Nuclear (PET/SPECT) and optical imaging probes targeting the CXCR4 chemokine receptor

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Nuclear(PET/SPECT)andopticalimagingprobestargetingtheCXCR4chemokinereceptor

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DOI:10.1039/C2MD20117H

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JamesCKnight

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Nuclear (PET/SPECT) and optical im

James C. Knight* and Frank R. Wuest

Received 8th February 2012, Accepted 6th July 2012

DOI: 10.1039/c2md20117h

The chemokine receptor CXCR4 has been found to be highly expressed in a wide variety of cancer

types, including breast, colorectal, melanoma, nasopharyngeal, oesophageal, osteosarcoma, and non-

small-cell lung carcinoma. It has been shown that these elevated expression levels are yet further

increased upon metastasis. This receptor therefore represents a highly attractive target which could

facilitate the diagnostic imaging of many aggressive cancers. Since 2005, there have been a wide

assortment of CXCR4-targeting imaging probes spanning both nuclear (PET/SPECT) and optical

imaging modalities. This review highlights a wide variety of pertinent examples from the recent

literature, placing special focus on the chemical aspects of probe design.

1. Introduction and scope

1.1 Chemokine receptor 4 (CXCR4) and its role in cancer

Chemokines are a family of small cytokines (60–100 aa) which

are known to activate G protein-coupled receptors (GPCRs)

thereby inducing cellular migration. This migration proceeds

through an increasing concentration gradient of the chemokine

towards the site of production in a process known as chemotaxis.

James C: Knight

Dr James C. Knight obtained a

BSc in chemistry from Cardiff

University in 2005 and subse-

quently a PhD in 2009 under the

supervision of Dr Angelo J.

Amoroso and Prof. Peter G.

Edwards. He subsequently spent

two years working between the

Cardiff School of Biosciences

and Chemistry departments

developing multimodal imaging

agents under the supervision of

Dr Richard W. E. Clarkson, Dr

Stephen J. Paisey and Prof.

Peter G. Edwards. Currently, he

is a postdoctoral fellow based at

the University of Alberta with Dr Frank Wuest. His work now

focuses on the development and pre-clinical evaluation of novel

agents for PET-based cancer imaging.

Department of Oncology, University of Alberta, Edmonton, AB, T6G 1Z2,Canada. E-mail: [email protected]; Tel: +1 780 432 8932

This journal is ª The Royal Society of Chemistry 2012

So far, approximately 50 chemokines and 20 chemokine recep-

tors have been identified which collectively form the human

chemokine system.1–3 All known chemokines can be categorised

into four distinct groups (CXC, CC, C, and CX3C) based on the

position of the first two of four cysteine residues.4–8

The human chemokine system is complex and chemokines are

known to serve a variety of functions, mostly involving the

regulation of cell trafficking.9–11 The dysregulation of either

chemokines or chemokine receptors has been linked with many

diseases, including cancer.12,13

Frank R: Wuest

Dr Frank Wuest earned his PhD

in chemistry in 1999 from the

Technical University of Dres-

den, Germany. He spent one

year as a postdoctoral fellow at

the School of Medicine in St.

Louis working on molecular

probe development in the labo-

ratory of Dr Michael J. Welch.

After his return to Germany he

became head of the PET-tracer

group of the Research Center

Dresden-Rossendorf. In 2008 he

started a new position as the

Dianne and Irving Kipnes Chair

in Radiopharmaceutical

Sciences at the Cross Cancer Institute in Edmonton. His current

research interests are focused on the design, synthesis and radio-

pharmacology of novel radiopharmaceuticals to optimize current

diagnosis and treatment of cancer.

Med. Chem. Commun., 2012, 3, 1039–1053 | 1039

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Chemokine receptor 4 (CXCR4) is a 352 amino acid GPCR

which is known to bind the chemokine CXCL12 (most commonly

referred to as stromal cell-derived factor-1, SDF-1).4,14 This

pairing serves a number of important roles,15most importantly (i)

controlling the homeostatic circulation of CXCR4+ haemato/

lymphopoitic cells as well as directing their passage to areas of

inflamed and/or damaged tissue,9–11 and (ii) directing the traf-

ficking of stem cells expressing CXCR4, for example during

embryo development. The lack of either CXCR4 or SDF-1 has

been found to lead to fetal mortality,16,17 and accordingly various

studies have demonstrated that CXCR4 is necessary for organo-

genesis, development, vascularisation and hematopoiesis.16–21

The CXCR4 receptor was originally identified as a co-receptor

for HIV entry,22 and subsequently its association with a variety

of diseases, particularly cancer, is now well-established.23–28 The

expression of CXCR4 is known to be promoted by hypoxia-

inducible factor-1 (HIF-1) which is commonly activated in the

hypoxic regions of certain tumour environments.29–31 Interest-

ingly, whilst CXCR4 expression is known to be lower in normal

tissues compared to tumours,32 CXCR4 expression in regions

immediately adjacent to tumours has been found to be minimal

and, in some cases, entirely absent.28,33,34 The presence of high

levels of CXCR4 in primary tumours is strongly associated with

tumour recurrence and low survival rates for a variety of cancer

types.32,35–38

In addition to primary tumours, the CXCR4/SDF-1 signalling

pathway has also been found to serve an important role in the

advancement of cancer metastasis.37,39–48 Accordingly, tumours

with high levels of CXCR4 have been linked with an aggressive

phenotype.35–37,49 Several groups have shown that metastases

frequently contain elevated levels of CXCR4 and proceed

through the circulatory system towards organs which secrete

SDF-1, such as lymph nodes, bone, liver and lung.28,33,50–60 These

properties make CXCR4 an attractive target, both for cancer

therapy and as a selective handle for cancer cell detection.

The CXCR4/SDF-1a pairing was long considered to be

monogamous,17,18,20,61 however several recent reports are now

shedding light on the involvement of an additional chemokine

receptor, CXCR7 (also formerly known as RDC-1).32,62–73 It has

been found that SDF-1 has approximately a ten-fold higher

binding affinity for CXCR7 compared with CXCR4.74,75 Whilst

SDF-1a is currently the only known ligand of CXCR4, CXCR7

is also known to bind an additional chemokine, CXCL11 (also

known as I-TAC) albeit with lower affinity.75 Like CXCR4,

CXCR7 is also expressed in a variety of tissues and is highly

expressed in some cancer cell types and human primary

tumours.62,63,67,75–78 Luker et al. have shown the ability of

CXCR4 and CXCR7 to form both homo- and heterodimers.79

This ability has since been proposed as a means by which

CXCR7 modulates the function of CXCR4.66,72,79 It has been

theorised that CXCR7 could function as a co-receptor for

CXCR4 resulting from heterodimerisation, thus augmenting

SDF-1a-mediated signalling.66,72 Another hypothesis is that

CXCR7 may function as a scavenger of SDF-1a, creating

gradients of this chemokine which would impact on the signal-

ling of CXCR4.80,81 It has also recently been shown that CXCR7

is internalized upon interaction with b-arrestin.69,82,83

A crucial recent advancement has been the determination of

the crystal structure of the CXCR4 receptor.84 In 2010, Wu et al.

1040 | Med. Chem. Commun., 2012, 3, 1039–1053

were able to obtain five crystal structures of CXCR4 bound to a

small molecule antagonist (IT1t) and a cyclic peptide (CVX15) at

2.5 to 3.2 �A resolution. Analysis of these structures revealed a

larger, more accessible cavity compared with other known

GPCR structures that is also located in closer proximity to the

extracellular surface. The information offered by these structures

now provides exciting opportunities (such as virtual screening85)

to develop tailored imaging probes with more favourable

CXCR4-targeting properties.86

The application of diagnostic probes with the ability to selec-

tively target CXCR4 could have many potential advantages,

including (i) the ability to evaluate primary tumours for elevated

levels of CXCR4 indicating the likelihood of recurrence and

potential for metastasis, (ii) the ability to track metastatic spread

throughout the body, facilitating rapid, targeted therapy, and

(iii) the guidance of surgical procedures, thereby ensuring

maximal tumour excision. A recent review by Woodard and

Nimmagadda gives a useful summary of imaging agents target-

ing CXCR4.87 We aim to build upon this by providing a

comprehensive overview of the application of CXCR4-targeted

imaging probes across both nuclear (PET/SPECT) and optical

modalities. This includes a detailed analysis of the chemical

aspects of probe design.

2. Nuclear (PET/SPECT) imaging probes targetingCXCR4

2.1 Overview of PET/SPECT imaging modalities

Positron emission tomography (PET) and single-photon emis-

sion computed tomography (SPECT) are important imaging

tools as they offer several advantages compared to other imaging

modalities, including the ability to track biochemical, physio-

logical, and pharmacological events with exquisite sensitivity

(10�11 to 10�12 mol L�1 and 10�10 to 10�11 mol L�1 for PET and

SPECT, respectively).88–90 Consequently, these techniques have

been widely used in biomedical and clinical settings to monitor

the progression of various malignancies, especially aggressive

cancers.91

The success of both PET and SPECT imaging modalities is

dependant upon the use of effective molecular probes and

therefore their development is currently an area of intense multi-

disciplinary investigation.92–97 In order to be successful, a

radiotracer designed to target a particular biomarker must have

several key characteristics, including: (i) high binding affinity for

its intended target, (ii) high binding specificity, (iii) high signal-

to-background ratio, (iv) high metabolic stability, and (v) low

immunogenicity and toxicity.98,99

At present, the vast majority of PET radiotracers are based on

small molecules incorporating non-metal radionuclides such as11C, 13N, 15O, 18F, and 124I. Of these, fluorine-18 is the most

widely used radionuclide as it has several desirable properties.

For example, (i) it can easily be produced on a cyclotron with

high specific activity, (ii) it has a convenient radioactive half-life

(t1/2 ¼ 109.8 min), and (iii) it has a low b+ energy (0.64 MeV)

which results in a short range in tissue (max ¼ 2.4 mm).100–102

Metal radionuclides such as 64Cu, 68Ga, 86Y and 89Zr for PET

and 99mTc for SPECT are also proving to be very useful tools in

diagnostic imaging applications,103–105 particularly as they

This journal is ª The Royal Society of Chemistry 2012

Fig. 1 The CXCR4 antagonist, T140, developed by Tamamura et al.

Nal ¼ L-3-(2-naphthyl)alanine, Cit ¼ L-citrulline.

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typically have longer half-lives which are compatible with the

long biological half-lives and extended blood clearance times of

larger targeting vectors such as antibodies.

Whilst the development of nuclear imaging agents directed at

the CXCR4 chemokine receptor is still an emerging area of

research, there is already a diverse selection of molecular probes

combining many different radionuclides with a variety of tar-

geting vectors. This section of the review aims to provide an

overview of what has so far been attempted in this endeavour.

2.2 Radiolabelled antibodies

To the best of our knowledge, there has been just one report of a

radiolabelled monoclonal antibody (mAb) being used for

imaging CXCR4 expression. This study focused on the evalua-

tion of a 125I-labelled anti-CXCR4 mAb, 125I-12G5, as a

potential SPECT agent.106 In vitro binding assays involving

human glioblastoma U87 and U87-stb-CXCR4 cell lines

confirmed that the specificity of 125I-12G5 mAb was not

impaired as a result of radioiodination. Furthermore, bio-

distribution analysis of 125I-12G5 mAb in severe combined

immunodeficient (SCID) BALB/C mice bearing U87 xenografts

revealed accumulation in the tumour at consistently higher levels

on comparison to a control radioiodinated antibody

(125I-IgG2A). A series of SPECT/CT scans showed clearly

distinguishable U87 tumour xenografts at 24 h post-injection

(p.i.), reaching the maximum tumour-to-nontumour ratio after

48 h. It should be noted, however, that this radiotracer also

exhibited rapid clearance from the blood which is atypical of

antibodies, indicating possible deiodination.

The excellent specificity and affinity that antibodies can afford

has made them highly attractive as targeting vectors for diag-

nostic imaging applications, particularly in PET/SPECT.

However, the use of antibodies for this purpose has been limited

by extended circulation times which often result in undesirable

high background activity. Therefore, antibodies are typically

used in conjuction with radionuclides which have long radioac-

tive half-lives (e.g. 64Cu, 68Ga, 89Zr, 99mTc, 111In and 125I). In

order to utilise long-lived metal radionuclides in conjunction

with antibodies, bifunctional chelating agents are normally

required.103,105,107 These agents contain (i) a reactive functional

group, (e.g. isothiocyanate, activated ester, maleimide) to form

covalent attachments with amine- or thiol-containing residues on

the antibody, and (ii) a chelating group to sequester the metal ion

within a thermodynamically stable and kinetically inert complex.

In this respect, there remains much scope for further develop-

ment in the application of radiolabelled antibodies for imaging

CXCR4 expression.

More favourable tumour targeting and clearance kinetics have

also been achieved by the use of lower molecular weight antibody

fragments and affibodies. The use of these smaller targeting

vectors in combination with radionuclides also offers an exciting

strategy for imaging CXCR4 expression in a highly sensitive

manner.

Fig. 2 The Ac-TZ14011 peptide has been modified with 111In–DTPA by

Hanaoka et al. in 2006 (ref. 119) and subsequently by 125I-IB byHan et al.

in 2010.120

2.3 Radiolabelled peptides derived from T140

In 1998, a low molecular weight peptidic inhibitor of CXCR4

named T140 (Fig. 1)108 was developed by Tamamura et al., and,

This journal is ª The Royal Society of Chemistry 2012

due to its impressive ability to bind CXCR4,108–115 has since been

used as a template for an array of CXCR4-targeted imaging

agents across both optical and nuclear imaging modalities. This

peptide contains 14-amino acid residues and a single disulphide

bridge forming a rigid b-hairpin conformation. T140 was origi-

nally evaluated as a potential anti-HIV compound as it was

found to have a high inhibitory activity against HIV-1 entry

(EC50¼ 3.5 nM).108 In addition, this peptide has also been shown

to have a strong binding affinity for CXCR4. In competitive

binding assays involving 125I-SDF-1, T140 has yielded IC50

values of 0.93–4 nM.116–118 In an attempt to identify the essential

pharmacophore of T140, Tamamura et al. found four amino acid

residues (Arg2, Nal3, Tyr5 and Arg14) which were shown to be

crucial in maintaining high activity.109 Unfortunately, T140

was found to be unstable in feline serum due to cleavage of

the indispensable C-terminal Arg14 residue.111 Consequently,

this group developed a series of peptide derivatives based on

T140, including TN14003 ([Cit6]-T140), TC14012 ([Cit6, D-Cit8]-

T140),111 Ac-TE1401,114 and 4F-benzoyl-TE14011-Me

(TF14013-Me).116 These peptides were found to have higher

biostability and improved anti-HIV activity compared to T140.

In 2006, Hanaoka et al. developed a 14-residue peptide, Ac-

TZ14011 (Fig. 2), which was derived from T140.119 This peptide

retained the four amino acid residues of T140 (Arg2, Nal3, Tyr5

and Arg14) which are crucial in forming strong interactions with

CXCR4. A useful feature of this peptide is the presence of a

single amino group located on D-Lys8 which is available for site-

specific functionalisation. In this example, the versatile chelating

group diethylenetriamine pentaacetic acid (DTPA) was chosen in

order to chelate the SPECT radionuclide 111In. The binding

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affinities (IC50) of both Ac-TZ14011 and In–DTPA–Ac-

TZ14011 for CXCR4 are 1.2 and 7.9 nM, respectively, indicating

that the incorporation of the In–DTPA moiety only resulted in a

minor reduction in ligand potency. In mice bearing CXCR4-

expressing ASPC-1 pancreatic carcinoma, 111In–DTPA–Ac-

TZ14011 (Fig. 2) was found to undergo rapid blood clearance

and after 1 h was mostly present in the kidney, liver and spleen.

The maximum tumour-to-muscle (T/M) and tumour-to-blood

(T/B) ratios were 4.43(1.89) (6 h) and 5.65(2.89) (24 h), respec-

tively. Whilst the tumour exhibited higher radiotracer accumu-

lation compared with the muscle and blood at all time points,

these values are quite modest in comparison to other radio-

labelled peptide conjugates and small molecules.

The Ac-TZ14011 peptide has also more recently been used by

Han et al. in their development of 125I-IB–Ac-TZ14011

(Fig. 2).120 In this case, the authors used N-succinimidyl-3-[125I]

iodobenzoate (125I-SIB) to label Ac-T14011 at the amino group

of the D-Lys8 residue. Incubation with MCF-7 breast cancer cells

resulted in high uptake of 125I-IB–Ac-TZ14011 which reached a

plateau at approximately 2 h. Analysis of the biodistribution in

non-tumour-bearing mice revealed that at 10 minutes p.i. the

highest proportion of radioactivity was located within the

kidneys. This observation, along with the low uptake observed in

the liver and intestines, indicates that the introduction of the

3-[125I]iodobenzoyl group did not significantly increase the

lipophilic character of the peptide.

In an effort to improve upon their previous syntheses involving111In–DTPA-conjugated peptides,119 Masuda et al. used a mon-

oreactive DTPA-based precursor to functionalise bromoacety-

lated lysine residues located on a series of peptides, in this case,

derived from TN14003 (Fig. 3).121 A selection of these peptides

were subsequently reacted with InCl3 and then evaluated in terms

of their ability to bind CXCR4 membrane extracts in a compe-

tition assay involving 125I-SDF-1. Two of the most promising

peptides, 111In–DTPA–Ac-TN14003 and 111In–DTPA–4F-Bz-

TN14003 (Fig. 3), yielded IC50 values of 1.60� 0.91, and 0.014�0.010 mM, respectively, which are in close agreement with those

previously established for the corresponding unlabelled

peptides.116

The peptide 4F-benzoyl-TN14003 (also referred to as

TF14016) was shown by Tamamura et al. to bind CXCR4 with

Fig. 3 A selection of peptide-conjugates based on

1042 | Med. Chem. Commun., 2012, 3, 1039–1053

high affinity and exhibited improved stability in mouse serum

and liver homogenate compared to the parent peptide,

T140.113,116 In 2010, Jacobson et al. evaluated the analogous18F-containing peptide, 4-18F-benzoyl-TN14003 (referred to as

4-18F-T140; Fig. 3), which was radiolabelled using the prosthetic

groupN-succinimidyl-4-[18F]fluorobenzoate (SFB).122An in vitro

competition assay revealed that the analogous cold peptide, 4-F-

T140 (4F-benzoyl-TN14003, TF14016), strongly competed

against 125I-SDF-1 (IC50 ¼ 2.5 nM). This is close to the value of

0.99 nM that Tamamura et al. had previously established for this

peptide.113 Biodistribution experiments using female nude mice

revealed no significant difference in uptake between CHO and

CHO–CXCR4 tumour xenografts. The authors suggested this

could be a result of the high uptake by red blood cells which

renders the probe largely unavailable. Accordingly, blocking

experiments involving excess amounts of the analogous cold

peptide resulted in substantially higher T/M and T/B ratios

(21.6 � 7.14 and 27.05 � 8.7, respectively). Moreover, PET

images acquired following co-injection of 4-18F-T140 and cold

peptide revealed clearly distinguishable CXCR4-positive but not

CXCR4-negative tumour xenografts.

In a recent follow-up study, this team resolved the issue of

binding to red blood cells in their development of two new PET

radiotracers, 64Cu–NOTA-NFB and 64Cu–DOTA-NFB (Fig. 3)

which are also derived from TN14003.123 The uncomplexed

(copper-free) DOTA-NFB and NOTA-NFB peptides yielded

IC50 values of 68 nM and 138 nM, respectively, representing a

reduction in binding affinity compared to T140 (IC50 ¼ 0.93–3.7

nM (ref. 116 and 124)). However, the 64Cu-labelled peptides

exhibited substantially higher uptake in the CHO–CXCR4

tumour compared to the control (CHO) tumour.At 4 h p.i., 64Cu–

NOTA-NFB and 64Cu–DOTA-NFB yielded T/M ratios of

39.30 � 2.26 and 19.32 � 2.35, respectively, and T/B ratios of

38.88� 3.91and14.50�0.82, respectively. Importantly, incontrast

with their previous study,122 there was very little binding to red

blood cells in both in vitro and in vivo experiments. These peptides

are therefore more viable as in vivo diagnostic probes for CXCR4.

In most of the studies highlighted in this section, the addition

of a blocking agent substantially reduced the T/M and T/B ratios

indicating that tumour uptake of these T140-derived peptide-

conjugates is largely mediated by CXCR4.

the potent CXCR4 antagonist TN14003.121–123

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A direct comparison of these probes (and, indeed, between

classes of probe) is difficult due to the use of different experi-

mental conditions and tumour models with varying degrees of

CXCR4 expression. Whilst more impressive T/M and T/B ratios

have been obtained with some radiolabelled small molecules (cf.

Section 2.6), the ability of peptide-conjugates derived from T140

to provide vivid images of CXCR4-expressing tumours is clearly

very promising. Furthermore, the apparent low uptake in the

liver could present some advantages for the detection of tumours

within the abdominal region compared with probes derived from

AMD3100.

Fig. 5 The 68Ga complex of CPCR4-2 exhibits favourable pharmaco-

kinetics and imaging performance.131,132

2.4 Radiolabelled cyclic pentapeptide frameworks

In 2003, Fujii et al. led the development of a new class of

CXCR4-targeting compounds based on a cyclic peptide frame-

work.117 This group utilised two orthogonal libraries of cyclic

pentapeptides incorporating the four known indispensible amino

acid residues (Arg2, Nal3, Tyr5 and Arg14) in combination to

effectively downsize T140 which led to the discovery of FC131

(Fig. 4). Like T140, this cyclic pentapeptide exhibits high affinity

for CXCR4 in competition studies with 125I-SDF-1, yielding IC50

values of 4–7.9 nM.117,125–127 Since their discovery, the impor-

tance of amino acid substitution,127–129 ring structure,126 and

backbone modifications118,130 upon bioactivity and binding

potency have been thoroughly examined. From these studies, it

has been concluded that Arg, Nal and D-Tyr are best situated in

positions 2, 3, and 5, respectively.127 All of the side-chain func-

tional groups of FC131 have also been shown to be important in

maintaining high binding potency, and Arg3 and Nal4 have been

shown to be particularly crucial in this respect.130 N-methylation

of some analogues, for example [cyclo(-D-Tyr1-D-MeArg2-Arg3-

Nal4-Gly5-), has also been shown to contribute to highly potent

binding behaviour.130 The effects of incorporating a 4-fluo-

rophenyl substituent within the cyclic pentapeptide framework

were also examined127 as this moiety was shown to be an effective

pharmacophore in previous studies focused on T140.113,116 As a

result, the peptide [D-Phe(4-F)1, Arg5]-FC131 was found to bind

CXCR4 with high potency (IC50 ¼ 35 nM). The favourable

properties possessed by cyclic pentapeptides have made them

excellent candidates for applications in imaging CXCR4

Fig. 4 The cyclic pentapeptide, FC131, developed by Fujii et al. has

been found to be a potent binder of CXCR4.

This journal is ª The Royal Society of Chemistry 2012

expression and significant progress has already been made in this

respect.

Recently, Demmer et al. developed a series of cyclic penta-

peptides which were optimised based on CXCR4 binding

affinity.131 These peptides were then modified by incorporating a

DOTA chelating group to facilitate labelling with radiometals,

in this case 68Ga and 111In. The Ga complex of one of these

cyclic pentapeptides (later referred to as CPCR4-2; Fig. 5)

(cyclo(D-Tyr1-D-[NMe]-Orn2-[AMBS-DOTA]-Arg3-2-Nal4-Gly5)

(AMBS ¼ 4-(aminomethyl) benzoic acid) yielded the highest

binding affinity (IC50 ¼ 5 � 1 nM). This radiotracer was evalu-

ated in mice bearing CXCR4-expressing OH-1 human small-cell

lung cancer (h-SCLC) xenografts. Promisingly, images obtained

Fig. 6 (a) PET summation images recorded at 90–110 minutes p.i. of

mice bearing OH1 h-SCLC tumours. Images reveal a clearly distin-

guishable tumour. (b) Co-injection with cyclo-(D-Tyr1-Arg2-Arg3-Nal4-

Gly5) results in significantly diminished tumour uptake. Reproduced

from ref. 131 with permission.

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from PET scans revealed a clearly distinguishable tumour

(Fig. 6).

Soon after, this group conducted more extensive studies

focused on further evaluating the ability of 68Ga–CPCR4-2 to

function as an in vivo diagnostic agent in mice bearing OH1

tumours.132 This radiotracer was found to clear rapidly from

non-target tissues and accumulate quickly in the tumour,

reaching maximum uptake at 1 h p.i. and yielding T/M and T/B

ratios of 16.6 � 3.8 and 5.8 � 0.9, respectively. These values are

significantly higher than found for both 111In–Ac-TZ14011 (ref.

119) or 18F-T140 (ref. 122), although it should be noted that

differences in experimental details such as the use of tumour

models with varying levels of CXCR4 expression render a direct

comparison difficult. Notably, this radiotracer exhibits only

minor uptake in organs known to express high levels of CXCR4

mRNA in mice, namely the liver and spleen. This is a very

important finding as many CXCR4 radiotracers (particularly

those based on small molecules) exhibit high uptake in the liver,

preventing acquisition of high contrast images in the abdominal

region.

The same group also reported a study focused on the gallium

and indium complexes of a series of symmetric dimers derived

from cyclic pentapeptides.133 These compounds were shown in

receptor binding assays to have high affinity for CXCR4 and a68Ga complex was selected for further in vivo radio-

pharmacological evaluation in mice bearing OH1 h-SCLC

tumours. Unfortunately, substantial nonspecific uptake was

found in the liver, rendering this probe unsuitable for the imaging

of primary tumours and metastases. The authors attributed this

unfavourable behaviour to the lipophilic character of the dimeric

radiotracer. It remains to be seen whether increasing the

hydrophilic character of this class of probes would reduce

accumulation in the liver, thereby rendering themmore viable for

imaging and therapeutic applications.

Another approach has been recently employed by �Aberg et al.,

leading to the development of the 18F-radiolabelled cyclic

pentapeptide [18F]CCIC-0007 (Fig. 7).134 In this example, the

Arg4 residue of FC131 was replaced with ornithine (Orn4)

without substantial loss of binding affinity (IC50 ¼ 19 nM). This

substitution facilitated the incorporation of a PEG linking group

with a terminal aminooxy functionality which was subsequently

reacted with the 18F-containing prosthetic group 4-[18F]fluo-

robenzaldehyde (18F-FBA). The IC50 value for 18F-CCIC-0007

was 0.80 mM on MDA-MB-231 cells, revealing that these

structural modifications have a significant detrimental effect on

binding affinity for CXCR4. The biodistribution of this radio-

tracer was evaluated in female non-tumour-bearing BALB/C

Fig. 7 A cyclic pentapeptide derived from FC131 has also been

successfully radiolabeled with 18F using the prosthetic group 18F-FBA.134

1044 | Med. Chem. Commun., 2012, 3, 1039–1053

mice. Whilst 18F-CCIC-0007 was rapidly cleared from the blood,

little uptake was observed in organs which are known to express

CXCR4, such as the spleen or bone marrow. These properties,

taken together, indicate low specific binding for CXCR4 and it

was suggested by the authors that structural modifications were

required to enhance the binding properties of this framework.

Even though there have been fewer reports of probes based

on cyclic pentapeptides compared with those derived from

T140, there have already been indications of their potential in

imaging CXCR4 expression. Some of these frameworks exhibit

similar affinity for CXCR4 compared to T140 and, furthermore,

one of these compounds, 68Ga–CPCR4-2, also reveals low

accumulation in the liver, potentially eliminating a problem

typically encountered with radiolabelled small molecules

(cf. Section 2.6).

2.5 Radiolabelled SDF-1a

In addition to the reports of radiolabelled T140-derivatives and

cyclic pentapeptides, the natural ligand of CXCR4, SDF-1a, has

also been successfully radiolabelled for diagnostic applications.

SDF-1, a small (68-residue) cytokine, has two isoforms, SDF-1a

and SDF-1b, which are encoded by a single gene, PBSF, and

result from alternative splicing. The two forms differ only by the

presence of four additional amino acids (RLKM) at the

C-terminus of SDF-1b.135–137 The crystal structure of a variant of

human SDF-1a, [N33a]SDF-1a, reveals a positively charged

surface which results from a grouping of the basic residues, Lys-

24, His-25, Lys-27, and Arg-41.138 This contributes to a strong,

complementary interaction with the negatively charged extra-

cellular loops of CXCR4. A study by Loetscher et al. demon-

strated the importance of the N-terminus of SDF-1a in binding

to CXCR4. This group tested a variety of N-terminal SDF-1

peptides and found an ability to bind and activate CXCR4.139

The high binding affinity of SDF-1a for CXCR4 has been

determined independently by several groups with IC50 values

mostly ranging between 0.08 and 39.6 nM.139–146 Whilst the use

of radiolabelled SDF-1a appears to be an attractive means of

imaging CXCR4 (and CXCR7) expression, the scarcity of

reports of this type could be due to the relatively poor in vivo

characteristics of this targeting vector. Recently, Antonsson et al.

established a SELDI-TOF mass spectrometry procedure for

analysing the processing of SDF-1a in vivo.147 This study

revealed that SDF-1a is largely processed, both at the C- and

N-termini, in the first 5 minutes following injection.

In 2008, Misra et al. used 99mTc-S-acetylmercaptoacetyl-

triserine-N-hydroxysuccinimide ([99mTc-MAS3]-NHS), to prepare

[99mTc-MAS3]-SDF-1a (Fig. 8) for SPECT imaging.148 This

group demonstrated that [99mTc-MAS3]-NHS has high stability

and resistance to transchelation. In a high-throughput homolo-

gous competition assay involving human prostate cancer (PC-3)

cells which had been transduced with CXCR4, and also neonatal

rat cardiomyocytes, [99mTc-MAS3]-SDF-1a exhibited high

binding affinity in the nanomolar range (1.0 � 0.1 and 2.9 �0.5 nM, respectively). The in vivo properties of this tracer were

also investigated in male rats. Following intravenous injection,

the radiotracer had a blood half-life of 25.8 � 4.6 minutes, and

after two hours 73.8 � 6.1% was found mostly in the urine

indicating predominantly renal clearance. In contrast to many of

This journal is ª The Royal Society of Chemistry 2012

Fig. 8 The native ligand for CXCR4, SDF-1, was modified by Misra

et al. using [99mTc-MAS3]-NHS, leading to anewSPECT imagingagent.148

Fig. 9 The small molecule AMD3100 is known to be a highly potent

binder of CXCR4. The corresponding mononuclear 64Cu complex has

since been investigated as a potential PET agent for imaging CXCR4

expression.39,170,171

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the other radiolabelled species discussed herein, 99mTc-SDF-1a

reveals only low accumulation in the liver.

Fig. 10 Volume-rendered whole body images of mice bearing U87 and

(solid arrow) and U87-stb-CXCR4 (unfilled arrow) tumours following

injection of [64Cu]AMD3100 (left). A blocking dose of unlabelled

AMD3100 (50 mg kg�1) was followed by the radiotracer (middle).

Injection of [64Cu]CuCl2 alone (right). Reprinted by permission from the

American Association for Cancer Research: Nimmagadda et al.,

Molecular imaging of CXCR4 receptor expression in human cancer

xenografts with [64Cu]AMD3100 positron emission tomography, Cancer

Res., 2010, 70(10), 3935–3944, DOI: 10.1158/0008-5472.CAN-09-4396.

2.6 Radiolabelled small molecules

The small bicyclam molecule ‘AMD3100’ (also called Mozobil,

Plerixafor and, formerly, JM3100) is known to bind to CXCR4

with high affinity and effectively inhibit the binding of the

natural chemokine ligand, SDF-1a.146,149–154 This compound was

originally discovered as an anti-HIV agent which specifically

blocks the CXCR4 co-receptor which is used by T-tropic (X4)

strains of HIV to enter the cell.151,155,156 In addition, AMD3100

has been shown to effectively mobilise hematopoietic stem cells

in both mice and healthy volunteers.157–160 Molecular modelling

experiments have revealed that this compound interacts with

three acidic residues within the ligand binding site of the CXCR4

receptor, namely Asp171 (AspIV:20), Asp262 (AspVI:23) and

Glu288 (GluVII:06).152,155,161,162 Further studies involving the

metal complexes of AMD3100 have determined that formation

of the mononuclear copper complex leads to a 7-fold enhance-

ment in overall binding affinity for CXCR4.163

The favourable binding properties of AMD3100 and its metal

complexes make these compounds excellent candidates for

applications in CXCR4-targeted diagnostic imaging. From a

radiolabelling perspective, the presence of the macrocyclic

cyclam chelating groups are an attractive feature as it allows for

the facile incorporation of a variety of radiometals such as

copper-64. However, whilst [Cu(cyclam)]2+ has remarkably high

thermodynamic stability (log KML ¼ 25.0–28.09),164–167 it should

be noted that cyclam is not an ideal chelating agent for radio-

metals as its metal complexes are not as kinetically inert

compared with those of other chelators (e.g. TETA, CB-

TE2A).168 This may increase the likelihood of complex dissoci-

ation and/or transchelation with, for example, superoxide dis-

mutase which is known to have a high affinity for copper.169

Due to the favourable CXCR4 binding properties of its ‘cold’

analogue, much recent attention has been focused on the devel-

opment and evaluation of 64Cu–AMD3100 (Fig. 9) as an in vivo

diagnostic probe of CXCR4 expression.39,170,171 An initial radi-

opharmacological investigation of this radiotracer was reported

by Jacobson et al. in 2009.170 In non-tumour-bearing mice,

uptake was observed in CXCR4-expressing organs and tissues,

including spleen, lymph nodes and bone marrow. Specific accu-

mulation was also observed in the liver.

Later, Nimmagadda et al.39 assessed the imaging potential of64Cu–AMD3100 in NOD/SCID mice bearing both U87 (low

This journal is ª The Royal Society of Chemistry 2012

CXCR4-expressing) and U87-stb-CXCR4 (high CXCR4-

expressing) glioblastoma xenografts. The radiotracer was found

to continuously accumulate in the U87-stb-CXCR4 tumour

(Fig. 10), reaching maximum T/M and T/B ratios after 90

minutes of 47.36 � 6.93 and 16.93 � 3.40, respectively. In

accordance with the observations made by Jacobson et al.,170

whole body images revealed the radiotracer was also present in

the liver, kidneys and bladder. This group also tested the efficacy

of 64Cu–AMD3100 in a MDA-MB-231-derived experimental

lung metastasis model. At 90 minutes p.i., biodistribution

analysis of mice both with and without lung metastases revealed

lung-to-muscle ratios of 22.99 � 2.50 and 14.32 � 2.67,

respectively.

Recently, Weiss et al. also reported a similar study focused on

evaluating the imaging performance of 64Cu–AMD3100 in mice

bearing CHO and CHO–XR4 tumours.171 This group established

Med. Chem. Commun., 2012, 3, 1039–1053 | 1045

Fig. 11 The 64Cu complex of monocyclam AMD3465 investigated by

De Silva et al. has exhibited superior imaging potential compared to64Cu–AMD3100.172

Fig. 13 The binuclear 99mTc complex of AMD3100 was evaluated by

Zhang et al. in 2010.173

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that the receptor binding affinity (IC50) of the analogous cold

Cu–AMD3100 complex was 84 nM. This represents a slightly

lower potency compared with the majority of peptide-based

CXCR4 imaging agents. As expected based on the previous

biodistribution studies involving this radiotracer, considerable

uptake was observed in the liver and, to a lesser extent, in the

kidneys. Almost no uptake was found in the blood or muscle

which yielded T/M and T/B ratios of 59.48 � 20 and 41.94 � 14,

respectively. This group also studied a mouse Lewis lung carci-

noma cell line (3LL-XR4) which was transfected with human

CXCR4. In this case, the T/M and T/B ratios were 50.29 � 13.8

and 23.43 � 6.4, respectively.

In each of these studies, blocking experiments supported the

CXCR4-mediated uptake of 64Cu–AMD3100.

A structurally related mono-cyclam, 64Cu–AMD3465

(Fig. 11), was recently reported by De Silva et al. and was shown

to have superior in vivo imaging performance compared to 64Cu–

AMD3100.172 The free ligand, AMD3465, has previously been

shown to bind CXCR4 with high affinity (IC50 ¼ 41.7 �1.2 nM).149 As this group previously examined the imaging

performance of 64Cu–AMD3100 using similar experimental

Fig. 12 PET/CT images of subcutaneous U87 (left flank, unfilled arro

[64Cu]–AMD3465. (A) Transaxial PET, CT, and fused sections recorded at

90minutes (left), 4 h (middle), and 8 h (right) post-injection. Reprinted by perm

Pullambhatla, M., Fox, J.J., Pomper, M.G. and Nimmagadda, S., Imaging C64Cu–AMD3465, J. Nucl. Med., 2011, 52(6), 986–993. Fig. 3.

1046 | Med. Chem. Commun., 2012, 3, 1039–1053

protocols,39 this allows for a reliable comparison between the two

probes. In a similar manner to 64Cu–AMD3100, 64Cu–

AMD3465 was found to continuously accumulate in U87-stb-

CXCR4 xenografts in NOD/SCIDmice, revealing the best image

contrast for U87-stb-CXCR4 tumours at 90 minutes post-injec-

tion (Fig. 12). This was in close agreement with ex vivo bio-

distribution analysis which at 90 minutes yielded the highest

U87-stb-CXCR4 T/M ratio of 362.56 � 153.51. Importantly, at

90 minutes p.i., the T/M and T/B ratios were 7- to 8-fold greater

for 64Cu–AMD3464 compared to 64Cu–AMD3100. Ex vivo

biodistribution analysis revealed that the highest uptake was

found in the U87-stb-CXCR4 tumour at all time points. In a

similar manner to 64Cu–AMD3100, significant uptake was also

observed in the liver, kidneys and bone marrow. It can be

concluded from this study that 64Cu–AMD3465 exhibits supe-

rior binding affinity and kinetics compared to 64Cu–AMD3100.

Using a slightly different approach, Zhang et al. reported their

initial findings into the potential SPECT imaging agent 99mTc–

AMD3100 (Fig. 13).173 Despite early reports indicating that

cyclam is an efficient chelating agent for 99mTc,174,175 the subse-

quent use of bifunctional cyclam agents (such as CPTA176) in99mTc radiolabelling applications reveals quite low radiolabelling

efficiency at physiological conditions, often requiring highly

w) and U87-stb-CXCR4 (right flank, solid arrow) xenografts using

90 minutes post-injection. (B) Volume-rendered whole body images at

ission of the Society of NuclearMedicine from: De Silva, R.A., Peyre, K.,

XCR4 expression in human cancer xenografts: evaluation of monocyclam

This journal is ª The Royal Society of Chemistry 2012

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basic conditions (pH ¼ 10) to achieve higher labelling

yields.177,178 Encouragingly, in this preliminary study, the radio-

labelling reaction was high yielding (98.5 � 1.2%) under mild

reaction conditions. In addition, this radiotracer was shown to be

very stable in both PBS (pH 7.4) and HAS at room temperature.

The biodistribution of this probe in mice bearing human liver

cancer (Hep-G2) xenografts was analysed by SPECT scintig-

raphy. The images obtained at 60 minutes p.i. revealed accu-

mulation at the tumour site, however considerable uptake was

also observed in the liver and kidneys.

Whilst this class of CXCR4-targeting probes evidently has a

tendency to accumulate in the liver and kidneys, the ability of

these radiotracers (particularly, 64Cu–AMD3465) to provide

high-contrast images of CXCR4-expressing tumours is very

promising. It is, however, important to note that AMD3100 has

also been found to behave as an allosteric agonist of the CXCR7

receptor.82 It is therefore plausible that metal complexes of

AMD3100 and its analogues could also have cross-reactivity

with this receptor.

3. Optical imaging probes targeting CXCR4

3.1 Application of fluorophores in receptor imaging

Fluorescent probes are now routinely used in a variety of disci-

plines. In particular, their applications in cell biology, molecular

biology, and immunology have played a crucial role in many of

the most important discoveries in these fields.179–184 Researchers

now have at their disposal fluorophores with a diverse array of

photophysical properties. Furthermore, the ready availability of

fluorophores with reactive functional groups means they can be

covalently attached to many different molecular species.

Recently, fluorescent probes have been applied in vivo as

diagnostic imaging tools with the potential to guide surgeons in

the resection of diseased tissue. For these purposes, fluorophores

which emit in the ultraviolet and visible regions are limited as

light scattering and absorption by endogenous chromophores

leads to considerable signal attenuation. However, near-infrared

Fig. 14 A variety of fluorescently tagged peptide-conjugates derived from Ac

This journal is ª The Royal Society of Chemistry 2012

fluorophores which emit in the range of 700–900 nm can pene-

trate more deeply through living tissue (several centimeters) and

are therefore more useful for in vivo applications.

This section of the review details what has so far been

accomplished in the effort to combine CXCR4-targeting vectors

with optical reporter groups.

3.2 Optical imaging probes derived from T140

Based on previous investigations focused on the development of

T140-based peptides,119 Oishi et al. derived a series of fluo-

rescently tagged peptides and evaluated their specificity and

affinity for CXCR4.185 The peptides were labelled with fluores-

cein (Exmax/Emmax ¼ 494/520 nm), Alexa Fluor� 488 (Exmax/

Emmax ¼ 495/519 nm) or biotin, either via acylation to the

a-amino group of the N-terminal arginine residue (Arg1) or to

the D-Lys8 residue via the 3-amino group (Fig. 14). It was found

that fluorescent labelling at the N-terminus typically resulted in

substantially lower binding affinities for CXCR4, whilst labelling

at the 3-amino group of the D-Lys8 residue caused only minor

reductions. Two of the most favourable peptides, modified with

fluorescein and Alexa Fluor� 488 fluorophores at the D-Lys8

residue retained high affinity for CXCR4, yielding IC50 values of

16� 0.8 nM and 8.1� 3.5 nM, respectively. This indicates only a

minor reduction in binding activity for CXCR4 compared to

T140 (IC50 ¼ 0.93–4 nM).117,118 A notable feature of these

peptides is that they did not appear to label CXCR7-expressing

HEK293 cells which supports the selectivity of these peptides for

CXCR4.

Two similar fluorescent derivatives of Ac-TZ14011, in this case

incorporating a hexamethylene spacer between each fluorescent

group and the peptide, were examined by Nomura et al. in 2008

(Fig. 14).124 These peptide conjugates were synthesised via

the covalent attachment of either fluorescein or TAMRA

(Exmax/Emmax ¼ 555/576 nm) to the D-Lys8 residue. The hex-

amethylene spacer was incorporated in order to distance the

bulky fluorescent group from the pharmacophore, thereby

limiting any potential loss of binding activity for CXCR4. The

-TZ14011 that have been evaluated as optical probes for CXCR4.124,185,186

Med. Chem. Commun., 2012, 3, 1039–1053 | 1047

Fig. 15 In 2011, Kuil et al. developed and evaluated a series of Ac-TZ14011 peptides derivatised with fluorescent iridium complexes.188

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binding affinities (IC50) of the fluorescein- and TAMRA-conju-

gated peptides were 11 and 14 nM, respectively. As these values

are in the same order compared with analogous peptide conju-

gates which do not contain a spacer group, it appears that

modifications of this type are not crucial in maintaining high

potency for CXCR4.

In addition, Ac-TZ14011 has also been labelled with carbox-

yfluorescein (to assist in the visualisation of bladder cancer

cells)187 and FITC (Fig. 14).186

A particularly innovative study focused on the development of

a series of iridium(III)-containing peptide conjugates was recently

conducted by Kuil et al. (Fig. 15).188 This work represents the

first application of neutral iridium(III)–peptide conjugates in

fluorescence lifetime imaging microscopy (FLIM) of a cell

membrane receptor associated with cancer. This group syn-

thesised three peptide–Ir(III) complex conjugates; Ir–(Ac-

TZ14011), Ir–(Ac-TZ14011)2, and Ir–(Ac-TZ14011)3, and

established dissociation constants for CXCR4 of 84.4 � 13.7,

254.4 � 81.3, and 66.3 � 28.0 nM, respectively. This group used

the crystal structure of CXCR4 (ref. 84) in the design process of

these peptides and concluded that the luminescent Ir(III) complex

would not be likely to interfere with the receptor binding of the

Ac-TZ14011 peptide. The luminescent lifetimes of the three Ir(III)

complexes containing either one, two or three b-alanine linkers

were measured and all found to be in the order of 200 ns. MDA-

MB-231CXCR4+ cells incubated with Ir–(Ac-TZ14011) were also

subjected to FLIM analysis. The long luminescent lifetimes of

the Ir(III) complex yielded images with good signal-to-noise.

All of the fluorescent peptides derived from Ac-TZ14011 and

modified at the D-Lys8 residue reveal an ability to selectively label

CXCR4-expressing cells. In most cases, this has been established

either by competition experiments with unlabelled CXCR4-

specific peptides (e.g. Ac-TZ14011 or SDF-1), or by measuring

enhanced signal on cells with high expression levels of CXCR4

compared against a control cell line. Rather than remaining on

the cell surface, many of these fluorescent peptides reveal

evidence of endocytotic internalisation in vesicular structures

within the cytoplasm.124,186,188

Only one of these studies has so far investigated the possibility

of CXCR7 binding which, importantly, found no interaction

with this receptor.185 This presents an advantage over probes

1048 | Med. Chem. Commun., 2012, 3, 1039–1053

derived from SDF-1a (and, potentially, AMD3100), which are

known to bind both CXCR4 and CXCR7. Whilst the ability to

probe CXCR7 expression will likely be a useful endeavour as the

role of this important pairing becomes better understood, the

present aim to selectively bind CXCR4 and quantify its expres-

sion without interference so far remains the most suitable

approach.

Most of the fluorophores used in these studies emit in the

visible region of the spectrum which, while useful for in vitro and

ex vivo experiments, presents some limitations for in vivo imaging

where near-infrared fluorophores are superior due to greater

tissue penetration and reduced signal attenuation. The combi-

nation of T140-based peptides or cyclic pentapeptides (e.g.

FC131) with fluorophores that emit in the near-infrared region

could afford useful in vivo diagnostic tools capable of yielding

highly sensitive and specific information.

These promising results exemplify the great potential of

CXCR4-specific peptides in the diagnosis and monitoring of

disease states. This has already been successfully applied in the

visualisation of bladder cancer cells187 and this approach could

also be applicable to other types of cancer in which CXCR4

expression is upregulated.

3.3 Optical imaging probes dervied from SDF-1

Some of the earliest fluorescently tagged CXCR4 targeting

vectors were derived from the natural ligand of CXCR4, SDF-

1.189,190 In 2005, Dar et al. utilised fluorescein isothiocyanate

FITC–SDF-1 in order to examine the CXCR4-dependent inter-

nalization of SDF-1 by bone marrow endothelial cells.189 Soon

after, in 2006, Kollet et al. also used FITC–SDF-1 in their study

into the role of bone-resorbing osteoclasts in homeostasis and

stress-induced mobilisation of progenitor cells.190

More recently, Meincke et al. evaluated a fluorescent deriva-

tive of SDF-1 conjugated with the near-infrared dye,

IRDye�800CW (Exmax/Emmax ¼ 778/794 nm).191 Two cell lines

were chosen for in vitro testing, these were MCF-7 cells which

express both CXCR4 and CXCR7 receptors, and the glioma cell

line A764 which expresses CXCR7 but not CXCR4. For both

cell lines, as few as 500 adherent cells could be detected following

incubation with SDF-1–IRDye�800CW. This supports, as

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expected, that SDF-1-derived probes bind to CXCR7 in addition

to binding CXCR4. In vivo evaluation in tumour-bearing mice

revealed the probe distributed rapidly and was almost entirely

cleared within 24 h by the kidneys. This is in accordance with

what was observed for the SPECT probe, 99mTc–SDF-1.148 After

1 h, the tumours were shown to be fluorescently labelled, and the

liver, skull and bone marrow were also visible. The background

signal diminished between 24 and 92 h, while the tumours could

still be easily detected.

3.4 Small molecule optical imaging agents

In 2007, Khan et al. developed a fluorescent small molecule with

a similar structural motif to that of AMD3100.192 This low

molecular weight compound contains a side-bridged cyclam

macrocycle and a fluorescent rhodamine group (Exmax/Emmax ¼570/595 nm) (Fig. 16). Both the free ligand and the corre-

sponding mononuclear copper(II) complex were evaluated in

vitro to assess their CXCR4 binding properties. The presence of

copper had a substantial (ca. 75%) quenching effect upon the

rhodamine fluorescence, however the remaining fluorescence

emission was still sufficient to be detected. In vitro studies

involving Jurkat T cells revealed that the free ligand had poor

binding characteristics while the copper complex was able inhibit

the binding of CXCR4-specific antibodies. Unfortunately, both

the free ligand and the copper complex revealed non-specific

cellular uptake which was shown by blocking studies to be via an

active transport process.

Another fluorescent small molecule (Fig. 17) derived from

AMD3100 and a series of transition metal complexes have also

recently been developed.193 The fluorescent group in this case was

Fig. 16 A rhodamine-containing small molecule with structural simi-

larities to AMD3100 was evaluated in vitro by Khan et al. in 2007.192

Fig. 17 Knight et al. evaluated an anthracenyl derivative of AMD3100

and its corresponding binuclear Zn2+ and Cu2+ complexes which were

shown to enhance and quench the fluorescence, respectively.193

This journal is ª The Royal Society of Chemistry 2012

anthracene which was situated between the two cyclam rings,

representing an extension of the para-xylyl group of the parental

structure, AMD3100. This modification was selected as it rep-

resented a relatively minor structural change compared to the

parent compound which is known to be a potent binder of

CXCR4. In a similar manner to the previous study, complexa-

tion with copper was shown to quench the fluorescence emission

(in this case, dramatically). In contrast, the binuclear zinc

complex revealed an enhancement in fluorescence intensity. The

binding affinities of the free ligand and the binuclear zinc

complex were substantially reduced compared to AMD3100 (0.6

and 3.4 mM, respectively), which limits their usefulness as fluo-

rescent probes. The free ligand exhibited modest specific binding

at 1 mM for cells stably transfected with CXCR4 (300-19(cl.5))

compared to a control cell line (300-19), however this specificity

was diminished at higher concentrations. This indicates that the

compound was either binding to non-specific sites at the cell

surface or was being internalised into the cell. The binuclear zinc

complex revealed a higher degree of specific binding and also

lower cytotoxicity compared to the free ligand. It was postulated

that the incorporation of anthracene contributes to the lipophilic

character of the molecule and facilitates its transport across the

plasma membrane. However, this effect is seemingly offset, at

least to a degree, by complexation to charged metal ions.

3.5 Metal nanoshells

A different approach aimed at quantifying CXCR4 expression

using optical imaging has recently been developed by Zhang

et al.194 This group synthesised metal nanoshells consisting of

silica cores (50 nm) with silver walls (10 nm) encapsulating ca.

120 fluorescent Ru(bpy)32+ complexes within each nanoshell

core. To each nanoshell, anti-CXCR4 monoclonal antibodies

were covalently attached via a condensation reaction with

carboxylate groups located in an organic monolayer on the outer

surface of the shell. These metal nanoshells exhibited strong

fluorescence emission and distinctive lifetimes which were easily

distinguishable from cellular autofluorescence. Using this

approach, this group were able to quantify the number of

CXCR4 receptors on CEM-SS cells derived from CD4-positive

T-lymphocytes.

4. Bimodal agents targeting CXCR4

The combination of two or more diagnostic modalities by uti-

lising bimodal, or indeed multimodal, imaging probes is an

exciting and rapidly developing area of research.195,196 This

approach has the potential to combine the inherent advantages

of each imaging modality in a synergistic manner whilst

decreasing the disadvantages. Whilst the incorporation of two or

more reporter groups within a selected targeting vector typically

necessitates more elaborate chemical modification compared to

monomodal probes (thus risking decreases in ligand potency for

the intended target), the use of suitably optimised ligands could

potentially enable a two-for-one approach.

An excellent example of a bimodal imaging agent has been

recently reported by Kuil et al. in their development of a dual

SPECT/fluorescence peptidic imaging agent (Fig. 18).197 This

group conjugated a multifunctional single-attachment-point

Med. Chem. Commun., 2012, 3, 1039–1053 | 1049

Fig. 18 A bimodal (SPECT/fluorescence) imaging agent developed by

Kuil et al. is capable of distinguishing between CXCR4 positive and

CXCR4 negative tumours in vivo.197

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(MSAP) reagent via an active NHS ester to the D-lysine residue of

Ac-TZ14011. The MSAP reagent contains both a CyAL-5.5

fluorescent group and a DTPA chelating moiety which was used

in this instance to coordinate indium.Whilst the receptor binding

affinity of this peptide-conjugate was reduced approximately 20-

fold compared to the parent Ac-TZ14011 peptide, both Ac-

TZ14011–MSAP and Ac-TZ14011–MSAP–In proved capable of

discriminating between MDA-MB-231CXCR4+ and basal MDA-

MB-231 cells with comparable performance to a phycoerythrin

(PE)-labelled anti-CXCR4 monoclonal antibody. The imaging

performance of the 111In-labelled probe was evaluated in mice

bearing CXCR4 positive MIN-O tumours. Promisingly, both

SPECT and fluorescence imaging modalities were able to confirm

high tumour uptake. Biodistribution studies on mice bearing

both MIN-O and CXCR4 negative 4T1 tumours were also

conducted, revealing T/M ratios of 4.55 � 0.68 and 1.20 � 0.12,

respectively. These results indicate that this peptide is clearly

capable of distinguishing CXCR4 positive tumours in vivo. This

combined approach enables the acquisition of vivid images

through SPECT, whilst the optical component could be used to

guide the surgical resection of tumours.

5. Summary and future perspectives

As the important role of CXCR4 in a number of diseases,

including cancer, is rapidly becoming clear, the development of a

wide variety of diagnostic probes targeted at this receptor has

also been advancing at a similar pace. Over the last five years,

much progress has been made combining an assortment of tar-

geting vectors with both nuclear and optical reporter groups.

Great progress has already been made in this respect and the

recent crystal structure determination of CXCR4 will undoubt-

edly facilitate the rational development of more tailored ligands

with yet higher binding affinities and specificity for this impor-

tant receptor.

Some particularly innovative research has recently led to the

development of a bimodal (SPECT/fluorescence) CXCR4

imaging agent. It seems likely that similar probes combining

multiple reporter groups, e.g. PET/SPECT, optical, and/or MRI,

1050 | Med. Chem. Commun., 2012, 3, 1039–1053

will be developed in the near future. The complementary data

acquired in this manner will help shed more light on the natural

function of this important receptor and its role in various

diseases, including cancer.

The application of other targeting systems, such as micelles,

nanoparticles, and dendrimers functionalised with multiple

CXCR4 targeting moieties, e.g.Ac-TZ14011, might also prove to

be an effective imaging strategy, particularly as this would enable

the delivery of multiple reporter groups to areas of disease with

high CXCR4 expression. Moreover, such targeting vectors could

also be labelled in parallel with a variety of other species capable

of confering yet more favourable properties, e.g. solubility, blood

clearance kinetics, tumour penetration, metabolic stability.

Clearly, there remains much scope for development in the

design of CXCR4-targeted imaging agents. This active area of

research will ultimately yield the ability to accurately quantify

CXCR4 expression in vitro, ex vivo and in vivo. Given the well-

documented involvement of the CXCR4 receptor in a variety of

cancer types, this could be of significant benefit for patients.

Acknowledgements

We thank the Natural Sciences and Engineering Research

Council of Canada (NSERC) and the Dianne and Irving Kipnes

Foundation for funding this work.

Notes and references

1 A. Viola and A. D. Luster,Annu. Rev. Pharmacol. Toxicol., 2008, 48,171–197.

2 E. J. Fernandez and E. Lolis, Annu. Rev. Pharmacol. Toxicol., 2002,42, 469–499.

3 A. Zlotnik, O. Yoshie andH. Nomiyama,Genome Biol., 2006, 7, 243.4 P. M. Murphy, M. Baggiolini, I. F. Charo, C. A. Hebert, R. Horuk,K. Matsushima, L. H. Miller, J. J. Oppenheim and C. A. Power,Pharmacol. Rev., 2000, 52, 145–176.

5 A. Zlotnik and O. Yoshie, Immunity, 2000, 12, 121–127.6 P. M. Murphy, Pharmacol. Rev., 2002, 54, 227–229.7 R. M. Ransohoff, K. Suzuki, A. E. Proudfoot, W. F. Hickey andJ. K. Harrison, Elsevier, Amsterdam, Universes in DelicateBalance: Chemokines and the Nervous System, 2002

8 W. Rostene, P. Kitabgi and S. M. Parsadaniantz, Nat. Rev.Neurosci., 2007, 8, 895–903.

9 P. Loetscher, B. Moser and M. Baggiolini, Adv. Immunol., 2000, 74,127–180.

10 M. Baggiolini, Nature, 1998, 392, 565–568.11 B. Moser and P. Loetscher, Nat. Immunol., 2001, 2, 123–128.12 M. Rotondi, L. Chiovato, S. Romagnani, M. Serio and

P. Romagnani, Endocr. Rev., 2007, 28, 492–520.13 S. Singh, A. Sadanandam and R. K. Singh, Cancer Metastasis Rev.,

2007, 26, 453–467.14 R. Fredriksson, M. C. Lagerstrom, L.-G. Lundin and H. B. Schioth,

Mol. Pharmacol., 2003, 63, 1256–1272.15 M. Kucia, K. Jankowski, R. Reca, M. Wysoczynski, L. Bandura,

D. J. Allendorf, J. Zhang, J. Ratajczak and M. Z. Ratajczak, J.Mol. Histol., 2004, 35, 233–245.

16 T. Nagasawa, S. Hirota, K. Tachibana, N. Takakura,S.-i. Nishikawa, Y. Kitamura, N. Yoshida, H. Kikutani andT. Kishimoto, Nature, 1996, 382, 635–638.

17 Y.-R. Zou, A. H. Kottmann, M. Kuroda, I. Taniuchi andD. R. Littman, Nature, 1998, 393, 595–599.

18 Q. Ma, D. Jones, P. R. Borghesani, R. A. Segal, T. Nagasawa,T. Kishimoto, R. T. Bronson and T. A. Springer, Proc. Natl.Acad. Sci. U. S. A., 1998, 95, 9448–9453.

19 K. E. McGrath, A. D. Koniski, K. M. Maltby, J. K. McGann andJ. Palis, Dev. Biol., 1999, 213, 442–456.

20 T. Nagasawa, K. Tachibana and T. Kishimoto, Semin. Immunol.,1998, 10, 179–185.

This journal is ª The Royal Society of Chemistry 2012

Publ

ishe

d on

23

July

201

2. D

ownl

oade

d by

Uni

vers

ity o

f O

xfor

d on

21/

11/2

014

10:5

3:11

. View Article Online

21 K. Tachibana, S. Hirota, H. Iizasa, H. Yoshida, K. Kawabata,Y. Kataoka, Y. Kitamura, K. Matsushima, N. Yoshida,S.-i. Nishikawa, T. Kishimoto and T. Nagasawa, Nature, 1998,393, 591–594.

22 Y. Feng, C. C. Broder, P. E. Kennedy and E. A. Berger, Science,1996, 272, 872–877.

23 B. Furusato, A. Mohamed, M. Uhl�en and J. S. Rhim, Pathol. Int.,2010, 60, 497–505.

24 A. Zlotnik, Contrib. Microbiol., 2006, 13, 191–199.25 J. A. Burger and T. J. Kipps, Blood, 2006, 107, 1761–1767.26 M. Kucia, R. Reca, K. Miekus, J. Wanzeck, W. Wojakowski,

A. Janowska-Wieczorek, J. Ratajczak and M. Z. Ratajczak, StemCells, 2005, 23, 879–894.

27 A. Zlotnik, Int. J. Cancer, 2006, 119, 2026–2029.28 Y.-X. Sun, J. Wang, C. E. Shelburne, D. E. Lopatin,

A. M. Chinnaiyan, M. A. Rubin, K. J. Pienta andR. S. Taichman, J. Cell. Biochem., 2003, 89, 462–473.

29 T. Schioppa, B. Uranchimeg, A. Saccani, S. K. Biswas, A. Doni,A. Rapisarda, S. Bernasconi, S. Saccani, M. Nebuloni, L. Vago,A. Mantovani, G. Melillo and A. Sica, J. Exp. Med., 2003, 198,1391–1402.

30 P. Staller, J. Sulitkova, J. Lisztwan, H. Moch, E. J. Oakeley andW. Krek, Nature, 2003, 425, 307–311.

31 D. Zagzag, B. Krishnamachary, H. Yee, H. Okuyama, L. Chiriboga,M. A. Ali, J. Melamed and G. L. Semenza, Cancer Res., 2005, 65,6178–6188.

32 D. G. Duda, S. V. Kozin, N. D. Kirkpatrick, L. Xu, D. Fukumuraand R. K. Jain, Clin. Cancer Res., 2011, 17, 2074–2080.

33 A. Muller, B. Homey, H. Soto, N. Ge, D. Catron, M. E. Buchanan,T. McClanahan, E. Murphy, W. Yuan, S. N. Wagner, J. L. Barrera,A. Mohar, E. Verastegui and A. Zlotnik, Nature, 2001, 410, 50–56.

34 C. J. Scotton, J. L. Wilson, D. Milliken, G. Stamp andF. R. Balkwill, Cancer Res., 2001, 61, 4961–4965.

35 B. A. Teicher and S. P. Fricker, Clin. Cancer Res., 2010, 16, 2927–2931.

36 K. E. Luker and G. D. Luker, Cancer Lett., 2006, 238, 30–41.37 O. Salvucci, A. Bouchard, A. Baccarelli, J. Deschenes, G. Sauter,

R. Simon, R. Bianchi and M. Basik, Breast Cancer Res. Treat.,2006, 97, 275–283.

38 L. V. Rhodes, S. P. Short, N. F. Neel, V. A. Salvo, Y. Zhu, S. Elliott,Y. Wei, D. Yu, M. Sun, S. E. Muir, J. P. Fonseca, M. R. Bratton,C. Segar, S. L. Tilghman, T. Sobolik-Delmaire, L. W. Horton,S. Zaja-Milatovic, B. M. Collins-Burow, S. Wadsworth,B. S. Beckman, C. E. Wood, S. A. Fuqua, K. P. Nephew, P. Dent,R. A. Worthylake, T. J. Curiel, M.-C. Hung, A. Richmond andM. E. Burow, Cancer Res., 2011, 71, 603–613.

39 S. Nimmagadda, M. Pullambhatla, K. Stone, G. Green,Z. M. Bhujwalla and M. G. Pomper, Cancer Res., 2010, 70, 3935–3944.

40 R. S. Taichman, C. Cooper, E. T. Keller, K. J. Pienta,N. S. Taichman and L. K. McCauley, Cancer Res., 2002, 62,1832–1837.

41 P. Kukreja, A. B. Abdel-Mageed, D. Mondal, K. Liu andK. C. Agrawal, Cancer Res., 2005, 65, 9891–9898.

42 M. Darash-Yahana, E. Pikarsky, R. Abramovitch, E. Zeira, B. Pal,R. Karplus, K. Beider, S. Avniel, S. Kasem, E. Galun and A. Peled,FASEB J., 2004, 18, 1240–1242.

43 T. Engl, B. Relja, D. Marian, C. Blumenberg, I. Mueller,W.-D. Beecken, J. Jones, E. M. Ringel, J. Bereiter-Hahn, D. Jonasand R. A. Blaheta, Neoplasia, 2006, 8, 290–301.

44 Y. Suzuki, M. Rahman and H. Mitsuya, J. Immunol., 2001, 167,3064–3073.

45 Y.-X. Sun, A. Schneider, Y. Jung, J. Wang, J. Dai, J. Wang,K. Cook, N. I. Osman, A. J. Koh-Paige, H. Shim, K. J. Pienta,E. T. Keller, L. K. McCauley and R. S. Taichman, J. Bone Miner.Res., 2005, 20, 318–329.

46 S. Singh, U. P. Singh, W. E. Grizzle and J. W. Lillard, Jr, Lab.Invest., 2004, 84, 1666–1676.

47 Y.-X. Sun, E. A. Pedersen, Y. Shiozawa, A. M. Havens, Y. Jung,J. Wang, K. J. Pienta and R. S. Taichman, Clin. Exp. Metastasis,2008, 25, 765–776.

48 Y. Xing, M. Liu, Y. Du, F. Qu, Y. Li, Q. Zhang, Y. Xiao, J. Zhao,F. Zeng and C. Xiao, Cancer Biol. Ther., 2008, 7, 1839–1848.

49 N. T. Holm, K. Byrnes, B. D. L. Li, R. H. Turnage, F. Abreo,J. M. Mathis and Q. D. Chu, J. Surg. Res., 2007, 141, 53–59.

This journal is ª The Royal Society of Chemistry 2012

50 H. Geminder, O. Sagi-Assif, L. Goldberg, T. Meshel, G. Rechavi,I. P. Witz and A. Ben-Baruch, J. Immunol., 2001, 167, 4747–4757.

51 C. Porcile, A. Bajetto, S. Barbero, P. Pirani and G. Schettini,Ann. N.Y. Acad. Sci., 2004, 1030, 162–169.

52 J. M. Hall and K. S. Korach, Mol. Endocrinol., 2003, 17, 792–803.

53 J. Libura, J. Drukala, M. Majka, O. Tomescu, J. M. Navenot,M. Kucia, L. Marquez, S. C. Peiper, F. G. Barr, A. Janowska-Wieczorek and M. Z. Ratajczak, Blood, 2002, 100, 2597–2606.

54 K. Jankowski, M. Kucia, M. Wysoczynski, R. Reca, D. Zhao,E. Trzyna, J. Trent, S. Peiper, M. Zembala, J. Ratajczak,P. Houghton, A. Janowska-Wieczorek and M. Z. Ratajczak,Cancer Res., 2003, 63, 7926–7935.

55 M. Allinen, R. Beroukhim, L. Cai, C. Brennan, J. Lahti-Domenici,H. Huang, D. Porter, M. Hu, L. Chin, A. Richardson, S. Schnitt,W. R. Sellers and K. Polyak, Cancer Cell, 2004, 6, 17–32.

56 C. L. Gladson and D. R. Welch, Cancer Biol. Ther., 2008, 7, 1849–1851.

57 M. Burger, A. Glodek, T. Hartmann, A. Schmitt-Graeff,L. E. Silberstein, N. Fujii, T. J. Kipps and J. A. Burger, Oncogene,2003, 22, 8093–8101.

58 J. H. Hwang, J. H. Hwang, H. K. Chung, D. W. Kim, E. S. Hwang,J. M. Suh, H. Kim, K.-H. You, O. Y. Kwon, H. K. Ro, D. Y. Jo andM. Shong, J. Clin. Endocrinol. Metab., 2003, 88, 408–416.

59 T. Kijima, G. Maulik, P. C. Ma, E. V. Tibaldi, R. E. Turner,B. Rollins, M. Sattler, B. E. Johnson and R. Salgia, Cancer Res.,2002, 62, 6304–6311.

60 C. C. Schimanski, R. Bahre, I. Gockel, A. Mueller, K. Frerichs,V. Hoerner, A. Teufel, N. Simiantonaki, S. Biesterfeld, T. Wehler,M. Schuler, T. Achenbach, T. Junginger, P. R. Galle andM. Moehler, Br. J. Cancer, 2006, 95, 210–217.

61 T. Nagasawa, T. Nakajima, K. Tachibana, H. Iizasa, C. C. Bleul,O. Yoshie, K. Matsushima, N. Yoshida, T. A. Springer andT. Kishimoto,Proc. Natl. Acad. Sci. U. S. A., 1996, 93, 14726–14729.

62 X. Sun, G. Cheng, M. Hao, J. Zheng, X. Zhou, J. Zhang,R. Taichman, K. Pienta and J. Wang, Cancer Metastasis Rev.,2010, 29, 709–722.

63 Z.Miao, K. E. Luker, B. C. Summers, R. Berahovich,M. S. Bhojani,A. Rehemtullat, C. G. Kleer, J. J. Essner, A. Nasevicius,G. D. Luker, M. C. Howard and T. J. Schall, Proc. Natl. Acad.Sci. U. S. A., 2007, 104, 15735–15740.

64 G. Valentin, P. Haas and D. Gilmour, Curr. Biol., 2007, 17, 1026–1031.

65 B. A. Zabel, Y. Wang, S. Lew�en, R. D. Berahovich,M. E. T. Penfold, P. Zhang, J. Powers, B. C. Summers, Z. Miao,B. Zhao, A. Jalili, A. Janowska-Wieczorek, J. C. Jaen andT. J. Schall, J. Immunol., 2009, 183, 3204–3211.

66 F. Sierro, C. Biben, L. Mart�ınez-Mu~noz, M. Mellado,R. M. Ransohoff, M. Li, B. Woehl, H. Leung, J. Groom,M. Batten, R. P. Harvey, C. Mart�ınez-A, C. R. Mackay andF. Mackay, Proc. Natl. Acad. Sci. U. S. A., 2007, 104, 14759–14764.

67 J. Wang, Y. Shiozawa, J. Wang, Y. Wang, Y. Jung, K. J. Pienta,R. Mehra, R. Loberg and R. S. Taichman, J. Biol. Chem., 2008,283, 4283–4294.

68 M. Thelen and S. Thelen, J. Neuroimmunol., 2008, 198, 9–13.69 S. Rajagopal, J. Kim, S. Ahn, S. Craig, C. M. Lam, N. P. Gerard,

C. Gerard and R. J. Lefkowitz, Proc. Natl. Acad. Sci. U. S. A.,2010, 107, 628–632.

70 T. N. Hartmann, V. Grabovsky, R. Pasvolsky, Z. Shulman,E. C. Buss, A. Spiegel, A. Nagler, T. Lapidot, M. Thelen andR. Alon, J. Leukocyte Biol., 2008, 84, 1130–1140.

71 B. Mazzinghi, E. Ronconi, E. Lazzeri, C. Sagrinati, L. Ballerini,M. L. Angelotti, E. Parente, R. Mancina, G. S. Netti,F. Becherucci, M. Gacci, M. Carini, L. Gesualdo, M. Rotondi,E. Maggi, L. Lasagni, M. Serio, S. Romagnani and P. Romagnani,J. Exp. Med., 2008, 205, 479–490.

72 A. l. Levoye, K. Balabanian, F. o. Baleux, F. o. Bachelerie andB. Lagane, Blood, 2009, 113, 6085–6093.

73 F. M. Decaillot, M. A. Kazmi, Y. Lin, S. Ray-Saha, T. P. Sakmarand P. Sachdev, J. Biol. Chem., 2011, 286, 32188–32197.

74 K. Balabanian, B. Lagane, S. Infantino, K. Y. Chow, J. Harriague,B. Moepps, F. Arenzana-Seisdedos, M. Thelen and F. o. Bachelerie,J. Biol. Chem., 2005, 280, 35760–35766.

75 J. M. Burns, B. C. Summers, Y. Wang, A. Melikian, R. Berahovich,Z. Miao, M. E. Penfold, M. J. Sunshine, D. R. Littman, C. J. Kuo,

Med. Chem. Commun., 2012, 3, 1039–1053 | 1051

Publ

ishe

d on

23

July

201

2. D

ownl

oade

d by

Uni

vers

ity o

f O

xfor

d on

21/

11/2

014

10:5

3:11

. View Article Online

K.Wei, B. E. McMaster, K. Wright, M. C. Howard and T. J. Schall,J. Exp. Med., 2006, 203, 2201–2213.

76 K. Hattermann, J. Held-Feindt, R. Lucius, S. S. M€uerk€oster,M. E. Penfold, T. J. Schall and R. Mentlein, Cancer Res., 2010,70, 3299–3308.

77 T. Goldmann, D. Dromann, J. Radtke, S. Marwitz, D. S. Lang,H. Schultz and E. Vollmer, Virchows Arch., 2008, 452, 347–348.

78 E. Schutyser, Y. Su, Y. Yu, M. Gouwy, S. Zaja-Milatovic, D. J. Vanand A. Richmond, Eur. Cytokine Network, 2007, 18, 59–70.

79 K. E. Luker, M. Gupta and G. D. Luker, FASEB J., 2009, 23, 823–834.

80 B. Boldajipour, H. Mahabaleshwar, E. Kardash, M. Reichman-Fried, H. Blaser, S. Minina, D. Wilson, Q. Xu and E. Raz, Cell,2008, 132, 463–473.

81 C. Dambly-Chaudiere, N. Cubedo and A. Ghysen, BMC Dev. Biol.,2007, 7, 23.

82 I. Kalatskaya, Y. A. Berchiche, S. Gravel, B. J. Limberg,J. S. Rosenbaum and N. Heveker, Mol. Pharmacol., 2009, 75,1240–1247.

83 K. E. Luker, M. Gupta, J. M. Steele, B. R. Foerster andG. D. Luker, Neoplasia, 2009, 11, 1022–1035.

84 B. Wu, E. Y. T. Chien, C. D. Mol, G. Fenalti, W. Liu, V. Katritch,R. Abagyan, A. Brooun, P. Wells, F. C. Bi, D. J. Hamel, P. Kuhn,T. M. Handel, V. Cherezov and R. C. Stevens, Science, 2010, 330,1066–1071.

85 B. K. Shoichet, Nature, 2004, 432, 862–865.86 W.-T. Choi, S. Duggineni, Y. Xu, Z. Huang and J. An, J. Med.

Chem., 2012, 55, 977–994.87 L. E. Woodard and S. Nimmagadda, J. Nucl. Med., 2011, 52, 1665–

1669.88 S. M. Ametamey, M. Honer and P. A. Schubiger, Chem. Rev., 2008,

108, 1501–1516.89 P. W.Miller, N. J. Long, R. Vilar and A. D. Gee, Angew. Chem., Int.

Ed., 2008, 47, 8998–9033.90 S. L. Pimlott and A. Sutherland,Chem. Soc. Rev., 2011, 40, 149–162.91 S. S. Gambhir, Nat. Rev. Cancer, 2002, 2, 683–693.92 M. E. Phelps, Proc. Natl. Acad. Sci. U. S. A., 2000, 97, 9226–9233.93 M. E. Phelps, J. Nucl. Med., 2000, 41, 661–681.94 T. F. Massoud and S. S. Gambhir, Genes Dev., 2003, 17, 545–580.95 A. M. J. Paans, A. van Waarde, P. H. Elsinga, A. T. M. Willemsen

and W. Vaalburg, Methods, 2002, 27, 195–207.96 T. J. McCarthy, S. W. Schwarz and M. J. Welch, J. Chem. Educ.,

1994, 71, 830.97 J. Czernin and M. E. Phelps, Annu. Rev. Med., 2002, 53, 89–112.98 K. Chen and X. Chen, Curr. Top. Med. Chem., 2010, 1227–1236.99 K. Chen and P. S. Conti, Adv. Drug Delivery Rev., 2010, 62, 1005–

1022.100 P. H. Elsinga, Methods, 2002, 27, 208–217.101 V. W. Pike, Drug Inf. J., 1997, 31, 997–1013.102 M. C. Lasne, C. Perrio, J. Rouden, L. Barr�e, D. Roeda, F. Doll�e,

C. Crouzel, Chemistry of b+-emitting Compounds Based onFluorine-18, in Topics in Current Chemistry, ed. W. Krauss,vol.222, Springer-Verlag, Berlin-Heidelberg, 2002, pp. 201–258.

103 B. M. Zeglis and J. S. Lewis, Dalton Trans., 2011, 40, 6168–6195.104 C. J. Anderson and M. J. Welch, Chem. Rev., 1999, 99, 2219–2234.105 T. J. Wadas, E. H.Wong, G. R.Weisman and C. J. Anderson,Chem.

Rev., 2010, 110, 2858–2902.106 S. Nimmagadda, M. Pullambhatla and M. G. Pomper, J. Nucl.

Med., 2009, 50, 1124–1130.107 M. W. Brechbiel, Q. J. Nucl. Med. Mol. Imag., 2008, 52, 166–173.108 H. Tamamura, Y. Xu, T. Hattori, X. Zhang, R. Arakaki,

K. Kanbara, A. Omagari, A. Otaka, T. Ibuka, N. Yamamoto,H. Nakashima and N. Fujii, Biochem. Biophys. Res. Commun.,1998, 253, 877–882.

109 H. Tamamura, A. Omagari, S. Oishi, T. Kanamoto, N. Yamamoto,S. C. Peiper, H. Nakashima, A. Otaka and N. Fujii, Bioorg. Med.Chem. Lett., 2000, 10, 2633–2637.

110 H. Tamamura, M. Sugioka, Y. Odagaki, A. Omagari, Y. Kan,S. Oishi, H. Nakashima, N. Yamamoto, S. C. Peiper,N. Hamanaka, A. Otaka and N. Fujii, Bioorg. Med. Chem. Lett.,2001, 11, 359–362.

111 H. Tamamura, A. Omagari, K. Hiramatsu, K. Gotoh,T. Kanamoto, Y. Xu, E. Kodama, M. Matsuoka, T. Hattori,N. Yamamoto, H. Nakashima, A. Otaka and N. Fujii, Bioorg.Med. Chem. Lett., 2001, 11, 1897–1902.

1052 | Med. Chem. Commun., 2012, 3, 1039–1053

112 W.-b. Zhang, J.-M. Navenot, B. Haribabu, H. Tamamura,K. Hiramatu, A. Omagari, G. Pei, J. P. Manfredi, N. Fujii,J. R. Broach and S. C. Peiper, J. Biol. Chem., 2002, 277, 24515–24521.

113 H. Tamamura, A. Hori, N. Kanzaki, K. Hiramatsu, M. Mizumoto,H. Nakashima, N. Yamamoto, A. Otaka and N. Fujii, FEBS Lett.,2003, 550, 79–83.

114 H. Tamamura, K. Hiramatsu, S. Kusano, S. Terakubo,N. Yamamoto, J. O. Trent, Z. Wang, S. C. Peiper, H. Nakashima,A. Otaka and N. Fujii, Org. Biomol. Chem., 2003, 1, 3656–3662.

115 H. Tamamura, H. Tsutsumi, H. Masuno, S. Mizokami,K. Hiramatsu, Z. Wang, J. O. Trent, H. Nakashima,N. Yamamoto, S. C. Peiper and N. Fujii, Org. Biomol. Chem.,2006, 4, 2354–2357.

116 H. Tamamura, K. Hiramatsu, M. Mizumoto, S. Ueda, S. Kusano,S. Terakubo, M. Akamatsu, N. Yamamoto, J. O. Trent, Z. Wang,S. C. Peiper, H. Nakashima, A. Otaka and N. Fujii, Org. Biomol.Chem., 2003, 1, 3663–3669.

117 N. Fujii, S. Oishi, K. Hiramatsu, T. Araki, S. Ueda, H. Tamamura,A. Otaka, S. Kusano, S. Terakubo, H. Nakashima, J. A. Broach,J. O. Trent, Z.-x. Wang and S. C. Peiper, Angew. Chem., Int. Ed.,2003, 42, 3251–3253.

118 H. Tamamura, K. Hiramatsu, S. Ueda, Z. Wang, S. Kusano,S. Terakubo, J. O. Trent, S. C. Peiper, N. Yamamoto,H. Nakashima, A. Otaka and N. Fujii, J. Med. Chem., 2005, 48,380–391.

119 H. Hanaoka, T. Mukai, H. Tamamura, T. Mori, S. Ishino,K. Ogawa, Y. Iida, R. Doi, N. Fujii and H. Saji, Nucl. Med. Biol.,2006, 33, 489–494.

120 Y. Han, D. Yin, M. Zheng, W. Zhou, Z. Lee, L. Zhan, Y. Ma,M. Wu, L. Shi, N. Wang, J. Lee, C. Wang, Z. Lee and Y. Wang,J. Radioanal. Nucl. Chem., 2010, 284, 279–286.

121 R. Masuda, S. Oishi, H. Ohno, H. Kimura, H. Saji and N. Fujii,Bioorg. Med. Chem., 2011, 19, 3216–3220.

122 O. Jacobson, I. D. Weiss, D. O. Kiesewetter, J. M. Farber andX. Chen, J. Nucl. Med., 2010, 51, 1796–1804.

123 O. Jacobson, I. D. Weiss, L. P. Szajek, G. Niu, Y. Ma,D. O. Kiesewetter, A. Peled, H. S. Eden, J. M. Farber andX. Chen, J. Controlled Release, 2012, 157, 216–223.

124 W. Nomura, Y. Tanabe, H. Tsutsumi, T. Tanaka, K. Ohba,N. Yamamoto and H. Tamamura, Bioconjugate Chem., 2008, 19,1917–1920.

125 H. Tamamura, K. Hiramatsu, S. Ueda, Z. Wang, S. Kusano,S. Terakubo, J. O. Trent, S. C. Peiper, N. Yamamoto,H. Nakashima, A. Otaka and N. Fujii, J. Med. Chem., 2004, 48,380–391.

126 H. Tamamura, T. Araki, S. Ueda, Z. Wang, S. Oishi, A. Esaka,J. O. Trent, H. Nakashima, N. Yamamoto, S. C. Peiper, A. Otakaand N. Fujii, J. Med. Chem., 2005, 48, 3280–3289.

127 H. Tamamura, A. Esaka, T. Ogawa, T. Araki, S. Ueda, Z. Wang,J. O. Trent, H. Tsutsumi, H. Masuno, H. Nakashima,N. Yamamoto, S. C. Peiper, A. Otaka and N. Fujii, Org. Biomol.Chem., 2005, 3, 4392–4394.

128 T. Tanaka, H. Tsutsumi, W. Nomura, Y. Tanabe, N. Ohashi,A. Esaka, C. Ochiai, J. Sato, K. Itotani, T. Murakami, K. Ohba,N. Yamamoto, N. Fujii and H. Tamamura, Org. Biomol. Chem.,2008, 6, 4374–4377.

129 T. Tanaka, W. Nomura, T. Narumi, A. Esaka, S. Oishi, N. Ohashi,K. Itotani, B. J. Evans, Z.-x. Wang, S. C. Peiper, N. Fujii andH. Tamamura, Org. Biomol. Chem., 2009, 7, 3805–3809.

130 S. Ueda, S. Oishi, Z.-x. Wang, T. Araki, H. Tamamura, J. Cluzeau,H. Ohno, S. Kusano, H. Nakashima, J. O. Trent, S. C. Peiper andN. Fujii, J. Med. Chem., 2007, 50, 192–198.

131 O. Demmer, E. Gourni, U. Schumacher, H. Kessler andH.-J. Wester, ChemMedChem, 2011, 6, 1789–1791.

132 E. Gourni, O. Demmer, M. Schottelius, C. D’Alessandria, S. Schulz,I. Dijkgraaf, U. Schumacher, M. Schwaiger, H. Kessler andH.-J. Wester, J. Nucl. Med., 2011, 52, 1803–1810.

133 O. Demmer, I. Dijkgraaf, U. Schumacher, L. Marinelli,S. Cosconati, E. Gourni, H.-J. Wester and H. Kessler, J. Med.Chem., 2011, 54, 7648–7662.

134 O. �Aberg, F. Pisaneschi, G. Smith, Q.-D. Nguyen, E. Stevens andE. O. Aboagye, J. Fluorine Chem., 2011, 135, 200–206.

135 M. D. L. L. Sierra, F. Yang, M. Narazaki, O. Salvucci, D. Davis,R. Yarchoan, H. H. Zhang, H. Fales and G. Tosato, Blood, 2004,103, 2452–2459.

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136 K. Tashiro, H. Tada, R. Heilker, M. Shirozu, T. Nakano andT. Honjo, Science, 1993, 261, 600–603.

137 M. Shirozu, T. Nakano, J. Inazawa, K. Tashiro, H. Tada,T. Shinohara and T. Honjo, Genomics, 1995, 28, 495–500.

138 C. Dealwis, E. J. Fernandez, D. A. Thompson, R. J. Simon,M. A. Siani and E. Lolis, Proc. Natl. Acad. Sci. U. S. A., 1998, 95,6941–6946.

139 P. Loetscher, J.-H. Gong, B. Dewald, M. Baggiolini and I. Clark-Lewis, J. Biol. Chem., 1998, 273, 22279–22283.

140 M. P. Crump, J.-H. Gong, P. Loetscher, K. Rajarathnam,A. Amara, F. Arenzana-Seisdedos, J.-L. Virelizier, M. Baggiolini,B. D. Sykes and I. Clark-Lewis, EMBO J., 1997, 16, 6996–7007.

141 J. Hesselgesser, M. Liang, J. Hoxie, M. Greenberg, L. F. Brass,M. J. Orsini,D. Taub andR.Horuk, J. Immunol., 1998, 160, 877–883.

142 S. K. Gupta, K. Pillarisetti, R. A. Thomas and N. Aiyar, Immunol.Lett., 2001, 78, 29–34.

143 C. Tudan, G. E. Willick, S. Chahal, L. Arab, P. Law, H. Salari andA. Merzouk, J. Med. Chem., 2002, 45, 2024–2031.

144 M. Lefrancois, M.-R. Lefebvre, G. Saint-Onge, P. E. Boulais,S. Lamothe, R. Leduc, P. Lavigne, N. Heveker and E. Escher,ACS Med. Chem. Lett., 2011, 2, 597–602.

145 N. Zhou, Z. Luo, J. Luo, X. Fan,M. Cayabyab,M. Hiraoka, D. Liu,X. Han, J. Pesavento, C.-Z. Dong, Y. Wang, J. An, H. Kaji,J. G. Sodroski andZ.Huang, J. Biol. Chem., 2002, 277, 17476–17485.

146 S. K. Gupta, K. Pillarisetti, R. A. Thomas and N. Aiyar, Immunol.Lett., 2001, 78, 29–34.

147 B. Antonsson, P. De Lys, V. Dechavanne, L. Chevalet andU. Boschert, Proteomics, 2010, 10, 4342–4351.

148 P.Misra, D. Lebeche, H. Ly,M. Schwarzkopf, G. Diaz, R. J. Hajjar,A. D. Schecter and J. V. Frangioni, J. Nucl. Med., 2008, 49, 963–969.

149 R. S. Y. Wong, V. Bodart, M. Metz, J. Labrecque, G. Bridger andS. P. Fricker, Mol. Pharmacol., 2008, 74, 1485–1495.

150 S. P. Fricker, V. Anastassov, J. Cox, M. C. Darkes, O. Grujic,S. R. Idzan, J. Labrecque, G. Lau, R. M. Mosi, K. L. Nelson,L. Qin, Z. Santucci and R. S. Y. Wong, Biochem. Pharmacol.,2006, 72, 588–596.

151 G. A. Donzella, D. Schols, S. W. Lin, J. A. Este, K. A. Nagashima,P. J. Maddon, G. P. Allaway, T. P. Sakmar, G. Henson, E. DeClercqand J. P. Moore, Nat. Med., 1998, 4, 72–77.

152 L. O. Gerlach, R. T. Skerlj, G. J. Bridger and T. W. Schwartz, J.Biol. Chem., 2001, 276, 14153–14160.

153 E. De Clercq, Nat. Rev. Drug Discovery, 2003, 2, 581–587.154 E. De Clercq, Mini-Rev. Med. Chem., 2005, 5, 805–824.155 D. Schols, S. Struyf, J. VanDamme, J. A. Este, G. Henson and E. De

Clercq, J. Exp. Med., 1997, 186, 1383–1388.156 D. Schols, J. A. Este, G. Henson and E. De Clercq, Antiviral Res.,

1997, 35, 147–156.157 S. Hatse, K. Princen, G. Bridger, E. De Clercq and D. Schols, FEBS

Lett., 2002, 527, 255–262.158 W. C. Liles, H. E. Broxmeyer, E. Rodger, B. Wood, K. H€ubel,

S. Cooper, G. Hangoc, G. J. Bridger, G. W. Henson, G. Calandraand D. C. Dale, Blood, 2003, 102, 2728–2730.

159 S. M. Devine, N. Flomenberg, D. H. Vesole, J. Liesveld,D. Weisdorf, K. Badel, G. Calandra and J. F. DiPersio, J. Clin.Oncol., 2004, 22, 1095–1102.

160 H. E. Broxmeyer, C. M. Orschell, D. W. Clapp, G. Hangoc,S. Cooper, P. A. Plett, W. C. Liles, X. Li, B. Graham-Evans,T. B. Campbell, G. Calandra, G. Bridger, D. C. Dale andE. F. Srour, J. Exp. Med., 2005, 201, 1307–1318.

161 M. M. Rosenkilde, L.-O. Gerlach, J. S. Jakobsen, R. T. Skerlj,G. J. Bridger and T. W. Schwartz, J. Biol. Chem., 2004, 279, 3033–3041.

162 S. Hatse, K. Princen, L.-O. Gerlach, G. Bridger, G. Henson, E. DeClercq, T. W. Schwartz and D. Schols, Mol. Pharmacol., 2001, 60,164–173.

163 L. O. Gerlach, J. S. Jakobsen, K. P. Jensen, M. R. Rosenkilde,R. T. Skerlj, U. Ryde, G. J. Bridger and T. W. Schwartz,Biochemistry, 2003, 42, 710–717.

164 R. J. Motekaitis, B. E. Rogers, D. E. Reichert, A. E. Martell andM. J. Welch, Inorg. Chem., 1996, 35, 3821–3827.

165 V. J. Thom, G. D. Hosken and R. D. Hancock, Inorg. Chem., 1985,24, 3378–3381.

This journal is ª The Royal Society of Chemistry 2012

166 I. Tabushi andM. Fujiyoshi, Tetrahedron Lett., 1978, 19, 2157–2160.167 M. Kodama and E. Kimura, J. Chem. Soc., Dalton Trans., 1977,

1473–1478.168 K. S. Woodin, K. J. Heroux, C. A. Boswell, E. H. Wong,

G. R. Weisman, W. Niu, S. A. Tomellini, C. J. Anderson,L. N. Zakharov and A. L. Rheingold, Eur. J. Inorg. Chem., 2005,2005, 4829–4833.

169 L. A. Bass, M. Wang, M. J. Welch and C. J. Anderson, BioconjugateChem., 2000, 11, 527–532.

170 O. Jacobson, I. D. Weiss, L. Szajek, J. M. Farber andD. O. Kiesewetter, Bioorg. Med. Chem., 2009, 17, 1486–1493.

171 I. D. Weiss, O. Jacobson, D. Kiesewetter, J. Jacobus, L. Szajek,X. Chen and J. M. Farber, Mol. Imag. Biol., 2011, 1–9.

172 R. A. De Silva, K. Peyre, M. Pullambhatla, J. J. Fox, M. G. Pomperand S. Nimmagadda, J. Nucl. Med., 2011, 52, 986–993.

173 J. M. Zhang, J. H. Tian, T. R. Li, H. Y. Guo and L. Shen, Chin.Chem. Lett., 2010, 21, 461–463.

174 D. E. Troutner, J. Simon, A. R. Ketring, W. Volkert andR. A. Holmes, J. Nucl. Med., 1980, 21, 443–448.

175 W. A. Volkert, D. E. Troutner and R. A. Holmes, Int. J. Appl.Radiat. Isot., 1982, 33, 891–896.

176 M. Struder and T. A. Kaden,Helv. Chim. Acta, 1986, 69, 2081–2086.177 A. Boschi, L. Uccelli, C. Bolzati, M. Marastoni, R. Tomatis,

S. Spisani, S. Traniello and A. Piffanelli, Nucl. Med. Biol., 2000,27, 791–795.

178 W. Stahl, G. Breipohl, L. Kuhlmann, A. Steinstraesser,H. J. Gerhards and B. A. Schoelkens, J. Med. Chem., 1995, 38,2799–2801.

179 G. D. Luker and K. E. Luker, J. Nucl. Med., 2008, 49, 1–4.180 S. R. Arridge, Inverse Probl., 1999, 15, R41–R49.181 A. B. Gibson, J. C. Hebden and S. R. Arridge, Phys.Med. Biol.,

2005, 50, R1–R43.182 J. G. Fujimoto, Nat. Biotechnol., 2003, 21, 1361–1367.183 A. Grinvald, R. D. Frostig, E. Lieke and R. Hildesheim, Physiol.

Rev., 1988, 68, 1285–1366.184 D. J. Hawrysz and E. M. Sevick-Muraca, Neoplasia, 2000, 2, 388–

417.185 S. Oishi, R. Masuda, B. Evans, S. Ueda, Y. Goto, H. Ohno,

A. Hirasawa, G. Tsujimoto, Z. Wang, S. C. Peiper, T. Naito,E. Kodama, M. Matsuoka and N. Fujii, ChemBioChem, 2008, 9,1154–1158.

186 N. S. van den Berg, T. Buckle, J. Kuil, J. Wesseling and F. W. B. vanLeeuwen, Transl. Oncol., 2011, 4, 234–240.

187 K. Nishizawa, H. Nishiyama, S. Oishi, N. Tanahara, H. Kotani,Y. Mikami, Y. Toda, B. J. Evans, S. C. Peiper, R. Saito,J. Watanabe, N. Fujii and O. Ogawa, Int. J. Cancer, 2010, 127,1180–1187.

188 J. Kuil, P. Steunenberg, P. T. K. Chin, J. Oldenburg, K. Jalink,A. H. Velders and F. W. B. van Leeuwen, ChemBioChem, 2011,12, 1897–1903.

189 A. Dar, P. Goichberg, V. Shinder, A. Kalinkovich, O. Kollet,N. Netzer, R. Margalit, M. Zsak, A. Nagler, I. Hardan,I. Resnick, A. Rot and T. Lapidot, Nat. Immunol., 2005, 6, 1038–1046.

190 O. Kollet, A. Dar, S. Shoham, A. Kalinkovich, K. Lapid,Y. Sztainberg, M. Tesio, R. M. Samstein, P. Goichberg,A. Spiegel, A. Elson and T. Lapidot, Nat. Med., 2006, 12, 657–664.

191 M. Meincke, S. Tiwari, K. Hattermann, H. Kalthoff andR. Mentlein, Clin. Exp. Metastasis, 2011, 1–8.

192 A. Khan, J. D. Silversides, L. Madden, J. Greenman andS. J. Archibald, Chem. Commun., 2007, 416–418.

193 J. C. Knight, A. J. Hallett, A. Brancale, S. J. Paisey,R. W. E. Clarkson and P. G. Edwards, ChemBioChem, 2011, 12,2692–2698.

194 J. Zhang, Y. Fu, G. Li, R. Y. Zhao and J. R. Lakowicz, J. Biomed.Opt., 2011, 16, 016011.

195 L. E. Jennings and N. J. Long, Chem. Commun., 2009, 3511–3524.196 H. Kobayashi, M. R. Longmire, M. Ogawa and P. L. Choyke,

Chem. Soc. Rev., 2011, 40, 4626–4648.197 J. Kuil, T. Buckle, H. Yuan, d. B. N. S. van, S. Oishi, N. Fujii,

L. Josephson and F. W. B. van Leeuwen, Bioconjugate Chem.,2011, 22, 859–864.

Med. Chem. Commun., 2012, 3, 1039–1053 | 1053