molecular imaging cxcr4 wester
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Nuklearmedizinische Klinik und Poliklinik, Klinikum rechts der Isar and Institut fr Radiochemie TUM Campus Garching
H.J. Wester
Molecular Imaging of CXCR4 Receptors
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Hypoxic Areas of Tumors:
CXCL12 expression by fibroblasts CXCR4 expression on tumor cells
tumor cell motility invasiveness
CXCL12: promotes tumor cell growth by
stimulating via CXCR4. induces recruitment of progenitors,
which allow for tumor angiogenesis
CXCR4: activation of CXCR4 leads to targeted
metastasis to the marrow or othersites of high CXCL12 expression.("hijacking" of circulating tumor cells)
Importance of CXCR4 and CXCL12
-
AIM:
to develop suitable ligands for CXCR4 for diagnosis, staging, therapy
monitoring
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TTopochemical exploration of potent compounds usingretro-enantiomer libraries of cyclic pentapeptides.
Tamamura H. Org Biomol Chem 2004, 2: 1255-57
T140 FC131
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Competitive binding curves of CPCR4 with 125I-CPCR4 and 125I-SDF-1 to Jurkatand CMS5/CXCR4 cells:
-11 -10 -9 -8 -7 -60
25
50
75
100125I-CPCR4125I-SDF-1
CMS-5/CXCR4
Jurkat
log [CPCR4]
b
o
u
n
d
t
r
a
c
e
r
[
%
]
Comparable IC50 values were determined for CPCR4 at both cell lines with both radioligands
125I-CPCR4 shows very low non-specific binding
Radioligand-Binding Studies (II)
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High tracer accumulation in the CXCR4-expressing tumor Low background accumulation Higher tracer accumulation only in the metabolic and excreting organs
(liver, intestine and kidneys)
0
5
10
15
20
25
30
35
Blood
Seru
m
Lung
Liver
Panc
reas
Splee
n
Intes
tine
Adre
nals
Kidne
ysMu
scle
Bone
CMS5
-Tu
mor
CMS5
/CXCR
4 Tum
or
%
I
D
/
g
30min 60min 120min
Biodistribution Analysis of 125I-CPCR4 in Mice
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Control(GFP)
CXCR4/Luc
Control(GFP)
CXCR4/Luc
Control(GFP)
CXCR4/Luc
(mirror image)Photo MRI -PET
Bio-luminescence
GFP-Fluorescence
PET and Bioluminescence Imaging:In vivo investigations of CXCR4 receptor status on tumors
C
P
C
R
4
i
n
v
i
v
o
Analysis of CXCR4/Luc expression and GFP control tumors:
Advanced Multimodal CXCR4-Tumor Imaging
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ex-vivo -Autoradiography: lung of a mouse 1h p.i. of nca. CPCR4 with a human SCLC (OH-1), primary tumor on the shoulder
Imaging CXCR4 Receptor Expression During Metastases
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thickness: 1mm
Imaging of CXCR4 expression in lung metastases-Autoradiography of lungs 1 h p.i. of 124I-CPCR4
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MicroImager analysis of the lung of mice with s.c. growing metastasized SCLC tumor at 1h p.i.. Quantitative activity profile along an arbitrary selected direction
Quantification of CPCR4-Binding ex-vivo
-
CXCR4 expression in lung metastases:
blockade
-Autoradiography of lungs 1 h p.i. of 124I-CPCR4 + xs CPCR4
124I-CPCR4 + xs CPCR4R = 6/1
-
Biodistribution 125I-CPCR4 2h p.i.
Biodistribution of 125I-CPCR4 in mice with OH-1 tumors 2 h p.i.
Blood
Hear
tLu
ngs
Musc
lePa
ncre
asSp
leen
Stom
ach
Intes
tine
Kidne
yLiv
erTu
mor
0
25
50
125I-CPCR4 2 h p.i.
u
p
t
a
k
e
(
%
I
D
/
g
)
-
Acylated Orn-analogs potential ligands for 18F-labelling-
Derivatized cyclic pentapeptides:
First candidate for18F-Radiolabeling
Determination of logP value: 1.07
High lipophilicity of [18F]-FB-OD25 will lead to high uptake in liver and intestineUnfavourable biodistribution restricts use for future tumor imaging;further modification necessary.
-
Synthesis of cyclic pentapeptideswith spacer at Orn
For 18F-labeling via aminoreactive prosthetic groups and simultaneous increase of hydrophilicity (e.g. PNA-linker):
NH2O
H
OOO
NH2
H
Ahx
PNA
IC50: ~ 80 nM
IC50: ~ 60 nM
O HN
ONH2
O HN
ONH
ONH2
OO
O OHN
O
O NH2
OO
O OO
O NH2OHN
O
O NH
-
Improved CPCR4 probe: 68Ga-DOTA-CPCR4-2.1
Biodistribution in mice with OH-1 tumors 2 h
Blood
Hear
tLu
ngs
Musc
lePa
ncre
asSp
leen
Stom
ach
Intes
tine
Kidne
yLiv
erTu
mor
0
5
10
u
p
t
a
k
e
(
%
I
D
/
g
)
ON
NON
O
N
O
O
N H
HH
H
HO
HN
HN
NH2
NH
ONH
O
NH
O N
N
N
N
O
O-
O
O-
O
Ga3+O-O
N
NON
O
N
O
O
N H
HH
H
HO
HN
HN
NH2
NH
ONH
O
NH
O N
N
N
N
O
O-
O
O-
O
Ga3+O-
-
Animal 1:
OH-1 SCLC,Injected withGa-68-CPCRx(scale max: 177)
Animal 2:
OH-1 SCLC,Injected withGa-68-CPCRx and xs cold peptide(scale: max 126)
max. intensityin kidney-slices
max. intensityin tumor-slices
max. intensityin kidney/bladder-slices
max. intensityin kidney-slices
max. intensityin tumor/bladder-slices
max. intensityin kidney/bladder-slices
68Ga-CPCR4-2.1: imaging in OH-1 tumor bearing mice
-
compd nIC50 [nM]a
a b c29 6 1512.3 26.522.6 30.36.530 5 121.717 40.921.62 14.2331 2 41.715.54 30.43.68 33.663.732 1 88.318.5 27.07.2 16.73.333 - 150.3 44.14.2 5.00.734 - 334.7 220162 11.54.435 - 903.05439.75 456.8 -36 - >1000 12325 89.7118.337 - 807.5477.4 - 288.9
ON
NON
O
N
O
O
N H
HH
H
HO
HN
HN
NH2
NH
O HN
ONH
O
ONH
ONH
O
O
HNO HN
O
O
R
R:
DOTA
DOTA
DOTA
29-32
HN DOTAn O
N
N
N
N
O
OH
O
OH
O
HO
O
N
N
N
N
O
O
O
O
O
OM
DOTA:
free: a
M: In3+ b Ga3+ c
33
34
35
36
DOTA 37
HN DOTA
Ga-68-labeled Ligands
-
SK
B
R
3
D
U
1
4
5
M
C
F
7
M
D
A
M
B
2
3
1
O
H
1
A
4
3
1
H
T
2
9
M
D
A
M
B
4
3
5
Selection of tumor models to prepare first clinical studies
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3D Surface Plot of Immunostained Section for CXCR4rabbit UMB-2 monoclonal antibody has been analyzed using NIH Image Software.
Distribution of the UMB-2 antibody around the membrane of positive OH-1 tumour cells. The intensity of the signal has been then plotted to create a 3D Surface Plot Image of the section
400x
-
Primary OH-1 Tumor Stained with UMB-2 Monoclonal Antibody
-
ON
NON
O
N
O
O
N H
HH
H
HONH2
HNNH
NH2
4
O HN
NON
O
N
O
O
N H
HH
H
HONH
HNNH
NH2
FC131
NH2HN
ON
NON
O
N
O
O
N H
HH
H
HO
HN
HN
NH2
X NH
O
NH
O
OOm m
nX: NH; active aX: O; inactive i
O
O
NH2
O
NH
NH2
R/LPeptide P: R:
P1 P2
Linker L:
Gua Ac
Gma
Dimeric DOTA labeled peptides
Demmer O. et al. (submitted)
-
Peptide LP1 P2 R m n IC50 [nM]a - - - - 9 1a a - 0 2 6 4a a - 0 3 4 2a a - 0 4 3 1a a - 0 6 3 1a a - 0 8 2 1a a - 0 11 4 7a a - 0 14 14.4
ON
N
O
N
O
NO
O NH
H
HH
OH
HN
NH
NH2HN
ON
N
O
N
O
NO
ONH
H
HH
OH
NH
HN
H2N NH
O
O
Dimeric DOTA labeled peptides
-
ON
N
O
N
O
NO
O NH
H
HH
OH
HN
O HN O
O
O N
N
N
NO
O
O
HO
NH
HN NH2NH
O
X
Ga
ON
N
O
N
O
NO
ONH
H
HH
OH
HN
H2N NH
X NH
n
O
comp X n M IC50 [nM]21 - 0 - 92 6
22 - 0 In3+ 29 1
23 - 1 - 25 13
24 - 1 In3+ 78 9
25 Gly 0 - 38 2
26 Gly 0 In3+ 14 2
27 Gly 1 - 12 2
28 Gly 1 In3+ 22 5
Dimeric DOTA labeled peptides
Demmer O. et al. (submitted)
-
NHO
NH
O
O
HN
HN
HN
O
O
HN
H2N NH
HN
OH
HN O
HN
O
O
NH
NH
HN
O
O
NH
NH2HN
NH
HO
9.5nM
Dimeric DOTA Complexes
NN
NN
O
O
O-
-OO
HN
O
NH
O
O
68Ga
Brohl F. et al. (in progress)
-
A Triazacyclononane-Based Bifunctional Phosphinate Ligand for thePreparation of Multimeric 68Ga Tracers for Positron Emission Tomography
Notni J et al. 2010. Chem. Eur. J. 2010
-
A Triazacyclononane-Based Bifunctional Phosphinate Ligand for thePreparation of Multimeric 68Ga Tracers for Positron Emission Tomography
Notni J et al. 2010. Chem. Eur. J. 2010
-
Immunoimaging of CXCR4 expression in brain tumor xenografts using SPECT/CT
(Nimmagadda et al, J Nucl Med, 2009)
SPECT/CT of CXCR4 expression levels in experimental brain tumors using 125I-labeled anti-CXCR4 mAbs, hCXCR4 (12G5) and the control IgG2A MAb
SPECT/CT of CXCR4 in U87 xenografts and MIP-image of 125I-12G5injected mouse at 48 h after injection.
Biodistribution in U87 tumor-bearing SCID mice. 74 kBq of 125I-12G5 or 125I-IgG2A at 24, 48, and 72 h
The feasibility of the RID of CXCR4 expression imaging of the tumor microenvironment with a MAb is possible.
37 MBq125I-12G5
37 MBq125I-IgG2A
-
U87-stb-CXCR4 mice(90 min p.i.)
Nimmagadda S, Cancer Res (2010)
-
MDA-MB-231 (solid arrow) and DU4475 mice (open arrow)
-
Nishizawa et al., Int.J Cancer 2010
Flourescent Imaging of bladder cancer
(Ac-Arg-Arg-Nal-Cys-Tyr-Cit-Arg-D-Lys*-Pro-Tyr-Arg-Cit-Cys-Arg-NH2; D-Lys* indicates the carboxyfluorescein-labeled D-LysIC50: 11nM
-
A CXCR4 antagonist CTCE-9908 inhibits primary tumor growth andmetastasis of breast cancer(Huang et al, Journal of Surgical Research, 2009)
KGVSLSYR
K-NH2
KGVSLSYRCTCE-9980 structure
Inhibition of CXCR4 reduced the primary tumor growth of MDA-MB-231 cells implanted into the inguinal mammary fat pad. (6 weeks)
provide an sensitive method to follow the progression of the primary and metastases over time
In this model, 1105 MDA-231-BSC12 cells were injected into the left cardiac ventricle to produce bone metastases.
(25 mg/kg, injected subcutaneously 5 d/wk
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