Molecular Imaging of CXCR4 Receptors

H.J. Wester
Nuklearmedizinische Klinik und Poliklinik, Klinikum rechts der Isar and Institut fr Radiochemie TUM Campus Garching

Importance of CXCR4 and CXCL12

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 other sites of high CXCL12 expression. ("hijacking" of circulating tumor cells)

AIM: to develop suitable ligands for CXCR4 for diagnosis, staging, therapy monitoring

Topochemical exploration of potent compounds using retro-enantiomer libraries of cyclic pentapeptides.

T140

FC131

Tamamura H. Org Biomol Chem 2004, 2: 1255-57

Radioligand-Binding Studies (II)

Competitive binding curves of CPCR4 with 125I-CPCR4 and 125I-SDF-1 to Jurkat and CMS5/CXCR4 cells:
125

100
125

I-CPCR4 I-SDF-1

CMS-5/CXCR4 bound tracer [%] 75 Jurkat

Comparable IC50 values were determined for CPCR4 at both cell lines with both radioligands
125I-CPCR4

50

shows very low

25

non-specific binding

0 -11 -10 -9 -8 -7 -6 log [CPCR4]

Biodistribution Analysis of 125I-CPCR4 in Mice

35 30min 30 25
%ID/g

60min

120min

20 15 10 5 0
Sp le en us cl e Li ve r es tin ne ys re as Se ru B lo B on Lu m or S5 -T u C M S5 /C al s Pa nc K id Ad r In t M XC R 4 Tu en m or od e m ng e C M

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)

Advanced Multimodal CXCR4-Tumor Imaging

Analysis of CXCR4/Luc expression and GFP control tumors: Photo MRI
(mirror image)

-PET

Bioluminescence

GFPFluorescence

Control (GFP)

CXCR4/ Luc

Control (GFP)

CXCR4/ Luc

Control (GFP)

CXCR4/ Luc

CPCR4 in vivo

PET and Bioluminescence Imaging: In vivo investigations of CXCR4 receptor status on tumors

Imaging CXCR4 Receptor Expression During Metastases

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 of CXCR4 expression in lung metastases

-Autoradiography of lungs 1 h p.i. of 124I-CPCR4

thickness: 1mm

Quantification of CPCR4-Binding ex-vivo

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

CXCR4 expression in lung metastases: blockade

-Autoradiography of lungs 1 h p.i. of 124I-CPCR4 + xs CPCR4

124I-CPCR4

+ xs CPCR4

R = 6/1

Biodistribution 125I-CPCR4 2h p.i.

Biodistribution of 125I-CPCR4 in mice with OH-1 tumors 2 h p.i.
125

I-CPCR4 2 h p.i.

50

uptake (%ID/g)

25

0
rt Lu ng s M us cl e Pa nc re as Sp le en St om ac h In te st in e K id ne y Li ve r Tu m or lo od B H ea

Acylated Orn-analogs potential ligands for 18F-labellingDerivatized cyclic pentapeptides:

First candidate for 18F-Radiolabeling

Determination of logP value: 1.07 High lipophilicity of [18F]-FB-OD25 will lead to high uptake in liver and intestine Unfavourable biodistribution restricts use for future tumor imaging; further modification necessary.

Synthesis of cyclic pentapeptides with spacer at Orn

For 18F-labeling via aminoreactive prosthetic groups and simultaneous increase of hydrophilicity (e.g. PNA-linker):
O O N H NH2

O NH2
O
H

H N O H N O

Ahx

NH2

IC50: ~ 60 nM

O
O O
H

O
O NH2

H N O

NH2 O

O O O

H N O

PNA

N H

NH2

IC50: ~ 80 nM

Improved CPCR4 probe: 68Ga-DOTA-CPCR4-2.1

Biodistribution in mice with OH-1 tumors 2 h
10
O NH HN NH O HO H N O H N O N O HO N H H N NH2 O NH ON Ga3+ N N N O OOO O

uptake (%ID/g)

N O

0
H ea rt Lu ng s M us cl e Pa nc re as Sp le en St om ac h In te st in e K id ne y od ve r Li Tu m B lo or

68Ga-CPCR4-2.1:

imaging in OH-1 tumor bearing mice

max. intensity in kidney/bladder-slices

max. intensity in kidney-slices

max. intensity in tumor/bladder-slices

Animal 1: OH-1 SCLC, Injected with Ga-68-CPCRx (scale max: 177)

max. intensity in kidney-slices

max. intensity in tumor-slices

max. intensity in kidney/bladder-slices

Animal 2: OH-1 SCLC, Injected with Ga-68-CPCRx and xs cold peptide (scale: max 126)

Ga-68-labeled Ligands
R HN

O R: O NH

HN DOTA n 29-32 N O

DOTA: O N N free: a OH OH O

HN DOTA 33

HO O

HO H N

O N O O H N N O H O N H H N NH 2 NH

DOTA NH 34 O

O N N M O DOTA O M: In3+ b Ga 3+ c N N

O O O O

O O HN O O O NH 36 35 HN

HN DOTA

DOTA

37

compd 29 30 31 32 33 34 35 36 37

n 6 5 2 1 -

a 1512.3 121.717 41.715.54 88.318.5 150.3 334.7 903.05439.75 >1000 807.5477.4

IC50 [nM]a b 26.522.6 40.921.62 30.43.68 27.07.2 44.14.2 220162 456.8 12325 -

c 30.36.5 14.23 33.663.7 16.73.3 5.00.7 11.54.4 89.7118.3 288.9

Selection of tumor models to prepare first clinical studies

MDAMB 231 MDAMB 435

SKBR3

DU145

MCF7

A431

HT29

OH1

3D Surface Plot of Immunostained Section for CXCR4

rabbit 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

Dimeric DOTA labeled peptides

HN NH 2 NH HO H N O H N N HO O N H HN NH NH 2 O H N O HO H N O H N N H O O N H HN NH NH 2 O N O NH 2

FC131

Peptide P: HN

R/L

R:

NH

Gua O O

Ac O

NH 2 HO H N O H N N O H O N H H N NH 2 X X: NH; active a X: O; inactive i O N O Linker L: O P1 O N H m O N n H P2 O m Gma NH 2

Demmer O. et al. (submitted)

Dimeric DOTA labeled peptides

OH H 2N HN H N N H O O H N O NH HN NH 2 OH O N H N O O N H O N H O N H O H O H N N NH

N H

Peptide P1 a a a a a a a a P2 a a a a a a a R m 0 0 0 0 0 0 0

L n 2 3 4 6 8 11 14 IC50 [nM] 91 64 42 31 31 21 47 14.4

Dimeric DOTA labeled peptides

OH

H 2N HN

NH

H N N H O O H N O NH O N H N X O HN O X n O N H O N

comp
O N H O N H H O H N N

X Gly Gly Gly Gly

n 0 0 1 1 0 0 1 1

M In3+ In3+ In3+ In3+

IC50 [nM] 92 6 29 1 25 13 78 9 38 2 14 2 12 2 22 5

21 22 23
O

N H

24 25 26

HN

NH2

O O HO O N Ga N N N

NH O

OH

27 28

O O

Demmer O. et al. (submitted)

Dimeric DOTA Complexes

HO O O NH O NH O H2N HN HN O NH O NH HN O HN O HN O NH
-O

N H

HN

H HN N O

O N N O O
68Ga

NH NH HN ONH2

9.5nM

H N

O OH

Brohl F. et al. (in progress)

A Triazacyclononane-Based Bifunctional Phosphinate Ligand for the Preparation of Multimeric 68Ga Tracers for Positron Emission Tomography
Notni J et al. 2010. Chem. Eur. J. 2010

A Triazacyclononane-Based Bifunctional Phosphinate Ligand for the Preparation 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)

37 MBq
125I-12G5

SPECT/CT of CXCR4 expression levels in experimental brain tumors using 125Ilabeled anti-CXCR4 mAbs, hCXCR4 (12G5) and the control IgG2A MAb

37 MBq
125I-IgG2A

SPECT/CT of CXCR4 in U87 xenografts and MIP-image of 125I-12G5injected mouse at 48 h after injection.

The feasibility of the RID of CXCR4 expression imaging of the tumor microenvironment with a MAb is possible.
Biodistribution in U87 tumor-bearing SCID mice. 74 kBq of 125I-12G5 or 125I-IgG2A at 24, 48, and 72 h

U87-stbCXCR4 mice (90 min p.i.)

Nimmagadda S, Cancer Res (2010)

MDA-MB-231 (solid arrow) and DU4475 mice (open arrow)

Flourescent Imaging of bladder cancer

Nishizawa et al., Int.J Cancer 2010

(Ac-Arg-Arg-Nal-Cys-Tyr-Cit-Arg-D-Lys*-Pro-Tyr-Arg-Cit-Cys-Arg-NH2; D-Lys* indicates the carboxyfluorescein-labeled D-Lys IC50: 11nM

A CXCR4 antagonist CTCE-9908 inhibits primary tumor growth and metastasis of breast cancer
(Huang et al, Journal of Surgical Research, 2009) (25 mg/kg, injected subcutaneously 5 d/wk

KGVSLSYR K-NH2 KGVSLSYR

CTCE-9980 structure

Inhibition of CXCR4 reduced the primary tumor growth of MDA-MB-231 cells implanted into the inguinal mammary fat pad. (6 weeks)

In this model, 1105 MDA-231-BSC12 cells were injected into the left cardiac ventricle to produce bone metastases.

provide an sensitive method to follow the progression of the primary and metastases over time