Clinical Applications of Radiolabeled Peptides for PET

ARTICLE IN PRESS

Clinical Applications of Radiolabeled Peptides for PET Isaac M. Jackson, BS, Peter J.H. Scott, PhD, and Stephen Thompson, PhD Radiolabeled peptides are a valuable class of radiotracer that occupies the space between small molecules and large biologics, and are able to exploit the advantages of both classes of compound. To date, radiolabeled peptides have mainly been utilized in oncology, where the same peptide can often be exploited for diagnostic imaging and targeted radiotherapy by simply varying the radionuclide. In this review, we introduce the main strategies used for synthesis of radiolabeled peptides, and highlight the state of the art for clinical imaging (and therapy) in oncology using the main classes of radiolabeled peptides that have been translated to date. Beyond oncology, radiolabeled peptides are also increasingly being used in other PET applications such as diabetes and cardiac imaging, and we review progress for the new applications. Semin Nucl Med ■■:■■–■■ © 2017 Elsevier Inc. All rights reserved.

Introduction

P

ositron emission tomography (PET) is a functional medical imaging technique that has revolutionized our understanding and approach to a diverse range of pathologies and physiological functions. An imaging modality popularized by the advent of [18F]FDG for imaging glucose metabolism, the field has grown to apply a wide range of radiopharmaceuticals to an array of pathologies. At present, the majority of Food and Drug Administration (FDA)-approved PET radiopharmaceuticals are small molecules, with the first peptide radiopharmaceutical for PET, [68Ga]DOTATATE (NETSPOT), having recently obtained regulatory approval.1 This milestone reflects a rapidly growing interest in the development and application of peptides as radiopharmaceuticals for imaging of a range of biological functions. Although applications of peptides to a host of imaging modalities such as scintigraphy and SPECT are well established, the superior qualitative and quantitative data available from PET have prompted a growth in interest in the development of PET radiolabeled peptides.2 Radiolabeled peptides occupy the space between small molecules and large biologics, and exploit the advantages of both classes of compounds. Radiolabeled peptides display pharmacokinetic properties similar to those of small molecules, Division of Nuclear Medicine, Department of Radiology, University of Michigan, Ann Arbor, MI.

This study received financial support from the U.S. Department of Energy (DE-SC0012484). Address reprint requests to Stephen Thompson, PhD, Division of Nuclear Medicine, Department of Radiology, University of Michigan, 1301 Catherine, Ann Arbor, MI 48109. E-mail: [email protected]

http://dx.doi.org/10.1053/j.semnuclmed.2017.05.007 0001-2998/© 2017 Elsevier Inc. All rights reserved.

such as fast clearance, and both classes of molecules tend to be relatively easy to synthesize and are generally stable to radiolabeling conditions. Like large biologics, radiolabeled peptides offer a high degree of selectivity for their receptor and are generally tolerant of structural modifications, so long as the binding motif is preserved.3 An additional consideration in the development of a radiolabelled peptide is its mode of receptor interaction (full or partial agonist or antagonist), however, the optimal approach tends to be target specific, warranting investigation on a case-by-case basis.

Methods for Radiolabeling Peptides Radiolabeled peptides used in the clinic are often based on modified variants of naturally occurring peptides. Of the commonly available radionuclides for PET, 18F, 64Cu, and 68Ga are most suited for radiolabeling peptides as their physical halflives are similar to the biological half-life of peptides. One problem posed by these radionuclides is that they require incorporation through the addition of synthetic functional groups to the native peptide. Although direct labeling is possible, the two most widely used strategies for introduction of the radionuclide into the peptide of choice use either bifunctional chelators (BFCs) or prosthetic groups (Fig. 1). Prosthetic groups are small, reactive entities that are first radiolabeled before being coupled to the peptide. BFCs are metal-binding motifs attached to the peptide of choice before radiolabeling with an appropriate radioactive metal ion (radiometal). As methods 1

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2

Figure 1 Strategies for radiolabeling peptides.

for radiolabeling proteins and peptides have been previously reviewed,4-10 included as part of this issue of Seminars (see “Small Prosthetic Groups in 18F-Radiochemistry: Useful Auxiliaries for the Design of 18F-PET Tracers” by Schirrmacher et al, Sem. Nucl. Med., 2017), only a brief discussion is included here.

Choice of Radionuclide Whereas radionuclide choice for labeling traditional small molecule PET radiotracers is dominated by 11C and 18F, peptide radiolabeling has favored the metal isotopes 68Ga and 64Cu using BFCs, with nontrivial prevalence of 18F and relatively no 11C work represented in the relevant literature. The use of radiometals offers certain advantages such as cyclotronfree generation of 68Ga, or the commercial availability and ability for wide distribution enabled by the long half-life of 64 Cu (t1/2 = 12.7 hours, Table). One advantage conferred by use of a radiometal such as 64Cu or 68Ga is the potential therapeutic applications achieved by exchanging the radiometal for a therapeutic α- (eg, 213Bi or 225Ac) or β−-emitting (eg, 90Y or 177Lu) radionuclide. This diagnostic and therapeutic (theranostic) pairing represents an attractive potential application of radiolabeled peptides found to selectively bind targets of interest. The choice of radiometal can be tailored for specific

biological contexts. For example, small peptides tend to show fast biodistribution and clearance, suitable for imaging with 68 Ga (half-life of 68 minutes), whereas large antibodies show slow distribution and clearance, making longer-lived 89Zr (halflife 3.7 days) an appropriate nuclide in this setting. The importance of radionuclide selection can be interpreted as a function of both half-life and mean free path, the average distance an emitted positron travels before annihilating with an electron. Longer half-life allows for longer and more complex tracer synthesis, as well as more time for in vivo biodistribution before image acquisition. Shorter mean free path can be seen to provide maximum theoretical image resolution. By these considerations, 18F would be expected to provide highest resolution, followed by 64Cu and finally 68 Ga. Mean free energy is a third nuclide dependent property impacting dosimetry, as this affects the amount of energy deposited in the surrounding tissues.

Prosthetic Groups A prosthetic group is usually a small molecule that bears two sites of reactivity, one for incorporation of the radionuclide and a second for reaction with the peptide itself. Prosthetic groups are used particularly to minimize the number of synthetic steps and to optimize yields when working with

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Table Physical Characteristics of Nuclides Important for Peptide Radiolabeling in Nuclear Medicine11-13 Nuclide

Half life

PET imaging isotopes 18 F 109.8 min 68 Ga 67.7 min 64 Cu 12.7 h

11

C Zr

89

20.4 min 3.3 d

Radiotherapeutic isotopes 90

Y 177 Lu

64 h 6.7 d

Production

Decay Mode

Mean Free Path (mm)

Mean Free Energy (keV)

18

β+ (97%) β+ (87%) β+ (17%) β− (39%) EC (44%) β+ (100%) β+ (23%) EC (77%)

0.6 3.5 0.7

250 844 278

1.2 1.3

386 396

β− (100%) β− (79%)

2.5 0.7

935 130

EC (100%)

Gamma energy (keV) 171 and 245

O(p,n)18F Ge(e−,ν)68Ga 64 Ni(p,n)64Cu 68

14

N(p,α)11C Y(p,n)89Zr

89

β − decay

Sr  90Y 176 Lu(n,γ)177Lu 90

−

β decay Yb(n,γ)177Yb  177Lu

176

Gamma imaging isotopes 111 In 2.8 d 123

I

13.2 h

99m

6.0 h

Tc

112

Cd(p,2n)111In 111 Cd(p,n)111In 124 Te(p,2n)123I β − decay 99 Mo  99mTc

expensive peptides. Amino acids contain several reactive functional groups including amines, carboxylic acids, thiols, and alcohols, and the inherent reactivity of these functional groups can be paired with a complementary radiolabeled prosthetic group to achieve radiolabeling of the peptide. A second strategy for radiolabeling involves chemoselective chemical modification of the peptide to bear an unnatural bioorthogonal functional group, such as an azide, for reaction with a suitable prosthetic group. This strategy is more selective than using native functionality on the peptide but more complex as it requires the incorporation of the unnatural group into the peptide. The most commonly utilized prosthetic groups for reaction with native peptide functionalities include radiolabeled activated esters or acids for reaction with N-terminal or free amines of lysine residues, and maleimides14 for reaction with free thiols of cysteine residues. Commonly used prosthetic groups include N-succinimydyl-4-[18F]fluorobenzoate15 and p-nitrophenyl 2-[18F]fluoropropionate.16 Introduction of the 2-[18F]fluoropropionate is often preferred to the introduction of aryl [18F]fluorides as it does not significantly alter the lipophilicity of peptides.17 Bio-orthogonal approaches have tended toward “click” chemistry as a strategy for radiolabeling, as the reactions are usually chemo- and regioselective, rapid, and take place under mild conditions. Click reactions between azides and alkynes (either copper catalyzed18 or strain promoted19) have been proven to be popular for radiolabeling peptides.10 The very rapid and selective inverse electron demand Diels-Alder reaction between a tetrazine and trans-cyclooctene (or cyclooctyne) has begun to emerge as a reaction suited for labeling biomolecules, particularly as a “pretargeting” strategy.20-22

Direct Labeling An alternative approach to prosthetic groups for radiolabeling of peptides has exploited the ability of fluoride to

EC (97%)

159

EC (89%)

140

form strong bonds to boron,23 silicon,24 and aluminum.25 Shirrmacher et al have developed an isotopic exchange reaction (silicon-based fluoride acceptor) between 19F-fluoride and [18F]fluoride at a sterically hindered aryl silicon fluoride already attached to a peptide of interest. The reaction takes place at room temperature in acetonitrile and provides an operationally simple method for incorporation of fluorine-18 into peptides. The method produces products of lower specific activity as the precursor and product cannot be separated, and the products tend to show greater hepatobiliary excretion because of the presence of the highly lipophilic silicon fluoride.26-28 Perrin et al29 have developed a similar strategy where fluorolysis of diaryl boronate esters in a potassium bifluoride or potassium [18F]fluoride generates aryl [18F]trifluoroborates. Although these complexes prove stable in vivo,30 the use of carrier still leads to production of low specific activity products.31 The most successful direct labelling strategy is the development of the aluminum-[18F]fluoride-chelator system by Goldenberg et al.32 The strategy centers around the use of fluorophilic aluminum chelate, which has the ability to sequester [18F]fluoride from ethanol-saline mixtures.33 This is an attractive strategy for the incorporation of fluorine-18 into biomolecules as the method is more akin to the wellestablished use of BFCs for incorporating radiometals such as copper-64, gallium-68, or zirconium-89. Because the actual metal in the chelator has minimal effect on the binding and biodistribution properties of peptides, peptides that were previously limited to radiolabeling with radioactive metal ions could now be radiolabeled with fluorine-18.25

Bifunctional Chelators (BFCs) Radioactive metal ions play a pivotal role in nuclear medicine, where technetium-99m-based gamma imaging accounts for a large proportion of procedures undertaken. Radiometals are most often attached to bioactive molecules though a BFC.

ARTICLE IN PRESS 4 BFCs have one site for chelating the chosen metal ion, and a second site for attachment of the BFC to a peptide or other biomolecule, with a linker between the two sites.34 Linkers can be chosen based on any number of factors, including ease of synthesis, solubility or lipophilicity, and in vivo stability. The site for attachment to a peptide is often a carboxylic acid or amine, as these are easily conjugated to peptides through peptide bonds before radiolabeling. Other attachment strategies often exploit bio-orthogonal reactions, such as the copper-catalyzed click reaction (Cu-catalyzed azidealkyne cycloaddition [CuAAC]) between an azide and alkyne, as reaction conditions are mild and the linkages often show higher in vivo stability. The site for metal coordination varies considerably.35 Some widely used macrocyclic chelators, such as 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and the smaller 1,4,7-triazacyclononane-1,4,7triacetic acid (NOTA), are promiscuous and bind to a range of metal ions. The cyclic topology of these chelators results in the formation of thermodynamically stable chelates with gallium, aluminum, zirconium, yttrium, and lutetium. Consequently, DOTA, NOTA, and derivatives thereof are the most widely used BFCs for radiolabeling peptide for PET. Usually, acyclic chelators are less common as they tend to leach the radiometal; however, bespoke acyclic chelators such as N,N'bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N'diacetic acid (HBED-CC),36 which has high affinity for Ga3+, have also been developed. Acyclic complexes are attractive as, unlike labeling with DOTA or NOTA, formation of the complexes does not require heating.

Radiolabeled Peptides in the Clinic To date, the bulk of peptide radiolabeling for PET imaging has been oriented toward oncological applications, an interest reflected by a substantial presence in literature reviews in recent years.5,37,38 The overexpression of various receptors in human cancer cells makes these receptors attractive targets for PET imaging, resulting in the synthesis and clinical application of numerous radiolabeled peptides in oncology imaging. Historically, the majority of peptide imaging agents target specific receptors, largely G protein-coupled receptors (GPCRs). Targets of particular interest include somatostatin receptors (SSTRs), prostate-specific membrane antigen (PSMA), bombesin (BBN) receptors, αvβ3 integrin receptors, and chemokine receptor 4 (CXCR4). In recent years, more examples of using radiolabeled peptides for PET imaging in oncology via targets beyond these traditional receptors have been reported. These further applications extend to the visualization of other physiological functions altered by cancer such as angiogenesis and apoptosis, as well as diseases like diabetes.

SSTR Imaging Neuroendocrine tumors (NETs) are a rare form of tumor most often found in the GI tract and are characterized by an

I.M. Jackson et al. overexpression of SSTRs. NETs are slow-growing tumors that preclude effective imaging with [18F]FDG, a tracer relying on the rapid growth of tumors coupled to upregulated metabolism to increase [18F]FDG avidity. Radiolabeled peptides offer an attractive solution to the challenge of imaging these slow-growing tumors. The SSTR family is a group of GPCRs normally distributed in the brain, pituitary, pancreas, thyroid, spleen, kidney, gastrointestinal tract, vasculature, and peripheral nervous system, and on immune cells.39 Natural somatostatin (SST, Fig. 2) is a 14- or 28-amino acid peptide that triggers a host of antiproliferative and antisecretory functions by binding to SSTRs.40 There are five known SSTR subtypes, with SSTR2 being the primarily expressed subtype in NETs. Beyond NETs, the SSTRs are expressed to different degrees in a variety of tumors including lymphoma, paraganglioma, carcinoids, breast cancer, brain cancer, renal cancer, small cell lung cancer (SCLC), and medullary lung cancer.41 This variance in distribution and density of SSTR subtypes across this wide range of cancers makes specificity of the desired imaging agent of crucial importance. Well-designed peptides offer selectivity for their targets, illustrated in the range of peptides developed for SSTRs, with different SST analogs displaying different affinity and selectivity for certain SSTR subtypes. Initial success at imaging of NETs via functional imaging techniques was performed via scintigraphy with SST analog [111In]In-DTPA-octreotide, an agent approved by the FDA as OctreoScan (Fig. 2).42 Quantitative limitations of [111In]In-DTPA-octreotide scintigraphy include intense uptake in the spleen, liver, and kidneys. This limitation provided impetus for the design of an alternative to this preliminary peptide imaging agent amenable to imaging with PET, which would be expected not to show the same physical limitations as scintigraphy.43 An initial response to this demand came in 2000 in the form of [68Ga]DOTA-D-Phe1-Tyr3-Octreotide ([68Ga]DOTATOC), an SST analog (TOC) with a DOTA chelating ligand amenable to coordination with PET radionuclide 68Ga.44,45 This peptide imaging agent exhibits high SSTR2 affinity, lower affinity for SSTR5, and enhanced kidney clearance. In this preliminary work, Hofmann et al have demonstrated the ability of [68Ga]DOTATOC to image smaller NETs than OctreoScan (Fig. 3). Additionally, the ability to interchange the radionuclide within the chelator pocket from diagnostic nuclide 68Ga to therapeutic radionuclides such as 90Y or 177Lu is key for theranostic applications of these peptides. A recent metaanalysis of [68Ga]DOTATOC pooled results seen across studies ultimately determining a pooled sensitivity and specificity of 92% and 82%, respectively, for the detection of NETs. The analysis went on to conclude that the tracer is useful in evaluating presence of NETs, in disease staging, and in guiding treatment decisions. Although [68Ga]DOTATOC is useful for locating the site of an unknown primary tumor in patients presenting with metastatic NETs, it is ill-suited for evaluating patients solely on the basis of symptoms and biomarkers with no definitive diagnosis. 46 Following preliminary studies with [68Ga]DOTATOC, a series of DOTA-tagged SST analogs were developed, with [68Ga]DOTANOC and [68Ga]DOTATATE (Fig. 2) being most promising. In comparison

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Figure 2 Structures of somatostatin (A); OctreoScan (B); and 68Ga-labeled somatostatin radiotracers (C). (Figure part C reprinted with permission under a Creative Commons Attribution [CC BY-NC] license from Velikyan I: Theranostics. 4:47-80, 2014. © The Author 2014)

to [68Ga]DOTATOC, [68Ga]DOTATATE exhibits 10-fold better binding affinity for SSTR 2 in vitro (0.2 ± 0.04 nM for DOTATATE and 2.5 ± 0.5 nM for DOTATOC) and no affinity for SSTR5.41 Despite this difference in affinity, in vivo studies show no significant difference in lesion detection rates or uptake between [68Ga]DOTATOC and [68Ga]DOTATATE.47 One headto-head comparison found maximum standardized uptake value (SUVmax) to be 16.0 ± 10.8 across 254 patients for [68Ga]DOTATATE, vs 20.4 ± 14.7 across 262 lesions for [68Ga]DOTATOC.48 While there was no significant difference in renal uptake, tumor-to-kidney ratio was found to significantly favor [68Ga]DOTATOC. Contrasting the very similar in vivo behavior seen between [68Ga]DOTATOC and [68Ga]DOTATATE, [68Ga]DOTANOC was found to display affinity for SSTR2, SSTR3, and SSTR5, with SSTR2 affinity exceeding that of [68Ga]DOTATATE. A major drawback of [68Ga]DOTANOC, however, is increased liver

accumulation as a result of the tracer being more lipophilic than [68Ga]DOTATATE and [68Ga]DOTATOC. Additionally, [68Ga]DOTANOC is seen to have a lower volume of distribution. Comparison of these three SST analogs across tumor types is not straightforward: no one of the three outperforms the other two in all scenarios, although the literature seems to reflect a preference for [ 68 Ga]DOTATOC and [68Ga]DOTATATE over [68Ga]DOTANOC. This is likely due to a need to minimize liver accumulation and a lack of any apparent advantage conferred by the general SSTR affinity of [68Ga]DOTANOC. In spite of this, the 2016 FDA approval of [68Ga]DOTATATE (NETSPOT; Advanced Accelerator Applications, New York, NY) is liable to further increase the preference for this peptide over the other two.1,49 SST analogs have been applied to the imaging of NETs in a variety of cancers. One such study of interest investigated the utility of [68Ga]DOTATATE PET/CT in imaging head and

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Figure 3 (A) Maximum intensity projections of a 54-year-old male patient (no. 1) suffering from multiple liver, lung, abdominal, and bone metastases of abdominal carcinoid. The scan was started 90 minutes following the intravenous injection of 220 MBq of [68Ga]DOTATOC, and six bed positions were acquired. (B) The planar whole-body scintigram acquired 24 hours after the injection of 110 MBq [111In]octreotide does not reveal the true extent of liver involvement as visualization is impaired by intensive renal accumulation of the tracer. Multiple bone marrow and lung metastases are not as clearly delineated as in the [68Ga]DOTATOC PET scan. BM, bone metastasis; DRV, view from the right; K, kidney; L, liver; LDR, posterior view; LM, liver metastasis; PG, pineal gland; RVL, anterior view; S, spleen; TG, thyroid gland; VLD, anterior view. (Reprinted from Hofmann et al: Eur. J. Nucl. Med. 2001;28:1751-1757, with permission from Springer. © Springer-Verlag 2001)

neck paraganglioma, asserting this method to be superior to presently employed imaging modalities and tracers.50 Other recent efforts to further build upon the knowledge base concerning utility of SST analogs in NET imaging include a novel correlational study from Tirosh et al, which explored the relationship between biochemical markers of NETs and total [68Ga]DOTATATE avid tumor volume. The work was motivated by a lack of consensus on the utility of biomarkers such as chromogranin A, pancreatic polypeptide, neuron-specific enolase, and urinary 24-hour 5-hydroxyindoleacetic acid in estimating total NET tumor burden in patients. Total [68Ga]DOTATATE uptake was quantified and compared to biomarker expression in a large cohort of NET patients to support use of one of seven different NET biomarkers based on primary tumor site in assessing the extent of disease.51 In addition to the application of these SST analogs, another current area of focus centers on diversification and improvement of these imaging agents themselves. For example, 18F-labeled radioligands are being explored,52 while a recent study from Johnbeck et al followed up on initial reports of [64Cu]DOTATATE imaging and provided a comparison of [64Cu]DOTATATE against popular radiopeptide [ 68 Ga]DOTATOC for imaging NETs. 53 The work was motivated by theoretically superior spatial resolution of [ 64 Cu]DOTATATE over [ 68 Ga]DOTATOC, because of a shorter positron mean free path and lower mean free

energy. This idea is supported by a comparative study with [111In]DTPA-octreotide showing [64Cu]DOTATATE to be superior in terms of both radiation dose and lesion detection rates across 112 patients.54 The work compared [64Cu]DOTATATE to [68Ga]DOTATOC in 22 patients and found that, although the two radiopeptides showed no difference in diagnostic performance, significantly more true-positive lesions were seen via [64Cu]DOTATATE. Pfiefer et al assert that differences between the two tracers are due to the difference in radionuclide not to difference in peptide, on the grounds that DOTATOC and DOTATATE are seen to behave similarly for a given radionuclide. The authors posit this increase in detected lesions to be due to the substantially shorter positron range of 64Cu, making it theoretically more amenable to detection of smaller lesions. It should be noted, however, that as this is the first use of this tracer in patients, clinical impact could not be assessed in the study in question. Finally, it is also noteworthy that the use of [64Cu]DOTATATE did result in a higher radiation dose to the patient (5.7-8.9 mSv) than [68Ga]DOTATOC (2.8-4.6 mSv), and this should be kept in mind when selecting one agent over the other. This work highlights the impact that radionuclide choice can have on image quality and tumor detection for a given peptide and chelating group.55 Another notable variation of the main three SST radiopeptides comes originally from Ginj et al in the form

ARTICLE IN PRESS Clinical applications of radiolabeled peptides of DOTA-iodo3Tyr-octreatate, an SST analog 68Ga-labeled and first imaged by Brogsitter et al under the moniker [ 68 Ga]HA-DOTATATE, and found to provide images comparable or superior in quality to those provided by [ 68 Ga]DOTATATE. 56 Motivated by this initial result, Brogsitter and Schottelius carried out respective clinical and preclinical comparisons of [ 68 Ga]DOTATATE and [68Ga]HA-DOTATATE. Preclinical studies from Schottelius found that [ 68 Ga]HA-DOTATATE demonstrated nearly 2× in vitro internalization, similar affinity for SSTR 1-4 , and >17-fold higher affinity for SSTR5 in comparison to [ 68 Ga]DOTATATE. Additionally, the two demonstrated roughly equivalent tumor targeting capabilities.57 In vivo studies from Brogsitter et al saw overall similar detection rates across 23 patients. Overall, [ 68Ga]HA-DOTATATE remains an attractive alternative to [ 68 Ga]DOTATATE, should the need for one arise, owing to similar tissue distribution, tracer uptake, and lesion detectability.58 Oncology imaging with 68Ga-labeled SST analogs extends beyond NETs, making them arguably the most widely used and well understood radiolabeled peptides to date.59 One recent study of interest comes from Walker et al and involves use of [68Ga]DOTATATE for indeterminate pulmonary lung nodules and non-neuroendocrine lung cancer, motivated by a desire for superior alternative to the current agent [18F]FDG, which is prone to false positives from active granulatomous nodules. Although [68Ga]DOTATATE was found to provide higher specificity, (94% vs 81%), lower sensitivity (73% vs 93%) resulted in overall equivalent diagnostic accuracy.60 Another interesting study from Gofrit et al applied [68Ga]DOTATATE to the imaging of neuroendocrine differentiation (NED) in castration-resistant prostate cancer (CRPC). NED is the transdifferentiation of epithelial cells known to be induced by castration, radiotherapy, chemotherapeutics abiraterone and docetaxel, and pharmaceutical enzalutamide. The study found uptake of [68Ga]DOTATATE in metastatic CRPC patients to be common, suggesting the regularity of NED in CRPC, and the potential utility of anti-NET therapy treatment for these patients.61

PSMA Imaging PSMA, the product of the FOLH1 gene, is an integral membrane glycosylated metalloenzyme, with a 19-amino acid intracellular N-terminal domain, 24-amino acid transmembrane helix, and a 707-amino acid extracellular C-terminal domain bearing two binding pockets and two zinc ions.62 PSMA is also known generally as glutamate carboxypeptidase II, reflecting its function in cleaving C-terminal glutamate residues from appropriate substrates. An N-terminal truncated variant of PSMA (known as PSM’) is expressed in the cytosol, whereas the membrane-anchored variant is found on the apical membrane of the epithelium lining the prostatic ducts in the healthy prostate.62,63 In addition, PSMA is also expressed in kidneys, liver, salivary glands, lachrymal glands, colon, duodenal brush border, and the nervous system.64 PSMA is of interest to the oncology community as the protein is overexpressed on the luminal surface of the prostatic ducts in increasing amounts as prostatic hyperplasia transitions to

7 neoplasia, then to primary and androgen-independent adenocarcinoma, where PSMA is overexpressed at levels 100to 1000-fold higher compared to those in normal prostate cells.65-68 PSMA levels have also been shown to correlate well with Gleason score.69 In an immunohistochemical study of 51 cases of metastatic prostate cancer, it was found that PSMA is expressed in more than 95% of primary tumors and more than 80% of metastatic lesions, whereas only in a single case were both the primary and metastatic lesions negative for PSMA.70 This combination of near-uniform expression of PSMA at greatly elevated levels in most cases of prostate cancer means that PSMA is an ideal marker for identifying and monitoring the progression of prostate cancer. A series of inhibitors of PSMA (also known as NAALADase) had been developed for neurologic applications before it was realized that PSMA was overexpressed in prostate cancer; however, application of these inhibitors to PSMA followed rapidly.71 A series of urea-linked dipeptide inhibitors of NAALADase were developed as analogs of the widely studied phosphonic acid derivative 2-(phosphonomethyl)pentanedioic acid.72 Although not true peptides in that they lack a peptide bond, the urea-linked diamino acid ligands developed by Kozikowksi et al were soon studied within the context of prostate cancer and were demonstrated to have high affinity for PSMA, and were primed to take advantage of the welldeveloped peptide radiolabeling strategies that already existed.73 Initial PET labeling and imaging of these constructs exploited a 11C-methylation of a cysteine residue and demonstrated specific binding of the radioligand in xenograft LNCaP PSMA-positive tumors in a mouse model, where tumor-to-muscle and tumor-to-blood ratios of 10.78 and 8.31, respectively, were observed. In non–PSMA expressing control human prostate cancer cell line (PC-3) xenografts, tumorto-muscle and tumor-to-blood ratios of 2.10 and 0.78 were observed, suggesting that such urea-based constructs were ideally suited for imaging PSMA overexpression in prostate cancer.74 Following this demonstration, a range of ureacoupled peptide ligands for PSMA were radiolabeled with a range of radioisotopes and were shown to generally be excellent agents for imaging PSMA-positive prostate cancers, in preclinical models. Progress in the development of imaging agents for PSMA occurred concurrently with the widespread adoption of commercial germanium-68/gallium-68 generators into PET imaging facilities.75 The significant logistical advantages of “ondemand” production of gallium-68 without the need for a cyclotron, coupled to improvements in processing and purification of the generator eluate, gave 68Ga-labeled agents a practical advantage over agents labeled with other isotopes.76,77 An early agent that made the successful transfer to the clinic was [68Ga]PSMA-HBED-CC ([68Ga]PSMA-11, Fig. 4A).78,79 [68Ga]PSMA-HBED-CC was developed in an effort to exploit the fact that PSMA has an active site with two binding pockets: one pocket binds the glutamate residues of the substrate and the second pocket is largely lipophilic,80 into which the lipophilic chelator HBED-CC was proposed to bind, before the entire assembly is internalized. The novel complex was found to bind with slightly higher affinity to PSMA than an analogous

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Figure 4 (A) [68Ga]PSMA-HBED-CC. (B) Maximum intensity projection of a patient with normal biodistribution of [68Ga]PSMA 1 hour after injection. Accumulation is seen in lacrimal and salivary glands, nasal mucosa, liver, spleen, bowel, kidneys, and bladder. (Reprinted from Afshar-Oromieh et al: Eur J Nucl Med Mol Imaging (2013) 40: 486-495, with permission from Springer. © Springer-Verlag Berlin Heidelberg 2012) (C) Patient 12 (a, b) and patient 18 (c, d). Red arrows point to a nodular pelvic wall metastasis (a, b, histologically confirmed) and to small lymph nodes (c, d), which present with clearly pathologic tracer uptake in [68Ga]PSMA PET/CT (b, d) only. Yellow arrows point to both catheterized ureters (c, d). Patient 12 presented with a minimal PSA value (0.01 ng/mL) despite visible tumor lesions. The PSMA ligand is therefore able to detect low differentiated PC. (a + c) Fusion of 18F-fluoromethylcholine PET and CT, (b + d) fusion of [68Ga]PSMA PET and CT. Color scales were automatically produced by the PET/CT machine. (Reprinted with permission under a Creative Commons Attribution [CC BY] license from Afshar-Oromieh et al: Eur. J. Nucl. Med. Mol. Imaging 2014;41:11-20. © The Author[s] 2013)

compound bearing a DOTA chelator; however, the [68Ga]PSMA-DOTA compound was shown to have high, nonspecific uptake in both PSMA-positive LNCaP and negative PC-3 cells. This contrasts with [68Ga]PSMA-HBED-CC which showed only specific uptake into LNCaP cells. An initial comparative clinical investigation of [68Ga]PSMA-HBED-CC against [18F]fluoroethylcholine ([18F]FECHL) in a single prostate cancer patient demonstrated that [68Ga]PSMA-HBED-CC was able to detect a bladder-adjacent lesion, which was not observed

with [18F]FECHL.79 Up to this point, choline-based PET imaging of phospholipid biosynthesis with [18F]FECHL, [ 18 F]fluoromethylcholine ([ 18 F]FCHL or [ 18 F]FCH) and [11C]choline ([11C]CHL) were the most commonly used agents for imaging prostate cancer,81 and this early success for [68Ga]PSMA-HBED-CC over [18F]FECHL was encouraging for improving the state of the art for imaging prostate cancer. In an initial study in 37 patients presenting with biochemical relapse of prostate cancer, imaging with [68Ga]PSMA-HBED-CC

ARTICLE IN PRESS Clinical applications of radiolabeled peptides revealed lesions in 31 patients, all detected just 1 hour after injection of the radiotracer, where a median tumor-tobackground ratio of 18.8 was observed (Fig. 4B).82 In a follow-up retrospective study, 37 patients with biochemical relapse of prostate cancer were imaged with both [18F]FCHL and [68Ga]PSMA-HBED-CC (Fig. 4C).83 Seventyeight lesions were detected in 32 patients upon imaging with [ 68 Ga]PSMA-HBED-CC; however, only 56 lesions were detected in 26 of the patients when imaged with [ 18 F]FCHL. The authors concluded that imaging with [68Ga]PSMA-HBED-CC was generally superior to [18F]FCHL, especially in detecting smaller metastases in lymph nodes, and when patients presented with low prostate serum antigen (PSA, a biomarker for prostate cancer) levels. The growth in imaging of prostate cancer with [68Ga]PSMA-HBED-CC expanded rapidly following these initial clinical investigations, and the history and current status of the field have been comprehensively reviewed.84-90 In short, imaging studies with [68Ga]PSMA-HBED-CC initially focused on locating metastatic lesions in secondary biochemical relapse of prostate cancer, characterized by rising serum PSA levels after radical prostatectomy, rather than staging the primary cancer.91 In a recent meta-analysis by Perera et al of 16 papers describing imaging of 1309 prostate cancer patients with [68Ga]PSMA-HBED-CC,92 14 studies were found to report data regarding imaging in patients with biochemical recurrence of prostate cancer. In these studies, a total of 76% of scans were judged positive, and it was found that pooled positivity varied with serum PSA levels, where PSA levels of 0-0.19, 0.20.99, 1.00-1.99, and >2.00 ng/mL corresponded to 42%, 58%, 76%, and 95% positivity, respectively. In comparison, for [11C]CHL PET scans in patients with PSA levels <1 ng/mL, positivity ranges between 20% and 36%, and increases to 63%-83% for patients with PSA levels >3 ng/mL.81 The metaanalysis found that in cases where histopathology of lesions was examined, imaging with [68Ga]PSMA-HBED-CC showed summary sensitivity of 80% and specificity of 97%. In a headto-head PET/CT comparison of imaging in the same patients with mean PSA levels of 11.1 ng/mL, [68Ga]PSMA-HBED-CC PET/CT identified at least one prostate cancer lesion in 86.5% of the patients, whereas [18F]FCHL PET/CT identified a positive lesion in only 70.3% of the patients. SUVmax was also higher for [68Ga]PSMA-HBED-CC in 79.1% of all lesions.83 A similar comparative study in patients with a much lower mean PSA level (2.2 ng/mL) showed detection rates of 32% for [18F]CHL and 66% for [68Ga]PSMA-HBED-CC.93 The data suggest therefore that [68Ga]PSMA-HBED-CC may be a superior prostate cancer imaging agent, especially in cases where serum PSA levels are low. Staging of primary prostate cancer using PET/CT or PET/ MRI has generally been proven to be more complicated than imaging recurrent prostate cancers. A meta-analysis revealed that [11C]CHL-PET has a pooled sensitivity of just 49.2% when imaging primary cancers. In a retrospective study,94 130 patients with suspected prostate cancer were scanned with [68Ga]PSMA-HBED-CC. After resection and histopathologic analysis of metastatic lymph nodes, per lesion sensitivity was found to be 74%, with a selectivity of 93%. A second study

9 examined 30 patients (mean PSA of 8.2 ng/mL) with a preoperative [68Ga]PSMA-HBED-CC PET scan, which was found to show per lesion sensitivity of 56% and specificity of 98% in detecting disease.95 A third, more cautious study reported a particularly high false negative rate of 66% in a cohort of 30 patients who had histopathologically confirmed lesions after a templated resection.96 The authors hypothesize that in primary prostate cancer cases, the pharmacokinetics of tracer biodistribution may lead to high uptake of [68Ga]PSMA-HBED-CC in the prostate itself, leaving little circulating tracer for binding to lymph node metastases, especially when these are small. It has been shown, however, that both serum PSA levels and Gleason score correlate with SUVmax, particularly when PSA levels are >10 ng/mL and Gleason score >7, and in these cases in particular, [68Ga]PSMA-HBED-CC may be useful in primary staging.97 Although data remain limited at this stage, PET imaging with [68Ga]PSMA-HBED-CC appears to offer improvement over other prostate cancer imaging methods for primary staging of prostate cancer; however, small lymphatic metastases remain difficult to detect in this setting.92 Although [68Ga]PSMA-HBED-CC is an excellent imaging agent for prostate cancer, the expression of PSMA in tissues other than prostate cancer and its metastases means that in such PET scans, physiological uptake in the kidneys, spleen, liver, parotid, submandibular and lachrymal glands, and the duodenum is also observed.98-100 These regions, however, are not common locations for the presence of metastatic prostate cancer and are thus unlikely to hamper imaging of such lesions.89 Indeed some researchers have taken advantage of PSMA overexpression in other cancers and have applied [68Ga]PSMA-HBED-CC PET imaging successfully in, for example, renal clear cell carcinoma101,102 and breast cancer.103,104 As more clinical data emerge concerning imaging of prostate cancer in various stages of the disease using a range of imaging modalities, 105-109 researchers and clinicians have begun to advise caution in the use of [68Ga]PSMA-HBED-CC PET imaging in the management of patients.110-112 Although [68Ga]PSMA-HBED-CC PET provides a wealth of biochemical information about the presence of likely prostate cancer and metastases, there is presently a lack of controlled, prospective clinical trials demonstrating how such information should impact treatment decisions by physicians, and whether such imaging affects patient outcomes. As this information slowly emerges,113-115 there is no doubt that [68Ga]PSMA-HBED-CC PET will continue to have a significant impact on the treatment and management of prostate cancer. Although [68Ga]PSMA-HBED-CC PET has enjoyed rapid translational success in the clinic and remains the most widely used agent for imaging PSMA localization in prostate cancer, two drawbacks of this agent have been identified. Firstly, the acyclic chelator HBED-CC, although ideal for gallium(III), is not suitable for radiolabeling with therapeutic radionuclides such as 177Lu, precluding the use of the PSMA-HBEDCC construct in theranostic applications. The second limitation arises from the decay characteristics of 68Ga itself. 68Ga decays to 68Zn, emitting a positron with a mean free energy of

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10 0.84 MeV (max. 1.90 MeV) and a maximum range in water of 10.3 mm compared to, for example, 0.25 MeV (max. 0.63 MeV) and a maximum range in water of 2.6 mm for positrons emitted by 18 F. 12 This maximum range defines a theoretical maximum resolution limit for imaging with each of these nuclides, where agents labeled with 18F would be expected to offer significant improvements in image resolution compared to agents labeled with 68Ga. The desire for a dual-imaging and therapy probe for PSMA led to the development of the ligand PSMA-I&T, which bears a 1,4,7,10-tetraazacyclododececane,1-(glutaric acid)-4,7,10triacetic acid BFC that is able to coordinate both 68Ga and 177 Lu, and a modified iodotyrosine peptidic linker between the chelator and the urea dipeptide pharmacophore.116 The 68 Ga and 177Lu variants of PSMA-I&T both show similar nM affinity for PSMA compared to [68Ga]PSMA-HBED-CC, and [177Lu]PSMA-I&T demonstrates particularly high cellular uptake in LNCaP cells compared to [68Ga]PSMA-I&T, a desirable characteristic for the therapeutic agent.117 [68Ga]PSMA-I&T has been demonstrated to show favorable high uptake in prostate cancer metastases in patients with biochemical recurrence and has demonstrated tumor-to-background ratios of 17.635.2. Compared to published data for [68Ga]PSMA-HBED-CC, [68Ga]PSMA-I&T demonstrated similar per lesion sensitivity when stratified by PSA levels; however, SUVmax in the lesions was found to be generally lower.118 Delayed imaging (3 hours post injection [p.i.]) has been demonstrated to detect lesions with increased contrast, and in some cases to detect lesions that were not evident at an early imaging time point (1 hour p.i.).119 [68Ga]PSMA-I&T has also been shown to be of use in the staging of primary prostate cancer in a cohort of 21 patients, where the agent was able to predict the spread of the disease before radical prostatectomy.120 To address the physical limitations of agents labeled with gallium-68, a series of PSMA-targeting agent bearing the urealinked dipeptide pharmacophore have been labeled with fluorine-18. First-generation compound [18F]DCFBC, bearing a [18F]fluorobenzyl group, was successfully used for imaging of prostate cancer but was found to have high blood pool activity.121-123 A second-generation compound, [18F]DCFPyL, bearing a 2-[18F]fluoropyridine group, has been proven to be more comparable to [68Ga]PSMA-HBED-CC for imaging of primary, recurrent, and biochemical relapsed prostate cancer.124 Comparisons between the two imaging agents have demonstrated that, in limited populations, [18F]DCFPyL performs as well as [68Ga]PSMA-HBED-CC and, in some cases, demonstrates higher average SUVmax in lesions and better tumor-to-background ratios. In some cases, imaging with [18F]DCFPyL was found to detect lesions not evident with [68Ga]PSMA-HBED-CC (Fig. 5).125-127 The authors are cautious in claiming that [18F]DCFPyL is a better agent than [68Ga]PSMA-HBED-CC, but maintain that images obtained from the two agents are at least comparable. The authors state that factors in the study design, including longer times between scanning and imaging, and the lack of histologic information about the lesions may be significant contributors to the improved imaging properties observed for [18F]DCFPyL. Finally, clinical data for [18F]PSMA-1007, a companion diagnostic for

therapeutic PSMA agent [177Lu]PSMA-617, have recently been reported, where the imaging and therapeutic agents have shown similar biodistribution and uptake properties, making this combination a promising diagnostic-therapeutic pair.128-130

Integrin Receptor Imaging The growth of a tumor mass is dependent on the supply of nutrients and oxygen,131 shuttled to the developing mass by diffusion when the tumor is small. However, once the tumor has reached a critical size, it outgrows its blood supply and diffusion can no longer deliver the requisite oxygen and nutrients at the required rate. At this point, the tumor cells initiate the growth of new vessels into the mass to maintain this supply through the process of angiogenesis. Integrins mediate this process and are consequently attractive targets for imaging of newly developing and established tumors.132,133 The integrins are a class of 24 heterodimeric membrane glycoproteins, made up of combinations of 18 α-subunits and 8 β-subunits, and are responsible for transduction of signals from the extracellular matrix to the interior of the cell. Overexpression of these receptors on the surface of vascular endothelial cells occurs during angiogenesis, and may be pathologic or benign (such as in wound healing or inflammation).134 The αVβ3 integrin, also known as the vitronectin receptor, is the most widely studied of the integrins. The binding of vitronectin to αVβ3 integrin is mediated by an Arg-GlyAsp (RGD) tripeptide motif on the surface vitronectin.135,136 This tripeptide motif acts as the core recognition motif for most integrins, and incorporating this tripeptide motif into smaller, more accessible peptides has resulted in a range of small peptides bearing the RGD sequence being developed as imaging biomarkers for angiogenesis.137 The most successful of these mimics are cyclic pentapeptides (cRGDs), which bind in a cleft between the α- and β-subunits138 and have been found to show an order of magnitude greater affinity for αVβ3 integrin while also demonstrating improved in vivo stability when compared to their linear analogs.139,140 The cRGDs have highest affinity when bearing a hydrophobic residue (such as D-phenylalanine, D-tyrosine or D-valine) adjacent to the Asp (D) residue, whereas the fifth residue could be substituted without loss of affinity for αVβ3 integrin.141 Consequently, prosthetic groups are incorporated into the motif through this fifth residue via the free thiol of cysteine (C) or the free amine of lysine (K) residue. Unlike most receptor-based imaging probes, the goal of integrin imaging with RGD-based peptides is to image the physiological process of angiogenesis, as opposed to the overexpression of integrin receptors on the surface of tumor cells. As such, the RGD peptides are used for imaging a wide range of cancers including non–small cell lung carcinoma, small cell head and neck cancers, breast cancers, and glioblastoma. However, some types of cancers are known to overexpress integrins on their surface,142 and as such, care needs to be taken when interpreting results from imaging with the RGD peptides to delineate imaging of integrins associated with vascular endothelial cells and tumor-associated angiogenesis vs benign angiogenesis vs imaging of tumor receptor overexpression of integrins.143 A second complication

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Figure 5 Patient with a rising PSA level of 3.87 ng/mL. In the past, the patient had retroperitoneal lymph node metastases, which were irradiated. On a Biograph mCT 128 scanner, comparison between (A) MIP with [18F]DCFPyL and (B) MIP with [68Ga]Ga-PSMA-HBED-CC. [18F]DCFPyL PET/CT clearly demonstrates several additional supradiaphragmatic PSMA-positive lesions. [68Ga]Ga-PSMA-HBED-CC PET/CT showed the supradiaphragmal lesion in the sternum. MIP, maximum intensity projection. (Reprinted with permission under a Creative Commons Attribution [CC BY] license from Dietlein et al: Mol. Imaging Biol. 2015;17:575-584. © The Author[s] 2015)

that arises when imaging integrins using radiolabeled RGDbased peptides is that these peptides generally show high physiological uptake in the liver, spleen, kidneys, and intestines, precluding their use in cancers that originate or have metastasized to these locations. These peptides also generally exhibit clearance through the renal system, complicating imaging of the kidneys, bladder, and other closely associated organs such as the prostate.144 Numerous factors have been exploited in the development of RGD-based clinical PET probes for visualizing angiogenesis (see Fig. 6 for examples), including the choice of radionuclide, BFC, pharmacokinetic modifier, and the specific integrin target (eg, αVβ3 vs αVβ5), and the use of multimerization to enhance binding properties; however, a full discussion of these factors is not within the scope of this article but has been extensively covered in a number of excellent reviews.144-149 [18F]Galacto-RGD (Fig. 7) is an αVβ3-selective cRGD peptide (IC50 = 5 nM) that bears a galactosamine sugar as a pharmacokinetic modifier and a (±)-2-[18F]fluoropropionyl prosthetic

group linked to the sugar via an amide bond.150 Although challenging to synthesize (multiple steps, 200-minute preparation time), [18F]galacto-RGD was the first of this class of tracer to be investigated in humans, where in a study of nine patients with a range of different types of cancers, investigators demonstrated successful clinical imaging of tumours using this tracer.151 The tracer showed accumulation in the tumors (tumor-to-muscle ratio of 8.8), which had been previously identified by biopsy, CT, or [18F]FDG-PET, as well as in the liver, kidneys, and colon. Importantly, immunohistochemistry of tumor sections from six patients all showed αVβ3 expression in the tumor vasculature, whereas two patients’ tumor sections presented with αVβ3 expression on the tumors themselves. Follow-up studies of this tracer have shown that the tracer shows variable uptake in primary and metastatic tumors but displays favorable pharmacokinetic properties,152 and that the standardized uptake value (SUV) of the tracer correlates well with both immunohistochemical staining and microvessel density in tumors biopsied after imaging.153 Successful PET imaging with [18F]galacto-RGD has been

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Figure 6 Biodistribution of clinically available RGD-based PET agents 1 hour after intravenous administration in healthy volunteers, except for [18F]Galacto-RGD PET, which is from a patient with osteomyelitis. All images are coronal views. High tracer retention is notable in urogenital tract because of predominant renal clearance. Intermediate uptake is found in the liver, spleen, and intestines. (Reprinted with permission under a Creative Commons Attribution [CC BY-NC] license from Chen et al: Theranostics 2016;6:78-92. © The Author[s] 2016)

undertaken in cases of squamous cell carcinoma of the head and neck,154 breast cancer,155 and glioblastoma,156 and Chen et al144 report a sensitivity for all lesions of 59%-92%, with sensitivity for primary lesions being 83%-100%. Highlighting the challenges of imaging more advanced disease, sensitivity for lymph node metastases and distant metastases were 33%-54% and 46%-78%, respectively. Direct comparison of [18F]galacto-RGD and [18F]FDG in the same patients showed that SUVs were higher for [18F]FDG and that [18F]galacto-RGD missed 14 lesions out of a total of 59.157 However, the authors do note that imaging with [18F]Galacto-RGD could be useful in low [18F]FDG-avid tumors, as has recently been demonstrated in a study in prostate cancer patients.158 Because of the complex synthesis of [18F]galacto-RGD, there was significant interest in the development of simpler routes to radiolabeled integrin-targeting radioligands. Progress toward a more streamlined synthesis of an integrin-targeting agent was reported by Siemens in the form of a CuAAC between an azido-modified cRGD and 5-[18F]fluoropentyne to access their target, [18F]RGDK5. The molecule of interest demonstrated high affinity and selectivity for αVβ3 (Kd = 7.9 nM)159 and similar pharmacokinetics, biodistribution, and dosimetry profile to other integrin-targeting PET agents.160 An initial comparison of [18F]RGDK5 to [18F]FDG in breast cancer

patients showed that 122 lesions out of a total of 157 identified by [ 18 F]FDG showed uptake of [ 18 F]RGDK5. 161 Disappointingly, immunohistochemistry of random areas of tumor biopsies from nine patients revealed no correlation with maximum vessel density, the number of microvessels, or vessel area, hypothesized to be due to [18F]RGDK5 binding to other integrins besides αVβ3. Most recently, a longitudinal pilot study comparing [18F]RGDK5 to [18F]FDG in nine patients with head and neck cancer following chemoradiotherapy demonstrated that changes in both [18F]RGDK5 and [18F]FDG] SUV in tumors 3 months post therapy were able to predict if patients would go into complete remission.162 Multimerization has been proven to be a popular strategy for RGD-based PET ligands to increase both the affinity and specificity with which they bind their integrin target.163-165 A variety of strategies combining 2, 4, or 8 cRGD units with PEG, polypeptide, or sugar-amino acid linkers have been employed to improve binding metrics (Kd or IC50), pharmacokinetics (urinary rather than hepatobiliary clearance), and imaging properties (faster washout and improved signal-tonoise ratios). An early example of such a multimeric RGD compound is [18F]FPRGD2 (Fig. 8), a glutamic acid-linked dimeric cRGD coupled to [18F]fluorobenzoate prosthetic group through a short PEG chain.166 Translation of this tracer into

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Figure 7 Comparison of different uptake patterns in [18F]FDG-PET and [18F]galacto-RGD PET. (A) Patient with non-SCLC of left upper lobe (arrow, closed tip) and multiple metastases to bone (arrow, open tip), liver, lymph nodes, and adrenal glands. Note intense uptake in all lesions in MIP of [18F]FDG-PET, whereas uptake in lesions in MIP of [18F]galacto-RGD PET is substantially lower. This typical uptake pattern is seen in most patients. (B) Patient with neuroendocrine tumor of bronchus in right lower lobe (arrow, closed tip) and multiple metastases to bone (arrows, open tip), liver, spleen, and lymph nodes. This patient shows more intense uptake in lesions on 18F-galacto-RGD PET compared with that on [18F]FDG-PET. MIP, maximum intensity projection. (Reprinted with permission from Beer et al: J. Nucl. Med. 2008;49:2229. © 2008 by the Society of Nuclear Medicine and Molecular Imaging, Inc)

the clinic in an initial investigation in 27 patients with suspicious renal masses revealed that [18F]FPRGD2 uptake significantly correlated with αVβ3 expression in malignant tumors. Biopsies of the tumors after resection showed that for clear cell renal cell carcinomas, [18F]FPRGD2 uptake correlated with expression of αVβ3 on the surface of the tumor cells, whereas in papillary renal cell carcinoma, uptake was correlated with tumor cell surface αVβ3 expression.167 A second pre- and postchemoradiotherapy PET study in 32 patients with locally advanced rectal cancer showed that uptake of the tracer, although inferior to uptake of [18F]FDG, correlated with tumor regression grade, a predictor of long-term cumulative incidence of metastases. It must be noted that overall specificity with this tracer was low in this patient group.168 An analogous dimeric cRGD, labeled with 2-[18F]fluoropropionate, [18F]FPPRGD2 has an IC50 of 51.8 nM, compared to 404 nM

for galacto-RGD in a cell-based assay of αVβ3 binding,169 and has also been evaluated in the clinic.170 The tracer was shown to induce no adverse effects, while being excreted through the urinary and hepatobiliary pathways,171 making this tracer especially suitable for investigating cancers above the diaphragm. Three recent studies in breast cancer patients (n = 8),172 recurrent glioblastoma multiforme patients (n = 17),173 and cervical and ovarian cancer (n = 6)174 showed the suitability of this tracer for imaging primary and metastatic disease, and for evaluating antiangiogenic treatment response and resulting prognosis in these groups. Comparison of the uptake of [18F]FPPRGD2 and [18F]FDG in 76 lesions in 26 patients with a variety of cancers revealed no statistical correlation between uptake of the two tracers, leading the authors to suggest that imaging with cRGD-based tracers offers complementary information to that of [18F]FDG (Fig. 8).175

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Figure 8 Comparison of [18F]FPPRGD2 and [18F]FDG uptake in the primary lung tumor lesion and mediastinal lymph node metastasis. Maximum intensity projection PET image and CT fused image of [18F]FPPRGD2 (A) and [18F]FDG (B) show positive uptake in the primary lung tumor lesion and mediastinal lymph node metastasis, but the uptake values (SUVmax) of the PET tracers in the primary lung tumor lesion ([18F]FPPRGD2 5.4, [18F]FDG 13.1) and mediastinal lymph node metastasis ([18F]FPPRGD2 3.2, [18F]FDG 12.3) were different. (Reprinted from Minamimoto et al: Eur. J. Nucl. Med. Mol. Imaging 2015;42:1850-1858, with permission of Springer. © Springer-Verlag Berlin Heidelberg 2015)

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Figure 9 2D projection images of [18F]Alfatide II PET (A) and [18F]FDG-PET (B) of a patient (no. 2) with metastatic adenocarcinoma of unknown primary site. [18F]Alfatide II PET demonstrated intense local accumulation of radioactivity in bone metastatic lesions located in thoracic vertebrae, sacrum and right scapula, and right clavicle with good background contrast, whereas [18F]FDG-PET only showed moderate uptake in some thoracic vertebrae and sacral lesions. The transaxial CT (C), [18F]alfatide II PET (D), and [18F]FDG-PET (E) were presented to focus on the lesions at sacrum. There was also bone metastasis with abnormal [18F]alfatide II uptake (G) but not visible by transaxial CT (F) or [18F]FDG-PET (H). (Reprinted with permission under a Creative Commons Attribution [CC BY-NC] license from Mi et al: Theranostics 2015;5:11151121. © The Author[s] 2015)

NOTA-aluminum [18F]fluoride complexes have also been applied to multimeric RGDs in an effort to reduce synthesis time, which could be reduced to as little as 40 minutes, while retaining high affinity (46 nM) of the multimers for αVβ3.176 Two such dimeric cRGD complexes differing mainly in linker topology have undergone translation into the clinic, alfatide I (AlF-NOTA-PRGD2) and alfatide II (Fig. 9). Alfatide I contains the AlF-NOTA chelator conjugated by a thiourea to a PEG chain bearing two cRGD units linked via a glutamic acid residue. Alfatide II bears the same AlF-NOTA chelator linked directly to a branching glutamic acid, which bears two PEG chains, each coupled to a cRGD. [18F]Alfatide I has been investigated in nine CT/[18F]FDG-PET-confirmed lung cancer patients, where tumor-to-blood and tumor-to-muscle ratios were 2.71 ± 0.92 and 5.87 ± 2.02, respectively, after 1 hour. In one patient, an additional lesion was identified by [18F]FDG, which was not observed with [18F]alfatide I. Immunohistochemistry in tumor specimens from these patients confirmed

the expression of αVβ3. An expanded study in a larger cohort (26 patients) with CT-suspected lung cancer was examined with [18F]alfatide I PET before surgery and biopsy. [18F]Alfatide I was found to correctly identify 17 cases of lung cancer, whereas 5 false positives as a result of inflammation were observed. 177 [ 18 F]alfatide I has also been examined in glioblastoma 178 and non-SCLC patients, 179 where PETderived metrics were found to be predictive of response to chemoradiotherapy. Developed as an analog of [18F]alfatide I, [18F]alfatide II was shown to have higher tumor uptake (2.92 vs 2.75 %ID/g at 60 minutes) and lower liver uptake (2.18 vs 1.81 %ID/g at 60 minutes) than its predecessor.180 Initial evaluation of [18F]alfatide II in five healthy volunteers and nine patients with brain metastases showed that the tracer was well tolerated.181 Of the 20 tumors identified by MRI or PET/CT, [18F]alfatide II was able to visualize all 20 lesions, whereas only 10 lesions were visible by [18F]FDG alone and 13 lesions were visible by CT alone. Tumor-to-background ratios were

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16 also far superior for [18F]alfatide II (18.9) vs [18F]FDG (1.5), suggesting that this peptide may be particularly suitable for imaging in regions that normally show high [18F]FDG uptake. [18F]Alfatide II has also been evaluated for the detection of skeletal metastases in 11 patients with 126 confirmed metastatic bone lesions.182 [18F]Alfatide II was found to be similar to [18F]FDG in the detection of osteolytic and mixed bone marrow metastases, and superior to [18F]FDG in detection of osteoblastic lesions (Fig. 9). As the popularity of gallium-68 as a nuclide for PET imaging has grown over the past few years, we have begun to see the introduction of 68Ga-labeled integrin-targeting radioligands to the clinic. As discussed previously, there is a range of BFCs available for gallium-68, and the availability of these chelators and the wide variety of existing 18F-radiolabeled monomeric and multimeric cRGD constructs have resulted in clinical translation of several similar 68Ga-labeled complexes. [68Ga]NOTA-RGD (IC50 = 218 nM) and [68Ga]NOTA-RGD2 (IC50 = 60.1 nM) are the monomeric and dimeric variants of [18F]alfatide I, containing cRGD units coupled through a thiourea linkage to a NOTA chelator either directly to the terminal lysine of the cRGD or through a glutamic acid residue bearing two cRGD peptides for the monomer and dimer, respectively.183,184 Initial studies of [68Ga]NOTA-RGD in cancer patients revealed no adverse effects upon administration,185 and a follow-up study in 43 breast cancer patients with invasive lobular carcinoma (ILC) demonstrated a significant difference in uptake of [68Ga]NOTA-RGD in HER2-positive lesions. This result highlights the complementary use of such tracers in combination with other imaging modalities in stratifying patient populations and providing minimally invasive prognostic information.186,187 [68Ga]NOTA-RGD2 has recently been examined in 91 lung cancer patients, where it was found to show lower sensitivity (80.9% vs 86.8%) but higher specificity (82.6% vs 69.6%) than [18F]FDG in diagnosing lung cancer. [68Ga]NOTA-RGD2 was shown to be especially suited in this sample for assessing lymph node metastases, where positive predictive value was improved from 30.2% with [18F]FDG to 90% with [68Ga]NOTA-RGD2.188 A recent report details the clinical evaluation of healthy volunteers and prostate cancer patients using a similar [68Ga]-NOTA dimeric compound, where one of the RGD units has been replaced with a BBN-targeting peptide.189 [68Ga]BBN-RGD was localized in three of four primary prostate cancer cases, and the case not seen with this tracer was found to be both integrin and gastrin-releasing peptide receptor (GRPR) negative. The heterodimeric tracer also performed as well as MRI and was found to be superior to [68Ga]BBN in identifying lymph node involvement, and outperformed both bone scanning and MRI in the detection of skeletal metastases. Two other 68Ga-labeled cRGD tracers, [68Ga]NODAGA-RGD and [68Ga]NODAGA-E(RGD)2, have recently been evaluated for safety in the clinic, and results from patient trials are eagerly awaited.190,191 Whereas most clinical RGD-based tracers target αVβ3, [18F]fluciclatide ([18F]AH111585) targets integrin αVβ5, showing 100× selectivity for αVβ5 over αVβ3 (IC50 0.1 nM vs 11.1 nM). An αVβ5-selective RGD-containing peptide was originally

discovered in a phage display library, and several modifications, including formation of the two rings and addition of PEG linkers, we made to improve pharmacokinetic properties and improve in vivo stability.192 Conjugation of the peptide to 18F-fluorinated aldehydes through an aminoxy functional group on the peptide provided a mild yet rapid route to radiolabeling the construct.193 Initial trials in seven breast cancer patients showed the tracer to be stable in vivo, well tolerated, and able to detect all 18 lesions previously detected by CT.194,195 As with other RGD-based tracers, high uptake was observed in the liver, where lesions were observed as hypointense compared to background. In a second study in 18 patients with either melanoma or renal cancers, all lesions showed [18F]fluciclatide uptake (SUV80%max of 6.4 for renal and 3.0 for melanoma), and positive correlation between SUV80%max and αVβ5 (and αVβ3) expression levels measured by immunohistochemistry in biopsy samples of the lesions (Fig. 10).196 Semiquantitative parameters derived from imaging with [18F]fluciclatide have also been shown to be reproducible in the same patients scanned on two separate days. This is an important property for widespread routine use of any tracer, particularly when used for evaluating treatment response, in this case antiangiogenic therapies.197

CXCR4 Imaging Chemokine receptors are involved in a range of cellular processes including cell adhesion, cytoskeletal rearrangement, and directional migration. CXCR4 is a seven-transmembrane domain GPCR normally expressed on T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, and hematopoietic stem or progenitor cells in bone marrow.198 The native CXCR4 ligand, stromal cell-derived factor-1 (CXCL12), is a 72-amino acid extracellular chemokine, and the CXCR4-CXCL12 interaction plays a vital role in chemotaxis, the migration of a variety of cells including stem, progenitor, and immune cells from their place of origin to final intended destination.199 Although CXCR4 initially gained attention as a research focus because of its identification as a coreceptor in HIV-1, more recent work has identified pathologic CXCR4 overexpression in upward of 30 different cancers including breast, pancreatic, ovarian, lung, prostate, colorectal and skin cancers, leukemia, and lymphoma.200,201 CXCR4 can enhance tumor cell survival and provide chemotherapy resistance and proliferation signals to cancer cells. Additionally, expression on primary tumor cells leads to organ-specific metastasis by directing circulating cancer cells to organs expressing CXCL12 such as the lungs, liver, and bone marrow.202 More generally, analysis via mRNA and immunohistochemistry has identified CXCR4 overexpression as an adverse prognostic factor in a variety of cancers. Identification of CXCR4 as an important target in oncology has led to a corresponding interest in PET, SPECT, and bioluminescence and fluorescence imaging, with these largely preclinical efforts being well represented in the review literature. 203-206 One of two clinically translated PET radiolabeled peptides designed for CXCR4 imaging, [68Ga]NOTA-NFB, was first imaged in a series of six healthy

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Figure 10 [18F]Fluciclatide PET/CT images in patient 4 (malignant melanoma.) Axial (A) sagittal (B) and coronal (C) images show focal radiotracer uptake within a left supraclavicular mass, with SUV80%max 6.5, as well as in other soft tissue nodules. (Reprinted from Mena et al: Eur. J. Nucl. Med. Mol. Imaging 2014;41:1879-1888, with permission of Springer. © Springer-Verlag Berlin Heidelberg [outside the United States] 2014)

volunteers and eight glioma patients by Wang et al in 2015.207 Because [18F]FDG imaging of brain pathologies is confounded by high physiological brain uptake and CXCR4 is known to be overexpressed in glioma, the authors sought to utilize a CXCR4-specific agent based on T140, a CXCR4 PET ligand. Biodistribution and dosimetry studies in healthy volunteers showed predominant accumulation in spleen and urinary bladder at 1 hour p.i., followed by liver, kidney, and red marrow. Imaging in glioma patients showed minimal tracer uptake in normal brain tissue including white and cortical gray matter (SUVmax 0.11 ± 0.02), contrasted with high [18F]FDG background signal as a result of high brain metabolism (SUVmax 9.63 ± 2.97). [68Ga]NOTA-NFB was also shown to specifically accumulate in glioma (SUVmax 4.11 ± 2.90) in contrast to [18F]FDG, which actually showed less accumulation in glioma than in normal brain tissue (SUVmax 7.34 ± 2.90). Although this work represents a successful effort in visualizing glioma via CXCR4 imaging, it should be noted that the authors acknowledge an inability to correlate CXCR4 expression with glioma grade. Consequently, further investigations with a larger cohort and variation in glioma grade are necessary to fully evaluate the potential of this tracer for glioma imaging.

A second and more thoroughly investigated clinical CXCR4 radiopeptide, [68Ga]CPCR4-2 ([68Ga]pentixafor, Fig. 11), was first synthesized and studied in tumor-bearing nude mice in 2011 by Demmer et al.208 The tracer showed predominant renal excretion because of high hydrophilicity but low kidney retention, possibly because of high metabolic stability and direct excretion into the urine. Furthermore, the tracer showed high tumor uptake and CXCR4 specificity. This is a notable advantage over numerous other radiopeptides targeting CXCR4, which have a tendency to also bind to CXCR7, a second chemokine receptor present in cancer cells.209 A followup study from 2015 provided further encouraging preclinical data confirming the high specificity of [68Ga]pentixafor for human CXCR4 (IC50 = 5.0 ± 0.7 nM), in addition to clinical data from four patients with varied lymphoproliferative cancers (Fig. 12).210 Patients include one with both CD30-positive aggressive T-cell lymphoma and non-SCLC, a patient with diffuse large B-cell lymphoma, a patient with multiple myeloma (MM), and a patient with chronic lymphocytic leukemia. Generally, results were positive and [68Ga]pentixafor was found to display comparable or superior imaging characteristics in comparison to [18F]FDG for localizing lesions.

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Figure 11 [68Ga]Pentixafor, [90Y]Pentixather and [177Lu]Pentixather.

An additional study from Phillip-Abbrederis et al in 2015 conducted a more in-depth visual comparison of [68Ga]pentixafor with [18F]FDG in 14 patients with histologically proven advanced MM.211 Of the 14 patients imaged, 9 (64%) were rated as positive with [18F]FDG, whereas 10 (71%) patients were rated as visually positive using [68Ga]pentixafor. Although these results show promise for the application of [68Ga]pentixafor toward MM imaging, the authors emphasize that the primary aim of the work was not to establish a diagnostic specifically for MM, but rather to study the application of [68Ga]pentixafor in diseases with high levels of CXCR4 on the surface of the cell membrane. Furthermore, the work suggests potential complementary benefits in utilizing both [18F]FDG and [68Ga]pentixafor in a disease with well-documented high levels of CXCR4 overexpression on the cell surface. Building on these promising diagnostic studies, a 2016 study from Herrmann et al applied CXCR4-targeted endoradiotherapy in combination with high-dose chemotherapy and autologous stem cell transplantation to three patients with advanced, pretreated MM.212 Before therapy, patients were imaged with [68Ga]pentixafor to confirm CXCR4 expression as well as [18F]FDG to assess metabolism in active myeloma lesions. Patients were treated with [M]pentixather, the therapeutic analog to diagnostic agent [68Ga]pentixafor. Two patients were treated with M = 177Lu and one with M = 90Y, and both therapeutic agents were found to be safe and well tolerated, and no adverse nonhematologic effects were observed. It should be noted that pentixather treatment led to myeloablation in all three patients, and may have contributed to the leukopenia or sepsis seen in one patient. Two of three patients were evaluated for response via [18F]FDG-PET/CT, with one found to show partial response and one found to show complete response of all extramedullary lesions via metabolic imaging. Although this initial

result serves as a promising proof of principle, further studies evaluating safety, toxicity, and a prospective clinical trial are necessary. Two studies from Lapa et al moved beyond MM to investigate applications of [68Ga]pentixafor toward imaging of CXCR4 expression in glioblastoma and SCLC.213,214 A 2015 study of [68Ga]pentixafor in 15 patients scheduled for surgical resection or biopsy of suspected primary or recurrent glioblastoma multiforme showed [68Ga]pentixafor retention in the majority of lesions, confirming the utility of the tracer for noninvasive visualization of intracerebral CXCR4 expression. Although it must be noted that [68Ga]pentixafor uptake did not correlate with histologic receptor expression, immunostaining verified that image-negative patients were also CXCR4 negative. A second study from the same group in 2016 applied [68Ga]pentixafor to imaging CXCR4 in 10 patients with lung cancer, motivated by a recent report demonstrating high CXCR4 expression in SCLC as characterized in biopsy samples of bronchopulmonary NETs. Interestingly, SCLC is distinguished from non-SCLCs by rapid doubling time, high growth fraction, and early development of metastases.215 Although not confirmed, the overlapping characteristics between high CXCR4 expression and frequent metastasis support a link between the two. The study in question observed eight patients with extensive disease and two with limited disease, and compared [ 68 Ga]pentixafor imaging of CXCR4 to [68Ga]DOTATOC imaging of SSTRs in a subset of five patients for the evaluation of SCLC. [68Ga]Pentixafor PET was found to clearly outperform [68Ga]DOTATOC on both a patient-by-patient basis and lesion-by-lesion basis in terms of general SCLC visualization. In all patients, lesions were identified visually, and all results could be compared to immunohistochemical staining of SSTR2a/5 and CXCR4. This work serves to highlight the utility of [68Ga]pentixafor in PET

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Figure 12 [68Ga]Pentixafor-PET/CT in patients with lymphoproliferative malignancies. (A) [68Ga]Pentixafor-PET/CT in a patient with relapsed diffuse large B-cell lymphoma (PET MIP) with (B) transaxial PET/CT image at the level of the brain. (C) [68Ga]Pentixafor-PET/MR in a patient with chronic lymphocytic leukemia and suspected transformation into aggressive B-cell lymphoma (PET MIP) with (D) transaxial PET/MRI image at the level of the kidneys. (E) [68Ga]Pentixafor PET/MR in a patient with multiple myeloma (PET MIP) with (F) transaxial image at the level of the maxilla demonstrated no uptake in the maxilla (green arrow). (G) Corresponding [18F]FDG-PET/MR (PET MIP) of the patient depicted in (E) and (F) with corresponding (H) [18F]FDG-PET/MR transaxial image at the level of the maxilla (red arrow, same region as depicted in F) showed [18F]FDG uptake in the maxilla or floor of the maxillary sinus, most likely caused by a dental infection. MIP, maximum intensity projection. (Reprinted with permission under a Creative Commons Attribution [CC BY-NC] license from Wester et al: Theranostics. 2015; 5(6): 618-630. © The Author[s] 2015)

imaging of CXCR4 expression in SCLC patients, as well as point to potential targeting of CXCR4 for therapeutic intervention. Finally, a much broader study from Vag et al looked at visualization of CXCR4 via [68Ga]pentixafor via PET/CT or PET/ MR in 21 patients with various histologically verified solid cancers.216 Of the total of 43 tumors imaged solely via [68Ga]pentixafor, 29 were detectable visually. A subset of 27 tumors were also visualized via [18F]FDG for comparison, with 27 of 27 being detectable via [18F]FDG and 19 of 27 being detectable via [68Ga]pentixafor. These initial results indicate that although [68Ga]pentixafor is not as suitable as [18F]FDG for use as a general oncological tool, it is still an imaging agent of value in a subset of these solid tumor types. It must be noted that limitations of the study include a heterogeneous patient cohort with a low number of patients representing each investigated tumor subtype, as well as a lack of immunohistochemical validation. The authors emphasize a need for further study on the basis of the observation that previously described in vitro CXCR4 expression profiles of solid cancers and metastases do not sufficiently match the in vivo

distribution revealed via PET. Taken in summation, clinical experiences with [68Ga]pentixafor indicate that, although the agent may not be suitable for use as a general oncological imaging tool, the agent holds promise for PET imaging of specific tumor types by means of visualizing CXCR4 expression. Furthermore, CXCR4 represents a potential therapeutic target for treatment in a manner analogous to the well-elucidated theranostic targeting of SSTRs.

GRPR Imaging Native to amphibians, BBN (Fig. 13) is a 14-amino acid homolog to mammalian gastrin-releasing peptide and represents another scaffold of interest in targeting a family of GPCRs relevant to oncology. The gastrin-releasing peptide receptor (GRPR) family is composed of four receptors: neuromedin B receptor (BBR1), GRPR (BBR2), orphan receptor (BBR3), and amphibious receptor (BBR4).217,218 Excepting BBR4, these three receptors are naturally expressed in both peripheral tissues and the central nervous system in mammals. The former three receptors are expressed in humans, with BBR1 and BBR2 in particular shown to be upregulated in cancer cells

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Figure 13 Bombesin and [68Ga]RM2 ([68Ga]BAY86-7548).

including breast, colon, lung, pancreatic, and prostate cancers.217 Preclinical research concerning application of BBN and BBN analogues toward nuclear imaging has been ongoing since the development of the first full-length 99mTc-labeled BBN analog, [99mTc]Lys3-BBN, in 1998 by Baidoo et al.219 Preclinical PET imaging and SPECT clinical imaging using BBN analogs is extensive, and a detailed discussion is outside of the focus of this review. However, one finding of interest was the human metabolic instability of full-length (14-amino acid) BBN analogs, and the related finding that removal of amino acid residues from the peptide increases in vivo stability as well as biological half-life.217 For this reason, C-terminal BBN(714) is a popular scaffold for development of PET GRPR imaging agents. Initially centered around preclinical work with 99mTc analogs, GRPR imaging first transitioned into PET in 2003 with work from Rogers et al detailing the synthesis and preclinical evaluation of [64Cu]DOTA-Aoc-BBN(7-14)NH2. This agent consists of a DOTA chelator group coupled to the N-terminus of the 8-amino acid C-terminal chain of BBN via an 8-aminooctanoic spacer.220,221 Initial results showed specific tumor uptake in human prostate cancer cell line PC-3 mouse xenograft models, indicating potential of BBN analogs in GRPR-positive tumor imaging. Studies with a second novel PET BBN analog, [68Ga]DOTA-PEG2-[D-Tyr6-β-Ala11,Thi13,Nle14]BBN(6-14) amide ([68Ga]BZH3), came from Schumacher et al and show high affinity targeting of BBR2-positive tumors in a xenograft mouse model.222 This BBN agonist represents one of the first GRPR-targeting radiopeptides translated to clinical studies, with initial studies

centered around a head-to-head comparison of [68Ga]BZH3 and [18F]FDG in both gastrointestinal stromal tumors and recurrent glioma. A comparison of gastrointestinal stromal tumors showed that whereas 25 of 30 lesions (14/17 patients) displayed enhanced [18F]FDG uptake, only 8 of 30 lesions (7/17 patients) displayed enhanced uptake of [68Ga]BZH3.223 A proof-of-principle study in patients with recurrent gliomas was motivated by the prevalence of glioma as the most common primary tumor of the central nervous system and a common lack of elevated [18F]FDG metabolism in lowgrade tumors. Furthermore, therapeutic management of glioma is dependent on tumor grade. Initial studies in 15 patients with recurrent gliomas of varied grades showed enhanced [68Ga]BZH3 tumor uptake and superior utility in tumor grading as compared with [ 18 F]FDG. 224 In a small patient set, [68Ga]BZH3 was seen to show enhanced uptake in three out of six patients with tumor grade World Health Organization (WHO) II, four out of six with WHO III, and three out of three with WHO IV. This finding can be contrasted with proper identification of tumor grade in three out of six WHO II patients, three out of six for WHO III, and zero out of six for WHO IV via [18F]FDG. Based on this comparison, preliminary results with [68Ga]BHZ3 show promise for further studies in glioma patients. A follow-up study compared scans from seven patients with gene array tumor data and demonstrated overexpression of BBR1 and BBR2, marking these BBN receptors as targets of interest in glioma imaging.225 The agent [68Ga]BZH3 is also of interest as an example of a bloodbrain barrier permeable 68Ga-chelated peptide, on the grounds that it shows brain uptake.

ARTICLE IN PRESS Clinical applications of radiolabeled peptides A second clinically translated BBN related peptide agonist, DO3A-CH2CO-G4-aminobenzyl-Gln-Trp-Ala-Val-Gly-HisLeu-Met-NH2 (AMBA) was first synthesized and characterized as [177Lu]AMBA by Lantry et al. for therapeutic use, with initial studies in the human prostate cancer PC-3 cell line, and in nude male xenograft tumor bearing mice. AMBA, was first synthesized and characterized as [177Lu]AMBA by Lantry et al for therapeutic use, with initial studies in PC-3, and in nude male xenograft tumor-bearing mice. [177Lu]AMBA was found to be specific for BBR1 and BBR2, with nanomolar affinity for BBR 2 . 226 Additionally, initial clinical results yielded promising preliminary images.227 The first studies involved administration of sufficient tracer for imaging rather than therapy, and lesions were seen in five out of seven patients. Three patients also received therapeutic doses of the agent, and uptake in these lesions persisted for up to 70 hours p.i. These initial diagnostic results led to implementation of [177Lu]AMBA in phase I clinical trials. Initial diagnostic studies with [68Ga]AMBA were also presented in 2007 by Baum et al, demonstrating utility of [68Ga]AMBA in imaging BBR2 expressing tumors in 10 patients.228 As BBN agonists are known to be internalized by target cells, it was initially expected that they would be more suitable for imaging and therapy applications than BBN antagonists.217 As a result, initial clinical translation of BBN analogs primarily focused on agonists. Surprisingly, however, BBN antagonists were found to demonstrate generally superior performance in visualizing BBR2-positive tumors in vivo.217,219 Additionally, antagonists were seen to demonstrate reduced physiological activity and thus fewer side effects.217,222 One early antagonist showing promise for imaging BBR2-positive tumors studied in prostate cancer patients in 2014 by Wieser et al is [64Cu]4,11-bis(carboxymethyl)-1,4,8,11-tetraazobicyclo(6.6.2) hexadecane-PEG4-D-Phe-Glu-Trp-Ala-Val-Gly-His-Sta-LeuNH2 ([64Cu]CB-TE2A-AR06/[64Cu]TE2A). An initial clinical study showed favorable biodistribution and high tumor uptake, with some advantages being low kidney and intestine uptakes, high BBR2 uptake, and rapid pancreas washout.229 A second BBN antagonist, [68Ga]DOTA-p-aminomethylanilinediglycolicacid-D-phe-Gln-Trp-Ala-Val-Gly-His-Leu-NHEt([68Ga]SB3), was translated to the clinic by Maina et al in 2016. This study of 17 patients (nine men and eight women) investigated the utility of [68Ga]SB3 in imaging advanced prostate or breast cancer. Fifty percent of breast cancer patients and 55% of prostate cancer patients showed enhanced [68Ga]SB3 uptake, indicating promise for further imaging and eventual translation to radionuclide therapy.230 New GRPR radiotracers such [68Ga]NeoBomb1231 and [68Ga] BAY86-7548232 continue to be developed. [68Ga] BAY867548, for example, was first translated to the clinic under the name [68Ga]DOTA-4-amino-1-carboxymethyl-pipiredine-D-PheGln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2 ([68Ga]BAY86-7548) in a 2013 clinical study by Kähkönen et al, following distribution, metabolism, and dosimetry studies in healthy patients. A BBN2-specific ligand, [68Ga]BAY86-7548, was imaged in 14 prostate cancer patients with special interest in intraprostatic lesions and metastatic lymph nodes. The tracer was found to be 83% accurate in the detection of organ-confined prostate

21 cancer and to show 70% sensitivity for lymph node metastasis. Additionally, the tracer was found to show high prostate cancer binding specificity, making it a promising and potentially more prostate cancer-specific alternative to the commonly used PET agents, [11C]CHL, [18F]FCHL, or [11C]acetate.233 More recently, [68Ga]BAY86-7546 has been rebranded as 68 [ Ga]RM2 (Fig. 13) and investigated by Minamimoto et al in a head-to-head comparison between [68Ga]RM2 and [68Ga] PSMA-HBED-CC in patients with biochemically recurring prostate cancer. Although the authors asserted that a priori expectations favored a significantly superior performance for [68Ga]PSMA-HBED-CC over [68Ga]RM2 in patients with biochemically recurrent prostate cancer, findings showed similar uptake between the two tracers in suspected lesions (Fig. 14). In areas of uptake beyond expected physiological distribution, SUVmean values for [68Ga]PSMA-HBED-CC and [68Ga]RM2 were found to be 7.1 ± 4.0 and 7.6 ± 3.8, respectively.234 Due in part to the fact that the two tracers target different biomarkers for the same pathology, results indicate that additional work is needed to understand expression of PSMA and GRPRs in prostate cancer. Reflecting this potential of [68Ga]RM2 in prostate cancer imaging, the authors intend to extend this dual tracer comparison to a larger cohort of patients. Beyond prostate cancer, [68Ga]RM2 ([68Ga]BAY86-7548) was extended to imaging of breast cancer in 2016 by Stoykow et al. In a study of 15 patients further divided into 14 “no special type” tumors, 3 ILCs, and 1 mucinous carcinoma, [68Ga]RM2 was found to visualize BBR2 expression with high contrast in 73% of patients.235 Of note, all ILCs were clearly visualized, a subtype often missed by [18F]FDG-PET/CT because of limited [18F]FDG avidity. Physiological uptake in muscle and fat tissue was also seen to be very low. Furthermore, a multivariate regression analysis identified estrogen as the primary predictor of [68Ga]RM2 uptake. This makes [68Ga]RM2 a promising diagnostic tool in the imaging of estrogen receptorpositive tumors, which represents the majority of breast cancer cases. On the basis of promising diagnostic value in breast cancer in addition to the potential to complement the already well-established [68Ga]PSMA in prostate cancer imaging, the BBN antagonist [68Ga]RM2 ([68Ga]BAY86-7548) can be seen as a very promising radiopeptide for clinical BBR2 imaging.

Glucagon-like Peptide Receptor 1 (GLPR1) Imaging Diabetes is a major chronic health care concern, with no existing cure for either the type-1 or type-2 variants. As a disease characterized by dysregulation of the balance between glucose and insulin levels, a means to visualize the mass of insulinsecreting beta cells in the islets of Langerhans in the pancreas would offer a unique insight into the disease. GLPR1 is found on the surface of beta cells, and is an ideal candidate for quantifying the beta-cell mass.236 The native ligand for GLPR1 is glucagon-like peptide 1 (GLP1); however, this peptide has a very short half-life in vivo and is not ideal for imaging.237 Exendin-4, isolated from the saliva of the Gila monster, is a more stable peptide analog of GLP1238 that has been proven to be amenable to radiolabeling. Exendin-4 has been shown

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Figure 14 Maximum intensity projection [68Ga]RM2 and [68Ga]PSMA-11 images of the seven enrolled patients. (Reprinted with permission from Minamimoto et al: J. Nucl. Med. 2016;57:557-562. © 2016 by the Society of Nuclear Medicine and Molecular Imaging, Inc)

to successfully quantify normal pancreatic beta-cell mass239 and has been proven to be useful in visualizing insulinoma,240 an often benign insulin-secreting tumor, in preclinical models.237,241-243 Clinical translation of such peptides initially used 111In-labeled exendin-4 for SPECT imaging of insulinoma,244 but with the explosion in use of gallium-68 generators, excedin-4 analogs labeled with this nuclide soon entered the clinic. A small prospective trial in five patients with suspected nonmalignant insulinoma compared [Nle 14 -Lys 40- (Ahx-DOTA- 68 Ga)-NH 2 ]exendin-4 ([68Ga]DOTA-exendin-4) to [111In]DOTA-exendin-4 using PET/ CT and SPECT/CT, respectively.245 Both imaging modalities showed distinct tumor foci; however, higher tumor-tobackground ratios were observed for [68Ga]DOTA-exendin-4 (Fig. 15). Histopathology in specimens collected after surgery in four out of the five patients confirmed the diagnosis of insulinoma. This study, combined with a growing number of imaging studies in insulinoma and other insulin-associated pathology case reports, highlights the potential use of this PET tracer in such patients.244,246,247 A similar tracer, [68Ga]NOTA-MAL-Cys40-exendin-4 ([68Ga]NOTA-exendin-4), has undergone a larger preliminary investigation in the clinic. In a trial of 43 patients with insulinomas, [68Ga]NOTA-exendin-4 PET/CT demonstrated a sensitivity of 97.7%, significantly higher than other commonly used imaging modalities.248 This tracer could detect very small lesions (<1 cm) and showed a mean SUV of 10.2 in lesions, with a mean tumor-to-pancreas ratio of 7.9. A third GLP1 receptor targeting tracer, [68Ga]DO3A-VS-Cys40-exendin-4, has also been utilized in a single clinical case of suspected insulinoma,249

and with dosimetry studies in place,250 a wider application of this tracer in the clinic is no doubt imminent.

Urokinase-type plasminogen receptor (uPAR) Imaging The uPAR is a glycosylphosphatidylinositol-anchored membrane receptor for urokinase-type plasminogen activator, a serine protease. Through an activation cascade, binding of urokinase-type plasminogen activator to uPAR activates the protease, which cleaves plasminogen to plasmin, which then activates proteolytic enzymes acting on the extracellular matrix and basal membranes.251 uPAR has been found to be overexpressed on the surface of a variety of tumor types. Considering its role in degrading the extracellular matrix, uPAR is linked to degree of aggression and metastatic spread of breast, prostate, pancreatic, and colorectal cancers.252-254 uPAR has therefore been identified as an attractive target for PET imaging of cancers, particularly because of its association with aggressiveness.255 Preclinical studies with radiolabeled peptide [64Cu]DOTA-AE105, a peptide identified from a phage display library,256 were found to show significantly higher uptake (10.8 ± 1.5% ID/g) in uPAR-positive xenograft tumors compared to uPAR-negative tumors (1.2 ± 0.6% ID/g). Following positive preclinical imaging results, including validation of uptake with expression levels of uPAR on tumor cells,257 PET imaging with [64Cu]DOTA-AE105 was undertaken in 10 patients with breast, prostate, and bladder cancers.258 No adverse effects were observed upon administration and activity was found to clear rapidly via the urinary system; however, high uptake was observed in the liver and bowel. Despite a short

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Figure 15 Coronal (A) and transaxial (B) PET/CT images from patient 4 obtained 2.5 hours after injection of 80 MBq of [68Ga]DOTA-exendin-4. Coronal (C) and transaxial (D) SPECT/CT images of same patient 72 hours after injection of 90 MBq of [111In]DOTA-exendin-4. Focal uptake of [111In]DOTA-exendin-4 and [68Ga]DOTA-exendin-4 is seen in pancreatic body (arrows); patient has refused surgery so far. (Reprinted with permission from Antwi et al: J. Nucl. Med. 2015;56:1075-1078. © 2015 by the Society of Nuclear Medicine and Molecular Imaging, Inc)

plasma half-life of 8.2 minutes, all primary tumors in breast and prostate cancer patients were all identified, while the primary tumor was identified in one of three bladder cancer patients (SUVmax 2.9-15.9). Histology-confirmed metastatic disease was also observed in one breast cancer patient and one prostate cancer patient. A second uPAR-targeting radiolabeled peptide, [68Ga]NOTA-AE105, has also recently been explored, again in a group of 10 patients with breast (Fig. 16), prostate, and bladder cancers.259 Similar to [64Cu]DOTA-AE105, [68Ga]NOTA-AE105 identified the primary tumors in all cases, and identified or confirmed metastatic disease in the breast and prostate cancer patients. Interestingly, in the bladder cancer patients who were in responsive treatment for their disease, liver metastases were identified by CT but were not observed in the PET scan. This finding was hypothesized to be the result of high physiological liver uptake masking the tumors, or due to the tumors responding to chemotherapy.

Caspase-3 Imaging Apoptosis, or preprogramed cell death, is an essential function, critical for maintaining populations of healthy cells within tissues, and can cause a range of diseases if disrupted, including cancers, neurodegenerative diseases, and atrophies.260 Caspase-3, a cysteine protease recognizing and cleaving at the C-terminal Asp residue of a -DEXD- sequence, plays a key role as an effector caspase in the apoptosis pathway.261 The presence of active caspase-3 is regarded as a hallmark for the process of apoptosis.262 As such, caspase-3 proved to be an attractive target for imaging of apoptosis and could be useful in monitoring therapies aimed to activate the apoptotic pathway as a method to reduce tumor burden. One successful strategy has used a radiolabeled pentapeptide, [18F]CP-18. The

peptide bears the requisite DEXD sequence, a PEG chain to aid cell membrane permeability, and an azido-modified sugar that is labeled through the CuAAC reaction with [18F]fluoropentyne to generate [18F]CP-18. [18F]CP-18 is a substrate for caspase-3, and preclinical imaging with this compound in an in vivo apoptosis model, where apoptosis was induced in the mice thymi using dexamethasone, showed a statistically significant increase in tracer accumulation in the apoptotic thymi.263 [18F]CP-18 has been investigated in seven healthy volunteers (Fig. 17), where the tracer remained stable in the blood. Uptake in the liver, heart, and testes was observed, along with accumulation of the tracer in the kidneys and bladder.264 The critical organ in terms of dosimetry was the bladder wall, because of predominant renal excretion of the tracer.

Nononcology Clinical Applications of Peptides Although the bulk of radiopeptide applications fall under the umbrella of oncology, multiple applications beyond oncology space including imaging pathologies such as diabetes and myocardial infarction can be found, with this apparent versatility in peptidic radioligands serving as a testament to the importance of this subset of molecular imaging. Nononcological radiopeptide PET imaging can be seen to harness targets already investigated in oncology as well as completely novel targets. Two well-studied targets with oncological applications, CXCR4 and αvβ3, have also been implicated in angiogenesis and inflammation-related pathologies such as myocardial infarction, atherosclerosis, Moyamoya disease, and rheumatoid arthritis. Radiotracers for both targets (eg, [18F]galacto-RGD for αvβ3 and [68Ga]pentixafor for CXCR4) have been used to image these conditions.265-274 Broadly, these efforts have shown

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Figure 16 uPAR PET imaging in breast cancer. (A) Representative transverse CT, PET, and coregistered PET/CT images of a primary tumor lesion with intense uptake of [68Ga]NOTA-AE105 (patient 1). (B) Images show a uPAR-positive axillary lymph node metastasis (red arrow) with significant uptake in the same patient. (Reprinted with permission from Skovgaard et al: J. Nucl. Med. 2017;58:379-386. © 2016 by the Society of Nuclear Medicine and Molecular Imaging, Inc)

Figure 17 Decay-corrected anterior maximum intensity projections of PET at 7, 46, 77, 144, and 179 minutes (from left to right) after injection of [18F]CP-18 in male volunteer. There was rapid clearance of activity in all organs. (Reprinted with permission from Doss et al: J. Nucl. Med. 2013;54:2087-2092. © 2013 by the Society of Nuclear Medicine and Molecular Imaging, Inc)

ARTICLE IN PRESS Clinical applications of radiolabeled peptides promising early clinical results and support further clinical investigation of the role of both CXCR4 and αvβ3 in angiogenesis and inflammation as they relate to these nononcological pathologies.

Conclusions Recent developments of radiolabelled peptides for PET imaging are diverse and allow for the assessment of previously difficult to understand pathologies. Although predominantly focused on oncology, these developments are beginning to have clinical impact in other areas, such as cardiovascular disease, and promising preclinical results are emerging in the use of radiolabeled peptides in diabetes, infection, and inflammation, and we eagerly anticipate their translation into the clinic. The continued application of well-established tracers such as [68Ga]DOTATATE, [68Ga]PSMA-HBED-CC, and the RGD peptides will continue to provide information as these tracers are explored in larger, more diverse patient populations. Applications of the newer radiolabeled peptides, as will many peptides currently in preclinical evaluation, will no doubt have a large clinical impact in coming years.

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