Therapeutic Performance Evaluation of 213Bi-Labelled Aminopeptidase N (APN/CD13)-Affine NGR-Motif ([213Bi]Bi-DOTAGA-cKNGRE) in Experimental Tumour Model: A Treasured Tailor for Oncology

Since NGR-tripeptides (asparagine-glycine-arginine) selectively target neoangiogenesis-associated Aminopeptidase N (APN/CD13) on cancer cells, we aimed to evaluate the in vivo tumour targeting capability of radiolabelled, NGR-containing, ANP/CD13-selective [213Bi]Bi-DOTAGA-cKNGRE in CD13pos. HT1080 fibrosarcoma-bearing severe combined immunodeficient CB17 mice. 10 ± 1 days after cancer cell inoculation, positron emission tomography (PET) was performed applying [68Ga]Ga-DOTAGA-cKNGRE for tumour verification. On the 7th, 8th, 10th and 12th days the treated group of tumourous mice were intraperitoneally administered with 4.68 ± 0.10 MBq [213Bi]Bi-DOTAGA-cKNGRE, while the untreated tumour-bearing animals received 150 μL saline solution. In addition to body weight (BW) and tumour volume measurements, ex vivo biodistribution studies were conducted 30 and 90 min postinjection (pi.). The following quantitative standardised uptake values (SUV) confirmed the detectability of the HT1080 tumours: SUVmean and SUVmax: 0.37 ± 0.09 and 0.86 ± 0.14, respectively. Although no significant difference (p ≤ 0.05) was encountered between the BW of the treated and untreated mice, their tumour volumes measured on the 9th, 10th and 12th days differed significantly (p ≤ 0.01). Relatively higher [213Bi]Bi-DOTAGA-cKNGRE accumulation of the HT1080 neoplasms (%ID/g: 0.80 ± 0.16) compared with the other organs at 90 min time point yields better tumour-to-background ratios. Therefore, the therapeutic application of APN/CD13-affine [213Bi]Bi-DOTAGA- cKNGRE seems to be promising in receptor-positive fibrosarcoma treatment.


Introduction
Angiogenesis/neo-angiogenesis has a central role in tumour development, tumour progression and metastatic spread [1,2]. Several biomarkers of tumour-associated angiogenesis are known from the literature, which can be targeted by different peptides (for example: vascular endothelial growth factor (VEGF) derivatives for vascular endothelial growth factor receptor (VEGFR), RGD for integrins and NGR for APN/CD13). Although NGR tripeptide sequence is currently the least investigated angiogenesis-connected agent, the ever-increasing importance of APN/CD13 in association with cancer-related processes, diagnostics or as a prognostic biomarker and therapeutic target brings NGR motif and its derivatives into the priority of today's science [3]. Former findings indicate that membranebound metalloprotease, aminopeptidase N (CD13/APN) takes part in the differentiation, movement, invasion, proliferation and apoptosis of the cells, as well as angiogenic processes [4]. In addition, tumour vascular endothelial cells and various cancer cells including prostate, pulmonary, gynaecological, breast and gastrointestinal ones could be characterised by CD13 upregulation [5][6][7][8][9][10]. Since neo-angiogenesis-associated exopeptidase CD13/APN is widely expressed on different neoplasms and angiogenic endothelial cells, it serves as a highly reliable diagnostic as well as prognostic biomarker of tumour-related angiogenic processes [1,11]. Therefore, CD13 selective imaging as well as therapeutic probes could be ground-breaking in early-stage tumour identification and personalised treatment of CD13pos. malignancies [12].
Ample evidence implies that asparagine-glycine-arginine sequence (tripeptide NGR)containing peptides exhibit high affinity to CD13/APN [13]. Hence, NGR motif-based peptide molecules could be essential in the targeted imaging of CD13 overexpressing neoplastic alterations. In this respect, nuclear medicine has a lot to offer. Prior literature data proved that radiolabelled NGR-based molecular agents make a leap forward in the non-invasive diagnostic assessment of cancers with high APN density [14,15]. Given the broad accessibility of different radiometals for NGR radiolabelling purposes such as copper-64 ( 64 Cu), gallium-68 ( 68 Ga), lutetium-177 ( 177 Lu) or rhenium-188 ( 188 Re) together with the high-quality and non-invasive imaging modalities, a rising number of research studies have been centred around the evaluation of APN-directed radio-appended peptide compounds in the recent years [16][17][18][19].
Previous in vivo investigations outlined the diagnostic feasibility of the following 64 Cu or 68 Ga radio-conjugated probes in receptor pos. experimental tumour models at translational level: 64 Cu-labelled NGR monomer and dimer attached to chelator DOTA (1,4,7,10- [17,20,21]. Out of the broad set of chelatorsincluding NOTA, NODAGA, DOTA, or 1,4,7,10-tetrakis(carboxymethyl)-1,4,7,10-tetraazacyclododecane glutaric acid (DOTAGA)-due to its high stability and the inertness of the complexes formed with either 68 Ga and 213 Bi radiometals, DOTA is recognised as the most promising macrocyclic chelator for diagnostic and therapeutic applications. To facilitate complexation with targeting peptides, several bifunctional derivatives of DOTA were developed, such as DOTAGA, by using an additional carboxy functionality added to the core of DOTA [22,23].
Although the past few decades are characterized by outstanding advancements in battling against tumourigenesis, cancer-associated death remains one of the major challenges of current healthcare systems worldwide [24]. To overcome the limitations of conventional anti-tumour treatment, targeted radionuclide therapy (TRNT) using alpha-emitting peptidespecific molecules represents a pioneering therapeutic approach in oncological fields [25,26]. The high linear energy transfer (LET: 50-230 keV/µm) and the short penetration range (0.1 mm) of alpha particles are well-suited for selective tumour-killing, bypassing adverse effects on neighbouring healthy tissues [27,28]. Produced by actinium-225/bismuth-213 ( 225 Ac/ 213 Bi) generator system, alpha-emitting 213 Bi (T 1 2 : 45.6 min, mean E α /MeV: 8.32, average soft tissue range: 78 µm) with such valuable physical properties seems to be a potent weapon in TRNT-based anti-tumour treatment [29,30].
In the present study we aimed at evaluating the therapeutic performance of a newly synthesised, [ 213 Bi]Bi-labelled, NGR-motif containing radiopharmaceutical in CD13/APNupregulated HT1080 fibrosarcoma-bearing mice. Prior to its therapeutic administration, positron emission tomography (PET) with 68 Ga-labelled CD13-targeting compound was applied for tumour verification.
The radiolabelling of DOTAGA-cKNGRE with 68 Ga was accomplished according to Gyuricza et al. [31]. This method is based on formerly published labelling procedures [32][33][34]. One mL (100-130 MBq) 68 GaCl 3 , eluted with 0.1 M u.p. HCl from a 68 Ge/ 68 Ga generator, was mixed with sodium acetate buffer (0.16 mL, 1 M) to obtain pH4. Then, 18 nmol of DOTAGA-cKNGRE was added to the solution and finally the mixture was incubated for 5 min at 95 • C. Then, [ 68 Ga]Ga-DOTAGA-cKNGRE was purified by solid-phase extraction (SPE) applying an Oasis HLB 1cc cartridge. Analitical radio-HPLC was applied for the quality control (QC) of the purified, 68 Ga-labelled complex. Analytical radio-HPLC was conducted using a KNAUER HPLC system, connected to an ATOMKI CsI scintillation detector (eluent A: 0.1% TFA in water and eluent B: 0.1% TFA in 95% ACN). The radiochemical purity (RCP) of the [ 68 Ga]Ga-DOTAGA-cKNGRE was higher than 95%. The distribution of the radioactivity in n-octanol as well as water was determined by Gyuricza et al. in order to define the LogP value of [ 68 Ga]Ga-DOTAGA-cKNGRE [31]. To have the two different layers separated, the mixture of 50 µL from the given radiotracer (approximately 2 MBq), 450 µL distilled water and 0.5 mL n-octanol was centrifuged for 5 min (9000× g). Then, the radioactivity was measured in 20 and 20 µL aliquots of noctanol and water, respectively, with a Perkin Elmer Packard Cobra gamma-counter for the determination of the partition coefficient of the investigated probe. The LogP value was −4.13 for [ 68 Ga]Ga-DOTAGA-cKNGRE.

Determination of LogP Value of [ 205/206 Bi]Bi-DOTAGA-cKNGRE
For the determination of LogP value we used 205/206 Bi isotopes as surrogates which were produced by GE PETtrace cyclotron using the method described by Lagunas-Solar et al. [35]. The radiolabelling of the precursor was carried out as follows: 50 µL of 3 M NH 4 OAc buffer (pH 4) and 15 µL of 6 mM DOTAGA-cKNGRE aqueous solution were added to 50 µL [ 205/206 Bi]BiCl 3 in 0.1 M HCl. The labelled complex was purified as described above. Then 10 µL of the purified [ 205/206 Bi]Bi-DOTAGA-cKNGRE in physiological saline (Salsol, TEVA, Debrecen, Hungary) was diluted with 0.49 mL water. It was then mixed with 0.5 mL n-octanol in a vial and shaken for 5 min. After centrifugation (6000 rpm, 5 min) the radioactivity of the sample from both the aqueous and n-octanol phase was measured with a Perkin Elmer Packard Cobra gamma counter. The LogP value was −2.59.

Serum Stability of [ 68 Ga]Ga-DOTAGA-cKNGRE
Following the addition of 50 µL of [ 68 Ga]Ga-DOTAGA-cKNGRE dissolved in physiological saline (Salsol, TEVA, Debrecen, Hungary) to 50 µL of mouse plasma, the mixture was incubated at 37 • C. With the application of radio-HPLC we evaluated the serum stability of the radiotracer 0, 30, 60 and 90 min post-incubation with mouse blood serum under predefined circumstances. [ 68 Ga]Ga-DOTAGA-cKNGRE demonstrated metabolic stability in serum after 90 min since the sample taken from the mixture showed an RCP above 95%, which meets the requirements for 68 Ga-labelled radiopharmaceuticals.

Serum Stability of [ 205/206 Bi]Bi-DOTAGA-cKNGRE
Fifty µL of [ 205/206 Bi]Bi-DOTAGA-Bn-cKNGRE in physiological saline (Salsol, TEVA, Debrecen, Hungary) was added to the solution of fifty µL of mouse plasma. The RCP of the samples was determined by radio-iTLC at 0, 30, 60 and 90 min time points using the same method as described above. After 90 min the [ 205/206 Bi]Bi-DOTAGA-Bn-cKNGRE remained intact as the RCP of the sample taken from the mixture was above 95%, which meets the requirements for 213 Bi-labelled radiopharmaceuticals.

Animal Housing
Twelve-week-old old severe combined immunodeficient CB17 (SCID) female mice (n = 35) were purchased from Charles River Laboratories (Sulzfeld, Germany). The experimental animals were bred and obtained under specific pathogen-free conditions in Individually Ventilated Cages (Sealsafe Blue line IVC system, Techniplast, Akronom Ltd., Budapest, Hungary) with regulated ambient temperature and relative humidity maintained at 24 ± 2 • C and 55 ± 10%, respectively. A circadian light/dark cycle of 12 h was provided for the study mice. All animals were kept on sterile semi-synthetic rodent maintenance chow (Akronom Ltd., Budapest, Hungary) and sterile drinking water ad libitum.
All procedures applied followed the applicable sections of the Hungarian Laws as well as the animal welfare directions and regulations of the European Union. For the accomplishment of the present study, the permission of the Ethics Committee for Animal Experimentation of the University of Debrecen, Hungary (ethical permission number: 16/2022/DEMÁB) was granted. The fulfilment of the 3R policy was our priority.

HT1080 Tumour Generation
Experimental tumour induction was carried out under inhalation anaesthesia with a dedicated small animal anaesthesia device (Tec3 Isoflurane Vaporizer, Eickemeyer Veterinary Equipment, Luton, UK) applying 3% isoflurane (Forane, AbbVie, Chicago, IL, USA), 0.4 L/min O 2 and 1.4 L/min N 2 O. This was followed by depilation and disinfection subcutaneous (s.c.) inoculation of 1.5 × 10 6 HT1080 (human fibrosarcoma) cells in 100 µL (1/3 part of Matrigel and 2/3 part of saline) into the left shoulder area of the CB17 SCID mice. For tumour verification the study mice underwent in vivo [ 68 Ga]Ga-DOTAGA-cKNGRE-based PET imaging 10 ± 1 days post tumour cell inoculation. To evaluate the organ distribution pattern of the 213 Bi-labelled therapeutic peptide compounds, ex vivo biodistribution studies were also executed 10 ± 1 days following the HT1080 cell transplantation at an average tumour volume of 155 ± 20 mm 3 . Thereafter, tumour sizes based on the largest and the smallest tumour diameters were registered ((largest diameter × smallest diameter 2 )/2). We used calliper measurements for tumour growth determination.

In Vivo MiniPET Imaging
CB17 SCID mice bearing a HT1080 fibrosarcoma in their left shoulder area were intravenously (i.v.) injected with 9.36 ± 1.24 MBq [ 68 Ga]Ga-DOTAGA-cKNGRE 10 ± 1 days after tumour generation at a tumour bulk of approximately 150 mm 3 . MiniPET scans were acquired 90 min post radiotracer administration with the application of a MiniPET scanner (University of Debrecen, Faculty of Medicine, Division of Nuclear Medicine and Translational Imaging). Isoflurane-based inhalation anaesthesia (AbbVie, Budapest, Hungary; OGYI-T-1414/01) was assured throughout the whole study employing the Tec3 Isoflurane Vaporizer small animal anaesthesia device (Eickemeyer Veterinary Equipment, Luton, UK). Three-dimensional ordered-subsets expectation-maximization line-of-response (3D OSEM-LOR) image reconstruction was performed to provide satisfactory PET images for interpretation. To determine the following uptake parameters, volume of interests (VOIs) were deposited around selected organs and tissues with the BrainCAD image analysis software: standardised uptake value (SUV), SUV mean and SUV max . Calculated by the following formula, SUV was used as a relative measure of radiopharmaceutical accumulation: SUV = [VOI activity (Bq/mL)]/[injected activity (Bq)/animal weight (g)]. The average radioactivity within the VOI is reflected by the SUV mean value, while SUV max refers to the highest radiotracer concentration of the region concerned.

Cancer Treatment Studies
Seven days after HT1080 cell inoculation the tumour-bearing animals were randomly classified into two subgroups: treatment-naïve group (control, n = 8) and [ 213 Bi]Bi-DOTAGA-cKNGRE-treated group (treated, n = 8). Approximately 5 MBq [ 213 Bi]Bi-DOTAGA-cKNGRE was intraperitoneally (i.p.) administered to the experimental mice of the treated cohort on the 7th, 8th, 10th and 12th experimental days. The control mice were i.p. injected with 150 µL saline solution applying the same time regime.

Ex Vivo Biodistribution Studies
To assess the organ distribution of [ 213 Bi]Bi-DOTAGA-cKNGRE, ex vivo investigations were performed. The HT1080 fibrosarcoma-bearing experimental mice were anaesthetised with Forane (5%) and were sacrificed at 30 and 90 min post administration of the radiopharmaceutical. After autopsy, selected tissues, organs and the tumour itself were removed, weighed wet and their radioactivity was measured on a calibrated gamma counter (Perkin-Elmer Packard Cobra, Waltham, MA, USA). Then, the activity was decay corrected to the time of the injection. The percent of administered dose per gram of tissue (%ID/g) was determined for all samples from the counts per minute values (CPM). We presented the ex vivo data as mean %ID/g ± SD.

Statistical Analyses
Statistical analyses were executed with the application of MedCalc 18.5 commercial software package (MedCalc 18.5, MedCalc Software, Mariakerke, Belgium). Statistical differences were determined applying two-tailed Student's t-test, two-way ANOVA and Mann-Whitney U-test. Data are shown as the mean ± SD. A p value of less than 0.05 was assumed to identify significant differences unless otherwise indicated.

[ 68 Ga]Ga-DOTAGA-cKNGRE MiniPET Imaging
In a bid to authenticate the presence of the HT1080 fibrosarcoma tumours developing in the left shoulder area of the study mice, in vivo whole body MiniPET images were acquired 90 min pi. of [ 68 Ga]Ga-DOTAGA-cKNGRE 10 ± 1 days after s.c. tumour induction. Within the framework of the in vivo studies, the tumour targeting potential of the CD13-affine 68 Ga-tagged peptide probe was also assessed. Figure 2A displays representative in vivo decay-corrected transaxial and coronal PET images of CB17 SCID mice bearing HT1080 tumour. SUV mean and SUV max -as quantitative uptake parameters-were registered as well (as seen in Figure 2B).
Upon qualitative assessment the s.c. progressing HT1080 tumours were clearly identified with the application of [ 68 Ga]Ga-DOTAGA-cKNGRE 90 min pi. and 10 ± 1 days following tumour cell implantation (presented in Figure 2A, red arrows). This considerable tumour uptake together with the discrete background activity led to the acquisition of high-contrast PET images. In accordance with the visual interpretation, quantitative SUV analyses also revealed meaningful radiopharmaceutical uptake in the neoplastic tissue with SUV mean and SUV max values of 0.37 ± 0.09 and 0.86 ± 0.14, respectively (as shown in Figure 2B). Taken together, our in vivo PET results confirm that the investigated 68 Ga-appended, cKNGRE-motif-containing imaging probe accumulates in the APN/CD13 expressing tumour. Accordingly, the tumour-specificity of dimeric cNGR radiolabelled with 68 Ga and conjugated to chelator DOTA ( 68 Ga-DOTA-cNGR 2 ) was strengthened in CD13 overexpressing ES2 ovarian cancer bearing nude mice by in vivo microPET imaging [36]. Added to this, reduced ES2 68 Ga-DOTA-c(NGR) 2 tracer uptake induced by the co-application of the cold peptide derivate also outlined the APN-selectivity of the assessed peptide probe. In a similar way, Szabó et al. further confirmed the diagnostic applicability of 68 Ga-tagged-c(NGR) linked to NOTA in the detection of subrenally growing primary mesoblastic nephrome (Ne/De) tumours (SUV mean : 4.12 ± 0.56; SUV max : 10.72 ± 1.85), and related metastases in the thoracic parathymic lymph nodes (PTLN; SUV mean : 0.72 ± 0.12; SUV max : 1.92 ± 0.58) in Fischer-344 rats [37]. In accordance with our results, high-contrast PET images of the PTLNs could be acquired with [ 68 Ga]Ga-NOTA-c(NGR) [37]. Obtaining scans with appropriate tumour-to-background ratios still represents a fundamental challenge in terms of image reporting since sharp distinction of the neoplastic tissue from the background activity is of paramount importance regarding precise lesion detection and localization. Corresponding to the findings of Szabó et al., in the experiments of Máté and co-workers, a high affinity of [ 68 Ga]Ga-NOTA-c(NGR) to ANP/CD13 pos. ortho and heterotopic transplanted Ne/De tumours was observed [16].
Given the aforementioned in vivo research findings, 68 Ga-labelled NGR sequencecontaining radiotracers have a promising role in the non-invasive detection of cancer-related neo-angiogenic processes as well as the timely identification of APN/CD13-rich tumours. Further, they could be also feasible in the follow-up of angiogenesis directed oncological treatments and the evaluation of therapeutic efficacy.

Performance Evaluation of [ 213 Bi]Bi-DOTAGA-cKNGRE Treatment: Effects on Body Weight and Tumour Volume
On the 7th, 8th, 10th and 12th days of the investigation at an average tumour volume of 26.56 ± 2.39 mm 3 CB17 SCID mice bearing HT1080 fibrosarcoma in their left shoulder area were i.p. injected with approximately 5 MBq [ 213 Bi]Bi-DOTAGA-cKNGRE. The treatment-naïve study mice i.p. received 150 µL saline solution on the same experimental days. In a bid to assess the therapeutic efficacy of the investigated probe, body weights (BW, expressed in grams/g ± SD) and tumour volume (mm 3 ± SD) of the small animals were registered. Calliper measurements were applied to evaluate tumour development.

Impact of [ 213 Bi]Bi-DOTAGA-cKNGRE Treatment on Body Weight
Rigorous measurements of BW took place on the 7th, 8th, 10th and 12th experimental days. As demonstrated on the follow-up curve of Figure 3A, no statistically significant change was pinpointed regarding the BW of the experimental small animals in either study group during the therapy (p ≤ 0.05). The BW in the untreated cohort was 19.84 ± 1.24, 19.99 ± 1.35, 18.82 ± 1.11, 18.31 ± 0.97 recorded on the 7th, 8th, 10th and 12th days, respectively. As for the [ 213 Bi]Bi-DOTAGA-cKNGRE-treated subclass the following values were obtained 7, 8, 10 and 12 days after the tumour cell inoculation: 20.12 ± 1.04, 20.93 ± 0.85, 19.98 ± 0.97 and 19.11 ± 1.07, respectively. Since the weight of the tumourous animals remained relatively stable throughout the whole examination period, we may presuppose that the currently applied [ 213 Bi]Bi-DOTAGA-cKNGRE treatment exerts no considerable effect either on tumour weight or BW. Hence, this alpha-emitting NGR-based anti-tumour molecule may constitute a novel, safe therapeutic alternative in the existing treatment avenue of fibrosarcoma. As the evolution of BW is dependent upon a myriad of factors; however, additional long-term studies are warranted to confirm our hypothesis.
A statistically meaningful disparity was observed between the tumour volumes of the untreated and the treated mice on the 9th, 10th and 12th days post-tumour cell implantation (p ≤ 0.01). Albeit, no remarkable disproportion was observed between the tumour progression of the two groups on the remaining experimental days (p ≤ 0.05, on day 7 and 8). As no significant alterations of tumour bulk were assessed during the administration of the first two therapies, we may draw the conclusion that at least two treatment regimes are necessary to induce measurable changes regarding tumour growth. Given the relatively permanent weight of the tumourous animals during the investigation and the experienced remarkable difference between the tumour volume of the treated and the untreated mice on the 9th, 10th, and 12th experimental days, we may draw the conclusion that the reduction of the tumour mass was not as significant in terms of affecting the BW of the study animals. In addition, the fact that the consistency of the tumours might become more compact may underpin why the meaningful tumour size reduction did not impact tumour weight. Taking into account the above remarked findings, application of [ 213 Bi]Bi-DOTAGA-cKNGRE appears to be an efficient tool in targeted fibrosarcoma treatment.
Although no previous research has been published so far on the administration of NGR-containing radioactive molecules in the treatment of fibrosarcoma, Maggiorella et al. dealt with the treatment of HT1080 tumours at preclinical level [38]. In their study, hafnium oxide-containing NBTXR3 nanopartices (NPs) were designed to broaden the therapeutic window of radiotherapy-delivered with a cobalt-60 source-in nude NMRI mice bearing HT1080 tumours. Notable enhancement (mean dose enhancement factor at 4 and 8 Gy above 1.5) in the radiation response of HT1080 tumourous mice was observed applying irradiation-activated NPs. Further no clonogenic toxicity associated with the intratumourally injected NPs was remarked in the tumour-bearing study mice.
To the best of our knowledge, this is the first study to evaluate the anti-cancer competence of radiolabelled, APN/CD13-affine NGR-based molecules. Peptide-selective therapeutic probes appended with alpha-emitting 213 Bi ensure directed tumour cytotoxicity sparing the surrounding intact tissue. Lack of unwanted side effects of the healthy organs together with no treatment-related BW reduction emphasise the safety of the labelled derivates. However, their future progression from bench-to-bedside could be hampered by some constraints around the usage of 213 Bi. Given the short half-life of the radiometal, problems with radiotracer transfer may arise. In addition, peptide selection must also be adjusted to the physiological half-life of 213 Bi. A constructive logistic approach is warranted to overcome these limitations to exploit the beneficial therapeutic effects of 213 Bi.
Relatively moderate tracer uptake registered at 30 min p.i. in the intestines showed a statistically significant decline until the termination of the ex vivo studies (p ≤ 0.01). The observed gastrointestinal (GIT) activity could be attributed to the physiological CD13 expression of the intestinal epithelial cells [40,41]. This uptake kinetics of the digestive organs may indicate the existence of a GIT way of elimination beyond renal clearance. We hypothesise that the rising radioactivity of the liver (0.72 ± 0.35 and 0.89 ± 0.64; 30 and 90 min after the injection, respectively) may be partially explained by reticuloendothelial cell (RES)-mediated radiotracer uptake and subsequent prolonged radiopharmaceutical retention in the parenchyma of the liver. Although no existing research findings support our assumption, we presume that the molecular size and the physicochemical properties of the investigated probes induce RES-directed tracer accumulation. We further suggest that hepatic vascularisation and factors associated with the metabolism of the liver could also underpin the reason behind the enhanced hepatic uptake. Albeit, long-term, unbiased future studies are warranted to strengthen our hypotheses. Moreover, excretion through the hepatobiliary system could also be supposed based on the elevated liver tracer accumulation. The statistically considerable drop of blood radioisotope uptake from 2.72 ± 0.22 to 0.38 ± 0.33 within 60 min refers to the rapid blood clearance of the investigated probe (p < 0.01). In a similar way, prior research study evaluating the ex vivo uptake kinetics of 68 Ga-appended NGR-containing radioligands-[ 68 Ga]Ga-NODAGA-NGR and [ 68 Ga]Ga-NOTA-(NGR) 2 -also encountered fast elimination from the blood pool [42].
In our study, the radioactivity was almost entirely cleared from the muscle within 1.5 h. We suppose that factors related to the vasculature could probably undermine the rapid clearance from the muscle tissue. Nevertheless, the experienced statistically (p < 0.01) remarkable decrease of the faint pancreatic radiopharmaceutical accumulation over the examination period could be connected to the tracer metabolism. In a like manner, former ex vivo research comparing the pancreatic uptake of [ 64 Cu]Cu-NOTA-RGD-NGR between a KCH genetically engineered mouse model bearing integrin α V β 3 and CD13pos. pancreatic ductal adenocarcinoma (PDAC) and healthy control counterparts with normal pancreas revealed insignificant radioactivity in the tumour naïve pancreas of the control cohort [1]. Our organ distribution analyses showed the second highest [ 213 Bi]Bi-DOTAGA-cKNGRE accumulation in the lungs. Relatively high activities of the lungs with uptake figures of 2.09 ± 0.74 and 0.71 ± 0.44 measured 30 and 90 min p.i., respectively, refer to the pulmonary retention of the labelled probe. Based on the research findings of Hajdu et al. we may suppose that the prolonged dissemination of the hydrophilic [ 213 Bi]Bi-DOTAGA-cKNGRE in pulmonary chambers with water content may underlie this notable tracer uptake [43]. The entrance of the probe to the central nervous system could be hampered by the bloodbrain barrier causing insignificant brain activity.
The tumour-targeting potential of [ 213 Bi]Bi-DOTAGA-cKNGRE was also evaluated in HT1080 tumour-bearing CB17 mice. The labelled derivate accumulated rapidly in the tumours producing images of adequate equality 1.5 h post-administration that confirmed the APN/CD13 affinity of the radio-conjugated derivative. Although the tumourous tracer activity decreased from 30 to 90 min post-administration, relatively higher accumulation of the HT1080 neoplasms compared with the other organs at 90 min time point yields better tumour-to-background (T/M) ratios and image contrast. The tumour-to-organs ratio is of particular concern in terms of diagnostics since more contrasted images ensure precise lesion identification as well as localisation. In accordance with our results, Shao et al. also observed tumour-specific prompt accumulation (4.96 ± 3.18 ID%/g) of CD13-selective [ 68 Ga]Ga-NOTA-G 3 -NGR with a comparable contrast level to that of ours investigating CD13pos. HT1080 fibrosarcoma-bearing nude mice with static microPET imaging [39]. Moreover, other studies also noted the diagnostic efficacy of NGR-based peptides radiolabelled with 68 Ga in well-differentiated SMMC 7721-based APNpos. hepatocellular carcinoma (HCC) and CD13 upregulated HT1080-bearing nude BALB/c mice (%ID/g: 2.17 ± 0.21 and 2.46 ± 0.23 for the HCC and the HT1080 tumours, respectively, (p = 0.18 > 0.05) [44]. Additional investigations also strengthened the imaging potential/diagnostic potential of 68 Ga-labelled NGR sequence-based PET probes linked to different chelators including DOTA, DOTAGA, NODAGA and N, N -bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N -diacetic acid (HBED-CC) in CD13pos. A549 (human lung small cell carcinoma) and HT1080 preclinical tumour models [45][46][47]. Based on these results we can conclude that these novel imaging agents selectively target APN regardless of the type of the chelator.
Since the overexpression of pro-angiogenic cell surface molecules such as CD13 on neoplastic as well as tumour vascular endothelial cells has a central role in cancer-related neo-angiogenesis, radiolabelled APN peptide-targeted ligands could represent a landmark in directed anti-tumour treatment [16]. Given the favourable accumulation profile of [ 213 Bi]Bi-DOTAGA-cKNGRE in HT1080 oncological models, it would be a promising therapeutic drug candidate for incorporation into standard-of-care fibrosarcoma treatment protocols. Beyond therapeutic purposes, concomitant gamma radiation of 213 Bi together with satisfactory image quality make the radiometal applicable for diagnostic usage as well.

Conclusions
Given the adequate binding competence of [ 68 Ga]Ga-DOTAGA-cKNGRE to APN/ CD13-overexpressing neoplasms, this PET imaging probe serves as a silver bullet for the precise identification of receptor pos. primary malignancies as well as pertinent metastases. Our results fuel the view that alpha-emitting 213 Bi-tagged, APN-targeting NGR-motif-[ 213 Bi]Bi-DOTAGA-cKNGRE-represents a breakthrough in current oncological therapies, leading to the ultimate goal of the establishment of bespoke, directed theranostic cancer treatment in the foreseeable future.