1. Development of smart contrast agents for MRI imaging of Alzheimer’s Disease
Brian Austen, Yeser Mohammed and Elliott Cheng
BMS, St George’s University, Cranmer Terrace, LONDON SW17 ORE
Imaging agents were synthesised by peptide synthesis,
incorporating a protected DOTA residue at the final step for binding to gadolinium.
After TFA deprotection and HPLC purification, Gd in 3 fold excess was incubated
with the reagent, and the GD complex purified by HPLC. Reagents were characterised by
maldi-mass spec R3 R2 R1
R1 +Gd
R2 + Gd
R3 + Gd
Many (35%) MRI scans on patients are performed with the use of contrast agents
which enhance selective tissues by increasing relaxation of adjacent water protons. Gd
is the most common contrast agent, chosen as it contains 7 unpaired electrons which increase
T1 longitudinal relaxation (protons realigning with the external field) and reduce T2
transverse relaxation (time for protons to exchange energy with other nuclei). Free Gd is
toxic unless complexed with a stable ligand.
The binding of reagents to Ab fibrils and oligomers was measured by ELISA,
using the biotin moities incorporated into the reagents. R2 bound more strongly to fibrils
than R1indicating that the hexa-DArg sequence applifies binding as well as acting
as a cell penetration sequence
That aggregation and deposition of residue peptides, b-amyloid,
in the brain of Alzheimer’s patients is associated with disease
The aim is to develop imaging agents that contain gadolinium, bind b-amyloid plaques,
and cross the blood-brain barrier.
Imaging plaques in brain by MRI will provide an early diagnosis of Alzheimer’s,
and permit monitoring of therapy designed to rid the brain of plaques.
Amyloid-binding peptides were derived from retroinverted forms of the aggregation domain in bAmyloid
NH VKMDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIATVIVITLVMLKK COOH
NL GQ VI X
Swedish double
mutation 670/671
Flemish
mutation
692
Florida
717
London
mutations
716
Australian
723
b-Secretase a-Secretase g-Secretase Ab
Extracellular domain
Cytoplasmic tail
APP K
Italian
Aggregation domain
Retroinversion FFVLK N C --
Membrane
FC uM R1-R4 binding to amyloid 1-40 fibrils
On a biacore, R2 and R3 bound more strongly to Ab fibrils
than R1 or the control peptide R4. On and off rates were fast
40 42 G
Dutch
mutation
692 F
DOTA- Gly-DArg-DPhe-DPhe-DVal-DLeu-DLys-Gly-DArg-Gly-Pentadiamine Gd
b-Amyloid-binding
Transport
Contrast R1
Synthesised MRI contrast agents for imaging Alzheimer’s pathology
DOTA –Gly-DArg-DPhe-DPhe-DVal-DLeu-DLys-DArg-rlsysrrrf-NH2
SynB
b-Amyloid binding R3 R2
DOTA –Gly-DArg-DPhe-DPhe-DVal-DLeu-DLys-Gly-hexaDArg
a-Synuclein binding R4
DOTA-Gly DArg-Vaa-Waa-Xaa-Yaa-Zaa-Gly-hexaDArg C D
AD section plus R2
Young control plus R2
AD section plus antibody
In AD pm sections, R2 stained plaques (A) and vascular amyloid (B),
in a similar fashion to Ab antibodies (D). Sections froma control young patient did not bind R2 B A D C
SW-APP 293 cells transfected with the amyloid precursor protein,
treated with proteasome inhibitor, lactalysin,to accumulate Ab,
took up and retained R2. Visulalised via incorporated biotin in R2
T2 image; plaques in CA3 of mouse hippocampus, not seen at 1hr
MRI of APP/PS-1 double transgenic mouse 1 hr after in injection
of R1 (0.5mg); thanks to Dr Mike Modo, Kings London