/2 was ?32h (young) and ?44-55h (elderly). BMS-

708163 reduced plasma Ab1-40 in a dose-dependent fashion. Conclusions:

BMS-708163 was safe and well tolerated at single doses of up to 200 mg

(young Japanese) and multiple doses up to 100 mg (young and elderly

Japanese), with no evidence of Notch-related dose-limiting toxicities. In

both studies, BMS-708163 demonstrated rapid absorption and dose-pro-

portional exposure. T1/2 was longer in elderly patients vs. younger patients

in the multiple-dose study. BMS-708163 produced dose-dependent

marked decreases in plasma Ab1-40 levels following single- and multi-

ple-dose administration.

P3-319 BRAIN INFILTRATION OF PERIPHERAL

MONOCYTES VIA AAV-9 DRIVEN CHEMOKINE

OVEREXPRESSION

Maj-Linda B. Selenica, Kevin Nash, Daniel C. Lee, Dave Morgan,

Marcia N. Gordon, Byrd Alzheimers Institute, University of South Florida,

Tampa, FL, USA. Contact e-mail: mselenic@health.usf.edu

Background: The role of glial cell activation in Alzheimer’s disease is mul-

tifaceted; evidence exists demonstrating both detrimental neurodegenerative

activity as well as neuroprotective actions. Activated microglia are increas-

ingly being phenotyped into classical (M1) or alternative (M2) activation

states. Moreover, the role of peripherally derived macrophages infiltrating

into the brain in response to glial activating stimuli is an area of considerable

interest. Chemokines and their receptors can regulate macrophage traffick-

ing during development, cell surveillance and inflammation. Methods: In

this study we investigate the hypothesis that delivery and over-expression

of chemokines via a recombinant adeno-associated virus serotype 9

(AAV-9) injections into brain will increase the infiltration of bone mar-

row-derived monocytes. Viral constructs driving over-expression of the che-

mokines CCL2, CCL5 or CX3CL1 under control of the chicken beta-actin

promoter were constructed with red flurorescent protein tags. Each virus was

injected bilaterally in the mouse hippocampus via convection enhanced de-

livery and expressed for 4 weeks.Histology and flow cytometry will be used

to measure infiltration and macrophage characterization. Results: Initial re-

sults indicate that chemokine over-expression in the hippocampus increased

immunostaining for the microglial markers Iba1 and lead to activation of

brain resident microglia as shown by CD45 staining of the brain tissue, es-

pecially around the region of injection. Interestingly, we observe that over-

expression of CCL2 in the brain can drive expression of the alternative

Poster Presentations P3S546

activation state markers, YM1 and arginase-1. We were unable to make the

same observation in mice over-expressing CCL5, suggesting that these che-

mokines might play different roles in activating endogenous microglia and/

or attracting exogenous cells of monocytic origin. Next, we will investigate

the efficacy of the different chemokines over-expressed by AAV-9 in in-

creasing the infiltration of GFP labeleld peripheral bone marrow derived

monocytes (selected by CD11b+ expression). Conclusions: Overall, these

experiments will attempt to identify conditions enhancing infiltration of cir-

culating monocytes to evaluate their role in modifying the pathogenesis in

AD. Furthermore, the phenotypic characterization of these cells will assess

the multiple microglial markers associated with the M1 or M2 activation

state.

P3-320 PHARMACOKINETIC AND PHARMACODYNAMIC

INVESTIGATION OF ELND006, A NOVEL

APP-SELECTIVE GAMMA-SECRETASE

INHIBITOR, ON AMYLOID-b CONCENTRATIONS

IN THE BRAIN, CSF AND PLASMA OF MULTIPLE

NONCLINICAL SPECIES FOLLOWING ORAL

ADMINISTRATION

Elizabeth Brigham, Kevin Quinn, Grace Kwong, Chris Willits,

Erich Goldbach, Ruth Motter, Michael Lee, Kang Hu, William Wallace,

Dora Kholodenko, Pearl Tang-Tanaka, Huifang Ni, Susanna Hemphill,

Xiao-hua Chen, Tovah Eichenbaum, Lany Ruslim, Lan Nguyen,

Pamela Santiago, Anna Liao, Michael Bova, Gary Probst, Michael Dappen,

Jacek Jagodzinski, Guriqbal Basi, Daniel Ness, Elan Pharmaceuticals, South

San Francisco, CA, USA. Contact e-mail: beth.brigham@elan.com

Background: Sequential processing of the beta-amyloid precursor protein

(APP) by beta-secretase and gamma-secretase produces Ab, a peptide that

is central to the pathogenesis of Alzheimer’s disease. We have identified

novel gamma-secretase inhibitors which demonstrate in vitro substrate se-

lectivity towards inhibiting APP cleavage compared to Notch. ELND006 is

one of these potent inhibitors and is currently being evaluated in Phase 1

human clinical trials. We present here the in vivo characterization of

ELND006 after oral administration in multiple preclinical models.

Methods: The potency of ELND006 for inhibiting Ab production result-

ing from gamma-secretase-mediated cleavage of APP and the selectivity

of ELND006 for inhibition of APP cleavage compared to Notch cleavage

was determined in enzymatic and cellular assays. Ab and compound levels

were analyzed in the brain, CSF and/or plasma of FVB wildtype mice,

PDAPP mice, Sprague Dawley rats and Hartley Guinea pigs after single

or repeat dose oral administration of ELND006 or vehicle. Compound

levels were determined by an LC-MS/MS assay. Ab levels (1-x, x-40 or

x-42) were determined by an ELISA assay. Results: ELND006 inhibited

APP and Notch cleavage with an IC50 ¼ 0.34 nM and 5.3 nM respectively.

ELND006 inhibited Ab production and Notch signaling in cells with an

IC50 ¼ 1.1 nM and 81.6 nM respectively. After oral administration of

ELND006 in multiple species, ELND006 penetrated the brain (Kp > 1)

and significantly reduced brain and CSF Ab at doses between 0.3 to 30

mg/kg. In all species tested, plasma ELND006 concentrations needed to re-

duce plasma Ab were higher than those needed to reduce brain Ab. Ele-

vated plasma Ab was observed under some ELND006 treatment

conditions, consistent with results achieved with other gamma-secretase in-

hibitors. Plasma ELND006 concentrations of 8-35 ng/mL were associated

with lowering of Ab in the brain of approximately 25%. Conclusions:

ELND006 is a potent, APP-selective gamma-secretase inhibitor and is

approximately 15- to 70-fold more selective towards inhibiting gamma-

secretase mediated cleavage of APP than Notch cleavage. ELND006

plasma concentrations required to reduce brain Ab were highly consistent

across species, offering an opportunity to translate these nonclinical data

into a clinical setting. Phase 1 clinical trials with ELND006 are in

progress.

P3-321 APP-SELECTIVE GAMMA SECRETASE INHIBITOR

ELND006 EFFECTS ON BRAIN PARENCHYMAL

AND VASCULAR AMYLOID BETA IN THE PDAPP

MOUSE MODEL OF ALZHEIMER’S DISEASE

Sally Schroeter, Elizabeth Brigham, Ruth Motter, Chris Nishioka,

Terry Guido, Karen Khan, Dora Kholodenko, Pearl Tanaka, Ferdie Soriano,

Kevin Quinn, Erich Goldbach, Dora Games, Daniel Ness, Elan Pharma-ceuticals, South San Francisco, CA, USA. Contact e-mail: sally.schroeter@

elan.com

Background: Cleavage of amyloid precursor protein (APP) by beta and

gamma secretases produces amyloid beta (Abeta), a protein with neurotoxic

properties that accumulates in the hallmark plaque pathologies of Alzheim-

er’s disease (AD). An APP-selective gamma secretase inhibitor, ELND006,

was tested to determine efficacy in reducing Abeta-associated pathologies

in aged PDAPP mice. Methods: Twelve month-old mice (limited, but es-

tablished pathology) were treated with 2 mg/kg QD or BID for 13 weeks,

and with 12.5 mg/kg/day QD for 13 and 26 weeks. Dosing paradigms were

previously shown to reduce brain Abeta 25% to >50% for partial or full

days in non-plaque-bearing PDAPP mice. Brain Abeta 1-x and x-42 levels

were determined by ELISA. Leptomeningeal vascular and parenchymal

amyloid burden(compact and diffuse) and neuritic dystrophy were assessed

by Abeta and APP immunohistochemistry. Results: Treatment with 12.5

mg/kg/day for 13 weeks significantly reduced hippocampal amyloid burden

and brain Abeta as measured by ELISA. Hippocampal neuritic burden,

a measure of dystrophic neuronal response to compacted Abeta plaques

and therefore an indirect measure of this plaque subtype, was not signifi-

cantly reduced. However, a significant change in density of neuritic sites

indicated an early effect on dystrophic neurites, consistent with a reduction

in the number of small neuritic sites. At the lower dose administered once

or twice daily for 13 weeks, a downward trend in hippocampal amyloid

burden and neuritic burden was observed, and vascular and cortical amy-

loid burden, and brain Abeta were not significantly reduced. After 26 weeks

of treatment at 12.5 mg/kg/day, the only dose tested at this timepoint, cor-

tical and vascular amyloid burden and hippocampal neuritic burden were

significantly reduced. There was a trend toward a decrease in brain Abeta

as measured by ELISA. Conclusions: ELND006 effectively decreased the

formation of, or prevented Abeta pathologies in the PDAPP mouse as as-

sessed by histopathology and ELISA measures. These results support that

a strategy for inhibiting gamma secretase may be efficacious in the treat-

ment of AD and also of cerebral amyloid angiopathy, which is present in

most AD patients.

P3-322 EFFECTS OF SINGLE AND MULTIPLE DOSE ORAL

ADMINISTRATION OF ELND006, A NOVEL APP-

SELECTIVE GAMMA-SECRETASE INHIBITOR, ON

AMYLOID-b CONCENTRATIONS IN THE BRAIN

AND CSF OF CYNOMOLGUS MONKEYS

Elizabeth Brigham1, Kevin Quinn1, Ruth Motter1, Wherly Hoffman1,

Erich Goldbach1, Dora Kholodenko1, Grace Kwong1, Chris Willits1,

Gary Probst1, Jane Gunther1, Eric Adams2, John-Michael Sauer1,

Gene Kinney1, Daniel Ness1, 1Elan Pharmaceuticals, South San Francisco,CA, USA; 2Northern Biomedical, Muskegon, MI, USA. Contact e-mail: beth.

brigham@elan.com

Background: ELND006 is a potent, APP-selective gamma-secretase inhib-

itor currently being evaluated in Phase 1 human clinical trials. We present

here the evaluation of ELND006 1) repeat-dose effects on beta-amyloid

(Ab) levels in the brain, CSF and plasma of cynomolgus monkeys enrolled

in nonclinical safety studies and 2) single-dose effects on Ab levels in the

CSF and plasma of lumbar cannulated cynomolgus monkeys. Methods:

For repeat-dose toxicology studies, cynomolgus monkeys were administered

ELND006 once daily by oral gavage for 3 months. Brain, CSF and plasma

samples were collected at termination, approximately 24 hours after the