[99mTc]Tc-DB15 in GRPR-Targeted Tumor Imaging with SPECT: From Preclinical Evaluation to the First Clinical Outcomes

Simple Summary Radiolabeled gastrin-releasing peptide receptor (GRPR)-antagonists have been proposed for diagnostic imaging and radionuclide therapy—theranostics—of human tumors, including prostate (PC) and breast cancer (BC). We herein present [99mTc]Tc-DB15, a SPECT radiotracer based on the potent GRPR-antagonist [DPhe6,LeuNHEt13]BBN(6-13). After Sar11/Gly11-replacement an acyclic tetraamine was coupled at the N-terminus via a spacer allowing for stable binding of the diagnostic radiometal Tc-99m. The forming [99mTc]Tc-DB15 radiotracer displayed high in vitro uptake in GRPR-expressing mammary (T-47D) and prostate cancer (PC-3) cells. Furthermore, it showed an attractive biodistribution profile in mice bearing T-47D or PC-3 xenografts. A pilot proof-of-principle study of [99mTc]Tc-DB15 in PC and BC patients applying SPECT is currently under way. Promising results from the first two BC patients are presented herein. Abstract Diagnostic imaging and radionuclide therapy of prostate (PC) and breast cancer (BC) using radiolabeled gastrin-releasing peptide receptor (GRPR)-antagonists represents a promising approach. We herein propose the GRPR-antagonist based radiotracer [99mTc]Tc-DB15 ([99mTc]Tc-N4-AMA-DGA-DPhe6,Sar11,LeuNHEt13]BBN(6-13); N4: 6-carboxy-1,4,8,11-tetraazaundecane, AMA: aminomethyl-aniline, DGA: diglycolic acid) as a new diagnostic tool for GRPR-positive tumors applying SPECT/CT. The uptake of [99mTc]Tc-DB15 was tested in vitro in mammary (T-47D) and prostate cancer (PC-3) cells and in vivo in T-47D or PC-3 xenograft-bearing mice as well as in BC patients. DB15 showed high GRPR-affinity (IC50 = 0.37 ± 0.03 nM) and [99mTc]Tc-DB15 strongly bound to the cell-membrane of T-47D and PC-3 cells, according to a radiolabeled antagonist profile. In mice, the radiotracer showed high and prolonged GRPR-specific uptake in PC-3 (e.g., 25.56 ± 2.78 %IA/g vs. 0.72 ± 0.12 %IA/g in block; 4 h pi) and T-47D (e.g., 15.82 ± 3.20 %IA/g vs. 3.82 ± 0.30 %IA/g in block; 4 h pi) tumors, while rapidly clearing from background. In patients with advanced BC, the tracer could reveal several bone and soft tissue metastases on SPECT/CT. The attractive pharmacokinetic profile of [99mTc]DB15 in mice and its capability to target GRPR-positive BC lesions in patients highlight its prospects for a broader clinical use, an option currently being explored by ongoing clinical studies.


Introduction
The expression of gastrin releasing peptide receptors (GRPRs) in a series of human tumors has provided the rationale for the application of anti-GRPR peptide radioligands but without notable improvement of end-pharmacokinetics [35,37]. Studying the profile of [ 99m Tc]Tc-DB15 in mice, we could confirm a high metabolic stability in the blood stream. We could also observe specific radioligand uptake in two GRPR-positive tumor animal models, namely, in human prostate PC-3 and breast T-47D cancer xenografts in immunosuppressed mice combined with a rapid background clearance. In a first pilot translational study, SPECT/CT diagnostic imaging was performed in two advanced BC patients, confirming the promising attributes of [ 99m Tc]Tc-DB15 for use in nuclear oncology.  11 replacing Gly 11 in the parent peptide sequence is highlighted in blue and bold.

Radiolabeling-Radioanalytical Control
For the preclinical studies, labeling of DB15 with Tc-99m was performed in alkaline pH and room temperature using SnCl2 as a reductant and citrate as a transfer ligand, following a published protocol [35]. Labeling yields and radiochemical purities were monitored by a combination of instant thin layer chromatography (ITLC) and high performance liquid chromatography (HPLC) adopting twin photometric and radiometric detection modes (HPLC equipment is detailed in Supplementary File), as previously described [35,36]. For patient use, this protocol was appropriately adopted as outlined in the Supplementary File. Radioiodination of [Tyr 4 ]BBN with I-125 was accomplished following a known procedure [35].
Handling of solutions containing beta-/gamma-emitting radionuclides was conducted by authorized personnel in compliance with European radiation safety guidelines.  in the acyclic tetraamine chain under formation of a monocationic octahedral radiometal-chelate; Sar 11 replacing Gly 11 in the parent peptide sequence is highlighted in blue and bold.

Radiolabeling-Radioanalytical Control
For the preclinical studies, labeling of DB15 with Tc-99m was performed in alkaline pH and room temperature using SnCl 2 as a reductant and citrate as a transfer ligand, following a published protocol [35]. Labeling yields and radiochemical purities were monitored by a combination of instant thin layer chromatography (ITLC) and high performance liquid chromatography (HPLC) adopting twin photometric and radiometric detection modes (HPLC equipment is detailed in Supplementary File), as previously described [35,36]. For patient use, this protocol was appropriately adopted as outlined in the Supplementary File. Radioiodination of [Tyr 4 ]BBN with I-125 was accomplished following a known procedure [35].
Handling of solutions containing beta-/gamma-emitting radionuclides was conducted by authorized personnel in compliance with European radiation safety guidelines. Licensed facilities were supervised by the Greek Atomic Energy Commission (GAEC, license #A/435/17092/2019).

Time-Dependent Uptake of [ 99m Tc]Tc-DB15 in PC-3 and T-47D Cells
One day before the experiment, PC-3 or T-47D cells were seeded in 6-well plates (≈1 × 10 6 cells per well). Next day, the cells were rinsed with ice-cold internalization medium (IM, RPMI-1640 GlutaMAX-I, supplemented by 1% (v/v) FBS). After adding fresh IM at 37 • C (1.2 mL), a further portion of IM (150 µL) was added in the upper wellrow and [Tyr 4 ]BBN solution in IM (150 µL) was added in the lower row (non-specific series). [ 99m Tc]Tc-DB15 (250 fmol total peptide in 150 µL 0.5% BSA-PBS) was added in all wells and the plates were incubated at for 15 min, 30 min, 1 h and 2 h at 37 • C in an Incubator-Orbital Shaker unit (MPM Instr. SrI, Bernareggio, MI, Italy). Cells were then placed on ice, the medium was collected, and the plates were rinsed with 0.5% BSA-PBS (1 mL). Membrane-bound fractions were collected after treatment in acid-wash solution (2 × 600 µL; 50 mM glycine buffer pH 2.8, 0.1 M NaCl). Internalized fractions were collected after treatment with 1 N NaOH (2 × 600 µL), as previously described [20,41]. After counting of radioactivity of all collected fractions in the gamma counter, the percentage of specific internalized and membrane-bound fractions per time point were calculated with Microsoft Excel and the respective curves were drawn. Specific internalized and membrane-bound counts were determined by subtracting the respective non-specific from the respective total counts. Results represent specific internalized ±SD of total added radioactivity per well from three experiments performed in triplicate.

In Vivo Metabolic Stability of [ 99m Tc]Tc-DB15 in Mice
Each of three healthy male Swiss albino mice (30 ± 5 g, NCSR "Demokritos" Animal House Facility) received a bolus of [ 99m Tc]Tc-DB15 (100 µL, 55.5-111 MBq, 3 nmol of total peptide in vehicle: saline/EtOH 9/1 v/v) injected in the tail vein together with vehicle (100 µL; controls). A parallel group of mice received the same bolus injected together with PA (100 µL of vehicle containing 300 µg PA; PA group). Animals were euthanized 5 min post-injection (pi) and blood (0.5-1 mL) was collected from the heart using a prechilled syringe, swiftly placed in pre-chilled Eppendorf Protein LoBind Tubes (Eppendorf, Wesseling-Berzdorf, Germany) containing EDTA. Samples were processed as previously described [26,36] and were then analyzed by radioanalytical HPLC (for HPLC equipment description see Supplementary File). A Symmetry-Shield RP18 (5 µm, 3.9 mm × 20 mm) column (Waters, Germany) was eluted at a 1 mL/min flow rate with the following gradient system: 100% A/0% B to 50% A/50% B in 50 min; A = 0.1% TFA in H 2 O and B = MeCN.

Biodistribution of [ 99m Tc]Tc-DB15 in PC-3 and in T-47D Xenograft-Bearing Mice
Biodistribution experiments were conducted in two sets of female severe combined immunodeficiency (SCID) mice 6-8 weeks of age, provided by the NCSR "Demokritos" Animal House Facility. In the first set (15.9 ± 1.8 g body weight, BW), animals were inoculated in their flanks with a freshly harvested sterile cell suspension of PC-3 cells (1.3 × 10 7 cells) in 150 µL physiological saline, as previously described [35]. Animals were kept under aseptic conditions for 3.5 weeks until tumors of adequate size (140 ± 50 mg) were developed at the inoculation site and biodistribution was conducted (vide infra). In the second animal set (16.3 ± 1.6 g BW) mice were treated with estrogens in their drinking water (4 mg/L of ß-estradiol, Sigma-Aldrich, St. Louis, MO, USA) while being kept in an aseptic environment for a period of one week. Next, inocula of freshly harvested T-47D cells in Matrigel (Corning Life Sciences, Inc., Bedford, MA, USA) were prepared (150 µL suspension of 1.2 × 10 7 cells) and subcutaneously (sc) injected in the flanks of mice. The animals were kept under estrogen treatment and aseptic conditions for further 9 weeks [11,41]. Within this period, they developed well-grown tumors (90 ± 30 mg) at the inoculation site and biodistribution was conducted as described below (the same procedure was followed for both sets of animals).
On the day of the experiment, mice in groups of four were injected through their tail vein with a [ 99m Tc]Tc-DB15 bolus (100 µL, 180-230 kBq, 10 pmol total peptide, in vehicle: saline/EtOH 9/1 v/v) together with either vehicle (100 µL; control groups-1, 4, and 24 h pi), or PA (300 µg PA dissolved in 100 µL vehicle; PA group-4 h pi), or excess [Tyr 4 ]BBN (40 nmol [Tyr 4 ]BBN dissolved in 100 µL vehicle; block group-4 h pi). At the selected time points animals were euthanized, dissected and blood, tumors and organs of interest were immediately collected, weighed and counted for their radioactivity content in the gamma counter along with appropriate standard solutions of the administered dose. Results were calculated as percent of administered dose per gram tissue (%IA/g) applying the Microsoft Excel program. Results were expressed as average %IA/g-values ± SD per time point.
All experiments involving mice were conducted in compliance with European and national regulations in licensed facilities (EL 25 BIO exp021). Applied protocols were approved by the Department of Agriculture and Veterinary Service of the Prefecture of Athens (protocol numbers #1609 for the stability and #1610 for the biodistribution studies).

Statistical Analyses
Statistical evaluation of results was accomplished adopting a two-way ANOVA with multiple comparisons applying Tukey's post hoc analysis (GraphPad Prism Software, San Diego, CA, USA). p-values of <0.05 were considered to be statistically significant.

Patient Studies
Two BC patients were tested for the potential of pathological lesion detection after injection of [ 99m Tc]Tc-DB15. The study protocol was approved by the Bioethical Committee of the Poznan University of Medical Sciences (decision no. 1153 issued on 16 January, 2020) and patients gave their informed consent for their participation in the study. Both patients had histologically confirmed advanced disease, with several lesions identified by conventional imaging modalities (MRI, [ 18 F]FDG PET/CT, mammography or breast USG), as detailed in the Supplementary File.
A single dose of [ 99m Tc]Tc-DB15 (5-mL, 700-800 MBq corresponding to 20 µg DB15) was administered as a slow intravenous (iv) injection over 1 min. Vital signs (pulse rate, systolic and diastolic BP) were measured prior to the injection of [ 99m Tc]DB15 as well as within 15 min pi. Likewise, ECGs were performed prior to the injection, continuously during the injection and for 15 min pi. ECG and vital signs were measured again after the last scan was completed and blood tests for hematology and biochemistry (glucose, creatinine, bilirubin, transaminases, sodium, potassium, calcium) were performed. The laboratory evaluation was repeated 7, 14 and 28 days after imaging. Adverse events (AEs) were assessed and graded according to National Cancer Institute Common Toxicity Criteria for Adverse Events (CTCAE) Version 3.0.
Gamma camera imaging was performed on a Symbia Intevo Bold dual-head SPECT/CT scanner (Siemens Healthineers AG; Erlangen, Germany) equipped with a 16-slice CT scanner. Planar imaging of the whole body and SPECT/CT images of the thorax were obtained 15 min, 1 h, 3 h, and 24 h pi. Planar and SPECT/CT images were inspected for increased uptake by two experienced nuclear medicine specialists.

Binding Affinity of DB15 for the Human GRPR
As shown in Figure  systolic and diastolic BP) were measured prior to the injection of [ 99m Tc]DB15 as well as within 15 min pi. Likewise, ECGs were performed prior to the injection, continuously during the injection and for 15 min pi. ECG and vital signs were measured again after the last scan was completed and blood tests for hematology and biochemistry (glucose, creatinine, bilirubin, transaminases, sodium, potassium, calcium) were performed. The laboratory evaluation was repeated 7, 14 and 28 days after imaging. Adverse events (AEs) were assessed and graded according to National Cancer Institute Common Toxicity Criteria for Adverse Events (CTCAE) Version 3.0.
Gamma camera imaging was performed on a Symbia Intevo Bold dual-head SPECT/CT scanner (Siemens Healthineers AG; Erlangen, Germany) equipped with a 16slice CT scanner. Planar imaging of the whole body and SPECT/CT images of the thorax were obtained 15 min, 1 h, 3 h, and 24 h pi. Planar and SPECT/CT images were inspected for increased uptake by two experienced nuclear medicine specialists.

Radiolabeling-Quality Control
Radiolabeling of DB15 with Tc-99m for preclinical testing (molecular activity of 20-30 MBq [ 99m Tc]Tc/nmol peptide) was accomplished by 30 min incubation of the radiolabeling reaction mixture, containing DB15, [ 99m Tc]TcO4 − , SnCl2 and citrate anions, at pH 11 and at room temperature. For clinical testing, the labeling reaction was modified to reach a higher molecular activity (greater than 50 MBq [ 99m Tc]Tc/nmol peptide).

GRPR-Specific Uptake of [ 99m Tc]Tc-DB15 by PC-3 and T-47D Cells
Time-dependent cell association curves of [ 99m Tc]Tc-DB15 in PC-3 and T-47D cells are shown in Figure 3. High and specific uptake was observed during 30 min incubation of [ 99m Tc]Tc-DB15 in PC-3 (13.1 ± 0.1%) and T-47D cells (24.2 ± 0.7%) at 37 • C. The majority of radioactivity was associated with the cell-membrane with a smaller portion (<6%) found within the cells, as expected for a GRPR-radioantagonist [20,35,41]. Cell association was banned (<0.9% at all time points, results not shown) in the presence of 1 µM [Tyr 4 ]BBN, consistent with a GRPR-mediated process.
Time-dependent cell association curves of [ Tc]Tc-DB15 in PC-3 and T-47D c shown in Figure 3. High and specific uptake was observed during 30 min incuba [ 99m Tc]Tc-DB15 in PC-3 (13.1 ± 0.1%) and T-47D cells (24.2 ± 0.7%) at 37 o C. The maj radioactivity was associated with the cell-membrane with a smaller portion (<6% within the cells, as expected for a GRPR-radioantagonist [20,35,41]. Cell associati banned (<0.9% at all time points, results not shown) in the presence of 1 µM [Ty consistent with a GRPR-mediated process.

Metabolic Stability of [ 99m Tc]Tc-DB15 in Mice
The stability of [ 99m Tc]Tc-DB15 in peripheral mouse blood was studied by HPL ysis of blood samples collected 5 min pi in two animal groups, in untreated contro mice treated with the NEP-inhibitor PA. As shown by the representative radiochr grams of Figure 4, [ 99m Tc]Tc-DB15 remained 76.4 ± 2.3% intact in controls (n = 3) minor but not statistically significant increase in the PA group (83.0 ± 2.3% intact, > 0.05).

Metabolic Stability of [ 99m Tc]Tc-DB15 in Mice
The stability of [ 99m Tc]Tc-DB15 in peripheral mouse blood was studied by HPLC analysis of blood samples collected 5 min pi in two animal groups, in untreated controls or in mice treated with the NEP-inhibitor PA. As shown by the representative radiochromatograms of Figure 4, [ 99m Tc]Tc-DB15 remained 76.4 ± 2.3% intact in controls (n = 3) with a minor but not statistically significant increase in the PA group (83.0 ± 2.3% intact, n = 3; p > 0.05).
shown in Figure 3. High and specific uptake was observed during 30 min incubation of [ 99m Tc]Tc-DB15 in PC-3 (13.1 ± 0.1%) and T-47D cells (24.2 ± 0.7%) at 37 o C. The majority of radioactivity was associated with the cell-membrane with a smaller portion (<6%) found within the cells, as expected for a GRPR-radioantagonist [20,35,41]. Cell association was banned (<0.9% at all time points, results not shown) in the presence of 1 µM [Tyr 4 ]BBN, consistent with a GRPR-mediated process.

Metabolic Stability of [ 99m Tc]Tc-DB15 in Mice
The stability of [ 99m Tc]Tc-DB15 in peripheral mouse blood was studied by HPLC analysis of blood samples collected 5 min pi in two animal groups, in untreated controls or in mice treated with the NEP-inhibitor PA. As shown by the representative radiochromatograms of Figure 4, [ 99m Tc]Tc-DB15 remained 76.4 ± 2.3% intact in controls (n = 3) with a minor but not statistically significant increase in the PA group (83.0 ± 2.3% intact, n = 3; p > 0.05).

Biodistribution of [ 99m Tc]Tc-DB15 in PC-3 and in T-47D Xenograft-Bearing Mice
Biodistribution results of [ 99m Tc]Tc-DB15 in SCID mice bearing PC-3 or T-47D xenografts at 1, 4 and 24 h pi are shown in Figure 5, as %IA/g±SD, n = 4. Results in numerical values are summarized in Tables S1 and S2, respectively, in the Supplementary File. The radioligand cleared fast from blood and background tissues via the kidneys into urine. [ 99m Tc]Tc-DB15 rapidly and specifically localized in the PC-3 (30.70 ± 2.76 %IA/g at 1 h pi) and T-47D (14.01 ± 2.87 %IA/g at 1 h pi) tumors showing good retention (PC-3: 17.79 ± 1.58 %IA/g / T-47D: 7.55 ± 1.81 %IA/g at 24 h pi). High uptake was also observed in the GRPR-expressing mouse pancreas (>130 %IA/g at 1 h pi), which however rapidly declined with time (~2 %IA/g at 24 h pi), as consistent with a radioantagonist profile [20,35,41]. Treatment of animals with PA did not induce any significant change in the tumor uptake, either for the PC-3 (controls: 25.56 ± 2.78 %IA/g vs PA-treated:  30.03 ± 3.90 %IA/g at 4 h pi; p > 0.05) or the T-47D implants (controls: 15.82 ± 3.20 %IA/g vs. PA-treated: 13.15 ± 1.55 %IA/g at 4 h pi; p > 0.05), in agreement with the minor impact of PA on the in vivo stability of [ 99m Tc]Tc-DB15 in peripheral mice blood. In all cases radioligand uptake was receptor-mediated, as suggested by the significant reduction (p < 0.001) of tumor and pancreas values during in vivo GRPR-blockade by co-injection with excess [Tyr 4 ]BBN. For example, the uptake in the PC-3 tumors was drastically reduced from 25.56 ± 2.78 %IA/g in controls to 0.72 ± 0.12 %IA/g in the block-group of mice at 4 h pi (p < 0.0001). radioligand cleared fast from blood and background tissues via the kidneys into urine. [ 99m Tc]Tc-DB15 rapidly and specifically localized in the PC-3 (30.70 ± 2.76%IA/g at 1 h pi) and T-47D (14.01 ± 2.87%IA/g at 1 h pi) tumors showing good retention (PC-3: 17.79 ± 1.58%IA/g / T-47D: 7.55 ± 1.81%IA/g at 24 h pi). High uptake was also observed in the GRPR-expressing mouse pancreas (>130%IA/g at 1 h pi), which however rapidly declined with time (~2%IA/g at 24 h pi), as consistent with a radioantagonist profile [20,35,41]. Treatment of animals with PA did not induce any significant change in the tumor uptake, either for the PC-3 (controls: 25.56 ± 2.78%IA/g vs PA-treated: 30.03 ± 3.90%IA/g at 4 h pi; p > 0.05) or the T-47D implants (controls: 15.82 ± 3.20%IA/g vs PA-treated: 13.15 ± 1.55%IA/g at 4 h pi; p > 0.05), in agreement with the minor impact of PA on the in vivo stability of [ 99m Tc]Tc-DB15 in peripheral mice blood. In all cases radioligand uptake was receptor-mediated, as suggested by the significant reduction (p < 0.001) of tumor and pancreas values during in vivo GRPR-blockade by co-injection with excess [Tyr 4 ]BBN. For example, the uptake in the PC-3 tumors was drastically reduced from 25.56 ± 2.78%IA/g in controls to 0.72 ± 0.12%IA/g in the block-group of mice at 4 h pi (p < 0.0001).

Tolerability
No adverse reactions were recorded after injection of [ 99m Tc]Tc-DB15. The patients did not report any specific signs, the heart rate and blood pressure were stable during and after injection. No significant changes in the hematology and biochemistry parameters were recorded at any time point after the imaging. Patient 1 complained of intensified bone pain (CTCAE grade 1) several hours after the scan, which however resolved after a few days and was attributed to the forced supine position during image acquisition.

Physiological Tissue Distribution
Planar and SPECT/CT images showed accumulation of [ 99m Tc]Tc-DB15 in the pancreas of both patients (Figures 6 and 7). The bulk of the radioactivity rapidly cleared via the kidneys into urine in the first 2 h, with a smaller portion being slowly eliminated via the liver and visible in the bowels at 24 h pi.

Pathological Findings
The planar and SPECT/CT images revealed increased uptake of [ 99m Tc]Tc-DB15 in the BC metastases in both patients. Patient history and further information concerning the  No adverse reactions were recorded after injection of [ 99m Tc]Tc-DB15. The patients did not report any specific signs, the heart rate and blood pressure were stable during and after injection. No significant changes in the hematology and biochemistry parameters were recorded at any time point after the imaging. Patient 1 complained of intensified bone pain (CTCAE grade 1) several hours after the scan, which however resolved after a few days and was attributed to the forced supine position during image acquisition.

Physiological Tissue Distribution
Planar and SPECT/CT images showed accumulation of [ 99m Tc]Tc-DB15 in the pancreas of both patients (Figures 6 and 7). The bulk of the radioactivity rapidly cleared via the kidneys into urine in the first 2 h, with a smaller portion being slowly eliminated via the liver and visible in the bowels at 24 h pi.

Pathological Findings
The planar and SPECT/CT images revealed increased uptake of [ 99m Tc]Tc-DB15 in the BC metastases in both patients. Patient history and further information concerning the study can be found in the Supplementary File. Patient 1 displayed increased [ 99m Tc]Tc-DB15 uptake in the bones (spine, sternum, ribs and pelvis), which correlated with osteosclerotic lesions on the CT scan (Figure 1 and Figure S3  study can be found in the Supplementary File. Patient 1 displayed increased [ 99m Tc]Tc-DB15 uptake in the bones (spine, sternum, ribs and pelvis), which correlated with osteosclerotic lesions on the CT scan (Figure 1 and Figure S3 in Supplementary File) as well as with bone lesions revealed by [ 18 F]FDG PET/CT ( Figure S4; Supplementary File). However, [ 99m Tc]Tc-DB15 failed to demonstrate intraperitoneal metastases during SPECT/CT which were identified by [ 18 F]FDG PET/CT and subsequently confirmed in histopathology (not including GRPR-expression status). In patient 2, increased tracer accumulation was visible in the thickened pleura of the right lung corresponding to pleural BC metastases. Additionally, increased uptake was noted in an enlarged right phrenic lymph node (Figure 7).   In patient 2, increased tracer accumulation was visible in the thickened pleura of the right lung corresponding to pleural BC metastases. Additionally, increased uptake was noted in an enlarged right phrenic lymph node (Figure 7).  In patient 2, increased tracer accumulation was visible in the thickened pleura of the right lung corresponding to pleural BC metastases. Additionally, increased uptake was noted in an enlarged right phrenic lymph node (Figure 7).

Discussion
As a part of our ongoing work on anti-GRPR radioligands for application in cancer theranostics [20,29,[34][35][36], we now present [ 99m Tc]Tc-DB15, a radiotracer based on the potent GRPR-antagonist [ D Phe 6 ,LeuNHEt 13 ]BBN (6)(7)(8)(9)(10)(11)(12)(13) [30,31] and, thus, associated with better safety for human use. The acyclic tetraamine framework positioned at the N-terminus allowed labeling with Tc-99m, a broadly available radiometal ideally suited for high-quality SPECT and SPECT/CT imaging [33]. Metabolic stability of peptide radioligands in the blood stream represents an important prerequisite for good tumor targeting by ensuring the sufficient delivery of intact molecules to tumor lesions expressing the target. Previous studies have shown that the fast in vivo degradation of BBN-like peptides is driven by neutral endopeptidase (NEP) [42,43]. Most importantly, it was demonstrated that in-situ inhibition of NEP induced by administration of appropriate inhibitors, such as phosphoramidon (PA) or Entresto ® , could provoke significant increases in the tumor uptake of several anti-GRPR radiopeptides through their stabilization in peripheral blood [25,26,36,44,45].
It is interesting to observe how the above promising qualities of [ 99m Tc]Tc-DB15 translated in biodistribution patterns in mice bearing GRPR-positive tumors. Firstly, the radiotracer displayed a high and GRPR-specific uptake in both the PC-3 and the T-47D xenografts at all time points. Secondly, the high %IA/g values at 24 h pi reveal the advantageous retention of [ 99m Tc]Tc-DB15 in the experimental tumors. Thirdly, background radioactivity declined rapidly, especially from the GRPR-rich mouse pancreas. As a result of the above, [ 99m Tc]Tc-DB15 displayed a quite attractive in vivo profile with tumor-tobackground ratios increasing with time. Thus, for example, the uptake of [ 99m Tc]Tc-DB15 in the PC-3 xenografts remained as high as 17.79 ± 1.58 %IA/g even at 24 h pi with the pancreatic uptake conversely declining to 2.07 ± 0.62 %IA/g, illustrating the excellent biodistribution pattern of the Sar 11 -radiotracer. It should be noted that the respective values for the non-modified Gly 11 -analog were previously reported to be 16.32 ± 1.82 %IA/g for the PC-3 tumors and 30.26 ± 14.65 %IA/g for the pancreas [35]. Prolonged retention in the tumor is an attractive quality for a theranostic GRPR-seeking radiolabeled probe, agonist, or antagonist, especially during radionuclide therapy. This fact has been illustrated in a recent report, whereby cysteine cathepsin inhibitors are coupled to GRPR-peptides leading to improved tumor retention via endolysosomal trapping [46]. Another interesting finding of the current biodistribution study has been the lack of improvements in the tumor uptake in the mice treated with PA vs. the untreated controls at 4 h pi. Indeed, no significant difference was observed in either the PC-3 or the T-47D xenografts during in-situ NEP-inhibition, concordant with findings from the in vivo stability study, which ruled out the involvement of NEP in the degradation of circulating [ 99m Tc]Tc-DB15.
The above promising preclinical properties of [ 99m Tc]Tc-DB15 prompted us to explore its clinical applicability in the detection of GRPR-positive lesions in BC and PC patients. Previous studies with [ 68 Ga]Ga-labeled GRPR-antagonist SB3 (SB3, [DOTA-p-AMA-DGA-D Phe 6 ,LeuNHEt 13 ]BBN(6-13)) revealed the safety and feasibility of detecting GRPR-expressing pathological lesions of advanced BC and PC patients applying [ 68 Ga]Ga-SB3 and PET/CT [29] with a more recent study in therapy-naïve PC patients revealing better results and reporting excellent correlation of imaging findings with GRPR-expression levels in the primary PC excised lesions [7].
Our It should be noted however that GRPR-expression levels were not determined in the samples acquired by laparotomy for histological confirmation of BC. In the second patient with advanced BC infiltrating in the pleura, as confirmed by histopathology, high uptake of [ 99m Tc]Tc-DB15 was shown on SPECT/CT in the lower lobe of the lung and additionally in an enlarged phrenic lymph node. The latter could not be confirmed histologically as a BC metastasis because of anatomical position restraining surgical intervention. Again, the GRPR-expression status was not determined in the samples taken from this patient either. The above preliminary clinical results are encouraging in terms of biosafety. They also seem rather positive with regards to efficacy, especially when the high heterogeneity of primary and metastatic BC, including GRPR-expression levels, is taken into account [9,10].
Yet, many open questions have to be rigorously addressed before confirming the diagnostic value of [ 99m Tc]Tc-DB15 in BC and potentially in other human cancers too. Firstly, we need to correlate imaging findings with histologically established data on GRPR-expression in a systematic way. Then, we need to understand if and to what extent additional parameters, such as BC type and stage along with preceding therapies, affect GRPR-expression levels on the lesions and thereby diagnostic accuracy. Hence, further clinical evaluation of [ 99m Tc]Tc-DB15 appears to be warranted.

Conclusions
We have introduced [ 99m Tc]Tc-DB15, a GRPR-antagonist based radiotracer, as a candidate for diagnostic imaging of GRPR-positive human tumors. In addition to the inherent biosafety of an antagonist, labeling with the preeminent nuclear medicine radionuclide Tc-99m allows for excellent quality images using broadly available SPECT and SPECT/CT instrumentation. Substitution of Gly 11 by Sar 11 in the peptide backbone, has led to high metabolic resistance to NEP, a major catabolizing protease of BBN-like peptides in vivo. Unlike previously attempted DAla 11 /Gly 11 substitutions, [ 99m Tc]Tc-DB15 retained high cell binding efficacy in both prostate PC-3 and in BC T-47D cells in vitro. Most interestingly, it displayed high uptake and prolonged retention in the respective PC-3 and T-47D xenografts grown in mice. These qualities combined with a rapid background clearance, resulted in an excellent pharmacokinetic profile. Translation in two advanced BC patients, resulted in no side effects, confirming previous observations on the biosafety of radiotracers based on the potent GRPR-antagonist [DPhe 6 ,LeuNHEt 13 ]BBN(6-13) and on GRPR-antagonist radioligands in general. Furthermore, it revealed the ability of [ 99m Tc]Tc-DB15 to detect several metastatic BC lesions, both in the skeleton and in soft tissues, but these findings need to be confirmed prospectively in a dedicated human study. In view of the above, further clinical evaluation seems to be warranted to establish the diagnostic value of [ 99m Tc]Tc-DB15 in BC, PC, and other GRPR-expressing human malignancies.