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Presented by Piero A. SALVADORI
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A collaborative research project of
CNR Institute of Clinical Physiology - Pisa
University of Pisa - Department of Pharmaceutical Sciences
CNR Institute of Biomolecular Chemistry - Pozzuoli
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Positron Emission Tomography and Amyotrophic Lateral Sclerosis:
Study of Cannabinoid subtype 2 receptor expression
in ALS experimental model
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• Why a proposal based on Positron Emission
Tomography
• Mining unexploited resources, merging hightech
technologies & skills
• Workplan
• Project update
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Outline:
• Scientific background of the project & aims
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Aim
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Development of PET tracers that selectively bind to the CB2R receptor and
highlight their upregulation in neuroinflammatory conditions.
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In vivo assessment of candidates by using an experimental model of ALS (mSOD1G93A transgenic mice)
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Understanding whether PET in association with CB2-ligands might be considered
for translational study to qualify as imaging biomarker for ALS in humans
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Background
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Evidence exists that ALS involves neuroinflammatory events.
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Cannabinoid type 2 Receptors (CB2R) are expressed in immune cells and tissues
but are weakly present in healthy CNS. However, in neuroinflammatory conditions
CB2R (but not CB1R) are upregulated in the activated microglia (MG).
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Activation of MG seems to precede symptoms onset in experimental models of ALS
and MG has been postulated to be involved in early motor neuron degeneration.
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PET allows the quantitation of the regional distribution of radiolabelled tracers in
vivo, dynamically and under real tracer conditions.
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Scientific background of the project & aims
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PET monitoring
of response to therapy
PET/FDG as biomarker
Courtesy of Peter MacCallum Cancer Institute
Shankar LK, et al., J.Nucl.Med. 2006; 47: 1059-66
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Why a proposal based on Positron Emission Tomography
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CT
MRI
MRS
SPET
PET
BIOIMAGES
anatomia
funzione
biochimica
biochemistry
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CT
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+/-
MRI
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+
+/-
MRS SPET
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+
+/-
Damage progression
Molecular imaging
Highlight an interaction occurring at the molecular level
while retaining its regional information
PET
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anatomy
function
PET/CT
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+
++
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US
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Why a proposal based on Positron Emission Tomography
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activity
PET
DYNAMIC IMAGING
Injection of
labelled drug
In vivo pharmacokinetic with PET/CT
time
….
Set of images (2D) from
Volume of Interest (3D)
t
Frame 1 (t=t0)
Frame 2 (t=t2)
Frame n (t=tn)
t
t
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Why a proposal based on Positron Emission Tomography
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Lead
Molecule
Discovery
500 - 3000
Patients
Product
launch
Clinical trials
Drug discovery
Target
ID
100 - 500
Patients
Preclinical
Research
Phase I
Animal research
investments
Phase II
Phase III
Product
Licencing
Industrial
Production
Marketing
Commercialisation
and
Return on investments
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Drug Development & Research flowchart
20 - 100
Volunteers
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Why a proposal based on Positron Emission Tomography
QuickTime™ e un
decompressore
sono necessari per visualizzare quest'immagine.
CNR
Institute of Biomolecular Chemistry - Pozzuoli
Dr. V Di Marzo
Structure-activity relationships & molecular pharmacology
Characterisation & development of bioactive molecules
Biomasses & biologically active substrates
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Radiopharmaceutical Chemistry & Imaging Biomarker development
Advanced biomedical technologies
In vivo/ex vivo imaging & pharmacology
University of Pisa
Department of Pharmaceutical Sciences
Prof. C. Manera
Computational Chemistry
New scaffolds & Analogue modification
Customised synthesis
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CNR
Institute of Clinical Physiology - Pisa
Dr. P.A. Salvadori
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Mining unexploited resources, merging hightech resources & skills
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Unsatisfactory
Biochemistry
Lead molecules, reference standard
& chemical precursors
QuickTime™ e un
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sono necessari per visualizzare quest'immagine.
Biochemical characterisation of lead compounds
Precursor design optimisation, test labelling,
purification & formulation
In vivo microPET & microCT,
dynamic imaging & biodistribution
Candidate selection by in vivo imaging
& proof-of-principle demonstration
Testing in disease model
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sono necessari per visualizzare quest'immagine.
Tissue hystology
& receptor analysis
Unsatisfactory
Radiochemistry
or Biology
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Workplan
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18F
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R1
A suitable "cold" precursor should
be designed so that the expected
active compound can be prepared
within the constraints of labelling
reaction (short time and high
specific-activity)
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R2
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Project update
General structure based on aromatic fused rings
Introduction of fluorine-18 label in different positions
R1, R2 and other structure modifiers to modulate SAR
compound
Ki (CB1)
Ki (CB2)
CB1/CB2
CB 83
>10000
370
> 27
CB 102
500
51.8
9.6
CB 92
9.6
0.7
14
CB 91
200
0.9
222
VL 22
11.6
0.2
48
AF 4
40
7.9
5
Warning:
Parent "cold" precursor should also be tested for biological
activity as it may compete with the tracer for the binding site
High specific activity: the mass associated to the radioactivity of tracer is
negligible (<< µmol) from the point of view of toxicity and macroscopic effect,
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Radiochemical
Radionuclide
production
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Project update
• Development of labelling chemistry
• Select precursor
• Test labelling (yield, time scale, bulk volume,
impurities & toxicity)
• Synthesis & purification (radiation protection,
radiochemical purity, specific activity)
Radiopharmaceutical
Radiochemistry
Image
acquisition
Microfluidic radiolabelling platform
Different routes for 18F "activation: counter
ions, criptands, phase-transfer agents, ..
Solvents, temperature, time of reaction
vary with substrates and labelling chemistry
<18F>
n OTs CH3CN
TsO
R1
R2
n OTs
18
F
R1
n = 2,3
R2
R3
Cl
N
R3
<18F>
DMSO
18
N
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R= morpholin (CB26), phenyl (C41)
N
N
R
R= morpholin (CB83), phenyl (C102)
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Examples of TAC on
µCT identified organs
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Project update
QuickTime™ and a
PNG decompressor
are needed to see this picture.
QuickTime™ and a
YUV420 codec decompressor
are needed to see this picture.
HIGH-RESOLUTION µCT and µPET
CD56 healthy mouse (wild type)
microPET/microCT image fusion
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Lead molecules, reference standard
& chemical precursors
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Project update
Project clock
START
APRIL 2011
Biochemical characterisation of lead compounds
ACTUALLY
ON-GOING
MID TERM
Precursor design optimisation, test labelling,
purification & formulation
In vivo microPET & microCT,
dynamic imaging & biodistribution
END
APRIL 2012
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sono necessari per visualizzare quest'immagine.
Candidate selection by in vivo imaging
& proof-of-principle demonstration
Testing in disease model
Control group (CD1 mice)
60-70 dd old
(young healthy)
120-140 dd old
(old healthy)
Disease developing group (mSOD1 mice)
Tissue hystology
& receptor analysis
60-70 dd old
(young asymptomatic)
90-100 dd old
(symptoms onset)
120-130 dd old
(serious symptoms)
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We are grateful to AriSLA
for supporting our research
Thank you for your kind attention
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