binding of a beta-amyloid 1-42 peptide to human albumin grifols®

Poster Presentations P3 P417

suggests that Enox may not act directly on brain Ab in this model. Finally,

since plasma Ab levels remained unchanged, we have no evidence that

Enox acts as a peripheral sink agent. Possibly, the mode of action of Enox

is by improving cerebrovascular perfusion.

P3-250 METALLOTHIONEIN-IIA BLOCKS COPPER

MEDIATED Ab1-40 NEUROTOXICITY

Claire Howells, Adrian K. West, Roger S. Chung, University of Tasmania,

Sandy Bay, Australia. Contact e-mail: howellsc@utas.edu.au

Background: The abnormal interaction of copper ions with the amyloid-b pep-

tide (Ab) is associated with aberrant Ab aggregation and reactive oxygen spe-

cies (ROS) production, both of which are toxic to neurons and potentiate the

progression of Alzheimer’s disease (AD). The endogenous astrocyte protein,

metallothionein (MT)-IIA, possesses numerous neuroprotective properties in-

cluding the ability to: sequester metal ions, such as Zn(II) and Cu(I) (Kelly et

al., 1996; Aschner et al., 1997); scavenge free radicals (Sato and Bremner,

1993; Kumari et al., 1998); promote neuroregeneration; and reduce proinflam-

matory responses (Chung et al., 2003). We examined the protective effect of

Zn7MT-IIA against Ab1-40-Cu(II) induced neurotoxicity using an in vitro

model. Methods: Freshly prepared Ab1-40-Cu(II) was applied to cultured rat

cortical neurons, in the presence or absence of different forms of MT. After

24 hours incubation, cellular viability was measured via the alamar Blue� met-

abolic reduction. Neuronal viability was determined as the % survival vs vehi-

cle treated cultures. Results: We determined that Zn7MT-IIA rescued the

neurons from Ab1-40-Cu(II) mediated toxicity (80% vs 30% neuronal via-

bility; p <0.05, ANOVA). The protective effect of MT-IIA was signifi-

cantly higher than Zn7MT-III (80% vs 50% neuronal viability; p < 0.05,

ANOVA). The application of Cu10MT-IIA and carboxyamidomethylated

MT-IIA failed to significantly protect the neurons from Ab1-40-Cu(II) in-

duced neurotoxicity (p>0.05, ANOVA). This indicates that a metal-medi-

ated interaction involving the removal of Cu from Ab is involved in the

protective effect of MT, an idea that has been suggested previously for

Zn7MT-III (Meloni et al., 2008). To investigate this protective mechanism

further, H2O2 was applied directly to cortical neurons (H2O2 is the direct

ROS product of the redox active copper within Ab1-40-Cu(II) (Huang et

al., 1999)). Marked cell death was observed after 30 minute exposure to

100mM H2O2, which could not be blocked by the presence of Zn7MT-

IIA, indicating that MTs protective effect is unlikely to be mediated by di-

rect protection from ROS. Conclusions: These results strongly suggest that

Zn7MT-IIA protects against Ab1-40-Cu(II) induced toxicity by a transmetal-

lation event between Ab1-40-Cu(II) and Zn7MT-IIA. The protection elicited

by Zn7MT-IIA, in addition to its neuroprotective properties, provide great

potential for its therapeutic use in AD.

P3-251 BINDING OF A BETA-AMYLOID 1-42 PEPTIDE TO

HUMAN ALBUMIN GRIFOLS�

Montserrat Costa, Anna Ma Ortiz, Juan Ignacio Jorquera, Instituto Grifols,

S.A., Parets del Valles, Spain. Contact e-mail: montse.costa@grifols.com

Background: Beta-amyloid (Ab) deposits may be the central pathological

process in Alzheimer’s disease. Since there seems to be an equilibrium be-

tween brain and plasma Ab and given that 90% of plasma Ab may be bound

to albumin (1), it might be possible to reduce plasma Ab levels by plasma

exchange with therapeutic albumin, which could in turn translate into brain

amyloid burden reduction (2, 3). The aim of this study is to demonstrate the

capacity of Human Albumin Grifols� to bind a synthetic peptide with the

human amyloid beta peptide 1-42 sequence (sAb1-42). Methods: The capac-

ity of Human Albumin Grifols� to bind human sAb1-42, has been measured

for three different batches, using Surface Plasmon Resonance (SPR). Kinet-

ics and equilibrium features of Human Albumin Grifols� / sAb1-42 peptide

interaction have been analyzed in a Biacore T100 by immobilizing sAb1-42

(Biosource) on a CM5 chip. Known quantities of these samples were ana-

lyzed to obtain kinetic constants for sAb1-42 binding and release. Moreover,

a qualitative comparison of the SPR response has been established by expos-

ing sAb1-42 to plasma samples. Results: Results obtained from three differ-

ent lots of Human Albumin Grifols� show that the association rate constant

(ka) for Human Albumin Grifols�/sAb1-42 is (1.53 6 0.11) x104 M-1s-1,

while the corresponding dissociation rate constant (kd) is (0.026 6 0.004)

s-1. Thus, the equilibrium constant KD (concentration at which 50% of

sAb1-42 is complexed) for Human Albumin Grifols�/sAb1-42 is on average

(1.72 6 0.24) x10-6 M, demonstrating the consistency of this interaction.

Likewise, as expected according to the literature indicating that human am-

yloid beta appears to be able to interact with several plasma proteins, the

SPR response obtained when different plasma samples were analysed was

higher than for isolated albumin. Conclusions: These results indicate that

Human Albumin Grifols� is able to bind to an Ab1-42 peptide with the hu-

man aminoacid sequence. (1): Biere AL et al J Biol Chem, 271: 32916,

1996. (2): Boada M et al Alzheimer’s & Dementia, 4 (Suppl 2): P4-355,

2008. (3): Roca I et al Alzheimer’s & Dementia, 4 (Suppl 2): P4-388,

2008.

P3-252 MICROENCAPSULATED CELLS EXPRESSING

VEGF REDUCE BRAIN AMYLOID LOAD AND

BEHAVIORAL IMPAIRMENT IN ALZHEIMER’S

DISEASE

Carlos Spuch1, Desiree Antequera1, Aitziber Portero2, Gorka Orive3,

Rosa Hernandez3, Jose A. Molina4, Felix Bermejo-Pareja4, Jose L. Pedraz3,

Eva Carro1, 1Research Center. Hospital 12 de Octubre, Madrid, Spain;2Laboratory of Pharmacy and Pharmaceutical Technology, Faculty of

Pharmacy. University of the Basque Country, Vitoria -Gasteiz, Spain;3Neurology service. Hospital 12 de Octubre, Madrid, Spain.

Contact e-mail: carlos.spuch@gmail.com

Background: Cerebrovascular dysfunction contributes to cognitive decline

and neurodegeneration in Alzheimer’s disease (AD). Vascular endothelial

growth factor (VEGF), an angiogenic protein with important neurotrophic

and neuroprotective actions, is under investigation as a therapeutic agent

for the treatment of neurodegenerative disorders. Cell microencapsulation

technology is a promising strategy for controlled, localised and long term

in vivo delivery of therapeutic peptides to the host. In the present approach,

we have evaluated the use of cell microencapsulation as a tool for the contin-

uous release of VEGF for the treatment of AD. Methods: Assuming that

VEGF plays key roles in brain angiogenesis, neuroprotection and cerebromi-

crovascular exchange of substrates and nutrients, we hypothesized that the

local and continuous release of this angiogenic factor could exert beneficial

effects in the pathogenesis of AD. We used double mutant amyloid precursor

protein/presenilin 1 (APP/Ps1) mice, as a transgenic model of AD, character-

ized by disturbed vessel homeostasis. Results: We report that, 3 months after

stereotaxy implantation of VEGF-microcapsules, brain Ab burden, hyper-

phosphorylated tau and cognitive impairment are attenuated in APP/Ps1

mice. Conclusions: The preliminary data presented herein suggests that mi-

croencapsulated cells secreting VEGF might be an alternative to treat disor-

ders characterized by vascular dysfunction such as AD.

P3-253 ACUTE ADMINISTRATION OF CHF5074, A NOVEL

GAMMA-SECRETASE MODULATOR, IMPROVES

CONTEXTUAL MEMORY IN A TRANSGENIC

MOUSE MODEL OF ALZHEIMER’S DISEASE

Bruno P. Imbimbo1, Luciana Giardino2, Alessandro Giuliani2,

Marco Gusciglio2, Luca Lorenzini2, Gino Villetti1, Laura Calza2, 1Chiesi

Farmaceutici, Parma, Italy; 2BioPharmaNet-DIMORFIPA, University of

Bologna, Ozzano Emilia, Italy. Contact e-mail: b.imbimbo@chiesigroup.com

Background: CHF5074 is a new gamma-secretase modulator that after

chronic administration has been shown to inhibit brain plaque deposition

and attenuate spatial memory deficit in two different transgenic mouse

models of Alzheimer’s disease (AD) (JPET 2007; 323: 822-30, Br J Pharma-

col 2009 in press). We evaluated the effects of single subcutaneous doses of

CHF5074 on contextual memory in transgenic mice overexpressing the

Swedish mutation of the human amyloid precursor protein. Methods:

Five-month old heterozygous transgenic female mice and aged-matched

transgene-negative littermates (n ¼ 16-27 per genotype per treatment) were

trained and tested on two consecutive days. Freezing behavior was recorded

by a computerized camera. Transgenic mice received two subcutaneous