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

H.J. Wester

Molecular Imaging of CXCR4 Receptors

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

TTopochemical exploration of potent compounds usingretro-enantiomer libraries of cyclic pentapeptides.

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

T140 FC131

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)

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

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

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

thickness: 1mm

Imaging of CXCR4 expression in lung metastases-Autoradiography of lungs 1 h p.i. of 124I-CPCR4

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

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