Radiolabeled PSMA Inhibitors

Simple Summary Prostate cancer remains one of the leading causes of cancer death in men worldwide. Despite the recent success in the development and clinical application of radiopharmaceuticals targeting the prostate-specific membrane antigen (PSMA) for diagnosis and endoradiotherapy of prostate cancer, more research is ongoing to further investigate and improve patient care and quality of life. Herein, an overview of novel developments and applications for small molecule and low-molecular weight radiolabeled PSMA inhibitors with an outlook to clinical translation is given. Abstract PSMA has shown to be a promising target for diagnosis and therapy (theranostics) of prostate cancer. We have reviewed developments in the field of radio- and fluorescence-guided surgery and targeted photodynamic therapy as well as multitargeting PSMA inhibitors also addressing albumin, GRPr and integrin αvβ3. An overview of the regulatory status of PSMA-targeting radiopharmaceuticals in the USA and Europe is also provided. Technical and quality aspects of PSMA-targeting radiopharmaceuticals are described and new emerging radiolabeling strategies are discussed. Furthermore, insights are given into the production, application and potential of alternatives beyond the commonly used radionuclides for radiolabeling PSMA inhibitors. An additional refinement of radiopharmaceuticals is required in order to further improve dose-limiting factors, such as nephrotoxicity and salivary gland uptake during endoradiotherapy. The improvement of patient treatment achieved by the advantageous combination of radionuclide therapy with alternative therapies is also a special focus of this review.


Quality Issues and Technical Improvements
Although radiolabeled PSMA inhibitors are used extensively for clinical applications, including radiotracers for which quality standards have been established by legally applicable specific monographs [43], special care needs to be taken during radiosynthesis and quality control of the radiopharmaceuticals. Iudicello et al. showed that PSMA-11, which is used as a reference standard and precursor for the synthesis of [ 68 Ga]Ga-PSMA-11, when dissolved in acidic aqueous solution forms a nat Fe-PSMA-11 complex (a side product with two diastereomeric forms similar to the ones previously reported for [ 68 Ga]Ga-PSMA-11) resulting thus, in lower radiochemical yield, chemical purity and radiochemical purity [91,92]. As a consequence, for storage of the PSMA-11 solution, EDTA was added to transchelate the Fe iron from the HBED-CC chelator.
Five-membered ring systems can be formed as radioactive side-products by a thermally mediated and pH-dependent condensation reaction of the Glu-urea-Lys binding motif during synthesis of [ 177 Lu]Lu-PSMA-617 [93]. These side-products did not show any binding affinity to PSMA and were rapidly excreted via the kidneys from the body of five patients. The synthesis of [ 177 Lu]Lu-PSMA-617 could be optimized by adjustment of pH and temperature and subsequent reduction of side-products, however, the risk of formation of unwanted radioactive side-products for PSMA inhibitors bearing the Glu-urea-Lys binding motif still remains [93].
Reaction temperature during radiolabeling plays an important role as shown above. Attempts have been made to replace DOTA, which requires time and heating for complexing radiometals, with a different chelator that allows low temperature radiolabeling in particular for the alpha-particle emitting isotope 225 Ac. Thiele et al. showed that the eighteen-membered macrocyclic chelator macropa is suitable to complex 225 Ac at room temperature (RT) in 5 min with high stability in human serum over 8 days [94]. Macropa was then conjugated to the PSMA/albumin binding ligand RPS-070 and radiolabeled with 225 Ac within 20 min at RT. [ 225 Ac]Ac-macropa-RPS-070 was tested in LNCaP tumor bearing mice where it showed high in vivo stability and high kidney uptake after 4 h (52 ± 16% ID/g), which then cleared rapidly, while tumor uptake (13 ± 3% ID/g) washout was slower. Reissig et al. expanded the "macropa"-principle by adding one or two alkyne moieties for conjugation of biomolecules using the copper-catalyzed azide-alkyne-cycloaddition [95]. A PSMA binding block was attached via click chemistry to obtain either the monomer mcp-M-PSMA or the dimer mcp-D-PSMA. Radiolabeling was done within 1 h at RT, both [ 225 Ac]Ac-mcp-M-PSMA and [ 225 Ac]Ac-mcp-D-PSMA were evaluated in vitro and in vivo using LNCaP cells and the corresponding xenograft mouse model. It is noticeable that the tumor uptake for the monomer was higher within the first hour and then decreased while the tumor uptake for the dimer constantly increased within 24 h making it more suitable for a therapy approach [95].
Radiopharmaceuticals are also prepared via so-called cold kits, for a long time established in Nuclear Medicine by reconstitution with the eluate of 99m Tc generators [96]. More recently, this approach has seen a wider application also for PET radiopharmaceuticals with three commercially available kits using either PSMA-11 (Illuccix and isoPROtrace-11) or THP-PSMA (GalliProst) for radiolabeling with the eluate of a 68 Ge/ 68 Ga generator [97]. Illuccix has been approved by the Australian Therapeutic Goods Administration (TGA) on 2nd November 2021 [98].

Salivary Gland Uptake
Uptake in the salivary glands is the dose-limiting factor during endoradiotherapy with small-molecule PSMA inhibitors, resulting in (partially reversible) xerostomia when applied with 177 Lu, and severe to persistent xerostomia when applied with 225 Ac [50,51]. The quality of life for patients can, therefore, be significantly impaired despite preventive strategies, e.g., injection of botulinum toxin, external cooling, or gustatory stimulation have been described [99][100][101][102][103]. The uptake mechanism of PSMA inhibitors in salivary glands has not been thoroughly investigated to date, a more comprehensive review concerning known mechanisms is described by Heynickx et al. [103]. Tönnesmann et al. showed in quantitative autoradiography experiments that in the salivary glands of pigs for smallmolecule and low-molecular weight PSMA inhibitors the uptake is highly non-specific and at the same time specific while the exact ratio is not yet understood [104]. Attempts to minimize the uptake of the PSMA-targeted radiopharmaceutical in the salivary glands have been made following the modifications at the inhibitor component. This resulted in low salivary gland uptake and very low tumor uptake [105]. Two independent preclinical studies demonstrated that additional substance amount-dependent administration of nonradioactive PSMA ligand (PSMA-11 or DCFPyL, respectively) and reduction of the effective molar activity significantly reduces the radioligand uptake ([ 177 Lu]Lu-PSMA-617 or [ 18 F]DCFPyL, respectively) in the salivary glands (Table 2) [106,107]. However, these promising results need to be confirmed in further preclinical and clinical studies.

PSMA Inhibitors for Radioguided Surgery
In addition to the properties for noninvasive single photon emission computed tomography (SPECT) or positron emission tomography (PET) imaging, the radiation exposed from the radionuclide bound to the PSMA inhibitor can also be used for the detection of tumor entities during intraoperative surgery using a gamma probe in a practice named radioguided surgery (RGS) [108].
As an undesirable consequence of RGS, personnel are typically exposed to constant radiation from the radiopharmaceutical. The occupational radiation exposure for workers involved in RGS using [ 99m Tc]Tc-PSMA I&S was determined with a maximum dose of 0.32 mSv/year for 100 procedures [118]. When laparoscopic gamma probes are used for sentinel lymph node dissection [119] they suffer from practical limitations, due to rotational impairment of the probe [120]. This limitation has been circumvented by the development of a drop-in probe, which can be controlled by grasps of the DaVinci robotic-assisted surgery system, thereby providing additional rotational freedom and reducing the radiation dose [121]. A first-in-human application has been reported for [ 99m Tc]Tc-PSMA I&S after preoperative [ 68 Ga]Ga-PSMA-11 PET/CT [122]. In an experimental study with seven patients, the drop-in concept was tested with [ 68 Ga]Ga-PSMA-11 using a beta probe instead of a gamma probe. After an additional dose of approximately 70 MBq [ 68 Ga]Ga-PSMA-11 per patient prior surgery, the obtained surgical specimens were evaluated ex vivo using a DaVinci robotic system equipped with the drop-in beta probe [123]. Radiation dose to workers limits the use to 62 procedures/year of this technique, but improvements towards a lower occupational radiation dose are feasible. Alternative imaging technologies focusing on beta-radiation emission, e.g., Cerenkov luminescence imaging using [ 68 Ga]Ga-PSMA-11 are currently under early clinical investigation [124][125][126][127][128][129][130].

PSMA Inhibitors for Fluorescence-Guided Surgery and/or Targeted Photodynamic Therapy
Aside from radionuclide detection for radioguided surgery, an attractive alternative for intraoperative guided surgery is radiolabeled molecules functionalized with fluorescent dyes for optical/fluorescence imaging [131][132][133][134]. A variety of commercially available dyes with different absorptions and emission spectra used for dual-labeled imaging agents has been reported [135]. Dual-labeled agents allow preoperative nuclear imaging (prestaging/staging) and subsequent treatment planning while fluorescence imaging makes surgical tumor resection possible and more accurate due to the high spatial resolution.
[ 68 Ga]Ga-Glu-urea-Lys-HBED-CC-IRDye800CW showed an increased tumor uptake in mice compared to [ 68 Ga]Ga-PSMA-11 (13.66 ± 3.73% ID/g vs. 4.89 ± 1.34% ID/g) [136,137]. Non-radiolabeled Glu-urea-Lys-HBED-CC-IRDye800CW has been investigated in healthy pigs using a DaVinci robotic system. A PSMA-specific fluorescence signal has been shown in vivo and ex vivo for both mice and pigs. More recently, the linker design of Glu-urea-Lys-HBED-CC-IRDye800CW was modified by insertion of histidine-and glutamic acid spacers ((HE) 3 ) near the PSMA binding motif to further reduce background uptake in non-target tissue and to accelerate excretion [138,139]. Glu-urea-Lys-(HE) 3 -HBED-CC-IRDye800CW (PSMA-914) was used in a proof-of-concept study, with a first, pre-operative PET/CT imaging, 1-day prior surgery in a patient with high-risk prostate carcinoma and a second PET/CT imaging, after the surgical removal of the primary tumor [140]. 68 Ga-radiolabeled PSMA-914 and PSMA-914 alone were administered 1-h prior to PET imaging and DaVinci robot-assisted prostatectomy respectively.
One limitation/challenge regarding the utilization of fluorescent dyes is that the detection efficacy of tumor lesions is limited to superficial tissue [142,143]. Certain dyes can also be used for photodynamic therapy [144]. In this approach, a dye can also simultaneously act as a photosensitizer which is selectively activated with long wavelength light, subsequently causing reactive oxygen species (ROS) formation leading to the induction of immediate cell death [108]. Derks et al. synthesized a variety of Glu-urea-Lys-based PSMA-targeting ligands conjugated to IRDye700DX [145]. The most promising candidate PSMA-N064 consists of a DOTAGA chelator, additional glutamic acid residues in the linker structure, a Glu-urea-Lys binding motif, and a fluorophore. After radiolabeling of PSMA-N064 with 111 In, an increased tumor uptake of up to 2 h post injection (13.1 ± 2.3% ID/g) was determined in mice bearing LS1754T colon carcinoma cells transfected with human PSMA (LS1754T-PSMA). The tumors could be visualized using SPECT/CT and near infrared fluorescence scanners. Interestingly, the incorporation of the fluorophore leads to a higher internalization in tumor cells than the corresponding ligand without the dye (15.1 ± 0.8% ID/g vs. 6.7 ± 1.1% ID/g). Accordingly, the fluorophore parts may have a positive impact on tumor uptake [136,145]. Samples obtained from biopsies of human normal tissue could be distinguished from tumor tissue after incubation with PSMA-N064 using fluorescence imaging. In a proof-of-concept study for targeted photodynamic therapy, the incubation of LS1754T-PSMA cells with PSMA-N064 and exposure to NIR light resulted in reduced cell viability (34 ± 3.2%).

PSMA + Albumin
PSMA inhibitors used for endoradiotherapy, such as PSMA-617 and PSMA I&T are rapidly excreted via the kidneys, increasing the radiation exposure to these organs [146]. Introducing albumin-binding groups into the chemical structure of small molecules can increase their blood circulation time, which can potentially lead to enhanced tumor uptake, while the reduction of injected activity results in lower non-target tissue doses [147,148].
The 4-(p-iodophenyl)acetic acid moiety was employed as an albumin-binding group by Kelly et al. in radiotracers bearing a DOTA chelator and a urea-based PSMA binding-motif radiolabeled with 131 I (RPS-025) and 177 Lu (RPS-063 & RPS-067). This modification lead to a 4-fold higher tumor uptake compared to [ 177 Lu]Lu-PSMA-617, however, kidney-uptake was still significantly high [149,150]. It was observed that the use of polyethylene glycol (PEG) linkers of varying lengths had a dramatic impact on PSMA binding affinity as well as blood clearance [150]. Replacement of the albumin-binding group 4-(p-iodophenyl)acetic acid moiety with 4-(p-iodophenyl)butanoic acid (RPS-072) further reduced radiation exposure to the kidney and blood clearance [151].
Benešová et al. used the 4-(p-iodophenyl)butanoic acid moiety as an albumin-binding group in a similar way and extended the linker entity with aspartate residues to counterbalance the lipophilicity of the albumin-binding group [152]. Three radioligands consisting of a DOTA chelator, the aforementioned linker, the albumin-binding group and a urea-based PSMA binding motif (PSMA-ALB-02, PSMA-ALB-05 and PSMA-ALB-97) were radiolabeled with 177 Lu and evaluated against [ 177 Lu]Lu-PSMA-617 in PC-3 PIP tumor bearing mice with all three ligands showing a high tumor-to-blood ratio but still high kidney uptake. These results were further optimized by the replacement of the 4-(p-iodophenyl)butanoic acid moiety with (p-tolyl)butanoic acid [153], as the latter demonstrated a reduced albuminbinding affinity [147]. The most promising candidate PSMA-ALB-56 was compared against PSMA-617 in a therapy study of PC-3 PIP tumor bearing mice.  [154]. In a recently published clinical study, ten patients with metastatic castration resistant prostate cancer (mCRPC) received [ 177 Lu]Lu-PSMA-ALB-56 endoradiotherapy with a higher absorbed dose in tumor lesions and similar salivary gland-uptake compared to PSMA-617 and PSMA I&T [155]. Nevertheless, kidney and red marrow uptake in these patients remains high, this demands further preclinical optimization of albumin-binding PSMA radioligands (e.g., upon utilization of ibuprofen as an albumin-binding entity) [156][157][158].
Finally, three additional bispecific heterodimers based on RM2 peptide sequence (D-Phe 6 -Gln 7 -Trp 8 -Ala 9 -Val 10 -Gly 11 -His 12 -Sta 13 -Leu 14 -NH 2 ) and PSMA-617 are reported [183]. The two pharmacophores were linked via the spacer: X-triazolyl-Tyr-PEG 2 , where X = 0 (Hd-12), PEG 2 (Hd-13), (CH 2 ) 8 (Hd-14). The resulting heterodimers were radio-iodinated and evaluated in vitro for binding specificity, cellular retention, and affinity. In vivo specificity and tumor uptake for all heterodimers was studied in PC-3 and LNCaP xenografts (Table 3), while [ 125 I]I-Hd-13 was also evaluated in PC-3 PIP xenografts where it showed high tumor accumulation (30-35% ID/g at 3 h p.i.). However, this was followed by high kidney radioactivity values like the first heterodimer: [ 68 Ga]Ga-Hd-1. Two heterobivalent imaging agents targeting PSMA and integrin-α v β 3 surface markers, both overexpressed in certain tumor epithelium and/or neovasculature were synthesized and evaluated by Shallal et al. [184]. Integrins α v β 3 are considered an ideal target for the development of radioligands, since they are overexpressed on tumor vasculature due to angiogenesis, and on the cell membrane in various tumors, i.e., ovarian cancer, neuroblastoma, breast cancer and melanoma [185]. The tripeptide RGD (arginine-glycineaspartic) amino acid sequence and its cyclic RGD (cRGD) analogs specifically bind to the integrin α v β 3 receptor and thus have provided the platform for the development of various radioligands [185]. The heterodimers consisted of Lys-CO-Glu-OH PSMA binding motif connected to the Lys of a cyclic cRGDfK peptide (cyclo-(Arg-Gly-Asp-D-Phe-Lys)) and coupled either to the DOTA chelator for radiolabeling  or to the dye IRDye800CW (Hd-16). In vitro testing in PC-3 PIP and U87-MG cells and in isolated proteins showed low affinity with high IC 50 values for the both PSMA and α v β 3 receptors, i.e., Hd-15: PC-3 PIP, IC 50 = 479 nM, U87-MG, IC 50 = 1536 nM, Hd-16: integrin α v β 3 IC 50 = 90 nM. In vivo, optical imaging and ex vivo biodistribution studies for Hd-16 showed specific tumor uptake for PC-3 PIP tumors expressing PSMA, and U87-MG tumors expressing integrin α v β 3 . Tumor uptake was dose-dependent, and even at the lowest dose (0.5 nmol) the tumors were clearly visible. However, this study did not include a metalated or a radiolabeled version of Hd-15 [184].
The isotopic exchange chemistry for the fluorine isotopes 19 F/ 18 F has been described by Schirrmacher et al. using silicon-fluoride-acceptor (SiFA) building blocks [190]. This concept has been adopted for PSMA inhibitors, which also introduce a chelator into the molecule in close proximity to the SiFA motif in order to increase hydrophilicity [191]. This permits the radiolabeling of the molecule with either 18 F at the SiFA building block or with a radiometal of choice (e.g., 68 Ga, 177 Lu, 225 Ac) at the chelator while the moiety not used for radiolabeling is occupied with the non-radioactive 19 F or metal, which has been called by the authors radiohybrid PSMA (rhPSMA) inhibitors. Wurzer et al. developed a series of these rhPSMA inhibitors, with rh-PSMA-7 being the most promising candidate being radiolabeled with 18 F, bearing nat Ga within the chelator and being clinically evaluated in a number of patients with primary and biochemical recurrence of PCa [191][192][193][194]. Further improvement of rh-PSMA-7 has been achieved by investigation of its four stereoisomers and their influence on pharmacokinetics [83]. The resulting lead candidate [ 18 F]Ga-rh-PSMA-7.3 showed the highest tumor accumulation and low uptake in non-target tissues. This was confirmed in healthy volunteers determining biodistribution and radiation dosimetry data (NCT03995888) and in a larger cohort of patients [84,[195][196][197]. The molar activity of [ 18 F]Ga-rh-PSMA-7.3 had a minor influence on the biodistribution [198,199]. Three clinical trials using [ 18 F]Ga-rh-PSMA-7.3 are either ongoing or will commence in the near future (NCT04978675/NCT04186819/NCT04186845). The therapeutic counterpart [ 177 Lu]Lu-rhPSMA-7.3 showed a higher anti-tumor effect and longer median survival in C4-2 xenograft-bearings SCIDS than [ 177 Lu]Lu-PSMA I&T [85] followed by a pre-therapeutic dosimetry study in six mCRPC patients confirming the preclinical results [86].
Another approach of isotopic exchange has been reported for eight PSMA inhibitors by substituting 19 F with 18 F at a trifluoroborate group [200]. This concept has been extended to the radiohybrid ligand DOTA-AMBF 3 -PSMA [201]. The PSMA-617 pharmacophore was used as a starting point, then a lysine-trifluoroborate moiety was introduced and linked to a DOTA-chelator allowing radiolabeling with either 18 F or a radiometal. DOTA-AMBF 3 -PSMA radiolabeled with 18 F/free, 18 F/ nat Cu, nat F/ 64 Cu and nat F/ 177 Lu was preclinically evaluated in LNCaP xenografts with tumor uptake > 10% ID/g for all radiotracers. [ 64 Cu]Cu-DOTA-AMBF 3 -PSMA showed increased liver uptake, most likely due to transchelation of 64 Cu from the DOTA-chelator which has also been observed for [ 64 Cu]Cu-DOTA and [ 64 Cu]Cu-PSMA-617 [202,203]. An increased liver uptake was seen in patients scanned with [ 64 Cu]Cu-PSMA-617 2 h and 22 h post injection as well as in a biodistribution and radiation dosimetry study using [ 64 Cu]CuCl 2 [204,205].

Potential Radionuclides for the Future Use of PSMA Inhibitors
While radionuclides like 68 Ga,18 F,or 177 Lu have become a sort of routine in the use of radiolabeled PSMA inhibitors, other radionuclides are gaining attention due to their specific properties [206][207][208]. For example, 225 Ac can play a pivotal role in targeted alpha therapy (TAT) of prostate cancer [48,51,78,[209][210][211][212][213][214][215], but additional preclinical studies and prospective clinical trials need to be performed to secure its safety and efficacy [216][217][218][219][220][221]. The importance of 225 Ac-labeled radiopharmaceuticals is affirmed by efforts to establish GMP-compliant procedures for production and quality control [222][223][224][225][226]. Regulatory aspects on the supply of radionuclides for routine clinical application represent a hurdle that needs to be considered for the translation of new radiopharmaceuticals [227,228].

211 Astatine
211 At is a radiohalogen with a half-life of 7.2 h suitable for TAT using small molecules. Several PSMA radioligands have been radiolabeled with 211 At and indicated improved survival rates in PC-3 PIP PSMA-positive tumor bearing mice [229,230]. Although the inclusion of albumin binding moieties increases blood retention and reduces kidney uptake [149], dehalogenation and nephrotoxicity have been the limiting factors for clinical translation. The latest development 211 At-3-Lu [230] showed improved toxicity data for kidney uptake and reduced off-target uptake in organs like the stomach and salivary glands. The infrastructure for the production of 211 At-labeled radiopharmaceuticals is complex though, efforts are made to make them more widely available [231,232], such as the NOAR COST action, which has the goal to create a European Network for Optimized Astatine labeled Radiopharmaceuticals.

203/212 Lead
203 Pb and 212 Pb form an ideal theranostic pair for SPECT imaging and TAT with half-lives of 51.9 h and 10.6 h respectively. The production methods for 203 /212 Pb have been improved making them available for potential clinical application [236]. Dos Santos et al. developed four PSMA inhibitors (CA008, CA009, CA011 and CA012) with p-SCN-Bn-TCMC and DO3AM chelators bearing the PSMA-617 binding motif and linker structure [237]. The best ligand PSMA-CA012 radiolabeled with 203 Pb showed similar tumor uptake like [ 68 [238,239]. Dose-dependent treatment efficiency of [ 212 Pb]Pb-NG001 was confirmed in multicellular C4-2 spheroids and the corresponding mouse model with no long-term toxicity effects [240]. Apart from DOTA and TCMC, pyridine-based cyclen analogs (DOTA-1Py, DOTA-2Py and DOTA-3Py) [236] may be a better chelator alternative for 212 Pb and its daughter isotopes for avoiding recoil effects [241].

149/152/155/161 Terbium
Terbium, also called the "Swiss Army Knife" of Nuclear Medicine with its quadruplet of radioisotopes [242] with suitable properties for imaging with PET ( 152 Tb/ 149 Tb) and SPECT ( 155 Tb) and endoradiotherapy with alpha ( 149 Tb) and beta − /Auger electrons ( 161 Tb), has been investigated by the group at Paul Scherrer Institute (PSI). Terbium radioisotopes have been used in combination with PSMA-617 in PSMA-positive PC-3 PIP tumor cells.
[ 152 Tb]Tb-PSMA-617 has been evaluated in vitro and in vivo and successfully applied firstin-human in a patient with metastatic castration-resistant prostate cancer [76]. Suppression of tumor growth has been demonstrated using [ 149 Tb]Tb-PSMA-617 in TAT in mice bearing PC-3 PIP tumor cells [243]. In a preclinical study, [ 161 161 Tb vs. 177 Lu [244,245] opening the way for clinical translation. The additional therapeutic effect of 161 Tb due to Auger electrons was confirmed by a computational approach using a microdosimetry model [246]. However, despite these very promising results, the production of terbium radioisotopes for clinical application remains challenging [247,248].

89 Zirconium
Vázquez et al., modified PSMA-617 by replacing DOTA with DFO as chelator making it suitable for radiolabeling with 89 Zr [249]. 89 Zr is a positron-emitter with a physical halflife of 3.27 days. The aim of this study was to detect low PSMA-expressing lesions which are negative on PSMA PET scans using short-lived radiotracers, such as [ 68  Noor et al. [251] conjugated the Lys-ureido-Glu binding motif to the desferrioxamine B squareamide ester derivative H 3 DFOSq, leading to two monovalent and two bivalent DFO-Sq-lysine-ureido-glutamate derivatives with different linker structures to be radiolabeled with 89 Zr and 68 Ga. The bivalent ligands performed superior in LNCaP tumor models compared to the monovalent ligands. The bivalent ligand bearing an aromatic hydrophobic linker (H 3 DFO-tren-bis(glut-PAMBA-Lys-ureido-Glu)) showed the highest tumor uptake when radiolabeled with 89 Zr and 68 Ga compared against [ 68 Ga]Ga-PSMA-11 [252] 1 h after injection (9.33 ± 0.33% IA/g vs. 10.8 ± 1.3% IA/g vs. 4.89 ± 1.34% IA/g).

Conclusions
The feasibility of theranostics with PSMA-targeting radiopharmaceuticals has been successfully demonstrated in the last decade, upon their radiolabeling using pairs of radionuclides with suitable properties for imaging with PET or SPECT and endoradiotherapy with alpha and beta − /Auger electrons. Radioguided and fluorescence-guided surgery should be an additional treatment option for PCa patients, in this direction PSMA ligands for radio-/fluorescence-guided surgery and/or targeted photodynamic therapy are being investigated. In order to further improve the efficiency and efficacy of the existing PSMA radioligands for PCa diagnosis and radionuclide therapy several structural modifications have been suggested for reducing their uptake in off-target tissues or utilizing multitargeting (i.e., albumin, GRPr and integrin α v β 3 ). Addressing additional targets like GRPr with one radiotracer could reduce the number of false-negative findings. New radionuclides in combination with PSMA-binding structures are also being explored, but care should be taken on their radiobiological effects, availability for mass production and cost.