The Theranostic Optimization of PSMA-GCK01 Does Not Compromise the Imaging Characteristics of [99mTc]Tc-PSMA-GCK01 Compared to Dedicated Diagnostic [99mTc]Tc-EDDA/HYNIC-iPSMA in Prostate Cancer

Purpose Radiolabeled PSMA-ligands play a major role in today’s nuclear medicine. Since approval of [177Lu]Lu-PSMA-617 for therapy of metastatic prostate cancer, availability of 177Lu became bottleneck of supply due to the high demand. Recently, a theranostic PSMA-ligand, PSMA-GCK01, was developed which can be labeled either diagnostically with 99mTc or therapeutically with 188Re with both nuclides available from well-known generator systems. This novel tracer might aid to overcome aforementioned supply limitations. In this investigation, the biodistribution and general imaging characteristics of [99mTc]Tc-PSMA-GCK01 were compared with the diagnostic reference compound [99mTc]Tc-EDDA/HYNIC-iPSMA in patients with advanced stage prostate cancer. In addition, the binding of both ligands to PSMA was analyzed at the molecular level using molecular docking. Procedures Two cohorts (n = 19 vs. n = 21) of patients with metastatic castration-resistant prostate cancer matched for age, tumor stage, and Gleason score underwent a planar gamma camera imaging with [99mTc]Tc-EDDA/HYNIC-iPSMA or [99mTc]Tc-PSMA-GCK01 prior to PSMA-ligand therapy for PSMA-phenotyping. The imaging data were retrospective analyzed for salivary gland, kidney, liver, soft tissue, and tumor uptake on a semi-automated ROI-analysis using HERMES Medical Solutions AB (HMS, Sweden). Results The data sets were semi-automated quantified on a ROI-based analysis. The tumor-to-background presented equal results of [99mTc]Tc-PSMA-GCK01 compared to [99mTc]Tc-EDDA/HYNIC-iPSMA. The physiological PSMA-positive organs like salivary gland presented also equal uptake in counts/MBq (salivary gland median 9.48 [99mTc]Tc-PSMA-GCK01 vs. median 9.11 [99mTc]Tc-EDDA/HYNIC-iPSMA), while liver-to-kidney ratio presented a slight shift to the liver parenchyma using [99mTc]Tc-PSMA-GCK01 (0.83) compared to [99mTc]Tc-EDDA/HYNIC-iPSMA (0.55) with no statistical significance. This is in agreement with the results from the docking study revealing only a minor difference in the docking scores for both ligands. Conclusions The novel theranostic tracer [99mTc]Tc/[188Re]Re-PSMA-GCK01 demonstrates comparable general imaging characteristic with the reference compound [99mTc]Tc-EDDA/HYNIC-iPSMA. These results pave the way for the PSMA-targeting imaging and theranostic agents for a broader, rather low-cost, generator applied radio-ligand therapy utilization.

Recently, a novel theranostic PSMA-ligand GCK01, that can be labeled with diagnostic 99m Tc and therapeutic 188 Re, has been introduced [13].The generator-based beta-emitter 188 Re may aid to overcome supply limitations of 177 Lu and decrease the production costs for PSMA-RLT in comparison to 177 Lu.In contrast to the previous Tc-labeled ligands, GCK01 had to be refined with regard to labeling yield under more challenging rhenium conditions as well as in terms of tracer uptake in the dose-limiting organs.In addition, the in vivo stability has been improved, which represents a pivotal cornerstone for the therapeutic approach.
The aim of this work is to evaluate whether the optimizations of PSMA-GCK01 for the therapeutic application might have introduced an unfavorable trade-off regarding biodistribution in normal organs and general imaging characteristics.

Patient Population
We retrospectively included two cohorts of 21 consecutive patients with mCRPC who were considered to obtain systemic therapy with [ 177 Lu]Lu-PSMA-617 and underwent whole body scans with [ 99m Tc]Tc-EDDA/HYNIC-iPSMA or [ 99m Tc]Tc-PSMA-GCK01 in the Department of Nuclear Medicine of University Hospital Heidelberg between 01/2021 and 02/2022.Two subjects from [ 99m Tc]Tc-EDDA/ HYNIC-iPSMA cohort were censored due to ineligibility for further quantitative assessment because of improper image acquisition with motion artifacts.Informed consent was obtained from all the patients.The exams were conducted according to national regulatory on the basis of compassionate use, when a PSMA-PET scan was not available within reasonable time and the patient had urgently to be evaluated regarding PSMA therapy.Prospective clinical trial registration is not required for compassionate care that is performed under individual medical indication.The Ethical Committee of the University Hospital Heidelberg approved the retrospective Evaluation (permission S-732-18).

Image Analysis
Round regions of interest (ROIs) (1.6 cm 2 ) were placed in the areas with physiological tracer uptake (parotid gland (Gl.parotis) and kidneys pairwise, liver, and in the right thigh as a background) in conjugate view of planar anterior and posterior whole-body scans of the respective subjects and the geometric mean was calculated (HERMES Medical Imaging Suite v6.1, HERMES Medical Solutions AB, Strandbergsgatan 16, 112 51 Stockholm, Sweden).In addition to the measurement of physiological biodistribution in the healthy organs, three random metastases were visually identified and quantified in the same way.

Statistical Analysis
We performed the analysis of tracer uptake (measured counts divided by the injected activity in MBq), tumor-tobackground, as well as liver-to-kidney ratios with descriptive statistics and box-plot analysis using SigmaPlot version 14.0 (Systat Software, Inc., San Jose, CA, USA).

Molecular Docking
The crystal structure of PSMA was retrieved from the Protein Data Bank (PDB #5O5T) and processed, i.e., hydrogen atoms were added to the protein, ligand, and water molecules, using the protein preparation wizard of Maestro, included in the Schrödinger 2022-3 package.
To simulate the environment of the binding site as realistically as possible, we kept the ions and all water molecules located in the buried part of the binding pocket.The compounds [ 99m Tc]Tc-PSMA-GCK01 and [ 99m Tc]Tc-EDDA/HYNIC-iPSMA were drawn using Marvin Sketch version 21.9 (https:// chema xon.com/ marvin) and exported in 3D to pdb format.The following preparation and docking steps were performed with the software of the ADFR suite version 1.0 (https:// ccsb.scrip ps.edu/ adfr/): First, Gasteiger charges were added to the protein, including the ions Zn 2+ , Ca 2+ , and Cl − and the water molecules, using the prepare_receptor script.Then the docking grid was created with AGFR using the co-crystallized ligand 9OT to determine the size and position of the grid box.The prepare_ligand script from the ADFR suite was used to add Gasteiger charges and to define rotatable bonds of the compounds [ 99m Tc]Tc-PSMA-GCK01 and [ 99m Tc] Tc-EDDA/HYNIC-iPSMA.Since Tc and Re are unfamiliar elements for Autodock, we changed the atom type Tc to Mn in the pdbqt files without affecting any atomic coordinates and assigned a charge of + 2 to Mn.Finally, we performed the docking run with 120 searches and a maximum number of 5 million evaluations per search using ADFR.

Results
Apart from the data of the two [ 99m Tc]Tc-EDDA/HYNIC-iPSMA patients that had to be removed, all other data sets were analyzed the way described above.Our subjects were matched in regard to age ([ 99m Tc]Tc-EDDA/HYNIC-iPSMA 71 ± 10 years; range 55-94 years or [ 99m Tc]Tc-PSMA-GCK01 73 ± 8 years; range 59-84 years), tumor stage, and total Gleason score so that we assume here a comparability of both cohorts (Table 1).

Molecular Docking Studies
In order to examine the binding of both ligands in the PSMA receptor pocket, we conducted molecular docking studies.As a measure for the binding affinity, docking scores of − 14.6 kcal/mol for [ 99m Tc]Tc-PSMA-GCK01 and − 13.6 kcal/mol for [ 99m Tc]Tc-EDDA/HYNIC-iPSMA were obtained for the top-ranked conformations (Fig. 4): Both ligands are deeply embedded in the binding pocket, with the urea group interacting with the zinc(II) active site and the glutamate moiety pointing toward the glutamate pocket, while the 2-naphtylalanine occupies a lipophilic cavity and the chelators stick out of the entrance funnel (Fig. 4).

Discussion
Prostate cancer has an increasing incidence in the world so that there is a need of cost-effective PSMA imaging and therapy [4,14].Besides PSMA PET tracers, several [ 99m Tc]Tc-labeled PSMA ligands have been developed in recent years [15].Contrary to PET, gamma scanners are broadly available throughout the world suggesting the usefulness of compatible radioligands [6,16].
The first human data with [ 99m Tc]Tc-MIP-1404 and [ 99m Tc]Tc-MIP-1405 showed very promising results [6,17].In 2017, Ferro-Flores et al. introduced [ 99m Tc]Tc-EDDA/HYNIC-iPSMA which offers a rapid blood clearance, the ability to detect prostate cancer, as well as its metastases, and has mainly kidney elimination with less liver uptake than [ 99m Tc]Tc-MIP-1404 [18].Further investigations with [ 99m Tc]Tc-EDDA/HYNIC-iPSMA demonstrated high detection rates for PSMA positive lesions, especially in the setting of biochemically recurrence of prostate cancer but a lower sensitivity compared with PET PSMA tracer [4,19].The main advantage of the new 99m Tc-labeled tracer group is the detection of PSMA positive lesions for planning radio-ligand therapy, for instance, with [ 177 Lu]Lu-PSMA-617 [8] (Fig. 5).A specific radioligand therapy of prostate cancer can only be performed in presence of PSMA-positive phenotype of tumor lesions [20].The sensitivity of PSMA imaging plays a subordinate role in this context, rather, the diagnostic and therapeutic compounds should be as similar as possible to predict kinetics and uptake of the latter [7,8].
The novel PSMA ligand [ 99m Tc]Tc-PSMA-GCK01 was optimized to provide a suitable basis for both 99m Tc-and 188 Re-labeling, thus, providing a seamless PSMA-targeted theranostic tandem [13].In this investigation we have analyzed the biodistribution of the diagnostic [ 99m Tc]Tc-PSMA-GCK01 in organs with physiologic PSMA-uptake and in metastases to assess the appropriateness for imaging, especially in comparison with [ 99m Tc]Tc-EDDA/HYNIC-iPSMA.The ROI-based analysis showed equal tracer uptake in salivary glands and background with [ 99m Tc]Tc-PSMA-GCK01 and [ 99m Tc]Tc-EDDA/HYNIC-iPSMA, respectively.
This observation is also in agreement with the results from our in silico docking study, revealing only minimal differences in the conformations of both molecules in the PSMA binding pocket, and, thus, only a minimal difference in the docking score of approx.1.0 kcal/mol.Considering the high structural similarity of both PSMA ligands, differing only in their chelator moiety and slightly in the linker region, this explains our earlier findings of a slightly higher affinity of [ 99m Tc]Tc-PSMA-GCK01 (Ki = 26 nm) toward PSMA when compared to [ 99m Tc]Tc-EDDA/HYNIC-iPSMA (Ki = 38 nm) [13].In the practical setting, both tracers had a relevant uptake in the kidneys and the liver.But in the case of [ 99m Tc]Tc-PSMA-GCK01, a minor shift of the uptake to the liver parenchyma was observed.This behavior is particularly interesting because in mice only minimal liver uptake was previously described [13].Differences in biodistribution between preclinical data and in humans have already been reported for PSMA tracers [21].Vallabhajosula et al. found a very high liver uptake for [ 99m Tc]Tc-MIP-1404 contrary to preclinical data [17].[ 99m Tc] Tc-EDDA/HYNIC-iPSMA was optimized for rapid blood clearance; however, more activity is cleared by the kidneys to the bladder [18].The rapid blood clearance of [ 99m Tc]Tc-EDDA/HYNIC-iPSMA is maintained with [ 99m Tc]Tc-PSMA-GCK01 but the slight predominant hepatobiliary route of excretion of [ 99m Tc]Tc-PSMA-GCK01 might allow a better diagnostic performance of local relapse or perivesical lymph node metastasis [22] since tracer retention in the bladder might complicate the proper discrimination of perivesical lesions.Further, the slight predominant hepatobiliary excretion could be particularly beneficial when the ligand is labeled with 188 Re in regard to radioligand therapy in terms of nephrotoxicity.
We have also compared the uptake of both tracers in the metastases.The analysis of tumor-to-background ratio presented equal results of [ 99m Tc]Tc-PSMA-GCK01 and [ 99m Tc] Tc-EDDA/HYNIC-iPSMA on the planar whole body projections.Thus, the slight preference for hepatobiliary clearance of [ 99m Tc]Tc-PSMA-GCK01 does not have a negative effect on the imaging properties of tumor lesions and rather offers an optimized biodistribution regarding radioligand therapy, as mentioned above.
The main advantage of [ 99m Tc]Tc-PSMA-GCK01 over [ 99m Tc]Tc-EDDA/HYNIC-iPSMA is its utilization for theranostic purposes by 188 Re labeling [13,15].This ensures the correlation between pre-therapeutic and therapeutic distribution and is expected to have a direct impact on the clinical routine.
Although PSMA PET radioligands and Tc-labeled PSMA SPECT tracers use the same target structure, there are differences in sensitivity [19] and lesion-to-liver ratio.The latter is important in case of eligibility for [ 177 Lu]Lu-PSMA therapy and needs to be adjusted if 99m Tc labeled tracers are used for the pre-therapeutic diagnostic [8,23].The tracer PSMA-GCK01 labeled with 99m Tc-/ 188 Re-theranostic tandem could solve this problem [13] without negatively affecting the biodistribution and general imaging characteristics compared with [ 99m Tc]Tc-EDDA/HYNIC-iPSMA.

Conclusions
[ 99m Tc]Tc-PSMA-GCK01 shows a similar uptake (counts/ MBq) in normal organs and background, consequently leading to equivalent contrast ratios and general uptake in tumor lesions compared to [ 99m Tc]Tc-EDDA/HYNIC-iPSMA. [ 99m Tc]Tc-PSMA-GCK01 presents a small shift from kidney to liver parenchyma in the clearance route, which is an appreciated characteristic regarding its therapeutic use but not a relevant factor for diagnostics.In sum, its additional appropriateness for use as a therapeutic agent does not compromise the general imaging characteristics of [ 99m Tc] Tc-PSMA-GCK01 compared to the already well-established [ 99m Tc]Tc-EDDA/HYNIC-iPSMA.

Fig. 5
Fig.5 Eighty-one-year-old male patient.This presents an extended prostate cancer undergoing a phenotyping for specific radio-ligand therapy with PSMA. a [ 99m Tc]Tc-MDP uptake in whole body projections, b [ 99m Tc]Tc-PSMA-GCK01 uptake in whole body projec-