REVIEW

Positron Emission Tomographic Imaging of CXCR4 in Cancer: Challenges and Promises Guillaume Pierre Charles George, Federica Pisaneschi, Quang-De´ Nguyen, and Eric Ofori Aboagye

Abstract Molecular imaging is an attractive platform for noninvasive detection and assessment of cancer. In recent years, the targeted imaging of the C–X–C chemokine receptor 4 (CXCR4), a chemokine receptor that has been associated with tumor metastasis, has become an area of intensive research. This review article focuses on positron emission tomography (PET) and aims to provide useful and critical insights into the application of PET to characterize CXCR4 expression, including the chemical, radiosynthetic, and biological requirements for PET radiotracers. This discussion is informed by a summary of the different approaches taken so far and a comparison of their clinical translation. Finally, our expert opinions as to potential future advances in the field are expressed.

CXCR4 The C–X–C chemokine receptor 4 (CXCR4, also known as fusin and CD184) is a seven-transmembrane domain G protein–coupled receptor (GPCR), the widely accepted sole ligand of which is the stromal cell–derived factor–1, the predominant isoform of which is a (SDF-1a, also known as CXCL12).1,2 Recent reports indicate, however, the existence of other ligands to CXCR4, such as macrophage migration inhibitory factor, high-mobility group protein 1, and extracellular ubiquitin.3–5 Moreover, an alternate receptor that binds SDF-1a, the C–X–C chemokine receptor 7 (CXCR7), has also been described.6 CXCR4 is expressed throughout development and adulthood, and the CXCR4/ SDF-1a axis has a fundamental role in hematopoiesis by regulating retention and homing of hematopoietic stem cells in the bone marrow.7–10 This axis also plays a crucial role in lymphocyte trafficking to sites of inflammation.11 In the physiologic context, CXCR4 is widely expressed on most leukocytes and on a variety of other cell types, such as endothelial and epithelial cells.12,13 Besides its expression in Comprehensive Cancer Imaging Centre, Imperial College London, Faculty of Medicine, Hammersmith Hospital Campus, London, UK; Department of Chemistry, Imperial College London, South Kensington Campus, London, UK. Address reprint requests to: Eric Ofori Aboagye, PhD, Comprehensive Cancer Imaging Centre, Imperial College London, Faculty of Medicine, Hammersmith Hospital Campus, Du Cane Road, London W12 0NN, UK; e-mail: [email protected].

DOI 10.2310/7290.2014.00041 #

2014 Decker Intellectual Properties

normal tissues, CXCR4 expression has also been associated with various diseases. CXCR4 was first reported as a coreceptor for CD4+ T-cell infection of human immunodeficiency virus (HIV) type I,14,15 and more recently, a role for CXCR4 was described in the pathogenesis of rheumatoid arthritis.16 In addition to its extensive physiologic roles and implication in immune and inflammatory disorders, CXCR4 has been found to be expressed by various human cancers.17,18

CXCR4 and Cancer Upregulation of CXCR4 has been reported in at least 23 different epithelial, mesenchymal, and hematopoietic cancers.19–21 The percentage of CXCR4-positive tumor cells in patient samples determined by immunohistochemistry ranged from 12% in early breast cancer22 to . 90% in ductal carcinoma in situ,23 non–small cell lung cancer,24,25 renal carcinoma,26 mesothelioma,27 gastric cancer,28 and prostate cancer.29 In metastatic lesions of breast cancer, non–small cell lung cancer, gastric cancer, and renal cell carcinoma, the percentage of CXCR4-positive cells ranged between 63.3 and 100%.29–32 CXCR4 overexpression in tumor tissues has been correlated to poor prognosis, tumor aggressiveness, increased risk of metastasis, and a higher probability of recurrence.33–35 CXCR4/SDF-1a signaling is involved in different aspects of tumor progression. Notably, the CXCR4-based chemotaxis acts directly on tumor cell migration and invasion toward an SDF-1a gradient. High expression levels of SDF-1a have been found at the most common sites of breast cancer metastases: axillary lymph nodes, lungs, liver, and bone marrow (Figure 1).36–39

Molecular Imaging, Vol 13, 2014: pp 1–19

1

2

George et al

Multiple studies also report the involvement of the CXCR4 signaling in promoting angiogenesis,40–43 tumor cell proliferation and survival,44 and resistance to chemotherapy.45–47 Therefore, due to its critical roles in cancer, CXCR4 has been designated and characterized as a potential therapeutic target, and a number of CXCR4 inhibitors have been developed, including small-molecule antagonists,48 antibodies,49–52 peptide antagonists,53–68 and glycosaminoglycan mimetics.69 These inhibitors have been investigated in preclinical models, and some of them have reached clinical trials48; the long-term

toxicity and off-target activity of these agents remain to be fully elucidated. A comprehensive description of the CXCR4 pharmacophore is outside the scope of this review and has been reported elsewhere.17,48,70

CXCR4-Targeting PET Agents It was proposed recently that these CXCR4-targeting molecules could also provide versatile platforms for the development of imaging agents. The evaluation of CXCR4

Figure 1. Schematic representation showing the role of the CXCR4/SDF-1a axis as a key actor in the tumor invasion and metastasis process. Molecular crosstalks between the microenvironment (including fibroblasts, lymphocytes, macrophages, neutrophils, transforming growth factor -b) and cancer cells trigger CXCR4 expression and activation, promoting a migratory and invasive phenotype and resulting in the formation of organ-specific metastasis (bone, lungs, liver, brain, and lymph nodes). The metastatic process involves a series of sequential steps, including local invasion, intravasation, extravasation, and colonization of target organs guided by an SDF-1a chemoattracting gradient.18,133

PET Imaging of CXCR4 in Cancer

expression is currently assessed by the means of tissue sample biopsies,70,71 which may not be representative of the entire primary and metastatic disease volume in the patient,72 in spite of the high clinical value of histologic assessment in the initial tumor diagnosis. The underlying rationale is that noninvasive imaging of CXCR4, with an ability to reflect heterogeneity of protein expression, could potentially be used as a complementary diagnostic or prognostic biomarker through the detection of tumors and highly aggressive subpopulations of tumor cells, as well as a repeated measure pharmacodynamic for CXCR4-targeted therapeutic interventions. Among the various strategies developed to image CXCR4 noninvasively, this review article focuses on the development of positron emission tomography (PET) radiotracers.70,73–75 PET can significantly contribute to the clinical management of a patient by providing early functional data on disease extent, therapy response, and identification of recurrence and can also be used for therapy planning. Technological advances in PET instrumentation have enabled the transition of human imaging capabilities to the scale of small animals,76–78 and PET imaging is now recognized as a major translational ‘‘bench to bedside’’ molecular imaging approach due to its high sensitivity, together with the large variety and increasing portfolio of tracers for the monitoring of key biological processes in cancer.72 The development of a CXCR4-targeting PET imaging agent, including the selection of the targeting probe, the selection of the positron-emitting isotope and its associated labeling strategy, and the validation of these agents using cellbased assays and preclinical models, provides a number of (unique and generic tracer development) challenges that are discussed below. Although several CXCR4-specific radiotracers have been developed to date and have shown feasibility in animal models, the promise of a translational agent for the clinic is yet to be met. To the best of our knowledge, only one tracer for PET, [68Ga]–CPCR4.2, has to date completed initial evaluation in humans. The result of the trial is discussed further below. Another tracer, [64Cu]– AMD3100, originally developed by Nimmagadda and colleagues,79 is recruiting within a phase 0 trial.80 This review article aims to provide some insights toward the establishment of the optimal translational CXCR4-specific PET agent.

Rational Design and Development of CXCR4Targeting PET Agents Positron Emission Tomography PET is a noninvasive molecular and functional imaging modality that measures the in vivo biodistribution of a

3

molecular probe labeled with a positron-emitting radionuclide. Thus, following intravenous administration, this technology provides essential insights in biological processes in intact entire (whole body) living subjects. PET scanners detect the 511-kiloelectron volt annihilation photons generated as a result of positron decay (b+ decay) by unstable proton-rich radionuclides. The detailed description of the basic principles of PET imaging is outside the scope of this review article and has been reported elsewhere.72,81,82 A wide array of compounds of biological interest (e.g., antibodies, peptides, and small molecules) have been exploited and radiolabeled with positron-emitting radionuclides for PET imaging studies of various biological processes (e.g., blood flow, metabolism, cell surface receptor expression, angiogenesis, gene expression proliferation, and apoptosis). The flexibility of labeling any molecule that targets a specific biological process or marker, as well as the ability to image the spatial distribution of the labeled molecule in vivo over time, opens up many potential uses for PET from basic and preclinical research in animal models through to clinical investigations of various human pathologies. These radiolabeled probes are administered at trace amounts—pico/ femtomolar range—thus, they do not perturb normal physiology to allow in vivo studies under physiologic conditions. PET therefore enables the investigation of substrate–target interactions or physiologic processes with the advantage of not having to administer an efficacious dose of a chemical compound, as with classic pharmacologic investigations. An important characteristic of PET imaging is that it enables the distribution of the radiotracer to be measured quantitatively, assuming that appropriate corrections are made for physical factors such as c ray attenuation and scattering in tissues. The quantitative nature of PET is largely independent of the thickness of the object and the depth of the source within the subject. As radiotracers act as substrates/ligands for normal physiologic processes, they need to undergo extensive in vitro and preclinical validation. Development of CXCR4-Targeting PET Radiotracers The desirable optimal features for a PET imaging agent targeting the CXCR4 receptor are discussed below and illustrated in Figure 2. It should be emphasized that, irrespective of probe type (peptide, small molecule), the ideal tracer should have a rapid and high-affinity engagement of receptor and sufficiently fast clearance to permit low uptake in background tissues and hence high contrast.

4

George et al

Figure 2. PET tracer screening cascade. Diagram describing the screening cascade and detailing the various chemical, radiochemical, biological, and clinical traits radiotracers need to present. GMP 5 good manufacturing practice; HPLC 5 high-performance liquid chromatography; IC50 5 half maximal inhibitory concentration; IMP 5 investigational medicinal products; MS 5 mass spectrometry; SDF1-a 5 stromal cell–derived factor–1a; SOP 5 standard operating procedure; UV 5 ultraviolet.

High Binding Affinity To achieve good contrast and to maximize tumor visualization, tracer needs to have high affinity and a slow rate of tracer–receptor complex dissociation in target tissue to preclude tracer washout. For CXCR4, a number of assays have been implemented to ensure this. Cell-based assays employing [125I]SDF-1a or fluorescently labeled compounds have been used to assess affinity. Reversal of ligand binding is usually complemented by additional studies to verify downstream biological activity, such as a cell migration, given the impact of the CXCR4–SDF-1a axis on migration. These assays underpin the initial screening cascade for novel imaging agents. Using these assays, nanomolar affinity was seen with a number of compounds developed as CXCR4 probes for PET.83,84 Affinity and docking studies in the case of the cyclopentapeptide reported by Demmer and colleagues indicated a two-site binding model with CXCR4.83

Specificity It is important that any tracer presents no or low affinity for other receptors, that is, the tracer must be specific to the targeted receptor. If this is not the case, then the binding of the candidate tracer to another target than initially intended will lead to false positives and/or low contrast. Routine lipid and protein kinase, as well as receptor screens, are available for ensuring high specificity, although this service has not been explored for CXCR4 antagonists. Thus, although CXCR4 imaging agents show specificity in blocking studies or when low- and high-expressing cell lines are used, selectivity against other targets remains to be determined for the imaging probes. With our increasing knowledge of chemokine–receptor interactions, it has become necessary to also consider a lack of CXCR7 interaction as part of the screening cascade, although this aspect has not been widely investigated to date. Cyclopentapeptides, tetradecapeptides, and AMD3100, like the native ligand SDF-1a, have been

PET Imaging of CXCR4 in Cancer

shown to bind CXCR7.85,86 In this specific respect, new scaffolds will be required to discriminate CXCR4 from CXCR7. Of note, the noncyclam CXCR4 inhibitor AMD11070 does not bind CXCR7,87 and it remains to be seen if monocyclam probes bind CXCR7. Good Penetration Owing to their size, high-molecular-weight tracers may penetrate slowly into solid tumors; poor tumor penetration results in low absolute uptake. Although a generic issue for the development of radiotracers for PET, this issue is not deemed acute for current CXCR4 tracers as most of these are either small organic molecules or peptides. The expected high penetration of these compounds compared to antibodies (which have high binding affinity but a long residence time) should allow for the use of optimal radiohalogen labeling with fluorine 18 (which has a short half-life, high photon flux, and low energy). This ideal is, however, not obvious in the literature, and lead radiotracers (discussed below) mainly employ radiometals such as copper 64 or gallium 68 for labeling. Although gallium 68 could be considered a good compromise for the expected rapid kinetics and tissue penetration of small-molecule organic compounds and small peptides, the same argument could not be made for copper 64. This limitation is partly due to difficulties in modifying bicyclams originally exploited for PET imaging to incorporate fluorine-containing moieties79; on the other hand, the ease of incorporating DOTA or NOTA moieties into peptide has opportunistically favored radiometals for labeling. Good Contrast Effective distinction between an overexpressing CXCR4 tumor and its background, namely muscles and blood, is essential. Conversely, nontargeted tissues should present no or low uptake. Contrast can be quantified as the signal to noise ratio. Rapid renal clearance enhances contrast in most cases but can in some instances decrease the overall intensity of tumor uptake. Thus, a balance of physicochemical properties must be achieved to ensure contrast. Broadly, common strategies to achieve this include designing tracers with low n-octanol–water distribution coefficient (logD), which are ideally charged. Conversely, tracers presenting high logD values are likely to be excreted by the hepatobiliary route and to pass the blood-brain barrier, both of which would result in nonspecific radioactivity uptake. High-molecular-weight radiotracers are also often eliminated by the hepatobiliary route. CXCR4 peptidic

5

tracer [68Ga]–CCIC16 shows a logDpH 7.4 of 23.58 6 0.15,88 whereas cyclopentapeptides display a variety of distribution coefficients, ranging from 1.09 6 0.02 ([18F]CCIC07)89 to 22.30 6 0.30 ([18F]CCIC30) (GPC George, F Pisaneschi, E Stevens, et al, unpublished work, February 2014). Monocyclam [64Cu]–AMD3465 displays a logP of 22.71 6 0.37,84 whereas bicyclam [64Cu]– AMD3100 shows a logP of 0.52 6 0.02.79 Appropriate Metabolism Metabolism has two facets: on the one hand, metabolism could be desirable when it improves excretion of the tracer, but on the other hand, it might produce metabolites that bind nonspecifically, thus reducing contrast. Consequently, a tracer that presents partial metabolic instability in vivo could still prove to be efficient provided that metabolites are quickly excreted. Ideally, however, every effort should be undertaken to reduce metabolism. Unfortunately, the structure–activity relationship (SAR) around CXCR4 small molecules and peptides with respect to metabolism is less advanced, precluding substantive activity on this issue to date. For example, whereas [68Ga]–CPCR4.2 is reported to be stable in vivo,90 a relatively similar cyclopentapeptide probe for PET, [18F]CCIC07, a 4-[18F]fluorobenzoimino PEG-functionalized cyclo(Nal1–Gly2–D-Tyr3–Orn4–Arg5) cyclopentapeptide, showed rapid biliary and renal elimination and metabolic instability (O A˚berg, Pisaneschi F, Smith G, et al. unpublished work, 2012). For radiometal-labeled bicyclams such as [64Cu]– AMD3100, the possibility of transchelation and hence loss of the radiometal is a concern. Thus, in vivo stability should be a key screening variable to be empirically examined early in the development of CXCR4 probes for imaging by PET. Careful Chemical Design This aspect brings together all the aforementioned desirable properties. Recent disclosure of the crystal structure of CXCR491 permits modeling/docking studies to optimize affinity and specificity. For instance, three of the four nitrogens of small-molecule IT1t make important salt bridges or polar interactions with the receptor, whereas both cyclohexyl rings fit in small hydrophobic subpockets. These observations exhort the medicinal chemist to conserve these essential pharmacophores to maintain affinity. On the other hand, and in accordance with the SAR analysis of a series of CVX15 analogues,64–68 hexadecapeptide CVX15 principally interacts via numerous hydrogen bonds and salt bridges with the strongly negatively charged receptor binding site, whereas CVX15’s b-turn points toward the

6

George et al

extracellular milieu, admonishing us to introduce derivatizations enabling radiolabeling on the b-turn’s residues.92–94 Although the spatial arrangement of the pharmacophores will mainly impact receptor binding and specificity, other characteristics, such as the overall size, charge, and logD of the molecule, will influence other pharmacokinetic behavior aspects, including excretion, metabolism, and tumor penetration. One example of this statement is the substitution of several arginine residues for citrullines in the tetradecapeptide class, which was aimed at reducing the cell toxicity associated with the high overall charge of the peptide.65 Finally, as emphasized later in this review, the chemical design of the radiotracer will dictate which radioisotopes are available for labeling and which radiochemical reactions can be performed. This, in turn, will influence the tracer’s PET and radiosynthetic properties. Therefore, the chemical design of the radiotracer is a nontrivial problem consisting of multiple variables for which a compromise between its synthetic, radiosynthetic, and pharmacokinetic features needs to be found. High Radiochemical Purity and Specific Activity At tracer level, it is unlikely that an antagonist could elicit a biological response and consequently adversely interfere with the biological system. However, radioactive byproducts can present nonspecific binding and therefore reduce contrast. The measured variable to describe this property is radiochemical purity, which is defined as the proportion of radioactivity emanating from the desired radiotracer. In addition, nonradioactive by-products present in the injected solution and traces of the nonradioactive isotopologue can potentially compete with the tracer for receptor binding. Such nonradioactive products must be kept to a minimum and are quantified in a ‘‘specific activity’’ parameter, which is defined as the ratio between the activity of the sample and the amount of material in the sample (typically given in GBq.mmol–1). In the CXCR4 context, the range of specific activities varies greatly depending on the molecule labeled, the radio emitter, and the type of radiosynthesis performed. [64Cu]–AMD3100 is reported with specific activities as high as 417 GBq/mmol–1,95 whereas the specific activities of the other tracers that allow for the visualization of tumors range between 5 and 30 GBq/mmol–1.84,93,96,97 Tumor Model Once the above parameters have been optimized, the choice of cell lines/tumor models used for preclinical in vitro and in vivo experiments carried out to determine

candidate tracer’s characteristics can strongly influence the results obtained. The properties of numerous CXCR4 models were recently reviewed by Kuil and colleagues.70 For a fair comparison of data from different studies, it is important to specify the cell model used. The in vitro characterization of the tracer, including affinity and specificity, is influenced by the cell model and the level of CXCR4 expression at the cell surface. In this regard, the two main cellular models used are CXCR4-transfected cells, which harbor a significant upregulation and expression of the receptor compared to the mock-transfected counterpart, and human cancer cells with intrinsic high expression of CXCR4.79,83,84,90,92,97–103 However, the level of CXCR4 expression at the membrane is not always reported, and for that reason, cross-comparison of data between these models has to be drawn with caution. Alternative models that recapitulate human cancers in their microenvironment, including patient-derived xenografts and genetically engineered mouse models, would potentially make a significant contribution within the experimental arsenal for the development of CXCR4specific imaging agents and are eagerly awaited. CXCR4-Targeting Agents The apparent importance of CXCR4 in different pathologies encouraged efforts toward discovery of CXCR4targeting therapeutics. A quick overview of a selection of classes of CXCR4-targeting chemicals is provided in Figure 3, but the interested reader is referred to the most recent reviews on the subject for more detail.48,104–108 The quest for CXCR4 antagonists started two decades before the determination of any crystal structures for this transmembrane receptor.91 Early attempts at discovering potent and clinically viable CXCR4 antagonists (which were referred to as HIV-1 entry antagonists until 1994, the year when CXCR4 was discovered109) were mainly focused on peptides. The discovery of tachyplesin I and polyphemusin II in the late 1990s62,63 and multiple SAR analyses based on their structures yielded a plethora of octadeca- to tetradecapeptidic CXCR4 inhibitors.64–68 Of interest, TN1400365 and its N-4-fluorobenzoyl derivative TF1401668 were found to be particularly active and stable in vivo and are close analogues of CVX15, which has been cocrystallized with CXCR4.91 Efforts at reducing the molecular weight of these tetradecapeptides were made as smaller peptides tend to ameliorate their undesired immunoresponse. These efforts resulted in a library of cyclopentapeptides and cyclopentapeptide mimics,53–61,110,111 among which FC131 (see

PET Imaging of CXCR4 in Cancer

7

Figure 3. Selected examples of CXCR4 antagonists. The classes that constitute the major focus of this review article are boxed. Cit 5 citrulline; Nal 5 2naphthylalanine.

Figure 3) and FC122 were found to be highly potent against HIV-1 entry and CXCR4. Numerous small molecules were also developed. The serendipitously discovered AMD3100, found as an active impurity during a research program aimed at finding an anti-HIV agent,112 was recently approved for clinical use to mobilize hematopoietic CD34+ stem cells from the bone marrow into the circulation. In oncology, numerous clinical trials involving AMD3100 in combination with conventional chemotherapies are still ongoing.113 In 2005, Hatse and colleagues developed the less charged monocyclam AMD3465, which proved to be 10-fold more potent than AMD3100 despite lacking the bicyclam structure that was believed to be a fundamental requisite

for receptor binding.114 Many more molecules were developed based on these structures and were thoroughly and recently reviewed by Debnath and colleagues.48 In addition to the above, IT1t is a small molecule that has been cocrystallized with CXCR4 and has been reported to have superior binding affinity to AMD3100.115 This molecule was developed by Novartis along with 20 other analogues published in the original article.115 Although no other instances of antagonists with structural similarities to IT1t have been published, it can be anticipated given its favorable pharmacokinetic profile and the availability of the IT1t/CXCR4 cocrystal structure91 that this scaffold may well attract increased attention in the future.

8

George et al

Radiochemical Design and Production of CXCR4-Targeting Agents Insights into the SAR of the class of molecule or a crystal structure of the receptor (ideally cocrystallized with a representative of the tracer class of interest) could lead to determining with confidence positions on putative probes that could be modified to allow the introduction of the radioelement. The PET tracer should also be easy to synthesize in good quality. Therefore, the radiolabeling strategy should be high yielding (. 5% non–decay-corrected radiochemical yield [ndc-RCY]) and should be ideally suitable for automation to reduce the radioactive dose to the radiochemist and improve the reproducibility of radiotracer production. The choice of the radioelement used to produce the tracer is also important as it influences the labeling step, the structure of the tracer, and some of its imaging properties (Table 1). 18 F is usually regarded as the radionuclide of choice when it comes to positron emission. This is not only because its relatively long half-life allows for more complex labeling processes, but also because it decays almost exclusively by emission of positrons of low kinetic energy, which translates into shorter acquisition times and high resolution. 18FLabeling can be performed via the use of [18F]fluoride (18F–) or [18F]fluoronium (18F+). [18F]F+-labeling (mainly by demetallation/fluorination) is limited by the ability to only achieve low specific activity.116 Conditions used for labeling performed with [18F]fluoride, which can deliver higher specific activities, are often harsh and involve the use of either aliphatic or aromatic nucleophilic substitution reactions. The use of prosthetic groups has attracted much interest in recent years and enabled radiochemists to label more complex and delicate structures. On the other hand, this strategy introduces considerable structural variation, especially in small molecules. Recently, new radiolabeling methodologies have started to appear in the literature.117–121 Although a revision of these methods is outside the scope of

this review, it is clear that the possibilities for 18F-labeling using these techniques are expanding and will doubtless give access to increasingly complex and efficient radiosyntheses. Radiometals offer the advantage of a fast and tractable radiolabeling step by chelation with a precursor adorned by a multidentate chelating moiety.122 Unfortunately, such chelating moieties are large, and their introduction in the structure of a tracer can greatly influence its affinity for the receptor. Their use is therefore limited to large tracers, such as peptides, for which such modifications are comparatively small. In addition, transchelation may be observed after administration, and it is therefore necessary to determine the exact in vivo behavior of such radiotracers.123 Two of such radiometals are 68Ga and 64Cu. Owing to the low kinetic energy of its emitted positron, 64Cu offers good spatial resolution. Its long half-life allows scanning of subjects several hours after injection of the tracer. This provides time for the body to clear most of the nonspecifically distributed tracer from the organism, thus yielding images of high contrast. Nevertheless, only 18% of the decay is available for PET, exposing the subject to high doses of ionizing b radiation over a long period; in fact, 64Cu-ATSM, a radiospecies design for the imaging of hypoxic tumors, was recently used in preclinical evaluation for radiotherapeutic use.124 On the other hand, 68 Ga is an efficient PET radioisotope as it has 89% positron decay. Moreover, this isotope is produced in a portable generator, making it readily available for hospitals devoid of bulky and expensive cyclotrons. Its shorter half-life means that the patient receives a lesser dose but also precludes benefiting from the improved contrast concomitant with postadministration deferment of data acquisition. Finally, 68Ga offers images of lower spatial resolution than 18F.

Depiction of CXCR4-Targeting PET Agents Developed to Date A variety of the previously mentioned CXCR4 antagonists have been radiolabeled for PET imaging application

Table 1. Comparison of the Characteristics of PET Radionuclides Used for the Labeling of CXCR4-Targeting Tracers Radionuclide 18

F

Half-life (T1/2) 109.8 min

Common Labeling Precursor 18

[ F]

–

68

Ga

67.7 min

[68Ga]Cl3

64

Cu

12.7 h

[64Cu]Cl2

Production* 18

18

O(p,n) F cyclotron 68 Ge(b,ne)68Ga generator 64 Ni(p,n)64Cu cyclotron

Decay Mode +

97% b 3% EC 89% b+ 11% EC 43% EC 39% b 18% b+

Positron Kinetic Energy Daughter Isotope 0.64 MeV

18

O O 68 Zn 68 Zn 64 Ni 64 Zn 64 Ni 18

1.83 MeV

0.65 MeV

b 5 beta (electron) decay; b+ 5 positron emission; EC 5 electron capture; PET 5 positron emission tomography. *According to the compact notation for nuclear reactions, Azb?czD is equivalent to A(b,c)D.

PET Imaging of CXCR4 in Cancer

following modification for the introduction of a radionuclide. These compounds are summarized in tabular form below and then discussed on a class-by-class basis (Table 2). Cyclams Efforts at labeling AMD derivatives with 64Cu afforded [64Cu]–AMD310079,95,101 and [64Cu]–AMD3465,84 the latter of which presented superior characteristics, such as tumor to muscle and tumor to blood ratios, which were seven- and eightfold higher at 1.5 hours postinjection, respectively. In vivo evaluation with three different cancer cell lines presenting various levels of expression of CXCR4 exhibited levels of tracer uptake varying in accordance with the expression of the receptor, namely U87.CD4.CXCR4 . HT-29 . U87.84 These results are interesting as they suggest that the in vivo expression of CXCR4 could be quantified by PET scanning, which in turn could be related to the aggressiveness of the tumor.125–128 The observation of a high uptake of radioactivity in liver and kidneys has been assumed to result at least partly from specific interactions with an unknown target. Although uptake in the CXCR4-expressing cell model is comparatively high and suggests that [64Cu]–AMD3465 could allow for the visualization of hepatic or renal tumors expressing high levels of CXCR4, the liver and the kidneys would be exposed to high doses of ionizing radiation. Cyclopentapeptides Cyclopentapeptides have also been derivatized to afford tracers, either based on the structure of FC131 for labeling with p-fluorobenzaldehyde89 or by ‘‘click’’ chemistry with 2fluoroethylazide,96 or based on the structure of FC122 for labeling with 68Ga.83,90,102 Whereas the fluorobenzaldehyde derivative [18F]CCIC07 showed no in vitro uptake and had an extremely high rate of metabolism, probably due to the presence of the polyethylene glycol linker (O A˚berg, Pisaneschi F, Smith G, et al, unpublished work, 2012), the triazole derivative [18F]CCIC15 presented adequate uptake in vitro (GPC George, F Pisaneschi, E Stevens, et al, unpublished work, February 2014). Although all published work has focused on substitution of Arg3 to enable the introduction of the radioelement as this residue is believed to contribute least to the binding affinity of this class of antagonist, we have shown that replacement of FC131’s DTyr residue can lead to improved affinity (GPC George, F Pisaneschi, E Stevens, et al, unpublished work, February 2014). The tracer [18F]CCIC30 has an IC50 value of 0.37 6

9

0.12 mM, which is lower than that for FC131 (IC50 5 0.65 6 0.22 mM) (GPC George, F Pisaneschi, E Stevens, et al, unpublished work, February 2014). This can perhaps be explained by the size of the triazole ring, which is smaller than that of the phenol unit in D-Tyr and which may therefore occupy the same pocket in CXCR4 and thus preserve all of the side-chain and backbone interactions of the unmodified cyclopentapeptide. It also cannot be excluded that one of the triazole’s nitrogen participates in the binding via hydrogen bond with CXCR4’s Tyr190 and the aromatic ring itself via hydrophobic contacts, mimicking D-Tyr’s interactions with the receptor. [18F]CCIC30 showed a favorable in vitro profile, with twofold higher uptake in the CXCR4-positive U87.CD4.CXCR4 cells compared to isogenic CXCR4-negative U87.CD4 cells (GPC George, F Pisaneschi, E Stevens, et al, unpublished work, February 2014). Unfortunately, this did not translate in vivo, where no difference in tumor uptake was seen between the two cell lines. Moreover, although the imaging data averaged over 5 to 30 minutes postinjection highlighted the tumor, at 60 minutes postinjection, no tumor uptake was detected. Most of the radioactivity was found in the bladder (GPC George, F Pisaneschi, E Stevens, et al, unpublished work, February 2014). This, together with evidence of metabolic stability by 60 minutes postinjection, led to the conclusion that the low tumor uptake was due to excessive renal excretion (GPC George, F Pisaneschi, E Stevens, et al, unpublished work, February 2014). In contrast to the above, [68Ga]–CPCR4.2 permitted efficient visualization of OH-1 tumors.90 Although the contrast was much lower than that achieved with [64Cu]– AMD3465, [68Ga]–CPCR4.2 presented limited uptake in the liver and the kidney. Along with the intrinsically superior PET characteristics of 68Ga compared to 64Cu (especially when the reconstruction algorithm considers the type of radioisotope), these excellent preclinical results encouraged a preliminary human study, which showed for the first time PET imaging of CXCR4 expression in human.129 In a clinical setting, [68Ga]–CPCR4.2 exhibited limited uptake in nontarget tissues and the ability to visualize certain tumors, including nodal lesions, from chronic lymphocytic leukemia and lymphoma, non–small cell lung adenocarcinoma lesions, and CD30+ aggressive Tcell lymphoma [mean SUV60 values: blood 1.83, muscle 0.77, lung 0.75, liver 1.46, spleen 5.69, bone marrow 4.00, kidney 5.06, bladder 36.4. nodal lesion in chronic lymphocytic leukemia: 5.05 (. FDG). Lymphoma and lung lesions in nonsmall cell lung adenocarcinoma with CD30+ aggressive T-cell lymphoma: 9.91 and 2.70, respectively].130 Further clinical results are required, but

10

George et al

Table 2. CXCR4-Targeting PET Imaging Agents to Date Small Molecules

Cyclopentapeptides

Tetradecapeptides

Radiometals R 5 4-fluorobenzoyl

2009 64

[ Cu]–AMD3100

2011 79,95,101

2011

68

[ Ga]–dim.cycPP

83

64

[ Cu]–T140-2D92

R5

2011 64

[ Cu]–AMD3465

2011 84

68

[ Ga]–CPCR4.2

2012 90,102,134

64

[ Cu]–NOTA-NFB100 R5

64

Cu–DO3A–

2012 64

[ Cu]–DOTA-NFB100 R 5 4-fluorobenzoyl

2012 68

[ Ga]–T140-DOTA93 R5

68

Ga–NO2A–

2013 68

[ Ga]–CCIC1688 18

F

—

R 5 4-[18F]fluorobenzoyl

2012 [18F]CCIC0789

2010 4-[18F]-T14097

11

PET Imaging of CXCR4 in Cancer

Table 2. Continued R 5 acetyl

2013 [18F]CCIC1596

2013 [18F]FB-Ac-TC1401294

R 5 acetyl

2013 [18F]CCIC30*

2013 [18F]FP-Ac-TC1401294

Cit 5 citrulline; DO3A 5 2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetyl; Nal 5 3-(2-naphthyl)alanine; NO2A 5 2-[4,7bis(carboxymethyl)-1,4,7-triazacyclononan-1-yl]acetyl. *GPC George, F Pisaneschi, E Stevens, et al, unpublished work, February 2014.

[68Ga]–CPCR4.2 is already a significant milestone in the quest for a clinically useful CXCR4-specific PET radiotracer. The superior characteristics of [68Ga]–CPCR4.2 compared to [18F]CCIC30 can be attributed to the unexpectedly favorable interaction of the DOTA chelator unit. According to the authors, the addition of the DOTA moiety to FC122 led to loss of potency, but the chelation of this molecule with natGa, affording natGa–CPCR4.2, increased CXCR4 affinity by a factor of 35.90 The authors attribute this observation to the fundamental structural differences, as well as the overall charge and charge distribution induced by the complexation of gallium. Tetradecapeptides Finally, tetradecapeptide TN14003 has generated the most interest from the radiochemistry community. Although the first derivatives presented unexpected red blood cell binding92,97 and/or required long (. 2.5 hours) and lowyielding (, 5% ndc-RCY) 18F-labeling procedures,94,97 it was later found that the addition of a chelating group at the peptide’s N-terminus allowed red blood cell binding to be circumvented, while allowing for chelation with 64Cu and retaining acceptable affinity for CXCR4.100 The fact that this derivatization could be tolerated was at first surprising as CVX15, a close derivative of TN14003 [CVX15 5 TN14003– Gly–D-Pro–OH], was cocrystallized with CXCR4 in a pose where both its N- and C-termini were buried in CXCR4’s helix bundle, filling the whole binding site.91 This result can, however, be rationalized by considering that the shorter

peptide, TN14003, allows for substantial derivatization at one of the peptide’s termini. High liver and kidney uptake of the 64Cu-DO3A-N-terminus labeled TN14003, [64Cu]– DOTA–NFB, led us to engineer a revised TN14003-based CXCR4-specific PET tracer using a smaller chelating moiety to reduce affinity loss for CXCR4 when labeling with 68Ga.88 This tracer, [68Ga]–CCIC16 (Table 3), showed improved tumor uptake in shorter incubation times and allowed for effective localization of the tumor within 1 hour postinjection in the CXCR4-overexpressing U87.CD4.CXCR4 xenograft–bearing mouse compared to published studies of [64Cu]–DOTA–NFB and [64Cu]–NOTA–NFB with the same tumor lines (Figure 4C). It was characterized by adequate contrast and promising in vivo stability and clearance, therefore showing the essential features for the identification of CXCR4-expressing tumors in a clinical setting. Advantages and Limitations In the interest of brevity, only the most recent and promising PET tracers are critically reviewed. A comparison of radiosynthesis and biological properties of three of the most recent and promising CXCR4 ligands from different structural classes—small-molecule bicyclam, cyclic pentapeptide, and 14–amino acid cyclic peptide—is considered in the following discussion (see Table 3 and Figure 4). From a radiochemistry point of view, radiometal chelation appears favorable for CXCR4 ligands; this is mainly due to the fact that most of the tracers radiosynthesized to date are peptidic in nature. Moreover,

12

George et al

Table 3. Comparison of Radiosynthetic and Biological Properties of the Most Recent and Promising Tracers in Their Class Radiotracer Labeling ndc-RCY SpecAc (GBq/mmol) Starting activity Cell model

In vitro IC50 (reference) Cell uptake (fold) In vivo Postinjection time point Tumor uptake (% ID/g) T/B T/M Uptake in selected organs (% ID/g)

[64Cu]–AMD3465

[68Ga]–CPCR4.2

[68Ga]–CCIC16

40 6 10%* 6.0 6 3.1 370–740 MBq U87.CD4.CXCR4 (++) HT-29 (+) U87 (2)

< 13% < 67 1.1–1.3 GBq OH1 (+)

52 8%6 2.8 6 0.6 60–70 MBq U87.CD4.CXCR4 (+) U87.CD4 (2)

—*

4.99 6 0.72 (FC131: 4.43 6 0.82) Not relevant

65.8 6 32.9 (TC14012: 23.1 6 11.1) 3

10–12* 1.5 h 96.29 6 13.98 138.78 6 30.87 362.56 6 153.51 L: 36.15 6 1.79 U: —* K: 37.93 6 3.57 B: 1.38 6 0.19 BM: 9.18 6 2.04 S: 4.28 6 1.00

1h 6.16 6 1.16 < 5.70 16.55 6 3.84 L: 1.85 6 0.24 U: — K: 3.06 6 0.63 B: — Lg: 1.41 6 0.26

2h 4.63 6 1.54 < 7.98 18.49 6 7.29 L: 1.48 6 0.31 U: — K: 2.07 6 0.46 B: — Lg: 2.85 6 2.69

1h 5.33 6 1.84 8.64 9.50 6 1.71 L: 21.03 6 6.15 U: 30.67 6 11.59 K: 24.00 6 4.92 B: 2.52 6 0.53 Lg: 3.10 6 0.51 S: 3.46 6 0.78 GB: 21.13 6 6.22

B 5 blood pool; BM 5 bone marrow; GB 5 gallbladder; IC50 5 half maximal inhibitory concentration; K 5 kidneys; L 5 liver; Lg 5 lungs; ndc-RCY 5 non–decay-corrected radiochemical yield; S 5 spleen; T/B 5 tumor to blood ratio; T/M 5 tumor to muscle ratio; U 5 urine. *Information obtained from the author for correspondence.

although small molecules are not usually labeled via radiometal chelation owing to the incompatibility of the size of the chelator moiety needed relative to that of the targeting small molecule, both AMD3100 and AMD3465 were successfully radiolabeled with 64Cu. This peculiarity can be explained by the unique nature of the cyclam present in these molecules, which offers the opportunity for chelation of 64Cu while concomitantly acting as a pharmacophore for binding to CXCR4. As previously described, attempts at labeling with 18F have also been described but have tended to result in long (. T1/2) and low-yielding (, 5% ndc-RCY) syntheses, as exemplified by the efforts to label TN14003 derivatives with 2-fluoropropionate or 4-fluorobenzoate.94,97 As a side note, the use of 4-[18F]fluorobenzaldehyde or 2-[18F]fluoroethyl azide allowed for labeling in short times (, T1/2) and with acceptable efficiencies (10–20% ndc-RCY), although the obtained tracers did not show adequate biological properties.89,96 The comparatively low radiochemical yield obtained for [68Ga]–CPCR4.2 can be accounted for by the additional separation of the tracer from the remaining

unreacted precursor by time-consuming semipreparative high-performance liquid chromatography (HPLC) and subsequent lyophilization. Combined with using relatively high starting activities, these protocols resulted in good specific activities, with the purpose of reducing undesired adverse effects. Biologically speaking, the three classes of compounds explored so far all showed potential for the imaging of CXCR4 expression. [64Cu]–AMD3465 and [68Ga]–CCIC16 showed adequate in vitro uptake difference between CXCR4expressing U87.CD4.CXCR4 and their isogenic CXCR4-low counterparts. On the other hand, Wester and colleagues chose to assess their tracer, [68Ga]–CPCR4.2, in the human small cell lung cancer cell line OH1, which endogenously expresses CXCR4.90 No adequate control cell line is available for this cell line. In vivo, [64Cu]–AMD3465 yielded extremely high contrast, as depicted by the tumor to muscle and the tumor to blood ratios; however, the tracer also exhibited levels of uptake in the liver and the kidneys that cannot be explained by their endogenous expression of CXCR4. This tracer is therefore deemed to distribute nonspecifically to

13

PET Imaging of CXCR4 in Cancer

Figure 4. Selected images from PET/ CT experiments with [64Cu]–AMD3465 (A), [64Cu]–CPCR4.2 (B), and [68Ga]– CCIC16 (C). White arrowheads indicate the CXCR4-overexpressing tumors. The hollow arrowhead indicates the CXCR4low tumor. See the original research in De Silva and colleagues and Gourni and colleagues.84,90 B 5 bladder; K 5 kidneys; L 5 liver.

liver tissue, which would query use in liver metastases. Similar behavior was observed with its congener [64Cu]– AMD3100. Despite these concerns, [64Cu]–AMD3100 has been chosen for a small clinical trial aimed at assessing the safety and efficacy of the tracer in patients with tumors equal to or greater than 2 cm in diameter found outside the lymph nodes, bone marrow, liver, gallbladder, kidney, bladder, and brain. The study is currently recruiting participants. These nonspecificity issues might be due to the relative simplicity of the structures of both of these tracers, and it could be expected that further modification of the molecule could improve the specificity of this type of compound, for instance, by gaining inspiration from the numerous other small molecules that have been derivatized from AMD3100.48 Moreover, the small-molecule nature of this class could allow for efficient labeling with 18F, a radioisotope that is intrinsically more suitable for PET imaging than 64Cu; to date, no 18F-labeled small molecule–based CXCR4-specific radiotracer has been published, as emphasized by Table 2. Conversely, [68Ga]–CPCR4.2 and [68Ga]– CCIC16 showed good imaging properties, such as fast renal clearance and limited background uptake. Compared to [64Cu]–AMD3465, these two tracers exhibited much lower contrast; they were, however, still found adequate for the imaging of tumors. Similarly, uptake in metabolic organs was found to be lower with these two gallium 68–labeled tracers than with [64Cu]–AMD3465, although to different extents. The uptake of [68Ga]–CPCR4.2 in nontumor tissues was almost negligible when compared to [68Ga]– CCIC16; it can be anticipated that such low uptake might allow for the imaging of hepatic tumors. Although [68Ga]– CPCR4.2’s urinary localization was not measured, it seems clear from the PET images that renal tumors cannot be imaged with this tracer. All in all, to date, [68Ga]–CPCR4.2 and [68Ga]–CCIC16 are the CXCR4-targeting PET tracers

that show the most promising results across all tumor sites, whereas [64Cu]–AMD3465 had the highest sensitivity.

Perspectives and Future Directions To date, peptidic tracers have attracted the most interesting scaffold for CXCR4-targeting radiotracers, undoubtedly because of their ease of synthesis and the consequent relative rapidity with which compounds of desirable pharmacokinetic properties can be developed. [68Ga]– CPCR4.2 is the most promising tracer of this kind, and preliminary human scans with this tracer underlie the possibility of imaging certain tumors by targeting CXCR4. We can expect to see reports of how this tracer performs in a clinical setting soon, which could enlighten us as to the suitability (sensitivity and specificity) of CXCR4 imaging for prognosis, diagnosis, and tumor phenotyping. This is particularly eagerly anticipated as although numerous factors indicate that CXCR4 is a PET target of high interest, tangible confirmation of this assumption is required. In the longer term, this could allow for patient stratification and personalized medicine with CXCR4targeting drugs or radiotherapy. Moreover, it is possible that chelation of CPCR4.2 to radiotherapeutic metals such as 90Y or 177Lu could afford a radiotherapeutic agent, for use in a theranostic approach. On the other hand, small molecules have so far enjoyed less attention as potential CXCR4-targeting PET agents. AMD3100 and AMD3465 are the only small molecules that have been radiolabeled for PET imaging purposes (see Table 2). However, these cyclam derivatives seem to lack specificity for CXCR4, as depicted by their high and blockable uptake in the liver and the kidneys. Given the pharmacologic attraction of CXCR4, the number of small-molecule CXCR4 antagonists being reported is rapidly increasing, and the

14

George et al

Figure 5. Chemical structures of three CXCR4 inhibitors that could be derivatized to afford novel PET radiotracers. EC50 5 half maximal effective concentration; IC50 5 half maximal inhibitory concentration.

emergence of radiolabeled analogues of these molecules is therefore to be expected. It is hoped that such new radioligands may circumvent the nonspecificity issues encountered with [64Cu]–AMD3100 and [64Cu]–AMD3465. Other potential examples are Liotta and colleagues’ 31k,131 GlaxoSmithKline’s 13,132 and Novartis’s IT1t115 (Figure 5). These compounds have high affinities for CXCR4 and could be derivatized to allow for their radiolabeling with 18 F. Para-xyxyl 31k131 contains two fluorine atoms, one of which could be replaced by a nickel-based substituent to undergo a palladium-mediated fluorination with aqueous [18F]fluoride118 and afford [18F]31k. Similarly, it can be anticipated that substituting an aromatic hydrogen of tetrahydroquinoline 13132 for an iodine could allow for the [18F]trifluoromethylation of the resulting precursor into the corresponding [18F]trifluoromethylated 13. A careful in silico docking study and an SAR analysis will nevertheless be needed to determine which aromatic position is more adequate for the introduction of such modification. As aforementioned, four cocrystal structures of the small molecule IT1t with CXCR4 (2.523.2 A˚, PDB: 3ODU, 3OE6, 3OE8, 3OE9) were elucidated in 2010 in a landmark study by Stevens and colleagues, along with a CVX15–CXCR4 cocrystal structure (PDB: 3OE0).91 Taking into consideration the interesting pharmacokinetic profile of IT1t, which is characterized by high affinity and specificity for CXCR4 and in vitro stability against various cytochrome P-450 enzymes,115 these x-ray data are expected to promote the molecular design of IT1t-based tracers. Crystal structures are convenient as they allow for the determination of the interactions that mediate the binding of a ligand to the receptor and can help inform the de novo design of new CXCR4 ligands or the improvement of existing structures. However, the function of GPCRs is to transmit a signal through the cell membrane via a change of conformation; this type of receptor is therefore highly flexible, as illustrated by the differences between the IT1tand CVX15-bound cocrystal structures.91 The flexibility of CXCR4’s structure admonishes the medicinal chemist to

proceed with care when trying to dock molecules in silico into the crystal structure. The use of high-throughput virtual screenings should especially be avoided as this technique relies on rigid docking, whereby the receptor– ligand interactions are modeled on the ‘‘key–lock’’ rather than on the more accurate ‘‘hand–glove’’ paradigm. Although challenging, the evaluation of IT1t-based tracers is particularly attractive as it would enable the adequacy of 18 F-labeled small molecules for the imaging of CXCR4 expression to be explored, an area still uncharted, as highlighted previously (see Table 2).

Conclusion Since 1994 and the discovery of CXCR4, there has been great progress toward the development of CXCR4targeting PET tracers. Various potent CXCR4 inhibitors have been discovered, and over the last 7 years, some of these have been engineered to allow for radiolabeling with 18 F, 68Ga, or 64Cu. Although there is no clear consensus as to the precise clinical application for imaging CXCR4 to date, the potential use of such PET tracers in cancer prognosis, cancer diagnosis, tumor phenotyping, and theranostics makes research to develop such probes of great interest. 64Cu-radiolabeled AMD3465 has to date proven to be the best radiotracer with respect to absolute tumor uptake. The preliminary clinical results obtained from the evaluation of [68Ga]–CPCR4.2 are particularly encouraging regarding low liver radiotracer localization. Finally, an 18F-labeled small molecule for the noninvasive imaging of CXCR4 expression by PET has yet to be disclosed but can be expected to emerge in the near future.

Acknowledgments We would like to thank Elizabeth Stevens for producing the [68Ga]–CCIC16 PET imaging data set. Financial disclosure of authors: This work was funded by Cancer Research UK–Engineering and Physical Sciences Research Council (in association with the Medical Research

PET Imaging of CXCR4 in Cancer

Council and Department of Health, England) grant, C2536/ A10337. E.O.A.’s laboratory receives core funding from the UK Medical Research Council (MC_A652_ 5PY80). Financial disclosure of reviewers: None reported.

References 1. Yu L, Cecil J, Peng SB, et al. Identification and expression of novel isoforms of human stromal cell-derived factor 1. Gene 2006;374: 174–9, doi:10.1016/j.gene.2006.02.001. 2. Janowski M. Functional diversity of SDF-1 splicing variants Cell Adh Migr 2009;3:243–9, doi:10.4161/cam.3.3.8260. 3. Bernhagen J, Krohn R, Lue H, et al. MIF is a noncognate ligand of CXC chemokine receptors in inflammatory and atherogenic cell recruitment. Nat Med 2007;13:587–96, doi:10.1038/nm1567. 4. Saini V, Marchese A, Tang WJ, Majetschak M. Structural determinants of ubiquitin-CXC chemokine receptor 4 interaction. J Biol Chem 2011;286:44145–52, doi:10.1074/jbc.M111.298505. 5. Yang H, Antoine DJ, Andersson U, Tracey KJ. The many faces of HMGB1: molecular structure-functional activity in inflammation, apoptosis, and chemotaxis. J Leukoc Biol 2013;93:865–73, doi:10. 1189/jlb.1212662. 6. Sierro F, Biben C, Martinez-Munoz L, et al. Disrupted cardiac development but normal hematopoiesis in mice deficient in the second CXCL12/SDF-1 receptor, CXCR7. Proc Natl Acad Sci U S A 2007;104:14759–64, doi:10.1073/pnas.0702229104. 7. Ma Q, Jones D, Borghesani PR, et al. Impaired B-lymphopoiesis, myelopoiesis, and derailed cerebellar neuron migration in CXCR4- and SDF-1-deficient mice, Proc Natl Acad Sci U S A 1998;95:9448–53, doi:10.1073/pnas.95.16.9448. 8. Tachibana K, Hirota S, Iizasa H, et al. The chemokine receptor CXCR4 is essential for vascularization of the gastrointestinal tract. Nature 1998;393:591–4, doi:10.1038/31261. 9. Zou YR, Kottmann AH, Kuroda M, et al. Function of the chemokine receptor CXCR4 in haematopoiesis and in cerebellar development. Nature 1998;393:595–9, doi:10.1038/31269. 10. Sharma M, Afrin F, Satija N, et al. Stromal-derived factor-1/ CXCR4 signaling: indispensable role in homing and engraftment of hematopoietic stem cells in bone marrow. Stem Cells Dev 2011; 20:933–46, doi:10.1089/scd.2010.0263. 11. Bleul CC, Fuhlbrigge RC, Casasnovas JM, et al. A highly efficacious lymphocyte chemoattractant, stromal cell-derived factor 1 (SDF-1). J Exp Med 1996;184:1101–9, doi:10.1084/jem. 184.3.1101. 12. Gupta SK, Lysko PG, Pillarisetti K, et al. Chemokine receptors in human endothelial cells. Functional expression of CXCR4 and its transcriptional regulation by inflammatory cytokines. J Biol Chem 1998;273:4282–7, doi:10.1074/jbc.273.7.4282. 13. Murdoch C, Monk PN, Finn A. Functional expression of chemokine receptor CXCR4 on human epithelial cells. Immunology 1999;98:36– 41, doi:10.1046/j.1365-2567.1999.00848.x. 14. Donzella GA, Schols D, Lin SW, et al. AMD3100, a small molecule inhibitor of HIV-1 entry via the CXCR4 co-receptor. Nat Med 1998;4:72–7, doi:10.1038/nm0198-072. 15. Tarasova NI, Stauber RH, Michejda CJ. Spontaneous and ligandinduced trafficking of CXC-chemokine receptor 4. J Biol Chem 1998;273:15883–6, doi:10.1074/jbc.273.26.15883.

15

16. Nanki T, Takada K, Komano Y, et al. Chemokine receptor expression and functional effects of chemokines on B cells: implication in the pathogenesis of rheumatoid arthritis. Arthritis Res Ther 2009;11:R149, doi:10.1186/ar2823. 17. Weitzenfeld P, Ben-Baruch A. The chemokine system, and its CCR5 and CXCR4 receptors, as potential targets for personalized therapy in cancer. Cancer Lett 2013, doi:10.1016/j.canlet.2013.10.006. 18. Sun X, Cheng G, Hao M, et al. CXCL12 / CXCR4 / CXCR7 chemokine axis and cancer progression. Cancer Metastasis Rev 2010;29:709–22, doi:10.1007/s10555-010-9256-x. 19. Balkwill FR. The chemokine system and cancer. J Pathol 2012;226: 148–57, doi:10.1002/path.3029. 20. Fulton AM. The chemokine receptors CXCR4 and CXCR3 in cancer. Curr Oncol Rep 2009;11:125–31, doi:10.1007/s11912-009-0019-1. 21. Zlotnik A, Burkhardt AM, Homey B. Homeostatic chemokine receptors and organ-specific metastasis. Nat Rev Immunol 2011; 11:597–606, doi:10.1038/nri3049. 22. Andre F, Xia W, Conforti R, et al. CXCR4 expression in early breast cancer and risk of distant recurrence. Oncologist 2009;14: 1182–8, doi:10.1634/theoncologist.2009-0161. 23. Schmid BC, Rudas M, Rezniczek GA, et al. CXCR4 is expressed in ductal carcinoma in situ of the breast and in atypical ductal hyperplasia. Breast Cancer Res Treat 2004;84:247–50, doi:10.1023/ B:BREA.0000019962.18922.87. 24. Na IK, Scheibenbogen C, Adam C, et al. Nuclear expression of CXCR4 in tumor cells of non-small cell lung cancer is correlated with lymph node metastasis. Hum Pathol 2008;39:1751–5, doi:10.1016/j.humpath.2008.04.017. 25. Otsuka S, Klimowicz AC, Kopciuk K, et al. CXCR4 overexpression is associated with poor outcome in females diagnosed with stage IV non-small cell lung cancer. J Thorac Oncol 2011;6: 1169–78, doi:10.1097/JTO.0b013e3182199a99. 26. Wehler TC, Graf C, Biesterfeld S, et al. Strong expression of chemokine receptor CXCR4 by renal cell carcinoma correlates with advanced disease. J Oncol 2008;2008:626340. 27. Li T, Li H, Wang Y, et al. The expression of CXCR4, CXCL12 and CXCR7 in malignant pleural mesothelioma. J Pathol 2011;223: 519–30, doi:10.1002/path.2829. 28. Lee HJ, Kim SW, Kim HY, et al. Chemokine receptor CXCR4 expression, function, and clinical implications in gastric cancer. Int J Oncol 2009;34:473–80. 29. Akashi T, Koizumi K, Tsuneyama K, et al. Chemokine receptor CXCR4 expression and prognosis in patients with metastatic prostate cancer. Cancer Sci 2008;99:539–42, doi:10.1111/j.13497006.2007.00712.x. 30. Chen G, Wang Z, Liu XY, Liu FY. High-level CXCR4 expression correlates with brain-specific metastasis of non-small cell lung cancer. World J Surg 2011;35:56–61, doi:10.1007/s00268-010-0784-x. 31. Ying J, Xu Q, Zhang G, et al. The expression of CXCL12 and CXCR4 in gastric cancer and their correlation to lymph node metastasis. Med Oncol 2012;29:1716–22, doi:10.1007/s12032-0119990-0. 32. D’Alterio C, Consales C, Polimeno M, et al. Concomitant CXCR4 and CXCR7 expression predicts poor prognosis in renal cancer. Curr Cancer Drug Targets 2010;10:772–81, doi:10.2174/15680091 0793605839. 33. Saur D, Seidler B, Schneider G, et al. CXCR4 expression increases liver and lung metastasis in a mouse model of pancreatic cancer.

16

34.

35.

36.

37. 38.

39.

40.

41.

42.

43.

44.

45.

46.

47.

48. 49.

George et al

Gastroenterology 2005;129:1237–50, doi:10.1053/j.gastro.2005.06. 056. Jiang YP, Wu XH, Shi B, et al. Expression of chemokine CXCL12 and its receptor CXCR4 in human epithelial ovarian cancer: an independent prognostic factor for tumor progression. Gynecol Oncol 2006;103:226–33, doi:10.1016/j.ygyno.2006.02.036. Liao WC, Wang HP, Huang HY, et al. CXCR4 expression predicts early liver recurrence and poor survival after resection of pancreatic adenocarcinoma. Clin Transl Gastroenterol 2012;3:e22, doi:10.1038/ ctg.2012.18. Gassmann P, Haier J, Schluter K, et al. CXCR4 regulates the early extravasation of metastatic tumor cells in vivo. Neoplasia 2009;11: 651–61. Mukherjee D, Zhao J. The role of chemokine receptor CXCR4 in breast cancer metastasis. Am J Cancer Res 2013;3:46–57. Kochetkova M, Kumar S, McColl SR. Chemokine receptors CXCR4 and CCR7 promote metastasis by preventing anoikis in cancer cells. Cell Death Differ 2009;16:664–73, doi:10.1038/cdd.2008.190. Muller A, Homey B, Soto H, et al. Involvement of chemokine receptors in breast cancer metastasis. Nature 2001;410:50–6, doi: 10.1038/35065016. Orimo A, Gupta PB, Sgroi DC, et al. Stromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis through elevated SDF-1/CXCL12 secretion. Cell 2005;121:335–48, doi:10.1016/j.cell.2005.02.034. Ping YF, Yao XH, Jiang JY, et al. The chemokine CXCL12 and its receptor CXCR4 promote glioma stem cell-mediated VEGF production and tumour angiogenesis via PI3K/AKT signalling. J Pathol 2011;224:344–54, doi:10.1002/path.2908. Liekens S, Schols D, Hatse S. CXCL12-CXCR4 axis in angiogenesis, metastasis and stem cell mobilization. Curr Pharm Des 2010; 16:3903–20, doi:10.2174/138161210794455003. Sun X, Charbonneau C, Wei L, et al. CXCR4-targeted therapy inhibits VEGF expression and chondrosarcoma angiogenesis and metastasis. Mol Cancer Ther 2013;12:1163–70, doi:10.1158/15357163.MCT-12-1092. Sehgal A, Keener C, Boynton AL, et al. CXCR-4, a chemokine receptor, is overexpressed in and required for proliferation of glioblastoma tumor cells. J Surg Oncol 1998;69:99–104, doi:10.1002/ (SICI)1096-9098(199810)69:2,99::AID-JSO10.3.0.CO;2-M. Singh S, Srivastava SK, Bhardwaj A, et al. CXCL12-CXCR4 signalling axis confers gemcitabine resistance to pancreatic cancer cells: a novel target for therapy. Br J Cancer 2010;103:1671–9, doi:10.1038/sj.bjc.6605968. Rhodes LV, Short SP, Neel NF, et al. Cytokine receptor CXCR4 mediates estrogen-independent tumorigenesis, metastasis, and resistance to endocrine therapy in human breast cancer. Cancer Res 2011;71:603–13, doi:10.1158/0008-5472.CAN-10-3185. Margolin DA, Silinsky J, Grimes C, et al. Lymph node stromal cells enhance drug-resistant colon cancer cell tumor formation through SDF-1alpha/CXCR4 paracrine signaling. Neoplasia 2011; 13:874–86. Debnath B, Xu S, Grande F, et al. Small molecule inhibitors of CXCR4. Theranostics 2013;3:47–75, doi:10.7150/thno.5376. Brelot A, Heveker N, Montes M, Alizon M. Identification of residues of CXCR4 critical for human immunodeficiency virus coreceptor and chemokine receptor activities. J Biol Chem 2000; 275:23736–44, doi:10.1074/jbc.M000776200.

50. Chabot DJ, Zhang PF, Quinnan GV, Broder CC. Mutagenesis of CXCR4 identifies important domains for human immunodeficiency virus type 1 X4 isolate envelope-mediated membrane fusion and virus entry and reveals cryptic coreceptor activity for R5 isolates. J Virol 1999;73:6598–609. 51. Brelot A, Heveker N, Adema K, et al. Effect of mutations in the second extracellular loop of CXCR4 on its utilization by human and feline immunodeficiency viruses. J Virol 1999;73:2576–86. 52. Kuhne MR, Mulvey T, Belanger B, et al. BMS-936564/MDX-1338: a fully human anti-CXCR4 antibody induces apoptosis in vitro and shows antitumor activity in vivo in hematologic malignancies. Clin Cancer Res 2013;19:357–66, doi:10.1158/1078-0432.CCR-12-2333. 53. Fujii N, Oishi S, Hiramatsu K, et al. Molecular-size reduction of a potent CXCR4-chemokine antagonist using orthogonal combination of conformation- and sequence-based libraries. Angew Chem Int Ed 2003;42:3251–3, doi:10.1002/anie.200351024. 54. Ueda S, Oishi S, Wang Z-X, et al. Structure-activity relationships of cyclic peptide-based chemokine receptor CXCR4 antagonists: disclosing the importance of side-chain and backbone functionalities. J Med Chem 2007;50:192, doi:10.1021/jm0607350. 55. Tanaka T, Nomura W, Narumi T, et al. Structure-activity relationship study on artificial CXCR4 ligands possessing the cyclic pentapeptide scaffold: the exploration of amino acid residues of pentapeptides by substitutions of several aromatic amino acids. Org Biomol Chem 2009;7:3805, doi:10.1039/b908286g. 56. Tanaka T, Tsutsumi H, Noruma W, et al. Structure-activity relationship study of CXCR4 antagonists bearing the cyclic pentapeptide scaffold: identification of the new pharmacophore. Org Biomol Chem 2008;6:4374, doi:10.1039/b812029c. 57. Tamamura H, Tsutsumi H, Nomura W, Fujii N. Exploratory studies on development of the chemokine receptor CXCR4 antagonists toward downsizing. Perspect Med Chem 2008;2:1. 58. Tsutsumi H, Tanaka T, Ohashi N, et al. Therapeutic potential of the chemokine receptor CXCR4 antagonists as multifunctional agents. Biopolymers 2007;88:279–89, doi:10.1002/bip.20653. 59. Tamamura H, Araki T, Ueda S, et al. Identification of novel low molecular weight CXCR4 antagonists by structural tuning of cyclic tetrapeptide scaffolds. J Med Chem 2005;48:3280, doi:10. 1021/jm050009h. 60. Tamamura H, Hiramatsu K, Ueda S, et al. Stereoselective synthesis of [L-Arg-L/D-3-(2-naphthyl)alanine]-type (E)-alkene dipeptide isosteres and its application to the synthesis and biological evaluation of pseudopeptide analogues of the CXCR4 antagonist FC131. J Med Chem 2005;48:380, doi:10.1021/jm049429h. 61. Tamamura H, Esaka A, Ogawa T, et al. Structure-activity relationship studies on CXCR4 antagonists having cyclic pentapeptide scaffolds. Org Biomol Chem 2005;3:4392, doi:10.1039/b513145f. 62. Nakamura T, Furunaka H, Miyata T, et al. Tachyplesin, a class of antimicrobial peptide from the hemocytes of the horseshoe crab (Tachypleus tridentatus). Isolation and chemical structure. J Biol Chem 1988;263:16709–13. 63. Miyata T, Tokunaga F, Yoneya T, et al. Antimicrobial peptides, isolated from horseshoe crab hemocytes, tachyplesin ii, and polyphemusins I and II: chemical structures and biological activity. J Biochem 1989;106:663–8. 64. Tamamura H, Xu Y, Hattori T, et al. A low-molecular-weight inhibitor against the chemokine receptor CXCR4: a strong antiHIV peptide T140. Biochem Biophys Res Commun 1998;253: 877–82, doi:10.1006/bbrc.1998.9871.

PET Imaging of CXCR4 in Cancer

65. Tamamura H, Omagari A, Hiramatsu K, et al. Development of specific CXCR4 inhibitors possessing high selectivity indexes as well as complete stability in serum based on an anti-HIV peptide T140. Bioorg Med Chem Lett 2001;11:1897–902, doi:10.1016/ S0960-894X(01)00323-7. 66. Masuda M, Nakashima H, Ueda T, et al. A novel anti-HIV synthetic peptide, T-22 ([Tyr5,12,Lys7]-polyphemusin II). Biochem Biophys Res Commun 1992;189:845–50, doi:10.1016/0006-291X (92)92280-B. 67. Nakashima H, Masuda M, Murakami T, et al. Anti-human immunodeficiency virus activity of a novel synthetic peptide, T22 ([Tyr-5,12, Lys-7]polyphemusin II): a possible inhibitor of viruscell fusion. Antimicrob Agents Chemother 1992;36:1249–55, doi: 10.1128/AAC.36.6.1249. 68. Tamamura T, Hiramatsu K, Mizumoto M, et al. Enhancement of the T140-based pharmacophores leads to the development of more potent and bio-stable CXCR4 antagonists. Org Biomol Chem 2003;1:3663–9, doi:10.1039/b306613b. 69. Friand V, Haddad O, Papy-Garcia D, et al. Glycosaminoglycan mimetics inhibit SDF-1/CXCL12-mediated migration and invasion of human hepatoma cells. Glycobiology 2009;19:1511–24, doi:10.1093/glycob/cwp130. 70. Kuil J, Buckle T, van Leeuwen FW. Imaging agents for the chemokine receptor 4 (CXCR4). Chem Soc Rev 2012;41:5239–61, doi:10.1039/c2cs35085h. 71. Kaifi JT, Yekebas EF, Schurr P, et al. Tumor-cell homing to lymph nodes and bone marrow and CXCR4 expression in esophageal cancer. J Natl Cancer Inst 2005;97:1840–7, doi:10.1093/jnci/dji431. 72. Nguyen QD, Aboagye EO. Imaging the life and death of tumors in living subjects: preclinical PET imaging of proliferation and apoptosis. Integr Biol 2010;2:483–95, doi:10.1039/c0ib00066c. 73. Weiss ID, Jacobson O. Molecular imaging of chemokine receptor CXCR4. Theranostics 2013;3:76–84, doi:10.7150/thno.4835. 74. Knight JC, Wuest FR. Nuclear (PET/SPECT) and optical imaging probes targeting the CXCR4 chemokine receptor. MedChemComm 2012;3:1039–53, doi:10.1039/c2md20117h. 75. Woodard LE, Nimmagadda S. CXCR4-based imaging agents. J Nucl Med 2011;52:1665–9, doi:10.2967/jnumed.111.097733. 76. Cherry SR, Gambhir SS. Use of positron emission tomography in animal research. ILAR J 2001;42:219–32, doi:10.1093/ilar.42.3.219. 77. Cherry SR. The 2006 Henry N. Wagner Lecture: Of mice and men (and positrons)—advances in PET imaging technology. J Nucl Med 2006;47:1735–45. 78. Cutler PD, Cherry SR, Hoffman EJ, et al. Design features and performance of a PET system for animal research. J Nucl Med 1992;33:595–604. 79. Nimmagadda S, Pullambhatla M, Stone K, et al. Molecular imaging of CXCR4 receptor expression in human cancer xenografts with [64Cu]AMD3100 positron emission tomography. Cancer Res 2010; 70:3935–44, doi:10.1158/0008-5472.CAN-09-4396. 80. Imaging CXCR4 expression in subjects with cancer using 64 CuPlerixafor. Available at: http://clinicaltrials.gov/show/NCT02069 080 (accessed June 2014). 81. Aboagye EO. Positron emission tomography imaging of small animals in anticancer drug development. Mol Imaging Biol 2005; 7:53–8, doi:10.1007/s11307-005-0886-2. 82. Smith G, Carroll L, Aboagye EO. New frontiers in the design and synthesis of imaging probes for PET oncology: current challenges

83.

84.

85.

86.

87.

88.

89.

90.

91.

92.

93.

94.

95.

96.

97.

98.

17

and future directions. Mol Imaging Biol 2012;14:653–66, doi: 10.1007/s11307-012-0590-y. Demmer O, Dijkgraaf I, Schumacher U, et al. Design, synthesis, and functionalization of dimeric peptides targeting chemokine receptor CXCR4. J Med Chem 2011;54:7648–62, doi:10.1021/ jm2009716. De Silva RA, Peyre K, Pullambhatla M, et al. Imaging CXCR4 expression in human cancer xenografts: evaluation of monocyclam 64Cu-AMD3465. J Nucl Med 2011;52:986–93, doi:10. 2967/jnumed.110.085613. Gravel S, Malouf C, Boulais PE, et al. The peptidomimetic CXCR4 antagonist TC14012 recruits b-arrestin to CXCR7: roles of receptor domains. J Biol Chem 2010;285:37939–43, doi:10. 1074/jbc.C110.147470. Kalatskaya I, Berchiche YA, Gravel S, et al. AMD3100 is a CXCR7 ligand with allosteric agonist properties. Mol Pharmacol 2009;75: 1240–7, doi:10.1124/mol.108.053389. Mosi RM, Anastassova V, Cox J, et al. The molecular pharmacology of AMD11070: an orally bioavailable CXCR4 HIV entry inhibitor. Biochem Pharmacol 2012;83:472–9, doi:10. 1016/j.bcp.2011.11.020. George GP, Stevens E, A˚berg O, et al. Preclinical evaluation of a CXCR4-specific 68Ga-labelled TN14003 derivative for cancer PET imaging. Bioorg Med Chem 2014;22:796–803, doi:10.1016/ j.bmc.2013.12.012. [Epub 2013 Dec 11] A˚berg O, Pisaneschi F, Smith G, et al. 18F-labelling of a cyclic pentapeptide inhibitor of the chemokine receptor CXCR4. J Fluorine Chem 2012;135:200–6, doi:10.1016/j.jfluchem.2011.11.003. Gourni E, Demmer O, Schottelius M, et al. PET of CXCR4 expression by a 68Ga-labeled highly specific targeted contrast agent. J Nucl Med 2011;52:1803–10, doi:10.2967/jnumed.111.098798. Wu B, Chien EYT, Mol CD, et al. Structures of the CXCR4 chemokine GPCR with small-molecule and cyclic peptide antagonists. Science 2010;330:1066–71, doi:10.1126/science.1194396. Jacobson O, Weiss ID, Szajek LP, et al. PET imaging of CXCR4 using copper-64 labeled peptide antagonist. Theranostics 2011; 251–62, doi:10.7150/thno/v01p0251. Hennrich U, Seyler L, Scha¨fer M, et al. Synthesis and in vitro evaluation of 68Ga-DOTA-4-FBn-TN14003, a novel tracer for the imaging of CXCR4 expression. Biorg Med Chem 2012;20:1502– 10, doi:10.1016/j.bmc.2011.12.052. Zhang X-X, Sun Z, Guo, et al. Comparison of 18F-labeled CXCR4 antagonist peptides for PET imaging of CXCR4 expression. Mol Imag Biol 2013, doi:10.1007/s11307-013-0640-0, 1-10. Jacobson O, Weiss ID, Szajek L, et al. 64Cu-AMD3100—a novel imaging agent for targeting chemokine receptor CXCR4. Bioorg Med Chem 2009;17:1486–93, doi:10.1016/j.bmc.2009.01.014. George GPC, Pisaneschi F, Stevens E, et al. Scavenging strategy for specific activity improvement: application to a new CXCR4-specific cyclopentapeptide positron emission tomography tracer. J Labelled Compd Radiopharm 2013;56:679–85, doi:10.1002/jlcr.3095. Jacobson O, Weiss ID, Kiesewetter DO, et al. PET of tumor CXCR4 expression with 4-18F-T140. J Nucl Med 2010;51:1796– 804, doi:10.2967/jnumed.110.079418. Hanaoka H, Mukai T, Tamamura H, et al. Development of a 111In-labeled peptide derivative targeting a chemokine receptor, CXCR4, for imaging tumors. Nucl Med Biol 2006;33:489–94, doi:10.1016/j.nucmedbio.2006.01.006.

18

George et al

99. Kuil J, Buckle T, Yuan H, et al. Synthesis and evaluation of a bimodal CXCR4 antagonistic peptide. Bioconjug Chem 2011;22: 859–64, doi:10.1021/bc2000947. 100. Jacobson O, Weiss ID, Szajek LP, et al. Improvement of CXCR4 tracer specificity for PET imaging. J Controlled Release 2012;157: 216–23, doi:10.1016/j.jconrel.2011.09.076. 101. Weiss ID, Jacobson O, Kiesewetter DO, et al. Positron emission tomography imaging of tumors expressing the human chemokine receptor CXCR4 in mice with the use of 64Cu-AMD3100. Mol Imaging Biol 2012;14:106–14, doi:10.1007/s11307-010-0466-y. 102. Demmer O, Gourni E, Schumacher U, et al. PET imaging of CXCR4 receptors in cancer by a new optimized ligand. ChemMedChem 2011;6:1789–91, doi:10.1002/cmdc.201100320. 103. van den Berg NS, Buckle T, Kuil J, et al. Immunohistochemical detection of the CXCR4 expression in tumor tissue using the fluorescent peptide antagonist Ac-TZ14011-FITC. Transl Oncol 2011;4:234–40, doi:10.1593/tlo.11115. 104. Choi W-T, Duggineni S, Xu Y, et al. Drug discovery research targeting the CXC chemokine receptor 4 (CXCR4). J Med Chem 2011;55:977–94, doi:10.1021/jm200568c. 105. Singh IP, Chauthe SK. Small molecule HIV entry inhibitors: part I. Chemokine receptor antagonists: 2004 – 2010. Expert Opin Ther Patents 2011;21:227–69, doi:10.1517/13543776.2011. 542412. 106. Tamamura H, Fujii N. The therapeutic potential of CXCR4 antagonists in the treatment of HIV infection, cancer metastasis and rheumatoid arthritis. Expert Opin Ther Targets 2005;9:1267– 82, doi:10.1517/14728222.9.6.1267. 107. Fujii N, Nakashima H, Tamamura H. The therapeutic potential of CXCR4 antagonists in the treatment of HIV. Expert Opin Investig Drugs 2003;12:185–95, doi:10.1517/13543784.12.2.185. 108. Mosley CA, Wilson LJ, Wiseman JM, et al. Recent patents regarding the discovery of small molecule CXCR4 antagonists. Expert Opin Ther Patents 2009;19:23–38, doi:10.1517/13543770802553483. 109. Loetscher M, Geiser T, O’Reilly T, et al. Cloning of a human seven-transmembrane domain receptor, LESTR, that is highly expressed in leukocytes. J Biol Chem 1994;269:232–7. 110. Tamamura H, Omagari A, Oishi S, et al. Pharmacophore identification of a specific CXCR4 inhibitor, T140, leads to development of effective anti-HIV agents with very high selectivity indexes. Bioorg Med Chem Lett 2000;10:2633–7, doi:10.1016/ S0960-894X(00)00535-7. 111. Cluzeau J, Oishi S, Ohno H, et al. Design and synthesis of all diastereomers of cyclic pseudo-dipeptides as mimics of cyclic CXCR4 pentapeptide antagonists. Org Biomol Chem 2007;5:1915, doi:10.1039/b702649h. 112. De Clercq E. The bicyclam AMD3100 story. Nat Rev Drug Discov 2003;2:581–7, doi:10.1038/nrd1134. 113. Domanska UM, Kruizinga RC, Nagengast WB, et al. A review on CXCR4/CXCL12 axis in oncology: no place to hide. Eur J Cancer 2013;49:219–30, doi:10.1016/j.ejca.2012.05.005. 114. Hatse S, Princen K, Clercq ED, et al. AMD3465, a monomacrocyclic CXCR4 antagonist and potent HIV entry inhibitor. Biochem Pharmacol 2005;70:752–61, doi:10.1016/j.bcp.2005.05.035. 115. Thoma G, Streiff MB, Kovarik J, et al. Orally bioavailable isothioureas block function of the chemokine receptor CXCR4 in vitro and in vivo. J Med Chem 2008;51:7915–20, doi:10.1021/ jm801065q.

116. Bergman J, Solin O. Fluorine-18-labeled fluorine gas for synthesis of tracer molecules. Nucl Med Biol 1997;24:677–83, doi:10.1016/ S0969-8051(97)00078-4. 117. Hollingworth C, Hazari A, Hopkinson MN, et al. Palladiumcatalyzed allylic fluorination. Angew Chem Int Ed 2011;50:2613– 7, doi:10.1002/anie.201007307. 118. Lee E, Hooker JM, Ritter T. Nickel-mediated oxidative fluorination for PET with aqueous [18F]fluoride. J Am Chem Soc 2012; 134:17456–8, doi:10.1021/ja3084797. 119. Lee E, Kamlet AS, Powers DC, et al. A fluoride-derived electrophilic late-stage fluorination reagent for PET imaging. Science 2011;334:639–42, doi:10.1126/science.1212625. 120. Gao Z, Lim YH, Tredwell M, et al. Metal-free oxidative fluorination of phenols with [18F]fluoride. Angew Chem Int Ed 2012;51:6733–7, doi:10.1002/anie.201201502. 121. Huiban M, Tredwell M, Mizuta S, et al. A broadly applicable [18F]trifluoromethylation of aryl and heteroaryl iodides for PET imaging. Nat Chem 2013;5:941–4, doi:10.1038/nchem.1756. 122. Wadas TJ, Wong EH, Weisman GR, Anderson CJ. Coordinating radiometals of copper, gallium, indium, yttrium, and zirconium for PET and SPECT imaging of disease. Chem Rev 2010;110: 2858–902, doi:10.1021/cr900325h. 123. Hueting R, Kersemans V, Cornelissen B, et al. A comparison of the behavior of 64Cu-acetate and 64Cu-ATSM in vitro and in vivo. J Nucl Med 2014, doi:10.2967/jnumed.113.119917. 124. Yoshii Y, Furukawa T, Kiyono Y, et al. Internal radiotherapy with copper-64-diacetyl-bis (N4-methylthiosemicarbazone) reduces CD133+ highly tumorigenic cells and metastatic ability of mouse colon carcinoma. Nucl Med Biol 2011;38:151–7, doi:10.1016/ j.nucmedbio.2010.08.009. [Epub 2010 Oct 27] 125. Sun Y-X, Wang J, Shelburne CE, et al. Expression of CXCR4 and CXCL12 (SDF-1) in human prostate cancers (PCa) in vivo. J Cell Biochem 2003;89:462–73, doi:10.1002/jcb.10522. 126. Salvucci O, Bouchard A, Baccarelli A, et al. The role of CXCR4 receptor expression in breast cancer: a large tissue microarray study. Breast Cancer Res Treat 2006;97:275–83, doi:10.1007/ s10549-005-9121-8. 127. Oda Y, Yamamoto H, Tamiya S, et al. CXCR4 and VEGF expression in the primary site and the metastatic site of human osteosarcoma: analysis within a group of patients, all of whom developed lung metastasis. Mod Pathol 2006;19:738–45, doi: 10.1038/modpathol.3800587. 128. Torregrossa L, Giannini R, Borrelli N, et al. CXCR4 expression correlates with the degree of tumor infiltration and BRAF status in papillary thyroid carcinomas. Mod Pathol 2012;25:46–55, doi:10.1038/modpathol.2011.140. 129. Wester H-J, Keller U, Beer AB. et al. [68Ga]CPCR4.2-PET for imaging of CXCR4-chemokine receptors opens a new and exciting field of clinical research. Presented in part at the FO Annual Congress of the European Association of Nuclear Medicine, Lyon, France, 2013. 130. First in human and clinical studies – multi-organ physiology and metabolism. Available at: http://www.wmis.org/abstracts/2013/ data/papers/SS112.htm (acessed August 23, 2014). 131. Zhan W, Liang Z, Zhu A, et al. Discovery of small molecule CXCR4 antagonists. J Med Chem 2007;50:5655–64, doi:10.1021/jm070679i. 132. Gudmundsson KS, Sebahar PR, Richardson LDA, et al. Amine substituted N-(1H-benzimidazol-2ylmethyl)-5,6,7,8-tetrahydro8-quinolinamines as CXCR4 antagonists with potent activity

PET Imaging of CXCR4 in Cancer

against HIV-1. Bioorg Med Chem Lett 2009;19:5048–52, doi:10. 1016/j.bmcl.2009.07.037. 133. Psaila B, Lyden D. The metastatic niche: adapting the foreign soil. Nat Rev Cancer 2009;9:285–93, doi:10.1038/nrc2621.

19

134. Martin R, Ju¨ttler S, Mu¨ller M, Wester H-J. Cationic eluate pretreatment for automated synthesis of [68Ga]CPCR4.2. Nucl Med Biol 2014;41:84–9, doi:10.1016/j.nucmedbio.2013. 09.002.