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Eur J Nucl Med Mol Imaging DOI 10.1007/s00259-013-2643-0 REVIEW ARTICLE Molecular imaging agents for SPECT (and SPECT/CT) Gopinath Gnanasegaran & James R. Ballinger Received: 11 November 2013 / Accepted: 12 November 2013 # Springer-Verlag Berlin Heidelberg 2013 Abstract The development of hybrid single photon emission computed tomography/computed tomography (SPECT/CT) cameras has increased the diagnostic value of many existing single photon radiopharmaceuticals. Precise anatomical localization of lesions greatly increases diagnostic confidence in bone imaging of the extremities, infection imaging, sentinel lymph node localization, and imaging in other areas. Accurate anatomical localization is particularly important prior to surgery, especially involving the parathyroid glands and sentinel lymph node procedures. SPECT/CT plays a role in characterization of lesions, particularly in bone scintigraphy and radioiodine imaging of metastatic thyroid cancer. In the development of novel tracers, SPECT/CT is particularly important in monitoring response to therapies that do not result in an early change in lesion size. Preclinical SPECT/CT devices, which actually have spatial resolution superior to PET/CT devices, have become essential in characterization of the biodistribution and tissue kinetics of novel tracers, allowing coregistration of serial studies within the same animals, which serves both to reduce biological variability and reduce the number of animals required. In conclusion, SPECT/CT increases the utility of existing radiopharmaceuticals and plays a pivotal role in the evaluation of novel tracers. Keywords Single photon emission computed tomography . Computed tomography . SPECT/CT . Radiopharmaceutical . Preclinical imaging G. Gnanasegaran : J. R. Ballinger (*) Department of Nuclear Medicine, Guy’s and St Thomas’ NHS Foundation Trust, Great Maze Pond, London SE1 9RT, UK e-mail: [email protected] J. R. Ballinger Division of Imaging Sciences and Biomedical Engineering, King’s College London, Westminster Bridge Road, London SE1 7EH, UK Introduction The development of hybrid SPECT/CT cameras has been an important advance in traditional nuclear medicine, much as PET/CT broadened the indications and acceptance of PET. However, there is one difference in that planar gamma camera imaging remains an important modality, whereas PET/CT has virtually replaced stand-alone PET. The availability of SPECT/CT has increased the diagnostic value of many existing single photon radiopharmaceuticals, some of which were on the verge of withdrawal from the market but have been given new life by SPECT/CT. Other papers in this special issue of the European Journal of Nuclear Medicine and Molecular Imaging address various clinical areas in great detail. We begin by briefly reviewing the contribution of SPECT/CT to existing radiopharmaceuticals, then we discuss the role of clinical and preclinical SPECT/CT in the development of novel tracers. Clinical role of SPECT/CT The combined functional and structural information provided by SPECT/CT has been shown to influence patient management in many cases. The clinical role of SPECT/CT can be divided into four areas: anatomical localization of a lesion for diagnostic purposes, localization as a guide to surgery, multimodal characterization of a lesion, and CT-based attenuation correction for quantification, with the last of these being covered by Bailey et al. in this special issue in more detail [1]. Localization for diagnosis Accurate localization of increased tracer uptake, especially while imaging the bone extremities such as the wrist, foot and ankle, is often challenging in planar radionuclide Eur J Nucl Med Mol Imaging 99m Tc-diphosphonate bone imaging due to the complex grouping of bony articulations in a small area [2]. In general, SPECT/CT is particularly useful in wrist/hand and foot/ankle pathology, especially after intervention/surgery, when metallic artefacts may limit imaging with MRI [2]. Further, SPECT/CT is useful in making a more specific diagnosis [2] (Fig. 1). In patients with low back pain, bone SPECT with 99mTcdiphosphonates is useful for diagnosing facet joint arthropathy in the spine, but the addition of CT makes it more useful in terms of accurately localizing the vertebral level. Radionuclide infection imaging is a widely used procedure involving radiolabelled leucocytes, 67Ga-citrate, radiolabelled antigranulocyte antibodies and 99mTc-diphosphonates [3]. However, conventional radionuclide techniques struggle to differentiate soft-tissue infection from osseous infection and to define the extent of disease. SPECT/CT in infection imaging has been reported to lead to more accurate diagnosis and precise localization [3–6] (Fig. 2). In patients with suspected infection, Bar-Shalom et al. evaluated the role of SPECT/CT as an adjunct to 67Ga-citrate or 111In-labelled white blood cell imaging for the diagnosis and localization of infection [4]. In their study, the results of conventional radionuclide scintigraphy (planar and SPECT) and SPECT/CT were concordant for the diagnosis and location of infection in 50 % of their patients Fig. 1 99mTc-MDP SPECT/CT imaging in a patient with pain in the right scaphoid 9 years after injury. a, b On the two-phase bone scan, there is low-grade blood pool activity with intense focal uptake of tracer in the right scaphoid bone which on the CT component of the study (d) corresponds to a fracture through the waist of the scaphoid which has partial bridging. c–e The scan findings are in keeping with pseudoarthrosis at the site of a partially united fracture through the waist of the right scaphoid (41/82) and, encouragingly, SPECT/CT was found to be beneficial in determining the precise anatomical location of infection in 85 % (35/41) of their discordant studies [4]. Furthermore, in a site-based analysis, conventional scintigraphy and SPECT/CT showed concordant results for the diagnosis and localization of infection in 50 % (49/98) of sites, and SPECT/CT was able to provide precise anatomical location of 44 % (43/98) of infectious sites which had been equivocal or erroneous on conventional scintigraphy [4]. Filippi et al. evaluated SPECT/CT for 99mTc-HMPAO-labelled leucocyte imaging in patients with diabetic foot infection and reported a change in the interpretation of the planar and SPECT images in 10 of 19 suspected sites (53 %) [5]. Localization of sentinel lymph nodes (SLN) in patients with breast cancer and melanomas by planar lymphoscintigraphy using 99mTc-labelled nanocolloids is well-accepted [7]. However, the major limitations include failure to detect SLNs and false-positive radiotracer uptake on planar scans [7–14]. Several studies have shown the usefulness of SPECT/CT with 99m Tc-radiocolloid in not only breast cancer and melanoma but also in head/neck malignancies, prostate cancer and gynaecological malignancies [7–14]. In SLN localization, SPECT/CT with 99mTc-radiocolloid may improve the accuracy of preoperative anatomical localization, detect hot lymph Eur J Nucl Med Mol Imaging the evaluation of 169 lesions in 81 patients with NET, In-pentetreotide SPECT/CT was found to have a significantly greater diagnostic accuracy than SPECT in both a patient-based analysis (93 % vs. 79 %, respectively) and a lesion-based analysis (96 % vs. 81 %, respectively) [19]. Overall, 111In-pentetreotide SPECT/CT was significantly more accurate for lesion localization than was SPECT (95 % vs. 46 %, respectively). In a study in 54 patients with NET, 111 In-pentetreotide SPECT/CT provided additional information in 46 % of the patients, anatomical localization improved in 37 % and tracer uptake considered as possible disease was excluded as physiological uptake in 9 %. 111In-Pentetreotide SPECT/CT altered the management in 26 % of the patients, changing the diagnostic strategy in 15 % and the treatment strategy in 11 % [20]. In general, there are fewer equivocal results/interpretation with SPECT/CT than with SPECT imaging alone in patients with NET. However, in the future SPECT/CT imaging of NET tumours will face competition from PET/CT with 68Ga-DOTA peptides and/or 18 F-fluorodopa [21]. Although 111In-capromab pendetide (Prostascint) has been on the market since 1997 in the US, interpretation of SPECT images was plagued by the lack of anatomical markers and inability to distinguish between pathological and physiological accumulation [22]. In the first study to combine SPECT and CT imaging with 111In-capromab, 46 % (74/161) of the sites interpreted as positive on SPECT alone turned out to be negative when examined by SPECT/CT [20]. More recent studies have confirmed the added value of CT in interpretation of 111In-capromab SPECT images, to the point that pretreatment 111In-capromab SPECT/CT has been shown to predict biochemical disease-free survival and disease-specific survival in primary prostate cancer and to be useful for defining biological target volumes for intensity modulated radiotherapy [23]. This is a clear example of SPECT/CT extending the scope of use of an existing radiopharmaceutical. 111 Fig. 2 111In-labelled WBC SPECT/CT in a patient with previous left quadriceps tendon repair with ongoing sinus; query left patella osteomyelitis. a There is a focal area of increased tracer uptake seen in the region of the left lower thigh on the whole body scan. b–d On SPECT/CT images the uptake is localized to soft-tissue uptake in the quadriceps femoral tendon. The scan findings are consistent with soft-tissue infection at the quadriceps femoral tendon which will correspond with the ongoing sinus problem. There is no evidence of patellar osteomyelitis nodes missed by conventional planar imaging, and exclude non-nodal false-positive sites of increased uptake [7–10] (Fig. 3). Radionuclide scintigraphy and therapy with 123I/131I is used in the treatment and follow-up of patients with differentiated thyroid cancer [15]. Radioiodine SPECT/CT has been reported to be useful in accurate localization of sites of pathological uptake such as metastasis or remnant/residual thyroid [15–17]. There are several studies evaluating the role of radiolabelled somatostatin analogues in imaging neuroendocrine tumours (NET) which have shown that the addition of SPECT/CT to conventional planar scintigraphy and SPECT, as well providing incremental value for lesion localization and characterization, increases diagnostic confidence, and has a positive clinical impact on the management of NET [18–20]. In Localization for surgery 99m Tc-Sestamibi SPECT/CT has proved to be a useful technique for preoperative localization of parathyroid adenomas, especially at ectopic sites and in patients with a history of previous neck surgery/interventions [24, 25]. However, the role 99mTc-sestamibi SPECT/CT in the routine investigation of eutopic sites is debatable [26, 27]. SLN radiocolloid imaging is performed in patients with melanoma, breast cancer and other malignancies such as vulval, penile, and prostate cancer [7–14]. Often, intraoperative SLN detection using the gamma probe can be challenging. In general, approximately 95 % of the injected activity may remain at the injection site, and SLN close to the injection site can be missed, both on planar images and when using hand-held gamma ray detection probes. Further, the SLN and second echelon nodes may not be Eur J Nucl Med Mol Imaging Fig. 3 99mTc-Nanocolloid SPECT/CT imaging in a patient with squamous cell carcinoma of the left lateral border of the tongue. 99mTcnanocolloid was injected around the left lateral tongue border. a, b On the planar images and SPECT images two focal areas of increased tracer uptake are seen on the left side of the neck. c On the fused SPECT/CT scan, the increased tracer uptake close to the injection site on the planar images localizes to a level 2a lymph node, and one lower to a level 3 lymph node accurately differentiated using intraoperative gamma probes. 99m Tc-Radiocolloid SPECT/CT improves the accuracy of preoperative anatomical localization of these nodes to help in planning the surgical approach [9, 13, 18]. Further, a few centres have reported the use of concomitant radionuclide and fluorescence-guided SLN biopsy using indocyanine green 99m Tc-nanocolloid, in which SPECT/CT is used for anatomical localization [9, 14]. result of upstaging or downstaging disease using radioiodine SPECT/CT [17]. To summarize the role of SPECT/CT with current clinical tracers, SPECT/CT adds incremental value to conventional radionuclide planar and SPECT imaging studies by improving lesion detection, localization and characterization, which translates into better diagnostic confidence, sensitivity and specificity (Table 1). These factors are essential components for optimal management of both oncological and nononcological patients. Examples of radiopharmaceuticals for which diagnostic utility has been enhanced by SPECT/CT are presented in Table 2. Characterization of lesions Radionuclide planar bone scintigraphy with 99m Tcdiphosphonates is highly sensitive for detecting bone metastases but its specificity is relatively variable for accurate differentiation of benign and malignant bone disease [2]. The addition of SPECT imaging has improved diagnostic accuracy to a certain extent, but is still inadequate for confirming or excluding metastases with a high degree of confidence [28–31]. The additional value of SPECT/CT compared with SPECT alone in differentiating benign and malignant bone lesions on 99mTc-diphosphonate bone scintigraphy has been shown in several studies and most of the papers agree that SPECT/CT is useful for the assessment of indeterminate lesions noted on both planar and SPECT imaging [28–31]. The consensus is that 99mTc-diphosphonate SPECT/CT improves diagnostic confidence in making a definitive diagnosis in most patients (Fig. 4). Radioiodine SPECT/CT is also useful in differentiating pathological from physiological tracer uptake [15]. Overall, radioiodine SPECT/CT has been reported to improve detection and accurate localization of lymph node metastases and distant metastases, compared with conventional whole-body imaging [16]. Further, Schmidt et al. have reported management changes in approximately one-quarter of patients as a Development of novel tracers The development of novel tracers generally involves a sequence of chemical and biological validations. Once the targeting and labelling chemistry have been worked out, the potential tracer is evaluated in an in vitro binding system. Only after that has been established is it ethical to evaluate localization and biodistribution in animal models. Biodistribution studies have traditionally involved the killing of groups of animals at a limited number of time-points after injection, removal of specific organs, and measurement of their radioactive content in a gamma counter. However, the information obtained is limited by the number of time-points, requires relatively large numbers of animals, and introduces variability since, for example, every experimental tumour will not be the same size. Moreover, transgenic animals can be extremely expensive. Thus, imaging techniques would allow serial studies in the same animal, improving reproducibility and reducing the number of animals required [32]. Eur J Nucl Med Mol Imaging Table 1 Contribution of SPECT/CT to interpretation of clinical radionuclide images Contribution Example Reference Anatomical localization of a lesion for diagnostic purposes Anatomical localization as a guide to surgery 99m [2] Tc-Diphosphonates in wrist/hand and foot/ankle pathology 99m Tc-Sestamibi for preoperative localization of parathyroid adenomas, especially at ectopic sites Multimodal characterization 131I-Iodide for detection and accurate localization of of a lesion lymph node metastases and distant metastases Attenuation correction CT component improves accuracy of quantification Fig. 4 99mTc-MDP SPECT/CT imaging in a patient with breast cancer with back pain, worse at night; query bone metastasis. a On the wholebody bone scan, there is a focal area of increased tracer uptake seen in the T8 vertebra, which corresponds to a sclerotic lesion at this site on the CT component (c). b–d The appearance of the scans is consistent with a solitary T8 vertebral metastasis. On the whole-body bone scan, the lowgrade increased tracer uptake in the upper thoracic spine is suggestive of degenerative disease Gamma cameras have been used to image research animals for many years, but there has always been uncertainty about the exact anatomical localization of sites of radiotracer accumulation. With SPECT, even in relatively large animals such as monkeys, the precise localization of brain regions has proven difficult in our experience. Dedicated preclinical SPECT devices were introduced about a decade ago, offering submillimetre resolution with advances in detector and collimator design [32]. Indeed, resolution is sufficient to allow discrimination of regions within the mouse brain or heart. Very quickly CT was incorporated into the devices, further reducing the uncertainty about the anatomical localization of radioactivity on the SPECT images [32]. SPECT/CT allows differentiation of pathological and physiological accumulation, [24] [16] [1] particularly in the organs of excretion, and is important for coregistration of serial studies within the same animals, making best use of the reduction in biological variability. With clinical equipment, the spatial resolution of SPECT is typically inferior to that of PET. However, the same does not hold true with preclinical equipment where SPECT can achieve higher spatial resolution than PET. Moreover, SPECT allows simultaneous imaging of multiple tracers with different gamma energies, which is not possible with PET where all radionuclides are detected via 511 keV annihilation photons. Also, most positron-emitting radionuclides have very short half-lives, making studies technically challenging. The easier availability and longer half-lives of most commonly used gamma-emitting radionuclides of medical interest allows longer experiments to be carried out. Thus, preclinical SPECT/CT has become of interest to the pharmaceutical industry as well as to radiopharmaceutical researchers [32]. We review a number of examples of novel tracers under development which will lead to improvements in current diagnostic targets or allow exploitation of new targets. Angiogenesis Reduction in angiogenesis is another early indicator of the effectiveness of novel therapies, particularly those which do not immediately result in a reduction in tumour size. Most angiogenesis markers have used the integrin αvβ3 binding sequence Arg-Gly-Asp (RGD). As reviewed recently by Gaertner et al. [33], 99mTc-maraciclatide (NC100692) has been used in clinical trials [34] but development of other RGD tracers continues, in particular to evaluate the role of multimers in enhancing accumulation in tumours. The recent clinical study by Zhao et al. provides evidence of the wisdom of this approach [35]. They used a high-affinity RGD dimer Eur J Nucl Med Mol Imaging Table 2 Examples of current radiopharmaceuticals used with SPECT/CT Contribution of SPECT/CT Application Radiopharmaceutical Anatomical localization of a lesion for diagnostic purposes Bone imaging Inflammation imaging Inflammation imaging 99m Anatomical localization as a guide to surgery Multimodal characterization of a lesion with PEG to fine-tune the pharmacokinetics and a Hynic group for attachment of 99mTc. The utility of this tracer for monitoring the response to antiantiogenic therapy has recently been demonstrated in a mouse model of glioma [36]. Moreover, 111In-labelled RGD has been coupled with a fluorescent dye to produce a hybrid tracer which will allow visualization of tumour margins during surgery as well as the detection of distant metastases [37]. However, SPECT/CT will face challenges from PET/CT, in which 18F fluciclatide and 18 F-galacto-RGD have already been widely used in patients [33, 38, 39]. Apoptosis Detection of apoptosis is an important goal in the assessment of the effectiveness of novel therapies. While the ideal agent for clinical use has not yet been identified, a variety of tracers have been evaluated preclinically. Annexin V was the original targeting agent, binding to phosphatidyl serine exposed at an early stage of apoptosis, but 99mTc-Hynic-annexin V does not have ideal biological properties [40]. Engineered variants of annexin V have been developed which incorporate a 99mTc binding domain or a His-tag for labelling with 99mTctricarbonyl, thus improving specific targeting and reducing renal accumulation [40, 41]. Another protein, the C2A domain of synaptotagmin I, has also been shown to bind to apoptotic cells [42]. Both of these classes of compounds are relatively large proteins which suffer the disadvantage of slow kinetics and high activity in the abdomen. It does not appear that gamma-emitting analogues of the malonate or isatin small molecules developed for PET imaging have been evaluated [43, 44]. However, recently a caspase-3-binding peptide consisting of a Phe-Gly-Cys 99mTc chelation sequence and Asp-Glu-Val-Asp targeting sequence has shown promising results [45]. It is not yet clear whether SPECT/CT or PET/ CT will predominate in the future for imaging apoptosis. SLN localization Thyroid cancer imaging Neuroendocrine tumour localization Prostate cancer detection Parathyroid gland localization SLN localization Bone imaging Thyroid cancer imaging Tc-Medronate In-Labelled white blood cells 99m Tc-Labelled white blood cells 99m Tc-Nanocolloid 131 I-Iodine 111 In-Pentetreotide 111 111 In-Capromab Tc-Sestamibi 99m Tc-Nanocolloid 99m Tc-Medronate 131 I-Iodine 99m Chemokine receptor 4 expression The chemokine receptor 4 (CXCR4) is overexpressed in a variety of cancers including cancer of the breast, prostate and ovary. Its expression in mammary ductal carcinoma in situ has been studied by Buckle et al. using an 111In-labelled peptide antagonist Ac-TZ140011 [46]. Early images (1 h after injection) showed that the peptide is delivered to both CSCR4expressing and non-expressing tumours (which is also similar for angiogenesis, as noted above) but the peptide is only retained (24 h after injection) in the CXCR4-expressing tumours as shown by serial SPECT/CT imaging [46]. Another group has labelled a non-peptide small-molecule antagonist AMD3100 with 99mTc and studied its biodistribution in athymic mice bearing human prostate cancer xenografts [47]. Tumours were visible over 2 h on SPECT imaging. Moreover, activity could be displaced from the tumours and other CXCR4-rich tissues by preinjection of an excess dose of unlabelled AMD3100 [47]. A recent review offers the opinion that PET/CT is more promising than SPECT/CT for imaging CXCR4 expression in patients, but fluorescent, paramagnetic and hybrid agents are also under development [48]. Epidermal growth factor receptor in breast cancer Members of the human epidermal growth factor (EGF) receptor family including HER2 and EGFR are overexpressed in many solid tumours including breast cancer. In particular, HER2/EGFR heterodimers are associated with a more aggressive tumour phenotype. Recently, a group from the University of Toronto has prepared a bispecific radioimmunoconjugate which is capable of binding to this heterodimer [49]. To prepare the bispecific agent, Fab fragments of trastuzumab (Herceptin) were coupled to human EGF using a polyethylene glycol linker, then a DTPA chelator was added for radiolabelling with Eur J Nucl Med Mol Imaging 111 In. In athymic mice bearing HER+/EGFR+ xenografts, accumulation of the radioimmunoconjugate seen on SPECT/CT imaging was maximal at 24–48 h and could be blocked by pretreatment with unlabelled trastuzumab Fab or EGF [49]. Folate receptor overexpression Folate is involved in many biosynthetic processes including nucleotide precursors of DNA. Rapidly dividing cells, including many cancers, consume higher amounts of folate. The folate receptor (transporter) is overexpressed on the surface of some epithelial tumours including ovarian (95 %) and lung (75 %) cancer [50]. Thus, folate receptor overexpression is an attractive target for selective delivery of chemotherapeutics and toxins. However, only patients who overexpress the receptor will benefit from these novel treatments so a “companion diagnostic” imaging agent is required for patient selection. That is what is being carried out in clinical trials of etarfolatide (EC20, FolateScan) prior to treatment with vintafolide (EC145), which consists of vinblastine coupled to a folate targeting moiety [51]. However, the use of folate targeting is complicated by accumulation of radiofolate in normal tissues, particularly the kidneys. Thus, pharmacological intervention is required to allow delivery of a sufficient fraction of the administered activity to the tumour. In the etarfolatide trials, folic acid is administered immediately before the tracer. A group at the Paul Scherrer Institute has been evaluating alternative means of achieving this, including the antifolate pemetrexed combined with thymidine as an antidote to the potential toxicity of pemetrexed, a chemotherapeutic agent in its own right [52–54]. Although trials of this combination have been promising, it remains to be seen whether the degree of protection of normal tissues is sufficient to allow therapy with folatecoupled alpha or beta particle-emitting radionuclides without unacceptable radiotoxicity to the kidneys. Although PET agents are under development for imaging the folate receptor, etarfolatide for SPECT/CT is the most advanced agent, being already under review for licensing by the European Medicines Agency as a theranostic together with vintafolide. Hypoxia Tumours which are hypoxic are less sensitive to radiation therapy and other treatments, and hypoxia tends to select a more aggressive phenotype. However, there are no structural or clinical predictors of the extent of hypoxia in a particular tumour. Moreover, hypoxia tends to be heterogeneous both spatially and temporally. Imaging of hypoxia has traditionally involved either nitroimidazole or bioreductive targeting, but clinically this is mainly the domain of PET/CT rather than SPECT/CT [55]. However, a novel alternative approach has been suggested recently that utilizes hypoxia-inducible factor 1α (HIF-1α) which has previously been used as an in vitro marker for hypoxia [56]. The oxygen-dependent degradation domain of HIF-1α was fused with the protein transduction domain and with streptavidin which will then react with radiolabelled biotin. Using preclinical SPECT coregistered with CT and MRI images, intratumoral heterogeneity in localization was visualized. Hypoxia imaging is a prime example of the need for hybrid imaging, since both functional and structural measurements are required to calculate the hypoxic fraction of a tumour – the fractional volume of the tumour in which the tissue oxygen concentration is below a particular threshold value. Prostate-specific membrane antigen Prostate specific membrane antigen (PSMA) is a cell surface antigen which is expressed at a basal level in prostate cells, but its expression increases progressively in higher grade tumours, metastatic disease, and hormone-refractory disease. Urea-based small-molecule ligands which bind to PSMA are being developed for SPECT/CT and PET/CT imaging and for molecular radiotherapy. Building on the success of 123 131 I/ I-labelled compounds (diagnostic/therapeutic), chemists at Molecular Insight Pharmaceuticals have developed 99m Tc/186/188Re-labelled analogues [57]. The Glu-urea-Glu pharmacophore has turned out to be relatively forgiving, and the second Glu can readily be conjugated or replaced with Lys derivatives containing tridentate chelating moieties for 99m Tc-tricarbonyl. All but one of the analogues tested has shown low nanomolar affinity for PSMA. Currently, one of these compounds, 99mTc-MIP-1404, is under evaluation in an international phase 2 study in men scheduled for radical prostatectomy at high risk of lymph node involvement [58]. However, SPECT/CT will face difficult competition from the 68 Ga-labelled ureas developed for PET/CT [58]. Bone imaging Although the 99mTc-labelled diphosphonates have been used for many years for bone imaging, and constitute the most widely used class of radiopharmaceuticals internationally, they are not optimal because the diphosphonate acts as both chelator and targeting group. Moreover, each role may compromise the other. Several research groups are developing bifunctional agents in which the diphosphonate targeting group is coupled to a chelator, separating the two functions [59, 60]. Preclinical SPECT/CT has been essential in characterizing the in vivo behaviour of these tracers. Torres et al. used alendronate, a clinically approved diphosphonate with high affinity for hydroxyapatite, coupled via the sidechain to a dipicolylamine group which serves as a chelator for 99mTc tricarbonyl [60]. The resulting complex is a single, chemically defined species, unlike current 99mTc- Eur J Nucl Med Mol Imaging diphosphonates, and shows excellent bone localization with good clearance from nontarget tissues (Fig. 5). Moreover, the same technology can be used to prepare a stable 188Re complex for palliative treatment of bone metastases [60]. Indeed, this theranostic application of bifunctional bone-seeking agents will be important if they are to compete with 18Ffluoride PET/CT bone imaging [61]. Perspectives The addition of hybrid CT improves confidence in spatial localization of the radioactive signal, accurate sizing of lesions, anatomically relevant placement of regions of interest, and precise repositioning for serial studies. The studies described above included examples of these properties. Buckle et al. used CT to determine experimental tumour volumes and stratify mice into early, intermediate or late stages of tumour development for analysis of SPECT images [46]. Serial studies have been performed over 24 h [46] or even 72 h [49] with CT being used for repositioning, allowing the time of peak accumulation to be determined following a single injection of a radiotracer with a half-life that is sufficiently long. Serial studies in the same animals with repeat administration of radiotracer have been performed over 18 days in a therapeutic study [36]. SPECT/CT is important for localization of radioactivity in the xenograft and metastases, but also in the organs of excretion which can be affected by coadministered drugs [52]. Excised tissues can be imaged with SPECT/CT ex vivo and the images superimposed on the in vivo images in order to study the influence of respiratory motion, scatter, and counting statistics [56]. However, preclinical SPECT/CT can require injection of relatively large quantities of radiotracer (e.g. 50 MBq) in order that the acquisition times are not unreasonably long; this may result in slight quantitative differences from low activity biodistribution (tissue counting) studies, in part due to specific activity effects [47, 56]. Despite PET being the current flavour of the month, there is a continuing need to develop tracers for SPECT, as elegantly argued by Mariani et al. [62]. The worldwide installed equipment base for SPECT is much greater than that for PET. The longer half-lives of many of the radionuclides used for SPECT make them much more convenient to supply compared to the short-lived PET radionuclides which often rely on a nearby cyclotron. The average cost of PET radiopharmaceuticals is about ten times greater than those for SPECT, although the difference may not be as great for newer agents. It is uncertain whether PET will be economically viable in some parts of the world. Table 3 Examples of novel radiopharmaceuticals being developed for SPECT/CT Application Radiopharmaceutical Reference Angiogenesis 99m Tc-Maraciclatide Tc-3P-RGD2 111 In-MSAP-RGD [34] [35] [37] 99m Tc(CO)3-His-annexin A5 C2AcH-[99mTc(CO)3] 99m Tc-(Me)FGCDEVD 111 In-DTPA-Ac-TZ14011 99m Tc-AMD3100 111 In-DTPA-Fab-PEG24-EGF [41] [42] [45] [46] [47] [49] 99m Tc-Etarfolatide In-DOTA-folate 123/125 I-IPOS 99m Tc-MIP1404 [51] [53] [56] [57] 99m [60] 99m Apoptosis Chemokine receptor 3 expression Fig. 5 SPECT (colour)/CT (greyscale) image of an adult Balb/C mouse acquired 4 h after intravenous injection of 99mTc(CO)3-DPA-alendronate. The image is a maximum intensity projection from a 30-min acquisition on a NanoSPECT/CT system (Bioscan Inc). There is high accumulation of 99mTc in the skeleton, particularly the joints, and good clearance from other tissues (courtesy of Dr. R. Torres and Prof. P. Blower, Division of Imaging Sciences and Biomedical Engineering, King’s College London) Epidermal growth factor receptor Folate receptor 111 Hypoxia Prostate-specific membrane antigen Bone imaging Tc(CO)3-DPA-alendronate Eur J Nucl Med Mol Imaging Summary SPECT/CT has become the state of the art technique for in vivo evaluation of novel gamma-emitting radiotracers (Table 3). Preclinical SPECT offers submillimetre resolution, accurate quantification and reasonable acquisition times, while taking advantage of the properties of gamma emitters—longer half-lives than most positron emitters, easier accessibility and the potential for simultaneous imaging of more than one radionuclide using different energy windows. In the clinic, SPECT/CT is revolutionizing radionuclide imaging by its incremental value providing accurate localization, diagnosis, characterization and surgical guidance. Further, SPECT/CT improves specificity and diagnostic confidence, which may reduce the number of equivocal reports. Overall, SPECT/CT is spectacular, especially in orthopaedic indications, and should be considered the standard modality with equivalent status to PET/CT. Acknowledgments The authors acknowledge financial support from the Department of Health via the National Institute for Health Research (NIHR) comprehensive Biomedical Research Centre award to Guy’s & St Thomas’ NHS Foundation Trust in partnership with King’s College London and King’s College Hospital NHS Foundation Trust. We would also like to thank Dr. R. Torres and Prof. P. Blower, Division of Imaging Sciences and Biomedical Engineering, King’s College London, for providing the preclinical SPECT/CT image. 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