Metal-Based PSMA Radioligands

Prostate cancer is one of the most common malignancies for which great progress has been made in identifying appropriate molecular targets that would enable efficient in vivo targeting for imaging and therapy. The type II integral membrane protein, prostate specific membrane antigen (PSMA) is overexpressed on prostate cancer cells in proportion to the stage and grade of the tumor progression, especially in androgen-independent, advanced and metastatic disease, rendering it a promising diagnostic and/or therapeutic target. From the perspective of nuclear medicine, PSMA-based radioligands may significantly impact the management of patients who suffer from prostate cancer. For that purpose, chelating-based PSMA-specific ligands have been labeled with various diagnostic and/or therapeutic radiometals for single-photon-emission tomography (SPECT), positron-emission-tomography (PET), radionuclide targeted therapy as well as intraoperative applications. This review focuses on the development and further applications of metal-based PSMA radioligands.


Introduction
Prostate cancer is the most common malignancy found in men and the second leading cause of cancer death in the Western world. Even though prostate cancer is one of the few slow-growing cancers, it becomes potentially lethal upon metastasis. Consequently, early detection is particularly important for effective treatment. Functional imaging methods to detect prostate cancer include magnetic resonance imaging (MRI), computed tomography (CT), single-photon-emission tomography (SPECT) and positron emission tomography (PET). Prostate cancer is a complex and biologically heterogeneous disease and cannot be fully assessed with conventional imaging alone. MRI has shown high sensitivity for localizing prostate cancer, although it's diagnostic performance and impact on the treatment regimen and varies in the patient populations studied so far. MRI is also not sensitive in detecting cancer in regions other than the peripheral zone of the prostate [1]. Both conventional imaging modalities, MRI and CT, focus on morphological changes, and their contribution to primary staging of prostate cancer is limited. Therefore, efforts to discover and evaluate new diagnostic and therapeutic biomarkers for prostate cancer continue. Radionuclide molecular imaging with SPECT or PET is poised to fill this unmet need through noninvasive detection of the multiple molecular and cellular processes that are active in prostate cancer patients [2]. Furthermore, the hybrid imaging techniques such as SPECT/CT and PET/CT combine functional and morphological information leading to high diagnostic accuracy. Nowadays, both hybrid imaging techniques are used in the clinical routine, not only as the primary staging tool in prostate cancer, but also in patients with suspected disease recurrence. The basis The NAALDase activity of PSMA has been extensively investigated for the development of PSMA-specific based ligands with the potential to be used for prostate cancer diagnosis and/or therapy. Briefly, the substrate, NAAG binds extracellularly to PSMA followed by hydrolysis of NAAG to glutamate and N-acetyl aspartate. Studies of the enzyme structure have shown the presence of a deep tunnel with a length of ~20 Å which connects the extracellular surface of PSMA with the active site of the enzyme, called the NAAG binding pocket. This binding pocket is also the binding site for the binding of the newly developed anti-PSMA ligands [9]. The proposed pharmacophore for PSMA active site can be divided into three parts: (i) three carboxylate groups; (ii) a carbonyl oxygen as part of the zinc complexation unit and (iii) nearby aromatic residues. More specifically, the crystal structure reveals two zinc atoms separated by approximately 3.2 Å. Each zinc atom in the PSMA active site is coordinated by three endogenous amino acids: a histidine (His-553 or -377), a terminal aspartate (Asp-453) or glutamate (Glu-425), and a bridged aspartate (Asp-387). The formed complex is finally surrounded by water molecules including conservative points [9,10]. Human PSMA has structural homology to another type II transmembrane glycoprotein, the transferrin receptor, which consists of 760 amino acids with a molecular weight of 190 kDa. Part of the extracellular domain of PSMA shows sequence similarity to the transferrin receptor (~54% similarity). The transferrin receptor however exists in dimeric form, whilst PSMA exists in dimeric/monomeric form although it is only enzymatically active as a dimer [11]. Both PSMA and transferrin receptor bind their ligands The NAALDase activity of PSMA has been extensively investigated for the development of PSMA-specific based ligands with the potential to be used for prostate cancer diagnosis and/or therapy. Briefly, the substrate, NAAG binds extracellularly to PSMA followed by hydrolysis of NAAG to glutamate and N-acetyl aspartate. Studies of the enzyme structure have shown the presence of a deep tunnel with a length of~20 Å which connects the extracellular surface of PSMA with the active site of the enzyme, called the NAAG binding pocket. This binding pocket is also the binding site for the binding of the newly developed anti-PSMA ligands [9]. The proposed pharmacophore for PSMA active site can be divided into three parts: (i) three carboxylate groups; (ii) a carbonyl oxygen as part of the zinc complexation unit and (iii) nearby aromatic residues. More specifically, the crystal structure reveals two zinc atoms separated by approximately 3.2 Å. Each zinc atom in the PSMA active site is coordinated by three endogenous amino acids: a histidine (His-553 or -377), a terminal aspartate (Asp-453) or glutamate (Glu-425), and a bridged aspartate (Asp-387). The formed complex is finally surrounded by water molecules including conservative points [9,10]. Human PSMA has structural homology to another type II transmembrane glycoprotein, the transferrin receptor, which consists of 760 amino acids with a molecular weight of 190 kDa. Part of the extracellular domain of PSMA shows sequence similarity to the transferrin receptor (~54% similarity). The transferrin receptor however exists in dimeric form, whilst PSMA exists in dimeric/monomeric form although it is only enzymatically active as a dimer [11]. Both PSMA and transferrin receptor bind their ligands followed by internalization of the PSMA-bound ligand complex through the clathrin-coated pits [11][12][13]. PSMA and transferrin receptor are expressed on various tumor cells and inhibiting their functions may lead to implications for cancer therapeutics.
In pathological situations, PSMA is primarily expressed in the human prostate epithelium, salivary and lacrimal glands and kidneys with enhanced expression by almost all prostate cancers and further up-regulation in poorly differentiated, metastatic and hormone-refractory carcinomas without being shed into the circulation [5,14]. These characteristics render PSMA a promising target for imaging and therapy of prostate cancer. In particular, it is overexpressed by almost all prostate cancers with an increased expression by a factor of about 1000 in poorly differentiated, metastatic, and hormone-refractory cases [6,15,16]. Given the overexpression of PSMA in advanced prostate cancer, several efforts have been made especially during the last decade for the development of anti-PSMA-based imaging agents for SPECT and PET imaging.

PSMA-Based Radioligands
As part of the ongoing efforts of the scientific community to develop new anti-PSMA-specific ligands, various radiotracers have been reported, with some of them having shown great promise not only preclinically, but also in the clinical assessment. The dual nature of PSMA to act not only as a receptor protein, but also as an enzyme, has paved the way for the establishment of several approaches for its targeting via radiolabeled molecules. Firstly, based on the macromolecular protein structure of PSMA, specific monoclonal antibodies and smaller molecules called aptamers have been developed. These molecules bind tightly, selectively and specifically to PSMA [17,18]. Secondly, the enzymatic activity of PSMA served as the trigger for the synthesis and the further evaluation of a variety of anti-PSMA inhibitors of low molecular weight, with the potential to be used as nuclear imaging probes [19].

Anti-PSMA Monoclonal Antibodies as Radiotracers for Prostate Cancer Imaging and Therapy
PSMA was originally targeted by the monoclonal antibody 7E11-C5 (capromab) functionalized with the chelator diethylenetriaminepentaacetic acid (DTPA) and labeled with 111 In to obtain 111 In-7E11-C5 (ProstaScint ® ; Cytogen Corporation, Princeton, NJ, USA) [20][21][22][23]. ProstaScint ® binds to the intracellular site of PSMA (the amino terminus) which is accessible on necrotic tumors only [24,25]. Therefore, this tracer fails to gain wide acceptance in the field of nuclear medicine for the detection of prostate cancer. Hence, the need for the development of monoclonal antibodies which bind to the extracellular site of PSMA was evident. Consequently, several monoclonal antibodies, such as the humanized monoclonal antibody J591 [26,27] and three others murine monoclonal antibodies, named 3/A12, 3/E7 and 3/F11, were generated. These antibodies exhibit high and specific binding to cell-adherent PSMA [28,29].
Contrary to 7E11-C5, J591 can recognize PSMA that is present on the surface of nearly all prostate cancer tumors and circulating tumor cells. J591 is the first monoclonal antibody targeting the extracellular domain of PSMA as well as the first humanized monoclonal antibody to PSMA to be tested in humans. J591 has been evaluated preclinically for SPECT, PET as well as radioimmunotherapy. Its functionalization has been performed using a variety of chelators, such as 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and deferoxamine (DFO) and was further labeled with 99m Tc, 111 In, 89 Zn, 64 Cu, 177 Lu, 90 Y [30][31][32][33][34][35][36][37][38][39][40][41]. PSMA SPECT imaging using 99m Tc-J591 depicts the presence of prostate cancer, and it can be also considered as a sensitive tool for the delineation of micro-invasion of the capsule, seminal vesicles or bladder neck. Although 99m Tc-J591 is of value in locating recurrence of the disease after radical prostatectomy, the results of the study indicated that extraprostatic cancer was not reliably detected. This difficulty may be due to the high activity of 99m Tc degradation products in the bladder [30]. 111 In-J591 was also evaluated for SPECT imaging preclinicaly and in clinical trials. In the preclinical settings, the pharmacokinetics, biodistribution, and tumor uptake of 111 In-J591 were compared with those of 111 In-7E11 in nude mice with PSMA-positive LNCaP tumors [41]. The most important findings were that the blood clearance of 111 In-J591 was relatively faster (8.98 ± 2.10, 4.78 ± 0.85, 2.52 ± 0.56 % IA/g at 2, 4 and 6 days p.i.) than that of 111 In-7E11 (7.22 ± 0.46, 5.69 ± 1.00, 4.16 ± 0.41 % IA/g at 2, 4 and 6 days p.i.), leading to higher tumor-to-blood ratios ( 111 In-J591: 1.62 ± 0.72, 3.35 ± 0.39, 6.07 ± 1.79, 111 In-7E11: 1.34 ± 0.24, 2.83 ± 0.51, 4.57 ± 0.31 at 2, 4 and 6 days p.i. respectively). Furthermore, in clinical trials, 111 In-J591 was found superior to ProstaScint ® , revealing most soft-tissue and bone metastases [31,39,40]. J591 has also been radiolabeled with 89 Zr, after its functionalization with 3.9 ± 0.3 accessible DFO chelators [34]. 89 Zr-DFO-J591 was produced in high radiochemical yield (>77%) with radiochemical purity >99% and a specific activity of 181.7 ± 1.1 MBq/mg. The radiolabeled immunoconjugate remained active for up to 7 days at 37 • C. In vivo biodistribution studies revealed high uptake of 89 Zr-DFO-J591 in LNCaP tumors after 24, 48, 96 and 144 h (34.4 ± 3.2 % IA/g; 38.0 ± 6.2 % IA/g; 40.4 ± 4.8 % IA/g; and 45.8 ± 3.2 % IA/g, respectively). The immunoPET imaging indicated that 89 Zr-DFO-J591 shows high potential as a radiotracer for specific, non-invasive delineation of PSMA positive prostate cancers in vivo. In localized human prostate cancer cases, 89 Zr-J591 bound to tumor foci in situ and PET identified primarily Gleason score 7 or greater and larger tumors. High grade tumors were generally better visualized with 89 Zr-J591. These clinical data provide a basis on which to investigate the various roles of 89 Zr-J591 PET in prostate cancer diagnosis, management and treatment [42]. The coupling of the chelator DOTA to J591 and the subsequent labeling with 64 Cu led to 64 Cu-J519, which was also evaluated for PET imaging preclinically [35]. 89 Zr-labeled rather than 64 Cu-labeled J591 was proved to successfully image PSMA-expression in preclinical prostate cancer models. J591 has also been adapted for radioimmunotherapy, and Ab-drug conjugates and therapeutic doses are well tolerated in patients. Multiple (two or three) administrations of 177 Lu-J591 (30-60 mCi/m 2 ) or 90 Y-J591 (17.5 mCi/m 2 ) over a 4-to 6-month period were tolerated by the patients with manageable thrombocytopenia. Furthermore, the clinical trials showed that even though a single large dose may deliver optimal radiation dose to kill a larger fraction of tumor cells, fractionated therapy offers the advantage of lower myelotoxicity and prolonged tumor response [33,36].
For the development of antibodies with a high cell binding activity which may have an important impact on antibody-based imaging and therapy, a series of monoclonal antibodies against PSMA, named 3/F11, 3/A12 and 3/E7, generated [28,29]. All three antibodies were able to bind efficiently to cell surface expressed PSMA. In addition, the in vivo behavior and tumor uptake of the 64 Cu-labeled monoclonal antibodies 3/A12, 3/F11 and 3/E7 were also investigated. Radiolabeling of 3/A12, 3/F11 and 3/E7 with 64 Cu through a conjugated DOTA moiety was accomplished without loss of immunoreactivity [43]. Biodistribution as well as PET imaging studies of the three 64 Cu-labeled antibodies showed specific tumor uptake in PSMA-positive C4-2 tumors, whereas the uptake of tracers in PSMA-negative DU 145 tumors was found to be at the background level. In the same study, the pharmacokinetics of the 64 Cu-DOTA-3/A12 Fab and 64 Cu-DOTA-3/A12 F(ab )2 fragments compared to that of 64 Cu-DOTA-3/A12 were also evaluated at 3, 24 and 48 h after the injection of the radiotracers. Only a faint uptake of 64 Cu-DOTA-3/A12 Fab and 64 Cu-DOTA-3/A12 F(ab )2 fragments in the PSMA-positive tumors was observed at all time points tested, indicating that the rate of clearance from blood was too fast to allow for sufficient tracer binding at the receptors. Furthermore, high kidney retention was also reported for both 64 Cu-labeled fragments [43]. Behe et al. was published the labeling of 3/F11 through a conjugated DOTA moiety with 177 Lu along with the in vitro and in vivo evaluation of 177 Lu-DOTA-3/F11 [44]. The biodistribution data showed that the tumor uptake of 177 Lu-DOTA-3/F11 gradually increased with time up to 72 h after the injection of the radiotracer. The high and persistent tumor uptake may be caused by a rapid internalization of the 177 Lu DOTA-3/F11-PSMA complex into the target cells after binding [45], followed by metabolism and trapping of the radioactivity at the tumor site [46]. The same study also revealed that the treatment of C4-2 bearing prostate cancer xenografts with a single dose of 1 MBq 177 Lu-DOTA-3/F11 led to a more than two-fold enhanced mean survival and a delay in tumor growth. However, this therapeutic window is small since mice treated with 2 MBq 177 Lu-DOTA-3/F11 apparently died of myelotoxicity, which is the predominant dose-limiting factor in radioimmunotherapeutic approaches with 177 Lu [36,47,48].
Despite the improved targeting of J591 and the subsequent generation of anti-PSMA monoclonal antibodies, the major impediment to the use of antibodies for diagnostic purposes continues to be the slow clearance from the non-target tissues. This, in most of the cases, is mitigated by introducing a time interval of several days between radiotracer administration and imaging. An alternative approach is to optimize the pharmacokinetics of the monoclonal antibodies by the generation of antibody fragments, such as single domain antibodies (V HH ), called nanobodies. The comparatively low molecular mass of nanobodies leads both to a better permeability in tissues, and a shorter plasma half-life [49,50]. Recently, the anti-PSMA nanobodies, JVZ-007-c-myc-his and JVZ-007-cys, were developed and the nanobody-DTPA conjugates were labeled with 111 In to obtain 111 In-JVZ-007-c-myc-his and 111 In-JVZ-007-cys [51]. 111 In-JVZ-007-c-myc-his was evaluated in mice bearing PC-310 (PSMA+) and PC-3 (PSMA-) tumors, showing good and specific tumor targeting already at 4 h after injection, with low background intensity. Only kidney tracer uptake was high (110.89 ± 7.35 % IA/g at 4 h p.i.) which was reduced by about 75% after the administration of gelofusin and lysine [52,53]. The introduction of a cysteine at the C terminus for site-specific coupling to maleimide-DTPA and the labeling with 111 In led to 111 In-JVZ-007-cys. The in vivo evaluation of 111 In-JVZ-007-cys in the same tumor model as described above revealed a further drop in renal uptake without loss of tumor targeting. These optimal characteristics of 111 In-JVZ-007-cys may pave the way for its applicability to radionuclide therapy. The engineering of the new nanobody construct, based on JVZ-007, in which the c-myc-his tag was substituted with Cys tag, may have a considerable impact on the clinical implementation of a wide range of nanobodies in the field of nuclear medicine.
In addition to nanobodies, a variety of small, genetically engineered immunological constructs have been developed for a broader range of in vivo applications. Examples of these constructs include Fab (fragment antigen-binding), F(ab )2, single-chain (sc) Fv, bis-scFV, diabodies and minibodies. As described above, their small size potentially gives them access to tissues that are poorly accessible by intact antibodies; rapid clearance from blood and non-targeted tissues, and at the same time they exhibit high affinity properties to antigens. As far the PSMA targeting concerns, an anti-PSMA minibody named IAB2M was genetically engineered from the parent antibody J591 and labeled with 89 Zr [54]. Preclinical studies demonstrated faster clearance and rapid in vivo biokinetics, with efficient target penetration, allowing for high-contrast images within a few hours after the injection [46,55,56]. 89 Zr-IAB2M [54] showed properties similar to that of 89 Zr-J591 [34], coupled with a significantly faster blood clearance and lower uptake at 24 h after the injection in PSMA-positive tissue compared to 89 Zr-J591, as shown by the PET images [57]. The latter finding can be attributed to the extended blood circulation of the intact antibody rather than loss of the affinity of IAB2M. Nevertheless, the longer circulation may benefit immunotherapies, but a key element for the successful diagnosis of cancer is a fast blood clearance in combination with high and retained tumor uptake, leading to high tumor-to-background ratios over time. The first-in-human imaging with 89 Zr-IAB2M in metastatic prostate patients showed that PET imaging is feasible. Furthermore, favorable biodistribution profile in patients with advanced prostate cancer was also revealed. In addition to these characteristics, targeting of both bone and soft tissue lesions was observed after the administration of 89 Zr-IAB2M [54].
Although immunoPET holds a very important role in the field of diagnostic imaging, the long blood circulation in combination with the nonspecific accumulation in normal tissues may lead to radiotoxicity of several vital organs such as the liver or the spleen. These obstacles could be overcome by generating smaller molecules which show fast blood clearance and retained radiotracer target specificity. The development of the genetically engineered immunological constructs properly modified in order to be used as nuclear probes can be considered as alternative imaging tools for cancer imaging.

PSMA Inhibitors of Low Molecular Weight
Since 2-PMPA (compound 1, Figure 2) was firstly reported as a potent PSMA inhibitor in 1996 [65], thorough efforts have been devoted to generating further molecules with inhibitory action towards PSMA. The main strategy for the discovery of those inhibitors was to find zinc-binding groups which are linked to a glutamate moiety. Three functionalities with affinity for zinc, including phosphonates (phosphates, phosphoramidates), thiols and ureas (compounds 2-6, Figure 2) have been identified [66][67][68][69][70][71]. In the field of radiopharmaceutical development, a great number of phosphoramidates and urea inhibitors have been synthesized and modified accordingly in order to be labeled with a variety of radiometals. In the following chapters we will have a closer look on the derived radiotracers from these two categories. low immunogenicity and toxicity, in addition to an increased half-life in the circulation, rendering them attractive molecules either for diagnostic or therapeutic purposes. In fact, a variety of aptamer-based conjugates accumulate in tumors, suggesting that with specific modifications they may be used for imaging and potentially therapeutic applications [18,[60][61][62] 64 Cu [64]. The NOTA and PCTA constructs appear to be the most promising for further studies of the chelator-aptamer derivatives; however, in vivo images or biodistribution data of these 64 Cu-labeled agents have so far not been reported.

PSMA Inhibitors of Low Molecular Weight
Since 2-PMPA (compound 1, Figure 2) was firstly reported as a potent PSMA inhibitor in 1996 [65], thorough efforts have been devoted to generating further molecules with inhibitory action towards PSMA. The main strategy for the discovery of those inhibitors was to find zinc-binding groups which are linked to a glutamate moiety. Three functionalities with affinity for zinc, including phosphonates (phosphates, phosphoramidates), thiols and ureas (compounds 2-6, Figure 2) have been identified [66][67][68][69][70][71]. In the field of radiopharmaceutical development, a great number of phosphoramidates and urea inhibitors have been synthesized and modified accordingly in order to be labeled with a variety of radiometals. In the following chapters we will have a closer look on the derived radiotracers from these two categories.

Phosphoramidate-Metal Based PSMA Inhibitors
Misra et al. reported the development of a series of irreversible phosphoramidate inhibitors ( Figure 3) that also target PSMA with high affinity and specificity [72]. The starting molecule for the synthesis of the new PSMA inhibitors was GPI (compound 7, Figure 3). The phosphoramidate-based PSMA inhibitors were shown to rapidly internalize into subcellular organelles of PSMA-positive (PSMA+) prostate tumor cells, presumably through the enzyme-inhibitor complex [73]. 99m Tc-labeling of

Phosphoramidate-Metal Based PSMA Inhibitors
Misra et al. reported the development of a series of irreversible phosphoramidate inhibitors ( Figure 3) that also target PSMA with high affinity and specificity [72]. The starting molecule for the synthesis of the new PSMA inhibitors was GPI (compound 7, Figure 3). The phosphoramidate-based PSMA inhibitors were shown to rapidly internalize into subcellular organelles of PSMA-positive (PSMA+) prostate tumor cells, presumably through the enzyme-inhibitor complex [73]. 99m Tc-labeling of the derived inhibitors was obtained using a simple, cartridge-based, solid phase prelabeled 2-mercaptoacetyl-Ser-Ser-Ser-(MAS 3 ) chelator. However, no in vivo imaging studies have been reported.  The modification of another irreversible phosphoramidate inhibitor, named CTT-54 (compound 11, Figure 4), for the delivery of 99m Tc(CO)3-DTPA-CTT-54, was reported by Nedrow-Byers et al. in 2012. 99m Tc(CO)3-DTPA-CTT-54 was evaluated in vitro and in vivo for its potential as a SPECT imaging nuclear probe for PSMA targeting [74]. Although DTPA may not be the ideal chelator for [ 99m Tc(CO)3] + , the results from the study justify a proof-of-concept evidence for the development of a next-generation of phosphoramidate-based PSMA inhibitors as SPECT imaging agents. A PET tracer, 64 Cu-ABN-1 (compound 12, Figure 5) was generated from the irreversible phosphoramidate inhibitor of PSMA CTT-1297 after its conjugation to the phosphonate-pendantarmed cross-bridged chelator, 1,4,8,11-tetraazacyclotetradecane-1-(methanephosphonic acid)-8-(methanecarboxylic acid) (CB-TE1A1P) [75], and the incorporation of 64 Cu [74]. 64 Cu-ABN-1 was evaluated for its selective uptake both in vitro and in vivo with PSMA-positive cells. A particular focus was given to assess the ability of this 64 Cu-labeled radiotracer to detect and distinguish elevated levels of PSMA in a panel of prostate tumor-bearing mouse models. The study showed that 64 Cu-ABN-1 demonstrated selective uptake in PSMA-positive cells and tumors, which was also correlated to the level of PSMA expression. 64 Cu-ABN-1 may serve as a non-invasive method to follow the progression of prostate cancer in the future. The modification of another irreversible phosphoramidate inhibitor, named CTT-54 (compound 11, Figure 4), for the delivery of 99m Tc(CO) 3 -DTPA-CTT-54, was reported by Nedrow-Byers et al. in 2012. 99m Tc(CO) 3 -DTPA-CTT-54 was evaluated in vitro and in vivo for its potential as a SPECT imaging nuclear probe for PSMA targeting [74]. Although DTPA may not be the ideal chelator for [ 99m Tc(CO) 3 ] + , the results from the study justify a proof-of-concept evidence for the development of a next-generation of phosphoramidate-based PSMA inhibitors as SPECT imaging agents.  The modification of another irreversible phosphoramidate inhibitor, named CTT-54 (compound 11, Figure 4), for the delivery of 99m Tc(CO)3-DTPA-CTT-54, was reported by Nedrow-Byers et al. in 2012. 99m Tc(CO)3-DTPA-CTT-54 was evaluated in vitro and in vivo for its potential as a SPECT imaging nuclear probe for PSMA targeting [74]. Although DTPA may not be the ideal chelator for [ 99m Tc(CO)3] + , the results from the study justify a proof-of-concept evidence for the development of a next-generation of phosphoramidate-based PSMA inhibitors as SPECT imaging agents. A PET tracer, 64 Cu-ABN-1 (compound 12, Figure 5) was generated from the irreversible phosphoramidate inhibitor of PSMA CTT-1297 after its conjugation to the phosphonate-pendantarmed cross-bridged chelator, 1,4,8,11-tetraazacyclotetradecane-1-(methanephosphonic acid)-8-(methanecarboxylic acid) (CB-TE1A1P) [75], and the incorporation of 64 Cu [74]. 64 Cu-ABN-1 was evaluated for its selective uptake both in vitro and in vivo with PSMA-positive cells. A particular focus was given to assess the ability of this 64 Cu-labeled radiotracer to detect and distinguish elevated levels of PSMA in a panel of prostate tumor-bearing mouse models. The study showed that 64 Cu-ABN-1 demonstrated selective uptake in PSMA-positive cells and tumors, which was also correlated to the level of PSMA expression. 64 Cu-ABN-1 may serve as a non-invasive method to follow the progression of prostate cancer in the future. A PET tracer, 64 Cu-ABN-1 (compound 12, Figure 5) was generated from the irreversible phosphoramidate inhibitor of PSMA CTT-1297 after its conjugation to the phosphonate-pendantarmed cross-bridged chelator, 1,4,8,11-tetraazacyclotetradecane-1-(methanephosphonic acid)-8-(methanecarboxylic acid) (CB-TE1A1P) [75], and the incorporation of 64 Cu [74]. 64 Cu-ABN-1 was evaluated for its selective uptake both in vitro and in vivo with PSMA-positive cells. A particular focus was given to assess the ability of this 64 Cu-labeled radiotracer to detect and distinguish elevated levels of PSMA in a panel of prostate tumor-bearing mouse models. The study showed that 64 Cu-ABN-1 demonstrated selective uptake in PSMA-positive cells and tumors, which was also correlated to the level of PSMA expression. 64 Cu-ABN-1 may serve as a non-invasive method to follow the progression of prostate cancer in the future. The irreversible binding profile of the phosphoramidate-based PSMA inhibitors is desirable because it induces a higher rate of internalization compared to the slowly released, but reversiblebinding of phosphate-based inhibitors and the reversible profile urea-and phosphonic-based inhibitors. The phosphoramidate-based PSMA inhibitors are effective in delivering a therapeutically relevant dose from a relatively low dose due to the higher internalization rate which reduces radiation exposure of patients. The further optimization of this class of PSMA targeting agents has the potential to enable efficient in vivo targeting of PSMA positive tumors either for radiodiagnostic or radiotherapeutic applications.

Urea-Based PSMA Inhibitors
In 2001 the Kozikowski group was the first to first synthesize and evaluate urea-based PSMA inhibitors [70]. In 2002, the Pomper group at John Hopkins School of Medicine (Baltimore, MD, USA) published the synthesis and preclinical evaluation of the first radiolabeled urea-based PSMA inhibitor, (N-(N-[(S)-1,3-dicarboxypropyl]carbomoyl)-S-11 C-methyl-L-cysteine) ( 11 C-DCMC), also referred to as 11 C-MeCys-C(O)-Glu ( 11 C-MCG) [75]. To date, a variety of urea-based PSMA inhibitors have been developed and labeled with different SPECT ( 99m Tc, 111 In), PET ( 68 Ga, 64 Cu), and therapeutic ( 177 Lu) radiometals using several chelators and showed great promise not only preclinicaly but also in the clinical assessment. Apart from the radiometals to be used as the cytotoxic units, a considerable amount of urea-based PSMA inhibitors labeled with 11 C, 125 I, 124 I, 131 I and 18 F, have also been reported [19,. These radiotracers were also able to successfully image PSMA-expressing xenografted mice, but since this is beyond the focus of this review no further information will be given here with regard to this topic.
The development of urea-based PSMA precursors for subsequent labeling with radiometals requires the attachment of a relatively bulky chelator to the peptidomimetic structure of the PSMA inhibitors. As it has been noticed earlier in this review, a ~20 Å deep tunnel connects the extracellular surface of PSMA with the active site of the enzyme, rendering difficult the entry of the radiometal-bearing chelator into the enzymatic binding site. Therefore, the insertion of a spacer between the urea-based motif and the chelator is essential. Although the binding affinity can be considered as the most crucial parameter which affects the tumor uptake, the overall pharmacokinetic performance of a radiotracer is determined by many other factors which certainly need to be taken into consideration during the development of potential radiopharmaceuticals. In particular, parameters such as the nature of the spacer, lipophilicity, charge, plasma protein binding and molecular weight also influence the pharmacokinetic performance of a radiotracer [97]. Indeed, the presence of the spacer not only greatly influences the affinity of the derived radiotracers towards PSMA but also the pharmacokinetic properties of the chelator-conjugated PSMA inhibitors [97].

99m Tc-Labeled Urea-Based PSMA Inhibitors
99m Tc remains the radionuclide of choice for SPECT applications and a variety of PSMA inhibitors have been modified to facilitate efficient labeling with 99m Tc. Banerjee et al. have published a series of PSMA inhibitors suitable for labeling with 99m Tc [98,99]. The influence of the spacer length as well as the nature of different chelators was thoroughly investigated with respect to the overall performance of the derived 99m Tc-labeled Lys-urea-Glu-based PSMA inhibitors (compounds 13-25, Figure 6, Table 1). In particular, the PSMA inhibitors are attached to hydrophilic ligands for radiolabeling with the 99m Tc(I)-tricarbonyl-labeled ([ 99m Tc(CO)3] + ) core, traditional NxSy-based chelator for The irreversible binding profile of the phosphoramidate-based PSMA inhibitors is desirable because it induces a higher rate of internalization compared to the slowly released, but reversiblebinding of phosphate-based inhibitors and the reversible profile urea-and phosphonic-based inhibitors. The phosphoramidate-based PSMA inhibitors are effective in delivering a therapeutically relevant dose from a relatively low dose due to the higher internalization rate which reduces radiation exposure of patients. The further optimization of this class of PSMA targeting agents has the potential to enable efficient in vivo targeting of PSMA positive tumors either for radiodiagnostic or radiotherapeutic applications.

Urea-Based PSMA Inhibitors
In 2001 the Kozikowski group was the first to first synthesize and evaluate urea-based PSMA inhibitors [70]. In 2002, the Pomper group at John Hopkins School of Medicine (Baltimore, MD, USA) published the synthesis and preclinical evaluation of the first radiolabeled urea-based PSMA inhibitor, [75]. To date, a variety of urea-based PSMA inhibitors have been developed and labeled with different SPECT ( 99m Tc, 111 In), PET ( 68 Ga, 64 Cu), and therapeutic ( 177 Lu) radiometals using several chelators and showed great promise not only preclinicaly but also in the clinical assessment. Apart from the radiometals to be used as the cytotoxic units, a considerable amount of urea-based PSMA inhibitors labeled with 11 C, 125 I, 124 I, 131 I and 18 F, have also been reported [19,. These radiotracers were also able to successfully image PSMA-expressing xenografted mice, but since this is beyond the focus of this review no further information will be given here with regard to this topic.
The development of urea-based PSMA precursors for subsequent labeling with radiometals requires the attachment of a relatively bulky chelator to the peptidomimetic structure of the PSMA inhibitors. As it has been noticed earlier in this review, a~20 Å deep tunnel connects the extracellular surface of PSMA with the active site of the enzyme, rendering difficult the entry of the radiometal-bearing chelator into the enzymatic binding site. Therefore, the insertion of a spacer between the urea-based motif and the chelator is essential. Although the binding affinity can be considered as the most crucial parameter which affects the tumor uptake, the overall pharmacokinetic performance of a radiotracer is determined by many other factors which certainly need to be taken into consideration during the development of potential radiopharmaceuticals. In particular, parameters such as the nature of the spacer, lipophilicity, charge, plasma protein binding and molecular weight also influence the pharmacokinetic performance of a radiotracer [97]. Indeed, the presence of the spacer not only greatly influences the affinity of the derived radiotracers towards PSMA but also the pharmacokinetic properties of the chelator-conjugated PSMA inhibitors [97].  [98,99]. The influence of the spacer length as well as the nature of different chelators was thoroughly investigated with respect to the overall performance of the derived 99m Tc-labeled Lys-urea-Glu-based PSMA inhibitors (compounds 13-25, Figure 6, Table 1).
In particular, the PSMA inhibitors are attached to hydrophilic ligands for radiolabeling with the 99m Tc(I)-tricarbonyl-labeled ([ 99m Tc(CO) 3 ] + ) core, traditional N x S y -based chelator for the 99m Tc-oxo ([ 99m TcO] 3+ ) core, and a 99m Tc-organohydrazine ( 99m Tc-hynic) core. It was proved that parameters such the charge, polarity and hydrophilicity are crucial and contribute not only to the efficient targeting of PSMA by the 99m Tc-labeled agent but also to its pharmacokinetics.
Molecules 2017, 22, 523 9 of 33 the 99m Tc-oxo ([ 99m TcO] 3+ ) core, and a 99m Tc-organohydrazine ( 99m Tc-hynic) core. It was proved that parameters such the charge, polarity and hydrophilicity are crucial and contribute not only to the efficient targeting of PSMA by the 99m Tc-labeled agent but also to its pharmacokinetics.     (25) HYNIC (2 mg/mL), 0.5 M NaHCO 3 , tricine (7 mg/mL), SnCl 2 (1 mg/mL), 99m TcO4 − , r.t., 30 min, yield: >80%, purity: >99% −3.05 (25) no clinical translation [99] The above tested low-molecular-weight, urea-based, 99m Tc-labeled, PSMA-targeted radiotracers, differ in the chelator and in the lipophilicity. Except for HYNIC-labeled 99m Tc-25 irrespective of charge and lipophilicity the radioligands 99m Tc- (13)(14) enabled visualization of PSMA positive tumors (>20 % IA/g at 2 h p.i.) and kidneys. More specifically, the study showed that the degree of lipophilicity had a considerable effect on binding affinity as well as on in vivo performance. The ligands which showed higher lipophilic character revealed higher inhibitory activity, lower tumor and higher background uptake. Indeed, compounds 13 to 15, which possess different degree of lipophilicity due to different number of Phe moieties (from zero (13) to three (15)) on the spacer, displayed considerable differences in tumor and background uptake as measured by SPECT/CT images from male SCID mice bearing PSMA+ PC3 PIP and PSMA− PC3 flu tumors at 2 h p.i. High gallbladder, liver, and gastrointestinal uptake were observed for 99m Tc-15 as compared to that of 99m Tc-13. Between the radioligands bearing the tricarbonyl core, 99m Tc-21 and 99m Tc-22, the neutral complex 99m Tc-22, exhibited favorable pharmacokinetics and promising SPECT/CT images, presumably because of the comparatively low background within the gastrointestinal tract relative to the positively charged 99m Tc-21 ( 99m Tc-21: small intestine: 1.29 ± 0.76 % IA/g, large intestine: 16.02 ± 12.39 % IA/g at 1 h p.i., 99m Tc-22: small intestine: 1.35 ± 0.19 % IA/g, large intestine: 0.29 ± 0.06 % IA/g at 1 h p.i.). In addition, 99m Tc-22, the most hydrophilic radioligand in the tricarbonyl series, had high, specific and retained tumor uptake in the PSMA+ PC3 PIP tumor (28.31 ± 4.38, 28.05 ± 2.04, 26.29 ± 7.45, 23.22 ± 6.02 % IA/g, at 0.5, 1, 2 and 5 h p.i. respectively). Another point which also needs to be pointed out is that the 99m Tc-oxo radioliagnds showed high and prolonged spleen uptake, which was not observed with the tricarbonyl-based radioligands. 99m Tc-22 proved superior with respect to tumor-to background ratios over time. Despite high tumor uptake, other compounds had either high gastrointestinal or spleen uptake, which cannot be easily attributed on their charge and lipophilicity. No clinical studies with of these 99m Tc compounds have been reported.
Six targeted radioimaging agents labeled with 99m Tc derived from the Lys-urea-Glu peptidomimetic structure functionalized with the chelator Dap-Asp-Cys through spacers of different lengths (compounds 26-31, Figure 7, Table 2) were investigated by Kularante et al. [100]. This study demonstrates the importance of the spacer between the urea-based PSMA inhibitors and the chelator which have been used for their functionalization. Docking studies revealed that coupling the urea analogue to an 8-aminooctanoic acid moiety (to avoid steric overlap within the narrow regions at the base of the tunnel) followed by two phenylalanine residues (for maximal interaction with hydrophobic pockets near the mouth of the tunnel) generated an agent (compound 28) with high affinity and specificity. Also the most lipophilic compound was found to be the most affine towards PSMA. The in vivo data were in accordance with the findings from in vitro studies, with remarkable tumor uptake upon injection in nu/nu mice xenografted with LNCaP tumor cells (average 9.8 ± 2.4 % IA/g at 1 h p.i.) was observed for radiotracers 27, 28, and 29 with little accumulation in other tissues except the kidneys. These ligands have so far not been transferred to clinical evaluation. Robu et al. recently published the synthesis of two 99m Tc-labeled anti-PSMA agents [101]. In this study the Lys-urea-Glu motif was coupled to the spacer D-Tyr-D-2-Nal-D-Lys-suberoyl (y-nal-k-Sub) to enhance interaction of the peptidic spacer with a remote arene binding site and the functionalization of the precursor took place through the conjugation of the chelators MAS3 (2-mercaptoacetyl-Ser-Ser-Ser-) (compound 32, Figure 8) and mas3 (2-mercaptoacetyl-D-Ser-D-Ser-D-Ser-) (compound 33, Figure 8). The derived conjugates labeled with 99m Tc (Table 3). The main focus of the study was to investigate whether or not the all-L-amino acid chelator MAS3 is susceptible towards proteolytic degradation. As anticipated, no detectable influence of chelator stereochemistry on the outcome of the 99m Tc-labeling reaction was observed, since the formation of the 99m TcO-MAS3/mas3-complex should be independent from the spatial orientation of the serine side chains. On the other hand, the MAS3-chelating 99m Tc-labeled tracer showed substantially decreased in vivo stability compared to the mas3-derivative 99m Tc-PSMA-I&S (PSMA-I&S for Imaging and Surgery), for which only intact tracer was detected in blood, urine and kidney at 1 h p.i. Furthermore, 99m Tc-PSMA-I&S uptake in kidney and LNCaP-tumor was found to be high (186 ± 23, 8.28 ± 3.27% IA/g at 1 h p.i., respectively) and specific, as shown by the blocking studies. The straightforward and reliable kit-production and the initial patient data indicate the potentiality of 99m Tc-PSMA-I&S as a SPECT imaging agent. In another study, Kimura et al. [102] synthesized and evaluated a novel anionic 99m Tctricarbonyl complex ( 99m Tc-TMCE) with high hydrophilicity due to strong polarity and electric Robu et al. recently published the synthesis of two 99m Tc-labeled anti-PSMA agents [101]. In this study the Lys-urea-Glu motif was coupled to the spacer D-Tyr-D-2-Nal-D-Lys-suberoyl (y-nal-k-Sub) to enhance interaction of the peptidic spacer with a remote arene binding site and the functionalization of the precursor took place through the conjugation of the chelators MAS 3 (2-mercaptoacetyl-Ser-Ser-Ser-) (compound 32, Figure 8) and mas 3 (2-mercaptoacetyl-D-Ser-D-Ser-D-Ser-) (compound 33, Figure 8). The derived conjugates labeled with 99m Tc (Table 3). The main focus of the study was to investigate whether or not the all-L-amino acid chelator MAS 3 is susceptible towards proteolytic degradation. As anticipated, no detectable influence of chelator stereochemistry on the outcome of the 99m Tc-labeling reaction was observed, since the formation of the 99m TcO-MAS 3 /mas 3 -complex should be independent from the spatial orientation of the serine side chains. On the other hand, the MAS 3 -chelating 99m Tc-labeled tracer showed substantially decreased in vivo stability compared to the mas 3 -derivative 99m Tc-PSMA-I&S (PSMA-I&S for Imaging and Surgery), for which only intact tracer was detected in blood, urine and kidney at 1 h p.i. Furthermore, 99m Tc-PSMA-I&S uptake in kidney and LNCaP-tumor was found to be high (186 ± 23, 8.28 ± 3.27 % IA/g at 1 h p.i., respectively) and specific, as shown by the blocking studies. The straightforward and reliable kit-production and the initial patient data indicate the potentiality of 99m Tc-PSMA-I&S as a SPECT imaging agent. Robu et al. recently published the synthesis of two 99m Tc-labeled anti-PSMA agents [101]. In this study the Lys-urea-Glu motif was coupled to the spacer D-Tyr-D-2-Nal-D-Lys-suberoyl (y-nal-k-Sub) to enhance interaction of the peptidic spacer with a remote arene binding site and the functionalization of the precursor took place through the conjugation of the chelators MAS3 (2-mercaptoacetyl-Ser-Ser-Ser-) (compound 32, Figure 8) and mas3 (2-mercaptoacetyl-D-Ser-D-Ser-D-Ser-) (compound 33, Figure 8). The derived conjugates labeled with 99m Tc (Table 3). The main focus of the study was to investigate whether or not the all-L-amino acid chelator MAS3 is susceptible towards proteolytic degradation. As anticipated, no detectable influence of chelator stereochemistry on the outcome of the 99m Tc-labeling reaction was observed, since the formation of the 99m TcO-MAS3/mas3-complex should be independent from the spatial orientation of the serine side chains. On the other hand, the MAS3-chelating 99m Tc-labeled tracer showed substantially decreased in vivo stability compared to the mas3-derivative 99m Tc-PSMA-I&S (PSMA-I&S for Imaging and Surgery), for which only intact tracer was detected in blood, urine and kidney at 1 h p.i. Furthermore, 99m Tc-PSMA-I&S uptake in kidney and LNCaP-tumor was found to be high (186 ± 23, 8.28 ± 3.27% IA/g at 1 h p.i., respectively) and specific, as shown by the blocking studies. The straightforward and reliable kit-production and the initial patient data indicate the potentiality of 99m Tc-PSMA-I&S as a SPECT imaging agent. In another study, Kimura et al. [102] synthesized and evaluated a novel anionic 99m Tctricarbonyl complex ( 99m Tc-TMCE) with high hydrophilicity due to strong polarity and electric In another study, Kimura et al. [102] synthesized and evaluated a novel anionic 99m Tc-tricarbonyl complex ( 99m Tc-TMCE) with high hydrophilicity due to strong polarity and electric charges with the intent to further decrease non-target tissue uptake and enhance renal clearance (compound 34, Figure 9, Table 4). 99m Tc-TMCE was obtained in very low radiochemical yield (12-17%), introducing the implementation of a HPLC for purification of the radioligand. Compared to the neutral and positively charged tricarbonyl core-bearing radioligands published by Banerjee et al. the in vivo performance of the anionic 99m Tc-tricarbonyl complex 99m Tc-TMCE is characterized by a relatively long blood circulation and fast washout from the tumor (blood: 11.4 ± 2.3, 3.2 ± 0.5, 0.6 ± 0.6 ± 0.1% IA/g and LNCaT-tumor: 4.0 ± 1.2, 12.8 ± 2.2 and 5.0 ± 2.7 % IA/g at 5, 30 and 120 min p.i. respectively) The outcome of the study demonstrated the effect of affinity, hydrophilicity, and electric charge in the pharmacokinetics of PSMA imaging probes. In particular, the hydrophilicity of both cationic and anionic charges led to rapid hepatobiliary clearance, although anionic charges might enhance renal clearance to a greater extent (kidney: 124.9 ± 26.2, 136 ± 6.4 and 56.8 ± 20.6 % IA/g at 5, 30 and 120 min p.i.). The implementation of this 99m Tc-labeled anti-PSMA radiotracer into the clinical pathway has not taken place yet. charges with the intent to further decrease non-target tissue uptake and enhance renal clearance (compound 34, Figure 9, Table 4). 99m Tc-TMCE was obtained in very low radiochemical yield (12-17%), introducing the implementation of a HPLC for purification of the radioligand. Compared to the neutral and positively charged tricarbonyl core-bearing radioligands published by Banerjee et al. the in vivo performance of the anionic 99m Tc-tricarbonyl complex 99m Tc-TMCE is characterized by a relatively long blood circulation and fast washout from the tumor (blood: 11.4 ± 2.3, 3.2 ± 0.5, 0.6 ± 0.6 ± 0.1% IA/g and LNCaT-tumor: 4.0 ± 1.2, 12.8 ± 2.2 and 5.0 ± 2.7% IA/g at 5, 30 and 120 min p.i. respectively) The outcome of the study demonstrated the effect of affinity, hydrophilicity, and electric charge in the pharmacokinetics of PSMA imaging probes. In particular, the hydrophilicity of both cationic and anionic charges led to rapid hepatobiliary clearance, although anionic charges might enhance renal clearance to a greater extent (kidney: 124.9 ± 26.2, 136 ± 6.4 and 56.8 ± 20.6% IA/g at 5, 30 and 120 min p.i.). The implementation of this 99m Tc-labeled anti-PSMA radiotracer into the clinical pathway has not taken place yet.  Four small-molecule inhibitors for PSMA labeled with 99m Tc via technetium tricarbonyl chemistry were reported by Hillier et al. [103]. The 99m Tc-labeled PSMA inhibitors derived from the glutamate-ureaglutamate or glutamate-urea-lysine pharmacophores after the incorporation of the chelators 2,2′-(2,2′-(azanediylbis(methylene))bis(1H-imidazole-2,1-diyl))diacetic acid (CIM) or 2,2′,2′′,2′′′-((2,2′-(2,2′-(azanediylbis(methylene))bis(1H-imidazole-2,1-diyl))bis(acetyl))bis(azanetriyl))tetraacetic acid (TIM) and the labeling with 99m Tc (compounds 35-38, Figure 10) were evaluated in vitro and in vivo (Table 5). The introduction of the chelators CIM and TIM to glutamate-urea-glutamate or glutamateurea-lysine pharmacophores and the subsequent labeling with 99m Tc did not influence the affinity of the derived radioligands towards PSMA (Table 5), as their KD values were found to be in the low nanomolar range. Furthermore, a side-by-side comparison of the affinity reveals a slightly higher affinity for PSMA of the glutamate-urea-glutamate-containing compounds, 99m Tc-MIP-1404 and Four small-molecule inhibitors for PSMA labeled with 99m Tc via technetium tricarbonyl chemistry were reported by Hillier et al. [103]. The 99m Tc-labeled PSMA inhibitors derived from the glutamate-ureaglutamate or glutamate-urea-lysine pharmacophores after the incorporation of the chelators 2,2 -(2,2 -(azanediylbis(methylene))bis(1H-imidazole-2,1-diyl))diacetic acid (CIM) or 2,2 ,2 ,2 -((2,2 -(2,2 -(azanediylbis(methylene))bis(1H-imidazole-2,1-diyl))bis(acetyl))bis(azanetriyl))tetraacetic acid (TIM) and the labeling with 99m Tc (compounds 35-38, Figure 10) were evaluated in vitro and in vivo (Table 5). The introduction of the chelators CIM and TIM to glutamate-urea-glutamate or glutamate-urea-lysine pharmacophores and the subsequent labeling with 99m Tc did not influence the affinity of the derived radioligands towards PSMA (Table 5), as their K D values were found to be in the low nanomolar range. Furthermore, a side-by-side comparison of the affinity reveals a slightly higher affinity for PSMA of the glutamate-urea-glutamate-containing compounds, 99m Tc-MIP-1404 and 99m Tc-MIP-1427. The formal addition of an extra carboxylic acid group to the CIM chelator, yielding the TIM chelator, resulted in improved pharmacokinetics of the derived radioligands. Indeed, 99m Tc-MIP-1404 and 99m Tc-MIP-1428 displayed lower uptake in and more rapid clearance from blood, liver, kidneys ( 99m Tc-MIP-1404: blood: 0.13 ± 0.03 and 0.02 ± 0, liver: 0.14 ± 0.03 and 0.07 ± 0.01, kindey: 105 ± 37 and 12 ± 7 % IA/g at 1 and 4 h p.i. respectively, 99m Tc-MIP-1428: blood: 0.44 ± 0.07 and 0.14 ± 0.03, liver: 0.27 ± 0.05 and 0.14 ± 0.04, kidney: 136 ± 6 and 63 ± 27 % IA/g at 1 and 4 h p.i. respectively), and most other nontarget tissues than did 99m Tc-MIP-1405 and 99m Tc-MIP-1427 ( 99m Tc-MIP-1405: blood: 0.72 ± 0.19 and 0.26 ± 0.03, liver: 0.43 ± 0.14 and 0.19 ± 0.05, kidney: 157 ± 69 and 14 ± 6 % IA/g at 1 and 4 h p.i. respectively, 99m Tc-MIP-1427: blood: 0.55 ± 0.08 and 0.28 ± 0.03, liver: 0.83 ± 0.24 and 0.76 ± 0.26, kidney: 149 ± 24 and 142 ± 39 % IA/g at 1 and 4 h p.i. respectively). The chelator TIM did not diminish uptake or retention in the LNCaP xenograft tumor, as there was no statistical difference between the tumor uptake of any of the compounds at either the 1 h or the 4 h time points. More specifically, uptake in LNCaP xenografts ranged from 9.3% to 12.4% injected dose per gram at 1 h after injection and from 7.2% to 11.0% at 4 h, with tumor-to-blood ratios ranging from 29:1 to 550:1 and tumor-to-muscle ratios ranging from 31:1 to 157:1 at 4 h. 99m Tc-MIP-1404 is currently under clinical investigation in a phase II trial.

68 Ga-Labeled Urea-Based PSMA Inhibitors
Because of its availability from the 68 Ge/ 68 Ga generator systems and the relative ease of labeling chemistry, the positron emitter 68 Ga (t½ = 68 min, β + max = 1899 keV) has gained increasing interest in the field of molecular imaging [104] and can considered a PET-applicable counterpart of 99m Tc.
Many efforts have been made with respect to the development of 68 Ga-PSMA-based imaging agents in the hope of developing a cyclotron-independent nuclear probe for prostate cancer imaging. A variety of PSMA-based inhibitors have been generated where several chelators suitable for labeling with 68 Ga as well as spacers have been investigated. In solution, gallium is most stable in the +3-oxidation state and forms stable complexes with several chelating agents. The HBED chelator (N,N′-bis [2-hydroxybenzyl] ethylenediamine-N,N′-diacetic acid) which contains an amine-phenol

68 Ga-Labeled Urea-Based PSMA Inhibitors
Because of its availability from the 68 Ge/ 68 Ga generator systems and the relative ease of labeling chemistry, the positron emitter 68 Ga (t 1 2 = 68 min, β + max = 1899 keV) has gained increasing interest in the field of molecular imaging [104] and can considered a PET-applicable counterpart of 99m Tc.
Many efforts have been made with respect to the development of 68 Ga-PSMA-based imaging agents in the hope of developing a cyclotron-independent nuclear probe for prostate cancer imaging. A variety of PSMA-based inhibitors have been generated where several chelators suitable for labeling with 68 Ga as well as spacers have been investigated. In solution, gallium is most stable in the +3-oxidation state and forms stable complexes with several chelating agents. The HBED chelator (N,N -bis [2-hydroxybenzyl] ethylenediamine-N,N -diacetic acid) which contains an amine-phenol backbone which renders it suitable for complexation with +3 metal ions, and it forms a thermodynamically stable complex with 68 Ga even at room temperature [105]. A representative example of PSMA-inhibitors associated with the chelator HBED, is the peptidomimetic structure Lys-urea-NH-Glu, published by Eder et al., when coupled to the spacer 6-aminohexanoic acid (Ahx) and functionalized with the chelator N,N -dis[2-hydroxy-5-(carboxyethyl)benzyl] ethylenediamine-N,N -diacetic acid (HBED-CC) to obtain HBED-CC-Ahx-Lys-urea-Glu (HBED-CC-PSMA or PSMA-11) (compound 39, Figure 11) [106]. HBED-CC has a dual nature, it also acts as a lipophilic moiety due to the presence of the two phenolic rings and therefore an aliphatic spacer such as Aca is adequate for providing the required lipophilicity and retaining the affinity of the derived ligand towards PSMA (K i = 12.1 ± 2.1 nM). Radiolabeling with 68 Ga was performed at pH 4.2 by incubating PSMA-11 in a mixture consisting of 50-100 MBq [ 68 Ga]Ga 3+ and HEPES. An amount of 0.1 nmol of PSMA-11 at a final concentration of 1.7 µM led to a radiochemical yield of more than 99% in less than 1 min at room temperature, with specific activities in the range og 500-1000 GBq/µmol. These are the highest specific activities which have been reported for 68 Ga-lebeled PSMA inhibitors. In order to achieve comparable high radiochemical yields with the DOTA-conjugated PSMA inhibitors, the compounds were incubated for 2 min at 80 • C using 1 nmol of precursor [106]. 68 Ga-PSMA-11 exhibited fast blood clearance, relatively low liver uptake (0.87 ± 0.05 % IA/g at 1 h p.i.), and high specific uptake in PSMA-expressing tissues and tumor (tumor uptake 7.7 ± 1.5 % IA/g at 1 h p.i.). The ability to image PSMA using 68 Ga-HBED-CC-PSMA showed great promise not only preclinically but also clinically . The dimerization of the same pharmacophore, Lys-Urea-Glu, coupled to the same spacer Ahx, through the chelator HBED-CC (PSMA-10) (compound 40, Figure 11) was also published by Schäfer et al. [130]. The dimer was also labeled with 68 Ga to obtain 68 Ga-PSMA-10 and evaluated in vitro and in vivo compared to 68 Ga-PSMA-11. Despite the fact that the preclinical evaluation revealed that 68 Ga-PSMA-10 was superior to 68 Ga-PSMA-11 in terms of binding affinity and tumor to background ratios (IC 50 = 3.9 ± 1.8 nM with a target to non-target ratio of 26.5 at 1 h p.i. as compared to a value of 9.2 for the monomer at the same time point), most of the clinical studies, so far, have been conducted using 68 Ga-PSMA-11. backbone which renders it suitable for complexation with +3 metal ions, and it forms a thermodynamically stable complex with 68 Ga even at room temperature [105]. A representative example of PSMA-inhibitors associated with the chelator HBED, is the peptidomimetic structure Lys-urea-NH-Glu, published by Eder et al., when coupled to the spacer 6-aminohexanoic acid (Ahx) and functionalized with the chelator N,N′-dis[2-hydroxy-5-(carboxyethyl)benzyl] ethylenediamine-N,N′-diacetic acid (HBED-CC) to obtain HBED-CC-Ahx-Lys-urea-Glu (HBED-CC-PSMA or PSMA-11) (compound 39, Figure 11) [106]. HBED-CC has a dual nature, it also acts as a lipophilic moiety due to the presence of the two phenolic rings and therefore an aliphatic spacer such as Aca is adequate for providing the required lipophilicity and retaining the affinity of the derived ligand towards PSMA (Ki = 12.1 ± 2.1 nM). Radiolabeling with 68 Ga was performed at pH 4.2 by incubating PSMA-11 in a mixture consisting of 50-100 MBq [ 68 Ga]Ga 3+ and HEPES. An amount of 0.1 nmol of PSMA-11 at a final concentration of 1.7 μM led to a radiochemical yield of more than 99% in less than 1 min at room temperature, with specific activities in the range og 500-1000 GBq/μmol. These are the highest specific activities which have been reported for 68 Ga-lebeled PSMA inhibitors. In order to achieve comparable high radiochemical yields with the DOTA-conjugated PSMA inhibitors, the compounds were incubated for 2 min at 80 °C using 1 nmol of precursor [106]. 68 Ga-PSMA-11 exhibited fast blood clearance, relatively low liver uptake (0.87 ± 0.05% IA/g at 1 h p.i.), and high specific uptake in PSMA-expressing tissues and tumor (tumor uptake 7.7 ± 1.5% IA/g at 1 h p.i.). The ability to image PSMA using 68 Ga-HBED-CC-PSMA showed great promise not only preclinically but also clinically . The dimerization of the same pharmacophore, Lys-Urea-Glu, coupled to the same spacer Ahx, through the chelator HBED-CC (PSMA-10) (compound 40, Figure 11) was also published by Schäfer et al. [130]. The dimer was also labeled with 68 Ga to obtain 68 Ga-PSMA-10 and evaluated in vitro and in vivo compared to 68 Ga-PSMA-11. Despite the fact that the preclinical evaluation revealed that 68 Ga-PSMA-10 was superior to 68 Ga-PSMA-11 in terms of binding affinity and tumor to background ratios (IC50 = 3.9 ± 1.8 nM with a target to non-target ratio of 26.5 at 1 h p.i. as compared to a value of 9.2 for the monomer at the same time point), most of the clinical studies, so far, have been conducted using 68 Ga-PSMA-11. In the field of radiopharmaceutical chemistry, the cyclic chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-triacetic acid (DOTA) has been used for complexation of +3 radiometal ions such as 68   In the field of radiopharmaceutical chemistry, the cyclic chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-triacetic acid (DOTA) has been used for complexation of +3 radiometal ions such as 68 Ga, 90 Y and 177 Lu. Banerjee et al. described two DOTA-conjugated PSMA inhibitors based on the Lys-Urea-Glu construct that confers PSMA specificity (compounds 41 and 42, Figure 12) and are suitable for labeling with 68 Ga [131]. The difference between those two tracers is confined to the spacer unit. More specifically, a benzyl group was inserted into compound 41 ( Figure 12) in order to provide a chromophore that facilitates purification. On the other hand, two phenylalanines were added to compound 42 ( Figure 12) in order to offset its high hydrophilicity and enable sustained retention and potentially higher absolute uptake in tumor [98]. Furthermore, in a recent study from the same group [132], a new radiotracer containing the macrocyclic chelator NOTA was prepared, since NOTA has been shown to be an effective chelator for 68 Ga (compound 43, Figure 13,). A head-to-head preclinical comparison of 68 Ga-42 with 68 Ga-43 ( Figure 12) was conducted. 68 Ga-PSMA-11, the radioligand which has been used throughout Europe in clinical trials, was also included in the study for comparison 68 Ga-41, 68 Ga-42 and 68 Ga-43 were directly labeled with the eluted 68 Ga from the 68 Ge/ 68 Ga generator without the use of a buffer solution within 10 min at 90-95 • C. The yields ranged from 60% to 70% with HPLC purification and the reported radiochemical purities were >99% with specific activities >168 GBq/µmol. Both 68 Ga-41 (−3.9) and 68 Ga-43 (−4.01 ± 0.16) were found to be 1 order of magnitude more hydrophilic than 68 Ga-42 (−3.0 ± 0.1). It is evident that 68 Ga-42 is more lipophilic than 68 Ga-41 and 68 Ga-43, which is reasonable because of the presence of two phenylalanine residues in the spacer. The corresponding metal-labeled compounds demonstrated high binding affinity to PSMA, with Ki values ranging from 0.33 to 29 nM (compound 41 was the less affine towards PSMA).
Both compounds, 68 Ga-41 and 68 Ga-42, exhibited PSMA specific tumor imaging in vivo. Nevertheless, the improved tumor-to-background ratios of 68 Ga-42 at later time points after injection renders it a more promising candidate for clinical translation. The PSMA+ PC3 PIP tumors as well as PSMA positive organs such as, kidney and urinary bladder were clearly delineated already at 15 min p.i. More specifically, the NOTA-conjugated radioligand 68 Ga-43 exhibited the highest tumor uptake with 42.2 ± 6.7 % IA/g at 1 h p.i. and the fastest background clearance. The PSMA+ PC3 PIP-to-PSMA− PC3 flu tumor ratios were 110 ± 22 at 1 h, 232 ± 26 at 2 h, and 182 ± 15 at 3 h p.i. Renal uptake for 68 Ga-43 was highest at 1 h, 106 ± 23 % IA/g, much higher than that seen for the DOTA-conjugated radioligand 68 Ga-42 (26.5 ± 6.9 % IA/g), and showed faster renal clearance, which decreased to 34.7 ± 5.7 % IA/g by 2 h p.i. There was no significant difference in PSMA+ PIP tumor uptake between 68 Ga-42 and 68 Ga-PSMA-11 or between 68 Ga-43 and 68 Ga-PSMA-11 (p > 0.05) at any time-point. 68 Ga-PSMA-11 demonstrated the highest and retained uptake in normal tissues, including kidney, blood, spleen, salivary glands and PSMA-negative PC3 flu tumors up to 3 h post-injection. This preclinical evaluation showed that 68 Ga-43 was superior for PSMA-targeted PET imaging in clinical settings. construct that confers PSMA specificity (compounds 41 and 42, Figure 12) and are suitable for labeling with 68 Ga [131]. The difference between those two tracers is confined to the spacer unit. More specifically, a benzyl group was inserted into compound 41 ( Figure 12) in order to provide a chromophore that facilitates purification. On the other hand, two phenylalanines were added to compound 42 ( Figure 12) in order to offset its high hydrophilicity and enable sustained retention and potentially higher absolute uptake in tumor [98]. Furthermore, in a recent study from the same group [132], a new radiotracer containing the macrocyclic chelator NOTA was prepared, since NOTA has been shown to be an effective chelator for 68 Ga (compound 43, Figure 13,). A head-to-head preclinical comparison of 68 Ga-42 with 68 Ga-43 ( Figure 12) was conducted. 68 Ga-PSMA-11, the radioligand which has been used throughout Europe in clinical trials, was also included in the study for comparison 68 Ga-41, 68 Ga-42 and 68 Ga-43 were directly labeled with the eluted 68 Ga from the 68 Ge/ 68 Ga generator without the use of a buffer solution within 10 min at 90-95 °C. The yields ranged from 60% to 70% with HPLC purification and the reported radiochemical purities were >99% with specific activities >168 GBq/μmol. Both 68 Ga-41 (−3.9) and 68 Ga-43 (−4.01 ± 0.16) were found to be 1 order of magnitude more hydrophilic than 68 Ga-42 (−3.0 ± 0.1). It is evident that 68 Ga-42 is more lipophilic than 68 Ga-41 and 68 Ga-43, which is reasonable because of the presence of two phenylalanine residues in the spacer. The corresponding metal-labeled compounds demonstrated high binding affinity to PSMA, with Ki values ranging from 0.33 to 29 nM (compound 41 was the less affine towards PSMA). Both compounds, 68 Ga-41 and 68 Ga-42, exhibited PSMA specific tumor imaging in vivo. Nevertheless, the improved tumor-to-background ratios of 68 Ga-42 at later time points after injection renders it a more promising candidate for clinical translation. The PSMA+ PC3 PIP tumors as well as PSMA positive organs such as, kidney and urinary bladder were clearly delineated already at 15 min p.i. More specifically, the NOTA-conjugated radioligand 68 Ga-43 exhibited the highest tumor uptake with 42.2 ± 6.7% IA/g at 1 h p.i. and the fastest background clearance. The PSMA+ PC3 PIP-to-PSMA− PC3 flu tumor ratios were 110 ± 22 at 1 h, 232 ± 26 at 2 h, and 182 ± 15 at 3 h p.i. Renal uptake for 68 Ga-43 was highest at 1 h, 106 ± 23% IA/g, much higher than that seen for the DOTA-conjugated radioligand 68 Ga-42 (26.5 ± 6.9% IA/g), and showed faster renal clearance, which decreased to 34.7 ± 5.7% IA/g by 2 h p.i. There was no significant difference in PSMA+ PIP tumor uptake between 68 Ga-42 and 68 Ga-PSMA-11 or between 68 Ga-43 and 68 Ga-PSMA-11 (p > 0.05) at any time-point. 68 Ga-PSMA-11 demonstrated the highest and retained uptake in normal tissues, including kidney, blood, spleen, salivary glands and PSMA-negative PC3 flu tumors up to 3 h post-injection. This preclinical evaluation showed that 68 Ga-43 was superior for PSMA-targeted PET imaging in clinical settings.  Benešová et al. [133], published the synthesis and preclinical evaluation a novel theranostic compound termed PSMA-617 (compound 44, Figure 13). In this case, the chelator DOTA, was conjugated to the pharmacophore Lys-urea-Glu via a naphthylic spacer. PSMA-617 was labeled with [ 68 Ga]Ga 3+ eluate in HEPES buffer, pH 4.0, within 15 min at 95 • C, with a radiochemical yield of more than 90% and a specific activity in the range of 14-140 GBq/µmol. The study showed that the presence of the naphthylic linker has a significant impact on the tumor-targeting as well as on the pharmacokinetics and the resulting imaging contrast. In fact, 68 Ga-PSMA-617 (K i = 6.40 ± 1.02 nM) was superior to 68 Ga-PSMA-11 (K i = 12.1 ± 2.1 nM) as far as the affinity towards PSMA and efficacy of internalization (up to 17.67 6 4.34 percentage injected activity/10 6 LNCaP cells) into the cancer cells concerns. Biodistibution studies upon injection of 68 Ga-PSMA-617 on LNCaP tumor bearing mice 1 h p.i. revealed a high specific uptake in LNCaP tumors (8.47 ± 4.09 % IA/g; 0.98 ± 0.32 % IA/g by coinjection of 2-PMPA) and in the kidneys (113.3 ± 24.4 % IA/g). Other organs such as the liver (1.17 ± 0.10 % IA/g), lung (1.41 ± 0.41 % IA/g), and spleen (2.13 ± 0.16 % IA/g) showed rather low uptake. Furthermore, 68 Ga-PSMA-617 dynamic PET imaging showed that the maximum kidney uptake was reached within 15 min after injection and decreased substantially as early as 20 min p.i. High and sustained tumor uptake was observed. The fast kidney clearance emboldened clinical translation of this compound.
Benešová et al. [133], published the synthesis and preclinical evaluation a novel theranostic compound termed PSMA-617 (compound 44, Figure 13). In this case, the chelator DOTA, was conjugated to the pharmacophore Lys-urea-Glu via a naphthylic spacer. PSMA-617 was labeled with [ 68 Ga]Ga 3+ eluate in HEPES buffer, pH 4.0, within 15 min at 95 °C, with a radiochemical yield of more than 90% and a specific activity in the range of 14-140 GBq/μmol. The study showed that the presence of the naphthylic linker has a significant impact on the tumor-targeting as well as on the pharmacokinetics and the resulting imaging contrast. In fact, 68 Ga-PSMA-617 (Ki = 6.40 ± 1.02 nM) was superior to 68 Ga-PSMA-11 (Ki = 12.1 ± 2.1 nM) as far as the affinity towards PSMA and efficacy of internalization (up to 17.67 6 4.34 percentage injected activity/10 6 LNCaP cells) into the cancer cells concerns. Biodistibution studies upon injection of 68 Ga-PSMA-617 on LNCaP tumor bearing mice 1 h p.i. revealed a high specific uptake in LNCaP tumors (8.47 ± 4.09% IA/g; 0.98 ± 0.32% IA/g by coinjection of 2-PMPA) and in the kidneys (113.3 ± 24.4% IA/g). Other organs such as the liver (1.17 ± 0.10% IA/g), lung (1.41 ± 0.41% IA/g), and spleen (2.13 ± 0.16% IA/g) showed rather low uptake. Furthermore, 68 Ga-PSMA-617 dynamic PET imaging showed that the maximum kidney uptake was reached within 15 min after injection and decreased substantially as early as 20 min p.i. High and sustained tumor uptake was observed. The fast kidney clearance emboldened clinical translation of this compound. When Lys-Urea-Glu (KuE) was coupled to the spacer Phe-Phe-Lysine-suberoyl (L-amino acid spacer, FFK-Sub) and functionalized with the chelator (1-(1,3-carboxypropyl)-4,7,10(carboxymethyl)-1,4,7,10 tetraazacyclododecane (DOTAGA) DOTAGA-FFK(Sub-KuE) was obtained [134]. HEPES buffer was used for the labeling with 68 Ga (pH 4.5). The reaction completed within 5 min at 95 °C and resulted in a radioligand with a specific activity of 250-300 GBq/μmol. However, a rapid in vivo metabolysis of the 68 Ga-labeled radiovector was demonstrated. In the same report by Weineisen et al. the inclusion of a D-amino acids spacer led to an in vivo metabolic stable radiotracer (DOTAGAffk(Sub-KuE)) (compounds 45-47, Figure 14).
In an attempt from the same group to further optimize this second-generation of the PSMA inhibitor with respect to its affinity towards the PSMA enzyme, additional modifications on the spacer unit were undertaken. [135]. For that purpose, D-Phe (f) was substituted with D-I-Tyr (I-y) and DOTAGA-(I-y)fk(Sub-KuE) was synthesized ( Figure 14). DOTAGA-(I-y)fk(Sub-KuE) is also termed PSMA I&T (PSMA I&T for Imaging &Therapy), since it can be labeled with both the diagnostic radionuclide 68 Ga and the therapeutic radionuclide 177 Lu. The necessary modifications for the generation of a metabolically stable radioligand did not alter the affinity of the derived ligands towards PSMA, since all the precursors as well as the corresponding metalloconjugates exhibited IC50 values within the range of 8 to 16 nM. 68 Ga-PSMA I&T is characterized by rapid tumor targeting (4.95 ± 1.57% IA/g at 1 h p.i.) and pharmacokinetics with high uptake in PSMA-positive organs such as the tumor and the kidneys (53.26 ± 9.02% IA/g at 1 h p.i.). 68 Ga-PSMA I&T is currently under clinical investigation. When Lys-Urea-Glu (KuE) was coupled to the spacer Phe-Phe-Lysine-suberoyl (L-amino acid spacer, FFK-Sub) and functionalized with the chelator (1-(1,3-carboxypropyl)-4,7,10(carboxymethyl)-1,4,7,10 tetraazacyclododecane (DOTAGA) DOTAGA-FFK(Sub-KuE) was obtained [134]. HEPES buffer was used for the labeling with 68 Ga (pH 4.5). The reaction completed within 5 min at 95 • C and resulted in a radioligand with a specific activity of 250-300 GBq/µmol. However, a rapid in vivo metabolysis of the 68 Ga-labeled radiovector was demonstrated. In the same report by Weineisen et al. the inclusion of a D-amino acids spacer led to an in vivo metabolic stable radiotracer (DOTAGA-ffk(Sub-KuE)) (compounds 45-47, Figure 14).
In an attempt from the same group to further optimize this second-generation of the PSMA inhibitor with respect to its affinity towards the PSMA enzyme, additional modifications on the spacer unit were undertaken. [135]. For that purpose, D-Phe (f) was substituted with D-I-Tyr (I-y) and DOTAGA-(I-y)fk(Sub-KuE) was synthesized ( Figure 14). DOTAGA-(I-y)fk(Sub-KuE) is also termed PSMA I&T (PSMA I&T for Imaging &Therapy), since it can be labeled with both the diagnostic radionuclide 68 Ga and the therapeutic radionuclide 177 Lu. The necessary modifications for the generation of a metabolically stable radioligand did not alter the affinity of the derived ligands towards PSMA, since all the precursors as well as the corresponding metalloconjugates exhibited IC 50 values within the range of 8 to 16 nM. 68 Ga-PSMA I&T is characterized by rapid tumor targeting (4.95 ± 1.57 % IA/g at 1 h p.i.) and pharmacokinetics with high uptake in PSMA-positive organs such as the tumor and the kidneys (53.26 ± 9.02 % IA/g at 1 h p.i.). 68 Ga-PSMA I&T is currently under clinical investigation.  Figure 15) [136]. 68 Ga-CC34 was obtained in high specific activity (75-80 MBq/nmol) and evaluated in vitro and in vivo in LNCaP tumor xenografts by biodistribution and PET imaging studies. 68 Ga-PSMA-11 was also evaluated for comparison. 68/natGa -CC34 exhibited high affinity for the LNCaP cells, with a KD value of 19.3 ± 2.5 nM. Tumor uptake of 68 Ga-CC34 (14.5 ± 2.9% IA/g) in LNCaP xenografts at 1 h p.i. was comparable to 68 Ga-PSMA-11 (15.8 ± 1.4% IA/g) (p = 0.67). The tumor-to-normal tissue ratios at 1 and 2 h p.i of 68 Ga-CC34 were also comparable to that of 68 Ga-PSMA-11 (p > 0.05). The spacer Phe-Phe-D-Lys(suberoyl) was included to obtain (R)-NODAGA-Phe-Phe-D-Lys(suberoyl)-Lys-urea-Glu (CC34) (compound 48, Figure 15) [136]. 68 Ga-CC34 was obtained in high specific activity (75-80 MBq/nmol) and evaluated in vitro and in vivo in LNCaP tumor xenografts by biodistribution and PET imaging studies. 68 Ga-PSMA-11 was also evaluated for comparison. 68/natGa -CC34 exhibited high affinity for the LNCaP cells, with a K D value of 19.3 ± 2.5 nM. Tumor uptake of 68 Ga-CC34 (14.5 ± 2.9 % IA/g) in LNCaP xenografts at 1 h p.i. was comparable to 68 Ga-PSMA-11 (15.8 ± 1.4 % IA/g) (p = 0.67). The tumor-to-normal tissue ratios at 1 and 2 h p.i of 68 Ga-CC34 were also comparable to that of 68 Ga-PSMA-11 (p > 0.05).  Figure 15) [136]. 68 Ga-CC34 was obtained in high specific activity (75-80 MBq/nmol) and evaluated in vitro and in vivo in LNCaP tumor xenografts by biodistribution and PET imaging studies. 68 Ga-PSMA-11 was also evaluated for comparison. 68/natGa -CC34 exhibited high affinity for the LNCaP cells, with a KD value of 19.3 ± 2.5 nM. Tumor uptake of 68 Ga-CC34 (14.5 ± 2.9% IA/g) in LNCaP xenografts at 1 h p.i. was comparable to 68 Ga-PSMA-11 (15.8 ± 1.4% IA/g) (p = 0.67). The tumor-to-normal tissue ratios at 1 and 2 h p.i of 68 Ga-CC34 were also comparable to that of 68 Ga-PSMA-11 (p > 0.05). In another recent preclinical study, the cyclohexyl-diethylene triamine pentaacetic acid (CHX-A″-DTPA) was used as the chelator for the Glu-urea-GLu-based peptide (Pep) bearing a 2-[3-(1,3dicarboxypropyl)-ureido]pentanedioic acid (DUPA) moiety to obtain CHX-A″-DTPA-DUPA-Pep (compound 49, Figure 16). The study describes the efficient labeling of CHX-A″-DTPA-DUPA-Pep In another recent preclinical study, the cyclohexyl-diethylene triamine pentaacetic acid (CHX-A -DTPA) was used as the chelator for the Glu-urea-GLu-based peptide (Pep) bearing a 2-[3-(1,3dicarboxypropyl)-ureido]pentanedioic acid (DUPA) moiety to obtain CHX-A -DTPA-DUPA-Pep (compound 49, Figure 16). The study describes the efficient labeling of CHX-A -DTPA-DUPA-Pep with 68 Ga, 90 Y and 177 Lu and the first in vitro characterization which shows high affinity of the tested radiotracers towards PSMA with K D values of ≤14.67 ± 1.95 nM. Labelling with 68 Ga was performed at room temperature under neutral conditions (HEPES, pH 7.4). Significant differences in radiochemical yields were observed. Radio labelling with 68 Ga succeeded in 10 min with high radiochemical yields; ≥95% when 50 µg (36 nM) of the peptide was used. However, in vivo images or biodistribution data of these agents have so far not been published [137].  68 Ga was performed at room temperature under neutral conditions (HEPES, pH 7.4). Significant differences in radiochemical yields were observed. Radio labelling with 68 Ga succeeded in 10 min with high radiochemical yields; ≥95% when 50 μg (36 nM) of the peptide was used. However, in vivo images or biodistribution data of these agents have so far not been published [137].

64 Cu-Labeled Urea-Based PSMA Inhibitors
Amongst the PET-radioisotopes, 64 Cu has gained particular attention because of its decay characteristics (t1/2 = 12.7 h; β + , Emax = 0.653 MeV [17.8%]; β − , Emax = 0.579 MeV [38.4%]) and the well-established coordination chemistry with a variety of chelators. The longer half-life of 64 Cu relative to 68 Ga (t1/2 = 67.8 min) and 18 F (t1/2 = 109.8 min) is particularly attractive, as it allows PET-images at later time points with improved tumor-to-background ratios [138]. Additionally, 64 Cu-labeled radiopharmaceuticals can be produced at a central facility and distributed to remote hospitals. The successful application of 64 Cu as a diagnostic agent may also lead to potential targeted radionuclide therapy with 67 Figure 17). The radiolabeling with 64 Cu was performed in acetate buffer and the conditions differed based on the chelator ( Table 6). The metal-free CB-TE2A-conjugated ligand 53 contained two nearly inseparable isomeric peaks by HPLC. This is most probably caused by the presence of an asymmetric center at the Phe moiety of the spacer, located very close to CB-TE2A. Radiolabeling of 53 resulted in two HPLC separable products, named 64 Cu-53A and 64 Cu-53B. All radioligands were obtained in specific activities in the range 2.9-9.1 GBq/μmol. 64 Cu-53A, 64 Cu-53B and 64 Cu-54 were found to be more hydrophilic despite the presence of two Phe moieties at the spacer. The diastereomeric metalloconjuagtes 53A and 53B were the ones with highest affinity for PSMA. PET imaging as well as ex vivo biodistribution studies showed that 64 Cu-50 and 64 Cu-53A/B (NOTA and CB-TE2A chelators, respectively) exhibited high in vivo stability as evidenced by their lower liver uptake than that of the other three conjugates (blood: 1.06 ± 0.29, 1.87 ± 0.6, 0.20 ± 0.03 and 0.20 ± 0.07% IA/g; liver: 8.63 ± 0.92, 17.04 ± 1.44, 1.68 ± 0.38 and 1.63 ± 0.72% IA/g for 64 Cu-50, 64 Cu-51, 64 Cu-53A and 64 Cu-53B at 2 h p.i.). An additional finding of this study was that liver uptake and blood concentration were much lower at all time points for 64 Cu-53A/B compared to that of 64 Cu-50, suggesting a higher in vivo stability of CB-TE2A-conjugated 64 Cu-53A/B than of NOTA-conjugated 64 Cu-50. That might also be related to the higher hydrophilicity of 64 Cu-53A/B compared to 64 Cu-50. On the other hand, the high liver uptake and the slow blood clearance for the 64 Cu-DOTA-conjugated radiotracer were indicative of free Cu(II), which is accumulated in liver [140,141]. Noteworthy is also the fact that negatively charged 64 Cu-labeled NOTA-conjugated PSMA inhibitor, 64 Cu-50, similarly to 99m Tc-oxo compounds [99],

64 Cu-Labeled Urea-Based PSMA Inhibitors
Amongst the PET-radioisotopes, 64 Cu has gained particular attention because of its decay characteristics (t 1/2 = 12.7 h; β + , Emax = 0.653 MeV [17.8%]; β − , Emax = 0.579 MeV [38.4%]) and the well-established coordination chemistry with a variety of chelators. The longer half-life of 64 Cu relative to 68 Ga (t 1/2 = 67.8 min) and 18 F (t 1/2 = 109.8 min) is particularly attractive, as it allows PET-images at later time points with improved tumor-to-background ratios [138]. Additionally, 64 Cu-labeled radiopharmaceuticals can be produced at a central facility and distributed to remote hospitals.
The successful application of 64 Cu as a diagnostic agent may also lead to potential targeted radionuclide therapy with 67 Cu (t 1/2 = 61.9 h; β − , E max = 0.141 MeV [100%]) providing a promising theranostic pair.
Banerjee et al. reported the synthesis and preclinical evaluation of a series of five PSMA inhibitors labeled with 64 Cu [139]. The main focus of the study was given on the effect of various chelators on PSMA-targeted ureas with respect to their pharmacokinetics for in vivo PET imaging. The investigated chelators are 1,4,7,10-tetraazacyclodoadecane-N,N ,N",N -tetraacetic acid, (DOTA), oxa-4,7,1-tetraazacyclododecane-4,7,10-triacetic acid, (Oxo-DO3A), 1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11-diacetic acid, (CB-TE2A), 3,6,9,15-tetraazabicyclo[9.3.1]-pentadeca-1(15),11,13-triene)-3,6,9-triacetic acid (PCTA) and 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) (compounds 50-54, Figure 17). The radiolabeling with 64 Cu was performed in acetate buffer and the conditions differed based on the chelator ( Table 6). The metal-free CB-TE2A-conjugated ligand 53 contained two nearly inseparable isomeric peaks by HPLC. This is most probably caused by the presence of an asymmetric center at the Phe moiety of the spacer, located very close to CB-TE2A. Radiolabeling of 53 resulted in two HPLC separable products, named 64 Cu-53A and 64 Cu-53B. All radioligands were obtained in specific activities in the range 2.9-9.1 GBq/µmol. 64 Cu-53A, 64 Cu-53B and 64 Cu-54 were found to be more hydrophilic despite the presence of two Phe moieties at the spacer. The diastereomeric metalloconjuagtes 53A and 53B were the ones with highest affinity for PSMA. PET imaging as well as ex vivo biodistribution studies showed that 64 Cu-50 and 64 Cu-53A/B (NOTA and CB-TE2A chelators, respectively) exhibited high in vivo stability as evidenced by their lower liver uptake than that of the other three conjugates (blood: 1.06 ± 0.29, 1.87 ± 0.6, 0.20 ± 0.03 and 0.20 ± 0.07 % IA/g; liver: 8.63 ± 0.92, 17.04 ± 1.44, 1.68 ± 0.38 and 1.63 ± 0.72 % IA/g for 64 Cu-50, 64 Cu-51, 64 Cu-53A and 64 Cu-53B at 2 h p.i.). An additional finding of this study was that liver uptake and blood concentration were much lower at all time points for 64 Cu-53A/B compared to that of 64 Cu-50, suggesting a higher in vivo stability of CB-TE2A-conjugated 64 Cu-53A/B than of NOTA-conjugated 64 Cu-50. That might also be related to the higher hydrophilicity of 64 Cu-53A/B compared to 64 Cu-50. On the other hand, the high liver uptake and the slow blood clearance for the 64 Cu-DOTA-conjugated radiotracer were indicative of free Cu(II), which is accumulated in liver [140,141]. Noteworthy is also the fact that negatively charged 64 Cu-labeled NOTA-conjugated PSMA inhibitor, 64 Cu-50, similarly to 99m Tc-oxo compounds [99], exhibited higher kidney and spleen uptake compared to 64 Cu-53A/B (kidney: 125 ± 42, 26 ± 9 and 25 ± 11 % IA/g; spleen: 13.42 ± 1.18, 0.39 ± 0.30 and 0.90 ± 0.23 % IA/g for 64 Cu-50, 64 Cu-53A and 64 Cu-53B at 2 h p.i.) Furthermore, although the PSMA-mediated renal uptake has been shown to be specific by several groups, it is interesting that variations are observed not only in regard to the absolute kidney uptake, but also with regard to the renal elimination.  64 Cu-53A and 64 Cu-53B at 2 h p.i.) Furthermore, although the PSMA-mediated renal uptake has been shown to be specific by several groups, it is interesting that variations are observed not only in regard to the absolute kidney uptake, but also with regard to the renal elimination.  The NODAGA-conjugated PSMA inhibitor CC34 (Figure 15), was also radiolabeled with 64 Cu in ammonium acetate buffer, pH 5.4, to completion within 30 min at 95 °C, and was used without further purification. The specific activity of 64 Cu-CC34 was 40 MBq/nmol and a logP of −3.01 ± 0.06. When CC34 was labeled with 64 Cu, the resulting tracer exhibited a slightly higher lipophilicity than did 68 Ga-CC34 (LogP = −3.54 ± 0.06) [136]. 64 Cu-CC34 was preclinically evaluated in LNCaP xenografted mice. A high affinity towards PSMA on LNCaP cells was shown (KD = 27.5 ± 2.7 nM). Furthermore, The NODAGA-conjugated PSMA inhibitor CC34 (Figure 15), was also radiolabeled with 64 Cu in ammonium acetate buffer, pH 5.4, to completion within 30 min at 95 • C, and was used without further purification. The specific activity of 64 Cu-CC34 was 40 MBq/nmol and a logP of −3.01 ± 0.06. When CC34 was labeled with 64 Cu, the resulting tracer exhibited a slightly higher lipophilicity than did 68 Ga-CC34 (LogP = −3.54 ± 0.06) [136]. 64 Cu-CC34 was preclinically evaluated in LNCaP xenografted mice. A high affinity towards PSMA on LNCaP cells was shown (K D = 27.5 ± 2.7 nM). Furthermore, the versatility of NODAGA was exploited for efficient labeling also with 64 Cu, which allows for the conduction of biodistribution/imaging studies at later time points compared to 68 Ga-CC34, and thus a complete assessment of the pharmacokinetics of the radiotracer. 64 Cu-CC34 is specifically taken up by the PSMA positive organs at early time points and washed out faster from the PSMA positive organs compared to uptake in tumor, leading to improved tumor to background ratios (tumor/blood: 41 ± 10 and 114 ± 17, tumor/muscles: 61 ± 18 and 103 ± 21 at 1 and 4 h p.i., respectively). The very low liver uptake of 64 Cu-CC34 at all time points in combination with the short blood circulation, comparable to what was also reported for the CB-2A-conjugated PSMA inhibitor from Banerjee et al. [139] is a strong indication of the excellent in vivo stability of the 64 Cu-NODAGA complex.

177 Lu-Labeled Urea-Based PSMA Inhibitors
The development of new radiolabeled specific biomarkers is mainly focused on the early diagnosis and prognostic prediction continuing with the adaptation of their therapeutic interventions that are individually optimized. Taking into account the theranostic approach, an ideal radiopharmaceutical should be able to combine the ability to be used for both diagnosis and therapy when labeled with a diagnostic or a suitable therapeutic radionuclide respectively. The beta emitter 177 Lu can be considered as an appropriate therapeutic unit during the construction of a therapeutic nuclear probe due to its favorable characteristics (t 1/2 = 6.73 days, E βmax = 497 keV, E γ = 113 keV (6.4%) and 208 keV (11%)).
The tailor-made DOTA-conjugated PSMA inhibitor, PSMA-617, has also been reported to be efficiently labeled with 177 Lu [133] in addition to 68 Ga. Radiolabeling with 177 Lu reached a radiochemical yield of greater than 99% at low amount of precursor (0.5 mg, 0.5 nmol) in sodium acetate buffer, pH 5. The specific activity was in the range of 4-40 GBq/mmol. 177 Lu-PSMA-617 exhibited high stability for at least 72 h, high binding affinity towards PSMA (K i = 6.91 ± 1.32 nM for nat Lu-PSMA-617) along with enhanced internalization rate into the LNCaP prostate cancer cells. 68 Ga-labeled PSMA-617 was specifically internalized up to 17.67 ± 4.34% injected activity/10 6 LNCaP cells and 177 Lu-labeled PSMA-617 up to 17.51 ± 3.99% injected activity/10 6 LNCaP cells, both at 37 • C. The in vivo performance of the 177 Lu-PSMA-617 was characterized by high and retained tumor uptake combined with a rapid clearance from the kidneys within 24 h p.i., which led to improved tumor to background ratios over time (1058 (tumor to blood) and 529 (tumor to muscle), 24 h p.i.). It is worth noting that 177 Lu-PSMA-617 exhibited similar pharmacokinetics as 111 In-PSMA-617 [131]. 177 Lu-PSMA-617 seems attractive for endoradiotherapy due to its higher tumor uptake at later time points, lower spleen accumulation, and the highly efficient clearance from the kidneys. The clinical evaluation of 177 Lu-PSMA-617 is under way.

SPECT Imaging with 99m Tc-Labeled PSMA Inhibitors
An exploratory investigational new drug application (IND) was implemented to bring the compounds MIP-1404 and MIP-1405 which been developed by the group at Molecular Insight Pharmaceuticals (MIP), and labeled with 99m Tc, into the clinic. Vallabhajosula et al. [143], reported the comparison of the pharmacokinetics, biodistribution, and tumor uptake of 99m Tc-MIP-1404 and 99m Tc-MIP-1405 in 6 healthy men and 6 men with radiographic evidence of metastatic prostate cancer.
SPECT imaging was performed between 3 and 4 h after injection of the radiotracers (Figure 18). Both radiotracers showed fast blood clearance persistent uptake in the salivary, lacrimal, and parotid glands. They localized in bone and lymph node lesions as early as 1 h. Because of the lower urinary activity of 99m Tc-MIP-1404 (7%) compared to 99m Tc-MIP-1405 (26%), a clear advantage for detecting prostate cancer in the gland and pelvis at early stages of the disease is indicated for this compound. 99m Tc-MIP-1404 was selected for phase II studies to determine sensitivity and specificity to detect prostate cancer in high-risk patients. Furthermore, the development and optimization of a "cold" kit for the preparation of 99m Tc-MIP-1404 using generator eluted pertechnetate has also been reported [144]. noting that 177 Lu-PSMA-617 exhibited similar pharmacokinetics as 111 In-PSMA-617 [131]. 177 Lu-PSMA-617 seems attractive for endoradiotherapy due to its higher tumor uptake at later time points, lower spleen accumulation, and the highly efficient clearance from the kidneys. The clinical evaluation of 177 Lu-PSMA-617 is under way. Under the same concept of the theranostic approach, the PSMA inhibitors DOTAGA-ffk (Sub-KuE) and DOTAGA-(I-y)fk(Sub-KuE) (also named PSMA I&T) were also labeled with 177 Lu and evaluated preclinically [135]. Radiolabeling with 177 Lu performed in ammonium acetate buffer, pH 5, and the radiolabeled products were achieved in a specific activity of more than 38 GBq/μmol. Both cold lutetium analogues exhibited high affinity towards PSMA (IC50: nat Lu-DOTAGA-ffk (Sub-KuE):13.1 ± 2.2 nM, nat Lu-PSMA I&T: 7.9 ± 2.4 nM). Compared with 177 Lu-DOTAGA-ffk (Sub-KuE), tumor targeting of 177 Lu-PSMA I&T was fast, with the highest uptake in tumor (7.96 ± 1.76% IA/g at 1 h p.i.) xenografts and kidneys (107 ± 16% IA/g at 1 h p.i.) (both PSMA-specific). 177 Lu-PSMA I&T exhibits suitable targeting and retention characteristics and an assessment of this compound for application in endoradiotherapy is currently ongoing.

SPECT Imaging with 99m Tc-Labeled PSMA Inhibitors
An exploratory investigational new drug application (IND) was implemented to bring the compounds MIP-1404 and MIP-1405 which been developed by the group at Molecular Insight Pharmaceuticals (MIP), and labeled with 99m Tc, into the clinic. Vallabhajosula et al. [143], reported the comparison of the pharmacokinetics, biodistribution, and tumor uptake of 99m Tc-MIP-1404 and 99m Tc-MIP-1405 in 6 healthy men and 6 men with radiographic evidence of metastatic prostate cancer. SPECT imaging was performed between 3 and 4 h after injection of the radiotracers (Figure 18). Both radiotracers showed fast blood clearance persistent uptake in the salivary, lacrimal, and parotid glands. They localized in bone and lymph node lesions as early as 1 h. Because of the lower urinary activity of 99m Tc-MIP-1404 (7%) compared to 99m Tc-MIP-1405 (26%), a clear advantage for detecting prostate cancer in the gland and pelvis at early stages of the disease is indicated for this compound. 99m Tc-MIP-1404 was selected for phase II studies to determine sensitivity and specificity to detect prostate cancer in high-risk patients. Furthermore, the development and optimization of a "cold" kit for the preparation of 99m Tc-MIP-1404 using generator eluted pertechnetate has also been reported [144].

PET Imaging with 68 Ga-Labeled PSMA Inhibitors
PET imaging of prostate cancer using PSMA-ligands has gained great attention during the last years. The numerous clinical trials evaluating 68 Ga-PSMA-11 PET-CT imaging in prostate cancer patients have proved that this tracer can be used for lesion characterization, staging, planning and monitoring of therapy as well as evaluation of recurrence. 68 Ga-PSMA-11 PET-CT imaging was firstly introduced in 2013 by Afshar-Oromieh et al. in 37 patients with relapsing prostate cancer ( Figure 19) [107,145]. Within a relatively short time frame, 68 Ga-PSMA-11 has been thoroughly clinically assessed by several research groups, indicative of the large number of publications the last four years . Although the background uptake of 68 Ga-PSMA-11 in salivary and lacrimal glands was abnormally high, PET-CT imaging was obtained leading to excellent detection of the metastatic lesions of prostate cancer.

PET Imaging with 68 Ga-Labeled PSMA Inhibitors
PET imaging of prostate cancer using PSMA-ligands has gained great attention during the last years. The numerous clinical trials evaluating 68 Ga-PSMA-11 PET-CT imaging in prostate cancer patients have proved that this tracer can be used for lesion characterization, staging, planning and monitoring of therapy as well as evaluation of recurrence. 68 Ga-PSMA-11 PET-CT imaging was firstly introduced in 2013 by Afshar-Oromieh et al. in 37 patients with relapsing prostate cancer ( Figure 19) [107,145]. Within a relatively short time frame, 68 Ga-PSMA-11 has been thoroughly clinically assessed by several research groups, indicative of the large number of publications the last four years . Although the background uptake of 68 Ga-PSMA-11 in salivary and lacrimal glands was abnormally high, PET-CT imaging was obtained leading to excellent detection of the metastatic lesions of prostate cancer. Sterzing et al. [111] reported that PET-CT imaging with 68 Ga-PSMA-11 may greatly contribute to the therapy planning of prostate cancer. The results of the study showed that 68 Ga-PSMA-11 PET-CT had impact on the therapy regimen in 50.8% of the cases.
In a retrospective study, Afshar-Oromieh et al. [110] demonstrated that tumor detection mediated by 68 Ga-PSMA-11 uptake was positively associated with prostate specific antigen (PSA) level and androgen deprivation therapy (ADT). Furthermore, 68 Ga-PSMA-11 PET-CT imaging in patients with biochemical recurrence was proved to be crucial for recurrence evaluation, restaging as well as early treatment [112].
The ability of 68 Ga-PSMA-11 to detect lesions of prostate cancer in patients with biochemical recurrence was also evaluated versus [ 18 F]fluoromethylcholine [108,111]. In both studies 68 Ga-PSMA-11 was found to be superior in detecting metastatic lesions associated with prostate cancer and displayed higher SUVmax values and superior tumor to background ratios. Another study by Dietlein et al. [113], PSMA-PET-CT imaging was conducting by applying 68 Ga-PSMA-11 and 18 F-DCFPyl in 14 patients with biochemical recurrence. 18 F-DCFPyl was found to be somehow superior Sterzing et al. [111] reported that PET-CT imaging with 68 Ga-PSMA-11 may greatly contribute to the therapy planning of prostate cancer. The results of the study showed that 68 Ga-PSMA-11 PET-CT had impact on the therapy regimen in 50.8% of the cases.
In a retrospective study, Afshar-Oromieh et al. [110] demonstrated that tumor detection mediated by 68 Ga-PSMA-11 uptake was positively associated with prostate specific antigen (PSA) level and androgen deprivation therapy (ADT). Furthermore, 68 Ga-PSMA-11 PET-CT imaging in patients with biochemical recurrence was proved to be crucial for recurrence evaluation, restaging as well as early treatment [112].
The ability of 68 Ga-PSMA-11 to detect lesions of prostate cancer in patients with biochemical recurrence was also evaluated versus [ 18 F]fluoromethylcholine [108,111]. In both studies 68 Ga-PSMA-11 was found to be superior in detecting metastatic lesions associated with prostate cancer and displayed higher SUV max values and superior tumor to background ratios. Another study by Dietlein et al. [113], PSMA-PET-CT imaging was conducting by applying 68 Ga-PSMA-11 and 18 F-DCFPyl in 14 patients with biochemical recurrence. 18 F-DCFPyl was found to be somehow superior compared to 68 Ga-PSMA-11 and could be considered as an alternative for PSMA-PET-CT imaging in relapsed prostate cancer.
The PSMA inhibitor PSMA-617 labeled with 68 Ga was also clinically assessed in the diagnosis of prostate cancer by PET-CT [146]. 68 Ga-PSMA-617 shows lesions of prostate cancer with high contrast, especially in late images ( Figure 20). Maximum contrast of tumor lesions was achieved between 3 and 4 h after injection. compared to 68 Ga-PSMA-11 and could be considered as an alternative for PSMA-PET-CT imaging in relapsed prostate cancer.
The PSMA inhibitor PSMA-617 labeled with 68 Ga was also clinically assessed in the diagnosis of prostate cancer by PET-CT [146]. 68 Ga-PSMA-617 shows lesions of prostate cancer with high contrast, especially in late images ( Figure 20). Maximum contrast of tumor lesions was achieved between 3 and 4 h after injection. First-in-human 68 Ga-PSMA I&T PET imaging allowed high-contrast detection of bone lesions, lymph node, and liver metastases [135]. In another study [143], 68 Ga-PSMA I&T PET delayed images were also systematically evaluated after forced diuresis in 66 prostate cancer patients. The study explored the rather complex influence of delayed imaging and timing of forced diuresis on image quality. The authors suggest diuretic administration at 60 min p.i. in combination with delayed imaging at 180 min p.i. in order to obtain the maximum information possible, at least for 68 Ga-PSMA I&T. However, the approach is time consuming [147].

PET Imaging with 64 Cu-Labeled PSMA Inhibitors
The first clinical use of the 64 Cu-labeled ligand PSMA-617 for PET imaging was conducted at two different centers in Austria and Germany [148]. 29 patients with either a high suspicion of recurrent disease were imaged in the context of therapy planning including surgery or PSMA radioligand therapy. The PET images showed an excellent resolution of the detected lesions with very high lesion to background contrast. An additional advantage of the 64 Cu-labeled radiopharmaceuticals is the long half-life of 64 Cu which allows distribution of the radiotracer to clinical PET centers where 68 Ga-PSMA ligands are not available. 177 Lu-labeled monoclonal antibody J591, which binds to the extracellular domain of PSMA, was firstly used for radionuclide therapy of prostate cancer [149]. Although the response to therapy was encouraging, the dose-limiting toxicity which is transient myelosuppression was the major reason of abandoning this therapeutic approach. In contrast, the development and preclinical evaluation of new PSMA ligands which could be labeled with therapeutic radionuclides such as 177 Lu, paved the way for the reinvestigation of the 177 Lu-based radionuclide therapy in patients with advanced prostate cancer. The radiotracers which have been used of the conduction of those studies are 177 Lu-PSMA-617 and 177 Lu-PSMA I&T ( Figure 21). The clinical trials reported so far demonstrate promising results [150][151][152][153][154][155][156] with a mean tumor dose 6 to 12 fold higher compared to other critical organs such as kidneys and salivary glands. The PSA levels were found to be reduced in 60-90% of the patients after a single dose of both 177 Lu-labeled PSMA-based radiotracers. The patients tolerated the therapy well, with no significant nephrotoxicity and just rarely occurring other severe side effects were observed. Although PSMA-directed radionuclide therapy bears a potential for implementation in routine clinical practice, the limited number of studies so far requires further controlled clinical trials for the establishment of the clinical value of 177 Lu-PSMA-617 and 177 Lu-PSMA First-in-human 68 Ga-PSMA I&T PET imaging allowed high-contrast detection of bone lesions, lymph node, and liver metastases [135]. In another study [143], 68 Ga-PSMA I&T PET delayed images were also systematically evaluated after forced diuresis in 66 prostate cancer patients. The study explored the rather complex influence of delayed imaging and timing of forced diuresis on image quality. The authors suggest diuretic administration at 60 min p.i. in combination with delayed imaging at 180 min p.i. in order to obtain the maximum information possible, at least for 68 Ga-PSMA I&T. However, the approach is time consuming [147].

PET Imaging with 64 Cu-Labeled PSMA Inhibitors
The first clinical use of the 64 Cu-labeled ligand PSMA-617 for PET imaging was conducted at two different centers in Austria and Germany [148]. 29 patients with either a high suspicion of recurrent disease were imaged in the context of therapy planning including surgery or PSMA radioligand therapy. The PET images showed an excellent resolution of the detected lesions with very high lesion to background contrast. An additional advantage of the 64 Cu-labeled radiopharmaceuticals is the long half-life of 64 Cu which allows distribution of the radiotracer to clinical PET centers where 68 Ga-PSMA ligands are not available.

PSMA Radionuclide Therapy Using 177 Lu-Labeled PSMA Inhibitors
177 Lu-labeled monoclonal antibody J591, which binds to the extracellular domain of PSMA, was firstly used for radionuclide therapy of prostate cancer [149]. Although the response to therapy was encouraging, the dose-limiting toxicity which is transient myelosuppression was the major reason of abandoning this therapeutic approach. In contrast, the development and preclinical evaluation of new PSMA ligands which could be labeled with therapeutic radionuclides such as 177 Lu, paved the way for the reinvestigation of the 177 Lu-based radionuclide therapy in patients with advanced prostate cancer. The radiotracers which have been used of the conduction of those studies are 177 Lu-PSMA-617 and 177 Lu-PSMA I&T ( Figure 21). The clinical trials reported so far demonstrate promising results [150][151][152][153][154][155][156] with a mean tumor dose 6 to 12 fold higher compared to other critical organs such as kidneys and salivary glands. The PSA levels were found to be reduced in 60-90% of the patients after a single dose of both 177 Lu-labeled PSMA-based radiotracers. The patients tolerated the therapy well, with no significant nephrotoxicity and just rarely occurring other severe side effects were observed. Although PSMA-directed radionuclide therapy bears a potential for implementation in routine clinical practice, the limited number of studies so far requires further controlled clinical trials for the establishment of the clinical value of 177 Lu-PSMA-617 and 177 Lu-PSMA I&T. 177 Lu-PSMA-617 recently received FDA approval for phase II clinical trials in the U.S., ("First U.S. multi-center investigational clinical trial of 177 Lu-PSMA-617 targeted radioligand therapy in metastatic castration resistant prostate cancer receives FDA clearance". Daily Tribune, SyndiGate Media Inc. (Houston, TX, USA); 7 February, 2017, as found on http://www.pharmacychoice.com/News/article.cfm?Article_ID=1690538).

Image-Guided Surgery of Prostate Cancer Lesions Using 111 In-Labeled PSMA Inhibitors
The promising results of the diagnostic and endoradiotherapeutic approaches which have been successfully applied into the clinical setting using PSMA-based radiotracers rendered several research groups to make use of suitable PSMA-based radiotracers for other therapeutic strategies, such as the image-guided surgery of prostate cancer lesions. 111 In-PSMA I&T 111 In-PSMA-617 are the probes which have been used in two different clinical trials for radio-guided surgery for the detection of lymph node metastasis [117,142,157]. Both trials showed that all PET-positive lesions were also detected by PSMA-radio-guided surgery. Furthermore, tracer uptake and median gamma probe counts were significantly different between regions with lymph node metastases and other normal tissues. Overall, these studies showed that the gamma probes measurements could be considered as an indicator for lymph node metastasis and that radio-guided surgery may consequently lead to improvements with respect to the resection of prostate cancer.

Conclusions
Targeting the integral membrane glycoprotein PSMA by using radiometal-based PSMA inhibitors shows a great promise for diagnosis, (re)staging and therapy of prostate cancer. Specific PSMA-based ligands show high potential for initial staging, lymph node staging, restaging and therapy of prostate cancer. Between the different groups of PSMA inhibitors which have been developed so far, the peptidomimetic structure Lys-urea-Glu is the most successful biomolecule for utilization in radiopharmaceutical development. Furthermore, the choice of the chelator significantly affects the stability, affinity, lipophilicity and ultimately pharmacokinetics of the final radiolabeled PSMA inhibitor.
At the moment, 99m Tc-MIP-1404 seems to be the tracer of choice for SPECT imaging of prostate cancer, while 68 Ga-PSMA-11 is one of the most promising tracers for PET imaging of patients with this disease. Due to the fact that 68 Ga-PSMA-11 can be used only for diagnostic purposes, two

Image-Guided Surgery of Prostate Cancer Lesions Using 111 In-Labeled PSMA Inhibitors
The promising results of the diagnostic and endoradiotherapeutic approaches which have been successfully applied into the clinical setting using PSMA-based radiotracers rendered several research groups to make use of suitable PSMA-based radiotracers for other therapeutic strategies, such as the image-guided surgery of prostate cancer lesions. 111 In-PSMA I&T 111 In-PSMA-617 are the probes which have been used in two different clinical trials for radio-guided surgery for the detection of lymph node metastasis [117,142,157]. Both trials showed that all PET-positive lesions were also detected by PSMA-radio-guided surgery. Furthermore, tracer uptake and median gamma probe counts were significantly different between regions with lymph node metastases and other normal tissues. Overall, these studies showed that the gamma probes measurements could be considered as an indicator for lymph node metastasis and that radio-guided surgery may consequently lead to improvements with respect to the resection of prostate cancer.

Conclusions
Targeting the integral membrane glycoprotein PSMA by using radiometal-based PSMA inhibitors shows a great promise for diagnosis, (re)staging and therapy of prostate cancer. Specific PSMA-based ligands show high potential for initial staging, lymph node staging, restaging and therapy of prostate cancer. Between the different groups of PSMA inhibitors which have been developed so far, the peptidomimetic structure Lys-urea-Glu is the most successful biomolecule for utilization in radiopharmaceutical development. Furthermore, the choice of the chelator significantly affects the stability, affinity, lipophilicity and ultimately pharmacokinetics of the final radiolabeled PSMA inhibitor.
At the moment, 99m Tc-MIP-1404 seems to be the tracer of choice for SPECT imaging of prostate cancer, while 68 Ga-PSMA-11 is one of the most promising tracers for PET imaging of patients with this disease. Due to the fact that 68 Ga-PSMA-11 can be used only for diagnostic purposes, two modified PSMA inhibitors, PSMA-617 and PSMA I&T, have been developed. The superiority of PSMA-617 and PSMA I&T compared to PSMA-11 is that they can be efficiently labeled with both 68 Ga for PET, as well as, 177 Lu for radionuclide therapy. Therefore, both have the potential to be used as theranostic pairs. Although PSMA-directed radionuclide therapy shows great promise for implementation in routine clinical practice, the limited number of studies so far emphasizes the need for further controlled clinical trials for assessing the clinical value of 177 Lu-PSMA-617 and 177 Lu-PSMA I&T. Finally, image-guided surgery of prostate cancer lesions using 111 In-labeled PSMA inhibitors seems to be an appealing approach for detection of lymph node metastasis.